WO2025061878A1

WO2025061878A1 - Molecular means to extend desiccation tolerance during seed germination

Info

Publication number: WO2025061878A1
Application number: PCT/EP2024/076324
Authority: WO
Inventors: Jérôme VERDIER; Naoto Sano; Jaiana MALABARBA
Original assignee: Institut National D'enseignement Superieur Pour L'agriculture L'alimentation Et L'environnement; Universite dAngers; Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Current assignee: Institut National D'enseignement Superieur Pour L'agriculture L'alimentation Et L'environnement; Universite dAngers; Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Priority date: 2023-09-19
Filing date: 2024-09-19
Publication date: 2025-03-27
Anticipated expiration: 2026-03-19

Abstract

Desiccation tolerance is a protective process that allows mature seeds to tolerate cellular water contents below 10%. This process is acquired during seed maturation and is usually lost during imbibition, prior to germination, what enhances seed sensitivity to drought and heat. The present invention shows that it is however possible to maintain desiccation tolerance mechanisms after germination by activating iron absorption in mature seed before germination begins. As shown in the present results, modulation of iron content in seeds can be performed by several means : (i) by genetic means, either by reducing the expression of genes involved in chromatin condensation (e.g., by reducing the expression of the clf, h2az or swn genes), or by over-expressing proteins known to enhance iron absorption (e.g., the IRONMAN or bHLHs proteins), or (ii) by chemical means, for example by applying synthetic IRONMAN peptides or simply their synthetic conserved domains issued thereof directly onto the mature seed. Thanks to these treatments, the present invention advantageously allows germinating seeds to tolerate strong heat/dehydration stress during a phase of high sensitivity of the young seedling, what will have a major impact on agricultural yields and production timing.

Description

Molecular means to extend desiccation tolerance during seed germination

SUMMARY OF THE INVENTION

Desiccation tolerance is a protective process that allows mature seeds to tolerate cellular water contents below 10%. This process is acquired during seed maturation and is usually lost during imbibition, prior to germination, what enhances seed sensitivity to drought and heat, in particular during the germination stage. The present invention shows that it is however possible to maintain desiccation tolerance mechanisms after germination by activating iron absorption in mature seed before germination begins. As shown in the present results, modulation of iron content in seeds can be performed by several means : (i) by genetic means, either by reducing the expression of genes involved in chromatin condensation (e.g., by reducing the expression of the elf, h2az or swn genes) or by reducing the expression of genes involved in the negative regulation of the iron accumulation (bts gene for BRUTUS or BRUTUS-LIKE genes), or by over-expressing proteins known to enhance iron absorption (e.g., the IRONMAN or bHLHs proteins), or (ii) by chemical means, for example by applying synthetic IRONMAN peptides or simply their synthetic conserved domains issued thereof directly onto the mature seed (or during seed priming). Thanks to these treatments, the present invention advantageously allows germinating seeds to tolerate strong heat/dehyd ration stress during a phase of high sensitivity of the young seedling, what will have a major impact on agricultural yields and production timing.

BACKGROUND OF THE INVENTION

Drought is one of the major abiotic stress factors limiting crop productivity worldwide. Global climate changes may further exacerbate the drought situation in major crop-producing countries. Although irrigation may in theory solve the drought problem, it is usually not a viable option because of the cost associated with building and maintaining an effective irrigation system, as well as other noneconomic issues, such as the general availability of water. Thus, alternative means for alleviating plant water stress and increasing desiccation tolerance are needed.

Mechanisms for selecting drought tolerant plants fall into three general categories. The first is called drought escape, in which selection is aimed at those developmental and maturation traits that match seasonal water availability with crop needs. The second is dehydration avoidance, in which selection is focused on traits that lessen evaporation water loss from plant surfaces or maintain water uptake during drought via a deeper and more extensive root system. The last mechanism is dehydration tolerance, in which selection is directed at maintaining cell turgor or enhancing cellular constituents that protect cytoplasmic proteins and membranes from drying. The genetic regulatory networks of drought stress tolerance have been reviewed recently (Shinozaki et al. 2006). Plant modification for enhanced drought tolerance is mostly based on the manipulation of either transcription and/or signaling factors or genes that directly protect plant cells against water deficit. Despite much progress in the field, understanding the basic biochemical and molecular mechanisms for drought stress perception, transduction, response and desiccation tolerance remains a major challenge in the field.

Desiccation tolerance (DT) is defined as a protective process enabling mature seeds to tolerate a water cellular content of less than 10%. This process is acquired during seed development, within the plant. It is maximal when seeds are mature, but it is lost once germination begins, as of the first imbibition of the seeds. This means that mature seeds become very sensitive to desiccation after their first imbibition, what triggers the necessity to continuously maintain sufficient water supply to the growing crops, once germination is induced.

Several studies of the acquisition of DT during seed development and on its loss upon germination have been reported. Interestingly, DT can be rescued in germinated seeds by the application of a mild osmotic stress. The re-induction of DT in germinated seeds by incubation in PEG was first reported by Bruggink and van der Toorn (1995). They showed that DT could be fully restored in germinated seeds of Cucumis sativus and Impatiens walleriana. These authors suggested that this approach could serve as a convenient model system in research studies to decipher DT molecular processes. This strategy to re-induce DT in germinated seeds has been confirmed in other species like Medicago (Medicago truncatula) and Tabebuia impetiginosa. The re-establishment of DT in primary roots of Medicago germinated seeds by a mild osmotic stress (-1 .5 MPa) treatment has been so far used to identify the transcriptome and (heat stable) proteome associated with DT. Furthermore, the application of a cold or heat shock prior to osmotic treatment improved desiccation tolerance in protruded radicles of T. impetiginosa [Vieira et al, 2010], Arabidopsis (Arabidopsis thaliana) is a well- known model system in plant biology. As other plant seeds, Arabidopsis seeds loose DT upon germination. Maia et al (2011) showed that DT can be re-induced in germinated seeds by treating them with PEG (-2.5 Mpa) during three days. The use of Arabidopsis for studying loss and reestablishment of DT in germinated seeds in combination with genetic and molecular tools developed for this model species, engenders a powerful model to further unravel DT in higher plants. According to Maia et al, four distinct developmental stages are critical for studying DT in these plants: stage I (testa rupture), stage II (seeds at radical protrusion), stage III (germinated seeds showing a primary root of 0.3-0.5 mm length) and stage IV (at the appearance of the first root hairs) (Figure 1 of Maia et al. 2011). According to these results, DT in seeds at earlier stages (i.e. stages I and II) can be better re-induced as compared to the two later stages. Yet, this DT enhancing method cannot be used in crops and fields because PEG is an ethoxylated compound not allowed in agricultural or alimentary plant cultures in fields. Moreover, those methods are not easily implementable under field conditions, and inducing or enhancing desiccation tolerance in agricultural seeds at stages II I III and IV post-germination is still a big challenge. DETAILLED DESCRIPTION OF THE INVENTION

In the context of global warming, it is more and more needed to render seeds, especially early germinating seeds at stages II / III and IV post-germination, more tolerant to water deficit.

To solve this need, the present inventors discovered for the first time that it is possible to maintain DT mechanisms active in Arabidopsis seeds even after germination is induced, by loss of function of chromatin condensation genes such as CLF, H2AZ and SWN, or loss of function of genes involved in the negative regulation of the iron accumulation such as BTS but also by modifying iron homeostasis, more precisely by enhancing iron accumulation and/or iron absorption in these seeds. In particular, they showed that DT is maintained at stage II post-germination when seeds are imbibed in a solution of Fe-EDTA (Figure 3). It was very surprising, since nobody ever previously suggested any link between iron content in seeds and their desiccation or drought tolerance.

To confirm this discovery, the inventors have then assayed several means to exogenously modulate iron homeostasis in Arabidopsis seeds:

Genetic means in order to either i) over-express in the seeds genes that are known to enhance iron accumulation and/or iron absorption, or ii) to under-express in the seeds genes that are known to negatively affect iron accumulation and/or iron absorption.

Chemical means by adding proteins or peptides (e.g synthetic peptides derived from proteins) known to enhance iron accumulation and/or iron absorption to the seeds before or at imbibition.

More precisely, they showed in the results presented below that it is possible to maintain DT in seeds until stage IV post-germination by several independent means:

By imbibing the dry seeds with IRONMAN polypeptides or a short peptide issued thereof (Figures 4-6) until germination procedure, in particular by soaking the seeds or by performing seed priming

By over-expressing the IRONMAN genes or the bHLH38/39 genes that are known to encode proteins involved in iron accumulation and/or iron absorption (Figure 2)

By mutating I inhibiting genes known to encode proteins inducing the condensation of chromatin in genes involved in DT and iron accumulation and/or iron absorption, said proteins being the swinger protein (SWN), the curly leaf protein (CLF) and the histone 2A protein (H2A.Z) (Figure 1)

By mutating/inhibiting genes known to encode proteins involved in the negative regulation of the iron accumulation, said proteins being the BRUTUS E3 ligase (BTS) (Figure 2). Any of these embodiments, along with an optional supply of exogenous iron, enables germinating seeds to tolerate heat stress and dehydration for a long time, even during late germination states where seeds are the most sensitive to these stressful conditions.

Thus, in a first aspect, the present application targets the use of a compound that activates iron accumulation and I or iron absorption in a seed of a target plant, to increase the desiccation tolerance of said seed. Preferably, the compound is present on the seed or in the seed before or when germination is induced, so that the iron content of the seed is significantly enhanced during germination, especially in post-germinating stages II, III and IV according to Maia et al. 2011. More preferably, the compound is present in the seed embryo of the target plant. The purpose of the present invention is generally to enhance the DT of seeds after germination. In a preferred embodiment, the compound of the invention is a protein or a peptide (e.g., an IRONMAN protein or peptide); in this case, the compound is preferably put in contact with the seed before germination occurs, in particular during the pre-germination process or using a seed priming treatment, or during the first imbibition of the seeds. In another preferred embodiment, the compound of the invention is a nucleic acid; in this case, it is preferably present in the seed before germination is induced, e.g. because the seed is produced by a transgenic plant, so as to overexpress or under-express proteins that are able to modulate iron homeostasis.

Target genes / proteins of the invention

As explained above, the compound of the invention can be a nucleic acid encoding for a protein eventually activating iron accumulation and I or iron absorption. The compound of the invention can also be the said protein itself. These proteins are herein called “proteins of the invention”. The genes encoding same are herein called “genes of the invention”.

A compound that activates iron accumulation and/or absorption according to the invention may be detected by using a staining solution on isolated mature/germinated embryos, and observing the images by a microscope coupled with a camera showing iron localization in the embryos, such as described by Roschzttardtz et al. (2009).

So, in a particular embodiment, a compound that activates iron accumulation and/or absorption for use according to the invention to increase the desiccation tolerance of said seed once germination is induced, is selected in an in vitro method comprising the following steps :

(i) Seeds are imbibed with water (control) or peptides solutions (the compounds to be tested), by incubation at room temperature, in particular 20°C, under continuous light,

(ii) Germinating embryos at stage II and IV are isolated/collected, in particular by manually removing envelopes (/.e. seed coat and endosperm),

(iii) The isolated mature/germinated embryos are brought into contact with a staining solution containing a potassium ferrocyanide, and (iv) A color change is observed, in comparison to the control (water), when the tested compound activates iron accumulation and/or absorption.

In fact, the potassium ferrocyanide (yellow) of the staining solution will react with iron (Fe3+) present in the solution, providing a blue color. The presence of Fe3+ is indicative of the effect of the compound on iron accumulation in the germinating embryos.

In a preferred embodiment, the isolated mature/germinated embryos were vacuum-infiltrated at room temperature with a staining solution at step (iii), in particular a Peris stain solution (4% (v/v) HCI and 4% (w/v) K-ferrocyanide). The iron localization in the embryos may further be observed by a microscope coupled with a camera.

In a first embodiment, the compound of the invention is an IRONMAN protein or a functional fragment peptide thereof. In a preferred embodiment, the compound of the invention is an IRONMAN protein expressed in seeds. Indeed, some IRONMAN proteins are expressed in seeds such as IMA1 , IMA2, IMA3 in Arabidopsis thaliana and IMA7 in Solanum lycopersicum, and other IRONMAN proteins are not expressed in seeds such as IMA4 to IMA8 in Arabidopsis thaliana and IMA1 to IMA6 and IMA8 and IMA9 in Solanum lycopersicum.

So, ini particular, the compound of the invention is an IRONMAN protein expressed in seeds and can be chosen in the group consisting of : IMA1 , IMA2, IMA3 or a functional fragment of any of them that is able to activate iron accumulation and I or iron absorption in seeds such as the ones not expressed in seeds or orthologs from other species (Grillet et al, 2018; Peng et al, 2022). IMA1 , IMA2, IMA3 in A. thaliana are also named AtIMAI , AtlMA2, AtlMA3, for Arabidopsis thaliana IMA1 , Arabidopsis thaliana IMA2 and Arabidopsis thaliana IMA3, respectively. In another particular embodiment, the compound of the invention is IMA7 in S. lycopersicum or a functional fragment that is able to activate iron accumulation and I or iron absorption in seeds such as the ones not expressed in seeds or orthologs from other species (Grillet et al, 2018). IMA7 in S. lycopersicum is also named SIIMA7.

The IMAI protein or "IRONMAN 1"

In Arabidopsis thaliana, the IMA1 protein has the reference number NP_175173 (SEQ ID NO:1) encoded by the gene whose reference number is AT1 G47400 (and whose mRNA is referenced as NM_103634 as depicted in SEQ ID NO:5).

In the context of the invention, it is preferred to use the IMA1 protein of the target plant species whose DT should be enhanced. This means that any orthologs of IMA1 in other plant species can be used, in order to enhance the DT of the corresponding plant seeds. For example, it is possible to overexpress any IMA1 ortholog proteins expressed in other plant species. Some of them are described in Grillet et al, 2018, on the supplementary Figure 4 and in Supplementary Table 2 which are incorporated herein by reference. In particular, in the methods of the invention, it is possible to overexpress any IMA1 brassica orthologs chosen in the group consisting of:AL1 G53650 from Arabidopsis lyrate; Carub.0001s3619 from Capsella rubella; Carub.0001s3620 from Capsella rubella; AT1 G47395 from Arabidopsis thaliana; Carub.0001s3617 from Capsella rubella; CARHR036980 from Cardamine hirsute; AL1 G53630 from Arabidopsis lyrate; Carub.0001s3618 from Capsella rubella; CARHR036960 from Cardamine hirsute; AL1 G53640 from Arabidopsis lyrate; A05p21330 from Brassica napus; BraA05t21041 Z from Brassica rapa; BraA08t32568Z from Brassica rapa; BcaC05g27603 from Brassica carinata; C05p35910 from Brassica napus; BolC5t32310H from Brassica oleracea; A08p05650 from Brassica napus; BcaB04g 17526 from Brassica carinata; BolC8t47478H from Brassica oleracea; Aa31 sc96G10,from Aethionema arabicum; C08p06320 from Brassica napus; and A08p05670 from Brassica napus.

It is in particular possible to use any IMA1 ortholog or variant protein whose sequence has at least 60%, 65%, 70%, 75%, or 80% identity with SEQ ID NO:1 , provided that it can activate iron accumulation and I or iron absorption in the target plant seed.

In a preferred embodiment, the IMA1 ortholog or variant proteins that can be used in the context of the invention contain the C-terminal amino acid consensus sequences highlighted in Figure 3 of Grillet et al, 2018, e.g., the polypeptide of SEQ ID NO:26 (GDDDDD) and/or the polypeptide of SEQ ID NO :27 (APAA). Also, the IMA1 ortholog or variant proteins that can be used in the context of the invention contain preferably a consensus sequence having at least 50%, 60%, or 70% identity with SEQ ID NO:4 or with SEQ ID NO:11.

The IMA2 protein or "IRONMAN 2"

In Arabidopsis thaliana, the IMA2 protein has the reference number NP_849780 (SEQ ID NO:2) encoded by the gene whose reference number is AT1 G47395 (and whose mRNA is referenced as NM_179449.2 as depicted in SEQ ID NO:6).

In the context of the invention, it is preferred to use the IMA2 protein of the target plant species whose DT should be enhanced. This means that any orthologs of IMA2 in other plant species can be used in order to enhance the DT of the corresponding plant seeds. For example, it is possible to overexpress any IMA2 ortholog proteins expressed in other plant species. Some of them are described in Grillet et al, 2018, on the supplementary Figure 4 and in Supplementary Table 2 which are incorporated herein by reference. In particular, in the methods of the invention, it is possible to overexpress any IMA2 brassica orthologs chosen in the group consisting of: AT1 G47395 from Arabidopsis thaliana, AL1 G53630 from Arabidopsis lyrata, AL1 G53640 from Arabidopsis lyrata, Carub.0001s3619 from Capsella rubella, Carub.0001s3617 from Capsella rubella, AL1 G53650 from Arabidopsis lyrate, CARHR036960 from Cardamine hirsuta, AT1 G47400 from Arabidopsis thaliana, CARHR036980 from Cardamine hirsuta, Carub.0001s3620 from Capsella rubella, Carub.0001s3618 from Capsella rubella, BraA08t32568Z from Brassica rapa, A08p05650 from Brassica napus, A05p21330 from Brassica napus, BraA05t21041 Z from Brassica rapa, BcaB04g17526 from Brassica carinata, BcaC05g27603 from Brassica carinata, C05p35910 from Brassica napus, BolC5t32310H from Brassica oleracea, A08p05670 from Brassica napus, BolC8t47478H from Brassica oleracea, C08p06320 from Brassica napus, BraA08t32572Z from Brassica rapa, C08p06340 from Brassica napus, and BolC8t47481 H from Brassica oleracea.

It is in particular possible to use any IMA2 ortholog or variant protein whose sequence has at least 60%, 65%, 70%, 75%, or 80% identity with SEQ ID NO:2, provided that it can activate iron accumulation and I or iron absorption in the target plant seed.

In a preferred embodiment, the IMA2 ortholog or variant proteins that can be used in the context of the invention contain the C-terminal amino acid consensus sequences highlighted in Figure 3 of Grillet et al, 2018, e.g., the polypeptide of SEQ ID NO:26 (GDDDDD) and/or the polypeptide of SEQ ID NO :27 (APAA). Also, the IMA2 ortholog or variant proteins that can be used in the context of the invention contain preferably a consensus sequence having at least 50%, 60%, or 70% identity with SEQ ID NO:4 or with SEQ ID NO:11.

The IMA3 protein or "IRONMAN 3"

In Arabidopsis thaliana, the IMA3 protein has the reference number NP_001318323 (SEQ ID NO:3) encoded by the gene whose reference number is AT2G30766 (and whose mRNA is referenced as NM_001336300 or NM_001336301 as depicted in SEQ ID NO:7 and SEQ ID NO:8 respectively).

In the context of the invention, it is preferred to use the IMA3 protein of the target plant species whose DT should be enhanced. This means that any orthologs of IMA3 in other plant species can be used in order to enhance the DT of the corresponding plant seeds. For example, it is possible to overexpress any AT2G30766 from Arabidopsis thaliana, AL4G25670 from Arabidopsis lyrate, CARHR124240 from Cardamine hirsuta, C04p59770 from Brassica napus, BolC4t27485H from Brassica oleracea, Carub.0004s1195 from Capsella rubella, A04p23460 from Brassica napus, BraA04t17873Z from Brassica rapa, BolC3t14404H from Brassica oleracea, and C03p20290 from Brassica napus.

It is in particular possible to use any IMA3 ortholog or variant protein whose sequence has at least 60%, 65%, 70%, 75%, or 80% identity with SEQ ID NO:3, provided that it can activate iron accumulation and I or iron absorption in the target plant seed.

In a preferred embodiment, the IMA3 ortholog or variant proteins that can be used in the context of the invention contain the C-terminal amino acid consensus sequences highlighted in Figure 3 of Grillet et al, 2018, e.g., the polypeptide of SEQ ID NO:26 (GDDDDD) and/or the polypeptide of SEQ ID NO :27 (APAA). Also, the IMA3 ortholog or variant proteins that can be used in the context of the invention contain preferably a consensus sequence having at least 50%, 60%, or 70% identity with SEQ ID NO:4 or with SEQ ID NO:11.

In the methods and compositions of the invention, it is preferred to use, among the IRONMAN proteins IMAs, the IMA3 protein of the target plant species, because even if independent overexpression of IMA1 , IMA2 or IMA3 re-induced DT, IMA3 appears to be slightly the most efficient one (Figure 2).

The conserved sequence of IRONMAN peptide of 17 aa

In an alternative embodiment, the compound of the invention is a functional fragment peptide issued of an IRONMAN protein as defined above. In particular, the compound of the invention can be the C-terminal sequence of 17 amino acids that is known to be conserved between various plant species and to activate iron accumulation and I or iron absorption in seeds (Grillet et al, 2018; Peng et al, 2022).

In a preferred embodiment, the compound of the invention is the C-terminal conserved sequence of 17 amino acids of IMA1 or of IMA2, whose sequence in Arabidopsis thaliana is ENGGDDD DSGYDYAPAA (SEQ ID NO: 11) or a functional variant thereof having at least 50%, 60%, 65%, 70%, 75%, or 80% identity with SEQ ID NO:11. Preferably, the sequence of this conserved IMA1 or of IMA2 C-terminal polypeptide contains the consensus sequences SEQ ID NO:26 (GDDDDD) and/or SEQ ID NO :27 (APAA).

In a more preferred embodiment, the compound of the invention is the C-terminal conserved sequence of 17 amino acids of IMA3, whose sequence in Arabidopsis thaliana is ENGGDDDDDD CDVAPAA (SEQ ID NO: 4) or a functional variant thereof having at least 50%, 60%, 65%, 70%, 75%, or 80% identity with SEQ ID NO:4. Preferably, the sequence of this conserved IMA3 C-terminal polypeptide contains the consensus sequences SEQ ID NO:26 (GDDDDD) and/or SEQ ID NO :27 (APAA). The peptide of SEQ ID NO:4 has been successfully used by the inventors in the results presented below (Figure 5).

In the context of the invention, it is preferred to use the C-terminal peptide of any IMA proteins of the target plant species whose DT should be enhanced. This means that the C-terminal peptide of any orthologs or variants of IMA1 , 2 and 3 from other plant species can be used as a compound of the invention in order to enhance the DT of the corresponding plant seeds, provided that it can activate iron accumulation and I or iron absorption in the target plant seed.

In another particular embodiment, it is also possible to use a generic C-terminal part of IMA peptide that contains the most conserved BID among the 131 IMA peptides identified in different species (described in Grillet et al, 2018, on the supplementary Figure 4 and in Supplementary Table 2) named as GenIMABID (GDDDDDGYDYAPAA, peptide SEQ ID NO: 29),

The IMA7 protein or "IRONMAN 7" in tomato

In Solanum Lycopersicum, the IMA7 protein (SIIMA7) is expressed in seeds and has the reference number A0A3Q7JRI4 (protein, SEQ ID NO:38) (Uniprot A0A3Q7JRI4, Solyc12g006760.1).

In the context of the invention, it is preferred to use the SIIMA7 protein of the target plant species whose DT should be enhanced. This means that any orthologs of IMA7 in other plant species can be used in order to enhance the DT of the corresponding plant seeds.

It is in particular possible to use any IMA7 ortholog or variant protein whose sequence has at least 60%, 65%, 70%, 75%, or 80% identity with SEQ ID NO:38, provided that it can activate iron accumulation and I or iron absorption in the target plant seed. In particular, it is also possible to use a SIIMA7BID peptide (CLDGDDDSDYDYAPAA, peptide SEQ ID NO: 31) that contain a conserved BRUTUS (BTS) interaction domain (BID), as designed and illustrated in the examples, which is a C- terminal BID part of tomato IMA7 peptide. As illustrated in Figure 9, adding SIIMA7BID synthetic peptide during germination allowed a reinduction of DT at stages 1 and 3mm of germinating seeds in tomato (but it is not as efficient as the specific Tomato SIIMA7BID peptide).

In another preferred embodiment, the compound of the invention is a nucleic acid encoding a protein that can activate iron accumulation and / or iron absorption in a seed. Such protein is for example chosen in the group consisting of: FIT, bHLH38, bHLH39, bHLH101 , PYE, MYB10, MYB72, 4CL1 , F6’H1 , S8H, CYP82C4, ABCG37, BGLU42, IRT1 , IRT2, FRO3, OPT3, NAS4, IMA1 , BTS, BTSL1 , BTSL2, IREG3, ZIF1 , WRKY22, NAC2, FER1 , FER3, FER4, SAPX, VTL1 , ZIP4, YSL3, HEMA1 , which are proteins known to influence iron homeostasis in plants (Kim et al, 2019).

The preferred proteins whose expression could be upregulated in the context of the invention are IMA1 , IMA2, IMA3, their consensus peptides, bHLH38, and bHLH39, whose effect is shown on Figure 2.

The IMA1 , IMA2 and IMA3 proteins have been described above.

The inventors have shown that up-regulating the genes encoding these proteins in plants of Arabidopsis thaliana enhances the DT of the seeds in stage II and also in stage IV post-germination (Figure 2).

Any other Arabidopsis IRONMAN proteins/peptides, which are not expressed in seeds, could potentially be overexpressed in or applied on seeds to increase iron accumulation and therefore reinducing desiccation tolerance in seedlings. Other Arabidopsis IRONMAN genes/proteins are AtlMA4 with reference number Uniprot Q1G327 (protein, SEQ ID NO: 43) (AT 1 G07367, mRNA, SEQ ID NO: 39), AtlMA5 (AT1 G09505, mRNA SEQ ID NO: 40), AtlMA6 (AT1 G07373, mRNA SEQ ID NO: 41), AtlMA7 (AT2G00920, mRNA SEQ ID NO: 42) and AtlMA8 (AT1 G47401).

In addition, any mixture of these eight Arabidopsis IMA peptides derived from the eight peptide sequences of the cited IRONMAN proteins could be used to treat seeds to increase the efficiency of a single peptide, leading to higher accumulation of iron and ultimately better reinduction of desiccation tolerance in seedlings.

In a particular embodiment, it is possible to use one or several IMA proteins expressed in the seeds, in mixture with one or several other IMA proteins not expressed in the seeds.

As an example, it is possible to use IMA3 protein in mixture with any one of IMA1 to IMA8 proteins in Arabidopsis thaliana.

Similarly in Tomato, any other IRONMAN proteins/peptides than SIIMA7 protein (SEQ ID NO:38) (Uniprot A0A3Q7JRI4, Solyc12g006760.1) not expressed in seeds could potentially be overexpressed in or applied on seeds to increase iron accumulation and therefore reinducing desiccation tolerance in seedlings. Other S. lycopersicum IRONMAN peptides are SIIMA1 with reference number Uniprot M1AZY7 (SEQ ID NO: 44), SIIMA2 with reference number Uniprot A0A3Q7HD18, Solyc07g044900.1 (SEQ ID NO: 45), SIIMA3 with reference number Uniprot A0A3Q7I6X1 , Solyc07g044910.1 (SEQ ID NO: 46), SIIMA4 with reference number Uniprot A0A3Q7J362, Solyc12g006720.1 (SEQ ID NO: 47), SIIMA5 with reference number Uniprot A0A3Q7J300, Solyc12g006730.1 (SEQ ID NO: 48), SIIMA6 with reference number Uniprot A0A3Q7J246, Solyc12g006750.1 (SEQ ID NO: 49), SIIMA8 with reference number Uniprot A0A3Q7J371 , Solyc12g006770.1 (SEQ ID NO: 50), SIIMA9 with reference number Uniprot A0A3Q7J308, Solyc12g006780.1 (SEQ ID NO: 51). In addition, any mixture of these nine IMA peptides derived from the nine peptide sequences of the cited IRONMAN proteins could be used to treat seeds to increase the efficiency of a single peptide, leading to higher accumulation of iron and ultimately better reinduction of desiccation tolerance.

As an example, it is possible to use SIIMA7 protein in mixture with any one of SIIMA1 , SIIMA2, SIIMA3, SIIMA4, SIIMA5, SIIMA6, SIIMA8, SIIMA9 proteins in Solanum lycopersicum. So, in a particular and preferred embodiment, the IRONMAN protein expressed in seeds is the protein IMA1 of SEQ ID NO:1 , IMA2 of SEQ ID NO:2 or IMA3 of SEQ ID NO:3 or a functional variant thereof, and wherein the consensus peptide thereof is a peptide having the sequence SEQ ID NO:4 or SEQ ID NO:11 or a functional variant thereof, or a generic IRONMAN peptide GenIMABID of SEQ ID NO: 29, or an IRONMAN protein SIIMA7 of SEQ ID NO: 38 or a synthetic peptide SIIMA7BID of SEQ ID NO:31 , optionally in mixture with one or several IRONMAN proteins not expressed in seeds, such as the ones of SEQ ID NO: 39 to SEQ ID NO: 43 in A. thaliana and SEQ ID NO: 44 to SEQ ID NO: 51 in S. lycopersicum.

The inventors have also shown that up-regulating the genes encoding two other proteins (namely bHLH38 and bHLH39) in plants of Arabidopsis thaliana enhances the DT of the resulting seeds in stage II and also in stage IV post-germination (Figure 2).

These two proteins are now described in more details.

The bHLH38 protein

This protein belongs to the basic helix-loop-helix (bHLH) DNA-binding superfamily protein. It is also known as “BHLH038”; “OBP3-RESPONSIVE GENE 2”; “ORG2”; “T8M16.7”.

In Arabidopsis thaliana, the bHLH38 protein has the reference number NP_191256 (depicted in SEQ ID NO:12) encoded by the gene whose mRNA is referenced as NM_115556.4 (as depicted in SEQ ID NO:13).

In the context of the invention, it is preferred to use the bHLH38 protein of the target plant species whose DT should be enhanced. This means that any orthologs of bHLH38 in other plant species can be used in order to enhance the DT of the corresponding plant seeds. For example, it is possible to overexpress any bHLH38 ortholog proteins expressed in other plant species.

It is in particular possible to use any bHLH38 ortholog or variant protein whose sequence has at least 70%, 75% or 80% identity with SEQ ID NO:12, provided that it can activate iron accumulation and I or iron absorption in the target plant seed.

The bHLH39 protein

This protein belongs to the basic helix-loop-helix (bHLH) DNA-binding superfamily protein. It is also known as “BHLH039”; “OBP3-RESPONSIVE GENE 3”; “ORG3”; “T8M16.8”.

In Arabidopsis thaliana, the bHLH39 protein has the reference number NP_191257 (SEQ ID NO:14) encoded by the gene whose mRNA is referenced as NM_115557 (SEQ ID NO:15). In the context of the invention, it is preferred to use the bHLH39 protein of the target plant species whose DT should be enhanced. This means that any orthologs of bHLH39 in other plant species can be used in order to enhance the DT of the corresponding plant seeds. For example, it is possible to overexpress any bHLH39 ortholog proteins expressed in other plant species.

It is in particular possible to use any bHLH39 ortholog or variant protein whose sequence has at least 70%, 75% or 80% identity with SEQ ID NO:14, provided that it can activate iron accumulation and I or iron absorption in the target plant seed.

In a particularly preferred embodiment, the compound of the invention is a nucleic acid encoding the proteins IMA1 of SEQ ID NO:1 , IMA2 of SEQ ID NO:2, IMA3 of SEQ ID NO:3, the consensus peptide of SEQ ID NO:4 or SEQ ID NO:11 , or any peptide containing the SEQ ID NO:26 and/or SEQ ID NO:27, or bHLH38 of SEQ ID NO:12, or bHLH39 of SEQ ID NO:14, or any ortholog or variant protein whose amino acid sequence shares at least 50%, 60%, 70%, or 80% identity with these sequences.

In another embodiment, the compound of the invention is a nucleic acid reducing the expression of at least one protein negatively affecting iron accumulation and I or iron absorption in plant seeds, for example, proteins known to induce chromatin condensation in genes involved in DT genes, or proteins known to be involved in the negative regulation of the iron accumulation, which also play a role in iron accumulation and I or iron absorption.

The inventors have shown that knocking-out the genes encoding the SWN, H2A or CLF or BTS proteins in plants of Arabidopsis thaliana enhances the DT of their mature seeds in stage II and also in stage IV post-germination (Figure 1 and Figure 2).

Thus, in a preferred embodiment, the compound of the invention is a nucleic acid reducing the expression of any of the SWN, H2A, CLF or BTS proteins in the target plant or directly in the target plant seeds.

The SWN protein

This protein is also known as the protein “swinger” or “SET domain-containing protein”.

In Arabidopsis thaliana, the SWN protein has for example the reference number NP_567221 (SEQ ID NO:9) encoded by the gene whose mRNA is referenced as NM_116433.3 (SEQ ID NO: 10). Other isoforms are also known for this protein:

- NP_001328437.1 encoded by NM_001340355.1

- NP_001328436.1 encoded by NM_001340356.1

- NP_001328435.1 encoded by NM_001340357.1

In the context of the invention, it is preferred to mutate or under-express the SWN protein of the target plant species whose post-germination DT should be enhanced. This means that any orthologs of SWN in other plant species can be mutated / under-expressed in order to enhance the DT of the corresponding plant seeds.

It is in particular possible to reduce the expression of any SWN ortholog or variant protein whose sequence has at least 70% or at least 80% identity with SEQ ID NO:9, provided that it can activate iron accumulation and I or iron absorption in the target plant seed without significantly affecting the agronomic properties of the plants.

The H2A.Z variant protein

The histone 2A variant Z protein (H2A.Z) is a variant of the H2A histone protein encoded by three genes, namely the HTA8, HTA9, and HTA11 genes.

In Arabidopsis thaliana, the HTA8 protein has for example the reference number NP_850299.1 (SEQ ID NO:16) which is encoded by the HTA8 gene whose mRNA is NM_179968.3 (SEQ ID NO:17).

In Arabidopsis thaliana, the HTA9 protein has the reference number NP_175683.1 (SEQ ID NO:18) which is encoded by the HTA9 gene whose mRNA is NM_104152.4 (SEQ ID NO:19).

In Arabidopsis thaliana, the HTA11 protein has for example the reference number NP_191019.1 (SEQ ID NQ:20) which is encoded by the HTA11 gene whose mRNA is NM_115313.5 (SEQ ID NO:21).

In the context of the invention, it is preferred to use a compound mutating (or mutate) or underexpressing any of the genes encoding the Histone 2A (H2A) protein, in particular those encoding the variant Z of H2A (H2A.Z) or a combination of any of these genes, provided that the phenotype of the transgenic plant is not affected in its agronomic properties. In a particular embodiment, only one of them is mutated I under-expressed. In another particular embodiment, two of them are mutated I under-expressed. In another embodiment, the three genes are mutated I under-expressed (as proposed in the results below).

Moreover, it is preferred to use a compound mutating or under-expressing the HTA8, HTA9, or HTA11 gene of the target plant species whose DT should be enhanced. This means that any orthologs of HTA8, HTA9, and HTA11 in other plant species can be mutated I under-expressed in order to enhance the post-germination DT of the corresponding plant seeds.

It is in particular possible to reduce the expression of any H2AZ ortholog proteins whose sequence has at least 70% or at least 80% identity with SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NQ:20, provided that this reduction eventually activates iron accumulation and I or iron absorption in the target plant seed, without significantly affecting the agronomic properties of the plants. The CLF protein

This protein is also known as the protein “curly leaf protein” or “SET domain-containing protein”.

In Arabidopsis thaliana, the CLF protein has the reference number NP_179919.1 (SEQ ID NO:22) or the reference number NP_001324816.1 (SEQ ID NO:24) encoded by the gene whose mRNA is referenced as NM_127902.6 (SEQ ID NO:23) or NM_001335847 (SEQ ID NO:25) respectively.

In the context of the invention, it is preferred to mutate I under-express the CLF protein of the target plant species whose post-germination DT should be enhanced. This means that any orthologs of CLF in other plant species can be mutated I under-expressed in order to enhance the DT of the corresponding plant seeds.

It is in particular possible to reduce the expression of any CLF ortholog or variant protein whose sequence has at least 70% or at least 80% identity with SEQ ID NO:22 or SEQ ID NO:24, provided that it can activate iron accumulation and I or iron absorption in the target plant seed without significantly affecting the agronomic properties of the plants.

In a particularly preferred embodiment, the compound of the invention is a nucleic acid reducing the expression of the SWN protein of SEQ ID NO:9, of the H2A 8 protein of SEQ ID NO:16, of the H2A 9 protein of SEQ ID NO: 18, of the H2A 11 protein of SEQ ID NQ:20, or of the CLF protein of SEQ ID NO:22 or SEQ ID NO:24 or any ortholog or variant protein whose amino acid sequence shares at least 80% identity with these sequences.

The BTSBTS protein

This protein, which is also known as the protein “brutus” (BTS) also known as EMB2454 (EMBRYO DEFECTIVE 2454) and its paralogs BTS-LIKE1 or BTS-LIKE2 (BRUTUS LIKE proteins) are known to encode negative regulators of iron deficiency response, responsible of regulating the iron accumulation.

In Arabidopsis thaliana, the BTS protein (AT3G18290) has the reference number NP_188457.1 (SEQ ID NO: 32), encoded by the gene whose mRNA is referenced as NM_112713.4 (SEQ ID NO: 33). The BTS-LIKE proteins (AT1 G74770 and AT1 G18910) with the Uniprot reference number F4HVS0 or RefSeq NP 177615.2 (SEQ ID NO:34) and with the Uniprot reference number F4IDY5 and RefSeq NP_173325.2 (SEQ ID NO:35) encoded by the gene whose mRNA is referenced as SEQ ID NO:36 and SEQ ID NO:37 respectively.

In the context of the invention, it is preferred to mutate I under-express the BTS or BTS-LIKE proteins of the target plant species whose post-germination DT should be enhanced to increase iron accumulation. This means that any orthologs of BTS in other plant species can be mutated I underexpressed in order to increase iron content to enhance the DT of the corresponding plant seeds. It is in particular possible to reduce the expression of any BTS ortholog or variant protein whose sequence has at least 70% or at least 80% identity with SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:35, provided that it can activate iron accumulation and I or iron absorption in the target plant seed without significantly affecting the agronomic properties of the plants.

In a particularly preferred embodiment, the compound of the invention is a nucleic acid reducing the expression of the BTS or BTS-LIKE proteins of SEQ ID NO:32, SEQ ID NO: 34 or SEQ ID NO: 35 respectively.

Methods / Uses of the invention

In a second aspect, the present invention targets methods for increasing the desiccation tolerance of germinating seeds of a target plant or for generating desiccation tolerant seedling from dry seeds of a target plant by either genetically modulating the expression of the proteins of the invention as defined above or by coating mature seeds with a chemical compound that enhance the iron accumulation I absorption in said mature seed.

Practically, these methods according to the invention can be reduced to practice at two different levels, i.e., directly at the seed level or at the plant level, according to the following embodiments:

I) contacting mature dry seeds of the target plant, before or during imbibition or using seed priming method, with a composition comprising a compound that activates iron accumulation I absorption in said mature seed, or

II) using a transgenic target plant comprising a compound that activates iron accumulation I absorption in mature seeds of said target plants, or

III) mutating I under-expressing in the target plant a gene encoding at least one protein negatively affecting iron accumulation and I or iron absorption in plant seeds, said protein preferably inducing the chromatin condensation in genes involved in DT genes including iron accumulation and I or iron absorption, said protein being more preferably chosen among: SWN, H2A.Z and CLF proteins; or said protein being involved in the negative regulation of the Fe accumulation such as BRUTUS protein or BRUTUS LIKE protein.

In the embodiment I, the contact or peptide treatment can be performed before, during or after imbibition of the seed, provided that it is done before stages II or IV of germination, i.e., when the seed is the more sensitive to drought. Preferably, the contact is performed before or during the first imbibition of the seed, so that the seeds are durably and efficiently protected. The duration of the contact depends on the plant species and need not to be specified here. The contact or peptide treatment can also be performed during the seed priming (industrial process implying seed imbibition then seed re-drying before radicle emergence). The concentration of the active compound (e.g., a peptide) in the composition of the invention is for example comprised between 1 pM and 200pM, preferably between 20pM and 150pM, more preferably between 50pM and 100pM (cf. Figure 4).

The methods of the invention can also comprise the step of contacting the mature seeds with a solution or a powder containing iron, so as to enhance the iron cellular content of the seeds. This step is preferably performed concomitantly with germination induction, e.g., by imbibing the seeds in a liquid solution containing Fe-EDTA. Afterwards, the seeds can be dried again, stored for several months without any risks of further germination, and eventually distributed to the farmers who will resume the germination of the seeds by imbibing them again and cultivate them as appropriate. Alternatively, it is possible to mix dry seeds with a powder containing the compounds of the invention and optionally an iron source such as iron dioxide. The compounds of the invention will enter into the seeds once they are then put in contact with water (imbibition).

The concentration of Fe²⁺ or Fe³⁺ in the methods of the invention can be comprised between 1 pM and 200pM, preferably between 20pM and 150pM, more preferably between 50pM and 100pM (cf. Figure 3).

The methods of the invention can also comprise the step of contacting the mature seeds with classical performance enhancing compounds well-known in the art.

When the seeds are directly treated to induce DT, the composition used in the method of the invention can contain an IRONMAN protein as defined above, or a functional fragment peptide thereof as defined above, preferably having the sequence SEQ ID NO:4, or a functional variant thereof having at least 50, 60, 70 or 80% identity with SEQ ID NO:4 and more preferably containing the SEQ ID NO:26 and/or SEQ ID NO:27.

In a particularly preferred embodiment, the method of the invention implies coating or contacting the seeds with proteins that modulates iron homeostasis, e.g. by activating iron accumulation I absorption in said mature seed. In that case, it can comprise the following steps : a) Providing non-germinated dry mature seeds of interest, b) Coating or contacting an IRONMAN protein as defined above, or a functional fragment peptide thereof as defined above, on or with said seeds, c) Optionally, contacting said coated seeds with a solution or powder containing iron, d) Optionally, coating said seeds with other performance enhancing compounds.

Preferably, in this seed coating method, the IRONMAN protein is the protein IMA1 of SEQ ID NO:1 , IMA2 of SEQ ID NO:2 or IMA3 of SEQ ID NO:3 or a functional variant thereof having at least 60, 70 or 80% identity with SEQ ID NO:1-3, and wherein the functional fragment peptide thereof contains the conserved region of SEQ ID NO:4 or a functional variant thereof having at least 50, 60, 70 or 80% identity with SEQ ID NO:4 and more preferably containing the SEQ ID NO:26 and/or SEQ ID NO:27, or a generic IRONMAN peptide GenIMABID of SEQ ID NO: 29, or an IRONMAN protein SIIMA7 of SEQ ID NO: 38 or a synthetic peptide SIIMA7BID of SEQ ID NO:31 , optionally in mixture with one or several IRONMAN proteins not expressed in seeds.

Indeed, in this seed coating method for tomato, the IRONMAN protein expressed in seeds is the protein IMA7 of SEQ ID NO: 38, or a SIIMA7BID peptide (CLDGDDDSDYDYAPAA, SEQ ID NO: 31) that contain a conserved BRUTUS (BTS) interaction domain (BID), or a functional variant thereof having at least 60, 70 or 80% identity with SEQ ID NO: 38 or SEQ ID NO:31 , optionally in mixture with one or several IRONMAN proteins not expressed in seeds.

In this seed coating method, the protein (be it an IRONMAN protein or another protein that activates iron accumulation / absorption in said mature seed) is applied on non-germinated seeds, or during first imbibition.

In a particular embodiment, it is possible to use an IRONMAN protein expressed in seeds, alone or in mixtures with other IRONMAN proteins not expressed in seeds, such as the ones disclosed above.

In a particular embodiment, the method of the invention involves to : a) Pre-germinate mature seeds with a soluble solution containing proteins as defined herein, preferably allowing them to reach stage II as defined in Maia et al, 2011 (seeds at radical protrusion), then b) Drying the thus obtained pre-germinated seeds, preferably at stage II, so as to obtain pre-germinated coated seeds having enhanced desiccation tolerance and faster plantlet establishment.

As explained above, the water solution of step a) may also contain Fe-EDTA and/or some performance enhancing compounds.

In this particular embodiment, the present invention requires to apply on the seed a composition containing a protein of the invention (IMA or peptide) and an agriculturally acceptable carrier. It may also contain a performance enhancing compound and/or a source of iron (Fe-EDTA, iron oxide, etc).

The composition of the invention may be applied to a seed in any physiological state, at any time between harvest of the mature seed and the sowing of the seed. It is preferred that the seed be in a sufficiently durable state that it incurs no or minimal damage, including physical damage or biological damage, during the treatment process. The composition may be applied to the seeds using conventional coating techniques and machines, such as fluidized bed techniques, the roller mill method, rotostatic seed treaters, and drum coaters. In order to apply an active ingredient to crops of useful plants as required by the methods of the invention, said active ingredient may be used in pure form or, more typically, formulated into a composition which includes, in addition to said active ingredient, a suitable inert diluent or carrier and optionally, a surface active agent (SFA). SFAs include non-ionic, cationic and/or anionic surfactants, as well as surfactant mixtures.

The compositions can be chosen from a number of formulation types, including dustable powders (DP), soluble powders (SP), water soluble granules (SG), water dispersible granules (WG), wettable powders (WP), granules (GR) (slow or fast release), soluble concentrates (SL), oil miscible liquids(OL), ultra low volume liquids (UL), emulsifiable concentrates (EC), dispersible concentrates (DC), emulsions (both oil in water (EW) and water in oil (EG)), micro-emulsions (ME), suspension concentrates (SC), aerosols, fogging/smoke formulations, capsule suspensions (CS) and seed treatment formulations. The formulation type chosen in any instance will depend upon the particular purpose envisaged and the physical, chemical and biological properties of the active ingredient (with this respect, see Shelar A. et al, 2023; Sun H. et al, 2022).

To achieve the same goal, it is also possible to contact the seeds with a composition containing a short complementary peptide (cPEP) as defined by Ormancey et al, 2023 the amino acid sequence of said cPEP being able to interact specifically with the mRNA of a protein enhancing iron accumulation and I or iron absorption (e.g., with any of the mRNAs mentioned above), in order to enhance its expression, as demonstrated in Ormancey et al, 2023. Preferably, in the context of the invention, the cPEP is able to interact with an IRONMAN transcript or with the bHLH38 or bHLH39 transcripts as defined above.

Alternatively, the method of the invention can be implemented by genetically engineering the target plants so that their seeds overexpress proteins that activates iron accumulation I absorption or underexpress proteins negatively affecting iron accumulation and I or iron absorption.

In that case, the target plants of the invention contain :

Ila) A nucleic acid coding for a protein enhancing iron accumulation and I or iron absorption in plant seeds as defined above, said protein being preferably, as defined above, an IRONMAN protein, a peptide issued thereof, or the bHLH38 protein or the bHLH39 protein or a functional variant thereof as defined above, or any proteins known to regulate iron homeostasis in plants such as FIT, bHLH101 , PYE, MYB10, MYB72, 4CL1 , F6’H1 , S8H, CYP82C4, ABCG37, BGLU42, IRT1 , IRT2, FRO3, OPT3, NAS4, BTS, BTSL1 , BTSL2, IREG3, ZIF1 , WRKY22, NAC2, FER1 , FER3, FER4, SAPX, VTL1 , ZIP4, YSL3, and HEMA1 , or lib) A nucleic acid reducing the expression of at least one protein negatively affecting iron accumulation and I or iron absorption in plant seeds as defined above, said protein preferably inducing the chromatin condensation in genes involved in DT activation including iron accumulation and I or iron absorption (said genes being as defined above), said protein being more preferably chosen among: SWN, H2A.Z and CLF protein as defined above, or said protein being involved in the negative regulation of the iron accumulation such as BRUTUS protein or BRUTUS LIKE protein. In a particularly preferred embodiment, in that case, the method of the invention can comprise the following steps : a) Introducing in said target plant a nucleic acid compound as defined in Ila) or lib) above, so as to produce a transgenic plant, preferably overexpressing the IRONMAN protein IMA1 of SEQ ID NO:1 , IMA2 of SEQ ID NO:2 or IMA3 of SEQ ID NO: 3, or the peptide of SEQ ID NO:4 or SEQ ID NO:11 or a functional variant thereof containing SEQ ID NO:26 and/or SEQ ID NO:27 these variants being as defined above, or under-expressing the SWN, H2A.Z, CLF or BTS proteins as defined above, b) Collecting the seeds of said plant, then drying them, c) Optionally, contacting said coated seeds with a solution or a powder containing iron, d) Optionally, coating said seeds with other performance enhancing compounds.

For tomato, the step a) will advantageously comprise introducing in said target plant a nucleic acid compound as defined in Ila) or lib) above, so as to produce a transgenic plant, preferably overexpressing the IRONMAN protein IMA7 of SEQ ID NO: 38 or a SIIMA7BID peptide (CLDGDDDSDYDYAPAA, SEQ ID NO: 31).

All the means and embodiments disclosed above concerning the proteins to be overexpressed, or their variants, or the solution or compounds to be used in steps c) or d) apply mutatis mutandis to perform this particular method.

Methods for obtaining transgenic plants use transformation techniques to introduce the gene or construct encoding the proteins of the invention, into plant cells Transformation can be accomplished by a variety of well-known methods including for example, Agrobacterium based systems, using either binary and/or cointegrate plasmids of A. tumefaciens and A. rhizogenes, (See e.g., U.S. Pat. No. 4,940,838, U.S. Pat. No. 5,464,763), the biolistic approach (See e.g., U.S. Pat. No. 4,945,050, U.S. Pat. No. 5,015,580, U.S. Pat. No. 5,149,655), microinjection, (See e.g., U.S. Pat. No. 4,743,548), direct DNA uptake by protoplasts, (See e.g., U.S. Pat. No. 5,231 ,019, U.S. Pat. No. 5,453,367) Any method forthe introduction of foreign DNA into a plant cell or seed and forexpression therein may be used within the context of the present invention. Plants that are capable of being transformed encompass a wide range of species, including but not limited to Brassicaceae, soybean, corn, wheat and other crops, or vegetables which are sawn See generally, Vain, P., Thirty years of plant transformation technology development, Plant Biotechnol J. 2007 March; 5(2):221-9.

Constructs for overexpressing a protein in a plant / seeds

In the particular case where the nucleic acid of the invention is aimed at overexpressing coding for a protein enhancing iron accumulation and I or iron absorption (e.g., a IMA protein or consensus peptide, or bHLH38 or bHLH39 as described above), this nucleic acid can be any recombinant DNA construct (typically, a vector or a plasmid containing a recombinant expression cassette) containing a polynucleotide encoding the protein of interest (e.g., a IMA protein or bHLH38 or bHLH39 as described above) under the control of an appropriate promoter.

"Over-expressing" a protein herein refers to artificially increasing its expression (for instance by adding at least one additional copy of a sequence encoding said protein) in plants which naturally express it.

The invention also provides means for carrying out said overexpression, in particular any recombinant DNA construct (typically, a vector or a plasmid containing a recombinant expression cassette) containing a polynucleotide encoding the protein of interest (e.g., a IMA protein or bHLH38 or bHLH39 as described above) under the control of an appropriate plant promoter. These DNA constructs can be obtained by well-known techniques of recombinant DNA and genetic engineering.

The promoter used in the recombinant DNA construct of the invention can be any promoter that is functional in a plant cell. The choice of the more appropriate promoter may depend in particular on the chosen host plant, on the organ (s) or tissue (s) targeted for expression, and on the type of expression (i.e. constitutive or inducible) that one wishes to obtain. A large choice of promoters suitable for expression of heterologous genes in plants is available in the art, either constitutive or tissue- organ-specific. In a preferred embodiment, one can use a promoter that is known to be efficient in plant seeds, e.g., a promoter from a gene involved in seed development, such as the promoter of oleosin, or of a seed storage protein, or any promoter from gene involved in germination. Other conventional promoters used in plants can also be used.

The selection of suitable vectors and the methods for inserting DNA constructs therein are well known to persons of ordinary skill in the art. The choice of the vector depends on the intended host and on the intended method of transformation of said host. A variety of methods for genetic transformation of plant cells or plants are available in the art for many plant species, dicotyledons or monocotyledons. By way of non-limitative examples, one can mention virus mediated transformation, transformation by microinjection, by electroporation, microprojectile mediated transformation, Agrobacterium mediated transformation, and the like. For instance, in the case of monocotyledons, one can advantageously use the method described by ISHIDA et al. (Nature Biotech. 14, 745-750, 1996).

Methods of the invention, aiming at producing a transgenic plant I plant seed having an increased tolerance to water deficit, can therefore contain the following steps:

- transforming at least one plant cell or plant seed with a vector containing an expression cassette expressing a protein enhancing iron accumulation and I or iron absorption (e.g., a IMA protein or consensus peptide or bHLH38 or bHLH39 as described above), and

- cultivating said transformed plant cell or plant seed in order to regenerate a plant having in its genome a transgene containing said expression cassette.

The transgenic plants and seeds of the present invention are described below.

Constructs for under-expressing a protein in plants / seeds

In the particular case where the nucleic acid of the invention is aimed at impairing or lowering the expression of a protein negatively affecting iron accumulation and I or iron absorption in plant seeds (e.g., altering the expression of the genes whose encoded proteins induce the chromatin condensation in genes involved in iron accumulation and / or iron absorption, notably the expression of the swn, hta8, hta9, hta11 and/or elf genes as described above), this nucleic acid can be any polynucleotide construct affecting the transcription, or translation of the protein encoded by the target genes of the invention, so that the levels of the corresponding proteins are rendered significantly lower than the levels of this protein would otherwise be (in the same conditions). To this end, either the coding or non-coding regions, or both, of the said target genes may be modified.

"Under-expressing" a protein herein refers to artificially reducing its expression in plants which naturally express it. In the context of the invention, it is recommended not to completely inhibit the expression of the target proteins, since they can have an essential function during plant development. Therefore, genetic tools for mildly silencing gene expression are preferred, such as RNA interference (using for example mi-RNAs or si-RNAs), or antisense DNA.

Short-interfering RNAs (si-RNAs) or other antisense nucleic acids can be used for inhibiting at least 50% of the expression of the target genes of the invention. These inhibitors of gene expression are for example anti-sense RNA molecules and anti-sense DNA molecules, act to directly block the transcription/translation of the mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the target proteins of the invention, and thus its activity, in the target plants or seeds. Antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding the targeted proteins can be used. They can be synthesized, e.g., by conventional phosphodiester techniques, and administered to the seeds, by e.g. during the imbibition step in a soluble solution. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131 ; 6,365,354; 6,410,323; 6,107,091 ; 6,046,321 ; and 5,981 ,732). Alternatively, under-expression may be obtained by mutating the target gene of the invention. A change in nucleotide sequence of the gene's coding region may lead to a different amino acid being added to the growing polypeptide chain, causing a change in protein structure and function. As example, when a mutation on the DNA strand creates a premature stop codon, the RNA template will not be completely translated, resulting in a protein with a lower molecular weight due to fewer amino acid residues. As a result, the truncated protein will also likely be nonfunctional.

Zinc-finger nucleases, nucleases, meganucleases (MNs), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) have emerged during the past decade as efficient tools for genome editing in many organisms. CRISPR-Cas9 is a simple two-component system that allows researchers to precisely edit any sequence in the genome of an organism (Nymark et al., 2017 CRISPR/Cas9-mediated genome editing is a simple and versatile tool for creating targeted genome modifications such as a frameshift introducing a stop codon in the targeted coding sequence or a deletion or a short insertion in this targeted coding sequence.

The present invention thus also relates to a plant recombinant DNA vector (or “plant DNA construct”) or a plant viral vector comprising a polynucleotide sequence encoding a functional iRNA against any of the genes of the invention, said functional iRNA being a dsRNA, a siRNA a miRNA or a synthetic inhibitor miRNA.

In another aspect, the present invention targets a recombinant construct comprising a polynucleotide encoding a long or short RNA affecting the expression of the swn, hta8, hta9, hta11 and/or elf genes, under control of a plant-appropriate promoter.

The vector of the invention can be prepared by conventional methods known in the art. For example, it can be produced by amplification of a nucleic sequence by PCR or RT-PCR, by screening genomic DNA libraries by hybridization with a homologous probe, or else by total or partial chemical synthesis. The recombinant vectors can be introduced into host cells by conventional techniques, which are known in the art.

Transgenic plants / plant cells of the invention

When the methods of the invention involve the genetic transformation of the plants of interest, they generate transgenic plants over-expressing proteins enhancing iron accumulation and I or iron absorption, or under-expressing the expression of at least one protein negatively affecting iron accumulation and / or iron absorption in their seeds. These proteins have been described in details above. They can be for example the IMA1 , IMA2, IM3, bHLH38, bHLH39, SWN, H2A or CLF proteins.

Therefore, in another aspect, the present invention targets a transgenic plant producing dry mature seeds able to tolerate desiccation at the post-germination stages, said plant containing a nucleic acid

Either over-expressing a protein enhancing iron accumulation and I or iron absorption, said protein being preferably an IRONMAN protein, a consensus peptide thereof, or the bHLH38 protein or the bHLH39 protein or a functional variant thereof,

Or under-expressing at least one protein negatively affecting iron accumulation and I or iron absorption in plant seeds, said protein preferably inducing the chromatin condensation in genes involved in iron accumulation and I or iron absorption, said protein being more preferably chosen among: SWN, H2A.Z and CLF protein, or said protein being involved in the negative regulation of the iron accumulation such as BRUTUS protein or BRUTUS LIKE protein

In a more preferred embodiment, the transgenic plants of the invention contain a nucleic acid coding for the IMA1 , IMA2, IMA3 protein or the consensus peptide as disclosed above.

In another embodiment fortomato, the transgenic plants of the invention contain a nucleic acid coding for the IMA7 protein as disclosed above.

The present invention also targets isolated cells, organs or tissues (such as fruits, seeds, leafs, pollen, flowers, roots, tubers) of the transgenic plants of the invention. They can be distinguished from prior art in that they containing an exogenous nucleic acid as defined above.

The over- or under-expression of the target proteins of the invention in said transgenic plants provides them with an increased tolerance to desiccation, when compared to a plant devoid of said transgene.

Seeds of the invention

In a further aspect, the present invention targets the seeds obtained by the methods of the invention. As exposed above, these seeds can either be coated with a chemical compound (usually a protein, or a short peptide), or primed with a chemical compound (usually a protein, or a short peptide) or genetically modified in order to over-express a protein enhancing iron accumulation and I or iron absorption or to under-express of at least one protein negatively affecting iron accumulation and I or iron absorption. In a preferred embodiment, the desiccation-tolerant mature seeds of the invention are seeds that are coated or have been imbibed with the IRONMAN protein IMA1 of SEQ ID NO:1 , IMA2 of SEQ ID NO:2 or IMA3 of SEQ ID NO:3 or with a functional variant thereof as defined above.

In a particularly preferred embodiment, the dry desiccation-tolerant mature seeds of the invention are coated or have been imbibed with the peptide having the sequence SEQ ID NO:4 or SEQ ID NO:11 , or a functional variant thereof as defined above, preferably one containing SEQ ID NO:26 and/or SEQ ID NO:27, or an IRONMAN protein SIIMA7 of SEQ ID NO: 38 or a synthetic peptide SIIMA7BID of SEQ ID NO:31 , optionally in mixture with one or several IRONMAN proteins not expressed in seeds.

In a particular embodiment for tomato, the desiccation-tolerant mature seeds of the invention are seeds that are coated or have been imbibed with the IRONMAN protein IMA7 of SEQ ID NO:38, or a SIIMA7BID peptide (CLDGDDDSDYDYAPAA, SEQ ID NO: 31) that contain a conserved BRUTUS (BTS) interaction domain (BID), or a functional variant thereof having at least 60, 70 or 80% identity with SEQ ID NO: 38 or SEQ ID NO:31 , optionally in mixture with one or several IRONMAN proteins not expressed in seeds.

They can be distinguished from prior art seeds in that they contain more IMA proteins on or inside the seeds, e.g., by performing mass chromatography, quantifying the amount of IMA proteins in the seed, and comparing this amount to the amount of the IMA proteins in untreated seeds of the same plant species. It is also possible to detect the presence of exogenous IMA proteins on or in target seeds by measuring and comparing the expression level of genes whose expression is known to be controlled by IMA proteins such as the IMAs themselves, PYE or bHLHs (Li et al. 2021). When the seeds have been treated with the consensus peptide as disclosed herein, it is easy to identify them by measuring the amount of said consensus peptide in or on the seed, e.g. by mass chromatography (non-treated seeds should not contain the consensus peptide).

The invention also targets seeds that have been coated and imbibed as explained above, until they reach the germination stage II, and have been dried afterwards. Consequently, the seeds of the invention can also be germinating seeds at stage II.

These dry desiccation-tolerant mature seeds of the invention can be optionally also coated with performance enhancing compounds and/or with iron (Fe²⁺ or Fe³⁺).

The invention is also drawn to desiccation-tolerant mature (dry) seeds in which iron accumulation and I or iron absorption is genetically enhanced, because they overexpress a protein enhancing iron accumulation and I or iron absorption. They are distinct from prior art seeds in that they contain an exogenous nucleic acid encoding at least one of the proteins of the invention, as defined above, namely an IRONMAN protein or the bHLH38 protein or the bHLH39 protein or a functional variant thereof. In an alternative embodiment, the desiccation-tolerant mature seed of the invention is a seed in which the swn, hta , elf or btsbts genes are mutated I inhibited so as to reduce the expression of the proteins SWN, H2A.Z.CLF or BTS.

DEFINITIONS

The singular forms “a,” “an,” and “the,” are used in the sense that they include plural reference of the referenced components, such as "at least one", "at least a first", "one or more" or "a plurality", unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound that activates iron accumulation and I or iron absorption in a seed of a target plant " includes one or several compounds, including mixtures thereof.

By “plants of interest” are herein encompassed any plant that produce seeds. In particular, the plant of interest can be of the Brassicaceae family which contains cruciferous vegetables, including species such as Brassica oleracea (cultivated as cabbage, kale, cauliflower, broccoli and collards), Brassica rapa (turnip, Chinese cabbage, etc.), Brassica napus (rapeseed, etc.), Raphanus sativus (common radish), Armoracia rusticana (horseradish), and the model organism Arabidopsis thaliana (thale cress). The methods of the invention could be immediately developed in any of the plant present in the list of 132 ortholog peptides disclosed in Grillet et al. 2018 (cf. supplementary Figure 4), or in any other plant in which IMA peptides are identified, including dicots such as tomato (as illustrated in Figure 9), bean, soybean, cacao, rapeseed, alfalfa but also monocots such as rice, wheat and Maize.

In a particular embodiment, the plant of interest or its seeds in the context of the invention is not rice.

The term “drought” as used herein refers to the set of environmental conditions under which a plant will begin to suffer the effects of moisture deprivation, such as decreased stomatai conductance and photosynthesis, decreased growth rate, loss of turgor (wilting), or ovule/pollen abortion. Plants experiencing drought stress typically exhibit a significant reduction in biomass and yield. Water deprivation may be caused by lack of rainfall or limited irrigation.

Water deficit may be caused by high temperatures, low humidity, saline soils, freezing temperatures, competition for limited moisture in the rooting zone, or damaged roots. Plant species vary in their capacity to tolerate water deficit and therefore the precise environmental conditions that cause drought stress cannot be generalized. However, drought tolerant plants produce higher biomass and yield compared to plants that are not drought tolerant under water limited conditions and may also exhibit enhanced survivability and/or delayed desiccation /permanent wilting point under water limited conditions. Differences in physical appearance, recovery, and yield can be quantified and statistically analyzed using known measurement and analysis techniques. The term “desiccation tolerance” (DT), or anhydrobiosis, is an extreme drought stress, which can be defined as the ability to survive, by reversible cessation of metabolism, the removal of almost all cellular free water when in equilibrium with moderately dry air and resume normal function when rehydrated (Phillips JR et al 2002). More precisely, desiccation tolerance is the ability of living organisms to deal with water losses below 0.1g H2O g^-1 dry weight (10%) and survive the re-hydration process without permanent damage (Oliver MJ et al, 2000). In other terms, desiccation tolerance enables the seeds to tolerate a water cellular content of less than 10%, without prejudice to the future plant.

The term “seed priming” used herein comprises a step of imbibing seeds, generally for a few days in an osmotic solution (osmopriming) or few hours in water (hydropriming) to avoid radicle emergence. Then imbibed seeds are followed by drying to quickly desiccate seeds, generally to less than 10% of water content. This empirical technique allows seeds to pre-initiate germination processes and provide commercial seed lots with higher germination vigor.

An “agriculturally acceptable carrier” includes adjuvants, mixers, enhancers, etc. beneficial for application of an active ingredient, such as molecules of the invention. Suitable carriers should not be phytotoxic to valuable crops, particularly at the concentrations employed in applying the compositions in the presence of crops, and should not react chemically with the compounds of the active ingredient herein, or other composition ingredients. Such mixtures can be designed for application directly to crops, or can be concentrates or formulations which are normally diluted with additional carriers and adjuvants before application. They may include inert or active components and can be solids, such as, for example, dusts, granules, water dispersible granules, or wettable powders, or liquids, such as, for example, emulsifiable concentrates, solutions, emulsions or suspensions. Suitable solid carriers may include talc, pyrophyllite clay, silica, attapulgus clay, kieselguhr, chalk, diatomaceous earth, lime, calcium carbonate, bentonire clay, Fuller's earth, cotton seed hulls, wheat flour, soybean flour, pumice, wood flour, walnut shell flour, lignin, and the like (Shelar A. et al, 2023).

For the present invention, an agriculturally acceptable carrier may also include non-pathogenic, attenuated strains of microorganisms, which carry the interfering RNA molecule. The microorganisms may be engineered to express a nucleotide sequence of a target gene to produce interfering RNA molecules comprising RNA sequences homologous or complementary to RNA sequences typically found within the targeted genes. Exposure of the plant to the microorganisms result in downregulation of expression of target genes mediated directly or indirectly by the interfering RNA molecules or fragments or derivatives thereof.

In another embodiment, the compounds of the invention may be encapsulated in a synthetic matrix such as a polymer and applied to the surface of a host such as a plant.

By “performance enhancing compounds” it is herein meant additives (binders, fillers or active ingredients) that can improve the biological performance of seeds (for example nutrients, symbionts, plant growth regulators, fertilizers, fertilizer enhancers, pesticides, nanomaterials such as calcium, silver, gold, copper, palladium, selenium, zinc oxide, magnesium oxide, titanium dioxide, chitosan, cellulose, lignin, starch, alginate, gum arabic, gelatin, soy protein, PVA nanofibers, etc. see Shelar A. et al, 2023; Sun H. et al, 2022). These compounds can be applied to the seeds by using a thin film (e.g. with a film-forming polymer suspension applied via a fluidized bed or a rotary coater), or by dipcoating or spray coating.

As used herein, the term “mature seed” designates the seed that has undergone the complete development process in plant so to be able to produce a dried seed that is able to germinate and produce a plant.

The term “transgenic plant” herein refers to a host plant into which a gene construct has been introduced. A gene construct, also referred to as a construct, an expression construct, or a DNA construct, generally contains as its components at least a coding sequence and a regulatory sequence. A gene construct typically contains at least on component that is foreign to the host plant. For purpose of this disclosure, all components of a gene construct may be from the host plant, but these components are not arranged in the host in the same manner as they are in the gene construct. A regulatory sequence is a non-coding sequence that typically contribute to the regulation of gene expression, at the transcription or translation levels. Examples of a regulatory sequence include but are not limited to a promoter, an enhancer, and certain post-transcriptional regulatory elements.

A “vector” is a composition for facilitating introduction, replication and/or expression of a selected nucleic acid in a cell. Vectors include, for example, plasmids, cosmids, viruses, yeast artificial chromosomes (YACs), etc. A “vector nucleic acid” is a nucleic acid vector into which heterologous nucleic acid is optionally inserted and which can then be introduced into an appropriate host cell. Vectors preferably have one or more origins of replication, and one or more sites into which the recombinant DNA can be inserted. Vectors often have convenient markers by which cells with vectors can be selected from those without. By way of example, a vector may encode a drug resistance gene to facilitate selection of cells that are transformed with the vector. Common vectors include plasmids, phages and other viruses, and “artificial chromosomes.” “Expression vectors” are vectors that comprise elements that provide for or facilitate transcription of nucleic acids which are cloned into the vectors. Such elements may include, for example, promoters and/or enhancers operably coupled to a nucleic acid of interest.

The percent identities referred to in the context of the disclosure of the present invention are determined on the after optimal alignment of the sequences to be compared, which may therefore comprise one or more insertions, deletions, truncations and/or substitutions. This percent identity may be calculated by any sequence analysis method well-known to the person skilled in the art. The percent identity may be determined after global alignment of the sequences to be compared of the sequences taken in their entirety over their entire length. In addition to manual comparison, it is possible to determine global alignment using the algorithm of Needleman and Wunsch (1970). 1 For nucleotide sequences, the sequence comparison may be performed using any software well- known to a person skilled in the art, such as the Needle software. The parameters used may notably be the following: “Gap open” equal to 10.0, “Gap extend” equal to 0.5, and the EDNAFULL matrix (NCBI EMBOSS Version NUC4.4). For amino acid sequences, the sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software. The parameters used may notably be the following: “Gap open” equal to 10.0, “Gap extend” equal to 0.5, and the BLOSUM62 matrix. Preferably, the percent identify as defined in the context of the present invention is determined via the global alignment of sequences compared over their entire length.

As mentioned above, it is possible to use in the methods and products of the invention any ortholog or variant protein whose sequence has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% identity with the reference sequences mentioned herein. It is understood that it is also possible to use in the methods and products of the invention any ortholog or variant protein whose sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95% identity with the reference sequences mentioned herein. It is important however to ensure that the ortholog or variant protein used in the invention can activate iron accumulation and I or iron absorption in the target plant seed.

So, the ortholog or variant protein according to the present invention, also named ‘functional variant’ in the description, can activate iron accumulation and I or iron absorption in the target plant seed, and may be detected by using a staining solution on isolated mature/germinated embryos, and observing the images by a microscope coupled with a camera showing iron localization in the embryos, as described above.

In a particular embodiment, a functional variant that activates iron accumulation and/or absorption for use according to the invention to increase the desiccation tolerance of said seed once germination is induced, is selected in an in vitro method comprising the following steps :

(i) Seeds are imbibed with water (control) or peptides solutions (the functional variant to be tested), by incubation at room temperature, in particular 20°C, under continuous light, or using seed priming,

(ii) Germinating embryos at stage II and IV are isolated, in particular by manually removing envelopes (i.e. seed coat and endosperm),

(iii) The isolated mature/germinated embryos are brought into contact with a staining solution containing a potassium ferrocyanide, and

(v) A color change is observed, in comparison to the control (water), when the tested functional variant activates iron accumulation and/or absorption.

By “enhancing iron accumulation”, it is herein meant that the total iron content in the treated seed is enhanced, or that the distribution of iron within seed tissues is modulated. Iron accumulation and I or iron absorption can be easily detected in seeds by conventional means. For example, by Inductively coupled plasma mass spectrometry (ICP-MS) or by coloration (see e.g., in Grillet et al, 2018). Any gene or protein that, when over-expressed or under-expressed by a conventional means as those mentioned above, can enhance iron accumulation and I or iron absorption in seeds by more than 50%, preferably by at least two folds, as compared with non-treated seeds, is encompassed within the present invention.

It is also important to ensure that the treatment of the plant or seeds does not significantly affect the agronomic properties of the resulting plant (e.g., its development, timing of production, yield, fruits, fertility, etc.).

Means inducing “under-expression” of a protein in a seed herewith encompassed tools that enable to reduce the amount of said protein in the seed, to a level significantly lower than in non-treated seeds, typically to a level that is less than 50%, preferably less than 25%, more preferably less than 10% of the protein content as compared to the level of the same protein in non-treated seeds. Conversely, means inducing “over-expression” of a protein in a seed herewith encompassed tools that enable to enhance the amount of said protein in the seed, to a level significantly higher than in non-treated seeds, typically to a level that is more than 25%, preferably more than 50%, more preferably more than 100% of the protein content as compared to the level of the same protein in non-treated seeds.

FIGURE LEGENDS

Figure 1A) explains how the desiccation tolerance tests were conducted and the three developmental stages studied during germination: mature dry seed (M), seed with radical protrusion (stage II) and seed with the first root hair appearances (stage IV); Figure 1 B) discloses the desiccation tolerance (DT) results obtained forthe wild-type Arabidopsis thaliana seeds and the three loss-of-function Arabidopsis thaliana mutants h2a.z, swn and elf. Survival of seedlings was scored 7 days after rehydration. Values are means ± SD of three replicates. Different letters indicate significant differences (P < 0.05, Tukey-Kramer tests).

Figure 2 shows the DT determined at three developmental stages during germination: mature dry seed (M), seed with radical protrusion (II) and seed with the first root hair appearances (IV) for wildtype Arabidopsis thaliana seeds or Arabidopsis thaliana seeds over-expressing the IRONMAN genes IMA1-OE, IMA2-OE, IMA3-OE, bHLH38-OE,bHLH39-OE and bts mutant (loss-of-function of BRUTUS gene known to be involved in iron homeostasis). Survival of seedlings was scored 7 days after rehydration. Values are means ± SD of three replicates. Different letters indicate significant differences (P < 0.05, Tukey-Kramer tests).

Figure 3 shows the dose effect of exogenously treated iron on DT. Seeds were imbibed with 0, 50, 100, 250, 500 and 1000 pM FeEDTA at three developmental stages during germination: mature dry seed (M), seed with radical protrusion (II) and seed with the first root hair appearances (IV). Survival of seedlings was scored 7 days after rehydration with deionized water. Values are means ± SD of three replicates. Different letters indicate significant differences (P < 0.05, Tukey-Kramer tests).

Figure 4 discloses the dose effect of the IMA3 full length peptide (exogenously applied) on DT. Seeds were imbibed with 0, 10, 50 and 100 pM IMA3 synthetic peptide and the DT was determined at three developmental stages during germination: mature dry seed (M), seed with radical protrusion (II) and seed with the first root hair appearances (IV). Survival of seedlings was scored 7 days after rehydration with deionized water. Values are means ± SD of three replicates. Different letters indicate significant differences (P < 0.05, Tukey-Kramer tests).

Figure 5 discloses the dose effect of the IMA3BID peptide (exogenously applied) on DT. Seeds were imbibed with 0, 10, 50 and 100 pM IMA3BID or 100 pM IMA3BID. Random synthetic peptide and the DT was determined at three developmental stages during germination: mature dry seed (M), seed with radical protrusion (II) and seed with the first root hair appearances (IV). Survival of seedlings was scored 7 days after rehydration with deionized water. Values are means ± SD ofthree replicates. Different letters indicate significant differences (P < 0.05, Tukey-Kramer tests).

Figure 6 shows the combined effect of applying exogenously both iron and the IMA3BID peptide on DT. Seeds were imbibed with combination of FeEDTA and IMA3BID synthetic peptide solution. (-)Fe: 0 pM FeEDTA, (+)Fe: 100 pM, (-)IMA3BID: 0 pM IMA3BID and (+)IMA3BID: 100 pM IMA3BID. The DT was determined at three developmental stages during germination: mature dry seed (M), seed with radical protrusion (II) and seed with the first root hair appearances (IV). Survival of seedlings was scored 7 days after rehydration with deionized water. Values are means ± SD of three replicates. Different letters indicate significant differences (P < 0.05, Tukey-Kramer tests).

Figure 7 shows the localization and over-accumulation of iron in germinating seeds reinducing DT, by Peris Staining of germinating Arabidopsis seeds. Localization of iron (Fe³⁺, dark staining) in Mature seed (M), seed with radical protrusion (II) and seed with the first root hair appearance (IV). White bar = 200 pm.

Figure 8 shows the effect of exogenously treated generic IMABID and IMA3BID peptides on DT- induction in Arabidopsis. Seeds were imbibed with water, 100 pM GenIMABID, 100 pM GenIMABID. Random, 100 pM IMA3BID or 100 pM IMA3BID. Random synthetic peptides and the DT was determined at three developmental stages during germination: mature dry seed (M), seed with radical protrusion (II) and seed with the first root hair appearance (IV). Survival of seedlings was scored 7 days after rehydration with deionized water. Values are means ± SD of three replicates. Different letters indicate significant differences (P< 0.05, Tukey-Kramer tests).

Figure 9 shows the effect of exogenously treated generic IMABID and SIIMA7BID peptides on DT- induction in tomato. Seeds were imbibed with water, 100 pM GenIMABID, 100 pM GenIMABID. Rand or 100 pM SIIMA7BID synthetic peptides and the DT was determined at three developmental stages during germination: mature dry seed (M), germinated seeds with radicles of 1 mm (1 mm) and germinated seeds with radicles of 3 mm (3 mm). Survival of seedlings was scored 14 days after rehydration with deionized water. Values are means ± SD of four replicates. Different letters indicate significant differences (P < 0.05, Tukey-Kramer tests).

EXAMPLES

By analyzing the transcriptome changes in the individual elf, swn and h2az mutants, many changes were identified in gene expressions belonging to a common pathway, the iron homeostasis, suggesting a link between iron accumulation/absorption and reinduction of DT in all these mutant lines. To validate this observation, a gene enrichment analysis (GEA) of the differentially expressed genes in the elf, swn and h2az mutant lines was performed and revealed that expressions of specific gene-sets related to functional classes of regulation of iron transport, cellular response to iron starvation and regulation of metal iron transport were statistically affected, suggesting a major modification of the iron homeostasis in these mutant lines re-inducing DT at stages II and IV. Therefore, the link between iron homeostasis and the re-induction of DT after germination was explored more in detail by modifying iron homeostasis directly via addition of iron molecules or using a genetic approach via some known genes regulating iron content such as bHLH38, bHLH39, IMA1, IMA2 and IMA3 genes.

Material and methods

Plant materials

The loss-of-function mutants h2a.z (CS69073; hta8-1 hta9-1 hta11-1), swn (SALK_109121 ; swn-4) and elf (SALK_021003; clf-29) and bts (SALK_087255; bts-1O)_ in Arabidopsis thaliana were isolated as described previously (Coleman-Derr & Zilberman 2012; Wang et al., 2006; Bouveret et al., 2006, Selote et al., 2015). The over-expressing lines IMA1-OE (JMA1oe-T), IMA2-OE (JMA2oe-6), IMA3- OE JMA3oe-2), bHLH38-OE (b38oe-2) and bHLH39-OE p39oe-5) in Arabidopsis were generated as described previously (Li et al., 2021 ; Cai et al., 2021). Arabidopsis natural accession Columbia-0 (Col-0) was used as the wild-type control for these mutants and over-expressing lines. For Arabidopsis initial seed production, seeds were sown in square pots of 6x6x6 cm filled with Tray substrate 75/25 (ref.092, Klasmann-Deilmann, Bourgoin Jallieu, France). Seedlings were grown in a growth chamber (22°C/18°C (day/night), 16 h photoperiod, 70 pmol m²s^-1 light intensity, 60-70% relative humidity) and plants were watered with a fertilizer Plant-Prod 15-30-15 (Master Plant-Prod Inc, Canada). Mature seeds were collected at plant maturity on dried plants. For tomato (S. lycopersicum L. cv. Micro-Tom) seed production, plants were grown in a greenhouse with standard conditions (23°C/19°C (day/night), a 16h photoperiod, 150 pmol m²s^-1 light intensity, 60-70% relative humidity, RH) and plants were watered with a fertilizer Plant-Prod 15-30-15 (Master Plant-Prod Inc, Canada). Seeds from mature fruits were collected, and locular tissues were removed by incubation in a pectolytic enzyme solution (Lafazym CL Laffort, France) for 1 h, followed by extensive washing with water to remove the remnants of fruit tissues. Thereafter, seeds were equilibrated and dried at 44% RH using a saturated solution of K2CO3 at 20°C for 3 days (d), then hermetically stored at 4°C prior to seed physiological analyses.

Physiological analysis (assessment of DT)

For assessment of desiccation tolerance (DT) in Arabidopsis, seeds were imbibed with sterile deionized water on a filter paper in a Petri dish at 4°C for 3 days in dark as a cold stratification followed by incubation at 20°C under continuous light (20 pmol m^-2s“¹) then germinating seed samples at each developmental stage were desiccated at 20°C for 3 days at 44% relative humidity using a saturated solution of K2CO3. Each developmental stage of germinating seeds (50 seeds in triplicate) was defined using a stereomicroscope as described previously (Maia et al., 2011): mature dry seed (M), stage II characterized by seed with radical protrusion (II) and stage IV characterized by seed with the first root hair appearances (IV). The desiccated samples were rehydrated with sterile deionized water on a filter paper (Whatman paper #1) in a Petri dish and incubated at 20°C under 16 h photoperiod with about 20 pmol m^-2s“¹ light intensity. Seedlings that continued their development with healthy green cotyledons at 7 days after incubation were considered desiccation tolerant and were scored as survival rate (%) at each germinating stage with 50 seeds in triplicate.

For assessment of DT in tomato, seeds were imbibed with sterile deionized water on filter paper in a Petri dish at 20°C for 3 d in the dark, then germinated seed samples (15 seeds in four replicates) with 1- and 3-mm radicles were desiccated at 20° C for 3 d at 44% RH using a saturated solution of K2CO3. For mature seeds, no additional dehydration treatment was performed as they had already been dried for at least 3 days using a saturated solution of K2CO3 prior their storage. The desiccated samples (mature seeds and germinated seeds) were then rehydrated with sterile deionized water on filter paper in a Petri dish and incubated in a growth chamber at 20°C under a 16 h photoperiod with 50 pmol m^-2s“¹ light intensity. Seedlings that continued their development with healthy cotyledons, hypocotyl and root at 14 days after incubation were considered desiccation tolerant and were scored as survival rate (%) at each germinating stage.

Chemicals/ peptide treatments to reinduce DT

FeEDTA was purchased from Sigma-Aldrich in USA and used for assessment of DT presented in Figs 3 and 6.

Full length of synthetic IMA3 peptide

(MAVVSHNNAEGRLYESTQTWPIAYLQIGGQENGGDDDDDDCDVAPAA - SEQ ID NO:3) and C- terminal part of IMA3 peptides that contain a conserved BRUTUS (BTS) interaction domain (BID), named as IMA3BID (ENGGDDDDDDCDVAPAA, SEQ ID NO:4) (Li et al., 2021) and a randomized peptide IMA3BID. Random (CPADNDEDADVDADGGD, SEQ ID NO:28) (containing randomized amino acid sequence of IMA3BID used as negative control) were purchased from Shanghai Royobiotech and used for assessment of DT in Arabidopsis presented in Figs 4, 5 and 6. To investigate the potential effects of peptides on DT in plant species other than Arabidopsis, a generic C-terminal part of IMA peptide that contains the most conserved BID among the 131 IMA peptides identified in different species (described in Grillet et al, 2018, on the supplementary Figure 4 and in Supplementary Table 2) named as GenIMABID (GDDDDDGYDYAPAA, SEQ ID NO: 29), its randomized sequence peptide GenIMABID. Random (DADYGDAGDPDYAD, SEQ ID NO: 30, used as negative control) and a C-terminal BID part of tomato IMA7 peptide named as SIIMA7BID (CLDGDDDSDYDYAPAA, SEQ ID NO: 31) were also designed and tested for assessment of DT in Arabidopsis and/or tomato._Chemicals were directly dissolved in sterile deionized water and each stock solution (100 mM FeEDTA, 200 pM IMA3 , 500pM IMA3BID,_590 pM IMA3BID, 590 pM IMA3BID. Random, 680 pM GenIMABID, 680 pM GenlMA3BID. Random and 580 pM SIIMA7BID respectively) was diluted to the tested concentrations for the experiments. For Arabidopsis, seeds were imbibed with either water, or water and peptides and/or iron for a cold stratification during 72 hours followed by incubation at 20°C under continuous light and were desiccated as described above. Sterile deionized water was used for all control experiments. For tomato, seeds were imbibed with the peptide solutions at 20°C and incubated for 3 d in dark without a cold stratification, then desiccated as described above. Sterile deionized water was used for all control experiments._The desiccated samples were rehydrated with sterile deionized water and survival rate of seedlings were scored on petri dishes as described above.

Visualization of iron localization by Peris staining

Peris staining in Arabidopsis germinating seeds were performed to detect iron (Fe³⁺)as described by Roschzttardtz et al. (2009). Seeds were imbibed with sterile deionized water or peptide solutions (100 pM IMA3BID and 100 pM IMA3BID. Random) for a cold stratification followed by incubation at 20°C under continuous light. Germinating embryos at stage II and IV were then manually isolated by removing envelopes (/.e. seed coat and endosperm) under a binocular microscope. For sampling embryos of mature dry seeds, seeds were soaked in water or in the peptide solutions for 2 h at 20°C under continuous light prior to the dissection, then embryos and envelopes were manually separated under a binocular microscope. The isolated mature/germinated embryos were vacuum-infiltrated at room temperature for 45 min with Peris stain solution (4% (v/v) HCI and 4% (w/v) K-ferro cyan ide) and images showing iron localization in the embryos were acquired using a stereo-microscope SZX16 with a DP71 digital camera (OLYMPUS).

Statistical analyses Tukey-Kramer tests were used to determine significant differences in multiple comparisons of seedling survival rate for assessment of DT.

Results

As shown on Figures 1-6, the present inventors herein show that it is possible to maintain the activation of desiccation tolerance mechanisms after germination by three different means:

(i) by delaying chromatin condensation with loss-of-fu notion mutants of elf, h2az and swn (figure 1),

(ii) by activating the iron accumulation pathway in seeds using plants over-expressing IRONMAN (35S::IMA) genes (figure 2) or the genes encoding the bHLH38 or bHLH39 proteins (figure 2), and

(iii) by applying an exogenous full-length IMA peptide (figure 4) or using the 17 amino acids synthetic peptide IMABID comprising only the conserved domain of the IMA proteins, cf. figure 5) on the mature seed.

It is noteworthy that the three ways means led to a re-induction of the desiccation tolerance during the germination (stage II) and young seedling establishment (stage IV) processes. This re-induction of DT at stages II and IV is visible by a higher viability of seeds following a post-germination desiccation treatment (see Material & Methods). It has also been observed that by modifying iron accumulation in seeds (by using mutant or over-expressed lines involved in iron homeostasis), DT was efficiently re-induced at stage II, which is reflected by a gain of more than 50 % seed viability (and up to 85% using the IMABID peptide) in comparison to wild-type seeds that showed about 10% viability. At stage IV, a gain in seed viability was also observed, but at lower level (up to 25% using the IMABID peptide in comparison to 0% in wild-type). However, at stage IV, it appeared that the swn loss-of-function was the most effective way to re-induce DT with about 50% of seed viability instead of 0% in wild-type.

The present results show that although germination has begun, the young seedlings, once treated with various agents that enhance iron accumulation or absorption in the seeds, are able to tolerate extreme loss of water by surviving the desiccation treatment and continuing germination afterward (stages II and IV, according to Maia et al, 2011).

As shown in Figure 1 , the chromatin condensation mutants (c/f, swn and h2az) display a clear increase in desiccation/drought tolerance at stages II and IV, potentially keeping the DT process (i.e., the core DT genes) in an open chromatin state allowing its re-activation after imbibition, whereas in the wild-type the core DT genes are located in a closed chromatin state. As shown in Figure 2, transformant lines over-expressing the bHLH38, bHLH39, IMA1, IMA2 and IMA3 genes and loss-of-function of btsbts gene (known to be involved in iron homeostasis) are able to reinduce DT at stages II and IV of germinating seeds, significantly increasing the seed viability after desiccation at stages II and IV in comparison to the wild-type (non-transformed lines).

The over-expressions of these genes were known to increase the iron content/absorption, so exogenous addition of iron was tested during wild-type seeds imbibition. As shown on Figure 3, adding different concentrations of soluble iron (Fe-EDTA) helped increasing the desiccation tolerance of the seeds at stages II and IV post-germination, but not as efficiently than in the different transgenic lines.

As shown in Figure 2, the over-expression of the full-length IRONMAN peptides were able to reinduce DT after germination. These IRONMAN peptides shared a conserved protein domain of about 17 amino acids called IMABID (see Grillet et al., 2018). Thus, it was decided to exogenously add two synthetic polypeptides :(i) the IMA3 full-length protein (Figure 4) and (ii) the IMA conserved domain of 17 amino acids (Figure 5) to the wild-type mature seed during imbibition. As shown in Figures 4 and 5 the exogenous addition of both the full-length and the conserved domain of 17 amino acids (IMABID) of the synthetic peptides mimicked the over-expression of IMA genes by sign ificatively enhancing the desiccation tolerance of stage II and stage IV post-germination in seeds in a dosedependent manner, whereas no reinduction of DT is observed using randomized peptide sequence (negative control). Interestingly, addition of the short peptide triggers a better effect on desiccation tolerance than the addition of the full-length polypeptide, especially at stage IV post-germination (Figure 4 versus Figure 5).

Finally, exogenous treatments were combined during imbibition of wild-type mature seeds by adding both soluble iron (FeEDTA) and the IMABID polypeptide. Adding soluble iron (Fe-EDTA) slightly increase the DT of germinating seed. However, adding soluble iron did not significantly enhance the desiccation tolerance of the seeds in the presence of the IMABID polypeptides of the invention (Figure 6).

As shown in Figure 7, the mutant h2a.z, swn or cld or over-expression lines IMAE-OE and IMA3BID allowing reinduction of DT during germination showed aa higher iron accumulation than wild-type mature seeds. And no reinduction of DT is observed using randomized peptide sequence IMA3BIDRandom (negative control).

As shown in Figure 8, using the most conserved generic sequence of IMABID derived from about 130 plant species is able to reinduce DT at stages II and IV of germinating seeds (but it is not as efficient as the specific Arabidopsis IMABID peptide). And no reinduction of DT is observed using randomized peptide sequence IMA3BIDRandom (negative control). As shown in Figure 9, adding generic or tomato specific IMABID synthetic peptide during germination allowed a reinduction of DT at stages 1 and 3mm of germinating seeds in tomato (but it is not as efficient as the specific Tomato SIIMA7BID peptide). And no reinduction of DT is observed using randomized peptide sequence IMA3BIDRandom (negative control).

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Claims

1 . Use of a compound that activates iron accumulation and / or iron absorption in a seed of a target plant, preferably in a seed embryo of a target plant, to increase the desiccation tolerance of said seed once germination is induced.

2. The use according to claim 1 , wherein said compound is an IRONMAN protein or a consensus peptide thereof or a functional variant thereof.

3. The use according to claim 1 , wherein said compound is a nucleic acid coding for a protein enhancing iron accumulation and I or iron absorption in plant seeds, said protein being preferably an IRONMAN protein such as IMA1 , IMA2 or IMA3 or a consensus peptide thereof, or the bHLH38 protein or the bHLH39 protein, or IMA7 protein in tomato.

4. The use according to claim 1 , wherein said compound is a nucleic acid reducing the expression of at least one protein negatively affecting iron accumulation and I or iron absorption in plant seeds, said protein preferably inducing the chromatin condensation in genes involved in iron accumulation and I or iron absorption, said protein being more preferably chosen among: SWN, H2A and CLF protein; or said protein being involved in the negative regulation of the iron accumulation such as BRUTUS (BTS) protein or BRUTUS-LIKE protein.

5. The use according to any of claim 1 , 2 or 4, wherein said compound is administered on mature dry seed before or when germination is induced, preferably along with a solution or powder containing iron.

6. A method for increasing the desiccation tolerance of germinating seeds of a target plant or for generating desiccation tolerant dry seeds of a target plant, said method comprising a step of:

I) contacting mature dry seeds of said target plant, before or during imbibition, with a composition comprising a compound that activates iron accumulation I absorption in said mature seed, or a step of :

II) contacting said target plant with a composition comprising a compound that activates iron accumulation / absorption in mature seeds of said target plant.

7. The method of claim 6, wherein the composition of step I) contains an IRONMAN protein or a consensus peptide thereof or a functional variant thereof.

8. The method of claim 6 or 7, comprising : a) Providing non-germinated dry mature seeds of the plant of interest, b) Coating an IRONMAN protein or a consensus peptide thereof on said seeds, c) Optionally, contacting said coated seeds with a solution or powder containing iron, and/or d) Optionally, coating said seeds with other performance enhancing compounds.

9. The use of claims 2-3 and the method of claims 7 or 8, wherein the IRONMAN protein is the protein IMA1 of SEQ ID NO:1 , IMA2 of SEQ ID NO:2 or lMA3 of SEQ ID NO:3 or a functional variant thereof, and wherein the consensus peptide thereof is a peptide having the sequence SEQ ID NO:4 or SEQ ID NO:11 or a functional variant thereof, or a generic IRONMAN peptide GenIMABID of SEQ ID NO: 29, or an IRONMAN protein SIIMA7 of SEQ ID NO: 38 or a synthetic peptide SIIMA7BID of SEQ ID NO:31 , optionally in mixture with one or several IRONMAN proteins not expressed in seeds.

10. The method of claim 6, wherein the composition of step II) contains a nucleic acid coding for an IRONMAN protein IMA1 of SEQ ID NO:1 , IMA2 of SEQ ID NO:2 or IMA3 of SEQ ID NO: 3 or a consensus peptide thereof or a functional variant thereof, or a generic IRONMAN peptide GenIMABID of SEQ ID NO: 29, or an IRONMAN protein SIIMA7 of SEQ ID NO: 38 or a synthetic peptide SIIMA7BID of SEQ ID NO:31 optionally in mixture with one or several IRONMAN proteins not expressed in seeds.

11. The method of claim 10, comprising: a) Introducing by genetic transformation in said target plant the nucleic acid compound, so as to produce a transgenic plant, preferably overexpressing the IRONMAN protein IMA1 of SEQ ID NO:1 , IMA2 of SEQ ID NO:2 or IMA3 of SEQ ID NO: 3, a consensus peptide thereof or a functional variant thereof, or the protein SIIMA7 of SEQ ID NO: 38, b) Collecting the seeds of said plant, then drying them, c) Optionally, contacting said coated seeds with a solution or powder containing iron, and/or d) Optionally, coating said seeds with other performance enhancing compounds.

12. A dry desiccation-tolerant mature seed which is genetically modified by genetic transformation to overexpress a protein enhancing iron accumulation and / or iron absorption, said protein being the bHLH38 protein or the bHLH39 protein or a functional variant thereof.

13. A dry desiccation-tolerant mature seed which is coated with an IRONMAN protein or with a functional variant thereof or with a consensus peptide thereof.

14. The dry desiccation-tolerant mature seed of claim 13, wherein it is coated with the IRONMAN protein IMA1 of SEQ ID NO:1 , IMA2 of SEQ ID NO:2 or IMA3 of SEQ ID NO:3 or with a functional variant thereof, or with a consensus peptide thereof having the SEQ ID NO:4 or SEQ ID NO:11 or with a functional variant thereof, preferably containing the SEQ ID NO:26 and/or SEQ ID NO:27, or a generic IRONMAN peptide GenIMABID of SEQ ID NO: 29, or an IRONMAN protein SIIMA7 of SEQ ID NO: 38 or a synthetic peptide SIIMA7BID of SEQ ID NO:31 , optionally in mixture with one or several IRONMAN proteins not expressed in seeds.

15. A transgenic plant producing dry desiccation-tolerant mature seeds, said plant overexpressing a protein enhancing iron accumulation and I or iron absorption, said protein being the bHLH38 protein or the bHLH39 protein or a functional variant thereof.