# Patent

## Title
Methods for altering the reactivity of plant cell walls

## Abstract

Methods and means are provided for the modification of the reactivity of plant cell walls, particularly as they can be found in natural fibers of fiber producing plants by inclusion of positively charged oligosaccharides or polysaccharides into the cell wall. This can be conveniently achieved by expressing a chimeric gene encoding an N-acetylglucosamine transferase, particularly an N-acetylglucosamine transferase, capable of being targeted to the membranes of the Golgi apparatus in cells of a plant.

## Background
This application is a Divisional of U.S. application Ser. No. 11/993,816, filed Dec. 21, 2007 now U.S Pat. No. 8,008,544, which is the U.S. National Stage filing of International Application No. PCT/EP2006/005853, filed Jun. 19, 2006, which claims priority to EP 05076488.5, filed Jun. 24, 2005; U.S. Provisional Patent Application No. 60/698,182, filed Jul. 11, 2005; and EP 06008463.9, filed Apr. 25, 2006, the disclosures of each of which are hereby incorporated by reference.


The present invention relates to the modification of the reactivity of plant cell walls, including secondary plant cell walls, particularly as they can be found in natural fibers of fiber producing plants. In particular, the present invention is related to cotton fibers with altered reactivity. The modified reactivity could be applied in methods for dyeing cell wail containing plant derived material such as natural fibers, using fiber-reactive dyes, to improve e.g. colorfastness, or to decrease the volumes of waste-water used during the dyeing process. The modified reactivity could also be applied to improve the reactivity of the natural fibers with reactants such as flame retardants, water, oil and soil repellents, anticrease agents, softeners, antistatic agents, fluorescent whitening agents etc.
BACKGROUND ART
Natural fibers, including cellulose containing natural fibers from plants, such as cotton and linen, have been used by mankind for more than 5000 years. Natural cellulose containing fibers, however, do not possess the chemical versatility of synthetic fibers, due to the relative inert nature of the cellulose consisting of β-1-4 linked glucose monomers.
This relative inert nature is e.g. apparent during the dyeing process of cotton fibers and fabrics. Generally two types of dyes are used to color cotton: direct dyes and fiber-reactive dyes, which are both anionic molecules. Cotton itself develops an anionic charge in water, so that without special treatment, the uptake of dye by the fiber or fabric is quite elaborate.
Direct dyes create a relatively weak hydrogen bond with the cellulose polymer forming a semi-permanent attachment. Direct dyes are easier to use and less expensive than fiber-reactive dyes, but do not withstand well washing. Fiber-reactive dyes are molecules that combine chromophores with a reactive group that forms strong covalent bonds with the fiber via reaction with hydroxyl groups. The covalent bonds provide a good resistance of the dyed fiber against laundring. Colorfastness can be improved using cationic fixatives.
During the dyeing process, large amounts of electrolytes are needed to shield the anionic dyes from the anionic fiber charges. Unreacted dyes (up to 40%) need to be removed by a washing step known as scouring, generating large volumes of wastewater, also containing the above mentioned electrolytes.
Providing the cellulose fiber with a positive electric charge, e.g. by incorporation of positively charged chemical compounds, could therefore improve the dyeability of natural cellulose fibers, as well as improve any chemical reaction of the modified cellulose fiber with negatively charged chemical compounds. It would also make the use of acidic dyes possible.
Several publications have described the incorporation into or coating of chitosan oligomers into cellulose fibers to make chitosan/cellulose blends, yarns or fabrics. Chitosan is a positively charged polymer of glucosamine, which can be obtained by deacetylation of chitin, e.g. by alkalic treatments. Chitin itself is a polymer of β-1-4 linked N-acetylglucosamine (GlcNAc).
US patent application US20031/0134120 describes the coating of natural fibers with chitosan.
Liu et al. (Carbohydrate Polymers 44 (2003) 233-238) describe a method for coating cotton fibers with chitosan, by oxidation of the cotton thread with potassium periodate at 60° C. in water and subsequent treatment with a solution of chitosan in aqueous acetic acid. With the chitosan coating, the cotton fiber surface became physiologically and biologically active. Since the chemical reactivity of the amino group is greater than the hydroxyl group of cellulose monomers, the fiber has more potential for further chemical modification. Moreover, the smooth surface of the cotton fiber became coarse, suggesting a greater potential for drug absorption and controlled release thereof.
Based on the physiological function of chitosan in inhibiting e.g. dermatophytes, many functional clothes, fabrics and fibers employ cellulose-chitosan blend fibers, cellulose fiber-chitosan conjugates and fabrics coated with chitosan-containing resins.
WO 00/09729 describe the expression of chitin synthase and chitin deacetylase genes in plants to alter the cell wall for industrial uses and improved disease resistance. Specifically cited uses are: to provide a single plant source of cellulose, chitin and chitosan, to increase tensile strength and to increase brittle snap. Specifically suggested chitin synthase genes are derived from fungal organisms. No experimental data are provided on the production of chitin or chitosan in plants, nor on the incorporation thereof in plant cell walls.
The prior art thus remains deficient in providing methods for obtaining plants from which plant cell walls, particularly secondary cell walls, such as natural fibers, can be isolated containing positively charged chemical groups and/or chemical groups which are more reactive than hydroxyl groups of cellulose. The prior art remains also deficient in providing fibers which can be directly harvested from plants and which contain positively charged chemical groups and/or group which are more reactive than hydroxyl groups of cellulose, which can be used directly without the need for further chemical treatment to introduce such chemical groups. These and other problems are solved as described hereinafter in the different embodiments, examples and claims.
SUMMARY OF THE INVENTION
Briefly, in one embodiment, the invention provides a method for increasing the amount of positively charged oligosaccharides or polysaccharides in the cell wall, particularly the secondary cell wall of a plant cell, comprising introducing a chimeric gene into the plant cell, whereby the chimeric gene comprises a plant-expressible promoter operably linked to a DNA region coding for an N-acetylglucosamine transferase, preferably wherein the N-acetylglucosamine transferase is targeted to the membranes of the Golgi-apparatus; and a transcription termination and polyadenylation region. In a particular embodiment, the plant is cotton, and the positively charged oligosaccharides or polysaccharides are incorporated in the secondary cell walls which make up the cotton fiber.
In another embodiment of the invention, a method is provided for increasing the amount of positively charged oligosaccharides or polysaccharides in the cell wall, particularly the secondary cell wall of a plant cell comprising introducing a chimeric gene into the plant cell, the chimeric gene comprising a plant-expressible promoter operably linked to a DNA region coding for an N-acetylglucosamine transferase of the NODC type; and a transcription termination and polyadenylation region. Again, in a specific embodiment, the plant is cotton, and the positively charged oligosaccharides or polysaccharides are incorporated in the secondary cell walls which make up the cotton fiber.
In yet another embodiment of the invention, a method is provided for increasing the amount of positively charged oligosaccharides or polysaccharides in the cell wall, particularly the secondary cell wall of a plant cell, the method comprising

    
    
        i. introducing a chimeric gene into the plant cell, said chimeric gene comprising the following operably linked DNA fragments:
        
            1. a plant-expressible promoter;
            2. a DNA region coding for chitin synthase; and
            3. a transcription termination and polyadenylation region.
        
        
        ii. applying to the transgenic plant cell an effective amount of N-acetylglucosamine, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylglucosamine-1-phosphate or UDP-N-acetylglucosamine.
    
    


The invention also provides plant cell walls, comprising an increased amount of polysaccharides or oligosaccharides, particularly positively charged oligosaccharides, such as oligo-N acetylglucosamines or oligo-glucosamines, preferably oligomers of N-acetylglucosamine or glucosamine with a polymerization degree between 3 and 10, particularly between 3 and 5. Such plant cell walls are obtainable by the methods of the invention. These plant cell walls may be subjected to further chemical modification.
In a specific embodiment, the invention provides cotton fibers comprising an increased amount of the positively charged oligosaccharides mentioned herein, and yarns, textiles which comprise such cotton fibers. The cotton fibers may be used as such or may have been subjected to further chemical modification, including dying. These cotton fibers can be recognized e.g. through their increased binding of anionic dyes, including congo red, through their increased binding of wheat germ agglutinin or through their increased reactivity with amine-reactive dyes when compared to cotton fibers obtained from cotton plants of a an isogenic line which does not contain a chimeric N-acetylglucosamine transferase gene as described herein. The presence and/or the amount of oligosaccharides in the cotton fibers can also be determined directly through e.g. high performance thin layer chromatography (HPTLC).
In another embodiment, the invention is directed towards the use of a DNA region coding for an N-acetylglucosamine transferase capable of being targeted to the Golgi apparatus of a plant cell to increase the amount of positively charged oligosacccharides in the cell wall of a plant cell or to increase the reactivity of plant cell walls for chemical modifications of such plant cell walls.
The invention also provides chimeric genes comprising the following operably linked DNA regions: a plant-expressible promoter; a DNA region coding for an N-acetylglucosamine transferase, said N-acetylglucosamine transferase when expressed in a plant cell, being targeted to the Golgi apparatus of said plant cell; and a transcription termination and polyadenylation region. The N-acetylglucosamine transferase may be an N-acetylglucosamine transferase of the NODC type or could also be a chitin synthase, particularly a chitin synthase which has been operably linked to a Golgi retention signal.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Alignment of the amino acid sequence of different NODC proteins. Amino acid residues conserved in all proteins are indicated in bold. ROT_NODC_RHILP: NODC protein from Rhizobium leguminosarum (biovar phaseoli) (SEQ ID No: 7); ROT_NODC_BRAJA: NODC protein from Bradyrhizobitun japonicum (SEQ ID No. 2); ROT_NODC_RHIS3 NODC protein from Rhizobium sp. (strain N33) (SEQ ID No: 8); ROT_NODC_RHISN: NODC protein from Rhizobium sp (SEQ ID No: 16); ROT_NODC_RHILV: NODC protein from Rhizobium leguminosarum (biovar viciae) (SEQ ID No: 4); and ROT_NODC_AZOCA: NODC protein from Azorhizobium caulinodans (SEQ ID No: 1).
FIG. 2: Alignment of the amino acid sequence of different NODC proteins. Amino acid residues conserved in all proteins are indicated in bold. ROT_NODC_BRAJA: NODC protein from Bradyrhizobium japonicum (SEQ ID No. 2); ROT_NODC_RHIS3 NODC protein from Rhizobium sp. (strain N33) (SEQ ID No: 8); ROT_NODC_RHISN: NODC protein from Rhizobium sp (SEQ ID No: 16); ROT_NODC_RHILV: NODC protein from Rhizobium leguminosarum (biovar viciae) (SEQ ID No: 4); and ROT_NODC_AZOCA: NODC protein from Azorhizobium caulinodans (SEQ ID No: 1).
FIG. 3: Photographs of fluorescent microscopy performed on root hair cells from hairy roots containing the 35S::NodC chimeric gene.
Panel A. Optical section of a root hair cell stained with Calcofluor; B: optical section of a root hair cell immunohistochemically stained for the presence of N-acetylglucosamine; C: superposition of the optical sections of panel A and panel B.
FIG. 4: Photographs of fluorescent microscopy performed on root hair cells from hairy roots containing the 35S::NODC-EGFP chimeric gene.
Panel A. superposition of the optical sections of panels B, C and D. B: Optical section of a root hair cell stained with Calcofluor; C: optical section of a root hair cell stained to visualize the Golgi apparatus; D: optical section of a root hair cell visualizing the fluorescence by EGFP.
FIG. 5: Photographs of fluorescent microscopy performed on root hair cells from hairy roots containing the 35S::chitin synthase chimeric gene.
Panel A: Optical section of a hairy root cultured in the presence of 50 mM N-acetylglucosamine, stained for the presence of N-acetylglucosamine; Panel B: Optical section of a hairy root cultured in the presence of no extra N-acetylglucosamine, stained for the presence of N-acetylglucosamine in the cell wall.
FIG. 6: High performance thin layer chromatogram of chito-oligomers from cell wall material isolated from Arabidopsis hairy roots.
The samples of the two outer lanes (1, 12) are standard solutions of N-acetylglucosamine, chitobiose, chitotriose, chitotetraose and chitopentaose. Lanes 2 to 5: cell wall material extracted from hairy root cultures initiated by Agrobacterium rhizogenes with a chimeric gene comprising a CaMV35S promoter linked to a nodC encoding region; lanes 6 to 9: cell wall material extracted from hairy root cultures initiated by Agrobacterium rhizogenes with a chimeric gene comprising a CaMV35S promoter linked to a nodC encoding region fused to eGFP; lanes 10 and 11, cell wall material extracted from hairy root cultures initiated by Agrobacterium rhizogenes with a chimeric gene comprising a CaMV35S promoter linked to a phosphinotricin acetyltransferase encoding region. TLC was performed in acetonitrile (76): water (24): 0.5% boric acid (10)
FIG. 7: Two dimensional HPTLC on cell wall material isolated from Arabidopsis hairy roots which has been subjected to chitinase digestion.
Control cell wall material (left panel) was extracted from hairy root cultures initiated by Agrobacterium rhizogenes with a chimeric gene comprising a CaMV35S promoter linked to a phosphinotricin acetyltransferase encoding region; experimental cell wall material (right panel) was extracted from hairy root cultures initiated by Agrobacterium rhizogenes with a chimeric gene comprising a CaMV35S promoter linked to a nodC encoding region fused to eGFP. Monomer saccharides (N-acetylglucosamine) and dimer chito-saccharides (chitobiose) can be detected in the experimental material, but not in the control material. TLC was performed in acetonitrile (76): water (24): 0.5% boric acid (10)
FIG. 8: HPTLC of cell wall material isolated from transgenic Arabidopsis plants.
Lane 1: standard solution chito-oligosaccharides; Lane 2: 0.1 glucosamine; lanes 3 and 4: 4 μl and 8 μl respectively of cell wall material isolated from control Arabidopsis shoots; lanes 5 and 6: 4 μl and 8 μl respectively of cell wall material isolated from transgenic Arabidopsis shoots comprising a CaMV35S::nodC chimeric gene; lanes 7 and 8: 4 μl and 8 μl respectively of cell wall material isolated from transgenic Arabidopsis shoots comprising a CaMV35S::nodC-eGFP chimeric gene. The dotted line indicates the increased amount of chito-triose particularly in landes 5 and 6.
FIG. 9: Fluorescence microscopy of cotton fibers reacted with wheat-germ agglutinin (WGA) conjugated to Alexa fluor 555.
Left panel: cotton fibers from transgenic cotton plants containing a CaMV35S::NodC gene. Right panel: cotton fibers from control cotton plants. Under UV-light, a bright fluorescence can be observed in the fibers from the transgenic cotton plants, indicating the presence of chito-oligomers in these fibers.



DETAILED DESCRIPTION OF DIFFERENT EMBODIMENTS OF THE INVENTIONS
The current invention is based on the finding that expression of N-acetylglucosamine transferase of the NODC type in plant cells leads to incorporation of N-acetylglucosamine oligomers into plant cell walls. The GlcNAc oligomers were unexpectedly associated very closely with the cell wall and were not dissolved from that cell wall by various treatments. Surprisingly, the synthesis of the GlcNAc oligomers did not require the external addition of GlcNAc to the growth medium, as was observed with other chitin synthases. Furthermore and equally surprising, the NODC protein was also closely associated with the membranes of the Golgi apparatus in addition to the association with the cell membrane as expected. When the N-acetylglucosamine transferase of the NODC type was expressed in cotton plants, the GlcNAc oligomers were incorporated into the cotton fibers, leading to more reactive cotton fibers.
Thus, in a first embodiment of the invention, a method is provided for increasing the amount of positively charged oligosaccharides in the cell wall, particularly the secondary cell wall of a plant cell, wherein the method comprises the step of introducing a chimeric gene into the plant cell, and the chimeric gene comprising the following operably linked DNA fragments:

    
    
        a plant-expressible promoter
        a DNA region coding for an N-acetylglucosamine transferase, wherein the N-acetylglucosamine transferase is of the NODC type; and
        a transcription termination and polyadenylation region.
    
    


Nodulation C protein (“NODC protein”) and its encoding gene are involved in the synthesis of the lipochitooligosacccharide signals or acetylated chitooligomers (Nod factors) which lead to the nodule formation typical of the symbiosis between Rhizobiaceae and leguminous plants.
The most crucial nod gene products required for the synthesis of these lipo-chito-oligosaccharides are NODA, NODB and NODC. In the absence of other nod gene products they can form a core signal consisting of oligomers of four or five N-acetylglucosamine residues carrying an N-linked acyl group. The function of each of the three proteins in the synthesis of nodulation factors is well known: NODC is an N-acetylglucosaminyl transferase which produces the chito-oligosacharide chain; the N-acetyl group from the non-reducing N-acetylglucosamine residue of the chito-oligosaccharide chain is removed by NODB, which acts as a chitin oligosaccharide deacetylase; NODA is involved in the attachment of the acyl chain to the free amino group generated by the action of NODB. Other Nod factors, encoded by other nod genes, provide for any of the decorating chemical groups discriminating the different nodulation factors. For the purposes of the present invention, only the NODC proteins and encoding genes are of relevance.
NODC protein is a well characterized protein (for a review see Kamst and Spaink, 1999, Trends in Glycoscience and Glycotechnology, 11, pp 187-199). It belongs to a family of β-polysaccharide synthase proteins that are involved in the synthesis of linear polysaccharides containing β-linked monosaccharide residues. The enzymes that are structurally most closely related to NODC are transferases involved in the synthesis of chitin (β-1-4 linked N-acetylglucosamines); cellulose (the polymer of β-1-4 linked glucose residues); hyaluronic acid (a co-polymer of N-acetylglucosamine and glucuronic acid) and chitin oligosaccharides produced during early development of zebrafish embryos. Six short regions conserved between these proteins can be recognized. For NODC proteins, these short sequences correspond to:
1) a K residue at position 23 of SEQ ID No 1 (NODC from Azorhizobium caulinodans)

    
    
        2) the sequence DDG at position 86-88 of SEQ ID No I
        3) the sequence VDSDT at position 137-141 of SEQ ID No 1
        4) the sequence GPCAMYR at position 207-213 of SEQ ID No 1
        5) the sequence GEDRHL at position 237-242 of SEQ ID No 1; and
        6) the sequence QQLRW at position 274-278 of SEQ ID No 1
    
    


However, it is important to realize that some NODC proteins or variants thereof may exist wherein one or more of the above mentioned consensus sequences are not absolutely conserved.
NODC proteins are also frequently characterized by hydrophobic stretches of amino acid residues representing transmembrane domains (Barney et al. 1996, Molecular Microbiology 19, pp 443-453). The N-terminal hydrophobic domain spans the bacterial membrane in a Nout-Cin orientation, with the adjacent large hydrophilic domain being exposed to the bacterial cytoplasm. This orientation appears to be dependent upon the presence of the hydrophobic region(s) near the C-terminus, potentially containing three membrane spans, such that the C-terminus of NODC is normally located in the bacterial periplasm.
The large hydrophilic loop of NODC also has other structural similarity to similar regions in the other β-glucosyl transferases. This region has been proposed to be made up of an A domain (which extends from about residue 45 to 140 in the sequence of SEQ ID No 4) consisting of alternating β-sheets and a-helices, and a B-domain (corresponding to residues 215-280 of SEQ ID No 4) thought to be responsible for the processivity of NODC. In the A-domain, two aspartate residues are conserved (residues 88 and 139 of SEQ ID No. 4); in the B-domain one aspartate residue and the motif QXXRW (residue 240 and 276-280 of SEQ ID No 4) are also conserved and thought to be crucial for catalytic activity.
When different NODC proteins are compared among themselves, amino acid sequences which are more conserved are revealed. FIG. 1 represents an alignment of different NODC proteins from SEQ ID No 1, 2, 8, 4, 7, 5 and indicates a number of conserved residues between the different NODC proteins including (in order):









(SEQ ID No: 17)



the sequence PXVDVIXPXXNE 



 



(SEQ ID No: 18)



the sequence VDDGSXN



 



(SEQ ID No: 19)



the sequence GDXXLDVDSDTXXXXDV



 



(SEQ ID No: 20)



the sequence GXXMGQ



 



(SEQ ID No: 21)



the sequence DMEYWLACNEERXXQXRFGXVMXCXGXCXMYR 



 



(SEQ ID No: 22)



the sequence FRTXYXPXAXAXTXVP



 



(SEQ ID No: 23)



the sequence YLXQQLRWARSTXRXTXL 



 



(SEQ ID No: 24)



the sequence QNXGXXLL



 



(SEQ ID No: 25)



the sequence RFXFXXXHXXXNXXXLXPLKXYALXT






FIG. 2 represents an alignment of a subset of different NODC proteins, showing even more conserved residues such as:







(SEQ ID No: 26)


the sequence 


WLTRLIDMEYWLACNEERXXQXRFGXVMCCCGPCAMYRRS


 


(SEQ ID No: 27)


the sequence 


LLXXYEXQXFXGXPSXFGEDRHLTILMLXAGFRTXYVPXAXAXTXVP


 


(SEQ ID No: 28)


the sequence YLRQQLRWARSTXRDTXLA






FIG. 2 represents an alignment of a subset of different NODC proteins, showing even more conserved residues such as:







the sequence 


WLTRLIDMEYWLACNEERXXQXRFGXVMCCCGPCAMYRRS


 


the sequence 


LLXXYEXQXFXGXPSXFGEDRHLTILMLXAGFRTXYVPXAXAXTXVP


 


the sequence YLRQQLRWARSTXRDTXLA






The length of the oligosaccharide backbone in lipo-chitin oligosaccharides produced by different Rhizobiaceae varies between two and six residues. It has been shown that the nodulation protein NODC is an important determinant of the chitin oligosaccharide chain length in the synthesis of the chito-oligosaccharide chain (Kamst et al., 1997, Journal of Bacteriology 179, p 2103-2108).
Coding regions coding for an N-acetylglucosamine transferase of the NODC type may be obtained directly from bacteria belonging to the genera Rhizobium, Azorhizobium, Bradyrhizobittm, Mesorhizobium, Ralstonia, Cupriavidus, Streptomyces, Burkholderia or Sinorhizobium. However, it will be immediately clear that such coding regions may also be made synthetically, even with a codon usage adapted to the plant, particularly the fiber producing plant into which the chimeric gene overexpresing the NODC type protein is introduced.
Different sequences for NODC proteins are available from databases such as the protein sequences identified by the following accession numbers: CAA67139, CAA608779, CAA51774, CAA51773, CAA25811, CAA25810, CAA25814, CAA68619, CAA2350, CAD31533, CACO5896, CAH04369, CAB56055, NP—629203, P26024, P17862, BAB524500, AAX30050, AAX30049, E38180, JQ0396, ZZZRC4, ZZZRCL, A95321, C23766, C26813, NP—659761, NP—443883, NP—106714, NP—768667, NP—435719, BAC47292, AAU 11365, AAU11364, AAU11363, AAU11362, AAU11361, AAU 11360, AAU11359, AAU11358, AAU11357, AAU11356, AAU11355, AAU11354, AAU11353, AAU11352, AAU11351, AAU11350, AAU114349, AAU11348, AAU11347, AAU11346, AAU11345, AAU11344, AAU11343, AAU11342, AAU11341, AAU11340, AAU11339, AAU11338, AAK65131, AAS91748, P04679—2, P04679—1, P04679, P72334, Q53513, P50357, P04678, P50536, P53417, Q07755, P04341, P04340, P24151, P04677, CAD90588, CAD90587, CAD90586, CAD90585, CAD90584, CAD90583, CAD90257, CAD43933, AAM54775, AAN62903, 534305, 509522, S07304, AAL88670, CAD29957, CAD29956, CAD29955, CAD29954, CAD29953, CAD 29952, CAD29951, CAD29950, CAD29949, CAC42489, AAK53549, AAK53548, AAK50872, AAK39967, AAK39966, AAK39965, AAK39964, AAK39963, AAK39962, AAK39961, AAK39960, AAK39959, AAK39958, AAK39957, AAK39956, AAG44125, AAK00157, AAG60998, AAB71694, AAB 16897, AAV80567, AAB95329, BAA24092, BAA06089, BAA06086, BAA06085, BAA06083, BAA06090, BAA06082, BAA06087, BAA06088, BAA06084, AAB91695, AAB51164, AAB47353, AAB34509, AAB24745, 1615305E, 1615305D, 165305C, CAA26311, CAA26310, CAA3731, AAA63602 or 26226 (incorporated herein by reference).
Other entries in the UNIPROT databases referring to full length NODC proteins are summarized in Table 1. All mentioned amino acid sequences referenced by the accession number are herein incorporated by reference.







TABLE 1







full length NODC proteins











UniProt




UniProt/UniParc ID
Accessions
Species Name
Length





NODC_BRAJA
P26024
Bradyrhizobium japonicum
485


NODC_AZOCA
Q07755
Azorhizobium caulinodans
395


Q6PTX8_9RHIZ
Q6PTX8
Rhizobium sp. SIN-1
408


Q70YC2_9BURK
Q70YC2
Cupriavidus taiwanensis
450


Q6EX51_SINSB
Q6EX51
Sinorhizobium sp.
452


NODC_RHIS3
P72334
Rhizobium sp.
450


NODC_RHILP
P24151
Rhizobium leguminosarum
428


Q8GNH5_RHIME
Q8GNH5
Rhizobium meliloti
421


Q53254_RHITR
Q53254
Rhizobium tropici
452


Q9AQ23_BRASW
Q9AQ23
Bradyrhizobium sp.
452


NODC_RHISN
P50357
Rhizobium sp.
413


Q8KLG3_RHIET
Q8KLG3
Rhizobium etli
443


Q9RAN5_MESS7
Q9RAN5
Mesorhizobium sp.
416


Q9Z3I6_BRASS
Q9Z3I6
Bradyrhizobium sp.
481


NODC_RHILO
P17862
Rhizobium loti
424


Q8KJI5_RHILO
Q8KJI5
Rhizobium loti
424


NODC_RHIGA
P50356
Rhizobium galegae
433


NODC_RHIME
P04341
Rhizobium meliloti
426


Q9R614_RHIME
Q9R614
Rhizobium meliloti
424


O52478_RHIME
O52478
Rhizobium meliloti
402


Q52971_RHIME
Q52971
Rhizobium meliloti
402


NODC_RHILV
P04340
Rhizobium leguminosarum
424









However, it will be clear that variants of NODC proteins, wherein one or more amino acid residues have been deleted, substituted or inserted, which can be deduced from the above mentioned amino acid sequences, can also be used to the same effect in the methods according to the invention, provided that the enzymatic activity has not changed. These variant NODC proteins may have about 95% sequence identity to any one of the herein mentioned NODC proteins. A method for determining enzymatic activity of NODC proteins in vitro has been described e.g. by Kamst et al., 1997 Journal of Bacteriology, 179, p2103-2108.
Thus, as used herein, an “N-acetylglucosamine transferase that is of the NODC type” is an N-acetylglucosamine transferase that catalyzes the transfer of the GlcNAc moiety from UDP-GlcNAc to a nascent chitin oligosaccharide. Preferably the protein contains the conserved regions which can be found by comparing the different NODC proteins.
Particularly suitable for the methods of the invention are the proteins listed in SEQ ID No 1 to SEQ ID No 9, particularly the protein listed in SEQ ID No 1, and the DNA fragments encoding such a protein.
It has been observed (see experimental section) that NODC proteins when expressed in plant cells are co-localized with the membranes of the Golgi apparatus, in addition to the co-localization with the plasmalemma. To arrive at the incorporation of chito-oligosaccharides into the plant cell wall, no feeding with GlcNAc is required. However, when chitin synthases of fungal origin are used, these proteins are not co-localized with the membranes of the Golgi apparatus, and feeding with GlcNAc is required to arrive at significant incorporation of chito-oligosaccharides into the cell walls. Without intending to limit the invention to a particular mode of action, it is thought that the transmembrane spanning domains of NODC proteins may be more apt to insertion into the membranes of the Golgi apparatus than those of the plasmalemma and that the location of these proteins circumvents the need for external feeding with GlcNAc. Modification of chitin synthase proteins, such as a chitin synthase of fungal origin, e.g a Neurospora crassa chitinsynthase, so as to relocate the chitin synthases to the membranes of the Golgi apparatus is sufficient to abolish the need of external GlcNAc feeding. Such relocation has been achieved by linking the chitin synthase protein to a signal anchor peptide targeting the linked protein to membranes of the Golgi apparatus.
Thus, in another embodiment of the invention, a method is provided for increasing the amount of positively charged oligosaccharides in the cell wall, particularly the secondary cell wall of a plant cell, comprising the step of introducing a chimeric gene into the plant cell, said chimeric gene comprising

    
    
        a plant-expressible promoter
        a DNA region coding for an N-acetylglucosamine transferase, wherein the N-acetylglucosamine transferase is targeted to the membranes of the Golgi-apparatus; and
        a transcription termination and polyadenylation region.
    
    


As used herein, the N-acetylglucosamine transferase is not limited to NODC type proteins, but also includes chitin synthases (Chitin UDP-acetyl-glucosaminyl transferases), such as the chitin synthases of fungal origin. Examples of amino acid sequences of such chitin synthases can be found in the different databases including amino acid sequences with the following identifiers (accession numbers): CHS1_AJECA (P30576) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Class-I chitin synthase 1) from Ajellomyces capsulata (Histoplasma capsulatum); CHS1_AJEDE (P30579) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Class-I chitin synthase 1) from Ajellomyces dermatitidis (Blastomyces dermatitidis); CHS1_ASPNG (P30581) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Class-I chitin synthase 1) from Aspergillus niger; CHS1_BOTCI (P49603) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Class-I chitin synthase 1) from Botrytis cinerea (Noble rot fungus) (Botryotinia fuckeliana); CHS1_CANAL (P23316) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1). from Candida albicans (Yeast); CHS1_CRYNV (013356) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Class-IV chitin synthase 1). {GENE: Name=CHS1}-Cryptococcus neoformans var. grubii (Filobasidiella neoformans var. grubii); CHS1_EMENI (P30583) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Class-I chitin synthase 1) (Fragment). {GENE: Name=chs1}-Emericella nidulans (Aspergillus nidulans); CHS1_EXODE (P30600) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Class-II chitin synthase 1). {GENE: Name=CHS1}-Exophiala dermatitidis (Wangiella dermatitidis); CHS1_EXOJE (P30585) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Class-I chitin synthase 1) (Fragment). {GENE: Name=CHS1}-Exophiala jeanselmei; CHS1_NEUCR (P29070) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Class-III chitin synthase 3). {GENE: Name=chs-1; ORFNames=B11H24.170, NCU03611.1}-Neurospora crassa; CHS1_PHAEX (P30590); Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Fragment). {GENE: Name=CHS1}-Phaeococcomyces exophialae; CHS1_PHYBL (P87073) Chitin synthase 1 (EC 2.4.1.6) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Class-II chitin synthase 1). {GENE: Name=chs1}-Phycomyces blakesleeanus; CHS1_RHIAT (P30592) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase I) (Class-I chitin synthase 1) (Fragment). {GENE: Name=CHS1}-Rhinocladiella atrovirens; CHS1_RHIOL (P30594) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase I). {GENE: Name=CHS1}-Rhizopus oligosporus; CHS1_RHIRA (Q12632) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Class-II chitin synthase 1). {GENE: Name=CHS1}-Rhizomucor racemosus (Mucor circinelloides f. lusitanicus); CHS1_SCHPO (P30596); Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Fragment). {GENE: Name=CHS1}-Schizophyllum commune (Bracket fungus); CHS1_SCHPO (P30597) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1). {GENE: Name=chs1; ORFNames=SPAC13G6. 12c, SPAC24B 11.01c}-Schizosaccharomyces pombe (Fission yeast); CHS1_TUBUN (P55003) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Fragment). {GENE: Name=CHS1}-Tuber uncinatum (Burgundy truffle); CHS1_USTMA (P30598) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Fragment). {GENE: Name=CHS1}-Ustilago maydis (Smut fungus); CHS1_XYLBA (P30603) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1) (Fragment). {GENE: Name=CHS1}-Xylohypha bantiana; CHS1_YEAST (P08004) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1). {GENE: Name=CHS1; Saccharomyces cerevisiae (Baker's yeast); CHS2_AJECA (P30577) Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2) (Class-III chitin synthase 2) Ajellomyces capsulata (Histoplasma capsulatum); CHS2_AJEDE (P30580) Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2) (Class-II chitin synthase 2) {GENE: Name=CHS2}-Ajellomyces dermatitidis (Blastomyces dermatitidis) CHS2_ASPNG (P30582); Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2) (Class-II chitin synthase 2) (Fragment). (GENE: Name=chs2}-Aspergillus niger; CHS2_CANAL (P30572) Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2). {GENE: Name=CHS2}-Candida albicans (Yeast); CHS2_EXODE (P30601) Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2) (Class-1 chitin synthase 2). {GENE: Name=CHS2}-Exophiala dermatitidis (Wangiella dermatitidis); CHS2_EXOJE (P30586) Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosarninyl transferase 2) (Fragment). {GENE: Name=CHS2}-Exophiala jeanselmei; CHS2_NEUCR (P30589); Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2). {GENE: Name=chs-2; ORFNames=NCU05239.1}-Neurospora crassa; CHS2_PARBR (Q92444) Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2) (Class-II chitin synthase 2). {GENE: Name=CHS2}-Paracoccidioides brasiliensis; CHS2_PHAEX (P30591); Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2) (Class-II chitin synthase 2) (Fragment). {GENE: Name=CHS2}-Phaeococcomyces exophialae; CHS2_RHIAT (P30593) Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2) (Class-III chitin synthase 2) (Fragment). {GENE: Name=CHS2}-Rhinocladiella atrovirens; CHS2_RHIOL (P30595) Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2). {GENE: Name-=CHS2}-Rhizopus oligosporus; CHS2_SCHPO (O74756) Chitin synthase-like protein 2. {GENE: Name=chs2; ORFNames=SPBC1709.01, SPBC1734.17}-Schizosaccharomyces pombe (Fission yeast); CHS2_USTMA (P30599) Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2) (Fragment). {GENE: Name=CHS2}-Ustilago maydis (Smut fungus); CHS2_XYLBA (P30604) Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2) (Class-II chitin synthase 2) (Fragment). {GENE: Name=CHS2}-Xylohypha bantiana; CHS2_YEAST (P14180); Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2). {GENE: Name=CHS2; OrderedLocusNames=YBR038W; ORFNames=YBR0407}-Saccharomyces cerevisiae (Baker's yeast); CHS3_AJECA (P30578) Chitin synthase 3 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 3) (Class-II chitin synthase 3) (Fragment). {GENE: Name=CHS3}-Ajellomyces capsulata (Histoplasma capsulatum); CHS3_CANAL (P30573) Chitin synthase 3 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 3) (Class-IV chitin synthase 3). {GENE: Name=CHS3}-Candida albicans (Yeast); CHS3_EXODE (P30602) Chitin synthase 3 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 3) (Class-III chitin synthase 3). {GENE: Name=CHS3}-Exophiala dermatitidis (Wangiella dermatitidis); CHS3_EXOJE (P30587); Chitin synthase 3 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 3) (Class-III chitin synthase 3) (Fragment). {GENE: Name=CHS3}-Exophiala jeanselmei; CHS3_NEUCR (P30588) Chitin synthase 3 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 3). {GENE: Name=chs-3; ORFNames=G65A3.040}-Neurospora crassa; CHS3_YEAST (P29465) Chitin synthase 3 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 3) (Class-IV chitin synthase 3). {GENE: Name=CHS3; Synonyms=CAL1, CSD2, DIT101, KIT2; Ordered Locus Names=YBRO23C; ORFNames=YBRO305}-Saccharomyces cerevisiae (Baker's yeast); CHS4_MAGGR (O13353); Chitin synthase 4 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 4) (Class-IV chitin synthase 4). {GENE: Name=CHS4}-Magnaporthe grisea (Rice blast fungus) (Pyricularia grisea); CHS4_NEUCR (Q01285) Chitin synthase 4 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 4) (Class-IV chitin synthase 4). {GENE: Name=chs-4; ORFNames=NCU09324.1}-Neurospora crassa; CHS5_USTMA (013394) Chitin synthase 5 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 5) (Class-IV chitin synthase 5). {GENE: Name=CHS5}-Ustilago maydis (Smut fungus); CHS6_USTMA (O13395) Chitin synthase 6 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 6) (Class-V chitin synthase 6). {GENE: Name=CHS6}-Ustilago maydis (Smut fungus); CHSA_AMPQU (Q12564); Chitin synthase A (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase A) (Class-I chitin synthase A). {GENE: Name=CHSA}-Ampelomyces quisqualis; CHSA_EMENI (P30584) Chitin synthase A (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase A) (Class-II chitin synthase A). {GENE: Name=chsA; Synonyms=chs2}-Emericella nidulans (Aspergillus nidulans); CHSB_EMENI (Q00757) Chitin synthase B (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase B) (Class-III chitin synthase B). {GENE: Name=chsB}-Emericella nidulans (Aspergillus nidulans); CHSC_ASPFU (Q92197) Chitin synthase C (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase C) (Class-III chitin synthase C). {GENE: Name=chsC}-Aspergillus fumigatus (Sartorya fumigata); CHSD_ASPFU (P78746) Chitin synthase D (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase D) (Class-VI chitin synthase D). {GENE: Name=chsD}-Aspergillus fumigatus (Sartorya fumigata); CHSD_EMENJ (P78611) Chitin synthase D (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase D) (Class-V chitin synthase D). {GENE: Name=chsD; Synonyms=chsE}-Emericella nidulans (Aspergillus nidulans); CHSG_ASPFU (P54267); Chitin synthase G (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase G) (Class-III chitin synthase G). {GENE: Name=chsG}-Aspergillus fumigatus (Sartorya fumigata); CHSX_USTMA (Q99126) Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 1). {GENE: Name=CHS1}-Ustilago maydis (Smut fungus); CHSY_USTMA (Q99127) Chitin synthase 2 (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase 2). {GENE: Name=CHS2}-Ustilago maydis (Smut fungus) or CHS_SAPMO (P48017) Chitin synthase (EC 2.4.1.16) (Chitin-UDP acetyl-glucosaminyl transferase). {GENE: Name=CHS}-Saprolegnia monoica. All sequences are incorporated herein by reference.
Chitin synthases should preferably be equipped with (heterologous) signal anchor sequences targeting the chitin synthase to the membranes of the Golgi apparatus. Such sequences are known in the art, including the sequences within and adjacent to the transmembrane segment of α-2,6-sialyltransferase (particularly the first 44 or 52 amino acids thereof; Munro et al. 1991, EMBO Journal, 10: 3577-3588); the signal anchor sequence from human galactosyl transferase (particularly the first 60 amino acids thereof) or the signal anchor sequence from the Arabidopsis homologue of the yeast HDEL receptor (AtERD2) (Saint-Jore et al., 2002, The Plant Journal, 29: 661-678), the signal anchor sequence from β1,2-xylosyltransferase protein (particularly the first 36 amino acids thereof; Pagny et al., 2003, The Plant Journal 33: 189-203) or the signal anchor sequences of N-acetyl-glucosaminyl transferase I (particularly the first 77 amino acids thereof; Essl et al. 1999, FEBS Lett. 453:169-173) (all publication incorporated herein by reference). Other Golgi targeting signals to be employed by fusion at the C-terminus of the N-acetylglucosamine transferase include the amino acid sequence “YYHDL” (SEQ ID NO: 29) as can be found in Arabidopsis DAGAT1 protein or “LKLEI” (SEQ ID NO: 30) as can be found in Arabidopsis DAGAT2. Fusion of such signal anchor sequences to chitin synthases by linking DNA fragments encoding the respective polypeptides can be achieved using standard recombinant DNA techniques. N-acetylglucosamine transferases of the NODC type may also be operably linked to signal anchor sequences targeting the Golgi apparatus.
In another embodiment of the invention, a method is provided for increasing the amount of positively charged oligosaccharides in the cell wall, particularly the secondary cell wall of a plant cell, comprising the step of introducing a chimeric gene into the plant cell, wherein the chimeric gene comprises the following operably linked DNA fragments

    
    
        a plant-expressible promoter;
        a DNA region coding for chitin synthase (chitin UDP-acetylglucosamine transferase), preferably of fungal origin; and
        a transcription termination and polyadenylation region

and further comprising the step of applying an effective amount of N-acetylglucosamine or N-acetylglucosamine-1-phosphate or N-acetylglucosamine-6-phosphate or glucosamine-6-phosphate to the plant cell or to the plant.

    
    


The chimeric genes according to the invention comprise a plant-expressible promoter. As used herein, the term “promoter” denotes any DNA which is recognized and bound (directly or indirectly) by a DNA-dependent RNA-polymerase during initiation of transcription. A promoter includes the transcription initiation site, and binding sites for transcription initiation factors and RNA polymerase, and can comprise various other sites (e.g., enhancers), at which gene expression regulatory proteins may bind.
As used herein, the term “plant-expressible promoter” means a DNA sequence which is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S, the subterranean clover virus promoter No 4 or No 7, or T-DNA gene promoters and the like.
A plant-expressible promoter that controls initiation and maintenance of transcription preferentially in fiber cells is a promoter that drives transcription of the operably linked DNA region to a higher level in fiber cells and the underlying epidermis cells than in other cells or tissues of the plant. Such promoters include the promoter from cotton from a fiber-specific (3-tubulin gene (as described in WO0210377), the promoter from cotton from a fiber-specific actin gene (as described in WO0210413), the promoter from a fiber specific lipid transfer protein gene from cotton (as described in U.S. Pat. No. 5,792,933), a promoter from an expansin gene from cotton (WO9830698) or a promoter from a chitinase gene in cotton (US2003106097) or the promoters of the fiber specific genes described in U.S. Pat. Nos. 6,259,003 or 6,166,294.
The invention further provides plant cell walls, comprising fibers including such cell walls obtained from plant cells using the methods according to the invention. Such plant cell walls comprise positively charged oligo- or polysaccharides, such as N-acetylglucosamine oligomers or chitin, embedded into the cellulose. These plant cell walls may be further modified, e.g. partly or completely deacetylated such that oligomers comprising glucosamine residues are obtained. The amino-group of the resulting glucosamines is chemically more reactive than the aminoacetyl group of N-acetylglucosamine or the hydroxyl group of cellulose.
The plant cell wall obtained according to the invention, particularly those which have been subjected to a deacetylation step, can be further chemically modified. Products containing such plant cell walls, such as fibers, yarns or fabrics have qualities resembling those of the cellulose-chitosan blends described in the art, including improved dyeability, improved inhibition of e.g. dermatophytes, controlled drug release etc.
In a specific embodiment, the invention provides cotton fibers obtained from or which can be obtained from cotton plants according to the methods of the invention. In other words, cotton fibers are provided from cotton plants comprising in the genome, such as the nuclear genome, of their cells a chimeric gene comprising a plant-expressible promoter operably linked to a DNA region coding for an N-acetylglucosamine transferase, wherein the N-acetylglucosamine transferase is targeted to the membranes of the Golgi-apparatus or cotton fibers are provided from cotton plants comprising in the genome, such as the nuclear genome, of their cells a chimeric gene comprising a plant-expressible promoter operably linked to a DNA region coding for an N-acetylglucosamine transferase of the NODC type, or cotton fibers are provided from cotton plants comprising in the genome, such as the nuclear genome, of their cells a chimeric gene comprising a plant-expressible promoter operably linked to a DNA region coding for a chitin synthase. Particularly in the latter case, it may be advantageous to apply to the plant cells or plants an effective amount of N-acetylglucosamine or N-acetylglucosamine-1-phosphate or N-acetylglucosamine-6-phosphate or glucosamine-6-phosphate. Particular embodiments of DNA coding regions or promoters comprised in the chimeric genes transferred into cotton plants are as described elsewhere in this document.
The cotton fibers according to the invention can be distinguished from naturally occurring cotton fibers, i.e. cotton fibers obtained from an isogenic line which does not comprise a chimeric gene according to the invention, by the capacity of such fibers for increased staining with anionic dyes (including e.g. Congo Red), by the capacity of such fibers for increased staining with amine-reactive dyes (including e.g. tetrafluorophenyl ester). The cotton fibers according to the invention also have the capacity of binding of Wheat germ agglutinin which binds chito-oligmers. The cotton fibers according to the invention can also be distinguished from naturally occurring cotton fibers by direct detection of the N-acetylglucosamine and GlcNAc oligmers, such as chitobiose, preferably after treatment of the fiber cell wall material with chitinase. The cotton fibers according to the invention may also be distinguished by their increased nitrogen content.
Cotton fibers according to the invention can also be distinguished from the chitosan coated fibers or from chitosan/cellulose blended yarns, in that the positively charged oligomers are more or less evenly distributed in the secondary plant cell walls making up the fibers. Accordingly, in microscopical sections of cotton fibers, stained e.g. with WGA or with congo red or with tetrafluorophenyl as described hereinafter, the dyes will be distributed more or less evenly throughout the cell walls making up the cotton fibers, whereas in chitosan-coated fibers, the staining will be concentrated at the coat of chitosan located as a sheet at the surface of the treated fibers.
Cotton fibers according to the invention can also be distinguished from other cotton fibers by detection of the N-acetylglucosamine transferase comprising chimeric genes in nucleic acids which remain in the plant material associated with cotton fibers.
The increased staining of the plant cell wall material according to the invention, by anionic dyes such as congo-red can be quantified e.g. by dying a uniform amount of material under standard conditions, spreading out the material over a standardized area (such as a well in a multiwell plate) digitalizing a picture of the area for the gray scale of the colored layer of material. The less gray, the more stained the plant cell wall material is. In this way, cotton fibers and cell wall material according to the invention showed an increase of at least about 5% in staining by congo-red compared to control cell wall material or fibers from isogenic plant lines without an N-acetylglucosamine transferase encoding gene.
The capacity of the novel cotton fibers to specifically bind wheat germ agglutin (detectable by the coupled flurophoric group) is a clear distinguishing feature of the provided novel cotton fibers over the naturally occurring cotton fibers. Except for a very low background fluorescence, naturally occurring cotton fibers do not stain/fluoresce when treated with WGA alexa fluor 488 or 555. The fluorescence of cotton fibers increases a least 5 times when chito-oligomers are present. Accordingly, the invention provides cotton fibers which are capable of specifically binding wheat germ agglutinin, or WGA coupled to a flurophore, such as WGA Alexa 488 or WGA Alexa 555 or which, when treated with WGA Alexa 488 or WGA Alexa 555 provide a bright fluorescence under UV light. This fluorescence is not restricted to the surface of the cotton fiber but is distributed throughout the cell wall of the fiber cells.
Plant cell wall material according to the invention, including cotton fibers typically possess chito-oligosaccharides in a concentration of at least 0.1 μg/mg cell wall material, preferably at least 1 μg/mg cell wall material, preferably at least 5 μg/mg cell wall material.
The invention also provides the chimeric genes as herein described, and plant cells or plants containing such chimeric genes.
Wherever the methods of the invention are directed to introduction of a chimeric gene in a plant cell, it will be clear that such methods can also be applied in cases whereby the plant cell is incorporated into a mature plant. E.g. transgenic cells may be regenerated into transgenic plants according to established methods.
Methods to transform plants cells and plants are well known in the art. Methods to transform cotton plants are also well known in the art. Agrobacterium-mediated transformation of cotton has been described e.g. in U.S. Pat. No. 5,004,863 or in U.S. Pat. No. 6,483,013 and cotton transformation by particle bombardment is reported e.g. in WO 92/15675.
The chimeric genes may be introduced by transformation in cotton plants from which embryogenic callus can be derived, such as Coker 312, Coker310, Coker 5Acala SJ-5, GSC25110, FiberMax 819, Siokra 1-3, T25, GSA75, Acala SJ2, Acala SJ4, Acaf a SJ5, Acala SJ-C1, Acala B1644, Acala B1654-26, Acala B1654-43, Acala B3991, Acala GC356, Acala GC510, Acala GAMI, Acala Cl, Acala Royale, Acala Maxxa, Acala Prema, Acala B638, Acala B1810, Acala B2724, Acala B4894, Acala B5002, non Acala “picker” Siokra, “stripper” variety FC2017, Coker 315, STONEVILLE 506, STONEVILLE 825, DP50, DP61, DP90, DP77, DES119, McN235, HBX87, HBX191, HBX107, FC 3027, CHEMBRED A1, CHEMBRED A2, CHEMBRED A3, CHEMBRED A4, CHEMBRED B I, CHEMBRED B2, CHEMBRED B3, CHEMBRED C1, CHEMBRED C2, CHEMBRED C3, CHEMBRED C4, PAYMASTER 145, HS26, HS46, SICALA, PIMA S6 and ORO BLANCO PIMA, Fibermax® FM5013, FM5015, FM5017, FM989, FM832, FM966 and FM958, FM989, FM958, FM832, FM99I, FM819, FM800, FM960, FM966, FM981, FM5035, FM5044, FM5045, FM5013, FM5015, FM5017 or FM5024 and plants with genotypes derived thereof.
“Cotton” as used herein includes Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium herbaceum or progeny from crosses between such species.
The methods and means of the current invention may also be employed for other plant species such as hemp, jute, flax and woody plants, including but not limited to Pinus spp., Populus spp., Picea spp., Eucalyptus spp. etc.
The obtained transformed plant can be used in a conventional breeding scheme to produce more transformed plants with the same characteristics or to introduce the chimeric gene according to the invention in other varieties of the same or related plant species, or in hybrid plants. Seeds obtained from the transformed plants contain the chimeric genes of the invention as a stable genomic insert and are also encompassed by the invention.
As used herein “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA region, which is functionally or structurally defined, may comprise additional DNA regions etc.
The following non-limiting Examples describe the methods for altering plant cell walls. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.
Throughout the description and Examples, reference is made to the following sequences represented in the sequence listing:

    SEQ ID No 1: Nodulation protein C of Azorhizobium caulinodans 
    SEQ ID No 2: Modulation protein C of Bradyrhizobium japonicum 
    SEQ ID No 3: Nodulation protein C of Rhizobium galegae 
    SEQ ID No 4: Nodulation protein C of Rhizobium leguminosartun (biovar viciae)
    SEQ ID No 5: Modulation protein C of Rhizobium meliloti 
    SEQ ID No 6: Nodulation protein C of Rhizobium tropici 
    SEQ ID No 7: Nodulation protein C of Rhizobium leguminosarum (biovar phaseoli)
    SEQ ID No 8: Nodulation protein of Rhizobium sp. Strain N33
    SEQ ID No 9: Nodulation protein of Rhizobium loti 
    SEQ ID No 10: T-DNA of pTGK42
    SEQ ID No 11: T-DNA of pTGK44
    SEQ ID No 12: T-DNA of pTDBI5
    SEQ ID No 13: T-DNA of pTDBI37
    SEQ ID No 14: T-DNA of pTDBI50
    SEQ ID No 15: Synthetic Chitin synthase linked to Golgi-targeting signal.


EXAMPLES
Example 1
Construction of Chimeric Plant-Expressible Genes Encoding a N-Acetylglucosamine Transferase Protein
Using standard recombinant DNA techniques, a plant expressible NODC chimeric gene was constructed containing the following operably linked DNA fragments:

    
    
        a 35S promoter region from CaMV
        a DNA fragment coding for an untranslated leader sequence (5′Cab22L)
        a DNA fragment coding for NODC of Azorhizobium caulinodans 
        a DNA fragment coding for EGFP (enhanced green fluorescent protein) cloned in frame with the NODC encoding ORF, such that a fusion protein is made comprising NODC and EGFP
        a transcription termination and polyadenylation signal from the 35S transcript of CaMV (3′ 35S)
    
    


The chimeric gene was introduced between T-DNA borders of a T-DNA vector together with a chimeric bar gene providing resistance to phosphinotricin. The resulting T-DNA vector was named pTGK44. The sequence of the T-DNA of this vector is provided in SEQ ID No 1 I. This T-DNA vector allowed histochemical analysis of the localization of the NODC-EGFP fusion protein.
Another chimeric gene was constructed containing the following operably linked DNA fragments:

    
    
        a 35S promoter region from CaMV
        a DNA fragment coding for an untranslated leader sequence (5′Cab22L)
        a DNA fragment coding for NODC of Azorhizobium caulinodans 
        a transcription termination and polyadenylation signal from the 35S transcript of CaMV (3′ 35S)
    
    


The chimeric gene was introduced between T-DNA borders of a T-DNA vector together with a chimeric bar gene providing resistance to phosphinotricin. The resulting T-DNA vector was named pTGK42. The sequence of the T-DNA of this vector is provided in SEQ ID No 10. This T-DNA vector allowed to express NODC in plant cells, to analyse whether chito-oligosaccharides were produced associated with the cell wall of such plant cells. A control chimeric gene encoding an N-acetylglucosamine transferase which is different from the NODC type protein was also constructed containing the following operably linked DNA fragments:

    
    
        a 35S promoter region from CaMV
        a DNA fragment coding for an untranslated leader sequence (5′Cab22L)
        a DNA fragment coding for chitin synthase of Neurospora crassa 
        a transcription termination and polyadenylation signal from the 35S transcript of CaMV (3′ 35S)
    
    


The chimeric gene was introduced between T-DNA borders of a T-DNA vector together with a chimeric bar gene providing resistance to phosphinotricin. The resulting T-DNA vector was named pTGK43. The sequence of the T-DNA of this vector is provided in SEQ ID No 12. This T-DNA vector allowed to express chitin synthase in plant cells, to analyse whether chito-oligosaccharides were produced associated with the cell wall of such plant cells.
The T-DNA vectors were introduced into Agrobacterium tumefaciens C58C1Rif(pEHA101). For control experiments, a T-DNA vector containing only a chimeric bar gene was introduced into the same Agrobacterium strain.
The A. tumefaciens strains were subsequently used to generate hairy root cultures from Arabidopsis thaliana by co-transformation of leaf disks with Agrobacterium rhizogenes ATCC15834 and the A. tumefaciens strains carrying the different T-DNA vectors according to the following protocol.
The following media were used:

    Germination medium: MS salts/2, B5 vitamines, 1.5% sucrose, pH 5.8, 0.7% agar (Difco)
    Standard medium: MS medium, 0.5 g/L MES, 2% glucose, pH 5.8, 0.7% agar (Difco)
    Callus inducing medium: MS medium, 0.5 gIL MES, 2% glucose, pH 5.8, 0.7% agar (Difco), 0.2 mg/L 2,4D and 0.2 mg/L kinetine
    Hairy root elongation medium: MS medium, 2% sucrose, pH 6.2, 0.7% agar (Difco)
    Root culture medium: B5 medium, 3% sucrose, pH 5.5.


In vitro Arabidopsis shoots were cultured from sterilized seeds in the following way.
Seeds were treated for 2 minutes with 70% EtOH, followed by a 10 minutes bleach with 6% active chlorine+0.05% Tween 20, and 5 washes with sterile tap water. Seeds were pregerminated in the light (30-50 μEinstein m-2 sec-1) for 24 hours at 24° C. in sterile tap water (about 10-12 mL tap water in 9 cm Falcon Optilux Petridish, nr. 1005).
Pregerminated seeds were put on germination medium and allowed to grow with the following light regime: 12 hours light/12 hours dark or 16 hours light/8 hours dark (30-50 μEinstein m-2 sec-1) at 23-24° C. for about 2-3 weeks. A. rhizogenes strains were grown on agar plates with YEB medium, while A. tumefaciens strains were grown on agar plates with minA medium supplement with antibiotics appropriate to select for the maintenance of the T-DNA vector.
For transformation, leaves were cut in two halves and placed on Callus inducing medium. A. rhizogenes and A. tumefaciens bacteria were resuspended in Standard medium to obtain an OD600 of about 0.2-0.3, mixed in a 1:1 ratio and used for incubation of the leaf pieces for about 5 minutes. Afterwards, the bacterial suspension was removed and infected leaf pieces were placed on Standard medium and incubated for about 3 days (23-24° C.; 30 μEinstein m-2 sec-1; 12 hours light/12 hours dark or 16 hours light/8 hours dark).
Thereafter, leaf explants were washed 3 times with ‘Standard medium’ containing 500 mg/L tricarcillin (Duchefa) and transferred to (20-30 mg/L gluphosinate) ‘Standard medium’ containing (20-30 mg/L gluphosinate and 500 mg/L tricarcillin and further cultured at 23-24° C.; 30 μEinstein m-2 sec-1; 12 hours light/12 hours dark or 16 hours light/8 hours dark. Leaf explants were transferred each week to fresh medium. After 3-4 weeks emerging roots were severed and transferred to ‘Hairy root elongation medium’. When the roots were a few centimeters, a hairy root culture in 250 mL erlenmeyers containing 50 mL ‘Root culture medium’+250 mg/L tricarcillin was started. The cultures were shaken (110 rpm) in the dark at 23-24° C. and subcultured every week. The tricarcillin concentration was reduced gradually. When the roots were growing well, the roots were broken into pieces of about 1.5 cm and the explants were distributed over several crlenmeyers.
Hairy root cultures could also be cultured on solid medium, whereby the cultures were transferred every two weeks to fresh ‘Hairy root elongation medium’
A similar protocol can be used to generate hairy root cultures from cotton.
Example 2
Histochemical Analysis of the Hairy Root Cultures
Root hairs of the different hairy root cultures obtained by co-infection between A. rhizogenes and A. tumefaciens carrying the different T-DNA vectors described in Example 1 were histochemically stained to visualize different compounds of the cells, and analyzed microscopically.
The localization of NODC-EGFP fusion protein can be visualized using the green fluorescence of the GFP part. N-acetylglucosamine can be either detected after immunological reaction with IgM monoclonal antibodies to N-acetylglucosamine (BIODESIGN) or using Wheat Germ Agglutin-Alexa Fluor 488. The endoplasmatic reticulum was stained using ER-Tracker Blue White DPX dye. The Golgi apparatus was visualized using BODIPY-TR. Cell walls were stained using Calcofluor White (Fluorescent brightener 28). Nuclei were stained using Hoechst 33342.
The histochemically stained root hair cells were examined by means of fluorescence microscopy, using an Axioplan 2 microscope (Zeiss, Jena, Germany) equipped with Apotome (Zeiss) to allow optical sections. Axio Vision 4.2 (Zeiss) was used for image processing.
The following protocols were used for the different histochemical methods:
A. Calcofluor Staining of Cell Walls.
Calcofluor White (or Fluorescent Brightener 28) is a colourless organic compound that fluoresces in a clear bluish color under ultraviolet radiation (λmax=350 nm).
The specimen to be stained is immersed for 15 to 30 minutes in culture medium or PBS (buffer solution) comprising Fluorescent Brightener 28 at 50 μg/mL final concentration. The specimen is then washed with medium or buffer, and samples are examined using a microscope equipped for fluorescence microscopy using Zeiss filter set 18. Cell walls fluoresce in a clear bluish color.
B. Histochemical Staining the Golgi Complex and Endoplasmatic Reticulum in Living Cells
For staining the Golgi complex, roots cultured for about 5 days in liquid root culture medium were used. These roots were rinsed with fresh root culture medium and incubated for about 30 minutes at 4° C. with 1 μM BODIPY TR C5-ceramide (Molecular probes, Cat No B-34400). The roots were rinsed a few times with root culture medium and incubated in fresh root culture medium at room temperature for 30 minutes with gentle shaking. The roots were then rinsed with fresh root culture medium and examined with a fluorescence microscope Axioplan 2 (Zeiss, Jena, Germany) using Filterset 00 (excitation: BP530/585; emission: LP615).
For staining the ER, roots cultured for about 5 days in liquid root culture medium were used. These roots were rinsed with fresh root culture medium and incubated with ER-Tracker Blue-White DPX (100 nM) dissolved in root culture medium for about 2 hours with gentle shaking. The roots were rinsed a few times with root culture medium and examined with a fluorescence microscope Axioplan 2 (Zeiss, Jena, Germany) using Filterset 02 (excitation: G365; emission: LP420).
C. Whole Mount Immunohistochemical Detection of Incorporated N-Acetylglucosamine in the Cell Wall of Roots (Root Hairs)
Roots of the different hairy root cultures were grown in liquid culture for 6 days, either supplemented with 50 mM GlcNAc or without any supplement. The roots were fixed and dehydrated, rehydrated and cell wall permeabilized in the following way.
When the sample has been incubated with N-acetylglucosamine the excess N-acetylglucosamine is washed away by incubating 4 times 10 min. with PBS solution.
Samples were fixed by incubation and vacuum infiltration of AA solution (incubation: four times 1 hr, each time followed by 5 min vacuum infiltration). AA solution contains 50% EtOH and 5% acetic acid.
In a next step the samples are dehydrated by rinsing with 50% EtOH and washing 2×30 min. with 50% EtOH, followed by 60 min. incubation in 70% EtOH. Samples can be stored at this stage at −20° C.
Subsequently, the samples were subjected to cell wall permeabilisation, by washing 5 min. with 50% EtOH, washing 2×5 min. with PBT (PBS with 0.1% Tween20), washing 2×5 min. with PBT+0.3% Triton X100 and finally washing 2×5 min. with PBS (150 mM NaCl; 10 mM Na-phosphate buffer; pH 7.4).
Permeabilized roots are transferred to a Petridish containing MQ-water, and mounted on a “Vectaboin-treated” microscope slide. The slides are baked on a TLC plate heater for 45 min at 55° C. A blocking step is performed by incubating the slides for one hour with blocking solution (1% BSA in PBT). Afterwards, the 1% blocking solution is replaced with about 400 μL IgM monoclonal antibody to N-acetylglucosamine (1 μg/mL in blocking solution; BIODESIGN, Cat No H67108M) and incubated for one hour. The slides are subsequently washed 3 times 5 to 10 minutes with blocking solution. The blocking solution is then replaced with about 400 μL Goat Anti-Mouse 1 gM antibody labelled with Alexa Fluor 488 (3 μg/mL in blocking solution; Molecular Probes, Cat No A-21042) and incubated for one hour. Thereafter, slides are washed 5 to 10 minutes with blocking solution, 2 times 5 to 10 minutes with PBT and a few times with PBS to remove Tween20. Results are evaluated by means of fluorescence microscopy with an Axioplan 2 microscope (Zeiss, Jena, Germany) using Filterset 38 (excitation: BP470/40; emission: BP525/50).
D. Wheat Aglutinin Mediated Detection of N-Acetylglusoamine
Roots of the different hairy root cultures were grown in liquid culture for 6 days, either supplemented with 50 mM GlcNAc or without any supplement. The roots were fixed and dehydrated, rehydrated and cell wall permeabilized as described in section C above.
Wheat germ agglutinin selectively binds to N-acetylglucosamine and N-acetylneuraminic acid residues. N-acetylneuraminic acid does not occur in plants. Therefore, in plants, wheat germ agglutinin can be used to specifically detect N-acetylglucosamine residues.
Permeabilized roots were placed in 9 cm Petri Dishes containing PBT. The roots were thereafter transferred to to the wells of 6-well culture plates containing about 1.7 μg/mL Wheat Germ Agglutinin labelled with Alexa Fluor 488 (Molecular Probes, Cat No W-11261) in PBT and incubated for one hour. The samples were subsequently washed for 3×10 minutes with PBT and twice for 5 minutes with PBS (to remove Tween20). The samples were placed on a a ‘Vectabond-treated’ or ‘Tissue Tack’ microscope slide in a drop of PBS. After removal of most of the PBS the coverslide was mounted. Results were evaluated by means of fluorescence microscopy using a fluorescence microscope Axioplan 2 (Zeiss, Jena, Germany) using Filterset 38 (excitation: BP470/40; emission: BP525/50).
E. GFP Analysis.
EGFP fluorescence was evaluated by means of fluorescence microscopy with an Axioplan 2 microscope (Zeiss, Jena, Germany) using Filterset 38 (excitation: BP470/40; emission: BP525/50).
Results
1. Localization of N-Acetylglucosamine in the Cell Wall

    
    
        Root hair cells comprising the chimeric NODC gene were immunohistochemically stained for the presence of N-acetylglucosamine and subsequently stained with Calcofluor to visualize the cell walls. FIG. 3 shows a representative set of photographs of optical sections using a fluorescence microscope, whereby panel A shows the (blue) fluorescence visualizing the cell wall, and panel B shows the (green) fluorescence visualizing N-acetylglucosamine. As can be seen from the superposition of both optical sections in panel C, the presence of N-acetylglucosamine is exclusively detected in the cell walls of the root hair cells.
    
    


2. Co-Localization of NODC Proteins and the Golgi Apparatus

    
    
        Root hair cells comprising the chimeric gene expressing the NODC-EGFP fusion protein were stained to visualize the Golgi apparatus and subsequently stained with Calcofluor to visualize the cell walls. FIG. 4 shows a representative set of photographs of optical sections using a fluorescence microscope, whereby panel B shows the (blue) fluorescence visualizing the cell wall, panel C shows the (red) fluorescence associated with the Golgi apparatus and panel D shows the (green) fluorescence visualizing the NODC-EGFP fusion protein. As can be seen from the superposition of the optical sections in panel A, the localization of the NODC-EGFP fusion protein coincides with the localization of the Golgi apparatus in the root hair cells.
    
    


3. Chitin Synthase Expression in Plant Cells Requires External Feeding with GlcNAc to Detect N-Acetylglucosamine in the Cell Walls.

    
    
        Roots expressing a chimeric chitin synthase from Neurospora crassa were cultured as described above in the presence or absence of externally added N-acetylglucosamine. After careful washing, the roots were histochemically stained to detect N-acetylglucosamine. In FIG. 5, panel A (hairy roots with external GlcNAc feeding) numerous green fluorescent spots can be detected, whereas in panel B (hairy roots without external GlcNAc feeding) very few green fluorescent spots could be detected.
    
    


Example 3
Biochemical Demonstration of Chitin-Like Oligomers in the Cell Wall of Arabidopsis p35S::NODC Hairy Roots
Arabidopsis thaliana (Col-0) hairy roots transgenic for p35S:NODC-p35S:bar, obtained using T-DNA vector pTGK42 and control Arabidopsis thaliana (Col-0) hairy roots transgenic for p35S:bar obtained using pTCO192 (control) were analyzed for the presence of N-acetylglucosamine using the Morgan-Elson assay.
To this end, about 100 mg of hairy roots were harvested in about 20 μL buffer (25 mM K-phosphate buffer pH 6.0) and ground with seasand, precipitated and the protein content of the extract determined (for standardization purposes). The supernatans was removed and the roots were resuspended in 100 μL buffer. I Unit cellulase (Cellulase “Onozuka R-10” from Trichoderma viride (Serva, Cat No 16419): 10 U/mL in buffer) or 1 Unit chitinase (Chitinase from Serratia marcescens (Sigma, Cat No C1650): 10 U/mL in buffer) or both were added to different samples and incubated for overnight at 25° C.
A Morgan-Elson asssay to measure N-acetylglucosamine was performed on the samples the next morning. In the employed colorimetric method, which is based on the Morgan-Elson reaction, the N-acetyl-glucosamine reducing end is successively transformed into the chromogens I and II under the alkaline conditions at 100° C. Subsequent treatment with a mixture of concentrated HCI and concentrated sulfuric acid results in elimination of water yielding the chromogen III of the Morgan-Elson reaction. In the final step of the reaction, chromogen III is allowed to react with DMAB, p-dimethylaminobenzaldehyde (Ehrlich's reagent) to form a red-colored product the concentration of which can be determined by measuring the absorption at 585 nm.
UDP N-acetylglucosamine, and N-acetylglucosamine-1-phosphate fail to give the test unless they are previously hydrolyzed with acid. The nucleotides can be hydrolyzed by heating at 100° in 0.01 N acid for 15 min. but the sugar phosphate requires more rigorous conditions, e.g. 5 minutes at 100° in 0.1 N HCl.
The results are summarized in Table 2.







TABLE 2







OD585 values after Morgan-Elson assay











Hairy root



Cellulase


culture
Buffer
Cellulase
Chitinase
Chitinase





Control
0.070
0.100
0.117
0.157


p35S:NODC
0.056
0.096
0.100
0.252









From these results it can be concluded that chitin-like polymers are embedded in the cell wall.
Example 4
Analysis of Chito-Oligo and Monosaccharides in Plant Cell Wall Material Using HPTLC
Cell walls of the Arabidopsis hairy roots of Example 1 (35S::NODC; 35S::NODC_EGFP) and 35S::bar control hairy roots were prepared according to the following protocol
Preparation of Cell Walls


    
    
        Harvest hairy roots; remove most of the medium with a tissue
        Wash tissue with PBS buffer
        Put about 1 g of tissue in a tube
        Freeze with liquid nitrogen
        Grind tissue in a mortar
        Transfer grinded tissue in a funnel with cheese cloth
        Wash with a few liters of demineralized water
        Seal the cheese cloth and transfer to 500 mL bottle containing 500 mL ethanol
        Wash 15 minutes with 500 mL of ethanol, refresh ethanol and wash for another 15 minutes
        Replace ethanol by 250 mL ether and wash 15 minutes.
    
    


The remaining material is the ‘cell wall material’. Dry and weigh the cell wall material and transfer it to a tube
Extraction of Chito-Oligos from Cell Wall Material


    
    
        Add 300 μL MQ-water to 10 mg of cell wall material (use a 25 or 50 ml tube), boil 3 minutes and incubate for 2 hours at 80° C. (shake). The cell wall material can be digested with chitinase and β-N-Acetylglucosaminidase Enzyme mixture: 0.5 U chitinase in 50 μL 125 mM Na-phosphate-2 mM CaCl2-pH 6 (Chitinase Sigma C7809 or C6137→digest (chito-oligo) saccharides very quickly to N-acetylglucosamine. Chitinase BioLabs P5206S→digest penta-N-acetylchitose (slowly) to di- and tri-chito-oligos)
        Add 100 μL enzyme mixture to about 5 mg cell wall material
        Incubate overnight at 25° C.
        The buffer can be separated from the cell wall material by centrifugation
    
    


After extraction, the buffer containing the chito-oligos is spotted on HPTLC plates (HPTLC plates NH2 (without fluorescent indicator) 10×20 cm (Merck, Art. 12572)) along with a mixture of chito-oligosaccharide standard solution in H2O. The standard solution comprises N-acetylglucosamine, chitobiose, chitotriose, chitotetraose and chitopentaose.
The following developing solvents can be used:

    
    
        n-butanol (70): acetic acid (20): H2O (10) (Buffer D)
        acetonitrile (76): H2O (24): 0.5% aqueous solution of boric acid (10) (Buffer A)
        acetonitrile (10): isopropanol (67): 50 mM KCl (23) (Buffer B)
        n-butanol (50): ethanol (30): H2O (20) (Buffer C)

Chromatography

        Clean plates by developing with methanol
        Spot 1(−2) μL of standard solutions (15 mm from bottom) and 1 to 5 μL of samples in bands of about 6 mm length
        Develop plates in ‘CAMAG Twin Through Chamber’: 7 to 7.5 cm migration distance
        Twin Through Chamber for: 10×10 cm plates→10 mL developing solution
        
            20×10 cm plates→20 mL developing solution
        
        
        Dry plates with ventilator
        Heat plates at about 150° C. for 20 minutes (TLC plate heater)
        Visualize sugars with UV (366 nm)

2D-Chromatography

        Clean plates by developing with methanol
        Spot 1 to 5 μL of samples in band of 3 mm: right angle of plate (15 mm from bottom and 15 mm from site)
        Develop plates in the first direction in ‘CAMAG Twin Through Chamber’: 7 to 7.5 cm migration distance
        Twin Through Chamber for: 10×10 cm plates→10 mL developing solution
        Dry plates with ventilator
        Develop plates in other direction
        Dry plates with ventilator
        Heat plates at about 150° C. for 20 minutes (TLC plate heater)
        Visualize sugars with UV (366 nm)
    
    


FIG. 6 shows the results of one dimensional HPTLC, whereby the cell wall material was extracted but not further digested with chitinase. FIG. 7 shows the results of a two dimensional HPTLC after digestion with chitinase. No chito-oligmers can be detected in the control plants, but significant amounts of chito-oligomers is present in plants comprising an N-acetylglucosamine transferase gene.
Transgenic Arabidopsis plants were also generated using the chimeric genes described in Example 1. This material is more uniform than the hairy root cultures described above. Cell wall material was prepared, chito-oligosaccharides extracted as described and HPTLC performed in Buffer A as herein described. The results are shown in FIG. 8.
Cell wall material from transgenic 35S::NodC Arabidopsis shoots showed a high amount of chito-triose, estimated by comparison to the standard solutions to be about 5 μg/mg cell wall material or 0.01% of the fresh leaf material.
Example 5
Staining of Plant Cell Wall Material from Arabidopsis Hairy Root Cultures
Arabidopsis hairy root cultures were generated as in Example 1, cell wall material thereof was prepared as described in Example 5 and stored at −20° C. The cell wall material was stained with an anionic dye (Congo Red) or an amino reactive dye (Alexa Fluor 488 tetrafluorophenyl ester.
A. Congo Red Staining

    
    
        Cell wall material stored at −20° C. from NODC hairy roots or control plants was rehydrated in acetate buffer pH 5 (50 mg cell wall material/tube)
        The material was stained with 0.03% Congo red dissolved in acetate buffer pH5
        The cell wall material was washed with acetate buffer pH5 and with PBS buffer for a few times
        All the cell wall material was transferred to the wells of an 48 multiwell plate
        Under standard illumination conditions, digital images were taken from individual wells and the mean gray value of the digital image was determined

Results:

Sample 1:

    
    


















Mean
Min
Max
IntDen
Median




















NodC
89.390
76
126
3460186
89


control
97.761
83
136
3784246.000
97









Sample 2

















Mean
Min
Max
IntDen
Median






















NodC
101.548
86
130
3479355
101



control
108.109
90
139
3704154
108









Sample 3

















Mean
Min
Max
IntDen
Median






















NodC
97.866
81
120
3650012
98



control
104.634
85
134
3902418
104









    
    
        Cell wall material from hairy roots containing a chimeric NodC gene reproducibly stained more intense than cell wall material from control plants. The gray values of the cell wall material was about 5-10% lower than that of control plants.
    
    


B. Alexa Fluor 488 Tetrafluorophenyl Ester Staining

    
    
        Cell wall material stored at −20° C. from NODC hairy roots or control plants was rehydrated in PBS buffer pH 5 (50 mg cell wall material/tube), and treated with proteinase K (100 μg/ml) overnight at 56° C.
        The material was intensively washed with PBS buffer and labelled with Alexa Fluor 488 tetrafluorophenyl ester. Alexa fluor 488 TFP ester is available as a kit (Alexa fluor 488 Monoclonal Antibody Labeling Kit (Molecular Probes, A-20181)
        The stained material can be examined using fluorescence microscopy e.g. with Zeiss filter 38.

Cell wall material from hairy roots containing a chimeric NodC gene reproducibly stained more intense than cell wall material from control plants.

    
    


Example 6
Transgenic Cotton Plants
Transgenic cotton plants comprising a chimeric NODC gene as described in Example 1, or a chimeric NODC gene under control of the F285 fiber-selective promoter (as described in US2003/106097) are generated using the method as described in U.S. Pat. No. 6,483,013.
Fibers from these transgenic cotton plants are isolated, and used to produce yarns and fabrics with improved reactivity, such as improved dyeability.
Example 7
Cotton Fibers with Increased Reactivity
Transgenic cotton plants comprising a chimeric NodC coding region operably linked to a CaMV35S promoter were generated as described in Example 6. Mature cotton fibers were harvested from these plants and stained with Congo Red or reacted with WGA-Alexa fluor 555.
A. Congo Red staining
Mature cotton fibers were harvested from transgenic cotton plants comprising a chimeric NodC gene, as well as from control plants not comprising a chimeric NodC gene. Lipids were removed from the fibers by washing with ethanol and ether. The cotton fibers were dryed.
Twenty-five mg of fibers were rehydrated in acetate buffer pH5 and stained with 0.03% Congo red dissolved in acetate buffer pH5. The cell wall material was washed with acetate buffer pH5 and with PBS buffer for a few times.
The stained fibers were analyzed by bright field microscopy and by fluorescence microscopy (Zeiss filter 18). Digital images of stained fibers in a 48 multiwell plate were also analyzed as described in Example 5A.
Under bright field microscopy, the cotton fibers harvested from the NodC transgenic cotton plants appeared more intense red than the cotton fibers from the non-transgenic plants. This difference was even more pronounced when analyzing the fibers under fluorescence microscopy.
The mean gray values obtained for the congo red stained cotton fibers from the transgenic NodC plants were also significantly lower than for the cotton fibers from non-transgenic plants, confirming the more intense staining by anionic dyes of the fibers of the transgenic cotton plants.

















Area
Mean
Min
Max
IntDen
Median







NodC
38958
81.388
72
179
3170716
80


control
38958
86.558
79
160
3372108
85









The difference in staining was maintained and even intensified when fibers were treated for at least one hour with hot NaOH (60% at 80° C.). This treatment removes proteins, pectic substances and waxes, and is capable of deacetylating the chito-oligomers.
The intensified congo-red stain was moreover distributed evenly in the cell wall as can be observed when virtual microscopic sections of individual fiber cells were made.
B. WGA-Alexa 555 Staining
Detection of N-acetylglucosamine oligomers in cotton fibers from transgenic NodC plants was done essentially as described in Example 2. Cotton fibers do not need to be dehydrated or permeabilized. Instead, lipids and waxes were removed by treating the fibers for 3 times 10 minutes in a chloroform: methanol mixture (1:1), follow by twice a treatment of 10 minutes in acetone and twice 5 minutes in ether. The fibers were allowed to air dry.
Fibers were stained with either WGA-Alexa555, WGA-Alexa488 or WGA-tetramethylrhodamine.
The fibers were placed in blocking solution (150 mM NaCL, 10 mM sodiumphosphate buffer pH 7.4; 0.1% Tween 20 and 1% bovine serum albumin) and incubated for one hour. Thereafter, the buffer was replaced by the same buffer containing WGA-fluorochrome and incubated for 4 hrs. The WGA-fluorochrome solution was replaced by blocking solution, washed 10 minutes, followed by 3 times 10 min washing with blocking solution without BSA, and 2 times 5 min washing with blocking solution without BSA and without Tween. The stained fibers were mounted on a microscope slide and evaluated by means of fluorescence microscopy (Axioplan 2 (Zeiss, Jena, Germany) using Filterset 38 (exitation: BP470/40; emission: BP525/50) for Alexa fluor 488 conugate or Filterset 20 (exitation: BP546/12; emission: BP575-640) for Alexa fluor 555 or tetramethylrhodamine conjugate.
Whereas no specific fluorescence could be detected in cotton fibers from non-transgenic plants, a bright fluorescence was detectable in cotton fibers from chimeric NodC gene comprising cotton plants (see FIG. 9). Virtual microscopic sections of the cotton fibers indicated that the WGA-fluor555 is evenly distributed throughout the secondary cell wall of the cotton fiber cells.
Example 8
Reactivity of Cell Walls of Arabidopsis Hairy Roots Comprising a Chitin Synthase from Neurospora crassa with Golgi-Targeting Signal
Using standard recombinant DNA techniques, a plant expressible N-acetylglucosamine transferase comprising a heterologous Golgi-targeting signal sequence, was constructed containing the following operably linked DNA fragments:

    
    
        a 35S promoter region from CaMV
        a DNA fragment coding for an untranslated leader sequence (5′Cab22L)
        a DNA fragment coding for the 35 N-terminal amino acids of β-1,2-xylosyltransferase from Arabidapsis thaliana 
        a DNA fragment coding for CHS2 (chitin synthase) of Neurospora crass cloned in frame with the previous DNA fragment
        a transcription termination and polyadenylation signal from the 35S transcript of CaMV (3′ 35S)
    
    


The chimeric gene was introduced between T-DNA borders of a T-DNA vector together with a chimeric bar gene providing resistance to phosphinotricin. The resulting T-DNA vector was named pTDBI37. The sequence of the T-DNA of this vector is provided in SEQ ID No 13.
The T-DNA vectors were introduced in A. tumefaciens and used to generate hairy root cultures as described in Example 1.
N-acetylglucosamine oligomers could be detected in the cell wall of the hairy root cultures after incubation with chitine binding domain conjugated to fluorescein, by fluorescence microscopy.
N-acetylglucosamine oligomers could also be detected in the cell wall of the hairy root cultures using WGA-Alexa555 as described in Example 2. In addition, fluorescence could also be observed associated with globules in the cytoplasm, corresponding to the Golgi-apparatus.
Example 9
Determination of the Nitrogen Content of Cotton Fibers
Mature cotton balls were harvested from the transgenic cotton plants of Example 7. From each ball, 20 mg of cleaned fibers were assayed. To this end, lipids and waxes were removed from the fibers by washing 3 times 20 minutes in a chloroform:methanol (1:1) mixture; 2 times 20 minutes in acetone; 2 times 5 minutes in ether and allowed to air dry.
The total nitrogen at the surface of fibers was measured using ‘Total Nitrogen’ analysis kit and C214 Multiparameter Bench Photometer of HANNA Instruments (Rhode Island, USA).
The following results were obtained:













Wild type
Transgenic



















Mean
 56 mg/L N
 85 mg/L N



Number of cotton balls
9
10



Standard error
2.6 mg/L N
5.1 mg/L N



t-test (α = 0.05)





P two tailed
2.2 × 10−4









The fibers from the cotton balls from the transgenic lines contained statistically significant more nitrogen at the surface than the fibers from wild type cotton balls.
Example 10
Fiber Specific Expression of a Chitin Synthase in Cotton
Using standard recombinant DNA techniques, a plant expressible N-acetylglucosamine transferase comprising a heterologous Golgi-targeting signal sequence, was constructed containing the following operably linked DNA fragments:

    
    
        a fiber specific promoter region from cotton
        a DNA fragment coding for an untranslated leader sequence (5′Cab22L)
        a DNA fragment coding for the 35 N-terminal amino acids of β-1,2-xylosyltransferase from Arabidopsis thaliana 
        a DNA fragment coding for CHS2 (chitin synthase) of Neurospora crassa cloned in frame with the previous DNA fragment
        a transcription termination and polyadenylation signal from the 35S transcript of CaMV (3′ 35S)
    
    


The chimeric gene was introduced between T-DNA borders of a T-DNA vector together with a chimeric bar gene providing resistance to phosphinotricin. The resulting T-DNA vector was named pTDBI50. The sequence of the T-DNA of this vector is provided in SEQ ID No 14.
The T-DNA vectors are introduced in A. tumefaciens and used to generate transgenic cotton. Fibers isolated from cotton balls of transgenic plants have an increased amount of N-acetylglucosamine oligomers, more or less evenly distributed throughout the cell wall.

## Claims

1. A chimeric gene, comprising the following operably linked DNA regions:
a.) a plant-expressible promoter;
b.) a DNA region coding for an N-acetylglucosamine transferase, wherein said N-acetylglucosamine transferase is a chitin synthase comprising a signal anchor sequence for targeting the membranes of the Golgi apparatus or wherein said N-acetylglucosamine transferase is of the NODC type, and wherein said N-acetylglucosamine transferase comprises the amino acid sequence of SEQ ID No. 15 from position 1 to position 35; and
c.) a transcription termination and polyadenylation region.

2. A plant cell wall obtained by a method comprising introducing the chimeric gene of claim 1 into a plant cell.

3. A cotton fiber comprising the chimeric gene of claim 1.

4. A plant cell comprising the chimeric gene of claim 1.

5. A plant comprising the plant cell of claim 4.

6. The plant of claim 5, wherein said plant is cotton.

7. A chimeric gene, comprising the following operably linked DNA regions:
a.) a plant-expressible promoter;
b.) a DNA region coding for an N-acetylglucosamine transferase, wherein said N-acetylglucosamine transferase is a chitin synthase comprising a signal anchor sequence for targeting the membranes of the Golgi apparatus or wherein said N-acetylglucosamine transferase is of the NODC type, and wherein said N-acetylglucosamine transferase comprises the amino acid sequence of SEQ ID No. 15; and
c.) a transcription termination and polyadenylation region.

8. The chimeric gene according to claim 1, wherein said NODC type N- acetylglucosamine transferase is obtainable from a Rhizobium species, an Azorhizobium species, a Bradyrhizobium species, a Mesorhizobium species, a Ralstonia species, a Streptomyces species, a Burkholderia species or a Sinorhizobium species.

9. The chimeric gene according to claim 1, wherein said NODC type N-acetylglucosamine transferases comprises the amino acid sequence of SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, or SEQ ID No 9.

10. The chimeric gene according to claim 1, wherein said promoter is a fiber-selective promoter of a beta tubulin gene from cotton, a fiber-specific promoter from an actin gene from cotton, a fiber specific promoter from a lipid transfer protein gene from cotton, a promoter from an expansin gene from cotton or a promoter from a chitinase gene in cotton.

11. A plant cell wall obtained by a method comprising introducing the chimeric gene of claim 7 into a plant cell.

12. A cotton fiber comprising the chimeric gene of claim 7.

13. A plant cell comprising the chimeric gene of claim 7.

14. The chimeric gene according to claim 7, wherein said NODC type N-acetylglucosamine transferase is obtainable from a Rhizobium species, an Azorhizobium species, a Bradyrhizobium species, a Mesorhizobium species, a Ralstonia species, a Streptomyces species, a Burkholderia species or a Sinorhizobium species.

15. The chimeric gene according to claim 7, wherein said NODC type N-acetylglucosamine transferases comprises the amino acid sequence of SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, or SEQ ID No 9.

16. The chimeric gene according to claim 7, wherein said promoter is a fiber-selective promoter of a beta tubulin gene from cotton, a fiber-specific promoter from an actin gene from cotton, a fiber specific promoter from a lipid transfer protein gene from cotton, a promoter from an expansin gene from cotton or a promoter from a chitinase gene in cotton.