WO2024008752A1 - Methods to increase iron content in plants - Google Patents

Abstract

The invention relates to genetically altered plants, parts thereof or plant cells, where the plants, parts thereof or plant cells are characterised by increased levels of iron. Also described are methods of increasing the levels of iron in a plant, particularly a crop plant as well as food compositions and nutritional supplements obtained from the plants or parts thereof.

Description

Methods to Increase Iron Content in Plants

FIELD OF THE INVENTION

The invention relates to genetically altered plants, parts thereof or plant cells, where the plants, parts thereof or plant cells are characterised by increased levels of iron. Also described are methods of increasing the levels of iron in a plant, particularly a crop plant as well as food compositions and nutritional supplements obtained from the plants or parts thereof.

BACKGROUND

Despite numerous interventions such as supplementation and fortification programmes, iron deficiency remains a significant global health problem. It detrimentally affects health outcomes for vulnerable populations including pregnant women and young children in developing countries with dire economic consequences.

More recently, focus has shifted to increasing iron levels in staple crops and algae as a more sustainable approach to address this number one micronutrient deficiency. While transgenic approaches to improve iron levels have been successful in crops such as wheat, rice, and cassava, the release of these lines has been hindered by regulatory barriers in response to a backlash against genetic modification. An alternative approach is to identify natural or induced variation leading to increased iron levels, which can then be directly deployed or used to inform targeted gene-editing approaches. However, because iron uptake is under strong homeostatic control, very little such variation has been found to date.

Only a small number of mutations in, to the best of our knowledge, four different plant genes are known to cause iron accumulation, but crucially, all the described mutations lead to severe growth defects, which is not the case for the dgl mutation in pea. The four genes and specific mutations in Arabidopsis are: FRD3 (AT3G08040; several loss of function mutants), OPT3 (AT4G16370; several loss of function mutants), BRUTUS/BTS (AT3G18290; RNA silencing and P1147L substitution in the E3 ligase domain) and IDT1/BHLH34 (AT3G23210; the A320V substitution, which is dominant). In the 1980s, W.G. Gottschalk identified an X-ray induced mutant in the pea (Pisum sativum) cultivar Dippes Gelbe Viktoria characterised by premature senescence of leaves and stipules, leading to the associated mutant name degenerative leaves (dgl). Often the leaves display brown spots, although not in all growth conditions. Further work on this mutant demonstrated that the brown spots were a symptom of excessive iron accumulation in the leaves and that the seed also accumulate iron. However, attempts to identify the mutation or at least map it to a specific chromosome have failed (Kneen et al., 1990) and consequently the causative mutation has not been identified to date. In fact, some studies have mis-characterised the dgl phenotype, for example it has been suggested that the dgl phenotype could be attributed to mutations in the OPT3 gene (Garcia et al., 2013), however, this cannot be the case as OPT3 mutants accumulate less iron in their seeds than wild-type plants.

There therefore exists a need to develop organisms, such as plants and algae, that have an increased level of iron. The present invention addresses this need.

SUMMARY OF THE INVENTION

We have identified a causal gene and mutation underpinning iron accumulation in plants. We also demonstrate that the dgl phenotype is caused by a short, in-frame deletion in the iron homoeostasis regulator BRUTUS (also referred to as BTS). Given that previous attempts to assign the dgl mutation to even a specific chromosome had failed, and the combination of iron hyper-accumulation and relatively healthy plant grown was unusual, it was not expected that BRUTUS would be the causative gene.

We have found that the dgl mutation affects a motif that is thought to mediate iron sensing, leading to at least 25-fold more iron in young pea shoots and at least four-fold more iron in seeds. Importantly, the plants have only minor growth defects, whereas null mutants in BTS homologs in model plant species are lethal. The BRUTUS polypeptide is highly conserved in both plants and algae. Therefore, introducing similar mutations into the BRUTUS polypeptide to reduce its iron-sensing function or reduce iron binding can be used to increase the iron content of any plant or alga.

In one aspect, there is provided a genetically altered organism, part thereof or cell, wherein the organism, part thereof or cell comprises one or more mutations in at least one nucleic acid sequence encoding a BRUTUS polypeptide, wherein when the organism is a plant, the BRUTUS polypeptide comprises an amino acid sequence as defined in SEQ ID NO: 4 or a functional variant or homologue thereof, wherein the functional variant has at least 30% overall amino acid sequence identity to SEQ ID NO: 4; and wherein when the organism is an alga, the BRUTUS polypeptide comprises an amino acid sequence as defined in SEQ ID NO: 15 or a functional variant or homologue thereof, wherein the functional variant has at least 30% overall amino acid sequence identity to SEQ ID NO: 15; and wherein the mutation reduces or abolishes binding of iron to the polypeptide, and wherein the organism is a plant or alga.

Preferably, the BRUTUS polypeptide comprises one or more hemerythrin domain(s), wherein there is at least one mutation in at least one hemerythrin domain, wherein the hemerythrin domain comprises a sequence selected from SEQ ID NO: 20, or 50 - 102 or a functional variant thereof. More preferably, the at least one mutation is in the first N- terminal hemerythrin domain.

Preferably, the at least one mutation is a deletion of one or more amino acids. More preferably, at least one mutation is a deletion of between 1 and 50 amino acids, preferably around 5 amino acids, or around 20 amino acids (specifically 22 amino acids in one embodiment). The mutation may be a deletion of SEQ ID NO: 1 , 22, 23, 24, 44, 47, 48, 49 or a variant thereof, wherein the variant has at least 80% overall sequence identity to SEQ ID NO: 1 , 22, 23,24, 44, 47, 48, or 49.

Preferably, the mutation does not significantly affect organism growth or yield.

In another aspect of the invention there is provided a method of increasing iron concentration in an organism, part thereof or cell, the method comprising introducing at least one mutation into at least one nucleic acid sequence encoding BRUTUS wherein when the organism is a plant, the BRUTUS polypeptide comprises an amino acid sequence as defined in SEQ ID NO: 4 or a functional variant or homologue thereof, wherein the functional variant has at least 30% overall amino acid sequence identity to SEQ ID NO: 4; and wherein when the organism is an alga, the BRUTUS polypeptide comprises an amino acid sequence as defined in SEQ ID NO: 15 or a functional variant or homologue thereof, wherein the functional variant has at least 30% overall amino acid sequence identity to SEQ ID NO: 15; and wherein the mutation reduces or abolishes binding of iron to the polypeptide, and wherein the organism is a plant or alga. In another aspect of the invention there is a method of producing an organism, part thereof or cell, wherein the organism, part thereof or cell has an increased iron content, wherein the method comprises introducing at least one mutation into at least one nucleic acid sequence encoding a BRUTUS polypeptide wherein when the organism is a plant, the BRUTUS polypeptide comprises an amino acid sequence as defined in SEQ ID NO: 4 or a functional variant or homologue thereof, wherein the functional variant has at least 30% overall amino acid sequence identity to SEQ ID NO: 4; and wherein when the organism is an alga, the BRUTUS polypeptide comprises an amino acid sequence as defined in SEQ ID NO: 15 or a functional variant or homologue thereof, wherein the functional variant has at least 30% overall amino acid sequence identity to SEQ ID NO: 15; and wherein the mutation reduces or abolishes binding of iron to the polypeptide, and wherein the organism is a plant or alga.

Preferably, the method comprises introducing a mutation into the BRUTUS polypeptide. More preferably the BRUTUS polypeptide comprises one or more hemerythrin domains, and the method comprises introducing at least one mutation in at least one hemerythrin domain, wherein the hemerythrin domain comprises a sequence selected from SEQ ID NO: 20, or 50 to 102 or a functional variant thereof. More preferably the method comprises introducing at least one mutation in the first N-terminal hemerythrin domain.

Preferably the method comprises introducing a mutation which results in a deletion of one or more amino acids. More preferably the method comprises introducing a mutation which results in a deletion of between 1 and 50 amino acids, preferably around 5 amino acids or around 20 amino acids (specifically 22 amino acids in one embodiment). Most preferably at least one mutation is a deletion of SEQ ID NO: 1 , 22, 23, 24, 44, 47, 48, 49 or a variant thereof, wherein the variant has at least 80% overall sequence identity to SEQ ID NO: 1 , 22, 23, 24, 44, 47, 48 or 49. Preferably the mutation does not significantly affect organism growth or yield.

Preferably when the method comprises an organism wherein said organism is a plant, part thereof or cell is a plant, the plant is selected from a monocot or dicot. More preferably the plant is selected from rice, wheat, maize, barley, Brassicas, Medicago soybean, potato and tomato, lettuce and beetroot. Preferably, when the organism is a plant, plant cell or plant part thereof, said plant is a monocot or dicot. More preferably, when the organism is a plant, plant cell or plant part thereof, said plant is selected from rice, wheat, maize, barley, Brassicas, Medicago, soybean, potato and tomato and beetroot. Preferably, the plant part thereof is a seed or a vegetative part of the plant, preferably selected from the root, shoot or leaves. The plant part of the invention may also comprise one or more mutations in a BRUTUS polypeptide as described herein. Preferably therefore the plant part has an increased iron content.

When the organism, part thereof or cell is an alga, preferably the alga is selected from Chlorella (such as Auxenochlorella protothecoides), Porphyra, Dulse, Laminaria, Alaria, Nostoc, Monostroma, Ulva, Enteromorpha, Caulerpa racemose and Durvillaea antarctica.

In another aspect of the invention there is a method of screening a population of plants and identifying and/or selecting an organism that will have an increased iron content, the method comprising detecting in the organism or organism germplasm at least one polymorphism in a BRUTUS gene, wherein the BRUTUS gene encodes a polypeptide as defined in SEQ ID NO: 4 or 15 or a functional variant or homologue thereof, wherein the functional variant has at least 30% overall sequence identity to SEQ ID NO: 4 or 15; and wherein the BRUTUS polypeptide comprises at least one hemerythrin domain, and wherein the at least one polymorphism is in at least one hemerythrin domain.

Preferably the polymorphism is a deletion of one or more, preferably around five amino acids, or around 20 amino acids (specifically 22 amino acids in one embodiment) in at least one hemerythrin domain of the BRUTUS polypeptide.

In another aspect of the invention there is a method of producing a food, vitamin or nutritional supplement, the method comprising producing a genetically altered organism, and producing a food composition, vitamin or nutritional supplement from the organism or part thereof.

In another aspect of the invention there is a food, vitamin or nutritional supplement obtained or obtainable by the method of the invention. In another aspect of the invention there is a method of treating anaemia in a patient in need of iron, the method comprising consuming the seed or other plant part of the genetically altered plant or the food, vitamin or nutritional supplement derived from the genetically altered plant, part thereof or plant cell.

DESCRIPTION OF THE FIGURES

The invention is further described in the following non-limiting figures:

Figure 1 shows the phenotypic characteristics associated with the dgl mutation. A. The pea cultivar Sparkle, which serves as the wild-type line (left) and the same cultivar but carrying the dgl mutation (right). Upper panel: 4-week-old plants grown on F2 Levington compost in the greenhouse. Lower panel: Leaves consisting of two leaflets of Sparkle wild type (left) and the dgl mutant (right). Note the brown spots due to hyperaccumulation of iron (toxicity). B. Iron staining using potassium ferrocyanide, known as Prussian Blue or Peris’ staining, in pieces of the leaf. C. Iron concentration in leaflets, measured 2 weeks apart. Leaflets were dried, weighed and digested in nitric acid and hydrogen peroxide. The digest was diluted with milliQ water to 5% nitric acid and the pH was brought to 7 with ammonium acetate. After reduction of iron with ascorbic acid, ferrous iron was quantitatively detected using the colorimetric chelator ferene. Similar results were obtained with Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES).

Figure 2 shows that the deletion is predicted to remove 5 amino acids from the Hr1 domain, likely affecting iron binding. A. The 15 nucleotide deletion is located in exon 2 of the BRUTUS gene and removes 5 semi-conserved amino acids in the first hemerythrin (Hr) domain of the polypeptide. Each of the three Hr domains of BRUTUS harbours a diiron centre. Two iron-binding ligands, histidine (H) and glutamate (E) residues, are close to this deletion. B. Modelling predicts that the position of 2 of the 7 iron ligands in the Hr1 domain are shifted, affecting the stability of the di-iron site.

Figure 3 shows that the expression and iron storage in ferritin is highly upregulated in dgl mutant leaves. A. Transcript abundance of the five ferritin genes found in the pea genome. No expression was observed for Psat15g171400 which is therefore likely to be a non-functional gene (pseudogene). The expression of the other 4 genes is significantly increased in dgl vs Sparkle leaves. B. Protein levels of ferritin detected by antibodies using Western blot analysis. The two bands around 25 kDa correspond to two different isoforms of the monomer, processed to slightly different molecular weights after import into the chloroplasts. Twenty-four monomers form a shell surrounding a core of iron, which is in the form of ferrihydrite (oxidized iron with water molecules). The bottom panel shows the total amount of protein in each lane on the blot; the most prominent band is Rubisco large subunit. C. Separation of iron-loaded ferritin and small molecular-weight iron species by native gel electrophoresis, stained for iron with potassium ferrous cyanide. The lower panel shows the total amount of protein in each lane, stained with Coomassie Brilliant Blue).

Figure 4 shows that a small deletion in BRUTUS co-segregates with the high-iron phenotype. A. Detail of the RNA-seq mapping data, showing a 15 nucleotide deletion at the 5’ end of exon 2 (red) in the BRUTUS gene, Psat1g036240. B. PCR analysis to detect the wild-type and dgl alleles. The shorter PCR product with the 15 nt deletion is associated with the dgl mutant and F2 segregates that displayed iron accumulation. A total of 44 sibling F2 plants were tested, of which 11 had the dgl phenotype and invariable the 15-nucleotide deletion in BRUTUS.

Figure 5 shows an amino acid alignment of part of the first hemerythrin domain in the BRUTUS protein in several crops; the high conservation of this domain and the specific 5 amino acids deleted in the dgl mutant makes it a gene-editing target for increased iron. The shown sequence fragments are from Arabidopsis (Athal), Brassica, pea (Psat), rice (Os), tomato (Solyc), barley (Hovul), wheat (Traes) and alga (Auxchl).

Figure 6 shows an amino acid alignment of BRUTUS proteins across different species. Sequences are shown for Arabidopsis (Athal), Brassica, pea (Psat), rice (Os), wheat (Traes) and green alga (chromochloris).

Figure 7 shows that the wild-type pea (Ps) BTS1 coding sequence genetically complements the iron accumulation phenotype of the Arabidopsis bts-3 mutant, but the pea BTS1-dgl sequence does not. A. Detail of rosette leaves stained for iron, imaged by light microscopy, scale bars are 0.2 mm. B. Image of agarose gel electrophoresis following genotyping PCRs confirms the presence of the bts-3 allele and the specific transgenes. Figure 8 shows the proposed mechanism of iron accumulation in dgl (bts1) mutants. Reduced function of the E3 ubiquitin ligase BRUTUS (BTS) leads to constitutive activity of the iron uptake pathway in roots. The pea BTS1 gene is expressed predominantly in shoots in agreement with Arabidopsis BTS promoter activity and high transcript levels in leaves, root stele and embryo cotyledons. Arabidopsis BTS interacts with the transcription factor ILR3 to target it for degradation. Decreased activity of BTS leads to increased levels (and transcriptional activity) of ILR3, which indirectly leads to enhanced transcription of iron uptake genes. Iron accumulates in minor and major veins and also in seeds.

Figure 9 shows an example of a gene-editing strategy in wheat based on the dgl mutation in pea. A. Two guide RNAs were designed, Guide 1 and Guide 2 (magenta), which are predicted to generate an in-frame deletion of 66 nucleotides in between histidine (H, orange) residues that bind the di-iron cofactor (based on homology) in the wheat HRZ1 protein, which is homologous to BTS. The target sequence of the guides is identical in all three gene copies of HRZ1 in bread wheat and is thus predicted to edit all three copies. B. 3D modelling using the neural network Alphafold2 shows that the deletion causes one of the histidine ligands to flip outwards, likely destabilizing the di-iron site (transparent pink spheres). In the pea dgl variant, two iron-binding ligands are predicted to rotated away (not shown here).

Figure 10 shows selected first-generation (TO) wheat plants transformed with the HRZ gene editing constructs which have increased levels of iron in leaves and grains, as well as increased zinc. A. Iron staining using the Peris’ method on leaf sectors and on mature grain cut transversely (top) and along the longitudinal plane (bottom). Iron is evenly distributed in leaves and barely detectable with Peris' staining in wild-type plants. The blue patches indicate significant iron accumulation which is only observed in a very limited number of iron homeostasis mutants. B. Iron and zinc concentrations measured by ICP-OES. The grain of gene-edited wheat plants often have increased zinc levels as well as increased iron. HRZ genes act upstream of the NAS genes which are known to enhance both iron and zinc concentrations when overexpressed. It is important to note that the TO lines are chimeric and that Cas9 activity continues until this is crossed out.

DETAILED DESCRIPTION The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics, which are within the skill of the art. Such techniques are explained fully in the literature.

As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or “gene sequence” is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.

The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

For the purposes of the invention, a “genetically altered organism” is a plant that has been genetically altered compared to a common, naturally occurring wild type (WT) plant. In one embodiment, a genetically altered organism is an organism that has been altered compared to the naturally occurring wild type (WT) plant using a mutagenesis method, such as any of the mutagenesis methods described herein. In one embodiment, the mutagenesis method is targeted genome modification or genome editing. In one embodiment, the genome has been altered compared to wild type sequences using a mutagenesis method. Such organisms have an altered phenotype as described herein, such as an increased iron content. Therefore, in this example, increased iron content is conferred by the presence of an altered genome, for example, the presence of at least one of the described mutations in the BRUTUS gene.

The aspects of the invention involve recombination DNA technology and exclude embodiments that are solely based on generating plants by traditional breeding methods.

The following embodiments apply to all aspects of the invention.

In one aspect of the invention, there is provided a genetically altered organism, part thereof or cell, wherein the organism, part thereof or cell comprises one or more mutations in at least one nucleic acid sequence encoding a BRUTUS polypeptide. Preferably, the mutation is in the endogenous gene. In a preferred embodiment, the organism is not a human.

As shown in the Examples, a plant comprising one of the described mutations in the BRUTUS polypeptide has an increased amount of iron, particularly in vegetative tissues such as the leaves and the roots or in the seeds. Accordingly, the genetically altered organism of the invention is characterised by having an increased amount of iron.

As shown in Example 2, a wheat plant altered to have one of the described mutations in the BRUTUS gene homolog (HRZ), or at least one of the three homeologsin hexapioid bread wheat, has an increased amount of iron and/or zinc, particularly in the grain. Accordingly, the genetically altered organism of the invention can be characterised by having an increased amount of zinc. In a further embodiment, said genetically altered organism has increased levels of iron and zinc.

In another aspect of the invention, there is provided a method of increasing the iron content in an organism, wherein the method comprises introducing at least one mutation into at least one nucleic acid sequence encoding a BRUTUS polypeptide. In a preferred embodiment, the organism is a plant or plant part thereof.

In another aspect of the invention, there is provided a method of increasing the zinc content in an organism, wherein the method comprises introducing at least one mutation into at least one nucleic acid sequence encoding a BRUTUS polypeptide. In a preferred embodiment, the organism is a plant or plant part thereof. In another aspect of the invention, there is provided a method of increasing the iron and zinc content in an organism, wherein the method comprises introducing at least one mutation into at least one nucleic acid sequence encoding a BRUTUS polypeptide. In a preferred embodiment, the organism is a plant or plant part thereof.

In another aspect of the invention, there is provided a method of producing an organism, part thereof or cell, wherein the organism, part thereof or cell has an increased iron content, and wherein the method comprises introducing at least one mutation into at least one nucleic acid sequence encoding a BRUTUS polypeptide. In a preferred embodiment, the organism is not a human

In another aspect of the invention, there is provided a method of producing an organism, part thereof or cell, wherein the organism, part thereof or cell has an increased zinc content, and wherein the method comprises introducing at least one mutation into at least one nucleic acid sequence encoding a BRUTUS polypeptide. In a preferred embodiment, the organism is not a human

In another aspect of the invention, there is provided a method of producing an organism, part thereof or cell, wherein the organism, part thereof or cell has an increased zinc and iron content, and wherein the method comprises introducing at least one mutation into at least one nucleic acid sequence encoding a BRUTUS polypeptide. In a preferred embodiment, the organism is not a human.

As used herein, the terms increased iron “content”, “level”, “concentration” or “amount” can be used interchangeably, and all refer to the amount of iron - preferably in the form of ferric or ferrous iron - or zinc in the genetically altered organism. The iron may be in the form of ferritin (ferric-oxyhydrite), as well as in other forms, for example ferric citrate2 malate2 and ferrous nicotianamine compounds. Where the genetically altered organism is a plant, the amount of iron and/or zinc is preferably increased in vegetative tissues. This includes the roots, stems, shoot buds and leaves, and in particular in the shoots and leaves. Alternatively, or additionally, the amount of iron and/or zinc in the seeds is increased. Iron and/or zinc from either the seeds or vegetative tissue can be extracted and used, for example, as a nutritional supplement or in a food or feed formulation. The amount of iron and/or zinc in the genetically altered organism is increased compared to the amount of iron and/or zinc in a control or wild-type organism, as defined below.

As used herein, an increase may be between 5 and 50-fold more than the amount of iron and/or zinc in the wild-type or control organism. Preferably, said increase is at least 5- fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold or at least 50-fold more than the amount of iron and/or zinc in the control or wild-type plant. In one embodiment, the level of increase is at least 5-fold in the leaves. In another embodiment, the level of increase is at least 2- fold in the seeds or part of the seeds. In one embodiment, said increase is around 25- fold as shown in Figure 1.

The amount of iron in the organism or any part of the organism (e.g. leaf, seed etc. where the organism is a plant) may be measured using standard techniques in the art. In one example, as described in Example 1 , an iron leaf staining assay can be used (as described below and in Meguro et al., 2007). Here, the leaf is incubated with potassium ferrous cyanide in an acid solution of 2% (weight/vol) hydrochloric acid, and left for 2 hours, after which leaf pigments such as chlorophyll are removed by incubation with ethanol. In another example, the first step is to digest the tissue in, for example, nitric acid and hydrogen peroxide, then either dilute the sample with water for Inductively Coupled Plasma-Optical Emission Spectroscopy, or neutralise the digested tissue with for example, ammonium acetate then reduce with, for example, ascorbic acid, and finally quantify the iron concentration with a colorimetric iron chelator and measure absorbance. This can be used to calculate the amount of iron in pg per g of dry weight. In a further example, ICP (Inductively Coupled Plasma-Optical Emission Spectroscopy) is used. This is used for a ‘broad-sweep’ element analysis (iron as well as other elements). Together with ICP-MS (mass spectrometry), ICP-OES is the gold standard for iron measurements in biological tissues. In further examples, atomic absorption spectroscopy (AAS) or X-ray fluorescence (XRF) may be used. Inductively Coupled Plasma-Optical Emission Spectroscopy can be used for a ‘broad-sweep’ element analysis to measure zinc as well as other elements.

In one embodiment, the methods of the invention may further comprise the step of assessing the phenotype of the genetically altered organism, and specifically measuring the amount of iron in the organism, or part thereof or cell, preferably compared to the level in a wild-type or control organism.

The BRUTUS polypeptide is an E3 ubiquitin ligase that negatively regulates the response to iron deficiency by promoting the degradation of iron-responsive transcription factors. BRUTUS polypeptides contain up to three iron-binding hemerythrin domains in the N- terminus and a CHY/RING-type Zn finger domain with ubiquitin E3 ligase activity in the C-terminus. Note that HRZ refers to a BRUTUS orthologue, and the terms BRUTUS and HRZ are therefore used interchangeably herein.

In one embodiment, the BRUTUS polypeptide is characterised by one or more, but preferably three hemerythrin domains, required for sensing the amount of iron in the cell. Each hemerythrin domain is believed to bind to two iron atoms Preferably, the hemerythrin domain or domains are found at the N-terminus of the protein. More preferably the domains are consecutive.

The sequence of the hemerythrin domains are highly conserved in both plants and algae, as shown in Figure 3. In one embodiment, the hemerythrin domain has four alpha-helices binding two iron ions. The sequence may be of a variable length, but contains histidine and glutamate residues required for iron binding spaced as follows: (H)-HxxxE-H-HxxxE.

In one embodiment, the sequence of the hemerythin domain may comprise the following sequence (from the Hr1 domain of the pea BRUTUS protein). Conserved residues are underlined.

Psat1g036240 Hr1 (SEQ ID NO: 20)

SPILIFSFFQKAIGNELDALHRLAMAFATGNCSDIQPLSERYHFLRSMYRHHSNAEDEV IFPALDKRVKNVAQTYSLEHKGESDLFDHLFELLNSSVDNDETFRRELASCTGALQTS LSQHMAKEQQQVFP

In another embodiment, the sequence of the hemerythin domain may comprise any of the sequences defined in SEQ ID NO: 50 to 102 or a functional variant thereof. Accordingly, a homologue or functional variant may be defined as comprising at least one hemerythin domain. This domain may comprise SEQ ID NO: 20, or any of SEQ ID NO: 50 to 102 or a functional variant thereof, preferably a sequence with at least 70%, more preferably 80%, even more preferably 90% or more overall sequence identity to SEQ ID NO: 20, or any of SEQ ID NO: 50 to 102. As used here, a functional variant of a hemerythrin domain is a domain that can bind iron as described herein.

The BRUTUS polypeptide may also comprise at least one CHY/RING-type Zn finger domain. Accordingly, a functional variant or homolog can be alternatively or additionally defined as comprising a CHY/RING-type Zn finger domain. In one embodiment, the CHY/RING-type Zn finger domain comprises the following sequence or a functional variant thereof:

CEHYKRNCKLRAACCDQLFTCRFCHDKVSDHSMDRKLVTEMLCMRCLKVQPVGPIC TTPSCDGFPMAKHYCSICKLFDDERAVYHCPFCNLCRVGEGLGIDFFHCMTCNCCLG MKLVNHKCLEKSLETNCPICCEFLFTSSEAVRALPCGHYMHSACFQAYTCSHYTCPIC GKSLGDMAVYFGMLDALLAAEELPEEYKNRCQDILCNDCERKGTTRFHWLYHKCGS CGSYNTR (SEQ ID NO: 21).

In an alternative embodiment the CHY/RING-type Zn finger domain may comprise the following sequence or a functional variant thereof:

CEHYKRNCKVRAACCGKLFTCRFCHDNNSSDHSMDRKATLEMMCMACMTIQPVGPI CTTPSCNGLSMAKYYCNICKFFDDERNVYHCPFCNICRVGQGLGIDYFHCMKCNCCV GIKSVSHKCLEKGLEMNCPICCDDLFTSSATVRALACGHYMHSSCFQAYTCSHYTCPI CSKSLGDMAVYFGMLDALLAAEQLPEEYRDRSQDILCHDCDRKGISHFHWLYHKCGF CGSYNTR (SEQ ID NO: 41).

As used herein, a functional variant of a CHY/RING-type Zn finger domain retains the ability of the domain to mediate E3 ubiquitin ligase activity.

In one embodiment, the nucleic acid or gene sequence of the BRUTUS gene may comprise or consist of a nucleic acid sequence encoding a polypeptide as defined in SEQ ID NO: 4 or a functional variant or homologue thereof. In one embodiment the sequence of the homologue comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 2 to 3 and 5 to 19 or a functional variant thereof. In one embodiment, the nucleic acid sequence may comprise or consist of SEQ ID NO: 42 or 43 or a functional variant or homologue thereof.

In another embodiment, the amino acid sequence of the BRUTUS protein comprises or consists of a sequence as defined in SEQ ID NO: 4 or a functional variant or homologue thereof. In an embodiment the sequence of the homologue comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 2, 3 or 5 to 19 or a functional variant thereof.

An ‘endogenous’ nucleic acid as used herein may refer to the native or natural sequence in the plant genome. In one embodiment, the endogenous sequence of the BRUTUS gene comprises a sequence that encodes an amino acid sequence as defined in SEQ ID NO: 4. Also included in the scope of this invention are functional variants (as defined herein) and homologs of the above-identified sequences. Examples of BRUTUS homologs are shown in SEQ ID NOs: 2, 3 and 5 to 19 or functional variants thereof. Accordingly, in one embodiment, the homologue is selected from one of the above homologous sequences.

The term “functional variant of a nucleic acid sequence” as used with reference to any SEQ ID NO described herein refers to a variant gene sequence or part of the gene sequence that retains the biological function of the full non-variant sequence (e.g. able to bind iron (e.g. one or more iron molecules) and/or has E3 ubiquitin ligase activity). A functional variant also comprises a variant of the gene of interest, which has sequence alterations that do not affect function, for example in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active. Alterations in a nucleic acid sequence that result in the production of a different amino acid at a given site that do not affect the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

A functional variant as used above also (or alternatively) refers to a variant sequence that has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,

37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %,

52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%,

67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,

97%, 98%, or at least 99% overall sequence identity to any of the SEQ ID NOs described herein, and that preferably is also able to bind iron (e.g. one or more iron atoms) and/or has E3 ubiquitin ligase activity. By “able to bind to iron” is meant that the iron-binding amino acids/motif (e.g. (H)-HxxxE-H-HxxxE) need to be present in the functional variant, and that preferably the amino acids of the iron-binding motif are spaced such that iron can bind. Iron binding can be measured as described below. The overall sequence identity of a variant can be determined using any number of sequence alignment programs known in the art. As an example, Emboss Stretcher from the EMBL-EBI may be used: https://www.ebi.ac.uk/Tools/psa/emboss stretcher/ (using default parameters: pair output format, Matrix BLOSUM62, Gap open = 1 , Gap extend = 1 for proteins; pair

output format, Matrix = DNAfull, Gap open = 16, Gap extend = 4 for nucleotides).

In one embodiment, the functional variant has at least 35 to 40% overall sequence identity to the non-variant sequence. In another embodiment, the functional variant has at least 70% overall sequence identity to the non-variant sequence.

The term homolog, as used herein, also designates a gene orthologue from other plant or algae species. A homolog may have, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,

41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%,

56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,

71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%,

86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the amino acid sequence represented by SEQ ID NO: 4 or to the nucleic acid sequences encoding said sequence. Again, the homolog may be functional, in that the homolog is able to bind iron (e.g. one or more iron atoms) and/or has E3 ubiquitin ligase activity and consequently negatively regulate iron responses. Functional variants of homologs as defined above are also within the scope of the invention.

Two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognised that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Non-limiting examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.

Suitable homologues can be identified by sequence comparisons and identifications of conserved domains, as described above. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function, for example when expressed in a plant and function is assessed (e.g. as described herein, some of the characterising features of BRUTUS include binding to iron and negatively regulating iron responses).

Thus, the nucleotide sequences of the invention and described herein can also be used to isolate corresponding sequences from other organisms, particularly other plants, particularly crop plants and algae. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein. Topology of the sequences and the characteristic domain structure can also be considered when identifying and isolating homologs. Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e. , genomic or cDNA libraries) from a chosen plant. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable group, or any other detectable marker. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

In a further embodiment, a variant as used herein can comprise a nucleic acid sequence encoding a BRUTUS polypeptide as defined herein that is capable of hybridising under stringent conditions, for example as described above, to a nucleic acid sequence as defined in SEQ ID NO: 4.

Therefore, there may be provided a genetically altered plant, part thereof or plant cell, wherein the plant, part thereof or plant cell comprises at least one mutation in at least BRUTUS gene, wherein the BRUTUS gene comprises or consists of a. a nucleic acid sequence encoding a polypeptide as defined in one of SEQ ID NO: 4 or a functional variant or homolog thereof; or b. a nucleic acid sequence as defined in one of SEQ ID NO:42, SEQ ID NO:43 or a functional variant or homolog thereof; or c. a nucleic acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to either (a) or (b); or d. a nucleic acid sequence encoding a BRUTUS polypeptide as defined herein that is capable of hybridising under stringent conditions as defined herein to the nucleic acid sequence of any of (a) to (c).

Preferably, the mutation is in one or more of the hemerythrin domains. For example, in one or more of the hemerythin domains described in SEQ ID NO: 20, or 50 to 102 and functional variants thereof. That is, the mutation is in the nucleic acid sequence that encodes one or more of the hemerythrin domains. Preferably, the one or more mutation prevents or reduces the binding of iron to the hemerythrin domain.

The ability to bind iron may be measured experimentally by (1) purifying the protein from the plant and determining bound iron by (i) UV-visible spectroscopy, in combination with (ii) a valid method to measure iron, such as published colorimetric reagents and ICP- OES or ICP-MS. The full length protein, or the full-length hemethryin domain sequences, can also be expressed in the bacterium Escherichia coli and purified, for example by incorporating an affinity tag and affinity chromatography. The same methods (i) and (ii) can then be applied. In both cases, the native sequence must be expressed and purified alongside the altered sequence, to show that the method itself does not affect iron binding.

“By at least one mutation in at least one copy” is meant that where the BRUTUS gene is present as more than one copy or homologue (with the same or slightly different sequence) there is at least one mutation in at least one gene. Preferably, only one copy of the gene is mutated. Alternatively, all genes are mutated.

The genetically altered organism with at least one mutation in at least one copy of the BRUTUS gene can also act as an environmental iron sensor whereby brown spots appear on an organism with a mutation in the BRUTUS gene when there is sufficient bioavailable iron in the soil and brown spots do not appear on the organism when there is very little bioavailable iron. Accordingly, in another aspect of the invention, there is provided the use of the genetically altered organism of the invention as a biosensor for iron.

In a preferred embodiment, the mutation that is introduced into the endogenous BRUTUS gene can be selected from the following mutation types

1. a "missense mutation", which is a change in the nucleic acid sequence that results in the substitution of an amino acid for another amino acid;

2. a "nonsense mutation" or "STOP codon mutation", which is a change in the nucleic acid sequence that results in the introduction of a premature STOP codon and, thus, the termination of translation (resulting in a truncated protein); plant genes contain the translation stop codons "TGA" (UGA in RNA), "TAA" (UAA in RNA) and "TAG" (UAG in RNA); thus any nucleotide substitution, insertion, deletion which results in one of these codons to be in the mature mRNA being translated (in the reading frame) will terminate translation.

3. an "insertion mutation" of one or more amino acids, due to one or more codons having been added in the coding sequence of the nucleic acid;

4. a "deletion mutation" of one or more amino acids, due to one or more codons having been deleted in the coding sequence of the nucleic acid; 5. a "frameshift mutation", resulting in the nucleic acid sequence being translated in a different frame downstream of the mutation. A frameshift mutation can have various causes, such as the insertion, deletion or duplication of one or more nucleotides.

6. a “splice site” mutation, which is a mutation that results in the insertion, deletion or substitution of a nucleotide at the site of splicing.

As described above, the mutation may be any of the above mutations that reduces or abolishes binding of iron to the polypeptide. In one example, if the hemerythrin domain normally binds two atoms of iron, the mutation may reduce the binding affinity, such that only one iron atom binds to the hemerythrin domain, or alternatively due to cooperativity no iron binds to the hemerythrin domain. In a preferred embodiment, the mutation is any mutation that prevents binding of iron to one or more hemerythrin domain(s). By “abolishing” is meant that the mutated hemerythrin domain of the BRUTUS polypeptide cannot bind iron, or that binding of iron cannot be detected. For example, if the hemerythrin domain normally binds 2 atoms of iron, the mutation may prevent binding of both atoms of iron to the hemerythrin domain. This may in turn result in a reduction in the amount of iron bound to a population of BRUTUS polypeptides. In other words, at the population level, the amount of iron bound to a population of BRUTUS polypeptides is reduced compared to the amount of iron bound to a population of BRUTUS polypeptides that do not contain one of the above-described mutations.

As such, by “reducing” is meant that the total amount of iron bound to a population of BRUTUS polypeptides is reduced by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more compared to the amount if iron bound in a population of BRUTUS polypeptides without one of the mutations described above.

In a preferred embodiment, the mutation is a deletion. As used herein, a “deletion” may refer to the deletion of at least one nucleotide. More preferably, the deletion is in the hemerythin domain. Again, the mutation is the deletion of one or more amino acids where the deletion is sufficient to reduce or abolish binding of iron to the hemerythrin domain(s). Alternatively, the mutation may be a substitution provided again that the substitution is sufficient to reduce or abolish binding of iron to the hemerythrin domain(s). In one embodiment the mutation is a deletion of at least one hemerythrin domain in its entirety. For example, the deletion of one more hemerythin domains as defined in any of SEQ ID NO: 20, or 50 to 102 and functional variants thereof. Alternatively, the mutation is the deletion of between 1 and 50 amino acid residues in at least one hemerythrin domain. For example, where the hemerythin domain is selected from any of SEQ ID NO: 20, or 50 to 102 and functional variants thereof. Preferably, the mutation is the deletion of between 5 and 25 amino acids. More preferably, the mutation is the deletion of around five amino acid residues or around 20 amino acids (in one embodiment 22 amino acid residues).

In one embodiment, the mutation is the deletion of one or more amino acids within the (H)-HxxxE-H-HxxxE motif. For example, as shown in Figure 5, the deletion may include the deletion of one or more of the H/E ligands. In another embodiment, the mutation may selected from one or more of the following: a deletion of one or more amino acids between the first H and the second H in the (H)-HxxxE-H-HxxxE motif; and/or a deletion of one or more amino acids between the first H and the first E in the (H)-HxxxE-H-HxxxE motif; and/or a deletion of one or more amino acids between the first H and the third H in the (H)-HxxxE-H-HxxxE motif; and/or a deletion of one or more amino acids between the first H and the fourth H in the (H)-HxxxE-H-HxxxE motif; and/or a deletion of one or more amino acids between the first H and the second E in the (H)-HxxxE-H-HxxxE motif; and/or a deletion of one or more amino acids between the second H and the first E in the (H)-HxxxE-H-HxxxE motif; and/or a deletion of one or more amino acids between the second H and the third H in the (H)-HxxxE-H-HxxxE motif; and/or a deletion of one or more amino acids between the second H and the fourth H in the (H)-HxxxE-H-HxxxE motif; and/or a deletion of one or more amino acids between the second H and the second E in the (H)-HxxxE-H-HxxxE motif; and/or a deletion of one or more amino acids between the third H and the fourth H in the (H)-HxxxE-H-HxxxE motif; and/or a deletion of one or more amino acids between the third H and the second E in the (H)-HxxxE-H-HxxxE motif; and/or a deletion of one or more amino acids between the fourth H and the second E in the (H)-HxxxE-H-HxxxE motif.

In a preferred embodiment, the mutation is the deletion of one or more amino acids between the third H and the fourth H in the (H)-HxxxE-H-HxxxE motif, as shown in Figure 10.

In one embodiment, the mutation is the deletion of the following amino acids in the hemerythrin domain(s):

QTS/TXS (SEQ ID NO: 103)

Where X can be any amino acid, but preferably selected form L, V or I.

In another embodiment, the mutation may be selected from the deletion of the following amino acid residues in one or more hemerythrin domains:

QTSLS (SEQ ID NO: 1). This is the dgl deletion; or

QTSVS (SEQ ID NO: 22); or

QTCLS (SEQ ID NO: 23); or

QTCLT (SEQ ID NO: 24);

QTFIT (SEQ ID NO: 47);

CTSIS (SEQ ID NO: 48);

RTFIT (SEQ ID NO: 49).

In an embodiment, when the mutation is a deletion of QTSVS (SEQ ID NO: 22) preferably, the plant is Brassica.

In an embodiment, when the mutation is a deletion of QTCLS (SEQ ID NO: 23), preferably the plant is rice.

In an embodiment, when the mutation is a deletion of QTCLT (SEQ ID NO: 24), preferably the plant is selected from barley or wheat. In an embodiment, when the mutation is a deletion of QTFIT (SEQ ID NO: 47), preferably the plant is rice.

In an embodiment, when the mutation is a deletion of CTSIS (SEQ ID NO: 48), preferably the plant is potato.

In an embodiment, when the mutation is a deletion of RTFIT (SEQ ID NO: 49), preferably the plant is selected from barley or wheat.

The dgl deletion identified in the pea ortholog of BRUTUS is located within the first hemerythrin domain, near to the second iron-binding HxxxE motif. The deletions described here are predicted to rotate this HxxxE motif away from the other two iron ligands, diminishing effective iron binding (Fig 2B). This in turn would directly or indirectly supress the ubiquitination activity of BRUTUS.

In another embodiment, the mutation may be the deletion of some or all of the following amino acids (or corresponding amino acids in a homologous sequence) in one or more of the hemerythrin domains:

LLALLQLDIQNDDALRRELASC (SEQ ID NO: 44).

Again, preferably this mutation disrupts the orientation of the iron binding ligands in at least one hemerythrin domain. Preferably, this diminishes effective iron binding.

In another preferred embodiment, the mutation disrupts the orientation of at least one histidine residue required for iron binding in the hemerythrin domain, preferably diminishing effective iron binding, as illustrated in Figure 6B.

In another preferred embodiment, the mutation leads to increased iron and/or zinc content in an organism.

In a further preferred embodiment, the mutation does not affect the expression levels or preferably, transcript levels of the BRUTUS polypeptide. In a preferred embodiment the mutation does not significantly affect organism growth or yield. Preferably an effect on organism growth (such as height) or yield (such as seed yield, thousand grain weight etc.) cannot be detected.

The term "yield" in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight. The actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square metres. As an example, yield may comprise a measurement of one or more of (a) biomass (weight) of one or more parts of a plant, aboveground (harvestable parts), or root biomass, root volume, root length, root diameter or root length or biomass of any other harvestable part (b) seed yield per plant, which may comprise one or more of seed biomass (weight) per plant or an individual basis, (c) seed filling rate, (d) number of filled seeds, (e) harvest index, which may be expressed as a ratio of the yield of harvestable parts such as seeds over the total biomass, (f) viability/germination efficiency, (g) number or size or weight of seeds or pods or beans or grain (h) seed volume (which may be a result of a change in the composition (i.e. lipid (also referred to herein as oil)), protein, and carbohydrate total content and composition), (i) (individual or average) seed area, (j) (individual or average) seed length, (k) (individual or average) seed width, (I) (individual or average) seed perimeter, (m) growth or branching, for example inflorescences, (n) fresh weight or grain fill, (o) ear weight; and (p) thousand kernel weight (TKW), which may be taken from the number of filled seeds counted and their total weight.

Importantly, it has been found that by introducing the mutations described above into the BRUTUS polypeptide, iron levels (in for example, the shoot, root and seed) are significantly increased without a significant effect on plant yield. Accordingly, in a further embodiment of the invention, the genetically altered organism of the invention comprising the described mutations in the BRUTUS polypeptide do not have a significant growth or yield penalty. In other words, the organisms are characterised by no or no significant loss or reduction in the growth of any part of the organism or the overall yield. Furthermore, it may be possible to overcome or minimise any growth or yield penalty caused by the mutation by growing the organism (e.g. plant) by vertical growing system and/or by using hydroponics. These systems have short production cycles and high harvestable yields, meaning that plants with a high iron content can be grown and harvested quickly before growth or yield penalties present. Accordingly, in one embodiment, the method of producing a plant with an increased iron content comprises growing the plant by vertical growing system and/or by using hydroponics. In an embodiment growing the plant by using hydroponics advantageously results in a plant with leaf iron concentrations of up to 5 mg/g dry weight.

In one embodiment, the mutation is introduced using mutagenesis or targeted genome editing. That is, in one embodiment, the invention relates to a method and an organism that has been generated by genetic engineering methods, and where the organism is a plant, does not encompass naturally occurring varieties.

Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events. To achieve effective genome editing via introduction of site-specific DNA DSBs, four major classes of customisable DNA binding proteins can be used: meganucleases derived from microbial mobile genetic elements, ZF nucleases based on eukaryotic transcription factors, transcription activator-like effectors (TALEs) from Xanthomonas bacteria, and the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats). Meganuclease, ZF, and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. Although meganucleases integrate nuclease and DNA-binding domains, ZF and TALE proteins consist of individual modules targeting 3 or 1 nucleotides (nt) of DNA, respectively. ZFs and TALEs can be assembled in desired combinations and attached to the nuclease domain of Fokl to direct nucleolytic activity toward specific genomic loci.

In a preferred embodiment, the genome editing method used according to the various aspects of the invention is CRISPR. The use of this technology in genome editing is well described in the art, for example in US 8,697,359 and references cited herein. In short, CRISPR is a microbial nuclease system involved in defence against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR- associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage (sgRNA). Three types (I- III) of CRISPR systems have been identified across a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers). The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer. In alternative embodiments Cas12a can be used to mediate cleavage of target DNA.

Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. For applications in eukaryotic organisms, codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, have been used. Alternatively, Cpf 1 , which is another Cas protein, can be used as the endonuclease. Cpf1 differs from Cas9 in several ways: Cpf1 requires a T-rich PAM sequence (TTTV) for target recognition, Cpf1 does not require a tracrRNA, (i.e. only a crRNA is required) and the Cpf 1 -cleavage site is located distal and downstream to the PAM sequence in the protospacer sequence (Li et al., 2017). Furthermore, after identification of the PAM motif, Cpf1 introduces a sticky-end-like DNA double-stranded break with several nucleotides of overhang. As such, the CRISPR/CPf1 system consists of a Cpf1 enzyme and a crRNA. In a further alternative embodiment, the nuclease may be MAD7.

The single guide RNA (sgRNA) is the second component of the CRISPR/Cas(Cpf or MAD7) system that forms a complex with the Cas9/Cpf1/MAD7 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5’end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20 bp. Examples of sgRNA sequences that can be used to generate genetically altered plants of the invention are described below.

CTTGAAATGACGATGCTCTTCGGA (SEQ ID NO: 25) AAACTCCGAAGAGCATCGTCATTT (SEQ ID NO: 26) CTTGCTGGGTGAGGCACGTTTGTA (SEQ ID NO: 27) AAACTACAAACGTGCCTCACCCAG (SEQ ID NO: 28) CTTGTTGTAATAAAGCAAGAAGCT (SEQ ID NO: 29) AAACAGCTTCTTGCTTTATTACAA (SEQ ID NO: 30) CTTGCGTTTGTATGGCTCCTGTGC (SEQ ID NO: 31) AAACGCACAGGAGCCATACAAACG (SEQ ID NO: 32) CTTGTTGACTCGAATATCGAGTGC (SEQ ID NO: 33) AAACGCACTCGATATTCGAGTCAA (SEQ ID NO: 34) CTTGCGTATTCCCTTGAGCACAAA (SEQ ID NO: 35) AAACTTTGTGCTCAAGGGAATACG (SEQ ID NO: 36) CTTGTATTCCCTTGAGCACAAAA (SEQ ID NO: 37) AAACTTTTGTGCTCAAGGGAATA (SEQ ID NO: 38) CTTGATTTTCCCTTTTGTGCTCAA (SEQ ID NO: 39) AAACTTGAGCACAAAAGGGAAAAT (SEQ ID NO: 40) AGCTTCTTGCTTTATTACAA (SEQ ID NO: 45) GCACAGGAGCCATACAAACG (SEQ ID NO: 46)

Cas9 (or Cpf1/MAD7) expression plasmids for use in the methods of the invention can be constructed as described in the art. Cas9 or Cpf1 or MAD7 and the one or more sgRNA molecules may be delivered as separate or as single constructs. Where separate constructs are used for the delivery of the CRISPR enzyme (i.e. Cas9 or Cpf1 or MAD7) and the sgRNA molecule (s), the promoters used to drive expression of the CRISPR enzyme/sgRNA molecule may be the same or different. In one embodiment, RNA polymerase (Pol) Il-dependent promoters or the CaMV35S promoter can be used to drive expression of the CRISPR enzyme. In another embodiment, Pol Ill-dependent promoters, such as U6 or U3, can be used to drive expression of the sgRNA.

Accordingly, using techniques known in the art (such as https://chopchop.cbu.uib.no/) it is possible to design sgRNA molecules that target a BRUTUS gene sequence as described herein. In one embodiment, the sgRNA molecules target a sequence selected from SEQ ID No: 20 or a variant thereof as defined herein. In one embodiment, the method described herein uses the sgRNA constructs defined in detail below to introduce a targeted mutation, such as the mutations described specifically above, into a BRUTUS gene.

Thus, aspects of the invention involve targeted mutagenesis methods, specifically genome editing, and in a preferred embodiment exclude embodiments that are solely based on generating plants by traditional breeding methods.

The genome editing constructs may be introduced into a plant cell using any suitable method known to the skilled person (the term “introduced” can be used interchangeably with “transformation”). In an alternative embodiment, any of the nucleic acid constructs described herein may be first transcribed to form a preassembled Cas9 (or other CRISP nuclease)-sgRNA ribonucleoprotein and then delivered to at least one plant cell using any of the above described methods, such as lipofection, electroporation, holistic bombardment or microinjection.

Specific protocols for using the above-described CRISPR constructs are well known to the skilled person. As one example, a suitable protocol is described in Ma & Liu (“CRISPR/Cas-based multiplex genome editing in monocot and dicot plants”) incorporated herein by reference.

The term "introduction" or "transformation" as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a CRISPR construct and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The construct may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed cell may then be used to regenerate a transformed organism.

Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may also be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation.

Optionally, to select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. In one example, plant embryos are subjected to transformation and plantlets are regenerated from the transformed embryos. In another example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. A suitable marker can be hygromycin, bar-phosphinothricin or PPT. Alternatively, the transformed plants are screened for the presence of a selectable marker, such as, but not limited to, GFP, GUS (P-glucuronidase). Other examples would be readily known to the skilled person. Alternatively, no selection is performed, and the seeds obtained in the above-described manner are planted and grown and the iron content measured. This alternative, which avoids the introduction of transgenes, is preferable to produce transgene-free plants. Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using PCR, to detect the presence of the mutation gene of interest. Alternatively or additionally, integration and expression levels of the newly introduced DNA may be monitored using Southern, Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.

The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.

In a further related aspect of the invention, there is also provided, a method of obtaining a genetically modified organism as described herein, the method comprising a. selecting a part of the organism; b. transfecting at least one cell of the part of the organism of paragraph (a) with at least one nucleic acid construct encoding a sgRNA or at least one sgRNA molecule, using the transfection or transformation techniques described above; c. regenerating at least one organism derived from the transfected cell or cells; d. selecting one or more organisms obtained according to paragraph (c) that have an increased iron and/or zinc content.

In another aspect of the invention, there is also provided, a method of increasing iron and/or zinc content in an organism, a part thereof or one or more cells, the method comprising a. selecting a part of the organism; b. transfecting at least one cell of the part of the organism of paragraph (a) with at least one nucleic acid construct encoding a sgRNA or at least one sgRNA molecule, using the transfection or transformation techniques described above; c. regenerating at least one organism derived from the transfected cell or cells; d. selecting one or more organisms obtained according to paragraph (c) that have an increased iron and/or zinc content. In a further embodiment, the method also comprises the step of screening the genetically modified plant for SSN (preferably CRISPR)-induced mutations in the BRUTUS gene sequence. In one embodiment, the method comprises obtaining a DNA sample from a transformed plant and carrying out DNA amplification to detect a mutation in at least one BRUTUS gene sequence.

An organism obtained or obtainable by the methods described above is also within the scope of the invention.

In a further embodiment of any of the above-described methods, the method may further comprise producing a food composition or food product, vitamin or nutritional supplement from the selected organism or part thereof. In one embodiment, the food composition is the seed. In an alternative embodiment, the food composition may be a flour of any product derivable from a seed. In another embodiment the food composition, vitamin or nutritional supplement is a vegetative part of the plant, or a composition comprising a vegetative part of the plant, or any product derivable from a vegetative part of the plant.

Accordingly, in a further aspect of the invention, there is provided a food, vitamin or nutritional supplement obtained or obtainable by the method of the invention.

In another example, the product derivable from the plant part is a plant-based meat.

In another aspect of the invention, there is provided a method of treating anaemia or iron-deficiency in an individual in need thereof, the method comprising consuming the food, vitamin or nutritional supplement as described above, or where the organism is a plant, by consuming the seed or vegetative part of the genetically altered plant such as the leaves.

In a further aspect of the invention, there is provided a method for screening a population of plants and identifying and/or selecting an organism that will have an increased iron and/or zinc content, the method comprising detecting in the organism germplasm at least one polymorphism in a BRUTUS gene, where the polymorphism is one of the abovedescribed mutations. Suitable tests for assessing the presence of a polymorphism would be well known to the skilled person, and include but are not limited to, Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats (SSRs-which are also referred to as Microsatellites), and Single Nucleotide Polymorphisms (SNPs). In one embodiment, Kompetitive Allele Specific PCR (KASP) genotyping is used.

In one embodiment, the method comprises a) obtaining a nucleic acid sample from the organism; and b) carrying out nucleic acid amplification of one or more of BRUTUS genes using one or more primer pairs.

In a further embodiment, the method may further comprise introgressing the chromosomal region comprising at least one of said BRUTUS polymorphisms or the chromosomal region containing the mutated residue as described above into a second organism germplasm to produce an introgressed germplasm, wherein said second organism will also display an increased iron and/or zinc content.

In a preferred embodiment, the organism is a plant or an alga.

A plant according to all aspects of the invention described herein may be a monocot or a dicot plant.

A dicot plant may be selected from the families including, but not limited to Apiaceae, Asteraceae, Brassicaceae (eg Brassica napus, Brassica oleracea), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from carrot, parsnip, beetroot, cassava, tomato, soybean, lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, potato, yam, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, field (fava) bean, pea, Medicago, lentil, peanut, chickpea, apricots, pears, peach, grape vine or citrus species. A monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop. Examples of a crop plant include wheat, rice, barley, maize, oat, sorghum, rye, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species, or a crop such as onion, leek, yam or banana.

In one embodiment, the plant is a legume or an edible legume. In another embodiment, the plant is a plant that produces edible seeds. Examples include pea, chickpea, lentil, legume pea or member of the bean family (optionally kidney, navy, pinto, black, cannellini), lupin, soybean, or the like. In one embodiment, the plant is a pea, but is not the dgl line from Pisum sativum, and in particular not from the Dippes Gelbe Viktoria cultivar or the Sparkle cultivar.

In another embodiment, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption. Examples of crop plant include rice, wheat, maize, barley, Brassicas, soybean, potato and tomato.

In another embodiment, the plant is a plant that can be grown in a vertical growing system. Examples of such plants that can be grown in these systems include bean sprouts, lettuce, kale, chard and collard greens, chives, mint and basil.

The term "plant" as used herein encompasses whole plants and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the nucleic acid constructs or mutations as described herein.

The invention also extends to harvestable parts of a plant of the invention as described herein, but not limited to seeds, grain, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. In a particularly preferred embodiment, the plant part is a legume seed. As used herein a “seed” and “bean” may be used interchangeably.

In a most preferred embodiment, the plant part or harvestable product is a seed or grain. Therefore, in a further aspect of the invention, there is provided a seed or grain produced from a genetically altered plant as described herein. Accordingly, in one aspect of the invention there is provided a seed, wherein the seed has an increased iron content as described herein (e.g. the seed contains one or more of the genetic alterations in BRUTUS as described herein). In another aspect of the invention there is provided a seed or grain (such terms are used interchangeably herein), wherein the seed or grain has an increased zinc content as described herein (e.g. the seed contains one or more of the genetic alterations in BRUTUS as described herein). In another aspect of the invention there is provided a seed or grain, wherein the seed or grain has an increased iron and zinc content as described herein (e.g. the seed contains one or more of the genetic alterations in BRUTUS as described herein). Also provided is a progeny plant obtained from the seed as well as seed obtained from that progeny.

In another embodiment, the plant part or harvestable product is the leaf or leaves. Accordingly, in one aspect of the invention there is provided a leaf, wherein the leaf has an increased iron and/or zinc content as described herein (e.g. the seed contains one or more of the genetic alterations described herein).

The aspects of the invention also extend to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, or proteins.

The aspects of the invention also relate to food products, vitamins and food supplements comprising or derived from the plant of the invention or the harvestable product. In one embodiment, the food product may be an animal feed. In another embodiment the food product is plant-based product, such as plant-based meat for human consumption. In another aspect of the invention, there is provided a product derived from a plant as described herein or from a part thereof.

In another aspect of the invention, there is provided a composition comprising the plant part thereof, such as a leaf or part thereof or extract from the plant of the invention and at least one vitamin. For example, the vitamin may be Vitamin C. The composition may be considered to be a nutraceutical. In one embodiment the plant is a legume or edible legume.

In another aspect of the invention, there is provided the use of an organism comprising a mutation in at least one nucleic acid encoding the BRUTUS polypeptide as described herein in agromining. In a further aspect of the invention, there is provided a method of agromining comprising growing an organism (e.g. plant) of the invention and extracting the iron and/or zinc. Agromining refers to the extraction of metals from soil. In the context of the present invention organisms comprising at least one mutation in the BRUTUS polypeptide can be used to extract iron and/or zinc from soil.

In an alternative embodiment, the plant part is pollen, a propagule or progeny of the genetically altered plant described herein. Accordingly, in a further aspect of the invention there is provided pollen, a propagule or progeny produced from a genetically altered plant as described herein.

A control plant as used herein according to all of the aspects of the invention is a plant which has not been modified according to the methods of the invention. In an alternative embodiment, the plant has not been genetically modified, as described above. In one embodiment, the control plant is a wild type plant. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant.

In another embodiment the organism is an alga. In one embodiment, the algae is selected from Chlorella (such as Auxenochlorella protothecoides), Porphyra, Dulse, Laminaria, Alaria, Nostoc, Monostroma, Ulva, Enteromorpha, Caulerpa racemose and Durvillaea antarctica.

Again, a control or wild-type alga is one that has not been modified according to the methods of the invention (e.g. does not contain one of the described mutations in a BRUTUS polypeptide).

While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

The foregoing application, and all documents and sequence accession numbers cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

The invention is now described in the following non-limiting examples.

EXAMPLES

Example 1 :

We obtained seeds for the dgl mutant, which had previously been backcrossed to the wild-type cultivar Sparkle, generating a near isogenic line. Initial phenotyping of the dgl lines confirmed the high-iron phenotype in the leaves, with the characteristic brown spots and iron accumulation (Fig 1 A, B).

We then carried out RNA-Seq on leaf tissue from the dgl and Sparkle lines, with the aim of determining both the transcriptional changes underpinning the high iron phenotype and identifying the causal mutation. Initial GO-term analysis of the genes differentially expressed between the wild type and dgl genotypes demonstrated a strong correlation between the dgl phenotype and changes in iron homoeostasis (Table 1). We identified a total of 86 differentially expressed genes including four of the five ferritin genes in pea, alongside other known iron-responsive genes such as orthologs of vacuolar iron transporters and genes involved in protein degradation. A gene involved in zinc detoxification (PCR2) was also upregulated, whereas various sugar metabolism genes were downregulated.

Table 1 : GO terms over-represented in genes differentially expressed between dgl and Sparkle leaf tissue.

GO term Description Ontology Category Over-represented q-value

G0:0006826 iron ion transport Biological Process 3.59E-07

G0:0006879 cellular iron ion homeostasis Biological Process 4.95E-06

G0:0008199 ferric iron binding Molecular Function 4.95E-06

G0:0055072 iron ion homeostasis Biological Process 4.95E-06

G0:0000041 transition metal ion transport Biological Process 1.28E-05

G0:0030003 cellular cation homeostasis Biological Process 2.25E-05

G0:0055082 cellular chemical homeostasis Biological Process 2.25E-05

G0:0006873 cellular ion homeostasis Biological Process 2.64E-05

G0:0050801 ion homeostasis Biological Process 8.74E-05

G0:0055080 cation homeostasis Biological Process 8.74E-05

G0:0048878 chemical homeostasis Biological Process 2.07E-04

G0:0030001 metal ion transport Biological Process 5.29E-03

Using the obtained RNA sequencing reads, we searched for any sequence polymorphisms within the expressed genes, which could account for the dgl phenotype. Identification of the mutation was not straight-forward because of a dense SNP region on chromosome 6, which suggested that the mutation was present there. Nonetheless, only one small, but high-quality deletion was eventually identified, located on Chromosome 1 in Psat1g036240 (Fig 2A). The 15-bp deletion, located in exon 2, causes an in-frame deletion of five amino acids. PCR marker analysis demonstrated that this deletion segregated consistently with the high iron phenotype in two separate F2 populations (44 and 48 individuals, respectively). We identified this protein as the BRUTUS polypeptide based on sequence homology with the BRUTUS protein from the model species Arabidopsis thaliana. BRUTUS is an E3 ubiquitin ligase, which negatively regulates the response to iron deficiency by promoting the degradation of iron-responsive transcription factors. The dgl deletion identified in the pea ortholog of BRUTUS is located within the first hemerythrin domain, near to the second iron-binding HxxxE motif. Lack of the amino acids is predicted to alter the position of the nearby His and Glu residues that provide 2 of the 7 amino acid ligands to the diiron centre. This in turn is predicted to rotate this HxxxE motif away from the other two iron ligands, diminishing effective iron binding (Fig 2B).

Recombinant expression of Hr1 from wild-type and dgl pea showed decreased stability of the dgl Hr1 polypeptide.

The BRUTUS protein is highly conserved in the green lineage, from algae to higher plants. In rice, lower transcript levels of the homologue OsHRZI led to miss-regulation of iron homeostasis genes, but also a 33% reduction in plant height and a drop in panicle weight per plant of around 90%. In contrast, the identified dgl mutation in PsBRUTUS leads to only a 28% decrease in seed number and 24% in seed weight while showing a 25-fold increase in shoot iron (Fig 1 B) and a four-fold increase in seed iron. This suggests that targeted deletions within the hemerythrin domains of BRUTUS and its orthologs are a useful approach for improving iron levels in crops without excessive effects on plant yields.

No other exome sequence polymorphisms were found in a close range of the 15 nucleotide deletion associated with dgl meaning that it is unlikely that a different yet genetically linked mutation causes the iron accumulating phenotype. Nevertheless, this was tested by complementing the Arabidopsis bts-3 mutant (which carries a point mutation in the E3 ligase domain and accumulates significant amounts of iron and has a severe growth defect) with the coding sequences for wild-type pea BTS1 or the dgl- associated mutant gene. Iron accumulation was suppressed in bts-3 plants transformed with AtBTS or pea BTS1, but not with the dg/-associated variant of BTS1 (Fig. 7B). This shows that pea BTS1 is a functional orthologue of Arabidopsis BTS and that the 15 nucleotide deletion is deleterious for BTS function. Ultimately, this provides evidence that BTS is highly conserved and performs the same function between different species.

The identification of the causal mutation in BRUTUS raises questions regarding the shoot-to-root iron responsive signal, first identified in grafting experiments using the dgl mutant lines. The in-frame deletion in BRUTUS is sufficient to cause constitutive activation of shoot-to-root signalling, inducing ferric reductase activity and root iron uptake despite iron accumulation in the shoot tissue. Perhaps the predicted changes to the iron binding site of the first hemerythin domain reduces or eliminates the responsiveness of BRUTUS protein to cellular iron levels. This would suppress the ubiquitination activity of BRUTUS, with lower turnover rates of transcription factors orchestrating iron uptake in the cell, thus promoting the shoot-to-root signal.

Example 2:

To increase iron content in wheat using genome editing guide RNAs were designed to generate an in frame 66 nucleotide deletion in Hr1 of the wheat HRZ1 gene (wheat BRUTUS homologue) that results in the deletion of 22 amino acids from the protein. These guide RNAs and the deletion they generate is shown in Fig.9A. 3D modelling showed that this 22 amino acid deletion would cause one of the histidine ligands to flip outwards, likely destabilising the di-iron site (Fig.9B).

After carrying out this 22 amino acid deletion in the HRZ gene levels of iron were measured. Levels of iron were noticeably increased in both the lower leaf and flag leaf of gene edited wheat. Notably, if levels of minerals are high in the flag leaf then these minerals will be relocated to the grain during senescence, this is shown in Fig.lOA. As well as increased iron, zinc levels were also elevated in wheat plants transformed with the construct to remove 22 amino acids from the first hemerythrin domain of the HRZ protein. This shows that mutating the HRZ/ BRUTUS gene can increase both iron and zinc accumulation in plants.

Materials and Methods

Plant growth

Pea seeds were grown on Levington’s F2 compost in a temperature-controlled greenhouse and watered as required.

Leaf iron staining and quantification Leaf samples were stained for iron using Peris’ reagent as previously described (Roschzttardtz et al., 2009). For measuring the iron concentrations, dried leaf samples were digested in 0.25 ml nitric acid (69%) and 0.25 ml hydrogen peroxide at 90°C. After neutralization with 15% (w/v) ammonium acetate, samples were reduced with ascorbic acid. Fe2+ was quantified using the colorimetric iron chelator ferene (3-(2-pyridyl)-5,6- bis-[2-(5-furyl-sulfonic acid)]-1 ,2,4-triazine and absorbance measurement at 593 nm.

RNA extraction and Illumina sequencing

Vegetative leaf tissue was sampled from three plants of each genotype (Sparkle and dgl) and snap frozen in liquid N2. The snap-frozen tissue was then ground to a fine powder before RNA extraction using TRIzol® Reagent (ThermoFisher) and DNase treatment with TURBO DNase (ThermoFisher). The quality and quantity of RNA was verified with the Agilent Bioanalyzer RNA 6000 Nano assay before cDNA library preparation (250- 300 bp insert) and Illumina Sequencing (PE 150, Novogene).

Differential expression analysis

Illumina reads were pseudo-aligned against the P. sativum reference transcriptome ( Kreplak et al., 2019) using Kallisto (Bray et al., 2016). Gene expression levels were determined using the R package Sleuth ( Pimentel et al ., 2017)) using the Wald test, where we considered genes to be differentially expressed between genotypes with q < 0.05. Expression levels of the FERRITIN genes were visualised using ggplot2 ( Wickham et al., 2016). Putative Arabidopsis orthologs were identified using NCBI blastp ( Altschul et al., 1990) of the P. sativum gene coding sequences against the Arabidopsis proteome. Enrichment of GO terms was calculated using the R package goseq (Young et al ., 2010).

Identification of deletions

Illumina reads were aligned against the P. sativum reference genome (Krepalk et al., 2019) using the software BWA-mem (Li. ,2013). The software translndel (Yang et al., 2018) was then used to identify the location of deletions and insertions using the default parameters except for DP = 1 (to capture all possible deletions, irrespective of gene expression level).

PCR marker analysis

PCR primers were designed to amplify across the region of Psat1g036240 which contains the identified five amino-acid deletion. DNA was extracted from individual F2 plants as previously (Pallotta et al., 2003). Amplified fragments were run on a 2% (w/v) agarose gel for 2 h at 70 V, allowing separation of the wild-type Sparkle band (334 bp) and the dgl band (319 bp) and thus identification of homozygous and heterozygous individuals.

Protein homology modelling

The first hemerythrin domain of Psat1g036240.1 (amino acids 55 - 184) was modelled against the mammalian FBXL5 hemerythrin domain (PDB 3V5Y) using Phyre² with default settings (Thompson et al., 2012 and Kelley et al., 2015). The protein model was visualised using Jmol (26).

Genetic complementation of Arabidopsis bts-3

Heterozygous bts-3 plants (Hindt et al, 2017) were transformed with plasmid plCSL869550OD (SynBio) carrying either the Arabidopsis BRUTUS coding sequence (BTS, AT3G18290); the pea BRUTUS1 coding sequence (Psat1g036240); or the pea dgl variant of BRUTUS1 (lacking nucleotides 487-510). The coding sequences were placed downstream of the Arabidopsis BRUTUS promoter, nucleotides -1904 to -1 , and upstream of the ocs terminator, using Golden Gate assembly. All constructs were verified by Sanger sequencing and the presence of the bts-3 allele and transgenes were confirmed by genotyping.

Medicago truncatula TILLING

A M2 population of EMS-mutagenized Medicago truncatula was screened for genetic polymorphisms in BTS1/Psat1g036240. For BTS1, primers MtBTS1-F1 and -R spanned exons 7 - 10 to maximize the ration of exomintron sequence. For the selected lines, seedlings were grown up and inspected for phenotypes and iron accumulation using Peris' staining.

SEQUENCE LISTING

SEQ ID NO: 1

QTSLS

SEQ ID N0:2 >Athal_BTS (Hemerythrin domains are highlighted) MATPLPDFETARGGGAVASSSTTVLPSSVSSSSSSSRPLPVANSFSDDAEEISPILIFL

FFHKAVCSELEALHRLALEFATGHHVDLRLLRERYRFLRSIYKHHCNAEDEVIFSALDI

RVKNVAQTYSLEHKGESNLFDHLFELLNSATETDESYRRELARSTGALQTSVSQHLAK

EQKQVFPLLIEKFKYEEQAYIVWRFLCSIPVNMLAVFLPWISSSISVDESKEMQTCLKKI

VPGEKLLQQVIFTWLGGKSNTVASCRIEDSMFQCCLDSSSSMLPCKASREQCACEGS

KIGKRKYPELTNFGSSDTLHPVDEIKLWHKSINKEMKEIADEARKIQLSGDFSDLSAFD

ERLQYIAEVCIFHSLAEDKIIFPAVDGEFSFSEEHDEEENQFNEFRCLIENIKSAGASST

SAAEFYTKLCSHADQIMETIQRHFHNEEIQVLPLARKNFSFKRQQELLYQSLCIMPLRLI

ERVLPWLTASLTEDEAKNFLKNLQAGAPKSDVALVTLFSGWACKGRKAGECLSPNGN

GLCPVKTLSNIKEVNLQSCNACASVPCTSRSTKSCCQHQDKRPAKRTAVLSCEKKTT

PHSTEVANGCKPSGNGRSCCVPDLGVNNNCLELGSLPAAKAMRSSSLNSAAPALNS

SLFIWEMDSNSFGTGHAERPVATIFKFHKAISKDLEFLDVESGKLIDCDGTFIRQFIGRF

HLLWGFYKAHSNAEDDILFPALESKETLHNVSHSYTLDHKQEEKLFGDIYSVLTELSIL

HEKLQSDSMMEDIAQTDTVRTDIDNGDCNKKYNELATKLQGMCKSIKITLDQHIFLEEL

ELWPLFDKHFSIQEQDKIVGRIIGTTGAEVLQSMLPWVTSALSEDEQNRMMDTWKQA

TKNTMFDEWLNECWKGSPDSSSTETSKPSPQKDNDHQEILDQSGELFKPGWKDIFR

MNQNELEAEIRKVYQDSTLDPRRKDYLVQNWRTSRWIAAQQKLPKEAETAVNGDVEL

GCSPSFRDPEKQIYGCEHYKRNCKLRAACCDQLFTCRFCHDKVSDHSMDRKLVTEM

LCMRCLKVQPVGPICTTPSCDGFPMAKHYCSICKLFDDERAVYHCPFCNLCRVGEGL

GIDFFHCMTCNCCLGMKLVNHKCLEKSLETNCPICCEFLFTSSEAVRALPCGHYMHS

ACFQAYTCSHYTCPICGKSLGDMAVYFGMLDALLAAEELPEEYKNRCQDILCNDCER

KGTTRFHWLYHKCGSCGSYNTRVIKSETIPPDCSTSS

SEQ ID NO: 3 >Brassica v1.0|Bol030932 (Hemerythrin domains are highlighted)

MATPLPDFEATRASSSSSTTVISSSVPSSSRPPASNSFSEDAEEISPILFFLFFHKAVCS

ELESLHRLALEFATGHHVDLRLLRERYRFLRSIYKHHCNAEDEVIFSALDIRVKNVAQT

YSLEHKGESTLFDHLFELLNPATEIDESYRRGLASSTGALQTSVSQHLAKEQKQVFPL

LIEKFKHEEQAYIVWRFLCSIPVNMLAVFLPWLASSISVDESKEMQTCLSKIVPDETLLQ

QVIFTWLGRKSDTAASCRIEDSLFQCCMDSSSSMLPCETSRAQCACEGSKVGKRKY

QELTSHDSFDAQMHPIDEIKLWHKSMNKEMKEIADEARKIQLSGDFSDLSAFDERLQY

IAEVCIFHSLAEDKIIFPAVDGEFSFSEEHDEEENQFNEFRCLIENIKSAGASSTSAAEFY

TKLCAHADQIMETIQRHFHNEEIQVLPLARKNFTFKRQQELLYQSLCIMPLRLIERVLP

WLAASLTEDEAKNFLKNLQAGAPKSDAALVTLFSGWACKGRKAGECLSPNGNGLCP

VKTLSNSEVYLQSCNASVSMPCSSRSIESCCQQQDKRPAKRTWSSSENNATPHSSE

GANGSNPSGNGRSCCVPDLGVNSDCLGLGSLPAAKAMRSSSLNSAAPALDSSLFGC EIDSNSFGTGNAERPVATIFAFHKAISKDLEFLDVESGKLIDCDETFIRQFMGRFHLLW GYYKAHSSAEDDILFPALESKEALHNVSHSYTLDHKQEEKLFEDIYSVLTELSMLHEKL

QSDSMMGGVTQTETVHTDIDSGDCKKKYNELATKLQGMCKSIKITLDQHIFLEELELW PLFDEHFSIQEQDKIVGRIIGTTGAEVLQSMLPWVTSALSEDEQNRMMDTWKQATKN

TMFDEWLNECWKGSPDSSSGEASKHSPQRDNDHQEIVDQTGQLFKPGWKDIFRMN QNELEAEIRKVYQDTTLDPRRKDYLVQNWRTSRWIAAQQKLPKETEIALNGDVTLGCT

PSFRDPEKQIFGCEHYKRNCKLRAVCCDQLFTCRFCHDKVSDHSMDRKLVTEMLCM RCLKVQPVGPICTTPSCDGFPMAKHYCSICKLFDDERAVYHCPFCNLCRVGEGLGIDY

FHCMTCNCCLGMKLVNHKCLEKSLETNCPICCEFLFTSSETVRALPCGHYMHSACFQ AYTCSHYTCPICGKTLGDMGVYFGMLDALLAAEELPEEYKDRCQDILCNDCQRKGTT

RFHWLYHKCGSCGSYNTRVIRSETTAQDCPTTS

SEQ ID NO: 4 >Pisum sativum; Psat1g036240.1 (Hemerythrin domains are highlighted) MATPLTGLQQHLDAGGGGVAVLSNLVSSSSPSSTSNGGGGFNRSSLSESPILIFSFFQ KAIGNELDALHRLAMAFATGNCSDIQPLSERYHFLRSMYRHHSNAEDEVIFPALDKRV KNVAQTYSLEHKGESDLFDHLFELLNSSVDNDETFRRELASCTGALQTSLSQHMAKE QQQVFPLLIEKFSVEEQASLVWQFLCSIPVNMMAEFLPWLSTSISPDESQDLRKCLSKI

VPEEKLLQKVIFTWMEGRSSANTAENCVDHSQVQCSACPLANQIEKIKCACESTVSG KRKYSASILDVPETMGSHPIDEILLWHNAIKKELNEIAVETRKIQHSGDYTNLSAFNERL

QFIAEVFIFHSIAEDKVIFPAVDGDFSFFQEHAEEESQFNDFRSLIERILSEEATSSSEVE LYSKLCSHADHIMETIQRHFHNEEVQVLPLARKHFSFKRQRELVYESLCMMPLKLIER

VLPWFVGSLTEDEAEIFLKNIQSAAPAIDSALVTLFSGWACKARKNGQCLSSSASRFC PAKKIGRSSCACALSGKDCSVLAESEGTQRSVKRSILELHKNGDVSKTTENECAQKP

CCGARSCCVPALGVSGNNLGLSSLSAAKSLRSFTSSAPSLNSSLFIWETDSSSCDMG SAERPIDTIFKFHKAIRIDLEYLDVESGKLCDGDGATIRQFTGRFRLLWGLYRAHSNAE

DDIVFPALESKETLHNVSHSYTLDHKAEEKLFEDISCVLSELSVLHEALQRTHMSEDLS ESNLGISEVNDSDDIRKYNELATKLQGMCKSIRVTLDQHIFREELELWPLFGKHFTVEE

QDKIVGRIIGTTGAEVLQSMLPWVTSALTQDEQNQMMDTWKQAAKNTMFNEWLTEC LIESPGSTSQTETSEHSTSQRGAEYQESLNLNDQMFKPGWKDIFRMNQNELESEIRK

VYRDSTLDPRRKAYLVQNLMTSRWIAAQQKLPKSQSGESSNKQIEGCVPSFRDPEKQ VFGCEHYKRNCKVRAACCGKLFTCRFCHDNNSSDHSMDRKATLEMMCMACMTIQP

VGPICTTPSCNGLSMAKYYCNICKFFDDERNVYHCPFCNICRVGQGLGIDYFHCMKC NCCVGIKSVSHKCLEKGLEMNCPICCDDLFTSSATVRALACGHYMHSSCFQAYTCSH

YTCPICSKSLGDMAVYFGMLDALLAAEQLPEEYRDRSQDILCHDCDRKGISHFHWLY HKCGFCGSYNTRVIKSETTNSSCP SEQ ID NO: 5 >Pisum sativum >Psat7gO10880.1 (Hemerythrin domains are highlighted) MATPLDGGGVAVLTNSANKVDSSSALNGGLKCSKLDSPILIFLFFHKAIRNELDVLHRL

AMAFATGNRSDIQPLFERYRFLSSIYRHHSNAEDEVIFPALDRRVKNVAKTYSLEHKG

ESNLFDHLFELLNSSIKNDESFPRELASCTGALQTSVSQHLAKEEEQVFPLLIEKFS.E

EQASLVWQFLCSIPVNMMAEFLPWLSTSISADESQDLRDFLVKIVPEERLLQKVVFTW

MEGRSSVNKIQSSADHSQVCCSSPLTHQAGRVNCVCESTTTGKRKHSGSMQDVSD

ATGTHPIDEILLWHNAIKKELSEIAVETRRIQRSGDFTDISAFNDRLQFIADVCIFHSIAED

KVIFPAVDGEFSFFQEHAEEESQFNDFRCLIESILSEGAASNSEVEFYSKLCSHADHIM

ETIQRHFHNEEVQVLPLARKHFSFRRQCELLYQSLCMMPLKLIERVLPWLVKSLTEEE

ANIFLRNMQFAAPTADSALVTLFSGWACKARNEGLCLSSGTSDCCPAQRLSDIEEDID

QPSCVCSSASSCRHCSVILESDGNKRPVKRNTLKLSNGDVPETLETESIPKQCFSPRS

CCVPGLGVNSNNLGLSSVSTTKSLRSLSFSSSAPSLNSSLFIWEAESSSCDVGSAERP

IDTIFKFHKAIRKDLEYLDVESGKLSDSDETVIRQFSGRFRLLWGLYRAHSNAEDDIVFP

ALESKEALHNVSHSYMLDHKQEEQLFEDISCVLSEFFVLHEALQLTHMAGDLIDSNFG

TSDTNDSDGVKKYNELATKLQGMCKSIRVTLDQHIFREECELWPLFGKHFTVEEQDKI

VGRIIGTTGAEVLQSMLPWVTSALTQDEQNKMMDTWKQATKNTMFNEWLNECWKE

SPESITQTETSHCSTSHRGSEYQECLDHNDQMFKPGWKDIFRMNQTELESEIRKVYR

DSTLDPRRKAYLVQNLLTSRWIASQQKSPKAPSEEGLSNGVEIEGHSPSFRDPRKLVF

GCEHYKRNCKLRAACCGKLFTCRFCHDNVSDHSMDRKATSEMMCMRCMNIQPIGSI

CMTPSCNALSMAKYYCSICKFFDDERNVYHCPFCNLCRVGRGLGIDYFHCMKCNCCL

GIKTSSHKCLEKGLEMNCPICCDDLFTSSATVRAQPCGHYMHSACFQAYTCSHYTCPI

CSKSLGDMAVYFGMLDALLAAEELPEEYRDRYQDILCNDCDRKGTSRFHWLYHKCG SCGSYNTRLIKRETR

SEQ ID NO: 6 Potato >S. tuberosum v6.1 |Soltu.DM.05G003350.2 (Hemerythrin domains are highlighted)

MATPLTTTGGGGIQGVGGGGGGGGGVAVMSGTTTVGHVEQSGTLNSSRAVGVKGS

SPIRIFLFFHKAIRKELDGLHRSAMAFATNQDTEIKPFMERCYFLRSIYKHHCNAEDEVI

FPALDIRVKNVARTYSLEHEGEGVLFDHLFALLDSDMQSEESYRRELASCTGALQTSI

SQHMSKEEEQVLPLLMEKFSFEEQASLVWQFLCSIPVNMMAEFLPWLSSSISADECK

DMHKCLHKVIPDEDLLQEIMFTWMDGKKLTNKRKACEESTTHNSSDSVVRGLIGQAE

NVPCPCESSRREFPVSNLDLKESTLNLPVDEILHWHKAIRKELNDITEAAREIKLRGDF

SDLSAFNQRLQFIAEVCIFHSIAEDKVIFPAVDAEISFAQEHAEEENEFDKFRCLIESVQ

SAGSNSTSVEFYSELCSQADHIMETVERHFCNEEAQVLPLARKHFSPKRQRELLYQS LCVMPLRLIECVLPWLVGSLSEEEARSFLQNMHMAAPASDTALVTLFSGWACKGRPA

DICLSSSVTGCCPAKILAGNQENLGKCCGTCTSSRIVKSSSSNGEQSNGERPTKRVNL

MSEEKCYRHDPSGGGKFRKGSTGNQSCCVPALGVVNSLAAAKSSRTFTTSAPSLNS

CLFNWNTSLTNAGYATRPIDNIFQFHKAIRKDLEFLDVESGKLTDCDETFLRKFCGRFR

LLRGLYKAHSNAEDDIVFPALESKETLHNVSHSYTLDHKQEEKLFEDISSALDELSQLR

ENLNGGSSVKGPCRNSGACDLHEYSRKYNELATKVQAMCKSIKVTLDQHVIREEVEL

WPLFDRHFSIEEQDKLVGRIIGTTGAEVLQSMLPWVTTALTQDEQNKMMETWKQATK

NTMFSEWLNEWWEGTPDGTSQASSSEDIVSRGCEFPESLEQSDSTFKPGWKDIFRM

NQNELESEIRKVSRDSSLDPRRKAYLIQNLMTSRWIAAQQESEARSVETSNGQDQIG

CSPSFRDPDKQVLGCEHYKRNCKLRAACCGKLFPCRFCHDKVSDHSMDRKATTEM

MCMNCLKVQPVGPTCTTPSCNGLSMAKYYCSSCKFFDDERTVYHCPFCNLCRLGQG

LGVDFFHCMTCNCCLGMKLVDHKCREKGLETNCPICCDFLFTSSETVRGLPCGHFMH

SACFQAYACTHYICPICSKSMGDMSVYFGMLDALMASEVLPEEFRNRCQDILCNDCG

KRGTAPFHWLYHKCASCGSYNTRVIKVETSPNCSS

SEQ ID NO: 7 Tomato: >S.lycopersicum ITAG4.0|Solyc03g119300.4.1 (Hemerythrin domains are highlighted)

MATQGREGGGGVAVLCGGGVNAVDSSASSSNGVLEKETGGKQESPILFFLFFHKAIR

LELDALHHSALAYATGQLEDIQPLLKRYRFLRSVYKHHCHAEDEVIFPALDIRVKNVAP

TYSLEHKGENDLFDHLFEILNSEKQNCERFPRELASCTGALQTSVSQHMSKEEEQVF

PLLTEKFSMDEQASLVWQFLCSIPVNMMKKFLPWLSSSISPDEHKDMQKCLSMIIPKE

KLLQQVIFSWMEGGKCVTAVGGHDVDADPPGSVDFNSVTETYASGNEKCVCESSSP

GKRKFRLKGDSFDTDSGNPIDEVLHWHNAIKRELDEIAAEARRIELAGELSSLTAFYAR

LQFIAQVCIFHSIAEDKVIFPAVDGGLSFFQEHAEEEIQFNELRCLIESIQCTEVNSTSAA

EFFSKLYSQADLIIETIKQHFHNEEVQVLPLARKHFTRDRQRKVLYQSLCLMPLKLMEQ

VLPWLVGALSEDEARSFLKNLQLAAPEADTALVTLLSGWACKGRTDGVCLSSSVTGC

CAVKRFADIEEYYTGAPCPCFLSVHSDDSKRPFKRNLNSLCSKDDTLDLSKGVNACNI

SCNDQSCRVPGLGVSDNNLVLTTISTPKSLRSLTFSSAAPSLESSLFVWETDCTSSQP

NHKVHPIDTIFKFHKAIQKDLEYLDVESGKLSDCPETFLRQFIGRFRLLWGLYRAHSNA

EDEIVFPELESKEALHNVSHSYMLDHKQEEKLFEDISSALTNLSELHKGLKEAYQKESG

SSILESTGLYDRDCKRKYNELATKVQGMCKSIRVSLDQHIFREEHELWPLFGKHFSME

EQDIIVGRIIGSTGAEVLQSMLPWVTSALTQDEQNKMMDTLKQATRNTMFSEWLNDC

WRRNPEVSSQSEALQNSYTNRGVDSHEGLDQSDHMFKPGWKDIFRMNQTELESEIR

KVHWDSTLDPRRKSYLIQNLMTSRWIASQQKSQASTEEISRSEDWGYSPSFRDKEK

QIFGCEHYKRNCKLLAACCGKLFACRFCHDEVSDHSMDRKATLEMMCMRCLKVQPI RPSCTTPSCNGFSMAKYYCSICKFFDDERPIYHCPSCNLCRVGHGLGIDFYHCMKCN

CCLGKGLVDHKCLEKALETNCPICCEFLFTSSATVRPLPCGHYMHSACFQEYASSNYI

CPICSKSMGNMAVYFGMLDALLANEVLPEEYRNHWQDILCNDCEQKCRTPFHWLYH KCGFCGSYNTRVITLPTTASDCPT

SEQ ID NO: 8 Lettuce: >L.sativa V8|Lsat_1_v5_gn_8_40260.1 (Hemerythrin domains are highlighted)

MATPLTGLQHRDVGGGGVAVMAASGGGAVNQMDPSSSNQSSKKQSSPIHIFLFFHK

AIRSELDALHRSAIAFATNSHVEIEPLLKRYHFLRSIYKHHCNAEDEVIFPALDIRVKNVA

RTYSLEHEGESVIFDQLFTLLDSDMQNEESFRRELASCTGALQTSINQHMSKEEEQVF

PLLVEKFSFEEQASLVWQFLCSIPVNMMAEFLPWLSASVSSDERHEMRNCLCKVIPE

EKLLQQIIFTWMDGANGFKKRKSSEECEEGHYCPCSSSRPKKRESFLRSIDDSTDSLH

DRPVDEILHWHKAIKKELIDIAEAARRIQLSGDFSDISAFNKRLQFIAEVCIFHSIAEDKVI

FPAVDAELSFAQEHAEEESEFDKFRCLIESIENDGANSSSSEFCSKLCSHADHIMSIIEK

HFKNEELQVLPLARKHFTPKRQRELLYQSLCVMPLRVIECVLPWLVGSLTEEESKSFL

HNMHMAAPPSDIALVTLFSGWACKGRPREICLSSGTTGCCPARAFLESNNGCNPSCC

AYNDMTSDDSGRSNKRSNSTPHQENKNHGSTRSKQSCCVPGLGMENNNLGTSSSK

SLRSLSFNPSSSTPSFSSSVFNWETGISLIDTEGNGRPIDTIFKFHKAIRKDLEFLDVES

GKLNETNESFLHQFNGRFRLLWGLYRAHSNAEDDIVFPALESKETLHNVSHSYTLDHK

QEEKLFEDISTSLFELCELHENLDMCDDSFRNYNELATKVQGMCKSIRVTLDQHILREE

LELWPLFDRHFSVEEQDKLVGRIIGTTGAEVLQSMLPWVTSVLTQEEQNKMMDTWK

QATKNTMFTEWLNEWWEGSSPSSEEASQTDANISQGGDVHEHEALDPNDYTFKPG

WKDIFRMNQNELESEIRKVSRDPTLDPRRKDYLIQNLMTSRWIAAQQKLPQGRKGET

SDSEGLHGFSPSFRDTEKQIFGCEHYKRNCKLRAACCQKLFTCRFCHDNVSDHTMD

RKATTEMMCMNCLKIQPVGPNCSTPSCNGLSMAKYYCSYCKFFDDERTVYHCPFCN

LCRLGKGLGVDFFHCMTCNYCLGIKLVDHKCREKGLETNCPICCDFLFTSSAAVRALP

CGHFMHSACFQAYACTHYICPICSKSMGDMSVYFGMLDALMASEELPEEYRNRCQDI

LCNDCDKKGNAPFHWLYHKCGRCGSYNTRVIKVDPIAPNCLN

SEQ ID NO: 9 Rice > OsHRZ1_Qs01g49470.1 (Hemerythrin domains are highlighted)

MATPTPMAGEGTLAAVMPRSPSPTASAAAGSAAEAPMLIFLYFHKAIRAELEGLHAAA

VRLATERAGDVGALAERCRFFVNIYKHHCDAEDAVIFPALDIRVKNVAGTYSLEHKGE

NDLFSQLFALLQLDIQNDDSLRRELASCTGAIQTCLSQHMSKEEEQVFPLLTKKFSYEE

QADLVWQFLCNIPVNMMAEFLPWLSSSVSSDEHEDIRSCLCKIVPEEKLLQQVVFAWI

EGKTTRKVTENSTKSNSEATCDCKDASSIDHADNHISSHEDSKAGNKKYAESIDGQVE RHPIDEILYWHNAIRKELIDIAEETRRMQQSGNFSDISSFNARLQFIADVCIFHSIAEDQV

VFPAVDSELSFVHEHAEEERRFNNFRCLIQQIQIAGAKSTALDFYSELCSHADQIMETI

EKHFCDEETKVLPQARMLFSPEKQRQLLYKSLCVMPLKLLERVLPWLVSKLSDEEAS

SFLENMRLAAPSSETALVTLFSGWACKARSEDKSNSGEYLCLTSGEMRCLLDEVDGL

EKCRPFCPCASRSNTDASLHPQTENGSRPGKRGNDAESVPGTNGSDLSQTDDTEAR

PCSKKPCCIPGLRVETGNLAISSSLASAKSFRSLSYNSSAPSLYSSLFSWETDASLSCS

DGISRPIDTIFKFHKAIRKDLEYLDVESGKLIDGDESCLRQFIGRFRLLWGLYRAHSNAE

DEIVFPALESRETLHNVSHSYTLDHKQEEQLFGDISDALAELSQLHERLTHPHIEVSEA

EKNDFNSSDEIDWTRKYNELATKLQGMCKSIRAALTNHVHREELELWPLFDEHFSVE

EQDKLVGRIIGSTGAEVLQSMLPWVTSALTQEEQNMMLDTWKQATKNTMFGEWLNE

WWKGAPTSSDSSEEASSAPEDSHLQDKIDQNDQMFKPGWKDIFRMNQSELEAEVRK

VSRDPTLDPRRKAYLIQNLMTSRWIAAQQKLPEPKSEECSEGAGIPGCAPSYRDQEK

QIFGCEHYKRNCKLVAACCNKLFTCRFCHDKISDHTMERKATQEMMCMVCLKVQPV

GPNCQTPSCNGLSMAKYYCNICKFFDDERTVYHCPFCNLCRLGKGLGVDFFHCMKC

NCCLGMKLTEHKCREKGLETNCPICCDFLFTSSAAVRALPCGHFMHSACFQAYTCSH

YTCPICCKSLGDMAVYFGMLDALLAAEELPEEYRDRCQDILCNDCERKGRSRFHWLY

HKCGSCGSYNTRVIKTDTADCSTPN

SEQ ID NO: 10: Barley >HovulHRZ1_HG0281210.1 (Hemerythrin domains are highlighted)

MATPTPMAGEGTLAAVMPLSPPPPAAAAGSAAEAPMLIFLYFHKAIRAELEGLHGAAV

RLATERAGDVDALAERCRFFVNIYKHHCDAEDAVIFPALDIRVKNVAGTYSLEHKGEN

DLFTQLLALLQLDIQNDDALRRELASCTGAIQTCLTQHMSKEEEQVFPLLTKKFSYEEQ

SDLVWQFLCNIPVNMLAEFLPWLSASVSSDEHEDIRNCLCKIVPEEKLLKQVIFTWIEG

KATREVAQSFVSDNLERSHCCKDASFVNQAEKLICPLEQSKVGHIKHAESNDGQADR

HPIDEILYWHNAIRKELNDIAEETRRMQQSGDFADISAFNARLQFIADVCIFHSIAEDQV

VFPAVNSELSFVLEHAEEERRFNNFRCLIQQIQMAGAKSTAAEFYSELCSHADQIMEAI

EKHFCNEETKVLPQARVLFSPEKQRELLYRSLCVMPLKLLERVLPWLVSKLSDEEASS

FLQNMRLAAPSSDTALVTLFSGWACKARSEDKSNSGEYICLTSGAARCLLDDVEELK

KCQSFCPCASRTSADIPLHLENENGSRPGKRGNDAESVPGTNGSHCSQIADTVARPC

SKKPCCIPGLRVDTSNLGIGSLPSAKSFLSLSYNSSAPSLYSSLFSWDTDTALSCSDGI

SRPIDTIFKFHKAIRKDLEYLDVESGKLIDGDESCLRQFIGRFRLLWGLYRAHSNAEDEI

VFPALESREPLHNVSHSYTLDHKQEEQLFEDISNVLCELSQLHESLNPAHTEANEAEK

HYFNSSNVIDSTRKYNELATKLQGMCKSIRVALSNHVHREELELWPLFDKHFSVEEQD

KLVGRIIGTTGAEVLQSMLPWVTSALNQEEQNKMLDTWKQATKNTMFGEWLNEWWK GVPTPSDSSSETSPIPEDSHSQDKLDQNDQMFKPGWKDIFRMNQSELEAEVRKVSR

DPTLDPRRKAYLIQNLMTSRWIAAQQKLPDPRSGECSEDAGIPGCCSSYRDQEKQVF

GCEHYKRNCKLVAACCNKLFTCRFCHDKVSDHTMERKATQEMMCMVCLKVQPVGP

NCQTPSCNGLSMAKYYCNICKFFDDERTVYHCPFCNLCRLGKGLGVDFFHCMKCNC

CLGMKLTEHKCREKGLETNCPICCDFLFTSSAAVRALPCGHFMHSACFQAYTCSHYT

CPICCKSLGDMAVYFGMLDALLAAEELPEEYRDRCQDILCNDCERKGRSQFHWLYHK CGSCGSYNTRVI KTDTADCSTPN

SEQ ID NO: 11 Wheat>Traes_HRZ1-A (Hemerythrin domains are highlighted)

MATPTPMAGEGTLAAVMPLLPPPPPAAEAAAGSAAEAPMLIFLYFHKAIRAELEGLHG

AAVRLATERAGDVGALAERCRFFVNIYKHHCDAEDAVIFPALDIRVKNVAGTYSLEHK

GENDLFTQLLALLQLDIQNDDALRRELASCTGAIQTCLTQHMSKEEEQVFPLLTKKFSY

EEQSDLVWQFLCNIPVNMLAEFLPWLSASVSSDEHQDIRNCLCKIVPEEKLLKQVVFT

WIEGKATREVAQSVVSDNLERSHCCKDASFVNQAEKLIYPLEQSKVGHIKYTESNDC

QADRHPIDEILYWHNAIRKELNDIAEETRRMQQSGDFADISAFNARLQFIADVCIFHSIA

EDQVVFPAVNSELSFVLEHAEEERRFNNFRCLIQQIQMAGAKSTAAEFYSELCSHADQ

IMEAIEKHFCNEETKVLPQARVLFSPEKQRELLYRSLCVMPLKLLERVLPWLVSKLSDE

EASSFLQNMRLAGHLFTLIILSYFLYTLYLALKFSLGIQCLLKDSCETAPSSDTALVTLFS

GWACKARSEDKSNSGEYICLTSGAARCLLDDVEELKKCQSFCPCASRISADVALHLE

NENGSRPGKRGNDAESVPGTNGSHCSQITDTVASPCSKKPCCIPGLRVDTSNLGIGS

LASAKSFLSLSYNSSAPSLYSSLFSWDTDTALSCSDGISRPIDTIFKFHKAIRKDLEYLD

VESGKLIDGDESCLRQFIGRFRLLWGLYRAHSNAEDEIVFPALESREPLHNVSHSYTL

DHKQEEQLFEDISNVLCELSQLHESLNQPHTEANEAEKHYLNSCNVIDSTRKYNELAT

KLQGMCKSIRVALSNHVHREELELWPLFDKHFSVEEQDKLVGRIIGTTGAEVLQSMLP

WVTSALNQEEQNKMLDTWKQATKNTMFGEWLNEWWKGVPTPSDSSSEASPIPEDS

HSQDKLDQNDQMFKPGWKDIFRMNQSELEAEVRKVSRDPTLDPRRKAYLIQNLMTS

RWIAAQQKLPDPRSEECSEGAGIPGCCSSYRDQEKQIFGCEHYKRNCKLVAACCNKL

FTCRFCHDKVSDHTMERKATQEMMCMVCLKVQPVGPNCQTPSCNGLSMAKYYCNI

CKFFDDERTVYHCPFCNLCRLGKGLGVDFFHCMKCNCCLGMKLTEHKCRXSAVTSY

SHQARQLELFLVVTSCIQLAFRHTLAVTTLVLSAANPWEIWRCTLACLMRCWLLKSFP

RNTAIGVRTFFVMTVKEKGDLSFIGCTTNAAPVVLTIPELSKLIRQIVLPQTSEGNILSFL

VFVSVNIVLQNLV

SEQ ID NO: 12 Rice >OsatKitaake_HRZ2_Qs05g252600.1 (Hemerythrin domains are highlighted) MATPLADEGSIAAAVMPRSPSPPAAAAGSAAEAPMLIFVYFHKAIRAELERLHAAAVRL

ATERSGDVGELERRCRFLFSVYRHHCDAEDAVIFPALDIRVKNVAGTYSLEHKGENDL

FAHLFSLLKLDVRNDDGLRRELASCTGAIQTFITQHMSKEEEQVFPLLIKKFSHEEQAD

LVWQFLCSIPVNMMAEFLPWLATSVSSDEHQDILNCLHKIVPDEKLLQQVVFAWIGGE

AVKTISHDFCSPCSKSNVRCKDAIDQTDKYGCSHEHFKTGKRKRAESSYSQLVMHPI

DEILCWHNAIRKELSDIVEETRRIQQSGDFSDISDFNVKLQFIADVCIFHSIAEDQVIFPA

VNDQVSFEQEHAEEERRFNKFRCLIEQIQITGARSTAVDFYSELCSQADQIMEKIERHF

KNEETKVLPQARIHFSSEKQRELLYKSLCVIPLKLLERVLPWFVSKLNDQDAEAFLQN

MFLAAPSSEAALVTLLSGWACKGRSKGTSNSGKFICLTPRALSSPLDENGFKDCQLC

PCSLQSDICSRPAKKWNDTESSNISNCSQTADIALTCKNRPCHIPGLRVEISNLAVNSF

ASAESFRSLSLNYSAPSLYSSLFSWETDAAFSGPDNISRPIDTIFKFHKAIRKDLEFLDV

ESRKLIDGDESSLRQFIGRFRLLWGLYRAHSNAEDEIVFPALESKETLHNVSHSYTLDH

KQEEELFKDISTILFELSQLHADLKHPLGGADAVGANHIHPYNRIDWSKKNNELLTKLQ

GMCKSIRVTLSNHVHREELELWPLFDKHFSVEEQDKIVGRIIGSTGAEVLQSMLPWVT

SALSLDEQNNMLDTWRQVTKNTMFDEWLNEWWKRSPTSSGPSSDASHPEEDHFQE

KFDQSEQMFKPGWKDIFRMNQSELEAEIRKVSRDSTLDPRRKAYLIQNLMTSRWIAA

QQKSPQPQSEDRNGCTVLPGCCPSYRDPENQIFGCEHYKRKCKLVAACCNKLFTCR

FCHDKVSDHTMERKATVEMMCMQCLKVQPVGPNCQTPSCNGLSMAKYYCSVCKFF

DDERSVYHCPFCNLCRLGQGLGIDFFHCMKCNCCLGMKLIEHKCREKMLEMNCPICC

DFLFTSSAAVKGLPCGHFMHSACFQAYTCSHYTCPICSKSLGDMTVYFGMLDGLLAA

EELPEEYRDRCQDILCNDCERKGRSRFHWLYHKCGFCGSYNTRVIKIDRADCSTSD

SEQ ID NO: 13 >HovulHRZ2_HG0083800.1 (Hemerythrin domains are highlighted)

MAPTPMAGDGPIAAVAPRTPPPPSSSAEASASGSGSGATAGSAAEAPVLIFVYFHKAI

RAELDRLHAAAVRLATERGGDGDVAALDTRCRFLFSVYRHHCDAEDAVIFPALDIRVK

NVAGTYSLEHKRENDLFSHLFALLQLDVHNNDGARREIASCTGAIRTFITQHMFKEEE

QVFPLLITKFSYEEQADLVWQFICNIPVNMMADFLPWLSSSVSSDEHQDILNCLQKIVP

QEKLLQQWFAWIGGKAITVVQDFDNPCTKGSYRCEGISYQTDKKICSHENSTIGKRK

YAESDHSQLVTHPIDEILYWHNAIREELSDIAEETRRIQQSGDFSNISAFNLRLQFIADV

CIFHSVAEDQVIFPAVDGEVSFEQEHAEQEQQFNKFRCLIEQIQTAGARSTAVDFYSE

LCSQADQIMEEIEKHFSNEETKVLPQARTNFSPEKQRELLYRSLCVMPLKLLEQVLPW

FVSKLDDVNGQSFLQNMCLAAPSCETALVTLLSGWACKGRFKDKSNLGKFICLPSGA

LSCPLDGDGLKRCQSFCPCSLASYGTFSAHLQTENGSRPVKRGNHAASSTNINGSHC

SQITDIEESRCGSKPCHIPGLRVESSNVVADSFASVNSFRSLSCSYSAPSLYSSLFSW

ETDTSFSSPDNISRPIDAIFKFHKAIRKDLEFLDAESGKLIDGDESCLRQFVGRFRLLWG LYRAHSNAEDEIVFPALESKDALHNVSHSYTLDHKQEEELFKDISIILLELSHLRDDSGH

PTDETDEAGKGHICSYSEIDWSRKHNELLTKLQGMCKSIRFTLSNHVHREELELWPLF

DKHFSVDDQDKIVGRIIGSTGAEVLQSMIPWVTSALSLDEQNKMLDTWKQASKNTMF

DEWLNEWWKSSPTSSGPSNESSPSEETQFEENLDQSDQMFKPGWKDIFRMNQSEL

EAEIRKVSQDSTLDPRRKAYLIQNLMTSRWIAAQQKSPQQRPEDHSGSTEIPGCSPSY

RDPEKQTFGCEHYKRNCKLVAACCNKLFTCRFCHDKVSDHTMERKATLEMMCMLC

MKVQPVSPNCRSPSCNGLSMAKYHCSICKFFDDERSVYHCPFCNLCRLGEGLGTDF

FHCMKCNCCLGMKLKEHNCREKMLEMNCPICCDFLFTSSAAVRGLPCGHFMHSACF

QAYTCSHYTCPICSKSLGDMTVYFGMLDGLLAAEELPEEYRNRCQDILCNDCGRKGL

SRFHWLYHKCGACGSYNTRVIKTEAPGCSTPN

SEQ ID NO: 14 Wheat>Traes_HRZ2-A (Hemerythrin domains are highlighted)

MAPTPMAGDGPIAAVAPRTPPPPSASAEAAASGSGSGTAGSAAEAPVLIFVYFHKAIR

AELDRLHAAAVRLATERGGDGDVAALDTRCRFLFSVYRHHCDAEDAVIFPALDIRVKN

VAGTYSLEHKRENDLFAHLFSLLQLDVHNDDGVRREVASCAGAIRTFITQHMFKEEEQ

VFPLLITKFSYEEQADLVWQFICNIPVNMMADFLPWLSSSVSPDEHQDILNCLHKIVPQ

EKLLQQWFAWIGGKAVTVAHNFDNSCSKGSYGCEDTSHQTDKKICSHENCKIGKRK

YAESNHSQLVTHPIDEILYWHDAIRKELSDIAEETRRIQQSGDFSNVSAFNVRLQFIADV

CIFHSIAEDQVIFPAVDGEVSFEQEHAEQEQRFNKFRCLIEEIQTAGARSTAVNFYSEL

CSQADQIMEEMEKHFNNEETKVLPQARINFSPEKQRELLYRSLCVMPLKLLEQVLPW

FVLKLDDANGQSFLQNMFLAAPSSETALVTLLSGWACKGRLKDTSNSGKFICLPSGA

QGCLLDGDELKNCQSFCPCSLASNGTFSVPLQTENGSRPVKRGKHAESITDRNHCS

QTTDIEESRCSKKPCHIPGLRVESSNFGADLFTSVNSFRSLSSSYSAPSLHSSLFSWE

TDMTFSSPDSISRPIDAIFKFHKAIRKDLEYLDVESGNLIDGDESCLRQFVGRFRLLWG

LYRAHSNAEDDIVFPALESKDALHNVSHSYTLDHKQEEELFKDISTILLELSHLRDDSA

HPVDEIDEAGKGHICSYSEIDWSRKHNELLTKLQGMCKSIRFTLSNHVHREELELWPL

FDKHFSVDDQDKIVGRIIGSTGAEVLQSMIPWVTSALTLDEQNKMMDTWKQATKNTM

FDEWLNEWWKSSPTSSGPSNEASSLSEESQENLEQSDQMFKPGWKDIFRMNQSEL

EAEIRKVSQDSTLDPRRKAYLIQNLMTSRWIAAQQKSPQPRSEDHNGSTVIPGCFPSY

RDPEKQILGCEHYKRNCKLVAACCNKLFTCRFCHDKV

SDHTMERKATLEMMCMLCMKVQPVGPNCQSPSCNGLSMAKYYCSICKFFDDERSVY

HCPFCNLCRLGEGLGTDFFHCMKCNCCLGLKLKEHKCREKMLEMNCPICCDFLFTSS

AAVRGLPCGHFMHSACFQAYTCSHYTCPICSKSLGDMTVYFGMLDGLLAAEELPEEY

RNRCQDILCNDCGRKGLSWFHWLYHKCGACGSYNTRVIKTEAPDCSTSN SEQ ID NO: 15 Algae: Auxenochlorella protothecoides >Auxchl_BTS (Hemerythrin domains are highlighted)

MTTIQAARPAGKLADGHEQPAPVFQPIHFLYTYLHEAIRHELALLSQSLQLLLAGTSTG

WPELRRRYIFLRDVYKYHSAAEDEVIYPALELEVANVTPSYSVEHEDEEHLLEEMVDL

LLATEQSPTKENLLAVRQLSWRIQTTVTKHLAKEEAQLLPLLLTHIPPREQGGLVAQFL

CCIPISTVARVLAWVKPHAAPADRAAIREALAGAVDDPLLRSLLLGWLEADEADGAGS

DGLARPGSPSSPGSLGDDEAAAAARSVADPPGPQSQREGRTDVPGSRQPAGPPPL

RSILHYHAAIRAALEGFAADARAVASAPGGPDPPALAALLVRHRFIRAVCAFHSAAEDE

VVFPALARLHTSQQEGGPAGEPGAAAGDPCASPSRASPGGDRLGAHAAPGPAAADT

GPSPPARHSPPPPPHDGCAGHGEEALRFDELQRLLGEVASCARRRCSRSGTASADL

VASADALAAAMAGHMEAEERGVLPALERLCPRPEQRALLWAMVRAMPLRLLERVMP

WIAARLAPAERRRWLADLARGAGRGDTTLVALVTGWAARGGEGGEAVAGEGDVSG

HVAGALGDVITGLQRCADPTRYSLLTCHPRPRPAADMRGVSEAIPPGPPPGGAERGA

AHLAPACPLPQPAPKRIRVGGPDHGAAAQRPAGAERLVTAAAFEAAPRGGGAAAQG

WAGPSPIDHIFQFHNALRKELAELEATIATCQQRLETAAELADTTEVMQRLAARFQFLR

GIYRAHSLSEDEVVFPALEGKKVLRNVSHAYTLDHEQEEMQFQTCSECLDRVMATGT

LDSRRQRLAELACYYSGVRGSLETHIRAEELELWPLFAEHFPVAEQEHLVGLIIGRTGA

DVLTSLLSWVRGAMTREEEDAMMLSIRSAASSTAFDAWLGAAMGQAMPDTPSGGA

GGGAGGEGATPRCGGGAGQAAEMRDTLAEVAGYLSSQGLLGAGAAAEAGAASAES

SLYTPGWGDIFRMNQKQLEAAVRRVSSDAGLEPGRKAYLIQHIMASRYVVAQQRLLA

GGAGDEGGGRAGGPAGERGGAQGGEAPGEGASASPAAASPPAAGGESSPPPRPP

AAGPAAGPLLHSARRYHDPKKGVLGCRHYRRAAQLVAPCCDRAYVCRFCHDEATDH

VLDRYAVCEMVCMECSLRQPVAGSCSGCGTCMSRYYCKVCHLFDDDPGKAIYHCPF

CNFCRQGKGPGIDSFHCMSCNACMSLELFNKHVCKEQSLKRDCPVCSDMLFESKHP

VKELPCGHFMHSHCFAAYTRYAYTCPLCFKSLGNLEMYWRMIDRLLEAEQLPAEYAG RRQSILCNDCGARSEVAFHFVYHRCASCKGYNTRIV

SEQ ID NO: 16: Chlamydomonas reinhardtii >cre05 . g248550 (Hemerythrin domains are highlighted)

MAGAAAQLVPRTAFLEPPTVSSAAAPARAFPPINFIYGHFHNSIRAELGLLAERVRSLE

APGEGVGEMLADLRERYKFLEQVYKYHSTVEDEVLYPVLDSKVRNVTLAYSIEHQDE

EILFEQLSKLISAALEEPEARRKCTIRTLICKVEEIHTTLRKHLAKEEEQLLPLLLQHFSFA

EQAELVAQFLYSIPLETVERVLSRLKPRIPRDEQERLLEEIQAVIPDNLLSQLLTTWLQP

EQQQALRMEREGLREAPAEAAEPQSRQSRQQGLGAGEGAGVGAGEDAGEDAGGW

KLQRFGLGGGGAGFTCCGNPAACTYSGSGVTAAGAEAGAEATGTGAGAGLAAAQR ADSDPAELSLPAASSPVCCAGADCGTACCDRTAAGGAAATPAAAPAAPAAPAAPGP

ALEEAVAAAAATATAGGAAAAGAAGGEAAAGASAAAARAPLQDIIHFHRSICASLVDF

AREARSLQAGREPITAGHLQSLLERHRFLRAVYVFHSISEEEVLFPEVQRLAAANVQL

AGGAAAAHQQQCEKDHAAELSSFEDLGRLLADVRAFARRGRKEVAGMLEKLCCSVE

A AASIEHHMQREEADVFPLLEAHLCQAQQRALLYRTIRAMPLRLLERVMPWVVSRL

DASAAAALLANIALGAPRSDQALVELLSRWARRGCRDRRHLTPPSRGLSPPGLLPPG

PPGPPGPLSGGGGGGFARASDGNDVWLGGGGGGAGGSPASDSAMLVGGEAQLG

QLGQQGQLGQQGQLGQQWQQQGQQLETAGSVGLRADSGDAPESAGGPGMGMGA

VAAGGGGAGGAAWGTAGPPQAGGCHVEVEGLADGWGDCTATCGARLLREAGVGC

AAAAAAAAGGAAGAAAGVAPGHGHVQVGYVTTAIAEDGADSKRRRISGEAAAAGTQ

PTMGVSRGGSGGGGGGAGAGGGGSGGGASMDAVSDAAAAAAAAAAGGCLYVMDL

DAGELSTAPRALLAAQAAGAAAADAMELERKASGRRASGTGYSGGGAAAAASGGGA

ELLPPTADSAGGAAAGGVDGAAAAELGGPGGGFNPIDHIFQFHKALRQELKQLEADV

MALEGAVQSVLRHMPASASQHNLAGLMGPGAAGAVGATGGAAGGGAGGARPATPT

SVGTPARGPRAAMPGSDGLQHNQQQPGQQPGQQQQGQQQQQGQQQQPGPHAH

LGVGWPAADGSGAAYTRGGQLAVVQHLHGRFQFLQGIYRAHSKSEDEIVFPALESKQ

ALRNVSHAYTLDHRQEEQLFADLEAVIDNLRAVDLTAQGADAELSRQVMAVRRMCAA

IRASLETHIRSEESELWPLFTEHFSREEQQYLVGVIIGRTGAQVLQALLPWVSETFSEE

EREAMMDSLREATRNTMFDQWLEAVQAGAVGPPGTAPGAAAAAAAAAAAAGPAAV

GDEYGTSQHQYQGYPGQGQQHQQGQQQQHQGPLPRLSHSQQQPLQHVAEYLAG

AGAGAGAGAGAEAPGSSAGPTAGTGTGAGTAPAAPRSIAAAAASAAAAPTSPTAPAT

RPSCATPPLPPHTPSAGAAGAGAGASASASGSSFRPGWEDIFRMNQQQLEAAIHRV

SNDPTLEPERKAYLMQNIMVSKYIVAQQRRMCESHGSSSSSSAAAGGCSSSSSTFGA

GAVAGSSSSIMSSGAAGATCGHGTPAAPEAAAAAAAAAAATVSPAAATATAAAAAAT

ATAAAGATAARQLHQTWHDAAGGVLGCGHYQRKCQLVAPCCGGVYTCRLCHDEAA

DHRMDRYAVSEMRCMSCGERQPVAAACRACAANMARYYCNICHLFDDEPGKDIYH

CPFCNVCRRGRGLGVDFFHCMNCNACMSLSLFSSHKCREKCIEGNCPVCHEALFDS

SQPIKELPCGHFMHSTCFSTYTRYNYTCPLCCKSVGDMSVYFQMLDSLLAAERLPPE

YSGRMQQVLCNDCGKMGFAPFHFVYHSCPHCRSYNTRVL*

SEQ ID No 17: >Beetroot EL10_Ac6g 14339.1 (Hemerythrin domains are highlighted)

MATPLTGLGHRDGGGVAIMAGPVNAVDPSPPSSSSSKVSLKNSAQNSPILIFLFFHKAI

KAELEGLHRAAIDFATNQGSDIKPLLERYHFLRCIYKHHCNAEDEVIFPALDIRVKNVAR

TYSLEHEGESVLFDELFELLNTTVRDEEAYRRELASRTGALQTSISQHMCKEEEQVFP

LLIEKFSFEEQASLVWRFLCSIPVNMMVEFLPWLSSSVSSDESQDMRKCLRKFIPNEV LLHEVIFTWMDGPKLDKFRTSCGSTLQSSIDSAACASVGQTKGVSCACESSQTGKRK RVKLSSSTADSDLSFPIDEILLWHKAINQELGEIAEAARRMQLNDEFTDLSAFNERLHF

VTEVCIFHSIAEDKVIFPAVDAELSFAQEHAEEESKLEKIRCLIENIQLANAESSLDDLYS

RLSSYADQIMDTIQKHFQNEEIQVLPLARKLFTPQRQRELLYQSLCVMPLKLIERVLPW

LVGSLGEEEAKCFLKNMHMAAPAADAALVTLFSGWACKGSKRNVCLSSSAIGCCPAR

LPSAEDNELPVSRCTFCLSNGDKSAIAQQDNEIRPSKRGNMTSREEVGKLEDERGDS

SQRISCGTISCCVPGLGVNNGHLVVGSLAAAKSLRSLSLSPAAPSLNSSLFNWETEISV

SDSGSTTRPIDNIFKFHKAIRKDLEYLDAESGKLSECNESFIRQFSGRFRLLWGLYKAH

SNAEDDIVFPALESKEALHNVSHSYTLDHKQEEMLFEDISSALSELSQLRGALSRSSTA

QDTLGFASNGGDSSDDLRKYHELATKLQGMCKSIRVTLDQHVFREELELWPLFDKHF

SVEDQDKIVGRIIGTTGAEVLQSMLPWVTSALTQEEQNKMMDTWKNATKNTMFSEWL

NEWWESAPPSQQCLTSETSSAQGHDICDALDSSDQTFKPGWKDIFRMNQNELESEI

RKVSRDSSLDPRRKDYLIQNLMTSRWIAAQQKSPHGSSGESSNGDGLLACSPSYRD

PEKKIFGCEHYKRNCKLRAACCGKLFACRFCHDKVSDHSMDRKMTSEMMCMLCCKI

QPVGPSCTTPSCHGLSMAHYYCNICKFFDDERSVYHCPFCNLCRVGKGLGSDFFHC

MTCNCCLSVKLIDHKCREKGLETNCPICCDFLFTSSASVRALPCGHYMHSACFQAYT

CSNYICPICSKSLGDMAVYFGMLDALLASEQLPEEYRDRCQDILCNDCGRKGTARFH WLYHKCGSCGSYNTRVIKIGLSDSNCPK

SEQ ID NO 18: >Z.mays RefGen_V4|Zm00001d043805_P001 (Hemerythrin domains are highlighted)

MATPTPMPGSEGTLVAVMPRSPSPTPAEAGTSATETPVLIFLYFHKAIRAELEALHGA

AVLLATERTGDVAALAERCRFFFSIYKHHCDAEDAVIFPALDIRVKNVAGTYSLEHKGE

SDLFSQLFDLLELDIQNDDALRRELASCTGAIQTCLSQHMSKEEEQVFPLLTKKFSCEE

QADLVWQFLCNIPVNMVAEFLPWLSTSVTSDEHQDIRDCLCKVVPDEKLLQQVVFTW

MEGKAAREVAESFATGNLVRNHSAEDVSDHGEIYVCSQQESKLGSKNCAESNGSQA

DRHPIDDILYWHNAIRMELHDIKKETRRVQQSGNFSDISAFNERLQFIADVCIYHSIAED

QVVFPAVDSELSFVQEHAEEEHRFNNFRCLIQQFQIAGAKSTALDFYSKLCSHADKILE

TIEKHFSNEETKVLPQARMFFSPEKQRELSYKSLCVMPLKLLERVLPWLVSKLSDEQA

TSFLQNIRLAASPSETALVTLISGWACKGRDKSKSGEYLCLTSGTARCLSDDVDDQGK

CRSFCPCASHNSSDLSLQLQTENGSRPGKRGKDAVSFPGTNGSYCSQTADIDASPC

SKKPCCIPGLRVKSSNLGIGSLASVKSFRSLPYNSTAPSIYSSLFSWETDASLSCSDGI SRPIDTIFKFHKAIRKDLEYLDVESGRLIDGDESCLRQFIGRFRLLWGLYRAHSNAEDEI

VFPALESRETLHNVSHSYTLDHKQEEQLFEDISDVLFQLSQLHDSQGHAQTKVNEVK

QSCFHSSNDVDFTRKYNELATKLQAMCKSIRVALTNHVHREELELWPLFDKHFSVEE QDKLVGRIIGSTGAEVLQSMVPWVTSALTQEEQNKMLDTWKQATKNTMFGEWLNEW

WKGAGTSDSSAEAPSAPEDSHLQDKLEQNDQMFKPGWKDIFRMNQSELEAEVRKV

SRDPTLDPRRKAYLIQNLMTSRWIAAQQKLPEPNSEECSDDASIPGCAPSYRDQEKEI

YGCEHYKRNCKLVAACCNKLFTCRFCHDKVSDHTMERSRKATQEMMCMVCLKIQPV

GPFCQTPSCNRLSMAKYYCNICKFFDDERTVYHCPFCNLCRLGKGLGVDFFHCMKC

NCCLGMKLTEHKCREKGLETNCPICCDFLFTSSAAVRALPCGHFMHSACFQAYTCSH

YTCPICCKSLGDMAVYFGMLDALLAAEELPEEYRDRCQDILCNDCERKGRCRFHWLY HKCGSCGSYNTRVIKTDTADCSTPN

SEQ ID NO: 19>Z.mays RefGen_V4|Zm00001d011622_P002 (Hemerythrin domains are highlighted)

MATPTPMPGGEGTLAAVMPRSPSPTPAEAGTSATETPVLIFLYFHKAIRAELEALHGA

AVLLATERTGDVEMLAKRCRFFFNIYKHHCDAEDAVIFPALDIRVKNVAGTYSLEHKGE

SDLFSQLFDLLQLDIHNDDGLRRELASCTGAIQTCLSQHMSKEEEQVFPLLTKKFSCE

EQADLVWQFLCNIPVNMVAEFLPWLSTSVTSDEHQDIRNCLCKVVPDEKLLQQVVFT

WMEGKATREVAESIAAGISARNNSVEDVPDQGKIHICLHHNSKLGSKNCGESNGPQA

DKHPIDDILYWHNAIRMELRDIKEETRRVQQSGDFSDISAFNERLQFIADVCIYHSIAED

QVVFPAVDSELSFVQEHAEEECRFNNFRCLIQQIQIAGAESTALDFYSKLCSHADKILE

AIEKHFCNEETKVLPQARMLFSLEKQRELSYKSLCVMPLKLLERVLPWLVSKLSDVQA

TSFLQNIRLAASPSETALVTLISGWACKGRDKSKDGEYLCLTSGAARCLSDDVDDLGK

CRSFCPCASPNSSDLSLQLHTENDSRPGKRGKDAVSFSHTNGIYCSQTADIEAIPCSK

KPCCIPGLRVESSNLGIGSLASAKSFHSLSYNSTAPSLYSSLFSWETDTSLSCSDSISR

PIDTIFKFHKAIRKDLEYLDVESGKLIDGNESCLRQFIGRFRLLWGLYRAHSNAEDEIVF

PALESRETLHNVSHSYTLDHKQEEQLFEDISNVLFQLSQLHDSQGHAQTEVNEVKKS

CFHSSNDVDFARKYNELATKLQGMCKSIRVALTNHVHREELELWPLFDKHFSVEEQD

KLVGRIIGSTGAEVLQSMLPWVTSVLTQEEQNKMLDMWKQATKNTMFGEWLNEWW

KGAGTASDSSAEASSAPEDSHLQDKLEQNDQMFKPGWKDIFRMNQSELEAEVRKVS

RDSTLDPRRKAYLIQNLMTSRWIAAQQKLPEPNSEECNHDASIPGCAPSYRDQEKQIY

GCEHYKRNCKLVAACCNKLFTCRFCHDKVSDHTMERKATQEMMCMVCLKIQPVGSF

CQTPSCNRLSMAKYYCNICKFFDDERTVYHCPFCNLCRLGKGLGVDFFHCMKCNCC

LGMKLTEHKCREKGLETNCPICCDFLFTSSAAVRALPCGHFMHSACFQAYTCSHYTC

PICCKSLGDMAVYFGMLDALLAAEELPEEYRDRCQDILCNDCERKGRCRFHWLYHKC

GSCGSYNTRVIKTATADCSTPN SEQ ID NO: 42> Pisum sativum; Psat1g036240.1

ATGGCGACTCCGTTAACGGGATTGCAGCAGCACTTAGACGCCGGCGGCGG

AGGAGTTGCCGTGCTGTCGAATCTCGTTTCCTCTTCTTCTCCTTCTTCCA

CCTCCAATGGTGGCGGCGGATTCAACCGTTCCTCTTTATCAGAATCGCCA

ATTTTGATTTTCTCGTTCTTCCAAAAAGCGATTGGGAACGAGCTAGATGC

GTTGCACCGATTAGCTATGGCGTTCGCTACCGGAAACTGCTCTGATATTC

AACCTCTCTCCGAACGTTACCATTTCCTTAGATCGATGTATAGACACCAT

TCCAATGCTGAAGATGAGATGGCGACTCCGTTAACGGGATTGCAGCAGCA

CTTAGACGCCGGCGGCGGAGGAGTTGCCGTGCTGTCGAATCTCGTTTCCT

CTTCTTCTCCTTCTTCCACCTCCAATGGTGGCGGCGGATTCAACCGTTCC

TCTTTATCAGAATCGCCAATTTTGATTTTCTCGTTCTTCCAAAAAGCGAT

TGGGAACGAGCTAGATGCGTTGCACCGATTAGCTATGGCGTTCGCTACCG

GAAACTGCTCTGATATTCAACCTCTCTCCGAACGTTACCATTTCCTTAGA

TCGATGTATAGACACCATTCCAATGCTGAAGATGAGGTGATTTTTCCAGC

TTTGGATAAGCGCGTGAAGAATGTAGCACAGACATATTCTCTTGAACATA

AGGGTGAAAGCGATCTTTTTGACCATTTGTTTGAGCTGTTAAACTCTTCA

GTTGATAACGATGAAACTTTCCGAAGGGAGCTAGCATCCTGCACAGGAGC

TCTGCAGACATCTCTTAGTCAACACATGGCTAAGGAGCAACAGCAAGTGA

TTTTTCCAGCTTTGGATAAGCGCGTGAAGAATGTAGCACAGACATATTCT

CTTGAACATAAGGGTGAAAGCGATCTTTTTGACCATTTGTTTGAGCTGTT

AAACTCTTCAGTTGATAACGATGAAACTTTCCGAAGGGAGCTAGCATCCT

GCACAGGAGCTCTGCAGACATCTCTTAGTCAACACATGGCTAAGGAGCAA

CAGCAAGTGTTTCCGCTGCTTATTGAGAAGTTCTCAGTGGAGGAACAGGC

ATCTTTAGTTTGGCAGTTTCTTTGCAGTATTCCAGTGAATATGATGGCAG

AATTTCTTCCATGGCTTTCAACATCCATCTCACCTGATGAATCTCAGGAT

TTACGGAAGTGCTTAAGCAAGATAGTGCCAGAGGAAAAGCTTCTTCAAAA

GGTGTTTCCGCTGCTTATTGAGAAGTTCTCAGTGGAGGAACAGGCATCTT

TAGTTTGGCAGTTTCTTTGCAGTATTCCAGTGAATATGATGGCAGAATTT

CTTCCATGGCTTTCAACATCCATCTCACCTGATGAATCTCAGGATTTACG

GAAGTGCTTAAGCAAGATAGTGCCAGAGGAAAAGCTTCTTCAAAAGGTTA

TCTTCACCTGGATGGAAGGGCGAAGTAGTGCTAACACAGCTGAAAATTGT

GTAGATCATTCTCAGGTTCAATGTAGTGCTTGTCCTTTAGCCAATCAGAT

TGAGAAAATAAAATGTGCATGCGAGTCCACTGTATCTGGGAAAAGAAAAT

ATTCTGCATCTATTCTAGATGTTCCAGAGACAATGGGTTCACATCCTATA

GATGAAATATTGCTATGGCATAATGCTATAAAAAAAGAATTAAATGAGAT AGCAGTGGAGACCAGAAAGATACAACACTCTGGAGATTATACCAATCTGT

CTGCTTTTAATGAAAGATTGCAGTTCATTGCTGAAGTTTTCATATTTCAC

AGGTTATCTTCACCTGGATGGAAGGGCGAAGTAGTGCTAACACAGCTGAA

AATTGTGTAGATCATTCTCAGGTTCAATGTAGTGCTTGTCCTTTAGCCAA

TCAGATTGAGAAAATAAAATGTGCATGCGAGTCCACTGTATCTGGGAAAA

GAAAATATTCTGCATCTATTCTAGATGTTCCAGAGACAATGGGTTCACAT

CCTATAGATGAAATATTGCTATGGCATAATGCTATAAAAAAAGAATTAAA

TGAGATAGCAGTGGAGACCAGAAAGATACAACACTCTGGAGATTATACCA

ATCTGTCTGCTTTTAATGAAAGATTGCAGTTCATTGCTGAAGTTTTCATA

TTTCACAGTATTGCTGAGGACAAGGTTATTTTTCCGGCAGTGGATGGGGA

TTTTTCTTTCTTTCAGGAGCATGCTGAAGAAGAAAGCCAATTTAATGACT

TCCGATCTTTGATTGAAAGAATTCTAAGTGAAGAAGCAACATCTAGTTCA

GAAGTTGAACTCTATTCCAAGTTGTGCTCGCATGCTGATCATATAATGGA

AACCATACAGAGGCATTTCCATAATGAAGAAGTTCAGTATTGCTGAGGAC

AAGGTTATTTTTCCGGCAGTGGATGGGGATTTTTCTTTCTTTCAGGAGCA

TGCTGAAGAAGAAAGCCAATTTAATGACTTCCGATCTTTGATTGAAAGAA

TTCTAAGTGAAGAAGCAACATCTAGTTCAGAAGTTGAACTCTATTCCAAG

TTGTGCTCGCATGCTGATCATATAATGGAAACCATACAGAGGCATTTCCA

TAATGAAGAAGTTCAGGTTCTTCCACTTGCACGGAAGCACTTTAGCTTCA

AAAGGCAACGTGAACTAGTGTATGAAAGCTTGTGCATGATGCCTCTAAAG

TTGATTGAGCGTGTCCTACCATGGTTTGTTGGATCATTAACTGAAGATGA

AGCAGAGATATTTCTGAAAAACATACAATCGGCAGGTTCTTCCACTTGCA

CGGAAGCACTTTAGCTTCAAAAGGCAACGTGAACTAGTGTATGAAAGCTT

GTGCATGATGCCTCTAAAGTTGATTGAGCGTGTCCTACCATGGTTTGTTG

GATCATTAACTGAAGATGAAGCAGAGATATTTCTGAAAAACATACAATCG

GCAGCTCCAGCAATAGATTCTGCTTTAGTCACTCTCTTCTCTGGTTGGGC

TTGCAAGGCTCGTAAAAATGGTCAGTGCTTGTCTTCAAGTGCATCACGTT

TCTGTCCTGCTAAAAAAATTGGTCGGTCTTCCTGTGCTTGTGCATTGTCT

GGTAAAGATTGCTCAGTGTTAGCTGAATCGGAAGGGACCCAAAGATCAGT

GAAGAGAAGCATATTGGAGCTGCACAAAAATGGAGATGTCTCAAAAACAA

CAGAAAATGAATGTGCCCAGAAACCATGTTGTGGTGCCCGGTCTTGTTGT

GTGCCAGCATTAGGAGTCAGCGGCAACAATTTGGGGCTGAGTTCACTTTC

TGCTGCCAAGTCCTTACGGAGCTTCACTTCTTCCGCCCCATCTCTTAATT

CCAGTCTTTTCATATGGGAAACAGACAGCAGCTCATGTGATATGGGCTCT

GCAGAAAGACCAATTGATACCATCTTTAAATTCCATAAGGCTATACGCAT AGATTTGGAGTATTTAGATGTAGAATCTGGAAAGCTTTGTGATGGTGATG

GAGCAACTATTCGACAATTTACTGGAAGATTCCGTCTTTTATGGGGTTTA

TACAGAGCTCATAGTAATGCTGAAGATGATATAGTTTTCCCCGCATTGGA

ATCCAAAGAGACACTTCATAACGTGAGCCATTCTTACACATTAGATCATA

AGGCGGAAGAGAAGTTATTTGAAGATATTTCTTGTGTTCTCTCCGAGCTT

TCTGTCCTTCATGAAGCATTGCAAAGGACCCATATGTCAGAGGATTTAAG

TGAAAGTAATTTGGGAATATCTGAAGTCAATGATAGTGATGATATCAGAA

AGTACAATGAGCTTGCAACTAAGCTTCAAGGAATGTGTAAATCTATACGA

GTGACCCTGGATCAGCATATTTTCAGGGAAGAGCTTGAACTCTGGCCGTT

GTTTGGGAAGCATTTCACTGTGGAAGAACAAGACAAGATAGTGGGCCGAA

TAATTGGAACTACAGGGGCCGAAGTTCTCCAATCAATGTTACCGTGGGTG

ACTTCTGCACTAACACAAGATGAACAGAACCAAATGATGGACACATGGAA

ACAAGCAGCTAAGAATACAATGTTCAATGAATGGCTAACTGAATGCTTGA

TAGAGAGTCCAGGATCTACATCACAGACAGAAACATCAGAACATAGCACT

TCTCAAAGAGCTCCAGCAATAGATTCTGCTTTAGTCACTCTCTTCTCTGG

TTGGGCTTGCAAGGCTCGTAAAAATGGTCAGTGCTTGTCTTCAAGTGCAT

CACGTTTCTGTCCTGCTAAAAAAATTGGTCGGTCTTCCTGTGCTTGTGCA

TTGTCTGGTAAAGATTGCTCAGTGTTAGCTGAATCGGAAGGGACCCAAAG

ATCAGTGAAGAGAAGCATATTGGAGCTGCACAAAAATGGAGATGTCTCAA

AAACAACAGAAAATGAATGTGCCCAGAAACCATGTTGTGGTGCCCGGTCT

TGTTGTGTGCCAGCATTAGGAGTCAGCGGCAACAATTTGGGGCTGAGTTC

ACTTTCTGCTGCCAAGTCCTTACGGAGCTTCACTTCTTCCGCCCCATCTC

TTAATTCCAGTCTTTTCATATGGGAAACAGACAGCAGCTCATGTGATATG

GGCTCTGCAGAAAGACCAATTGATACCATCTTTAAATTCCATAAGGCTAT

ACGCATAGATTTGGAGTATTTAGATGTAGAATCTGGAAAGCTTTGTGATG

GTGATGGAGCAACTATTCGACAATTTACTGGAAGATTCCGTCTTTTATGG

GGTTTATACAGAGCTCATAGTAATGCTGAAGATGATATAGTTTTCCCCGC

ATTGGAATCCAAAGAGACACTTCATAACGTGAGCCATTCTTACACATTAG

ATCATAAGGCGGAAGAGAAGTTATTTGAAGATATTTCTTGTGTTCTCTCC

GAGCTTTCTGTCCTTCATGAAGCATTGCAAAGGACCCATATGTCAGAGGA

TTTAAGTGAAAGTAATTTGGGAATATCTGAAGTCAATGATAGTGATGATA

TCAGAAAGTACAATGAGCTTGCAACTAAGCTTCAAGGAATGTGTAAATCT

ATACGAGTGACCCTGGATCAGCATATTTTCAGGGAAGAGCTTGAACTCTG

GCCGTTGTTTGGGAAGCATTTCACTGTGGAAGAACAAGACAAGATAGTGG

GCCGAATAATTGGAACTACAGGGGCCGAAGTTCTCCAATCAATGTTACCG TGGGTGACTTCTGCACTAACACAAGATGAACAGAACCAAATGATGGACAC

ATGGAAACAAGCAGCTAAGAATACAATGTTCAATGAATGGCTAACTGAAT

GCTTGATAGAGAGTCCAGGATCTACATCACAGACAGAAACATCAGAACAT

AGCACTTCTCAAAGAGGTGCTGAATATCAAGAAAGCTTGAACCTGAATGA

TCAGATGTTTAAGCCGGGTTGGAAAGACATATTCCGGATGAATCAGAATG

AACTTGAGTCAGAAATCCGGAAAGTTTATCGCGACTCAACCCTTGACCCA

AGGAGAAAGGCATATCTAGTGCAGAATCTGATGACAAGGTGCTGAATATC

AAGAAAGCTTGAACCTGAATGATCAGATGTTTAAGCCGGGTTGGAAAGAC

ATATTCCGGATGAATCAGAATGAACTTGAGTCAGAAATCCGGAAAGTTTA

TCGCGACTCAACCCTTGACCCAAGGAGAAAGGCATATCTAGTGCAGAATC

TGATGACAAGTCGCTGGATAGCTGCCCAACAGAAGTTACCTAAATCTCAA

TCTGGGGAATCATCTAATAAACAAATAGAAGGGTGTGTACCTTCATTTCG

GGACCCAGAGAAACAAGTATTTGGTTGTGAGCACTATAAGAGAAATTGCA

AAGTTCGAGCTGCTTGTTGTGGGAAGTTATTTACATGCAGATTTTGTCAT

GACAACAATTCAAGTGATCACTCAATGGATAGTCGCTGGATAGCTGCCCA

ACAGAAGTTACCTAAATCTCAATCTGGGGAATCATCTAATAAACAAATAG

AAGGGTGTGTACCTTCATTTCGGGACCCAGAGAAACAAGTATTTGGTTGT

GAGCACTATAAGAGAAATTGCAAAGTTCGAGCTGCTTGTTGTGGGAAGTT

ATTTACATGCAGATTTTGTCATGACAACAATTCAAGTGATCACTCAATGG

ATAGAAAAGCAACATTGGAAATGATGTGTATGGCCTGCATGACTATACAG

CCAGTTGGGCCCATATGCACAACCCCTTCTTGTAATGGACTTTCAATGGC

AAAATACTACTGTAACATTTGCAAATTTTTTGATGATGAAAGAAAAGCAA

CATTGGAAATGATGTGTATGGCCTGCATGACTATACAGCCAGTTGGGCCC

ATATGCACAACCCCTTCTTGTAATGGACTTTCAATGGCAAAATACTACTG

TAACATTTGCAAATTTTTTGATGATGAAAGGAATGTTTACCATTGCCCAT

TTTGCAATATATGCCGTGTTGGACAAGGGCTTGGGATTGATTACTTTCAT

TGTATGAAATGCAATTGTTGCGTGGGGATAAAATCAGTGTCTCACAAGTG

CCTGGAGAAAGGCTTAGAAATGAACTGCCCAATTTGCTGTGACGACTTGT

TCACATCAAGTGCAACGGTCAGAGCTCTTGCTTGTGGGCATTACATGCAT

TCATCTTGCTTTCAGGAATGTTTACCATTGCCCATTTTGCAATATATGCC

GTGTTGGACAAGGGCTTGGGATTGATTACTTTCATTGTATGAAATGCAAT

TGTTGCGTGGGGATAAAATCAGTGTCTCACAAGTGCCTGGAGAAAGGCTT

AGAAATGAACTGCCCAATTTGCTGTGACGACTTGTTCACATCAAGTGCAA

CGGTCAGAGCTCTTGCTTGTGGGCATTACATGCATTCATCTTGCTTTCAG

GCATACACTTGCAGTCACTACACATGTCCAATCTGCAGCAAGTCATTGGG AGATATGGCGGCATACACTTGCAGTCACTACACATGTCCAATCTGCAGCA

AGTCATTGGGAGATATGGCGGTTTACTTTGGTATGCTTGACGCGCTGTTG

GCTGCTGAGCAGCTTCCTGAAGAGTACAGGGACCGCTCTCAGGTTTACTT

TGGTATGCTTGACGCGCTGTTGGCTGCTGAGCAGCTTCCTGAAGAGTACA

GGGACCGCTCTCAGGACATACTCTGCCATGACTGTGATAGAAAGGGAATC

TCACACTTCCACTGGTTATATCATAAATGTGGATTTTGTGGTTCATATAA

TACTCGGGTGATCAAGAGTGAGACAACAAATTCCAGCTGCCCTTGAGACA

TACTCTGCCATGACTGTGATAGAAAGGGAATCTCACACTTCCACTGGTTA

TATCATAAATGTGGATTTTGTGGTTCATATAATACTCGGGTGATCAAGAG

TGAGACAACAAATTCCAGCTGCCCTTGA

SEQ ID NO: 43> Psat7g010880.1

ATGGCGACGCCATTAGACGGCGGAGGAGTGGCGGTTCTTACGAACTCGGC

GAACAAAGTCGATTCCTCTTCGGCGCTTAATGGCGGTTTGAAGTGCTCCA

AACTCGACTCGCCGATTTTGATTTTCTTGTTCTTTCATAAAGCGATTCGG

AATGAACTTGACGTTTTACACCGATTAGCCATGGCATTCGCTACCGGTAA

TCGCTCTGATATTCAGCCACTTTTCGAGCGTTACCGTTTTCTCAGTTCCA

TTTATAGACACCACTCCAATGCTGAAGATGAGATGGCGACGCCATTAGAC

GGCGGAGGAGTGGCGGTTCTTACGAACTCGGCGAACAAAGTCGATTCCTC

TTCGGCGCTTAATGGCGGTTTGAAGTGCTCCAAACTCGACTCGCCGATTT

TGATTTTCTTGTTCTTTCATAAAGCGATTCGGAATGAACTTGACGTTTTA

CACCGATTAGCCATGGCATTCGCTACCGGTAATCGCTCTGATATTCAGCC

ACTTTTCGAGCGTTACCGTTTTCTCAGTTCCATTTATAGACACCACTCCA

ATGCTGAAGATGAGGTGATTTTTCCCGCTCTAGATAGACGTGTGAAGAAT

GTTGCAAAAACTTATTCTCTTGAGCACAAGGGTGAAAGCAATCTTTTTGA

TCATCTTTTTGAGTTGTTAAATTCTTCCATCAAGAATGACGAAAGTTTTC

CAAGAGAATTAGCATCTTGCACAGGAGCCTTACAGACATCAGTTAGTCAA

CACTTGGCAAAGGAAGAGGAACAGGTGATTTTTCCCGCTCTAGATAGACG

TGTGAAGAATGTTGCAAAAACTTATTCTCTTGAGCACAAGGGTGAAAGCA

ATCTTTTTGATCATCTTTTTGAGTTGTTAAATTCTTCCATCAAGAATGAC

GAAAGTTTTCCAAGAGAATTAGCATCTTGCACAGGAGCCTTACAGACATC

AGTTAGTCAACACTTGGCAAAGGAAGAGGAACAGGTTTTTCCTCTGCTTA

TTGAAAAGTTCTCTCTTGAGGAACAAGCATCTTTAGTTTGGCAGTTTCTT

TGCAGTATTCCTGTGAACATGATGGCAGAATTTCTTCCCTGGCTTTCAAC

ATCTATATCAGCAGATGAATCTCAGGATTTACGAGATTTCTTAGTCAAGA TTGTGCCAGAGGAAAGGCTTCTTCAAAAGGTTTTTCCTCTGCTTATTGAA

AAGTTCTCTCTTGAGGAACAAGCATCTTTAGTTTGGCAGTTTCTTTGCAG

TATTCCTGTGAACATGATGGCAGAATTTCTTCCCTGGCTTTCAACATCTA

TATCAGCAGATGAATCTCAGGATTTACGAGATTTCTTAGTCAAGATTGTG

CCAGAGGAAAGGCTTCTTCAAAAGGTTGTTTTCACTTGGATGGAAGGGAG

AAGTAGTGTTAATAAAATTCAAAGTAGTGCGGATCATTCTCAAGTATGTT

GTTCCAGCCCGTTAACCCATCAGGCTGGAAGAGTCAATTGTGTATGCGAA

TCCACGACAACTGGAAAAAGGAAACATTCTGGATCTATGCAAGATGTTTC

TGATGCCACCGGAACACACCCTATAGATGAGATATTGCTCTGGCATAATG

CAATAAAAAAAGAGCTTAGTGAGATAGCAGTGGAGACCAGAAGGATACAA

CGTTCCGGAGATTTTACTGACATATCAGCTTTTAATGACAGATTGCAATT

CATTGCTGACGTTTGCATATTTCATAGGTTGTTTTCACTTGGATGGAAGG

GAGAAGTAGTGTTAATAAAATTCAAAGTAGTGCGGATCATTCTCAAGTAT

GTTGTTCCAGCCCGTTAACCCATCAGGCTGGAAGAGTCAATTGTGTATGC

GAATCCACGACAACTGGAAAAAGGAAACATTCTGGATCTATGCAAGATGT

TTCTGATGCCACCGGAACACACCCTATAGATGAGATATTGCTCTGGCATA

ATGCAATAAAAAAAGAGCTTAGTGAGATAGCAGTGGAGACCAGAAGGATA

CAACGTTCCGGAGATTTTACTGACATATCAGCTTTTAATGACAGATTGCA

ATTCATTGCTGACGTTTGCATATTTCATAGTATTGCGGAGGACAAGGTTA

TTTTTCCGGCGGTAGATGGAGAGTTTTCTTTCTTTCAGGAGCATGCTGAA

GAAGAAAGCCAATTTAATGACTTTCGGTGTTTGATTGAAAGTATTCTAAG

TGAAGGAGCGGCATCTAATTCAGAAGTTGAATTTTATTCCAAGTTATGCT

CACATGCTGATCATATAATGGAAACCATACAGAGGCATTTCCATAATGAA

GAAGTTCAGTATTGCGGAGGACAAGGTTATTTTTCCGGCGGTAGATGGAG

AGTTTTCTTTCTTTCAGGAGCATGCTGAAGAAGAAAGCCAATTTAATGAC

TTTCGGTGTTTGATTGAAAGTATTCTAAGTGAAGGAGCGGCATCTAATTC

AGAAGTTGAATTTTATTCCAAGTTATGCTCACATGCTGATCATATAATGG

AAACCATACAGAGGCATTTCCATAATGAAGAAGTTCAGGTTCTTCCACTT

GCAAGAAAACACTTCAGCTTTAGAAGACAGTGTGAACTTCTGTACCAAAG

CTTATGCATGATGCCTCTGAAATTGATAGAGAGAGTCCTACCATGGTTGG

TAAAATCTTTAACAGAAGAAGAAGCAAATATATTTCTGAGAAACATGCAA

TTTGCAGGTTCTTCCACTTGCAAGAAAACACTTCAGCTTTAGAAGACAGT

GTGAACTTCTGTACCAAAGCTTATGCATGATGCCTCTGAAATTGATAGAG

AGAGTCCTACCATGGTTGGTAAAATCTTTAACAGAAGAAGAAGCAAATAT

ATTTCTGAGAAACATGCAATTTGCAGCTCCAACAGCAGATTCTGCTCTGG TCACCCTCTTCAGTGGTTGGGCATGCAAGGCTCGTAATGAAGGCCTGTGT

CTGTCTTCAGGCACATCAGACTGCTGTCCTGCTCAAAGACTTTCTGATAT

TGAAGAAGATATTGATCAGCCATCCTGTGTTTGTTCCTCTGCATCATCTT

GCAGACATTGCTCAGTAATACTTGAGTCAGATGGCAACAAAAGACCAGTC

AAGCGAAACACATTGAAATTGAGCAATGGAGATGTACCTGAAACTTTGGA

GACTGAAAGTATCCCGAAACAGTGCTTTAGTCCTCGGTCTTGTTGTGTAC

CAGGTTTAGGAGTAAACAGTAACAATTTGGGGCTTAGTTCAGTTTCGACA

ACCAAGTCCTTACGCTCCTTGTCTTTCAGCTCTTCTGCTCCTTCTCTTAA

TTCCAGTCTTTTCATATGGGAAGCAGAGAGCAGCTCATGTGATGTTGGCT

CTGCAGAAAGACCAATTGATACCATATTTAAATTCCATAAAGCTATACGC

AAAGACTTGGAGTATCTAGATGTTGAATCTGGAAAGCTGAGTGATAGTGA

CGAGACAGTTATTAGGCAATTTAGTGGAAGATTTCGTCTTTTGTGGGGTT

TATATAGAGCTCATAGTAATGCAGAAGATGATATAGTATTTCCAGCATTA

GAATCCAAAGAGGCCCTTCACAATGTGAGCCATTCATACATGTTGGACCA

TAAGCAAGAAGAACAATTGTTTGAAGATATTTCTTGCGTGCTTTCTGAGT

TTTTTGTCCTTCATGAAGCCTTGCAGTTGACCCATATGGCAGGGGATTTG

ATTGATAGTAATTTTGGAACTTCTGATACAAATGATAGTGATGGTGTCAA

GAAGTATAACGAACTTGCAACAAAGCTTCAGGGGATGTGCAAATCCATAA

GAGTGACCCTGGATCAGCATATTTTCAGGGAAGAGTGTGAACTGTGGCCA

TTGTTTGGGAAACATTTCACTGTGGAAGAACAAGATAAGATAGTAGGCCG

GATAATTGGAACTACAGGTGCTGAAGTTCTCCAGTCAATGTTACCTTGGG

TAACTTCTGCACTTACTCAGGATGAGCAGAATAAAATGATGGATACATGG

AAGCAGGCAACTAAGAATACTATGTTCAATGAATGGCTTAATGAATGCTG

GAAAGAGAGTCCGGAATCTATAACACAGACAGAAACGTCACATTGTAGCA

CTTCTCACAGAGCTCCAACAGCAGATTCTGCTCTGGTCACCCTCTTCAGT

GGTTGGGCATGCAAGGCTCGTAATGAAGGCCTGTGTCTGTCTTCAGGCAC

ATCAGACTGCTGTCCTGCTCAAAGACTTTCTGATATTGAAGAAGATATTG

ATCAGCCATCCTGTGTTTGTTCCTCTGCATCATCTTGCAGACATTGCTCA

GTAATACTTGAGTCAGATGGCAACAAAAGACCAGTCAAGCGAAACACATT

GAAATTGAGCAATGGAGATGTACCTGAAACTTTGGAGACTGAAAGTATCC

CGAAACAGTGCTTTAGTCCTCGGTCTTGTTGTGTACCAGGTTTAGGAGTA

AACAGTAACAATTTGGGGCTTAGTTCAGTTTCGACAACCAAGTCCTTACG

CTCCTTGTCTTTCAGCTCTTCTGCTCCTTCTCTTAATTCCAGTCTTTTCA

TATGGGAAGCAGAGAGCAGCTCATGTGATGTTGGCTCTGCAGAAAGACCA

ATTGATACCATATTTAAATTCCATAAAGCTATACGCAAAGACTTGGAGTA TCTAGATGTTGAATCTGGAAAGCTGAGTGATAGTGACGAGACAGTTATTA

GGCAATTTAGTGGAAGATTTCGTCTTTTGTGGGGTTTATATAGAGCTCAT

AGTAATGCAGAAGATGATATAGTATTTCCAGCATTAGAATCCAAAGAGGC

CCTTCACAATGTGAGCCATTCATACATGTTGGACCATAAGCAAGAAGAAC

AATTGTTTGAAGATATTTCTTGCGTGCTTTCTGAGTTTTTTGTCCTTCAT

GAAGCCTTGCAGTTGACCCATATGGCAGGGGATTTGATTGATAGTAATTT

TGGAACTTCTGATACAAATGATAGTGATGGTGTCAAGAAGTATAACGAAC

TTGCAACAAAGCTTCAGGGGATGTGCAAATCCATAAGAGTGACCCTGGAT

CAGCATATTTTCAGGGAAGAGTGTGAACTGTGGCCATTGTTTGGGAAACA

TTTCACTGTGGAAGAACAAGATAAGATAGTAGGCCGGATAATTGGAACTA

CAGGTGCTGAAGTTCTCCAGTCAATGTTACCTTGGGTAACTTCTGCACTT

ACTCAGGATGAGCAGAATAAAATGATGGATACATGGAAGCAGGCAACTAA

GAATACTATGTTCAATGAATGGCTTAATGAATGCTGGAAAGAGAGTCCGG

AATCTATAACACAGACAGAAACGTCACATTGTAGCACTTCTCACAGAGGT

TCTGAGTATCAGGAATGCTTGGACCACAATGATCAAATGTTCAAGCCAGG

TTGGAAAGACATATTCCGGATGAATCAGACTGAACTTGAGTCAGAGATTC

GGAAGGTTTATCGCGATTCGACTCTTGATCCAAGGAGAAAGGCATATCTT

GTGCAGAATCTTTTGACAAGGTTCTGAGTATCAGGAATGCTTGGACCACA

ATGATCAAATGTTCAAGCCAGGTTGGAAAGACATATTCCGGATGAATCAG

ACTGAACTTGAGTCAGAGATTCGGAAGGTTTATCGCGATTCGACTCTTGA

TCCAAGGAGAAAGGCATATCTTGTGCAGAATCTTTTGACAAGTCGTTGGA

TAGCTTCCCAGCAGAAATCACCTAAAGCTCCATCTGAAGAAGGATTATCA

AATGGTGTTGAAATAGAAGGACACTCACCATCATTTCGGGACCCTAGGAA

ACTTGTATTTGGGTGTGAGCACTATAAGAGAAATTGCAAGCTTCGAGCTG

CATGTTGTGGCAAGTTATTTACTTGCAGATTTTGTCATGACAATGTGAGC

GATCACTCAATGGATAGTCGTTGGATAGCTTCCCAGCAGAAATCACCTAA

AGCTCCATCTGAAGAAGGATTATCAAATGGTGTTGAAATAGAAGGACACT

CACCATCATTTCGGGACCCTAGGAAACTTGTATTTGGGTGTGAGCACTAT

AAGAGAAATTGCAAGCTTCGAGCTGCATGTTGTGGCAAGTTATTTACTTG

CAGATTTTGTCATGACAATGTGAGCGATCACTCAATGGATAGAAAAGCAA

CATCAGAAATGATGTGTATGCGCTGCATGAATATACAACCAATTGGGTCT

ATATGCATGACACCTTCATGTAACGCACTTTCAATGGCAAAGTACTATTG

CAGTATATGCAAATTTTTTGATGATGAAAGAAAAGCAACATCAGAAATGA

TGTGTATGCGCTGCATGAATATACAACCAATTGGGTCTATATGCATGACA

CCTTCATGTAACGCACTTTCAATGGCAAAGTACTATTGCAGTATATGCAA ATTTTTTGATGATGAAAGGAATGTATACCATTGCCCATTTTGCAATTTAT

GCCGTGTTGGACGAGGTCTAGGGATTGATTATTTTCATTGCATGAAATGC

AATTGCTGCCTGGGGATTAAAACATCATCTCATAAGTGCCTAGAGAAAGG

TTTAGAAATGAACTGCCCAATATGCTGTGACGACTTATTCACGTCAAGTG

CTACAGTCAGAGCTCAGCCCTGTGGCCACTACATGCATTCTGCTTGCTTT

CAGGAATGTATACCATTGCCCATTTTGCAATTTATGCCGTGTTGGACGAG

GTCTAGGGATTGATTATTTTCATTGCATGAAATGCAATTGCTGCCTGGGG

ATTAAAACATCATCTCATAAGTGCCTAGAGAAAGGTTTAGAAATGAACTG

CCCAATATGCTGTGACGACTTATTCACGTCAAGTGCTACAGTCAGAGCTC

AGCCCTGTGGCCACTACATGCATTCTGCTTGCTTTCAGGCCTACACTTGT

AGTCACTACACGTGTCCAATCTGCAGCAAGTCATTGGGTGATATGGCGGC

CTACACTTGTAGTCACTACACGTGTCCAATCTGCAGCAAGTCATTGGGTG

ATATGGCGGTTTATTTTGGTATGCTTGATGCCTTATTGGCTGCAGAGGAG

CTCCCTGAAGAGTATAGAGACCGCTATCAGGTTTATTTTGGTATGCTTGA

TGCCTTATTGGCTGCAGAGGAGCTCCCTGAAGAGTATAGAGACCGCTATC

AGGACATACTTTGCAATGACTGTGATCGAAAGGGAACTTCCCGCTTCCAC

TGGTTGTATCACAAATGTGGGTCTTGTGGCTCTTACAATACTCGGTTGAT

CAAGCGCGAGACACGCTGAGACATACTTTGCAATGACTGTGATCGAAAGG

GAACTTCCCGCTTCCACTGGTTGTATCACAAATGTGGGTCTTGTGGCTCT

TACAATACTCGGTTGATCAAGCGCGAGACACGCTGA

SEQ ID NO: 50 >Athal HR1

SFSDDAEEISPILIFLFFHKAVCSELEALHRLALEFATGHHVDLRLLRERYRFLRSIYKHH

CNAEDEVIFSALDIRVKNVAQTYSLEHKGESNLFDHLFELLNSATETDESYRRELARST GALQTSVSQHLAKEQKQVFPLLIEKFKYEEQAYIVWRFLCSIPVNMLAVFLPWISSSISV DESKEMQTCLKKIVPGE

SEQ ID NO: 51 > Atal HR2

SSDTLHPVDEIKLWHKSINKEMKEIADEARKIQLSGDFSDLSAFDERLQYIAEVCIFHSL

AEDKIIFPAVDGEFSFSEEHDEEENQFNEFRCLIENIKSAGASSTSAAEFYTKLCSHAD QIMETIQRHFHNEEIQVLPLARKNFSFKRQQELLYQSLCIMPLRLIERVLPWLTASLTED EAKNFLKNLQAGAPKSDVAL

SEQ ID NO: 52 > Atal HR3 ERPVATIFKFHKAISKDLEFLDVESGKLIDCDGTFIRQFIGRFHLLWGFYKAHSNAEDDI

LFPALESKETLHNVSHSYTLDHKQEEKLFGDIYSVLTELSILHEKLQSDSMMEDIAQTD

TVRTDIDNGDCNKKYNELATKLQGMCKSIKITLDQHIFLEELELWPLFDKHFSIQEQDKI

VGRIIGTTGAEVLQSMLPWVTSALSEDEQNRMMDTWKQATKNT

SEQ ID NO: 53 > Brassica v1.0|Bol030932 HR1

SFSEDAEEISPILFFLFFHKAVCSELESLHRLALEFATGHHVDLRLLRERYRFLRSIYKH

HCNAEDEVIFSALDIRVKNVAQTYSLEHKGESTLFDHLFELLNPATEIDESYRRGLASS

TGALQTSVSQHLAKEQKQVFPLLIEKFKHEEQAYIVWRFLCSIPVNMLAVFLPWLASSI

SVDESKEMQTCLSKIVPDE

SEQ ID NO: 54 > Bras v1.0|Bol030932 HR2

FDAQMHPIDEIKLWHKSMNKEMKEIADEARKIQLSGDFSDLSAFDERLQYIAEVCIFHS

LAEDKIIFPAVDGEFSFSEEHDEEENQFNEFRCLIENIKSAGASSTSAAEFYTKLCAHA

DQIMETIQRHFHNEEIQVLPLARKNFTFKRQQELLYQSLCIMPLRLIERVLPWLAASLTE

DEAKNFLKNLQAGAPKSDA

SEQ ID NO: 55 > Bras v1.0|Bol030932 HR3

ERPVATI FAFH KAISKDLEFLDVESGKLI DCDETFI RQFMGRFH LLWGYYKAHSSAEDD

ILFPALESKEALHNVSHSYTLDHKQEEKLFEDIYSVLTELSMLHEKLQSDSMMGGVTQ

TETVHTDIDSGDCKKKYNELATKLQGMCKSIKITLDQHIFLEELELWPLFDEHFSIQEQD

KI VGRI IGTTGAEVLQSM LPWVTSALSEDEQN RMM DTWKQATKN

SEQ ID NO: 56 > Pisum sativum; Psat1g036240.1 HR1

GFNRSSLSESPILIFSFFQKAIGNELDALHRLAMAFATGNCSDIQPLSERYHFLRSMYR

HHSNAEDEVIFPALDKRVKNVAQTYSLEHKGESDLFDHLFELLNSSVDNDETFRRELA

SCTGALQTSLSQHMAKEQQQVFPLLIEKFSVEEQASLVWQFLCSIPVNMMAEFLPWL

STSISPDESQDLRKCLSKIVPEE

SEQ ID NO: 57 > Pisum sativum; Psat1g036240.1 HR2

ETMGSHPIDEILLWHNAIKKELNEIAVETRKIQHSGDYTNLSAFNERLQFIAEVFIFHSIA

EDKVIFPAVDGDFSFFQEHAEEESQFNDFRSLIERILSEEATSSSEVELYSKLCSHADHI

METIQRHFHNEEVQVLPLARKHFSFKRQRELVYESLCMMPLKLIERVLPWFVGSLTED

EAEIFLKNIQSAAPAIDS SEQ ID NO: 58 Pisum sativum; Psat1g036240.1 HR3

ERPIDTIFKFHKAIRIDLEYLDVESGKLCDGDGATIRQFTGRFRLLWGLYRAHSNAEDDI

VFPALESKETLHNVSHSYTLDHKAEEKLFEDISCVLSELSVLHEALQRTHMSEDLSESN

LGISEVNDSDDIRKYNELATKLQGMCKSIRVTLDQHIFREELELWPLFGKHFTVEEQDK I VGRI IGTTGAEVLQSM LPWVTSALTQDEQNQMM DTWKQAAKN

SEQ ID NO: 59 > Pisum sativum >Psat7gO10880.1 HR1

LKCSKLDSPILIFLFFHKAIRNELDVLHRLAMAFATGNRSDIQPLFERYRFLSSIYRHHS

NAEDEVIFPALDRRVKNVAKTYSLEHKGESNLFDHLFELLNSSIKNDESFPRELASCTG

ALQTSVSQHLAKEEEQVFPLLIEKFSLEEQASLVWQFLCSIPVNMMAEFLPWLSTSISA DESQDLRDFLVKIVPEE

SEQ ID NO: 60 > Pisum sativum >Psat7gO10880.1 HR2

DATGTHPIDEILLWHNAIKKELSEIAVETRRIQRSGDFTDISAFNDRLQFIADVCIFHSIAE

DKVIFPAVDGEFSFFQEHAEEESQFNDFRCLIESILSEGAASNSEVEFYSKLCSHADHI

METIQRHFHNEEVQVLPLARKHFSFRRQCELLYQSLCMMPLKLIERVLPWLVKSLTEE EANIFLRNMQFAAPTADS

SEQ ID NO: 61 > Pisum sativum >Psat7gO10880.1 HR3

ERPIDTIFKFHKAIRKDLEYLDVESGKLSDSDETVIRQFSGRFRLLWGLYRAHSNAEDD

IVFPALESKEALHNVSHSYMLDHKQEEQLFEDISCVLSEFFVLHEALQLTHMAGDLIDS

NFGTSDTNDSDGVKKYNELATKLQGMCKSIRVTLDQHIFREECELWPLFGKHFTVEE QDKIVGRIIGTTGAEVLQSMLPWVTSALTQDEQNKMMDTWKQATKN

SEQ ID NO: 62 > Potato >S. tuberosum v6.1 |Soltu.DM.05G003350.2 HR1

SRAVGVKGSSPIRIFLFFHKAIRKELDGLHRSAMAFATNQDTEIKPFMERCYFLRSIYK

HHCNAEDEVIFPALDIRVKNVARTYSLEHEGEGVLFDHLFALLDSDMQSEESYRRELA

SCTGALQTSISQHMSKEEEQVLPLLMEKFSFEEQASLVWQFLCSIPVNMMAEFLPWL SSSISADECKDMHKCLHKVIPDE

SEQ ID NO: 63 > Potato >S. tuberosum v6.1|Soltu.DM.05G003350.2 HR2

ESTLNLPVDEILHWHKAIRKELNDITEAAREIKLRGDFSDLSAFNQRLQFIAEVCIFHSIA

EDKVIFPAVDAEISFAQEHAEEENEFDKFRCLIESVQSAGSNSTSVEFYSELCSQADHI

METVERHFCNEEAQVLPLARKHFSPKRQRELLYQSLCVMPLRLIECVLPWLVGSLSE EEARSFLQNMHMAAPASDT SEQ ID NO: 64 > Tomato: >S.lycopersicum ITAG4.0|Solyc03g119300.4.1 HR1

EKETGGKQESPILFFLFFHKAIRLELDALHHSALAYATGQLEDIQPLLKRYRFLRSVYKH

HCHAEDEVIFPALDIRVKNVAPTYSLEHKGENDLFDHLFEILNSEKQNCERFPRELASC

TGALQTSVSQHMSKEEEQVFPLLTEKFSMDEQASLVWQFLCSIPVNMMKKFLPWLSS SISPDEHKDMQKCLSMIIPKE

SEQ ID NO: 65 > Tomato: >S.lycopersicum ITAG4.0|Solyc03g119300.4.1 HR2

DTDSGNPIDEVLHWHNAIKRELDEIAAEARRIELAGELSSLTAFYARLQFIAQVCIFHSIA

EDKVIFPAVDGGLSFFQEHAEEEIQFNELRCLIESIQCTEVNSTSAAEFFSKLYSQADLII

ETIKQHFHNEEVQVLPLARKHFTRDRQRKVLYQSLCLMPLKLMEQVLPWLVGALSED

EARSFLKN LQLAAPEADT

SEQ ID NO: 66 > Tomato: >S.lycopersicum ITAG4.0|Solyc03g119300.4.1 HR3

HPIDTIFKFHKAIQKDLEYLDVESGKLSDCPETFLRQFIGRFRLLWGLYRAHSNAEDEIV

FPELESKEALHNVSHSYMLDHKQEEKLFEDISSALTNLSELHKGLKEAYQKESGSSILE

STGLYDRDCKRKYNELATKVQGMCKSIRVSLDQHIFREEHELWPLFGKHFSMEEQDII

VGRI IGSTGAEVLQSM LPWVTSALTQDEQN KM M DTLKQATRN

SEQ ID NO: 67 > Lettuce: >L.sativa V8|Lsat_1_v5_gn_8_40260.1 HR1

SNQSSKKQSSPIHIFLFFHKAIRSELDALHRSAIAFATNSHVEIEPLLKRYHFLRSIYKHH

CNAEDEVIFPALDIRVKNVARTYSLEHEGESVIFDQLFTLLDSDMQNEESFRRELASCT

GALQTSINQHMSKEEEQVFPLLVEKFSFEEQASLVWQFLCSIPVNMMAEFLPWLSAS VSSDERHEMRNCLCKVIPE

SEQ ID NO: 68 > Lettuce: >L.sativa V8|Lsat_1_v5_gn_8_40260.1 HR2

DSLHDRPVDEILHWHKAIKKELIDIAEAARRIQLSGDFSDISAFNKRLQFIAEVCIFHSIAE

DKVIFPAVDAELSFAQEHAEEESEFDKFRCLIESIENDGANSSSSEFCSKLCSHADHIM

SIIEKHFKNEELQVLPLARKHFTPKRQRELLYQSLCVMPLRVIECVLPWLVGSLTEEES

KSFLHNMHMAAPPSDI

SEQ ID NO: 69 > Lettuce: >L.sativa V8|Lsat_1_v5_gn_8_40260.1 HR3

GRPIDTIFKFHKAIRKDLEFLDVESGKLNETNESFLHQFNGRFRLLWGLYRAHSNAED

DIVFPALESKETLHNVSHSYTLDHKQEEKLFEDISTSLFELCELHENLDMCDDSFRNYN ELATKVQGMCKSIRVTLDQHILREELELWPLFDRHFSVEEQDKLVGRIIGTTGAEVLQS

M LPWVTSVLTQEEQN KM M DTWKQATKN

SEQ ID NO: 70 > Rice > QsHRZ1_Qs01g49470.1 HR1

SAAAGSAAEAPMLIFLYFHKAIRAELEGLHAAAVRLATERAGDVGALAERCRFFVNIYK

HHCDAEDAVIFPALDIRVKNVAGTYSLEHKGENDLFSQLFALLQLDIQNDDSLRRELAS

CTGAIQTCLSQHMSKEEEQVFPLLTKKFSYEEQADLVWQFLCNIPVNMMAEFLPWLS

SSVSSDEHEDIRSCLCKIVPEE

SEQ ID NO: 71 > Rice > QsHRZ1_Qs01g49470.1 HR2

GQVERHPIDEILYWHNAIRKELIDIAEETRRMQQSGNFSDISSFNARLQFIADVCIFHSIA

EDQVVFPAVDSELSFVHEHAEEERRFNNFRCLIQQIQIAGAKSTALDFYSELCSHADQI

METIEKHFCDEETKVLPQARMLFSPEKQRQLLYKSLCVMPLKLLERVLPWLVSKLSDE

EASSFLENMRLAAPSSET

SEQ ID NO: 72 > Rice > QsHRZ1_Qs01g49470.1 HR3

RPIDTIFKFHKAIRKDLEYLDVESGKLIDGDESCLRQFIGRFRLLWGLYRAHSNAEDEIV

FPALESRETLHNVSHSYTLDHKQEEQLFGDISDALAELSQLHERLTHPHIEVSEAEKND

FNSSDEIDWTRKYNELATKLQGMCKSIRAALTNHVHREELELWPLFDEHFSVEEQDKL

VGRIIGSTGAEVLQSMLPWVTSALTQEEQNMMLDTWKQATKN

SEQ ID NO: 73 > Barley >HovulHRZ1_HG0281210.1 HR1

AAAGSAAEAPMLIFLYFHKAIRAELEGLHGAAVRLATERAGDVDALAERCRFFVNIYKH

HCDAEDAVI FPALDI RVKN VAGTYSLEH KGEN DLFTQLLALLQLDIQN DDALRRELASC

TGAIQTCLTQHMSKEEEQVFPLLTKKFSYEEQSDLVWQFLCNIPVNMLAEFLPWLSAS

VSSDEHEDIRNCLCKIVPEE

SEQ ID NO: 74 > Barley >HovulHRZ1_HG0281210.1 HR2

GQADRHPIDEILYWHNAIRKELNDIAEETRRMQQSGDFADISAFNARLQFIADVCIFHSI

AEDQVVFPAVNSELSFVLEHAEEERRFNNFRCLIQQIQMAGAKSTAAEFYSELCSHAD

QIMEAIEKHFCNEETKVLPQARVLFSPEKQRELLYRSLCVMPLKLLERVLPWLVSKLSD

EEASSFLQNMRLAAPSSDT

SEQ ID NO: 75 > Barley >HovulHRZ1_HG0281210.1 HR3 SRPIDTIFKFHKAIRKDLEYLDVESGKLIDGDESCLRQFIGRFRLLWGLYRAHSNAEDEI VFPALESREPLHNVSHSYTLDHKQEEQLFEDISNVLCELSQLHESLNPAHTEANEAEK HYFNSSNVIDSTRKYNELATKLQGMCKSIRVALSNHVHREELELWPLFDKHFSVEEQD KLVGRIIGTTGAEVLQSMLPWVTSALNQEEQNKMLDTWKQATKN

SEQ ID NO: 76 > Wheat>Traes_HRZ1-A HR1

AAAGSAAEAPMLIFLYFHKAIRAELEGLHGAAVRLATERAGDVGALAERCRFFVNIYKH HCDAEDAVI FPALDI RVKN VAGTYSLEH KGEN DLFTQLLALLQLDIQN DDALRRELASC TGAIQTCLTQHMSKEEEQVFPLLTKKFSYEEQSDLVWQFLCNIPVNMLAEFLPWLSAS VSSDEHQDIRNCLCKIVPEE

SEQ ID NO: 77 > Wheat>Traes_HRZ1-A HR2

CQADRHPIDEILYWHNAIRKELNDIAEETRRMQQSGDFADISAFNARLQFIADVCIFHSI AEDQVVFPAVNSELSFVLEHAEEERRFNNFRCLIQQIQMAGAKSTAAEFYSELCSHAD QIMEAIEKHFCNEETKVLPQARVLFSPEKQRELLYRSLCVMPLKLLERVLPWLVSKLSD EEASSFLQNMRLAGHLFTLIILSYFLYTLYLALKFSLGIQCLLKDSCETAPSSDT

SEQ ID NO: 78 > Wheat>Traes_HRZ1-A HR3

SRPIDTIFKFHKAIRKDLEYLDVESGKLIDGDESCLRQFIGRFRLLWGLYRAHSNAEDEI VFPALESREPLHNVSHSYTLDHKQEEQLFEDISNVLCELSQLHESLNQPHTEANEAEK HYLNSCNVIDSTRKYNELATKLQGMCKSIRVALSNHVHREELELWPLFDKHFSVEEQD KLVGRIIGTTGAEVLQSMLPWVTSALNQEEQNKMLDTWKQATKN

SEQ ID NO: 79 > Rice >QsatKitaake_HRZ2_Qs05g252600.1 HR1

AAAGSAAEAPMLIFVYFHKAIRAELERLHAAAVRLATERSGDVGELERRCRFLFSVYR HHCDAEDAVIFPALDIRVKNVAGTYSLEHKGENDLFAHLFSLLKLDVRNDDGLRRELA SCTGAIQTFITQHMSKEEEQVFPLLIKKFSHEEQADLVWQFLCSIPVNMMAEFLPWLA TSVSSDEHQDILNCLHKIVPDE

SEQ ID NO: 80 > Rice >QsatKitaake_HRZ2_Qs05g252600.1 HR2

SQLVMHPIDEILCWHNAIRKELSDIVEETRRIQQSGDFSDISDFNVKLQFIADVCIFHSIA EDQVIFPAVNDQVSFEQEHAEEERRFNKFRCLIEQIQITGARSTAVDFYSELCSQADQI MEKIERHFKNEETKVLPQARIHFSSEKQRELLYKSLCVIPLKLLERVLPWFVSKLNDQD AEAFLQNMFLAAPSSE SEQ ID NO: 81 Rice >OsatKitaake_HRZ2_Os05g252600.1 HR3

SRPIDTIFKFHKAIRKDLEFLDVESRKLIDGDESSLRQFIGRFRLLWGLYRAHSNAEDEI

VFPALESKETLHNVSHSYTLDHKQEEELFKDISTILFELSQLHADLKHPLGGADAVGAN

HIHPYNRIDWSKKNNELLTKLQGMCKSIRVTLSNHVHREELELWPLFDKHFSVEEQDK IVGRIIGSTGAEVLQSMLPWVTSALSLDEQNNMLDTWRQVTKN

SEQ ID NO: 82 > HovulHRZ2_HG0083800.1 HR1

ATAGSAAEAPVLIFVYFHKAIRAELDRLHAAAVRLATERGGDGDVAALDTRCRFLFSV

YRHHCDAEDAVIFPALDIRVKNVAGTYSLEHKRENDLFSHLFALLQLDVHNNDGARRE

IASCTGAIRTFITQHMFKEEEQVFPLLITKFSYEEQADLVWQFICNIPVNMMADFLPWLS SSVSSDEHQDILNCLQKIVPQE

SEQ ID NO: 83 > HovulHRZ2_HG0083800.1 HR2

SQLVTHPIDEILYWHNAIREELSDIAEETRRIQQSGDFSNISAFNLRLQFIADVCIFHSVA

EDQVIFPAVDGEVSFEQEHAEQEQQFNKFRCLIEQIQTAGARSTAVDFYSELCSQAD

QIMEEIEKHFSNEETKVLPQARTNFSPEKQRELLYRSLCVMPLKLLEQVLPWFVSKLD DVNGQSFLQNMCLAAPSCET

SEQ ID NO: 84 > HovulHRZ2_HG0083800.1 HR3

SRPIDAIFKFHKAIRKDLEFLDAESGKLIDGDESCLRQFVGRFRLLWGLYRAHSNAEDE

IVFPALESKDALHNVSHSYTLDHKQEEELFKDISIILLELSHLRDDSGHPTDETDEAGKG

HICSYSEIDWSRKHNELLTKLQGMCKSIRFTLSNHVHREELELWPLFDKHFSVDDQDK IVGRIIGSTGAEVLQSMIPWVTSALSLDEQNKMLDTWKQASKN

SEQ ID NO: 85 > Wheat>Traes_HRZ2-A HR1

SGTAGSAAEAPVLIFVYFHKAIRAELDRLHAAAVRLATERGGDGDVAALDTRCRFLFS

VYRHHCDAEDAVIFPALDIRVKNVAGTYSLEHKRENDLFAHLFSLLQLDVHNDDGVRR

EVASCAGAIRTFITQHMFKEEEQVFPLLITKFSYEEQADLVWQFICNIPVNMMADFLPW LSSSVSPDEHQDILNCLHKIVPQ

SEQ ID NO: 86 > Wheat>Traes_HRZ2-A HR2

SQLVTHPIDEILYWHDAIRKELSDIAEETRRIQQSGDFSNVSAFNVRLQFIADVCIFHSIA

EDQVIFPAVDGEVSFEQEHAEQEQRFNKFRCLIEEIQTAGARSTAVNFYSELCSQADQ

IMEEMEKHFNNEETKVLPQARINFSPEKQRELLYRSLCVMPLKLLEQVLPWFVLKLDD ANGQSFLQNMFLAAPSSET SEQ ID NO: 87 > Wheat>Traes_HRZ2-A HR3

SRPIDAIFKFHKAIRKDLEYLDVESGNLIDGDESCLRQFVGRFRLLWGLYRAHSNAEDD

IVFPALESKDALHNVSHSYTLDHKQEEELFKDISTILLELSHLRDDSAHPVDEIDEAGKG HICSYSEIDWSRKHNELLTKLQGMCKSIRFTLSNHVHREELELWPLFDKHFSVDDQDK IVGRIIGSTGAEVLQSMIPWVTSALTLDEQNKMMDTWKQATKN

SEQ ID NO: 88 > Algae: Auxenochlorella protothecoides >Auxchl_BTS HR1

KLADGHEQPAPVFQPIHFLYTYLHEAIRHELALLSQSLQLLLAGTSTGWPELRRRYIFL

RDVYKYHSAAEDEVIYPALELEVANVTPSYSVEHEDEEHLLEEMVDLLLATEQSPTKE

NLLAVRQLSWRIQTTVTKHLAKEEAQLLPLLLTHIPPREQGGLVAQFLCCIPISTVARVL AWVKPHAAPADRAAI REALAGAVDD

SEQ ID NO: 89 > Algae: Auxenochlorella protothecoides >Auxchl_BTS HR2

QPAGPPPLRSILHYHAAIRAALEGFAADARAVASAPGGPDPPALAALLVRHRFIRAVCA

FHSAAEDEVVFPALARLHTSQQEGGPAGEPGAAAGDPCASPSRASPGGDRLGAHAA

PGPAAADTGPSPPARHSPPPPPHDGCAGHGEEALRFDELQRLLGEVASCARRRCSR SGTASADLVASADALAAAMAGHMEAEERGVLPALERLCPRPEQRALLWAMVRAMPL RLLERVMPWIAARLAPAERRRWLADLARGAGRGDTT

SEQ ID NO: 90 > Algae: Auxenochlorella protothecoides >Auxchl_BTS HR3

PSPIDHIFQFHNALRKELAELEATIATCQQRLETAAELADTTEVMQRLAARFQFLRGIY

RAHSLSEDEVVFPALEGKKVLRNVSHAYTLDHEQEEMQFQTCSECLDRVMATGTLDS RRQRLAELACYYSGVRGSLETHIRAEELELWPLFAEHFPVAEQEHLVGLIIGRTGADVL TSLLSWVRGAMTREEEDAMMLSIRSAASS

SEQ ID NO: 91 > Chlamydomonas reinhardtii >Cre05.g248550 HR1

SAAAPARAFPPINFIYGHFHNSIRAELGLLAERVRSLEAPGEGVGEMLADLRERYKFLE

QVYKYHSTVEDEVLYPVLDSKVRNVTLAYSIEHQDEEILFEQLSKLISAALEEPEARRK CTI RTLICKVEEI HTTLRKH LAKEEEQLLPLLLQH FSFAEQAELVAQFLYSI PLETVERVL SRLKPRIPRDEQERLLEEIQAVIPD

SEQ ID NO: 92 > Chlamydomonas reinhardtii >Cre05.g248550 HR2

SAAAARAPLQDIIHFHRSICASLVDFAREARSLQAGREPITAGHLQSLLERHRFLRAVY

VFHSISEEEVLFPEVQRLAAANVQLAGGAAAAHQQQCEKDHAAELSSFEDLGRLLAD VRAFARRGRKEVAGMLEKLCCSVEAVAASIEHHMQREEADVFPLLEAHLCQAQQRAL

LYRTIRAMPLRLLERVMPWVVSRLDASAAAALLANIALGAPRSDQAL

SEQ ID NO: 93 > Chlamydomonas reinhardtii >Cre05.g248550 HR3

GFNPIDHIFQFHKALRQELKQLEADVMALEGAVQSVLRHMPASASQHNLAGLMGPGA

AGAVGATGGAAGGGAGGARPATPTSVGTPARGPRAAMPGSDGLQHNQQQPGQQP

GQQQQGQQQQQGQQQQPGPHAHLGVGWPAADGSGAAYTRGGQLAWQHLHGRF QFLQGIYRAHSKSEDEIVFPALESKQALRNVSHAYTLDHRQEEQLFADLEAVIDNLRAV DLTAQGADAELSRQVMAVRRMCAAIRASLETHIRSEESELWPLFTEHFSREEQQYLV GVIIGRTGAQVLQALLPWVSETFSEEEREAMMDSLREATRN

SEQ ID NO: 94 > Beetroot EL10_Ac6g 14339.1 HR1

VSLKNSAQNSPILIFLFFHKAIKAELEGLHRAAIDFATNQGSDIKPLLERYHFLRCIYKHH

CNAEDEVIFPALDIRVKNVARTYSLEHEGESVLFDELFELLNTTVRDEEAYRRELASRT GALQTSISQHMCKEEEQVFPLLIEKFSFEEQASLVWRFLCSIPVNMMVEFLPWLSSSV SSDESQDMRKCLRKFIPNE

SEQ ID NO: 95 >Beetroot EL10_Ac6g 14339.1 HR2

DSDLSFPIDEILLWHKAINQELGEIAEAARRMQLNDEFTDLSAFNERLHFVTEVCIFHSI

AEDKVIFPAVDAELSFAQEHAEEESKLEKIRCLIENIQLANAESSLDDLYSRLSSYADQI MDTIQKHFQNEEIQVLPLARKLFTPQRQRELLYQSLCVMPLKLIERVLPWLVGSLGEE EAKCFLKNM HMAAPAAD

SEQ ID NO: 96 > Beetroot EL10_Ac6g 14339.1 HR3

TRPIDNIFKFHKAIRKDLEYLDAESGKLSECNESFIRQFSGRFRLLWGLYKAHSNAEDD IVFPALESKEALHNVSHSYTLDHKQEEMLFEDISSALSELSQLRGALSRSSTAQDTLGF ASNGGDSSDDLRKYHELATKLQGMCKSIRVTLDQHVFREELELWPLFDKHFSVEDQD KI VGRI IGTTGAEVLQSM LPWVTSALTQEEQN KMM DTWKNATKN

SEQ ID NO: 97 > Z.mays RefGen_V4|Zm00001d043805_P001 HR1

AEAGTSATETPVLIFLYFHKAIRAELEALHGAAVLLATERTGDVAALAERCRFFFSIYKH HCDAEDAVIFPALDIRVKNVAGTYSLEHKGESDLFSQLFDLLELDIQNDDALRRELASC TGAIQTCLSQHMSKEEEQVFPLLTKKFSCEEQADLVWQFLCNIPVNMVAEFLPWLSTS VTSDEHQDIRDCLCKVVPDE SEQ ID NO: 98 > Z.mays RefGen_V4|Zm00001d043805_P001 HR2

SQADRHPIDDILYWHNAIRMELHDIKKETRRVQQSGNFSDISAFNERLQFIADVCIYHSI

AEDQVVFPAVDSELSFVQEHAEEEHRFNNFRCLIQQFQIAGAKSTALDFYSKLCSHAD

KILETIEKHFSNEETKVLPQARMFFSPEKQRELSYKSLCVMPLKLLERVLPWLVSKLSD EQATSFLQNIRLAASPSET

SEQ ID NO: 99 > Z.mays RefGen_V4|Zm00001d043805_P001 HR3

SRPIDTIFKFHKAIRKDLEYLDVESGRLIDGDESCLRQFIGRFRLLWGLYRAHSNAEDEI

VFPALESRETLHNVSHSYTLDHKQEEQLFEDISDVLFQLSQLHDSQGHAQTKVNEVK

QSCFHSSNDVDFTRKYNELATKLQAMCKSIRVALTNHVHREELELWPLFDKHFSVEE QDKLVGRIIGSTGAEVLQSMVPWVTSALTQEEQNKMLDTWKQATKN

SEQ ID NO: 100 > Z.mays RefGen_V4|Zm00001d011622_P002 HR1

AEAGTSATETPVLIFLYFHKAIRAELEALHGAAVLLATERTGDVEMLAKRCRFFFNIYKH

HCDAEDAVIFPALDIRVKNVAGTYSLEHKGESDLFSQLFDLLQLDIHNDDGLRRELASC

TGAIQTCLSQHMSKEEEQVFPLLTKKFSCEEQADLVWQFLCNIPVNMVAEFLPWLSTS VTSDEHQDIRNCLCKVVPDE

SEQ ID NO: 101 > Z.mays RefGen_V4|Zm00001d011622_P002 HR2

PQADKHPIDDILYWHNAIRMELRDIKEETRRVQQSGDFSDISAFNERLQFIADVCIYHSI

AEDQVVFPAVDSELSFVQEHAEEECRFNNFRCLIQQIQIAGAESTALDFYSKLCSHAD

KILEAIEKHFCNEETKVLPQARMLFSLEKQRELSYKSLCVMPLKLLERVLPWLVSKLSD VQATSFLQNIRLAASPSET

SEQ ID NO: 102 > Z.mays RefGen_V4|Zm00001d011622_P002 HR3

SRPIDTIFKFHKAIRKDLEYLDVESGKLIDGNESCLRQFIGRFRLLWGLYRAHSNAEDEI

VFPALESRETLHNVSHSYTLDHKQEEQLFEDISNVLFQLSQLHDSQGHAQTEVNEVKK

SCFHSSNDVDFARKYNELATKLQGMCKSIRVALTNHVHREELELWPLFDKHFSVEEQ DKLVGRIIGSTGAEVLQSMLPWVTSVLTQEEQNKMLDMWKQATKN

Claims

CLAIMS:

1. A genetically altered organism, part thereof or cell, wherein the organism, part thereof or cell comprises one or more mutations in at least one nucleic acid sequence encoding a BRUTUS polypeptide, wherein the BRUTUS polypeptide comprises an amino acid sequence as defined in SEQ ID NO: 4 or 15 or a functional variant or homologue thereof, wherein the functional variant has at least 30% overall amino acid sequence identity to SEQ ID NO: 4 or 15; and wherein the mutation reduces or abolishes binding of iron to the BRUTUS polypeptide, and wherein the organism is a plant or alga.

2. The genetically altered organism, part thereof or cell of claim 1 , wherein the BRUTUS polypeptide comprises one or more hemerythrin domain(s), and wherein the at least one mutation is in at least one hemerythrin domain, wherein the hemerythrin domain comprises a sequence as defined in at least one of SEQ ID NO: 20, or SEQ ID NO: 50 to 102 or a functional variant thereof.

3. The genetically altered organism, part thereof or cell of claim 2, wherein the at least one mutation is in the first N-terminal hemerythrin domain.

4. The genetically altered organism, part thereof or cell of claim 2 or 3, wherein the at least one mutation is a deletion of one or more amino acids, wherein preferably the mutation is within the (H)-HxxxE-H-HxxxE motif of the hemerythrin domain, and more preferably encompassing the third H and the fourth H in the (H)-HxxxE- H-HxxxE motif.

5. The genetically altered organism, part thereof or cell of claim 4, wherein the at least one mutation is a deletion of SEQ ID NO: 1 , 22, 23, 24, 44, 47, 48, or 49 or a variant thereof, wherein the variant has at least 80% overall sequence identity to SEQ ID NO: 1 , 22, 23, 24,44, 47, 48 or 49.

6. The genetically altered organism, part thereof or cell of any preceding claim, wherein the at least one mutation does not significantly affect organism growth and/or yield. 7. The genetically altered organism of any preceding claim, wherein the organism, part thereof or cell is a plant, and wherein the plant is a monocot or dicot.

8. The genetically altered organism of claim 8, wherein the plant is selected from rice, wheat, maize, barley, Brassicas, soybean, potato and tomato, lettuce, Medicago, and beetroot.

9. The genetically altered organism of any preceding claim, wherein the part thereof is a seed, and wherein the seed has an increased iron and/or zinc content.

10. The genetically altered organism of any of claims 1 to 8, wherein the part thereof is a vegetative part, preferably selected from the root, shoot or leaves, and wherein the vegetative part has an increased iron and/or zinc content.

11. The genetically altered organism of any of claims 1 to 6, wherein the organism, part thereof or cell is an alga, part thereof or algal cell, wherein preferably the alga is selected from Chlorella (such as Auxenochlorella protothecoides), Porphyra, Dulse, Laminaria, Alaria, Nostoc, Monostroma, lllva, Enteromorpha, Caulerpa racemose and Durvillaea antarctica.

12. A method of increasing iron and/or zinc concentration in an organism, part thereof or cell, the method comprising introducing at least one mutation into at least one nucleic acid sequence encoding a BRUTUS polypeptide wherein the BRUTUS polypeptide comprises an amino acid sequence as defined in SEQ ID NO: 4 or 15 or a functional variant or homologue thereof, wherein the functional variant has at least 30% overall amino acid sequence identity to SEQ ID NO: 4 or 15; and wherein the mutation reduces or abolishes binding of iron to the BRUTUS polypeptide, and wherein the organism is a plant or alga.

13. A method of producing an organism, part thereof or cell, wherein the organism, part thereof or cell has an increased iron and/or zinc content, wherein the method comprises introducing at least one mutation into at least one nucleic acid sequence encoding a BRUTUS polypeptide wherein the BRUTUS polypeptide comprises an amino acid sequence as defined in SEQ ID NO: 4 or 15 or a functional variant or homologue thereof, wherein the functional variant has at least 30% overall amino acid sequence identity to SEQ ID NO: 4 or 15; and wherein the mutation reduces or abolishes binding of iron to the BRUTUS polypeptide, and wherein the organism is a plant or alga.

14. The method of claim 12 or 13, wherein the BRUTUS polypeptide comprises one or more hemerythrin domains, and wherein the at least one mutation is in at least one hemerythrin domain, wherein the hemerythrin domain comprises a sequence selected from at least one of SEQ ID NO: 20, or SEQ ID NO: 50 to 102 or a functional variant thereof.

15. The method of claim 14, wherein the at least one mutation is in the first N-terminal hemerythrin domain.

16. The method of any of claims 12 to 15, wherein the at least one mutation is a deletion of one or more amino acids, wherein preferably the mutation is within the (H)-HxxxE-H-HxxxE motif of the hemerythrin domain, and more preferably encompassing the third H and the fourth H in the (H)-HxxxE-H-HxxxE motif.

17. The method of any of claims 12 to 16, wherein the at least one mutation is a deletion of SEQ ID NO: 1 , 22, 23, 24, 44, 47, 49 or 49 or a variant thereof, wherein the variant has at least 80% overall sequence identity to SEQ ID NO: 1 , 22, 23, 24, 44, 47, 48 or 49.

18. The method of any of claims 12 to 17, wherein the organism, part thereof or cell is a plant, plant part thereof or plant cell, wherein preferably the plant is selected from a monocot or dicot.

19. The method of claim 18, wherein the plant is selected from rice, wheat, maize, barley, Brassicas, soybean, potato and tomato, lettuce, Medicago and beetroot.

20. The method of any of claims 12 to 19, wherein the part thereof is a seed, and wherein the seed has an increased iron and/or zinc content. The method of any of claims 12 to 19, wherein the part thereof is a vegetative part preferably selected from the root, shoot or leaves, and wherein the vegetative part has an increased iron and/or zinc content. The method of any of claims 12 to 21 , wherein the organism, part thereof or cell is an alga, part thereof or algal cell, wherein preferably the alga is selected from Chlorella (such as Auxenochlorella protothecoides), Porphyra, Dulse, Laminaria, Alaria, Nostoc, Monostroma, lllva, Enteromorpha, Caulerpa racemose and Durvillaea Antarctica. A method of screening a population of plants and identifying and/or selecting an organism that will have an increased iron content, the method comprising detecting in the organism or organism germplasm at least one polymorphism in a BRUTUS gene, wherein the BRUTUS gene encodes a polypeptide as defined in SEQ ID NO: 4 or 15 or functional variant or homologue thereof, wherein the functional variant has at least 30% overall sequence identity to SEQ ID NO: 4 or 15; and wherein the BRUTUS polypeptide comprises at least one hemerythrin domain, and wherein the at least one polymorphism is in at least one hemerythrin domain. The method of claim 23, wherein the polymorphism is the deletion of one or more, amino acids in at least one hemerythrin domain, wherein preferably the polymorphism is the deletion of one or more amino acids, within the (H)-HxxxE- H-HxxxE motif of the hemerythin domain, and more preferably encompassing the third H and the fourth H in the (H)-HxxxE-H-HxxxE motif. A method of producing a food, vitamin or nutritional supplement, the method comprising producing the genetically altered organism according to any of claims 1 to 12, and producing a food composition, vitamin or nutritional supplement from the organism or part thereof. A food, vitamin or nutritional supplement obtained or obtainable by the method of claim 25.

27. A method of treating anaemia in a patient in need thereof, the method comprising consuming the seed of claim 9 or the food, vitamin or nutritional supplement of claim 26. 28. A composition comprising the seed or vegetative part of claim 10 or 11 , wherein the vegetative part is preferably a leaf or leaves or part(s) thereof, and at least one vitamin, wherein preferably the vitamin is vitamin C.