Patent Application: US-43391606-A

Abstract:
the present invention relates generally to isolated genes which encode polypeptides involved in cellulose biosynthesis in plants and transgenic plants expressing same in sense or antisense orientation , or as ribozymes , co - suppression or gene - targeting molecules . more particularly , the present invention is directed to a nucleic acid molecule isolated from arabidopsis thaliana , oryza sativa , wheat , barley , maize , brassica spp ., gossypium hirsutum and eucalyptus spp . which encode or an enzyme which is important in cellulose biosynthesis , in particular the cellulose synthase enzyme and homologues , analogues and derivatives thereof and uses of same in the production of transgenic plants expressing altered cellulose biosynthetic properties .

Description:
the arabidopsis thaliana rsw1 mutant was produced in a genetic background comprising the ecotype columbia . the altered root cell - shape and temperature sensitivity of the root morphology of the arabidopsis thaliana mutant rsw1 are disclosed , among other morphological mutants , by baskin et al . ( 1992 ). as shown in fig1 , the present inventors have shown that the rsw1 mutant exhibits the surprising phenotype of having reduced inflorescence height when grown at 31 ° c ., compared to wild - type columbia plants grown under similar conditions . in contrast , when grown at 21 ° c ., the inflorescence height of rsw1 is not significantly different from wild type plants grown under similar conditions , indicating that the shoot phenotype of rsw1 is conditional and temperature - dependent . furthermore , cryo - scanning electron microscopy of the epidermal cells of the rsw1 mutant indicates significant abnormality in cell shape , particularly in respect of those epidermal cells forming the leaves , hypocotyl and cotyledons , when the seedlings are grown at 31 ° c . ( fig2 ). rosettes ( terminal complexes ) are the putative hexameric cellulose synthase complexes of higher plant plasma membranes ( herth , 1985 ). freeze - fractured root cells of arabidopsis thaliana rsw1 plants grown at 18 ° c . show cellulose microfibrils and rosettes on the pf face of the plasma membrane that resembles those of wild - type a . thaliana and other angiosperms . transferring the rsw1 mutant to 31 ° c . reduces the number of rosettes in the mutant within 30 min , leading to extensive loss after 3 hours . plasma membrane particles align in rows on prolonged exposure to the restrictive temperature . in contrast , there is no change in the appearance of cortical microtubules that align cellulose microfibrils , or of golgi bodies that synthesise other wall polysaccharides and assemble rosettes . the effect of mutations in the rsw1 gene on the synthesis of cellulose and other carbohydrates was assessed by measuring in vivo incorporation of 14 c ( supplied as uniformly labeled glucose ) into various cell wall fractions . wild type ( rsw1 ) and homozygous mutant rsw1 seed were germinated at 21 ° c . on agar containing hoagland &# 39 ; s nutrients and 1 % ( w / v ) unlabelled glucose . after 5 d , half of the seedlings were transferred to 31 ° c . for 1 d while the remainder was maintained at 21 ° c . for the same time . seedlings were covered with a solution containing hoagland &# 39 ; s nutrients and 14 c - glucose and incubated for a further 3 h at the same temperature . rinsed roots and shoots were separated and frozen in liquid nitrogen . tissue was homogenised in cold , 0 . 5 m potassium phosphate buffer ( 0 . 5m kh2po4 , ph7 . 0 ) and a crude cell wall fraction collected by centrifugation at 2800 rpm . the wall fraction was extracted with chloroform / methanol [ 1 : 1 ( v / v )] at 40 ° c . for 1 hour , followed by a brief incubation at 150 ° c ., to remove lipids . the pellet was washed successively with 2 ml methanol , 2 ml acetone and twice with 2 ml of deionised water . finally , the pellet was extracted successively with dimethyl sulphoxide under nitrogen to remove starch ; 0 . 5 % ammonium oxalate to remove pectins ; 0 . 1 m koh and 3 mg / ml nabh 4 and then with 4 m koh and 3 mg / ml nabh 4 to extract hemicelluloses ; boiling acetic acid / nitric acid / water [ 8 : 1 : 2 ( v / v )], to extract any residual non - cellulosic carbohydrates and leave crystalline cellulose as the final insoluble pellet ( updegraph , 1969 ). all fractions were analysed by liquid scintillation counting and the counts in each fraction from the mutant were expressed as a percentage of the counts in the wild type under the same conditions . as shown in table 3 , mutant and wild type plants behave in quite similar fashion at 21 ° c . ( the permissive temperature ) whereas , at the restrictive temperature of 31 ° c ., the incorporation of 14c into cellulose is severely inhibited ( to 36 % of wild type ) by the rsw1 mutation . the data in table 3 indicate that cellulose synthesis is specifically inhibited in the rsw1 mutant . the wild type rsw1 gene is therefore involved quite directly in cellulose synthesis and changing its sequence by mutation changes the rate of synthesis . in homozygous mutant rsw1 plants , the pectin fraction extracted by ammonium oxalate contained abundant glucose , atypical of true uronic acid - rich pectins . the great majority of the glucose remained in the supernatant when cetyltrimethylammonium bromide precipitated the negatively charged pectins . the quantity of cellulose and the quantity of a non - crystalline β - 1 , 4 - glucan recovered from the ammonium oxalate fraction were determined for seedlings of wild type columbia and for backcrossed , homozygous rsw1 that were grown for either 7 days at 21 ° c . or alternatively , for 2 days at 21 ° c . and 5 days at 31 ° c ., on vertical agar plates containing growth medium ( baskin et al ., 1992 ) plus 1 % ( w / v ) glucose , and under continuous light ( 90 μmol m − 2 s − l ). roots and shoots were separated from about 150 seedlings , freeze - dried to constant weight and ground in a mortar and pestle with 3 ml of cold 0 . 5 m potassium phosphate buffer ( ph 7 . 0 ). the combined homogenate after two buffer rinses ( 2 ml each ) was centrifuged at 2800 × g for 10 min . after washing the pellet fraction twice with 2 ml buffer and twice with 2 ml distilled water , the pellet , comprising the crude cell wall fraction , and the pooled supernatants , comprising the phosphate buffer fraction were retained . the crude cell wall pellet fraction was stirred with two 3 ml aliquots of chloroform / methanol [ 1 : 1 ( v / v )] for 1 hour at 40 ° c ., 2 ml of methanol at 40 ° c . for 30 min , 2 ml of acetone for 30 min , and twice with water . the whole procedure repeated in the case of shoots . combined supernatants were dried in a nitrogen stream . the pellet was successively extracted with : ( i ) 3 ml of dmso - water 9 : 1 [ v / v ], sealed under nitrogen , overnight with shaking , followed by two 2 ml extractions using dmso / water and three 2 ml water washes ; ( ii ) 3 ml of ammonium oxalate ( 0 . 5 %) at 100 ° c . for 1 hour , followed by two water washes ; ( iii ) 3 ml of 0 . 1 m koh containing 1 mg / ml sodium borohydride , for 1 hour at 25 ° c . ( repeated once for root material or twice for shoot material ), with a final wash with 2 ml water ; ( iv ) 3 ml of 4 m koh containing 1 mg / ml sodium borohydride , for 1 hour at 25 ° c . ( repeated once for root material or twice for shoot material ). the final pellet was boiled with intermittent stirring in 3 ml of acetic acid - nitric acid - water [ 8 : 1 : 2 ( v / v )] ( updegraph 1969 ), combined with 2 water washes , and diluted with 5 ml water . the insoluble residue of cellulose was solubilised in 67 % ( v / v ) h 2 so 4 , shown to contain greater than 97 % ( w / v ) glucose using gc / ms ( fisons as800 / md800 ) of alditol acetates ( doares et al ., 1991 ) and quantified in three independent samples by anthrone / h 2 so 4 reaction . results of gc / ms for pooled replica samples are presented in table 4 . the non - crystalline β - 1 , 4 - glucan was recovered as the supernatant from the ammonium oxalate fraction when anionic pectins were precipitated by overnight incubation at 37 ° c . with 2 % ( w / v ) cetyltrimethylammonium bromide ( ctab ) and collected by centrifugation at 2800 × g for 10 min . the glucan ( 250 μg / ml ) or starch ( sigma ; 200 μg / ml ) were digested with mixtures of endocellulase ( ec 3 . 2 . 1 . 4 ; megazyme , australia ) from trichoderma and almond β - glucosidase ( ec 3 . 2 . 1 . 21 ; sigma ), or bacillus sp . α - amylase ( ec 3 . 2 . 1 . 1 ; sigma ) and rice α - glucosidase ( ec 3 . 2 . 1 . 20 ; sigma ). the material recovered in the supernatant from the ammonium oxalate fraction was shown to contain a pure β - 1 , 4 - glucan by demonstrating that : ( i ) only glucose was detectable when it was hydrolysed by 2 m tfa in a sealed tube for 1 h at 120 ° c . in an autoclave , the supernatant ( 2000 g for 5 min ) was dried under vacuum at 45 ° c . to remove tfa and glucose was determined by gc / ms ; ( ii ) methylation ( needs and selvendran 1993 ) gave a dominant peak resolved by thin layer chromatography and by gc / ms that was identical to that from a cellulose standard and so indicative of 1 , 4 - linked glucan ( fig3 ); and ( iii ) the endo - cellulase and β - 1 , 4 - glucosidase mixture released 83 % of the tfa - releasable glucose from the glucan produced by rsw1 at 31 ° c . while the α - amylase / α - glucosidase mixture released no glucose from the glucan . conversely , the α - amylase / β - glucosidase mixture released 95 % of the tfa - releasable glucose from a starch sample , while the endo - cellulase / β - 1 , 4 - glucosidase mixture released no glucose from starch . extractability of the glucan using ammonium oxalate , and the susceptibility of the glucan to endocellulase / β - glucosidase and tfa hydrolysis indicate that the glucan in the rsw1 mutant is not crystalline , because it is the crystallinity of glucan which makes cellulose resistant to extraction and degradation . table 4 shows the quantity of glucose in cellulose determined by the anthrone / h 2 so 4 reaction and the quantity in the non - crystalline glucan after tfa hydrolysis , for shoots of wild type and mutant rsw1 arabidopsis plants . the data indicate that the production of cellulose and of the non - crystalline β - 1 , 4 - glucan can be manipulated by mutational changes in the rsw1 gene . the quantity of starch recovered in the dmso fraction from roots in the experiment described above was also determined by the anthrone / h 2 so 4 extraction ( table 5 ). as shown in table 5 , the level of starch deposited in the rsw1 mutant is 4 - fold that detectable in the roots of wild - type plants at the restrictive temperature of 31 ° c . a similar rise in starch is also seen if the data are expressed as nmol glucose per plant . there is no detectable difference in deposition at starch between rsw1 plants and wild - type plants at 21 ° c . the composition of cell walls in the rsw1 mutant plant compared to wild type plants at the restrictive temperature of 31 ° c ., is summarised in table 6 . the rsw1 locus in the mutant arabidopsis thaliana plant described in example 1 above was mapped to chromosome 4 of a . thaliana using rflp gene mapping techniques ( chang et al ., 1988 ; nam et al ., 1989 ) to analyse the f 2 or f 3 progeny derived from a columbia ( co )/ landsberg ( ler ) cross . in particular , the rsw1 mutation was shown to be linked genetically to the ga5 locus , which is a chromosome 4 visual marker in a . thaliana . based on an analysis of map distances and chromosomal break points in 293 f 2 or f 3 progeny derived from a columbia ( co )/ landsberg ( ler ) cross , rsw1 was localised to an approximately 2 . 1 cm region between the rflp markers g8300 and 06455 , approximately 1 . 2 cm south of the caps ( cleaved amplified polymorphic sequence ; konieczny and ausubel , 1993 ) version of the g8300 marker ( fig4 ). the interval between g8300 and 06455 in which rsw1 residues was found to be spanned by an overlapping set of yeast artificial chromosome ( yac ) clones . the clones were obtained from plant industry , commonwealth scientific and industrial research organisation , canberra , australia . the yacs were positioned in the g8300 / 06455 interval by hybridisation using known dna molecular markers ( from within the interval ) and dna fragments from the ends of the yacs . the length of the interval was estimated to comprise 900 kb of dna . refined gene mapping of recombinants within the region spanned by yac clones established the genetic distance between the rflp marker g8300 and the rsw1 locus . the combination of genetic map distance data and the mapping of yac clones within the region further localised the rsw1 locus to the yac clone designated yup5c8 . an arabidopsis thaliana cdna clone designated t20782 was obtained from the public arabidopsis resource centre , ohio state university , 1735 neil avenue , columbus , ohio 43210 , united states of america . the t20782 cdna clone was localised broadly to the dna interval on arabidopsis chromosome 4 between the two markers g8300 and 06455 shown in fig4 . using a polymerase chain reaction ( pcr ) based approach dna primers ( 5 ′- agmcagcagatacacgga - 3 ′ seq id no : 15 and 5 ′- ctgmgmggctggacmt - 3 ′, seq id no : 16 ) designed to the t20782 cdna nucleotide sequence were used to screen arabidopsis yac clone libraries . the t20782 cdna clone was found to localise to yacs ( cic1f9 , cic10e9 , cic11d9 ) identified on the arabidopsis chromosome 4 g8300 and 06455 interval ( fig4 ). the same approach was used to further localise clone t20782 to yac clone yup5c8 , the same yac designated to contain the rsw1 locus in the same chromosome interval ( fig4 ). furthermore , amplification of the yac clone yup5c8 using primers derived from t20782 produces a 500 bp fragment containing two putative exons identical to part of the t20782 nucleotide sequence , in addition to two intron sequences . the cdna t20782 was considered as a candidate gene involved in cellulose biosynthesis . the nucleotide sequence of the cdna clone t20782 is presented in seq id no : 1 . the nucleotide sequence was obtained using a dye terminator cycle sequencing kit ( perkin elmer cat . # 401384 ) as recommended by the manufacturer . four template clones were used for nucleotide sequencing to generate the sequence listed . the first template was the cdna clone t20782 . this template was sequenced using the following sequencing primers : a ) 5 +- caatgcattcatagctccagcct - 3 + ( seq id no : 17 ) b ) 5 +- aaaaggctggagctatgaatgcat - 3 + ( seq id no : 18 ) c ) 5 +- tcaccgacagattcatcatacccg - 3 + ( seq id no : 19 ) d ) 5 +- gacatggaatcaccttaactgcc - 3 + ( seq id no : 20 ) e ) 5 +- ccattcagtcttgtcttcgtaacc - 3 + ( seq id no : 21 ) f ) 5 +- ggttacgaagacaagactgaaatgg - 3 + ( seq id no : 22 ) g ) 5 +- gaacctcataggcattgtgggctgg - 3 + ( seq id no : 23 ) h ) 5 +- gcaggctctatatgggtatgatcc - 3 + ( seq id no : 24 ) i ) standard m13 forward sequencing primer . j ) standard t7 sequencing primer . the second template clone ( t20782 sphi deletion clone ) was constructed by creating a dna deletion within the t20782 clone . the t20782 clone was digested with the restriction enzyme sphi , the enzyme was heat - killed , the dna ligated and electroporated into nm522 e . coli host cells . the t20782 sphi deletion clone was then sequenced using a standard m13 forward sequencing primer . two other deletion clones were made for dna sequencing in a similar fashion but the restriction enzymes ecori and smai were used . the t20782 ecori deletion clone and the t20782 smai deletion clone were sequenced using a standard t7 sequencing primer . the dna sequence shown in seq id no : 1 is for one dna strand only however those skilled in the art will be able to generate the nucleotide sequence of the complementary strand from the data provided . the amino acid sequence encoded by clone t20782 was derived and is set forth in seq id no : 2 . the t20782 clone encodes all but the first aspartate ( d ) residue of the d , d , d , qxxrw ( seq id no : 37 ) signature conserved in the general architecture of β - glycosyl transferases . in particular , t20782 encodes 5 amino acid residues of the d , d , d , qxxrw signature , between amino acid positions 109 and 370 of seq id no : 2 . the conserved aspartate , aspartate , glutamine , arginine and tryptophan amino acid residues are shown below , in bold type , with the local amino acid residues also indicated : 1 . amino acid residues 105 to 113 of seq id no : 2 : llnv d cdhy ; 2 . amino acid residues 324 to 332 of seq id no : 2 : svte d iltg ; and 3 . amino acid residues 362 to 374 of seq id no : 2 : drln q vl rw algs . it must be noted that these invariable amino acids merely indicate that the t20782 derived amino acid sequence belongs to a very broad group of glycosyl transferases . some of these enzymes such as cellulose synthase , chitin synthase , alginate synthase and hyaluronic acid synthase produce functionally very different compounds . the presence of the conserved amino acid residues merely indicates that the t20782 clone may encode a β - glycosyl transferase protein such as the cellulose gene product , cellulose synthase . the fact that the clone localises in the vicinity of a gene involved in cellulose biosynthesis is the key feature which now focus interest on the t20782 clone as a candidate for the rsw1 ( cellulose synthase ) gene . clone 23h12 contains approximately 21 kb of arabidopsis thaliana genomic dna in the region between the left border and right border t - dna sequences , and localises to the rsw1 candidate yac yup5c8 . clone 23h12 was isolated by hybridisation using est20782 insert dna , from a genomic dna library made for plant transformation . cosmid 12c4 was also shown to hybridize to the cdna clone t20782 , however this cosmid appears to comprise a partial genomic sequence corresponding to the related ath - a cdna sequence set forth in seq id no : 7 , for which the corresponding amino acid sequence is set forth in seq id no : 8 . a restriction enzyme map of clone 23h12 is presented in fig5 . nucleotide sequence of 8411 bp of genomic dna in the binary cosmid clone 23h12 was obtained ( seq id no : 3 ) by primer walking along the 23h12 template , using a dye terminator cycle sequencing kit ( perkin elmer cat . # 401384 ) as recommended by the manufacturer . the following primers at least , were used for dna sequencing of the 23h12 clone dna : a ) cs1 - r 5 ′- caatgcattcatagctccagcct - 3 ′ ( seq id no : 17 ) b ) cs1 - f 5 ′- aaaaggctggagctatgaatgcat - 3 ′ ( seq id no : 18 ) c ) up 5 ′- agaacagcagatacacgga - 3 ′ ( seq id no : 25 ) d ) ve76 - r2 5 ′- atccgtgtatctgctgttcttacc - 3 ′ ( seq id no : 26 ) e ) est1 - r 5 ′- aatgctcttgttgccaaagcac - 3 ′ ( seq id no : 27 ) f ) sve76 - f 5 ′- attgtccagccttcttcagg - 3 ′ ( seq id no : 28 ) g ) ve76 - r 5 ′- ctgaagaaggctggacaatgc - 3 ′ ( seq id no : 29 ) h ) b12 - r1 5 ′- aggtaagcatagctgaaccatc - 3 ′ ( seq id no : 30 ) i ) b12 - r2 5 ′- agtagattgcagatggttttctac - 3 ′ ( seq id no : 31 ) j ) b12 - r3 5 ′- ttcaatgggtccactgtactaac - 3 ′ ( seq id no : 32 ) k ) b12 - r4 5 ′- attcagatgcaccattgtc - 3 ′ ( seq id no : 33 ) the structure of the rsw1 gene contained in cosmid clone 23h12 is also presented in fig5 . as shown therein , coding sequences in 23h12 , from the last 12 bp of exon 7 to the end of exon 14 , correspond to the full t20782 cdna sequence ( i . e . seq id no : 1 ). the nucleotide sequences of the rsw1 gene comprising exons 1 to 8 were amplified from a . thaliana columbia double - stranded cdna , using amplification primers upstream of the rsw1 start site and a primer internal to the est clone t20782 . the exons in the rsw1 gene range from 81 bp to 585 bp in length and all 5 □ and 3 ′ intron / exon splice junctions conform to the conserved intron rule . the rsw1 transcript comprises a 5 ′- untranslated sequence of at least 70 bp in length , a 3243 bp coding region and a 360 bp 3 ′- untranslated region . northern hybridization analyses indicate that the rsw1 transcript in wild - type a . thaliana roots , leaves and inflorescences is approximately 4 . 0 kb in length , and that a similar transcript size occurs in mutant tissue . the derived amino acid sequence of the rsw1 polypeptide encoded by the cosmid clone 23h12 ( i . e . the polypeptide set forth in seq id no : 6 ) is 1081 amino acids in length and contains the entire d , d , d , qxxrw ( seq id no : 37 ) signature characteristic of β - glycosyl transferase proteins , between amino acid position 395 and amino acid position 822 . the conserved aspartate , glutamine , arginine and tryptophan residues are shown below , in bold type , with the local amino acid residues also indicated : 1 . amino acid residues 391 to 399 of seq id no : 6 : yvsd d gsam 2 . amino acid residues 557 to 565 of seq id no : 6 : llnv d cdhy ; 3 . amino acid residues 776 to 784 of seq id no : 6 : svte d iltg ; and 4 . amino acid residues 814 to 826 of seq id no : 6 : drln q vl rw algs . the second and third conserved aspartate residues listed supra , and the fourth conserved amino acid sequence motif listed supra ( i . e . qvlrw ) are also present in the cdna clone t20782 ( see example 4 above ). the complementation of the cellulose mutant plant rsw1 is the key test to demonstrate the function of the clone 23h12 gene product . complementation of the rsw1 phenotype was demonstrated by transforming the binary cosmid clone 23h12 , or a derivative clone thereof encoding a functional gene product , into the arabidopsis thaliana cellulose mutant rsw1 . two dna constructs ( 23h12 and prsw1 ) were used to complement the rsw1 mutant plant line . clone 23h12 is described in example 5 and fig5 . the 23h12 construct has an insert of about 21 kb in length . to demonstrate that any complementation of the phenotype of the rsw1 mutation is the result of expression of the gene which corresponds to seq id no : 3 , a genetic construct , designated as prsw1 , comprising the putative rsw1 gene with most of the surrounding dna deleted , was produced . a restriction enzyme ( re ) map of the rsw1 gene insert in prsw1 is provided in fig5 . to produce prsw1 , the rsw1 gene was subcloned from cosmid 23h12 and cloned into the binary plasmid pbin19 . briefly , escherichia coli cells containing cosmid 23h12 were grown in lb medium supplemented with tetracyclin ( 3 . 5 mg / l ). plasmid dna was prepared by alkaline lysis and digested sequentially with restriction enzymes pvuii and sali . two co - migrating fragments of 9 kb and 10 kb , respectively , were isolated as a single fraction from a 0 . 8 % ( w / v ) agarose gel . the rsw1 gene was contained on the 10 kb pvuii / sali fragment . the 9 kb fragment appeared to be a pvuii cleavage product not comprising the rswi gene . the restriction fragments were ligated into pbin19 digested with smai and sali . an aliquot of the ligation mix was introduced by electroporation into e . coli strain xlb1 . colonies resistant to kanamycin ( 50 mg / l ) were selected and subsequently characterised by restriction enzyme analysis to identify those clones which contained only the 10 kb pvuii / sali fragment comprising the rsw1 gene , in pbin19 . transfer of the 23hi2 and prsw1 constructs to agrobacterium tumefaciens cosmid 23h12 was transferred to agrobacterium by triparental mating , essentially as described by ditta et al . ( 1980 ). three bacterial strains as follows were mixed on solid lb medium without antibiotics : strain 1 was an e . coli helper strain containing the mobilising plasmid prk2013 , grown to stationary phase ; strain 2 was e . coli containing cosmid 23h12 , grown to stationary phase ; and strain 3 was an exponential - phase culture of a . tumefaciens strain agl1 ( lazo et al ., 1991 ). the mixture was allowed to grow over night at 28 ° c ., before an aliquot was streaked out on solid lb medium containing antibiotics ( ampicillin 50 mg / l , rifampicin 50 mg / l , tetracyclin 3 . 5 mg / l ) to select for transformed a . tumefaciens agl1 . resistant colonies appeared after 2 - 3 days at 28 ° c . and were streaked out once again on selective medium for further purification . selected colonies were then subcultured in liquid lb medium supplemented with rifampicin ( 50 mg / l ) and tetracyclin ( 3 . 5 mg / l ) and stored at − 80 ° c . plasmid prsw1 ( initially designated as p2029 ) was introduced into a . tumefaciens strain agl1 by electroporation . the rsw1 plant line was transformed with constructs 23h12 and prsw1 using vacuum infiltration essentially as described by bechtold et al . ( 1993 ). complementation of the radial swelling ( rsw ) phenotype , which is characteristic of the rsw1 mutant plant , was assayed by germinating transformed ( i . e . t1 seed ) rsw1 seeds obtained as described supra on hoaglands plates containing 50 μg / ml kanamycin . plates containing the transformed seeds were incubated at 21 ° c . for 10 - 12 days . kanamycin - resistant seedlings were transferred to fresh hoaglands plates containing 50 μg / ml kanamycin and incubated at 31 ° c . for 2 days . following this incubation , the root tip was examined for a radial swelling phenotype . under these conditions , the roots of wild - type plants do not show any radial swelling phenotype however , the roots of rsw1 plants show clear radial swelling at the root tip and also have a short root compared to the wild - type plants . as a consequence , determination of the radial swelling phenotype of the transformed plants was indicative of successful complementation of the rsw1 phenotype . the kanamycin - resistant seedlings were maintained by further growth of seedlings at 21 ° c ., following the high temperature incubation . once plants had recovered , the seedlings were transferred to soil and grown in cabinets at 21 ° c . ( 16 hr light / 8 hr dark cycle ). t2 seed was then harvested from mature individual plants . using the 23h12 construct for rsw1 transformation , a total of 262 kanamycin - resistant seedlings were obtained . all of these transformants were tested for complementation of the root radial swelling phenotype . a total of 230 seedlings showed a wild type root phenotype , while only 32 seedlings showed the radial swelling root phenotype characteristic of rsw1 plants . by way of example , fig6 shows the phenotypes of transformed seedlings compared to untransformed wild - type and rsw1 seedlings , following incubation at 31 ° c . as shown in fig6 , there is clear complementation of the radial swelling phenotype in the transformed seedlings , with normal root length being exhibited by the transformed seedlings at 31 ° c . using the prsw1 construct for transformation , a total of 140 kanamycin - resistant seedlings were obtained . all of the 11 seedlings tested for complementation of the root radial swelling phenotype showed a wild type root phenotype and none of the seedlings showed any signs of radial swelling in the roots . further characterisation of the complemented rsw1 plants has shown that other morphological characteristics of rsw1 have also been restored in the transgenic lines , for example the bolt ( inflorescence ) height , and the ability of the plants to grow wild type cotyledons , leaves , trichomes , siliques and flowers at 31 ° c . t2 seed from transformations using cosmid 23h12 as described supra or alternatively , using the binary plasmid pbin19 which lacks any rsw1 gene sequences , was sown on hoagland &# 39 ; s solid media containing kanamycin ( 50 μg / ml ), incubated for 2 days at 21 ° c . and then transferred to 31 ° c . for 5 days . wild - type a . thaliana columbia plants were grown under similar conditions but without kanamycin in the growth medium . kanamycin resistant t2 seedlings which have at least one copy of the 23h12 cosmid sequence , and wild - type seedlings , were collected and frozen for cellulose analysis . cellulose levels were determined as acetic - nitric acid insoluble material ( updegraph , 1969 ) for 10 lines of kanamycin - resistant t2 plants transformed with the 23h12 cosmid sequence , and compared to the cellulose levels in rsw1 mutant plants , wild - type a . thaliana columbia plants and a . thaliana columbia plants transformed with the binary plasmid pbin19 . the results are provided in table 7 . as shown in table 7 , the cellulose levels have been significantly elevated in the complemented rsw1 ( t2 ) plants , compared to the cellulose levels measured in the rsw1 mutant parent plant . in fact , cellulose levels in the 23h12 - transformed plants , expressed relative to the fresh weight of plant material or on a per seedling basis , are not significantly different from the cellulose levels of either wild - type arabidopsis thaliana columbia plants or a . thaliana columbia transformed with the binary plasmid pbin19 . these data indicate that the 23h12 cosmid is able to fully complement the cellulose - deficient phenotype of the rsw1 mutant . homozygous t3 lines are generated to confirm the data presented in table 7 . furthermore , data presented in table 7 indicate that there is no difference in the rate of growth of the t2 transformed rsw1 plants and wild - type plants at 31 ° c ., because the fresh weight of such plants does not differ significantly . in contrast , the fresh weight of mutant rsw1 seedlings grown under identical conditions is only approximately 55 % of the level observed in t2 lines transformed with 23h12 ( range about 30 % to about 80 %). these data support the conclusion that cellulose levels have been manipulated in the complemented rsw1 ( t2 ) plants . furthermore , the rate of cellulose synthesis in 23h12 - transformed plants and wild - type plants at 31 ° c ., as measured by 14c incorporation is also determined . furthermore , the β - 1 , 4 - glucan levels and starch levels in the 23h12 transformant lines are shown to be similar to the β - 1 , 4 - glucan and starch levels in wild - type plants . approximately 100 , 000 cdna clones in an unamplified cdna library were screened under standard hybridization conditions at 65 ° c ., using a probe comprising 32 p - labeled dna amplified from double stranded cdna . to prepare the hybridization probe , the following amplification primers were used : ( see seq id no : 3 ) 1 . 2280 - f : 5 ′ gaatcggctacgaatttccca 3 ′ ( see seq id no : 3 ) 2 . 2370 - f : 5 ′ ttggttgctggatcctaccgg 3 ′ ( see seq id no : 1 ) 3 . csp1 - r : 5 ′ ggt tct aaa tct tct tcc gtc 3 ′ wherein the primer combinations were either 2280 - f / csp1 - r or 2370 - f / csp1 - r . the primer 2280 - f corresponds to nucleotide positions 2226 to 2246 in seq id no : 3 , upstream of the translation start site . the primer 2370 - f corresponds to nucleotide positions 2314 to 2334 in seq id no : 3 , encoding amino acids 7 through 13 of the rsw1 polypeptide . the primer csp1 - r comprises nucleotide sequences complementary to nucleotides 588 to 608 of the t20782 clone ( seq id no : 1 ) corresponding to nucleotides 6120 to 6140 of seq id no : 3 . the hybridization probes produced are approximately 1858 nucleotides in length ( 2280 - f / csp1 - r primer combination ) or 1946 nucleotides in length ( 2370 - f / csp1 - r primer combination ). five hybridizing bacteriophage clones were identified , which were plaque - purified to homogeneity during two successive rounds of screening . plasmids were rescued from the positively - hybridizing bacteriophage clones , using the stratagene excision protocol for the zapexpress ™ vector according to the manufacturer &# 39 ; s instructions . colony hybridizations confirmed the identity of the clones . isolated cdna clones were sequenced by primer walking similar to the method described in examples 4 and 5 supra . a full - length wild - type rsw1 nucleotide sequence was compiled from the nucleotide sequences of two cdna clones . first , the 3 ′- end of the cdna , encoding amino acids 453 - 1081 of rsw1 , corresponded to the nucleotide sequence of the est clone t20782 ( seq id no : 1 ). the remaining cdna sequence , encoding amino acids 1 - 654 of rsw1 , was generated by amplification of the 5 ′- end from cdna , using primer 2280 - f , which comprises nucleotide sequences approximately 50 - 70 bp upstream of the rsw1 translation start site in cosmid 23h12 , and primer csp1 - r , which comprises nucleotide sequences complementary to nucleotides 588 to 608 of the t20782 clone ( seq id no : 1 ). several amplified clones are sequenced to show that no nucleotide errors were introduced by the amplification process . the 5 ′ and 3 ′ nucleotide sequences are spliced together to produce the complete rsw1 open reading frame and 3 ′- untranslated region provided in seq id no : 5 . those skilled in the art will be aware that the 5 ′- end and 3 ′- end of the two incomplete cdnas are spliced together to obtain a full - length cdna clone , the nucleotide sequence of which is set forth in seq id no : 5 . of the remaining cdna clones , no isolated cdna clone comprised a nucleotide sequence which precisely matched the nucleotide sequence of the rsw1 gene present in cosmid 23h12 . however , several clones containing closely - related sequences were obtained , as summarised in table 8 . the nucleotide sequences of the ath - a and ath - b cdnas are provided herein as seq id nos : 7 and 9 , respectively . fig1 a schematic representation of the important features of the rsw1 polypeptide which are conserved within a . thaliana and between a . thaliana and other plant species . in addition to the species indicated in fig1 , the present inventors have also identified maize , wheat , and barley and brassica spp . cellulose biosynthetic genes by homology search . accordingly , the present invention extends to cellulose genes and cellulose biosynthetic polypeptides as hereinbefore defined , derived from any plant species , including a . thaliana , cotton , rice , wheat , barley , maize , eucalyptus spp ., brassica spp . pinus spp ., populus spp ., picea spp ., hemp , jute and flax , amongst others . arabidopsis thaliana double - stranded cdna and cdna libraries were prepared using the capfinder cdna kit ( clontech ). rna was isolated from arabidopsis thaliana columbia rsw1 mutant plants grown in sterile conditions for 21 days . the full - length rsw1 mutant nucleotide sequence was generated by sequencing two amplified dna fragments spanning the rsw1 mutant gene . the 5 ′- end sequence of the cdna ( comprising the 5 ′- untranslated region and exons 1 - 11 ) was amplified using the primer combination 2280 - f / csp1 - r ( example 7 ). the 3 ′- end sequence was amplified using the primers est1 - f and cs3 - r set forth below : ( see seq id no : 5 ) 1 . primer est1 - f : 5 ′ aatgcttcttgttgccaaagca 3 ′ ( see seq id no : 5 ) 2 . primer cs3 - r : 5 ′ gacatggaatcaccttaactgcc 3 ′ wherein primer est1 - f corresponds to nucleotide positions 1399 - 1420 of seq id no : 5 ( within exon 8 ) and primer cs3 - r is complementary to nucleotides 3335 - 3359 of seq id no : 5 ( within the 3 □- untranslated region of the wild - type transcript ). the full - length sequence of the mutant rsw1 transcript is set forth herein as seq id no : 11 . whilst not being bound by any theory or mode of action , a single nucleotide substitution in the rsw1 mutant nucleotide sequence ( nucleotide position 1716 in seq id no : 11 ), relative to the wild - type rsw1 nucleotide sequence ( nucleotide position 1646 in seq id no : 5 ), resulting in ala549 being substituted with val549 in the mutant polypeptide , may contribute to the altered activity of the rsw1 polypeptide at non - permissive temperatures such as 31 ° c . additional amino acid substitutions are also contemplated by the present invention , to alter the activity of the rsw1 polypeptide , or to make the polypeptide temperature - sensitive . one example of transgenic plants in which cellulose production is inhibited is provided by the expression of an antisense genetic construct therein . antisense technology is used to target expression of a cellulose gene ( s ) to reduce the amount of cellulose produced by transgenic plants . by way of exemplification , an antisense plant transformation construct has been engineered to contain the t20782 cdna insert ( or a part thereof ) in the antisense orientation and in operable connection with the camv 35s promoter present in the binary plasmid prd410 ( datla et al . 1992 ). more particularly , the t20782 cdna clone , which comprises the 3 ′- end of the wild - type rsw1 gene , was digested with xbai and kpni and cloned into the kanamycin - resistant derivative of pgem3zf (−), designated as plasmid , pjkkmf (−). the rsw1 sequence was sub - cloned , in the antisense orientation , into the binary vector prd410 as a xbai / saci fragment , thereby replacing the β - glucuronidase ( gus or uida ) gene . this allows the rsw1 sequence to be transcribed in the antisense orientation under the control of the camv 35s promoter . the antisense rsw1 binary plasmid vector was transferred to agrobacterium tumefaciens strain agl1 , by triparental mating and selection on rifampicin and kanamycin , as described by lazo et al . ( 1991 ). the presence of the rsw1 insert in transformed a . tumefaciens cells was confirmed by southern hybridization analysis ( southern , 1975 ). the construct was shown to be free of deletion or rearrangements prior to transformation of plant tissues , by back - transformation into escherichia coli strain jm101 and restriction digestion analysis . eight pots , each containing approximately 16 a . thaliana ecotype columbia plants , were grown under standard conditions . plant tissue was transformed with the antisense rsw1 binary plasmid by vacuum infiltration as described by bechtold et al . ( 1993 ). infiltration media contained 2 . 5 % ( w / v ) sucrose and plants were infiltrated for 2 min until a vacuum of approximately 400 mm hg was obtained . the vacuum connection was shut off and plants allowed to sit under vacuum for 5 min . approximately 34 , 000 t1 seed was screened on ms plates containing 50 μg / ml kanamycin , to select for plants containing the antisense rsw1 construct . of the t1 seed sown , 135 kanamycin - resistant seedlings were identified , of which 91 were transferred into soil and grown at 21 ° c . under a long - day photoperiod ( 16 hr light ; 8 hr dark ). of the 91 transgenic lines , 19 lines were chosen for further analysis which had anther filaments in each flower which were too short to deposit pollen upon the stigma and , as a consequence , required hand - pollination to obtain t2 seed therefrom . t2 seed from 14 of these 19 lines was plated out onto vertical hoaglands plates containing kanamycin to determine segregation ratios . between five and ten seed were plated per transgenic line . control seeds , including a . thaliana columbia containing the binary vector pbin19 ( bevan , 1984 ) and segregating 3 : 1 for kanamycin resistance , and the rsw1 mutant transformed with the nptii gene , also segregating 3 : 1 for kanamycin resistance , were grown under the same conditions . kanamycin - resistant plants were transferred to soil and grown at 21 ° c . under long days , until flowering . untransformed arabidopsis thaliana columbia plants were also grown under similar conditions , in the absence of kanamycin . a comparison of the morphology of antisense rsw1 plants grown at 21 ° c ., to mutant rsw1 plants grown at the non - permissive temperature ( i . e . 31 ° c .) has identified a number of common phenotypes . for example , the antisense plants exhibit reduced fertility , inflorescence shortening and have short anthers , compared to wild - type plants , when grown at 21 ° c . these phenotypes are also observed in mutant rsw1 plants grown at 31 ° c . these results suggest that the antisense construct in the transgenic plants may be targeting the expression of the wild - type rsw1 gene at 21 ° c . fig7 shows the reduced inflorescence ( bolt ) height in antisense 35s - rsw1 plants compared to wild - type a . thaliana columbia plants grown under identical conditions . t3 plants which are homozygous for the 35s - rsw1 antisense construct are generated and the content of cellulose therein is determined as described in example 1 . plants expressing the antisense construct are shown to have significantly less cellulose in their cell walls , compared to wild - type plants . additionally , the levels of non - crystalline β - 1 , 4 - glucan and starch are elevated in the cells of antisense plants , compared to otherwise isogenic plant lines which have not been transformed with the antisense genetic construct . total rna was extracted from 0 . 2 g of leaf tissue derived from 33 kanamycin - resistant t1 plants containing the antisense 35s - rsw1 genetic construct , essentially according to longemann et al . ( 1986 ). total rna ( 25 μg ) was separated on a 2 . 2m formaldehyde / agarose gel , blotted onto nylon filters and hybridized to a riboprobe comprising the sense strand sequence of the cdna clone t20782 . to produce the riboprobe , t7 rna polymerase was used to transcribe sense rna from a linearised plasmid template containing t20782 , in the presence of [ α - 32 p ] utp . hybridizations and subsequent washes were performed as described by dolferus et al . ( 1994 ). hybridized membranes were exposed to phosphor screens ( molecular dynamics , usa ). the levels of expression of the rsw1 antisense transcript were determined and compared to the level of fertility observed for the plant lines . as shown in table 9 , the level of antisense gene expression is correlated with the reduced fertility phenotype of the antisense plants . in 13 lines , a very high or high level of expression of the 35s - rsw1 antisense gene was observed and , in 11 of these lines fertility was reduced . only lines 2w and 3e which expressed high to very high levels of antisense mrna , appeared to be fully fertile . in 12 lines which expressed medium levels of antisense mrna , approximately one - half were fertile and one - half appeared to exhibit reduced fertility . in contrast , in 8 plant lines in which only a low or very low level of expression of the antisense 35s - rsw1 genetic construct was observed , a wild - type ( i . e . fertile ) phenotype was observed for all but one transgenic line , line 2r . data presented in table 9 and fig7 indicate that the phenotype of the cellulose - deficient mutant rsw1 may be reproduced by expressing antisense rsw1 genetic constructs in transgenic plants . to confirm reduced cellulose synthesis and / or deposition in transgenic plants expressing the antisense rsw1 gene , the level of cellulose is measured by the 14 c incorporation assay or as acetic / nitric acid insoluble material as described in example 1 and compared to cellulose production in otherwise isogenic wild - type plants . cellulose production in the transgenic plants is shown to be significantly reduced compared to wild - type plants . the severity of phenotype of the transgenic plants thus produced varies considerably , depending to some extent upon the level of inhibition of cellulose biosynthesis . to identify rsw1 related nucleotide sequences in rice , a genetic sequence database was searched for nucleotide sequences which were closely - related to one or more of the arabidopsis thaliana rsw1 nucleotide sequences described in the preceding examples . rice est s0542 ( maff dna bank , japan ) was identified , for which only a partial nucleotide sequences was available . additionally , before the instant invention , there was no probable function attached to the rice est s0542 sequence . the present inventors have obtained the complete nucleotide sequence of clone s0542 and derived the amino acid sequence encoded therefor . the s0542 cdna is only 1741 bp in length and appears to be a partial cdna clone because , although it comprises 100 bp of 5 ′- untranslated sequence and contains the atg start codon , it is truncated at 3 ′- end and , as a consequence encodes only the first 547 amino acid residues of the rice rsw1 or rsw1 - like polypeptide . based upon the length of the corresponding arabidopsis thaliana rsw1 polypeptide ( 1081 amino acids ), the rice rsw1 sequence set forth in seq id no : 14 appears to contain approximately one - half of the complete amino acid sequence . the n - terminal half of the rice rsw1 amino acid sequence is approximately 70 % identical to the arabidopsis thaliana rsw1 polypeptide set forth in seq id no : 6 , with higher homology ( approximately 90 %) occurring between amino acid residues 271 - 547 of the rice sequence . these data strongly suggest that s0542 is the rice homologue of the a . thaliana rsw1 gene . alignments of rice , a . thaliana and cotton rsw1 amino acid sequences are presented in fig9 and 10 . to isolate full - length cdna clones and genomic clone equivalents of s0542 ( this study and maff dna bank , japan ) or d48636 ( pear et al ., 1996 ), cdna and genomic clone libraries are produced using rice mrna and genomic dna respectively , and screened by hybridisation using the s0542 or d48636 cdnas as a probe , essentially as described herein . positive - hybridising plaques are identified and plaque - purified , during further rounds of screening by hybridisation , to single plaques . the rice clones are sequenced as described in the preceding examples to determine the complete nucleotide sequences of the rice rsw1 genes and derived amino acid sequences therefor . those skilled in the art will be aware that such gene sequences are useful for the production of transgenic plants , in particular transgenic cereal plants having altered cellulose content and / or quality , using standard techniques . the present invention extends to all such genetic sequences and applications therefor . a 32 p - labeled rsw1 pcr fragment was used to screen approximately 200 , 000 cdna clones in a cotton fibre cdna library . the rsw1 pcr probe was initially amplified from arabidopsis thaliana wild type cdna using the primers 2280 - f and csp1 - r described in the preceding examples , and then re - amplified using the primer combination 2370 - f / csp1 - r , also described in the preceding examples . hybridisations were carried out under low stringency conditions at 55 ° c . six putative positive - hybridising plaques were identified in the first screening round . using two further rounds of screening by hybridisation , four of these plaques were purified to single plaques . three plaques hybridise very strongly to the rsw1 probe while the fourth plaque hybridises less intensely . we conclude that the positive - hybridising plaques which have been purified are strong candidates for comprising cotton rsw1 gene sequences or rsw1 - like gene sequences . furthermore , the cotton cdnas may encode the catalytic subunit of cellulose synthase , because the subunit protein architecture of cellulose synthase appears to be highly conserved among plants as highlighted in the preceding example . furthermore , a southern blot of cotton genomic dna digested with bglii was hybridised with the 5 ′ end of the rsw1 cdna , under low stringency hybridisation conditions at 55 ° c . results are presented in fig1 . these data demonstrate that rsw1 - related sequences exist in the cotton genome . the cotton cdna clones described herein are sequenced as described in the preceding examples and used to produce transgenic cotton plants having altered fibre characteristics . the cdnas are also used to genetically alter the cellulose content and / or quality of other plants , using standard techniques . putative eucalyptus spp . cellulose synthase catalytic subunit gene fragments were obtained by amplification using pcr . dna primers were designed to conserved amino acid residues found in the arabidopsis thaliana rsw1 and 12c4 amino acid sequences . three primers were used for pcr . the primers are listed below : ( seq id no : 34 ) pcsf - i 5 ′- a a / g a a g a t i g a c / t t a c / t c / t t i a a a / g g a c / t a a - 3 ′ ( seq id no : 35 ) pcsr - ii 5 ′- a t i g t i g g i g t i c g / t a / g t t c / t t g a / t / g / c c t / g a / t / c / g c c - 3 ′ ( seq id no : 36 ) pcsf - ii 5 ′- g c i a t g a a a / g a / c g i g a i t a c / t g a a / g g a - 3 ′ using standard pcr conditions ( 50 ° c . annealing temperature ) and solutions , the primer sets pcsf - i / pcsr - ii and pcsf - ii / pcsr - ii were used to amplify genetic sequences from pooled eucalyptus spp . cdna . in the first reaction primers pcsf - i and pcsr - ii were used to generate a fragment approximately 700 bp in length . in the second pcr reaction , which used primers pcsf - ii and pcsr - ii , a fragment estimated to 700 bp was obtained . the sizes of the pcr fragments are within the size range estimated for the corresponding arabidopsis thaliana sequences . we conclude that the amplified eucalyptus spp . pcr fragments are likely to be related to the arabidopsis thaliana rsw1 gene and may encode at least a part of the eucalyptus spp . cellulose synthase catalytic subunit . the eucalyptus spp . pcr clones described herein are sequenced as described in the preceding examples and used to isolate the corresponding full - length eucalyptus spp . cdnas and genomic gene equivalents . those skilled in the art will be aware that such gene sequences are useful for the production of transgenic plants , in particular transgenic eucalyptus spp . plants having altered cellulose content and / or quality , using standard techniques . the present invention extends to all such genetic sequences and applications therefor . the properties of plant cell walls depend on the carbohydrates , proteins and other polymers of which they are composed and the complex ways in which they interact . increasing the quantities of non - crystalline β - 1 , 4 - glucan in cell walls affects those wall properties which influence mechanical , nutritional and many other qualities as well as having secondary consequences resulting from the diversion of carbon into non - crystalline glucan at the expense of other uses . to illustrate one of these effects , we investigated the ability of the non - crystalline glucan to hydrogen bond to other wall components particularly cellulose in the way that has been shown to be important for wall mechanics . hemicelluloses such as xyloglucans cross - link cellulose microfibrils by hydrogen bonding to the microfibril surface ( levy et al ., 1991 ). since the β - 1 , 4 - glucan backbone of xyloglucan is thought to be responsible for hydrogen bonding ( with the xylose , galactose and fucose substitutions limiting the capacity to form further hydrogen bonds ) we can expect the non - crystalline β - 1 , 4 - glucan also to have a capacity to hydrogen bond and cross link cellulose . the effectiveness of strong alkalis in extracting xyloglucans is thought to relate to their disruption of the hydrogen bonds with cellulose ( hayashi and maclachlan , 1984 ). to demonstrate that the non - crystalline β - 1 , 4 - glucan forms similar associations with the cellulose microfibrils , we examined whether the 4 m koh fraction , extracted from shoots of the rsw1 mutant and from wild type rsw1 plants , contained non - crystalline glucan in addition to xyloglucan . the non - crystalline glucan was separated from xyloglucan in the 4 m koh extract by dialysing the neutralised extract against distilled water and centrifuging at 14000 g for 1 hour . the pellet was shown to be a pure β - 1 , 4 - glucan by using the methods for monosaccharide analysis , methylation analysis and enzyme digestion used to characterise the glucan in the ammonium oxalate fraction ( see example 1 ). table 10 shows the presence of substantial quantities of glucan recovered in pure form in the pellet from 4 m koh fractions extracted from the overproducing rsw1 mutant of arabidopsis thaliana . these data also demonstrate the presence of smaller quantities of non - crystalline β - 1 , 4 - glucan in the 4 m koh fraction from wild type plants , compared to rsw1 , particularly when grown at 31 ° c . the monosaccharide composition of the supernatant remaining after centrifugation was determined after tfa hydrolysis . these data , and data from methylation analysis , are consistent with the supernatant being a relatively pure xyloglucan . the supernatant was free of glucan , because no glucose could be released by the endocellulase / β - glucosidase mixture that released glucose from β - 1 , 4 - glucan . the presence of both non - crystalline β - 1 , 4 - glucan and xyloglucan in the 4 m koh fraction , when taken together with the implications from structural predictions ( levy et al . 1991 ), is consistent with some of the non - crystalline β - 1 , 4 - glucan in the wall hydrogen bonding to cellulose microfibrils in similar fashion to the β - 1 , 4 - glucan backbone of xyloglucan . the cross linking provided when xyloglucans and other hemicelluloses bind to two or more microfibrils is an important determinant of the mechanical properties of cellulosic walls ( hayashi , 1989 ). the effects of increasing the amounts of non - crystalline β - 1 , 4 - glucan in walls are likely to be greatest in walls which otherwise possess relatively low levels of cross linking as a result of high ratios of cellulose : hemicelluloses . such conditions are common in secondary walls including those of various fibres , and the cellulose : hemicellulose ratio is particularly high in cotton fibres . the effects on wall mechanical properties of overproducing non - crystalline glucan are shown by transforming plants with the mutant allele of rsw1 ( seq id no : 11 ) operably under the control of either the rsw1 promoter derived from seq id no : 3 or seq id no : 4 or alternatively , an appropriate constitutive promoter such as the camv 35s promoter . production of non - crystalline glucan is quantified by fractionating the cell walls using the methods described above to show in particular that non - crystalline glucan is recovered in the 4 m koh fraction . mechanical properties of the cell walls are measured using standard methods for fibre analysis to study parameters such as stress - strain curves , and breaking strain , amongst other properties . three strategies are employed to over - express cellulose synthase in arabidopsis thaliana plants . in the first strategy , the camv 35s promoter sequence is operably connected to the full - length cellulose synthase cdna which is obtainable by primer extension of seq id no : 1 . this is achievable by cloning the full - length cdna encoding cellulose synthase , in the sense orientation , between the camv 35s promoter or other suitable promoter operable in plants and the nopaline synthase terminator sequences of the binary plasmid pbi121 . in the second strategy , the coding part of the genomic gene is cloned , in the sense orientation , between the camv 35s promoter and the nopaline synthase terminator sequences of the binary plasmid pbi121 . in the third strategy , the 23h12 binary cosmid clone or the derivative prsw1 , containing the cellulose synthase gene sequence operably under the control of the cellulose synthase gene promoter and terminator sequences is prepared in a form suitable for transformation of plant tissue . for agrobacterium - mediated tissue transformation , binary plasmid constructs discussed supra are transformed into agrobacterium tumefaciens strain agl1 or other suitable strain . the recombinant dna constructs are then introduced into wild type arabidopsis thaliana plants ( columbia ecotype ), as described in the preceding examples . alternatively , plant tissue is directly transformed using the vacuum infiltration method described by beshtold et al . ( 1993 ). the transgenic plants thus produced exhibit a range of phenotypes , partly because of position effects and variable levels of expression of the cellulose synthase transgene . cellulose content in the transgenic plants and isogenic untransformed control plants is determined by the 14 c incorporation assay or as acetic / nitric acid insoluble material as described in example 1 . in general , the level of cellulose deposition and rates of cellulose biosynthesis in the transgenic plants are significantly greater than for untransformed control plants . furthermore , in some cases , co - supression leads to mimicry of the rsw1 mutant phenotype . the nucleotide sequence of the rsw1 gene contained in 23h12 is mutated using site - directed mutagenesis , at several positions to alter its catalytic activity or substrate affinity or glucan properties . in one example , the rsw1 gene is mutated to comprise one or more mutations present in the mutant rsw1 allele . the mutated genetic sequences are cloned into binary plasmid described in the preceding examples , in place of the wild - type sequences . plant tissue obtained from both wild - type arabidopsis thaliana ( columbia ) plants and a . thaliana rsw1 plants is transformed as described herein and whole plants are regenerated . control transformations are performed using the wild - type cellulose synthase gene sequence . plants transformed with genetic constructs described in example 15 ( and elsewhere ) are categorised initially on the basis of number of transgene copies , to eliminate variability arising therefrom . plants expressing single copies of different transgenes are analysed further for cell wall components , including cellulose , non - crystalline β - 1 , 4 - glucan polymer , starch and carbohydrate content . cellulose content in the transgenic plants is determined by the 14 c incorporation assay as described in example 1 . cell walls are prepared , fractionated and the monosaccharide composition of individual fractions determined as in example 1 . transgenic plants expressing the rsw1 mutant allele exhibit a higher level of non - crystalline , and therefore extractable , β - 1 , 4 - glucan in cell walls compared to plants expressing an additional copy of the wild - type rsw1 allele . thus , it is possible to change the crystallinity of the β - 1 , 4 - glucan chains present in the cell wall by mutation of the wild - type rsw1 allele . transgenic plants are also analysed to determine the effect of mutagenesis of the rsw1 gene on the level of starch deposited in their roots . the quantity of starch present in material prepared from the crude wall fraction is determined using the anthrone / h 2 so 4 method described in example 1 . the data show that mutating the rsw1 gene to the mutant rsw1 allele increases starch deposition . this demonstrates that the gene can be used to alter the partitioning of carbon into carbohydrates other than cellulose . the cell wall composition of transgenic plant material is also analysed . wild type and rsw1 and transgenic seedlings are grown for 2 d at 21 ° c . and then kept for a further 5 d at either 21 ° c . or 31 ° c . with transfer to 31 ° c . when the seed has scarcely germinated , the wall composition at final harvest largely reflects the operation of the mutated rsw1 gene product at its restrictive temperature . cell wall fractionation is carried out in similar fashion to that described for the 14 c - experiment ( example 1 ) and the monosaccharide composition of each fraction is quantified by gc / ms after hydrolysis with trifluoroacetic acid or , in the case of crystalline cellulose , h 2 so 4 . in some transgenic plants in which the rsw1 gene is mutated , the monosaccharide composition is comparable to that observed for homozygous rsw1 plants , at least in some cases , confirming that there is a major reduction in the quantity of crystalline cellulose in the final , acid insoluble fraction . thus , mutation of the rsw1 gene can be performed to produce changes in the composition of plant cell walls . chemical modification of the rsw1 gene to manipulate cellulose production and plant cell wall content as demonstrated in the preceding examples , the rsw1 gene is involved in cellulose production and the manipulation of cell wall content . in the present example , to identify novel phenotypes and gene sequences important for the normal functioning of the cellulose synthase gene , the rsw1 gene is modified in planta , using the chemical mutagen ems . the mutant plants are identified following germination and the modified rsw1 genes are isolated and characterised at the nucleotide sequence level . a sequence comparison between the mutant gene sequences and the wild type sequence reveals nucleotides which encode amino acids important to the normal catalytic activity of the cellulose synthase enzyme , at least in arabidopsis thaliana plants . this approach thus generates further gene sequences of utility in the modification of cellulose content and properties in plants . five pieces of evidence make a compelling case that the rsw1 gene product encodes the catalytic subunit of cellulose synthase : 1 . the rsw1 mutation selectively inhibits cellulose synthesis and promotes accumulation of a non - crystalline β - 1 , 4 - glucan ; 2 . the rsw1 mutation removes cellulose synthase complexes from the plasma membrane , providing a plausible mechanism for reduced cellulose accumulation and placing the rsw1 product either in the complexes or interacting with them ; 3 . the d , d , d , qxxrw ( seq id no : 37 ) signature identifies the rsw1 gene product as a processive glycosyl transferase enzyme ( saxena , 1995 ); 4 . the wild type allele corrects the temperature sensitive phenotype of the rsw1 mutant ; and 5 . antisense expression of the rsw1 in transgenic plants grown at 21 ° c . reproduces some of the phenotype of rsw1 which is observed following growth at 31 ° c . consistent with the plasma membrane location expected for a catalytic subunit , the putative 122 kda rsw1 product contains 8 predicted membrane - spanning regions . six of these regions cluster near the c - terminus ( fig1 ), separated from the other two by a domain that is probably cytoplasmic and has the weak sequence similarities to prokaryotic glycosyl transferases ( wong , 1990 ; saxena , 1990 ; matthyse , 1995 ; sofia , 1994 ; kutish , 1996 ). rsw1 therefore qualifies as a member of the large family of arabidopsis thaliana genes whose members show weak similarities to bacterial cellulose synthase . rsw1 is the first member of that family to be rigorously identified as an authentic cellulose synthase . among the diverse genes in a . thaliana , at least two genes show very strong sequence similarities to the rsw1 gene and are most likely members of a highly conserved sub - family involved in cellulose synthesis . the closely related sequences come from cosmid 12c4 , a partial genomic clone cross - hybridising with est t20782 designated ath - a , and from a full length cdna designated ath - b . ath - a resembles rsw1 ( seq id no : 5 ) at its n - terminus whereas ath - b starts 22 amino acid residues downstream [ fig8 and fig9 a - 9 j ]. closely related sequences in other angiosperms are the rice est s0542 [ fig9 a - 9 j ], which resembles the polypeptides encoded by rsw1 and ath - a and the cotton cela1 gene ( pear , 1996 ) at the n - terminus . the arabidopsis thaliana , rice and cotton genes have regions of very high sequence similarity interspersed with variable regions ( fig9 a - 9j and 10 ). most of the highest conservation among those gene products occurs in their central cytoplasmic domain where the weak similarities to the bacterial cellulose synthase occur . the n - terminal region that precedes the first membrane spanning region is probably also cytoplasmic but shows many amino acid substitutions as well as sequences in rsw1 that have no counterpart in some of the other genes as already noted for cela . an exception to this is a region comprising 7 cysteine residues with highly conserved spacings ( fig1 ). this is reminiscent of regions suggested to mediate protein - protein and protein - lipid interactions in diverse proteins including transcriptional regulators and may account for the striking sequence similarity between this region of rsw1 and two putative soybean bzip transcription factors ( genbank soystf1a and 1b ). in conclusion , the chemical and ultrastructural changes seen in the cellulose - deficient mutant combine with gene cloning and complementation of the mutant to provide strong evidence that the rsw1 locus encodes the catalytic subunit of cellulose synthase . accumulation of non - crystalline β - 1 , 4 - glucan in the shoot of the rsw1 mutant suggests that properties affected by the mutation are required for glucan chains to assemble into microfibrils . whilst not being bound by any theory or mode of action , a key property may be the aggregation of catalytic subunits into plasma membrane rosettes . at the restrictive temperature , mutant synthase complexes disassemble to monomers ( or smaller oligomers ) that are undetectable by freeze etching . at least in the shoot , the monomers seem to remain biosynthetically active but their β - 1 , 4 - glucan products fail to crystallise into microfibrils probably because the chains are growing from dispersed sites . crystallisation into microfibrils , with all its consequences for wall mechanics and morphogenesis , therefore may depend upon catalytic subunits remaining aggregated as plasma membrane rosettes . those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described . it is to be understood that the invention includes all such variations and modifications . the invention also includes all of the steps , features , compositions and compounds referred to or indicated in this specification , individually or collectively , and any and all combinations or any two or more of said steps or features . 2 . ausubel et al . 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