Role of Granule-bound Starch Synthase in Determination of Amylopectin Structure and Starch Granule Morphology in Potato
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m111579200
ISSN1083-351X
AutoresDaniel C. Fulton, Anne Edwards, Emma Pilling, Helen L. Robinson, Brendan Fahy, Robert Seale, Lisa Kato, Athene M. Donald, Peter Geigenberger, Cathie Martin, Alison M. Smith,
Tópico(s)Phytase and its Applications
ResumoReductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring. Reductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring. Little is known about the processes that determine the morphology of starch granules, but important clues have been gained from studies of mutant plants with altered granule morphology. Many such plants carry mutations in genes encoding isoforms of starch synthase and starch-branching enzyme responsible for the synthesis of amylopectin, the branched α1,4, α1,6 glucan, which forms the semi-crystalline matrix of the granule. For example, mutations in pea affecting starch-branching enzyme A (at the r locus (1.Bhattacharyya M.K. Smith A.M. Ellis T.H.N. Hedley C. Martin C. Cell. 1990; 60: 115-121Abstract Full Text PDF PubMed Scopus (322) Google Scholar)) and starch synthase II (SSII, 1The abbreviations used are: SSISSII, and SSIII, soluble starch synthases I, II, and IIIGBSSgranule-bound starch synthaseFAGEfluorophore-assisted polyacrylamide gel electrophoresisHPLChigh performance liquid chromatographySAXSsmall-angle x-ray scatteringdpdegree of polymerizationFWTfresh weight 1The abbreviations used are: SSISSII, and SSIII, soluble starch synthases I, II, and IIIGBSSgranule-bound starch synthaseFAGEfluorophore-assisted polyacrylamide gel electrophoresisHPLChigh performance liquid chromatographySAXSsmall-angle x-ray scatteringdpdegree of polymerizationFWTfresh weight at the rug5 locus (2.Craig J. Lloyd J.R. Tomlinson K. Barber L. Edwards A. Wang T.L. Martin C. Hedley C.L. Smith A.M. Plant Cell. 1998; 10: 413-426Crossref PubMed Scopus (162) Google Scholar)) convert the normally ovoid granules of the embryo into deeply fissured, multilobed structures and highly twisted, contorted structures, respectively (2.Craig J. Lloyd J.R. Tomlinson K. Barber L. Edwards A. Wang T.L. Martin C. Hedley C.L. Smith A.M. Plant Cell. 1998; 10: 413-426Crossref PubMed Scopus (162) Google Scholar, 3.Hedley C.L. Smith C.M. Ambrose M.J. Cook S. Wang T.L. Ann. Bot. (Lond.). 1986; 58: 371-377Crossref Scopus (62) Google Scholar). Mutations in maize affecting starch-branching enzyme IIb (at the amylose-extender locus (4.Kim K.N. Fisher D.K. Gao M. Guiltinan M.J. Plant Mol. Biol. 1998; 38: 945-956Crossref PubMed Scopus (50) Google Scholar)) convert the normally polyhedral granules of the endosperm into irregular, elongated structures (5.Boyer C.D. Daniels R.R. Shannon J.C. Crop Sci. 1976; 16: 298-301Crossref Google Scholar).The altered granule morphology of the mutants presumably results from alterations in the structures and relative amounts of the glucan polymers of which the granule is comprised. Most mutations affecting isoforms of starch synthase and starch branching enzyme affect both of these parameters. The r and rug5 mutations of pea and the amylose-extender mutation of maize affect the proportion of short and long chains in amylopectin, the chain-length distribution within the short-chain population, and the ratio within the granule of amylopectin to amylose, the essentially linear glucan that makes up 20–30% of storage starches (2.Craig J. Lloyd J.R. Tomlinson K. Barber L. Edwards A. Wang T.L. Martin C. Hedley C.L. Smith A.M. Plant Cell. 1998; 10: 413-426Crossref PubMed Scopus (162) Google Scholar, 6.Boyer C.D. Garwood D.L. Shannon J.C. Starch/Staerke. 1976; 12: 405-436Crossref Scopus (50) Google Scholar, 7.Colonna P. Mercier C. Carbohydr. Res. 1984; 126: 233-247Crossref Scopus (82) Google Scholar, 8.Wang Y.J. White P. Pollak L. Jane J. Cereal Chem. 1993; 70: 171-179Google Scholar, 9.Tomlinson K.L. Craig J. Smith A.M. Plant J. 1997; 11: 31-43Crossref Scopus (61) Google Scholar). However, it is not known which of these changes in granule composition and polymer structure leads to the changes in granule morphology.Transgenic potato plants with reduced activity of isoforms of starch synthase provide a good system for elucidation of the relationship between granule morphology and glucan structure. Plants expressing antisense RNA for granule-bound starch synthase I (GBSS antisense plants), the isoform exclusively responsible for the synthesis of amylose, have much less amylose in the tuber starch but normal granule morphology (10.Kuipers A.G.J. Jacobsen E. Visser R.G.F. Plant Cell. 1994; 6: 43-52Crossref PubMed Google Scholar). Plants expressing antisense RNA for starch synthase III (SSIII antisense plants), the major isoform responsible for amylopectin synthesis, have a markedly altered chain-length distribution among the short chains of amylopectin and an increased proportion of very long chains (11.Edwards A. Fulton D.C. Hylton C.M. Jobling S.A. Gidley M. Roessner U. Martin C. Smith A.M. Plant J. 1999; 17: 251-261Crossref Scopus (184) Google Scholar, 12.Lloyd J.R. Landschütze V. Kossmann J. Biochem. J. 1999; 38: 515-521Crossref Google Scholar). Whereas granules from wild-type and GBSS antisense plants have a smooth, ovoid profile, those from SSIII antisense lines are deeply internally fissured and are frequently multilobed (13.Marshall J. Sidebottom C. Debet M. Martin C. Smith A.M. Edwards A. Plant Cell. 1996; 8: 1121-1135Crossref PubMed Scopus (110) Google Scholar). We reasoned that the altered granule morphology in SSIII antisense lines could be caused either by the altered chain-length distribution of short chains of amylopectin, or by the increased proportion of very long chains in amylopectin, or by the interaction of amylose molecules within the granule with one or both of these abnormal features of amylopectin.To distinguish between these possibilities, we have generated transgenic lines down-regulated for both SSIII and GBSS simultaneously (SSIII/GBSS antisense lines), and compared the starch of control and SSIII, GBSS, and SSIII/GBSS antisense lines with respect to amylopectin structure, amylose content, and granule morphology.EXPERIMENTAL PROCEDURESMaterialsPotato plants (Solanum tuberosum L. cv. Desiree and transgenic plants derived from it) were grown from shoots propagated in tissue culture (14.Edwards A. Marshall J. Sidebottom C. Visser R.G.F. Smith A.M. Martin C. Plant J. 1995; 8: 283-294Crossref PubMed Scopus (110) Google Scholar) then transferred to soil-based compost in a greenhouse at a minimum temperature of 12 °C with supplementary lighting in winter. Tubers for enzymes assays, native gel electrophoresis, and starch preparations were used immediately after harvest. Tuber tissue for analysis of starch content was frozen at −20 °C for up to 4 months before use. For a given set of experiments, control and transgenic plants were grown in the same greenhouse at the same time.MethodsConstruction of Antisense Binary VectorThe 2.2-kb NcoI fragment encoding the full-length cDNA of potato GBSS (15.Dry I. Smith A. Edwards A. Bhattacharrya M. Dunn P. Martin C. Plant J. 1992; 2: 193-202PubMed Google Scholar) was subcloned, in the antisense orientation, into the NcoI site of pRAT3. pRAT3 contains a 1.1-kb fragment of the potato SSIII cDNA in antisense orientation between the cauliflower mosaic virus double 35S promoter and terminator (13.Marshall J. Sidebottom C. Debet M. Martin C. Smith A.M. Edwards A. Plant Cell. 1996; 8: 1121-1135Crossref PubMed Scopus (110) Google Scholar). The resulting [promoter]-[SSIII antisense (1 kb)]-[GBSS antisense (2.2 kb)]-[SSIII antisense (0.1 kb)]-[terminator] chimera was excised as a KpnI/XhoI fragment and ligated between the KpnI/SalI sites of plant transformation vector pBIN19 (16.Bevan M. Nucleic Acids Res. 1984; 12: 8711-8721Crossref PubMed Scopus (1806) Google Scholar) to give plasmid pROT1.Transformation of PotatoTransformation and preparation of Agrobacterium inoculum carrying the antisense construct, inoculation of tuber discs of potato, and regeneration of shoots were all done as described previously (13.Marshall J. Sidebottom C. Debet M. Martin C. Smith A.M. Edwards A. Plant Cell. 1996; 8: 1121-1135Crossref PubMed Scopus (110) Google Scholar, 14.Edwards A. Marshall J. Sidebottom C. Visser R.G.F. Smith A.M. Martin C. Plant J. 1995; 8: 283-294Crossref PubMed Scopus (110) Google Scholar).Enzyme Assays and Native GelsAssays for starch synthases were done according to Refs. 13.Marshall J. Sidebottom C. Debet M. Martin C. Smith A.M. Edwards A. Plant Cell. 1996; 8: 1121-1135Crossref PubMed Scopus (110) Google Scholar and 14.Edwards A. Marshall J. Sidebottom C. Visser R.G.F. Smith A.M. Martin C. Plant J. 1995; 8: 283-294Crossref PubMed Scopus (110) Google Scholar. Assays for starch-branching enzyme and ADP-glucose pyrophosphorylase were according to Refs. 17.Smith A.M. Planta. 1988; 175: 270-279Crossref PubMed Scopus (141) Google Scholar and 18.Sowokinos J. Plant Physiol. 1976; 57: 63-68Crossref PubMed Google Scholar, respectively. Native gels for starch synthase and starch-degrading enzymes were prepared according to Refs. 13.Marshall J. Sidebottom C. Debet M. Martin C. Smith A.M. Edwards A. Plant Cell. 1996; 8: 1121-1135Crossref PubMed Scopus (110) Google Scholar and 11.Edwards A. Fulton D.C. Hylton C.M. Jobling S.A. Gidley M. Roessner U. Martin C. Smith A.M. Plant J. 1999; 17: 251-261Crossref Scopus (184) Google Scholar, respectively.Assay and Extraction of Starch and Analyses of Granule-bound ProteinsStarch contents of tuber tissue were measured according to Ref. 17.Smith A.M. Planta. 1988; 175: 270-279Crossref PubMed Scopus (141) Google Scholar. Starch was purified from tubers according to Ref. 14.Edwards A. Marshall J. Sidebottom C. Visser R.G.F. Smith A.M. Martin C. Plant J. 1995; 8: 283-294Crossref PubMed Scopus (110) Google Scholar. Starch used in the compositional and structural analyses described below was from tubers harvested from mature, senescing plants. Granule-bound proteins were extracted from purified starch and subjected to SDS-polyacrylamide gel electrophoresis according to Ref.11.Edwards A. Fulton D.C. Hylton C.M. Jobling S.A. Gidley M. Roessner U. Martin C. Smith A.M. Plant J. 1999; 17: 251-261Crossref Scopus (184) Google Scholar.Visualization of Growth RingsApproximately 0.3 g of starch was suspended in 1 ml of water, frozen in liquid nitrogen in a mortar, and ground thoroughly to crack granules. After thawing, granules were recovered by centrifugation and incubated with occasional stirring at 37 °C for 7 h in 50 mm Mes-KOH (pH 5.6) at 0.3 mg/ml, with 4 units of α-amylase (porcine pancreas) mg−1 starch. Granules were then washed three times by resuspension and centrifugation in acetone at −20 °C, and dried. Dry starch samples were brushed onto the surface of double-sided, carbonated sticky stills attached to SEM stubs (Agar Scientific, Cambridge, UK), coated with gold for 3 min at 15 mA in argon using a Polaran SEM coating unit (Polaran Equipment, Watford, UK), and viewed in a Philips XL30 Field Emission Gun SEM (Philips, Eindhoven, The Netherlands).Small-angle X-ray Scatter AnalysisAnalyses were performed according to Refs. 19.Jenkins P.J. Cameron R.E. Donald A.M. Starch/Staerke. 1993; 45: 417-420Crossref Scopus (316) Google Scholar, 20.Jenkins P.J. Donald A.M. Int. J. Biol. Macromol. 1995; 17: 315-321Crossref PubMed Scopus (382) Google Scholar, 21.Cameron R.E. Donald A.M. Polymer. 1992; 33: 2628-2635Crossref Scopus (219) Google Scholar.Analysis of Starch CompositionThe size distribution and relative amounts of amylose and amylopectin in starch and starch fractions were examined by low pressure gel permeation chromatography. Samples of starch (20 mg), amylopectin (15 mg), or amylose (5.2 mg) were suspended in 1 m NaOH (0.4 ml) and diluted with 1 ml of dH2O and boiled for 5 min. After cooling, a further 1 ml of dH2O was added. The resulting solution was loaded onto two 1-m (i.d. 15 mm) Sepharose CL-2B columns connected in series, equilibrated in 10 mm NaOH, and eluted in a descending mode at 0.16 ml/min. Fractions of 6 ml were collected, and samples were mixed with acidified iodine (Lugol's) solution and monitored at 595 nm.Partial Purification of Amylose and AmylopectinAmylose and amylopectin were prepared by butanol precipitation. Starch samples (1.33 g) were suspended in 100 ml of H2O and boiled for 1 h, adjusted to pH 5.9–6.3 with 100 mm phosphate buffer, autoclaved (100 kPa, 1 h), and heated under reflux conditions at 100 °C for 1 h. Butanol (25 ml) was added, and the mixture was refluxed for 1 h then allowed to cool very slowly from boiling point to ambient temperature (over a period of 36–48 h). The amylose precipitate was collected by centrifugation, and the amylopectin-containing supernatant was again subjected to the refluxing procedure described above. The combined amylopectin-containing supernatants and the combined amylose-containing precipitates were lyophilized. Amylopectin was also prepared by gel permeation chromatography, as described above. Fractions from the amylopectin peak were neutralized with 2 m HCl, desalted, and lyophilized.Analysis of Chain-length Distribution of AmylopectinAnalysis of the chain-length distribution of the shorter chains of amylopectin was achieved by using fluorophore-assisted polyacrylamide gel electrophoresis (FAGE) of debranched starch using an Applied Biosystems 373A DNA sequencer (11.Edwards A. Fulton D.C. Hylton C.M. Jobling S.A. Gidley M. Roessner U. Martin C. Smith A.M. Plant J. 1999; 17: 251-261Crossref Scopus (184) Google Scholar). Analysis of the chain-length profile across the full range of chain lengths was achieved by HPLC. For routine analysis, amylopectin obtained either by the butanol precipitation method or by gel permeation chromatography, was suspended in boiling water (10 mg/ml) then autoclaved (100 kPa, 30 min). For some analyses amylopectin was instead suspended in 90% (v/v) Me2SO (10 mg/ml) and stirred in a boiling water bath for 15 min before precipitation with four volumes of methanol. The precipitate was redissolved in a one-fifth volume of water. Amylopectin suspensions obtained by either of the above methods were mixed with 1/20 volume of 220 mmsodium acetate (pH 4.8) and 4 × 104 units of isoamylase (Sigma Chemical Co.), incubated at 37 °C for 16 h then autoclaved (100 kPa, 30 min). A sample of 100 μl was immediately loaded onto the HPLC system, which consisted of guard columns and three HPLC columns connected in series (SecurityGuard (4 × 3 mm), Biosep-Sec-S 2000 guard column (75 × 7.8 mm), two Biosep-Sec-S 2000 columns (600 × 7.8 mm) and a Biosep-Sec-S 3000 column (600 × 7.8 mm); all from Phenomenex, Torrance, CA). The columns and sample injector were heated to 37 °C. The system was eluted with water at 0.28 ml/min, and 0.56-ml fractions were collected. To a sample (0.28 ml) of each fraction was added 1/20 volume of 220 mmsodium acetate (pH 4.8) and 0.28 units of amyloglucosidase. The mixture was incubated at 37 °C overnight then assayed enzymatically for glucose. Two checks were made on the authenticity of the long chains detected in amylopectin. First, results were compared from samples of amylopectin prepared from the same batch of starch either by gel permeation chromatography or by the butanol precipitation method. Second, results were compared from samples of the same batch of amylopectin prepared either in water or in Me2SO, as described above. Pullulan standards were from Polymer Laboratories Ltd., Church Stretton, Shropshire, UK.Measurement of Sugar Nucleotides, ATP and ADPRapid sampling and extraction of tuber tissue was according to Ref. 22.Merlo L. Geigenberger P. Hajirezaei M. Stitt M. J. Plant Physiol. 1993; 142: 392-402Crossref Scopus (101) Google Scholar. Assay of sugar nucleotides, ATP and ADP, was by HPLC according to Ref.23.Geigenberger P. Reimholz R. Geiger M. Merlo L. Canale V. Stitt M. Planta. 1997; 201: 502-518Crossref Scopus (184) Google Scholar, the authors of which provide evidence of the reliability of the methods of extraction and analysis for the metabolites.RESULTSGeneration of Plants in Which Activities of both SSIII and GBSS Are Significantly ReducedTo achieve a simultaneous reduction of both GBSS and SSIII isoforms, tubers of Desiree were transformed with plasmid pROT1, a construct that expresses a chimeric antisense RNA of SSIII and GBSS under the control of the cauliflower mosaic virus double 35S promoter. The transgenic lines thus produced are referred to as SSIII/GBSS antisense lines. The fragments of the GBSS and SSIII cDNAs used in the construct had both been used previously to obtain plants expressing antisense RNA for either SSIII (11, 13: referred to as SSIII antisense lines) or GBSS (24: referred to as GBSS antisense lines). In subsequent experiments, the SSIII/GBSS lines were compared with three SSIII and three GBSS antisense lines described previously (11.Edwards A. Fulton D.C. Hylton C.M. Jobling S.A. Gidley M. Roessner U. Martin C. Smith A.M. Plant J. 1999; 17: 251-261Crossref Scopus (184) Google Scholar, 13.Marshall J. Sidebottom C. Debet M. Martin C. Smith A.M. Edwards A. Plant Cell. 1996; 8: 1121-1135Crossref PubMed Scopus (110) Google Scholar, 24.Tatge H. Marshall J. Martin C. Edwards E.A. Smith A.M. Plant Cell Env. 1999; 22: 543-550Crossref Scopus (68) Google Scholar) and with control lines that were either untransformed Desiree or a line transformed with the vector pBIN19 alone.Developing tubers of primary transformants were assayed for activity of soluble and granule-bound starch synthase. Of the 35 independent transformants examined, seven had reductions of 80% or more in granule-bound activity relative to a control line, and five of these also had reductions of 50% or more in soluble starch synthase activity. The latter reductions were shown to be specific for the SSIII isoform by native gel electrophoresis of tuber extracts followed by staining for starch synthase activity (Fig. 1A). The intensity of the band representing SSIII was greatly reduced in transgenic plants with low soluble starch synthase activity, but the intensities of bands representing SSII (11.Edwards A. Fulton D.C. Hylton C.M. Jobling S.A. Gidley M. Roessner U. Martin C. Smith A.M. Plant J. 1999; 17: 251-261Crossref Scopus (184) Google Scholar) and SSI (25.Kossmann J. Abel G.J.W. Springer F. Lloyd J. Willmitzer L. Planta. 1999; 208: 503-511Crossref PubMed Scopus (62) Google Scholar) were unaffected. In two of the plants, soluble and granule-bound starch synthase activities were reduced to levels comparable with those in the most extreme SSIII and GBSS antisense lines characterized previously. These lines (Rot1.1 and Rot1.4) were propagated for further study.Soluble starch synthase activity in the SSIII/GBSS antisense tubers was reduced by 3.4-fold (Rot1.1) and 4.9-fold (Rot 1.4) relative to the control line (Table I), compared with reductions of between 2.5- and 5.5-fold in the SSIII antisense lines (11.Edwards A. Fulton D.C. Hylton C.M. Jobling S.A. Gidley M. Roessner U. Martin C. Smith A.M. Plant J. 1999; 17: 251-261Crossref Scopus (184) Google Scholar, 13.Marshall J. Sidebottom C. Debet M. Martin C. Smith A.M. Edwards A. Plant Cell. 1996; 8: 1121-1135Crossref PubMed Scopus (110) Google Scholar). Thus the reductions in SSIII activity in Rot1.1 and Rot1.4 are comparable with those in the SSIII antisense lines. Granule-bound starch synthase activity was reduced by 8-fold (Rot 1.1) and 14-fold (Rot 1.4) compared with a reduction of 7- to 12-fold at a comparable developmental stage in the most extreme GBSS antisense lines studied previously (lines 5 and 8 (24.Tatge H. Marshall J. Martin C. Edwards E.A. Smith A.M. Plant Cell Env. 1999; 22: 543-550Crossref Scopus (68) Google Scholar)). Confirmation that the extent of reduction in GBSS was comparable in the SSIII/GBSS and the most extreme GBSS antisense lines, was obtained by probing blots of granule-bound proteins with an antiserum against GBSS (Fig. 1B).Table IActivities of enzymes of starch synthesisEnzymeControl line (Desiree)SSIII/GBSSAntisense line Rot1.1Antisense line Rot1.4μmolSoluble starch synthase (nmol/min/g FWT)63.6 ± 4.218.7 ± 2.013.1 ± 0.7GBSS (nmol/min/g FWT)114.8 ± 5.114.9 ± 1.58.1 ± 0.6ADP-glucose pyrophosphorylase (nmol/min/g FWT)651 ± 78795 ± 81551 ± 78Starch-branching enzyme (μmol/min/g FWT)35.5 ± 3.245.9 ± 5.5 (3)35.7 ± 3.4Soluble starch synthase, ADP-glucose pyrophosphorylase, and starch-branching enzyme were assayed in the soluble fraction of crude extracts of freshly harvested tubers. Starch-branching enzyme was measured by the phosphorylase stimulation assay. Activity is expressed as micromoles of glucose from glucose 1-phosphate incorporated into glucan/min/g fresh weight (FWT). Granule-bound activity (GBSS) was estimated by subtracting soluble starch synthase activity from starch synthase activity in the crude, uncentrifuged homogenate. With the exception of the starch-branching enzyme activity for line Rot1.4, values are means ± S.E. of measurements on five extracts, each from a different tuber, using tubers from at least three plants. The value for starch-branching enzyme activity for line Rot1.4 is the mean of measurements on three extracts, each from a different tuber. Open table in a new tab To check whether activity of enzymes of starch metabolism other than SSIII and GBSS were affected in the SSIII/GBSS antisense tubers, we assayed activities of ADP-glucose pyrophosphorylase and starch-branching enzyme and analyzed starch-degrading activities on native, amylopectin-containing gels. There were no major differences between the SSIII/GBSS antisense lines and untransformed Desiree with respect to these activities (Table I, Fig. 1C).Granule Morphology and AnatomyStarch contents (per gram of fresh weight) of mature tubers of the transgenic lines were not reduced relative to those of the control line. In three harvests, the starch contents of SSIII and GBSS antisense lines and the SSIII/GBSS antisense line Rot1.4 were not statistically significantly different from the control line. The starch content of the SSIII/GBSS antisense line Rot 1.1 was about 30% higher than that of the control line in two harvests and not statistically significantly different from the control line in the third harvest (data not shown).There were striking differences in the morphology of starch granules from mature tubers of the GBSS, SSIII, and SSIII/GBSS antisense lines. Granules of the GBSS antisense lines were ovoid and unfissured like those of untransformed Desiree (Fig. 2, A and B). Granules of SSIII antisense lines were deeply internally fissured with fissuring centered on the hilum (origin of growth), or were multilobed or clustered structures. This was true of lines with reductions in SSIII activity of as little as 2.5- to 3-fold, as well as of lines with greater reductions in activity (11, 13; Fig. 2C). Fissures developed as SSIII granules matured, rather than being formed continuously from the start of granule synthesis. Small granules from young, developing tubers were not fissured (Fig. 2D). Thus fissures represent a fracturing of a previously continuous matrix rather than the non-continuous development of the matrix. Granules of the SSIII/GBSS antisense lines were not fissured. Some appeared as clusters of small granules. Mature granules were more spherical and had a more central hilum than those of untransformed Desiree and the GBSS antisense lines (Fig. 2E).Figure 2Morphology and internal structure of starch granules. A–F, light micrographs of intact, hydrated starch granules. Granules in B and F were stained with dilute iodine solution (1/20 Lugol solution), granules in other panels are unstained. Bars represent 50 μm. G–J, scanning electron micrographs of granules after mechanical cracking and incubation with α-amylase to reveal growth rings. Bars represent 2 μm. A, control line (Desiree). B, GBSS antisense line 8. C, SSIII antisense line 18: starch from mature tubers. D, SSIII antisense line 18, starch from young, developing tubers (of ∼0.5 g fresh weight). E and F, SSIII/GBSS antisense line Rot1.1. G, control line (Desiree). H, GBSS antisense line 8. I, SSIII/GBSS antisense line Rot1.1. J, SSIII antisense line 18.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The internal growth rings of granules also differed between the lines. The growth rings represent the alternation of semi-crystalline and amorphous zones within the matrix, with a repeat distance of some hundreds of nm. Growth rings were visualized by digesting internal surfaces with α-amylase, which preferentially digests material in amorphous zones. Rings were distinct and evenly spaced in granules from the control line and from the GBSS and SSIII/GBSS antisense lines (Fig. 2, G–I). However, growth rings in granules from SSIII antisense plants were much less distinct and regular in appearance (Fig. 2J). This indicates some disruption of the normal organization of the semi-crystalline zones of the granule.To investigate further the disruption of the semi-crystalline zones in granules of SSIII antisense lines, the small-angle x-ray scattering (SAXS) patterns of starch preparations were examined. Starch granules from many different plants, including potato, show a characteristic peak in the SAXS pattern at a q-spacing of about 0.06 Å−1, where q is the angular distance in the scattering pattern. This peak represents a repeat distance of 9 nm within the semi-crystalline zones of the granule matrix. It is believed that this repeat represents the clustering of the shorter chains of amylopectin at regular intervals along the axis of amylopectin molecules. Adjacent chains within clusters form double helices. The packing of the helices in regular arrays produces alternating crystalline and amorphous lamellae (19.Jenkins P.J. Cameron R.E. Donald A.M. Starch/Staerke. 1993; 45: 417-420Crossref Scopus (316) Google Scholar).The SAXS peak for SSIII antisense starch occurred at a markedly lower q-value and was broader than that for untransformed Desiree starch, whereas the q-value for peaks for SSIII/GBSS antisense and GBSS antisense starches was similar to that of untransformed Desiree (Fig. 3). This indicates that the periodicity of the semi-crystalline repeat is greater and the packing is less well organized in starch from SSIII antisense tubers than in starch from control, SSIII/GBSS, and GBSS antisense tubers.Figure 3Small-angle x-ray scatter analysis of starches. Radiation with a wavelength of 1.5 Å was focused onto starch samples in the form of 50% (w/w) slurries in water. A gas-filled proportional wire chamber quadrant detector was used to collect the diffraction pattern. Experiments were carried out at the Synchrotron Radiation Source, Daresbury Laboratory, UK. The symbols used indicate starches from a control line transformed with BIN19 alone (open triangles), SSIII antisense line 18 (closed triangles), GBSS antisense line 6 (closed squares), and SSIII/GBSS antisense line Rot1.1 (open squares).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Granule CompositionThe amylose content of starch was much lower in GBSS and SSSIII/GBSS antisense lines than in the control line. Amylose was almost undetectable in starch from both the most extreme GBSS antisense line and the SSIII/GBSS antisense lines when analyzed by gel permeation chromatography (Fig. 4A). Co
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