MXPA00006668A - Method for detecting growth and stress in plants and for monitoring textile fiber quality - Google Patents

Abstract

A method of detecting environmental stress in plants, particular water stress in cotton plants is based on a hot dilute acid extraction of plant tissues such as cotton fibers. The extracts are analyzed by high pH anion exchange chromatography to separate and characterize the carbohydrates. This method extracts a characteristic series of carbohydrate multimers containing galactose, mannose and glucose. The pattern of multimers is indicative of growth stress during the formation of the plant tissue. In addition, similar multimers can be extracted from textiles and are indicative of textile wear and can be used to determine which manufacturing treatment will improve fabric life. In addition the multimers are shown to contain a protein component. Chemical agents that cross-link the protein component alter the extractability of the multimers. Thus, cross-linking of this type can be used to alter favorably the resistance of fabric to washing induced wear. Finally, a sequential enzymatic extraction method for producing high purity cellulose is disclosed.

Description

METHOD FOR DETECTING GROWTH AND TENSION IN PLANTS AND TO MONITOR THE QUALITY OF TEXTILE FIBER BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a method for detecting environmental stress in terrestrial green plants, particularly in agricultural crops, so that production can be optimized by relieving stress before permanent damage to the plants occurs. In particular, this application describes biochemical methods for assessing the quality of cotton fibers.

DESCRIPTION OF THE RELATED ART In the background document of this application the present inventor described his surprising discovery that it was possible to extract a carbohydrate-containing fraction from properly prepared plant material by a simple process with cold water. Essentially, the plant tissue is prepared by rapid freezing (preferably by the use of liquid nitrogen or solid carbon dioxide) and is then lyophilized and stored at temperatures below freezing. As described in the basic application cited above, fractions of the cell wall containing carbohydrate can easily be extracted from lyophilized tissue by cold aqueous extraction; then, the greatly improved techniques of high pressure liquid chromatography (CLAP) allow the resolution of the aqueous extract in mono and poly saccharide constituents, # c which can be subsequently hydrolyzed to identify the constituent monosaccharides. The use of high pH anion exchange chromatography with pulsed amperometric detection (HPACS-PAD) makes it possible to unambiguously identify the constituents of the cell wall. In the HPAEC a salt gradient (such as '.0 a sodium acetate gradient) is applied to a column of special ion exchange resin held at a high pH to sequentially elute various mono and polysaccharides. Essentially, the hydroxyl groups of the sugars act as extremely weak acids that become deprotonated at high pH, joining the ion exchange matrix until they are eluted by the gradient. While there are a number of vendors of HPAEC materials, the current invention has employed products and systems produced by Dionex Corporation of Sunnyvale, California. These products and systems are fully explained in the • technical notes of Dionex, particularly in the technical notes of 20 and 21. The fractions of carbohydrates isolated before the cell walls of the plant were analyzed using Dionex columns.

CarboPac PA1 and PA-100. Both of these columns contain cross-linked polystyrene / divinylbenzene latex microbeads (350 nm diameter) with quaternary amine functional groups. The columns were put into operation under the pressure conditions recommended by the manufacturer (281 kg / cm2, 4000 psi maximum = 4.75 x 107 Pa) in sodium hydroxide eluted with an elution gradient of sodium acetate. When necessary, the sugar alcohols were analyzed using a CarboPac MAl column containing porous beads (8.5 microns in diameter) of chloride vinylbenzene / divinylbenzene with alkyl quaternary ammonium functional groups. The polysaccharides analyzed in the present invention are appropriately referred to as "glucoconjugates" because they comprise a monosaccharide conjugated to at least one additional monosaccharide (ie, to form an oligo or polysaccharide) and optionally a protein or a lipid. As will be discussed below at least some of the glycoconjugates comprise polysaccharides conjugated to a portion of protein. To summarize, glucoconjugates can are polysaccharides, polysaccharides containing a portion of protein, polysaccharides containing a portion of lipid and / or some combination thereof. In the present application only polysaccharides and polysaccharides containing a portion of protein have been identified in a non-proprietary manner. ambiguous. In any case HPAEC-PAD characterizes a glucoconjugate based on the carbohydrate component of the glycoconjugate. In the original application (see O-A-97/06668) two # t groups of polysaccharides were specially designated and described by their position in the HPAEC separations; These groups were identified as GC-1 and GC-2. In the present the composition of these groups is further elucidated and other important polysaccharides (glucoconjugates) are discussed. 10 BRIEF DESCRIPTION OF THE DRAWINGS The objects and features of the present invention, which are believed to be novel, are set forth in the claims with particularity attached. The present invention, insofar as both its organization and the manner of its operation, together with additional objects and advantages, can be better understood by reference to the following description, taken in connection with the accompanying drawings. Figure 1 shows a HPAEC-PAD chromatogram of a cold aqueous extract of cotton fibers according to the present invention indicating the position and identity of a number of carbohydrates; Figure 2 shows a typical chromatogram of HPAEC-PAD from a cold aqueous extract to show the position of the carbohydrates of GC-1 and GC-2; Figure 3 shows an alkaline degradation experiment of multimers extracted from plant tissues of # c according to the present invention; Figure 4 shows the effect of incubating the cold aqueous extract with fibers that grow normally where the insoluble enzymes cause changes in the carbohydrate profile (arrows); the control uses boiled fibers to inactivate the enzymes; Figure 5 shows the effect of incubating the cold aqueous extract with fibers stressed by drought (without irrigation) where the insoluble enzymes cause changes in the carbohydrate profile (arrows); the control uses boiled fibers to inactivate the enzymes; Figure 6 shows cold aqueous extracts of fibers taken at different times of the day (early [A], midday [B] and late [C] to illustrate that the carbohydrate pattern varies predictably with the time of day; Figure 7 shows the effect on the carbohydrate profile of incubating cotton fibers that grew normally (harvested according to the method of the invention) with a number of different aggregated substrates: A) boiled control to inactivate the enzymes, B) inositol aggregate; C) glycerol added; D) added sucrose; and E) inositos, glycerol, sucrose and arabinose aggregates; Figure 8 shows the effect on the carbohydrate profile of incubating cotton fibers stressed by # 1 drought (not irrigated) (harvested according to the method of the invention) with a number of different substrates added: A) boiled control to inactivate the enzymes; B) added inositol; C) added glycerol; D) added sucrose, and E) inositol, glycerol, sucrose and arabinose aggregates; Figure 9 shows carbohydrate multimers extracted by HCl according to the present invention from cotton fibers in the range from 15 to 39 days post-anthesis; these multimeros are made of cotton capsules of a normally cultivated plant and exhibit an extremely regular periodic pattern; Figure 10 shows carbohydrate multimers extracted by HCl according to the present invention from cotton fibers in the range from 12 to 36 days post anthesis; these multimeros are of cotton capsules of a stunted plant that grows in a portion of the field that receives a sub-optimal irrigation, and exhibits an irregular pattern particularly -between 15 and 20 minutes of retention; Figure 11 compares multimers extracted from a normal cotton fiber with multimers extracted from portions of beet root to show that some of these Carbohydrates are found in the cell walls of widely divergent plants; Figure 12 shows multimers extracted from fibers #t normal (p) and abnormal "white spots" (); Figure 13 shows an enlarged view of the multimer profile of Figure 12 showing that the white spot fibers (w) have the increased arabinose (Ara) on the normal fibers (p); Figure 14 shows the multimers extracted from normal fibers after incubation with a number of different combinations of substrates (identified in Table 1); Figure 15 shows the multimers extracted from a cotton towel not dyed (A) and dyed (B), in each case the upper trace is an extract of the new towel and the trace of the lower part is an extract after a laundry service; Figure 16 shows multimers extracted from a pillowcase (top) and a towel (part lower old, with many laundry services; Figure 17 shows a flow chart for an experiment with a proteolytic enzyme with cotton fibers of early harvest, mid-day and late harvest; Figure 18 shows the extracted multimers 25 using the scheme of Figure 17 for early fibers (A) of half day (B) and late (C); In addition to the carbohydrate multimers, the protein is also shown (A28o); Figure 19 shows the three multimer extracts of Figure 18 treated with protease; trypsin (T), chymotrypsin (CT) or 0.1 N HCl to generate multimers (HCl). Figure 20 shows the effect of boiling the cotton fibers before extracting the adhesive matrix. Figure 21a shows the boiled versus non-boiled extractions as in Figure 20 performed on fibers harvested at noon. Figure 21b shows the boiled versus unboiled extractions as in Figure 20 performed on fibers harvested in the morning; Figure 22 shows the results of the acid hydrolysis of the matrix of the adhesive extracted in Figure 20. Figure 23 shows the composition of the adhesive matrix extracted either passed (filtered) or retained (retained material) by several cutting filters of molecular weight; from the top to the bottom: material retained in the 30 kilodalton filter; material retained in the 10 kilodalton filter; material retained in the 4 kilodalton filter; and filtering the 4 kilodalton filter.

Figure 24 shows the composition of extracts of 8 DPA fibers collected in three times (morning, midday or night) and extracted in one of three temperatures: 37 ° C, 25 ° C t t or 4 ° C. Figure 25 compares the composition of fiber carbohydrates are "white spots" associated with the same fibers. Figure 26 shows the effects of incubating various substrates (CB = cellobiose; Raf = raffinose) with fibers during the extraction process and the effect of tunicamycin (T) added in the process. Figure 27 shows the effect of cellulase or ß-glucosidase on isolated multimers; the control is isolated multimeros without enzymatic treatment. Figure 28 shows the carbohydrates extracted from the first incubation of fibers treated with protease first (chymotricin) or cellulase first; PMSF = phenylmethylsulfonyl fluoride, a serine protease inhibitor. Figure 29 shows the carbohydrates released to from the second incubation of the fibers; the cellulase fibers had a first incubation with chymotrypsin and the chemotropin fibers had a first incubation with cellulase. Figure 30 shows the multimers extracted from the fibers following the two extractions of figures 28 and 29; CT = chymotrypsin, PMSF = phenylsulfonyl fluoride. Figure 31 shows the carbohydrates released by the cellulase alone (1) or following the crosslinking with either 125 mM (2) or 250 mM carbodiimide (3). Figure 32 shows carbohydrates released by chymotrypsin alone (1) or following cross-linking with either 125 mM (2) or 250 mM carbodiimide (3). Figure 33 shows the carbohydrates released by a cellulase treatment (following a first treatment with chymotrypsin) alone (1) or following the crosslinking with either 125 mM (2) or 250 mM carbodiimide (3). Figure 34 shows carbohydrates released by treatment with chymotrypsin (following a first treatment with cellulase) alone (1) or following cross-linking with either 125 mM (2) or 250 mM carbodiimide (3). Figure 35 shows the absorbance at 280 nm of the carbohydrates released by chymotrypsin, indicating the presence of a protein or glycoprotein. Figure 36 shows the products of hydrolysis of the white particle (presumably cellulose) left following the enzymatic digestions of figures 31-34.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The following description is provided to enable any person skilled in the art. making and using the invention and exposing the best modes contemplated by the inventor to carry out his invention. Several modifications, however, will be easily > C evident to those skilled in the art, since the general principles of the present invention have been defined herein specifically to provide methods for determining the growth of plants and the tension and quality of plant materials, especially cotton fibers, through the analysis of certain polysaccharide fractions. As already discussed, glucoconjugates are carbohydrates bound together. covalently to other carbohydrates, proteins or lipids. The glycoconjugates monitored in the present study appear function as precursors of the cell wall or intermediaries in the biosynthetic processes that produce the cell wall. Cotton fibers are unique as plant cells in that their primary function is the synthesis of material for the cell wall. A progression of appearance and The disappearance of specific glycoconjugates has been observed when developing cotton fibers under "normal" conditions. Developing cotton fibers obtained from plants subjected to various forms of stress, which negatively impacted the development of fiber, demonstrate a pattern abnormal or altered appearance and disappearance of monitored glycoconjugates. The analysis of glucoconjugates is a sensitive method of monitoring the synthesis of the cell wall, which is directly - coupled with cell growth. This analysis is applicable to roots, stems, leaves and fruits. In the present case, the analysis has been applied to a fruit. The presence of these glucoconjugates has been demonstrated in a range of different plants, which leads to the conclusion that they will be found in virtually all cells "" 0 vegetables. In addition to monitoring the growth of the fibers and their development, the analysis of the glucoconjugates will demonstrate the presence of trehalulosa or melisitosa, oligosaccharides present in the ligamasa of the whitefly, if present. Thus, the method is also useful for monitor insect pests.

SACAROSYL OLIGOSACCHARIDES (GLUCOCONJUGATES OF GC-1) The GC-1 series of glucoconjugates described in detail in the basic application is shown here that is composed of molecules in the raffinose oligosaccharide series, also known as the secarosyl oligosaccharides. Raffinose is a non-reducing trisaccharide consisting of D-galactose, D-glucose and D-fructose and galactose and glucose are linked by a bond glycosylic α-1,6, and the fructose bound to glucose with a glucosilic bond a, ß-l, 2. That is, raffinose comprises a unit of galactose attached to sucrose (more fructose glucose). It is believed that raffinose is synthesized by transferring a galactose unit of galactinol to sucrose. Galactinol is produced by transferring a galactose unit of UDP-galactose to myo-inositol. Successive members of the raffinose series (stachyose, verbascose, and higher homologs) are produced by gradual addition of galactose units. Thus, stachyose has two units of galactose attached to sucrose and verbascose has three, etc. At each stage a galactenol molecule provides a galactose unit and a myo-inositol free molecule. Related sugars include melibiose (galactose plus glucose) and maninotriose (galactose plus galactose plus glucose). Until now, most of the research on saccharosyl polysaccharides has focused on their synthesis and the apparent role as a storage product in seeds. However, it is likely that these oligosaccharides serve as glycosyl donors for the synthesis of polysaccharides in cell walls (eg, cotton fibers). There is a large body of literature that describes invertases of the cell wall in a wide variety of plants. However, no obvious function has been proposed for cell wall invertases different from that for cells in suspension culture. A report of the invertases in developing cotton fibers (Buchala, A.J., 1987, ß-fructofuranoside m - '(invertase) acid in developing cotton fibers (Gossypium arboreum L.) and its relation to the synthesis of ß-glucan from sucrose fed to the apoplast of the fiber. J. Plant Physiol. 127: 219-230) compared the activity of cell wall invertases on sucrose, raffinose and stachyose. As might be expected from considerations kinetics, activity decreased with increasing molecular weight. It seems likely that the cell wall invertases (insoluble) are hydrolyzing fructose from the compounds of the raffinose series and transferring these sugars to other carbohydrates that comprise part of the structure of the complex cell wall. In an experiment it was possible to verify that insoluble invertases convert verbascose (galactose, galactose, galactose, sucrose) to verbascotetraose (galactose, galactose, galactose, glucose). Presumably the hydrolyzed fructose is added to some other carbohydrate. The first compounds of GC-1 (14) in Figure 2 were defined as a group of carbohydrates that run in about 15 minutes in the standard separations described in the standard separations described in FIG. basic request. The figure shows a portion of a chromatogram similar to that of figure 2, except that the various carbohydrate maxima have been identified through hydrolysis experiments and through running • c known standards. Significantly, the GC-1 compounds are identified as raffinose, stachyose and verbascose. Preliminary results indicate that samples with additional GC-1 maxima (eg, Figure 2) have additional higher homologs from the raffinose series. The significant point is that "the carbohydrates from the raffinose series "is a more accurate term that can be substituted for carbohydrates from GC-1 in the basic application methods .. From the quantitative interrelationships shown in the basic application it is believed that the carbohydrates from GC-1 are precursors to carbohydrates of GC-2. However, while the carbohydrates from the raffinose series are non-reducing, current experiments have shown that GC-2 carbohydrates are reducing. Thus, GC-1 compounds are preferably not directly converted to GC-2 compounds. Rather, carbohydrates are probably transferred (perhaps by the agency of an invertase of the cell wall) to other carbohydrate molecules to form GC-2 compounds. An extremely exciting and unexpected discovery of the present study is that synthetic enzymes necessary to do the interconversions from GC-1 to GC-2 (and presumably other stages of cell wall synthesis) are preserved by lyophilization. If the aqueous extract is cold, it is allowed to react with the fibers (which contain # c non-soluble enzymes), the carbohydrate profile of the extract changes over time as simple sugars are used and more complex carbohydrates appear in place. Figure 4 shows the results of incubating a cold aqueous extract of cotton normally grown with the fibers from which the extract was made. Figure 4a shows the control (ie the aqueous extract) while Figure 4b shows the results of the incubation in which the aqueous extract was added back to the fibers and incubated for 1 hour at 37 ° C. Note the disappearance of the carbohydrate at the point of 10 minutes and the appearance of several new fifteen . carbohydrates Those in about 15 minutes are composed of GC-1 and those of about 20 minutes are composed of GC-2. If the aqueous extract is boiled before being added back to the fibers, the results do not change. If the fibers are boiled before incubation, there is no change in carbohydrates during incubation. This clearly shows that the reaction is driven by insoluble enzymes in the fibers. If the fibers are dried and weighed following the incubation, a significant increase in weight is detected. This proves that the insoluble carbohydrates are added to the fibers (that is, to cell walls); The changes seen in soluble carbohydrates are probably simply incidental to the additions of the cell wall. Figure 5 shows a similar experiment carried out using fibers from non-irrigated plants that were suffering from drought stress. The parent application demonstrates that the GC-1 and GC-2 compounds are sensitive stress indicators. Here we see that these differences are also demonstrated by the in vitro incubation experiments. 10 One may wonder whether differences in drought stress are due to changes in insoluble enzymes, changes in the availability of carbohydrate precursors, or both. It is well known that carbohydrates are involved in the response to the drought of plants stressed. For example, glycerol or trehalose can accumulate in response to drought and act as protective agents to maintain the integrity of cells and membranes. Clearly if carbohydrates are diverted to produce protective materials, the carbohydrate set available for the synthesis of the cell wall can be altered. The present study has also shown that carbohydrate patterns fluctuate during the day when the speed of photosynthesis increases (early to midday) and then decreases (half a day to late). This is shown in Figure 6, showing the aqueous extracts taken from fibers harvested at three different times of day (early, within 1 hour of sunrise, half day, within 1 hour of noon, and late, within 1 hour of nightfall). Of course, the activities of several enzymes of the cell wall can also vary during the day, further complicating the picture by means of this. The likelihood that stress-induced changes in cell wall carbohydrates are due to shifts in the carbohydrate pool rather than to changes in wall enzymes has also been demonstrated by combining aqueous extracts of normal fibers with plant fibers. stressed by drought and vice versa. In any case the resulting profiles are greatly controlled by the source of the extract. That is, the extracts of cultured fibers normally give essentially normal profiles when they are incubated with fibers from either normal or stressed plants. Similarly, extracts from stressed plants give abnormal profiles when they are incubated with fibers from either normal or stressed plants. However, adding known substrates to normal or stressed fibers gives some indication that the fibers also control the final soluble carbohydrate profiles. Figure 7 shows a series of supernatants incubated with fibers from irrigated plants. In these experiments a known concentration of substrate (25 mM inositol, 25 mM glycerol, 25 mM sucrose and / or 25 mM arabinose) was incubated with a known fiber weight. He * ~ control contains the normal fiber extract but boiled 5 to inactivate the enzymes; in the other cases normal soluble carbohydrates were supplemented with the indicated substrates. Note that different substrates produce different profiles - indicating that changes in the overall size greatly affect the profile of "Or carbohydrates last, for example, the addition of inositol greatly enhances the consumption of sucrose (a large maximum close to 10 minutes in the control.) The addition of glycerol, inositol and arabinose to sucrose results in an increased consumption of sugar. sucrose, Figure 8 shows the same experiment performed with fibers of a plant stressed by drought. While it is similar to Figure 7, there are clearly differences. The total level of Soluble carbohydrates are lower, but sucrose is clearly consumed. Part of the difference may be that the stressed fibers have a different ratio of enzymes to dry weight than the normal fibers and these experiments were normalized by weight of added fiber. If anything, fibers stressed by drought seem to metabolize almost all carbohydrates added to insoluble compounds. While this does not prevent there being a Different relationship in the various enzymes, certainly indicates the presence of active enzymes in the stressed material. l i MULTIMERS THAT CAN BE EXTRACTED BY ACID Perhaps the most exciting and unexpected discovery of the present investigation was that following the aqueous extraction it is possible to extract a boiling multimer fraction for 30 minutes in diluted 0.1 M HCl. In the past, several acid and basic extraction schemes have been used to separate and quantitate polysaccharide fractions from the plant cell wall (see, for example, SU-A-1 744 648). However, past extraction schemes have not revealed the polysaccharide patterns seen with the present invention. Figure 9 shows this unique pattern of multimers extracted from cotton buds from 15 to 39 days after anthesis. Older capsules have exactly the same multimer, but less material is extracted on a dry weight basis in mg. Presumably these multimeros represent some component that connects the paracrystalline cellulose in the wall. Like the GC-2 compounds, the multimers are reducing sugars that indicate an atypical glycan binding in the polymers (see Figure 3 which shows multimer profiles before and after the alkaline degradation by boiling in 0.1 N NaOH per 10 minutes to destroy reducing sugars, thus indicating that the binding between the multimers and the peptide is probably not a glycosyl link through C-1). The < Hydrolysis of individual maxima has been shown to contain galactose, glucose and mannose. In classical plant cell wall research, diluted mineral acids are sometimes used to extract peptinas "peptic materials" which, by definition, contain residues of galacturonic acid. Clearly, the multimers are not pectins or pectic materials. In addition, it is necessary first to carry out the cold accusation extraction so that the multimeros are not obscured by the GC-1 and GC-2 compounds. Figure 10 shows the HCl multimers extracted from fibers in a stress-stressed plant. Clearly the multimer pattern is interrupted particularly in about 15-10 minutes of retention time. The interruption of the multimer patterns is a very sensitive voltage detector, and obviates the need for quantitative comparisons since it is frequently required when doing the detection of stress based on GC-1 versus GC-2. Further analysis of the multimers of normal fibers has revealed that the main difference between successive multimers is in the addition of glucose units. That is, successive multimers in a series have quantities comparable galactose and mannosa but different amounts of glucose. It is not yet known if abnormal multimers of plants stressed by drought follow this pattern. It seems true that many of these same multimers are found / «In a variety of cell walls. Figure 11 shows that HCl extracts from beet root tissue contain a series of multimers where several of the compounds exactly overlap some of the cotton multimers. The extraction of multimeros is ideally suitable for evaluating samples of cotton fibers to determine a number of defects that plague the textile industry. The specks are short and immature fibers that diminish the quality of the cotton. Although its presence can be evaluated by microscopic inspection of the fibers, also give a unique carbohydrate pattern that allows the determination of speck contamination. Even more important is the presence of fibers with "white spots" which are abnormal fibers that do not normally pick up the dye. Although this defect can be evaluated by dyeing By inspecting the fibers, the analysis of HCl multimers provides an easy way to evaluate the presence of fibers with white spots. As shown in Figure 12 and Figure 13 the individual white spot (H) HCl extracts show a ratio of arabinose to glucose significantly greater than the extracts (p) of fibers normal. Interestingly, aqueous extracts of plant fibers extremely stressed by drought show some of the multimers. Preliminary experiments have indicated that these multimers are similar but identical to those released by treatment with dilute HCl. The real question is because they are released by a simple aqueous treatment. One can make the theory that multimers are part of a hemicellulosic "adhesive" that holds the cellulose microfibrils of the precellular together. Under drought stress conditions, the shortage of carbohydrates and / or enzymatic defects prevent the proper assembly of the cell wall components. In such a case the "adhesive" does not adhere properly and is very easily washed off the walls. As will be demonstrated below, there are proteins associated with at least some of the multimers (which produce a special glycoconjugate). These proteins may very well be responsible for producing some of the bonds that keep multimers in the wall. Certainly, gentle extraction with HCl would be adequate to partially denature the proteins and deny their intended binding. Figure 14 shows the multimers extracted from fibers incubated with the substrates shown in Table 1. The number associated with the particular trace is related to the aggregated substrates. Point important is that the addition of certain combinations of substrates (notice traces 2 and 8, for example) seem to reduce the extraction of multimer. The control (not adding exogenous substrates) indicates the normal extraction capacity of the multimers. Presumably certain combinations of substrates produce a more closely crosslinked product so that very few multimers can be easily extracted. Table 1 Another surprising discovery is that multimers can be extracted from cotton fabric finished as well as fibers carefully harvested as shown above. Figure 15 shows multimers extracted for 30 minutes of boiling in 0.1 M HCl from an undyed cotton towel (whitish or ivory) and a cotton towel dyed (green) in each case the upper trace represents the extraction of a towel new and the second trace (bottom) shows the removal of a towel that has been washed once. Attempts were made to standardize the amount of tissue removed. Note that the extracted multimers seem to be very similar to those extracted from specially prepared fibers. In this case the processing of the fabric has eliminated all the GC-1 and GC-2 compounds so that an aqueous previous extraction is not necessary - there is no danger that the GC compounds may obscure the multimers. The differences in quality and quantity of the extracted multimers are due either to differences in the starting cotton or the processing of the textile between the two different tissues. The experiments with treated cotton "permanent ironing" indicate that such treatments significantly alter the quantity and quality of the extracted multimers. Another important discovery is that cotton fabrics are capable of providing multimers even after prolonged wear and tear. Figure 16 shows multimers taken from an old towel and from an old pillowcase in the house. the inventors. These tissues They had been washed dozens of times and still produced similar multimers. Clearly, multimer analysis can be used to measure changes related to wear in cotton fabrics and to analyze various tissue treatments to determine their long-term effects on tissue wear. Any treatment that inhibits the release of multimeros will probably extend the useful life of the tissue. Although a dilute acid wash is the preferred way to extract multimers for analysis it has been found that prolonged aqueous extraction (several days) at elevated temperatures also releases the multimers. Presumably prolonged exposure to hot water gradually hydrates the paracrystalline portions of the cell wall and allows the multimers to be released. This strongly suggests that these materials are gradually released during washing; undoubtedly the loss of these "adhesive" elements results in a weakened tissue. Traditionally it has been believed that weakened tissues with age was merely a mechanical effect of wear and washings. These findings suggest that the washes effectively remove a vital binding component of the cotton. Treatments that slow this removal will extend the life of the cotton fabric. Another practical use of multimer extraction is the determination of the types of cotton used in a given fabric. The extraction of a range of different varieties of cotton has shown reproducible differences of multimer among some varieties. In particular, certain high-grade cottons are derived from different cotton species. It can be very beneficial to have a simple test to detect the adulteration of these premium quality cottons with less expensive "ordinary" cottons.

PROTEIN ADHESIVE AND MULTIMERS Figure 18 shows the flow diagram of a proposed experiment to determine what part, if any, the protein plays in the cell wall phenomenon discussed above. The fibers were hydrated and then "boiled to denature any enzyme and kill any microorganism (also toluene was added) to further ensure sterility." The fibers were then incubated at 37 ° C for 72 hours either with or without proteolytic enzyme (pronase). 1 mg / ml) At the end of this time the fibers were separated from the supernatant by centrifugation.The supernatant was then passed through a 0.22 μm pore filter to remove any particulate material (this is a standard procedure to protect the columns chromatographies Surprisingly, supernatants that were not treated with pronase capped the filters and they remained on the surface of the filters as a viscous material (retained material). The amount of this material depended strongly on the time of the day when the fiber sources were isolated. The early fibers (7 AM) showed a maximum amount of this material; those of the half-day fibers showed an intermediate amount; while those of the late fibers (7 PM) showed a minimum amount. It is believed that this viscous retained material represents the "adhesive" that holds cellulose in the cell wall. Obviously, the speeds of the synthesis of the cell wall vary with the time of day, the speed of synthesis can affect the extraction capacity of the adhesive material. If the filtrate (primarily from the samples treated with pronase) is treated with HCl, a typical multimer pattern is generated. Significantly, if the retained material is treated with HCl, or with proteolytic enzyme, multimers are generated. This indicates that long-term aqueous extraction removes a component of the cell wall that includes the multimers. This material is macromolecular and forms a viscous gel. If the material is treated with a proteolytic enzyme, the gel is destroyed and the multimers become soluble. The fact is that this gel is kept together with proteolytic enzyme sensitive bonds strongly suggest that proteins are important to adhere the cell wall together.

Figure 19 shows the multimers produced from the retained material treated with HCl from the early fiber (upper graph), half day (medium) and late v * (lower part). Each graph shows carbohydrates and proteins (A28o) • Note that certain of the multimeros are clearly associated with proteins. In addition, the precise nature of the proteins changes with the time of day. The early and late graphs show a maximum protein triplet between 20 and 25 minutes while the graph of the half day shows a maximum of prominent protein in about 35 minutes. As shown in Figure 20, the treatment of the samples with either high purity trypsin or high purity chymotrypsin removes the protein components and causes the maximum protein / carbohydrate together disappear or change in shape or position. The boiling of the cotton fibers before the removal of the adhesive resulted in a significant difference in the multimer pattern that was obtained. With fibers collected in the morning (7 AM) boiling resulted in the absence of 2 or 3 peaks or maxima as shown in Figure 20. However, with capsule fibers collected at noon, boiling resulted in the omission of almost all but the first multimer in the series, which was relatively more abundant on a fiber basis by milligram. This is shown in Figure 21. Fibers collected at night (7 PM) produced a somewhat intermediate multimer pattern but more like the pattern of the • C half day fiber. These results are consistent with the fact that most cell wall synthesis occurs at night. The fibers of the morning and the remaining material of the synthesis of the wall, the fibers of the middle day represent the lowest point of the synthesis of the wall and the fibers of the night are representative of the beginning of the Synthetic process of the wall. The chromatograms of the multimers are characteristic of having a slight tail edge of the maxima that eluted earlier. This is suggestive of an incomplete resolution of the maxima. This was investigated doing a time course of the hydrolysis of the adhesive as shown in Figure 22. The time point of 5 minutes shows that these early maxima consist of two small maximums which then, with longer hydrolysis time result in a maximum big with a tail edge. This indicates that there are two maxima eluting very close to each other, for one is much more abundant than the other. This apparently incomplete resolution of the maxima was also investigated by holding the 30-minute hydrolyzate to filtration in filter cuttings from molecular weight (MWCO). This result is shown in the figure 23 in which 30,000, 10,000 and 4,000 MW filters were used. The main portions of the multimeros were obtained in the retained material of 10,000 MW or in the filtrate of • C 4,000 MW. Cotton fibers (25 DPA) were extracted with water at three temperatures, 37, 25 and 4 ° C for up to 30 days. The extraction tubes were subjected to sonication for 15 minutes, and the filtrates were removed every day and subjected to centrifugation to provide white pellets in 0 particles. It was observed that between days 3 and 9 of extraction the fibers extracted at 4 ° C were characterized by an obvious difference. Before the sonication all the tubes seemed similar. But following sonication the No. 4 tubes became cloudy, indicative of a suspension very fine particulate. Still in the centrifugation, the pellets obtained from all the tubes were similar in the amount of precipitate and in the multimer pattern obtained (figure 24). This result is indicative of a temperature-dependent process and thus presumably enzyme that produces larger particles at higher temperatures.

WHITE HOJUELAS During the development of cotton fibers 25 there are some structures. presents that look like flakes or white flakes in the dry material. It is presumed that this material results in much of the drying of the liquid within the development carpel; however, the scales may also be apparent in recently opened capsules. The scales are obvious up to at least 39 DPA in many cases, but disappear in the later stages of development, and are eliminated by the time the capsules open at maturity. Although many researchers have mentioned white scales informally, there does not appear to be any investigation of them in the literature. I have bisected the white scales of the fibers and observed them independently to determine the soluble oligosaccharides. I have done the diluted acid extraction to obtain the multimeros. On a dry weight basis, the white flakes release at least 5-10 times the amount of multimeros that the fibers. This is shown in Figure 25. Since these white flakes contain multimers that eventually end up in the fibers, the obvious conclusion is that the white flakes contain precursors for the fiber wall. Therefore, all the material of the fiber wall in development does not originate within that particular fiber. At this time I do not know if the white scales originate from a particular population of fibers, other cells in the lining of the developing seed or other tissues of the carpel wall inside.

EXPERIMENT WITH TUNICAMICINE • c The effect of tunicamycin on the multimers obtained from cotton fibers was investigated. The fibers were incubated with water for two days to reduce the endogenous substrates and then incubated for another 24 hours with and without tunicamycin (10 micrograms / milliliters) both with and without added substrates. Tunicamycin specifically inhibits the formation of the bond between asparagine and N-acetylglucosamine in N-linked glycoproteins. Without added substrates, tunicamycin had no appreciable effect but with added substrates such as cellobiose and raffinose, the effect was dramatic since tunicamycin inhibited the The amount and pattern of the extracted multimers as shown in Figure 26. In other experiments, not shown here, the effect of tunicamycin was variable if the endogenous substrates were not reduced before the addition of tunicamycin. 20 TREATMENTS WITH ENZYMES The extracted multimers were subjected to incubation with a cellulase (Trichoderma reesei) or a β-glucosidase (almond emulsion) .The effect of the β-glucosidase appeared increase the height of the maximums of the multimer significantly and generate an additional small maximum with a retention time slightly longer than 20 minutes. Presumably this is the result of eliminating terminal glucose units which results in a compound with an increased response to the detector. The cellulase gave a very different result since it resulted in the almost elimination of many maxima and a large reduction in many heights of the maxima with a large increase in the height of the maximums of the first maximum in the multimer series, as shown in Figure 27. The result of the cellulase, with the exception of the maximum in 11 minutes (related to celubose), was very similar to the profile of the fibers of the atrophied plant. Based on the results of the treatment of the multimeros isolated with enzymes, it was decided to try to modify the multimers in situ by holding the fibers to a sequential treatment of enzymes. The goal was to be able to specifically eliminate the multimeros by the chemically soft and specific enzymatic means. If this could be achieved then one could make a convincing argument for the multimers as a specific component of the cell wall of the fiber. Fibers (25 DPA) were attached to a 24 hour incubation with trypsin, chymotrypsin, proteinase K or pepsin followed by a second 24 hour incubation at 37 ° C with cellulase or β-glucosidase.

Alternatively, a duplicate set of samples was subjected to the same enzymes but in the reverse order. This is the cellulase or ß-glucosidase first and then the second protease. The final fiber / residual material was then subjected to extraction with dilute acid to remove the multimers before the HPAEC-PAD. As shown in Figures 28-30, material was released by both the proteases and the cellulase or the β-glucosidase. The multimers extracted from the final released material (Figure 30) indicates that the multimers could be extracted from the control fibers or fibers attached to protease first followed by cellulase, but no multimers were obtained from the material attached to the cellulase first followed by the protease. In this case, chymotrypsin was the most effective protease just as it was for the degradation of the adhesive. However, the most surprising observation of the cellulase-treated fibers followed by protease lost their structural integrity and simply fell apart or were sucked into the pasteur pipette when the extract was removed. When mature fibers of open capsules were subjected to the same cellulase procedure followed by protease, very little happened, so the procedure was repeated a second time. At the end of the second cycle, the fibers completely lost their structural integrity and only a precipitate of very small particles remained. These particles were then washed, subjected to digestion either in dilute HCl, in 2N trifluoroacetic acid or in 6N HCl. Effective digestion occurred only in 6N HCl, and the resultant monosaccharides obtained appeared to be in excess of 99 °. of glucose. This indicates that the sequential treatment with cellulase followed by protease is an excellent method to produce cellulose of extremely high purity. This result is surprising since it provides evidence for the significant modification of the fiber walls associated with the opening and maturity of the capsule. This means that even through the cellulose fiber wall is deposited in daily growth rings, there is obviously a very significant post-depositional modification process that drastically alters the properties of the fiber wall.

PROTEIN ADHESIVE AND RETICULATION The probability that the cellulose microfibrils of the plant cell wall are embedded in a matrix that "adheres" them together has been proposed by a number of researchers over the years. The nature of such an adhesive matrix has been the subject of considerable discussion, but there has not been a characterization of such matrix material. The presence of cell wall subunits, cotton fibers, was proposed by W. Lawrence Balls (Balls, W. Lawrence, 1928, Studies of Quality in Cotton, Macmillan &Co., London). The present work (see above) on the "adhesive" matrix of the cell wall is an extension of the work in my laboratory to characterize soluble oligosaccharides and saccharosyl polysaccharides, in particular those that seem to be involved in the changes in the development of the cotton fiber. The mature fibers of open capsules were extracted with cold water and the extract was removed. The crosslinking was then achieved using carbodiimide soluble in undamped water. The pH of the reaction mixture was measured and determined to be between 5.0 and 5.2. Two concentrations of water soluble carbodiimide, 125 mM and 250 mM were used. The crosslinking reaction was carried out for two hours at room temperature followed overnight at 4 ° C. The reaction mixture was washed from the fibers and then followed by enzymatic digestion. The fibers were incubated with cellulase (T. Reesei) (1 mg / ml) for 24 hours, followed by chymotrypsin (CT) (1 mg / ml) and then the incubation sequence was repeated. The results are shown in figures 31-34. In all cases, sample number 1 is the control; No. 2 are the fibers of the 125 mM carbodiimide reaction and the No. 3 are the fibers of the carbodiimide reaction 250 mM. Under the reaction conditions, carbodiimide would be expected to promote the formation of the amide bond between the amino acids, while having an insignificant effect on the formation of the ester bond, between carbohydrates. Carbodiimides have been used by others to modify the properties of polyester textiles (see US-A-5, 246,992) and to bond materials covalently to fibers (see JP-A-58 083 964). However, within the inventor's knowledge, carbodiimides have not previously been used on cellulose textiles in the same manner as in the present invention. It has been possible to extract a series of oligomers (multimeros) of cotton fibers in development by both chemical and enzymatic methods. These multimeros have retention times in HPAEC-PAD of 14 minutes and greater under the conditions analyzed. The regular spacing of the maxima is indicative of a series of oligosaccharides that vary by one monomer unit in size. These multimers are heteropolymers with a repeating glucan unit, which extend from a core glycan peptide structure. Previous work in the laboratory has shown that 25 DPA cotton fibers can be degraded by a sequential enzymatic treatment with a cellulase followed by a protease, but the reverse sequence does not achieve the same result. The fibers completely lose their structural integrity. When the capsule fibers that have been opened »* Are subject to the same sequence, they do not lose their integrity 5 unless the process is repeated a second time. Following the second protease treatment, there is a white particulate precipitate in the lower part of the tube. Quantitatively the constituents released by the enzymatic treatments consist mainly of glucose (Glc) and cellobiose (CB). The carbohydrates released by the first cellulase treatment are shown in Figure 31, which shows that both concentrations of carbodiimide dramatically reduced the amount of glucose or cellobiose released by the cellulase treatment. He maximum in the retention time of 3.5 minutes is arabinose. Many more of the maxima in the range of 14-20 minutes are released by the cellulase from the control fibers than from the treated fibers. It is very significant to note that the main maximum with a retention time of approximately 14.5 minutes released from the control fibers has a distinctly shorter retention time than the main maximum at about 14.65 minutes released from the treated fibers. This is a significant difference and is only demonstrable in the first cellulase extract.

The carbohydrates released by the first Treatment with chymotrypsin is shown in Figure 32. More maxima in the range of 14-20 minutes are released from the control fibers than from the treated fibers in addition to plus the large amounts of glucose and cellobiose released from both treated and treated fibers. of control. This pattern is consistent for the carbohydrates released by the second treatment with cellulase (currently a treatment with cellulase following a treatment with chymotrypsin) (figure 33) and the "second" treatment with chemotrypress (effectively a treatment of chymotrypsin following a treatment with cellulase) (figure 34). The carbohydrate maxima released with retention times between 14 and 20 minutes also contain a constituent that absorbs at 280 nm as shown in Figure 35. The absorbance at 280 nm is usually due to phenolic amino acids, phenylalanine and tyrosine in proteins, although other compounds can also absorb at 280 nm. Based on these results together with the material released by the proteases, it is concluded that the maximum carbohydrates in this 14-20 minute interval are glycoproteins. The fact that the binding with a carbodiimide makes these carbohydrates more resistant to release with protease, substance further the conclusion that they are, in fact, glycopeptides. The fact that digestion with protease significantly increases the release of glucose and cellobiose confirms that the cellulosic constituents of the wall are crosslinked by a protease sensitive component (i.e., a protein or glycoprotein). Enzymes such as cellulases have previously been used to "polish" textile fibers (see WO-A-93/05225), to alter the staining properties of cotton textiles (see WO-A-90/07569), as " post-treatments "to clean textiles (see GB-A-2 258 655) but these treatments did not result in the total degradation of the fiber structure. In addition, enzymes have been used to remove lignin from wood pulp (see US-A-5, 374, 55). But again, this treatment does not result in the total degradation of the cellulosic fibers. The rod-like white particles released by the enzymatic degradation of the fibers were subjected to further purification. They were treated with 0.1 N HCl for 30 minutes in a boiling water bath that could not be solubilized. The residue was also not soluble in 2N trichloroacetic acid at 100 ° C for two hours, but was completely dissolved by a treatment with 6N HCl at 100 ° C for two hours.The hydrolyzate of 6N HCl was then subjected to chromatography under NOH conditions. 15 mM, which resolves the monosaccharides.The result is shown in figure 36, which shows a single maximum with a retention time identical to that of glucose.It is not possible at this time determine if the base line unstable before the glucose peak and after it is significant. The decline in approximately 15 minutes is due to dissolved oxygen and is # 1 a well-known phenomenon. This subject should be further investigated to determine if there is any significance for those unstable baseline regions-for example, if minor constituents of additional carbohydrates are present. At this time it seems that the white particles are essentially pure cellulose, and only provide The glucose with the hydrolysis is 6N HCl. As detailed above, it has been possible to obtain the oligomers (multimers) of a large molecular complex which is secreted by the fibers, in vitro, by a temperature-dependent mechanism. The The relative distribution of the multimers may vary depending on the exogenous substrates incubated with the fibers and the time of day in which the capsules were collected. Under optimal conditions I have been able to demonstrate the presence of the multimer in an initial soluble fraction, a fraction that will not pass through a 0.2 micron filter, the precipitate of the aqueous extract and the fibers themselves. Multimerics seem to play a structural role in the integrity of cotton fiber since recent experiments to extract multimers using specific enzymes have resulted in a remarkable loss of the physical integrity of cotton fibers. The experiments just described demonstrate the A2so profile of the material released by the treatment - Sequential of mature cotton fibers with cellulase, 5 chymotrypsin, cellulase and then chymotrypsin again. These profiles indicate that multimers are linked to proteins. When the fibers are treated with a water-soluble carbodiimide, to form amide bonds between the carboxyl and amino groups of the constituents of amino acid, the fibers become more resistant to enzymatic degradation. This result demonstrates that bifunctional reagents have applications in textiles and give rise to ways to improve the quality (e.g. durability) of cotton fabric. Above all I have shown that normal cotton textiles continuously disperse water-soluble multimer during the life of the fabric. This suggests that tissue wear is at least partially due to loss of soluble material during washing. Chemical crosslinking is one way to reduce this loss and therefore extend the life of cotton fabrics. Although this test used carbodiimide, any of a large number of known bifunctional reagents could be used to react with the amino groups. These reagents are well known to an ordinary person expert in the technique of the. protein chemistry. Point significant is that my experiment is the first demonstration that protein cross-linking reagents are useful for altering the properties of cotton # c and other plant-based textiles. The hydrolysis experiments on the white particulate material which remains following the enzymatic digestion of the fibers are consistent with the fact that these particles are perhaps very highly crystalline cellulose. This result is consistent with the prediction by Balls (Balls, W. Lawrence, 1928, Studies of Quality in Cotton, Macmillan &Co., London) that the fiber wall is made of small domino or brick-like structures that hold together and allow the fiber be flexible. It is likely that the material that holds the "bricks" together is the "adhesive" matrix described in part herein with the multimers attached to a protein backbone. This result is consistent with the fact that plant breeders directly select varieties with different fiber properties that include resistance. Is likely that a matrix protein is a primary genetic product while a polysaccharide such as cellulose is the product of a number of genes. Thus, direct selection and manipulation by genetic engineering should be more successful on the matrix protein than on the protein complex. enzymes needed to synthesize cellulose.

In addition to the equivalents of the claimed elements, obvious substitutions known to one ordinarily skilled in the art are defined to be within the scope of the defined elements. Thus it is to be understood that the claims include what is illustrated and described specifically above, what is conceptually equivalent, and what can be obviously substituted. The illustrated embodiment has been set forth only for the purposes of example and should not be taken as limiting the invention. Therefore, it is to be understood that, within the scope of the appended claims, the invention may be practiced in a different manner than as specifically described herein.

Claims (7)

1. CLAIMS 1. A method for evaluating vegetable materials containing carbohydrates, characterized in that it comprises: # c freezing and freeze-drying the vegetable material; make a cold aqueous extract of the plant material; reextract the previously extracted plant materials with diluted boiling hydrochloric acid; analyze the hydrochloric acid extract using 10 high pH anion exchange chromatography to reveal a series of carbohydrate-containing compounds including oligosaccharides, polysaccharides, carbohydrate-protein conjugates and multimers thereof.
2. 2. The method of compliance with the claim 15 1, characterized in that the pattern of the oligosaccharides, polysaccharides, conjugates of carbohydrate-protein and multimers is used to determine the presence or absence of tension during the growth of the plant material.
3. 3. The method of compliance with the claim 20 1, characterized in that the freezing and leophilization steps are optional, where the plant material consists of textiles and where the pattern of the oligosaccharides, polysaccharides, carbohydrate-protein conjugates and multimeros is used to predict the effect of 25 different stages of manufacturing on the quality of the textiles determining what stages of manufacturing later the pattern.
4. 4. The method of compliance with the claim # c 1, characterized in that the freezing and leophilization steps are not optional, where the plant material consists of samples of cotton textiles and where the pattern of the oligosaccharides, polysaccharides, carbohydrate-protein conjugates and multimer predicts the presence of Abnormal cotton fibers that have abnormal dyeing properties by detecting those samples that have a significantly higher ratio of arabinose to glucose than samples that show normal dyeing properties.
5. 5. A method to alter cotton textiles to reduce the extraction capacity of carbohydrates that 15 comprises treating the cotton fibers with a water-soluble crosslinking agent which forms covalent bonds between the amino groups present in proteins within the cotton fibers and the carboxyl groups within the cotton fibers.
6. 6. The method according to claim 5, characterized in that the chemical reagent is a water-soluble carbodiimide.
7. 7. A method to enzymatically degrade cotton fibers to completely destroy the structure of the 25 fiber provided pure cellulose, comprising the steps of sequentially treating the fibers first with cells and then with protease.