BACKGROUND
cereal grains contain a good balance of proteins, fats, carbohydrate, vitamins and minerals required for human growth and health.
unlike other cereals, wheat is rarely consumed in an unprocessed form but prepared into a wide range of end products.
common wheat  is used in the preparation of bread, noodles, biscuits, and cakes.
durum wheat  is used mainly for pasta production but also in an array of other regional foods in italy, north africa and west asia .
the processing and end-use quality of wheat-based products depends on different factors such as protein content and composition, grain hardness and starch composition.
starch, the most important polysaccharide in human diet and is the major component of the wheat kernel, representing more than 70% of its dry weight.
as well as its importance in the food industry, starch is also used as a raw material for the production of non-food products in the paper, plastic, adhesive, textile, medical and pharmaceutical industries  <cit> .
reserve starch is accumulated in the amyloplast organelles and is composed of two different glucosidic polymers, amylose and amylopectin.
the main differences between these polymers are the degree of polymerization and the number of side branches.
amylose is a linear chain of d-glucose molecules with a low degree of polymerization , whereas amylopectin shows a higher degree of polymerization  and which has important implications for function.
amylopectin is the major constituent of starch in wheat endosperm and comprises about 70-80%; with amylose constituting the remaining 20-30%.
amylose and amylopectin are synthesized by two different pathways having a common substrate .
a granule bound starch synthase  is involved in amylose synthesis, whereas amylopectin is produced by the concerted action of starch synthases , starch branching enzymes  and starch debranching enzymes of isoamylase- and limit dextrinase-type   <cit> .
sbes are transglycosylase enzymes that catalyze the formation of α- <dig>  linkages within the polymer by cleaving an internal alpha- <dig>  linkage.
in monocots, three starch branching isoforms are found: sbei, sbeiia and sbeiib.
in maize, rice and pea, suppression of sbeiib leads to amylose-extender  phenotype, with a very high amylose content   <cit> , in contrast suppression of sbei or sbeiia has no effect on the amount of amylose  <cit> .
in wheat sbeiia and sbeiib genes have been characterized and found to be located on the long arm of the homoeologous group  <dig> chromosomes  <cit> .
regina et al.
<cit>  demonstrated that wheat sbeiia gene is syntenic to the corresponding gene in other cereals, in contrast the sbeiib gene is not in a syntenic position.
in wheat, sbeiia is the predominant isoform present in the soluble phase of the endosperm  <cit> , whereas in maize and rice endosperm sbeiib is the predominant isoform involved in amylopectin biosynthesis  <cit> .
the role of sbeiia and sbeiib isoforms in bread wheat endosperm has been investigated by rna interference technology  <cit> .
in contrast to other cereals, the silencing of sbeiib genes has no effect on amylose content and starch granule shape; whereas silencing of sbeiia genes results in a strong increase in amylose content  and granule deformation.
there is increasing interest in the manipulation of starch composition in wheat due to the recognition of its important role in food and non food applications and its uses in industry.
in addition, the research is also focusing on the production of high amylose starch flours because derived foods have an increased amount of resistant starch which has been shown to have beneficial effects on human health.
resistant starch refers to the portion of starch that resists digestion in the stomach and small intestine and it can act as a substrate for microbial fermentation in the large intestine, the end-products being hydrogen, carbon dioxide, methane and short chain fatty acids   <cit> .
the nutritionists believe that the resistant starch has a role similar to dietary fibre inside the intestine, protecting against diseases as colon cancer, type ii diabetes, obesity and osteoporosis  <cit> .
a recent study demonstrated that high-amylose flours might be used to substitute for up to 50% normal wheat flour to make bread with both an acceptable bread quality and a significantly higher amount of resistant starch  <cit> .
furthermore, pasta produced with semolina containing a higher amylose content shows good cooking resistance and firmness, satisfying consumer preferences  <cit> .
in the present study, a transgenic approach was used to increase the amylose content in durum wheat seeds.
in particular, the effects of sbeiia silencing were investigated in terms of amylose content, transcript accumulation and protein profile of the enzymes involved in starch biosynthesis.
RESULTS
biolistic transformation and selection of transgenic plants
in total,  <dig> immature embryos obtained from the triticum durum cv.
svevo were co-transformed using prdpt + sbeiia and bar selectable marker plasmids.
forty eight t <dig> independent transgenic lines resistant to bialaphos and containing the rnai cassette were obtained, with a co-transformation efficiency of  <dig> %.
the presence of the transgene was verified by polymerase chain reaction  analysis of genomic dna from regenerated plants, by using two primer pairs, one specific for the promoter of the construct  and another for the bar gene , to produce amplicons of  <dig> bp and  <dig> bp, respectively .
no significant differences in morphology and growth were observed between transgenic lines and either the corresponding "null" genotype that had lost the transgene by segregation , or untransformed plants.
agrobacterium-mediated transformation and selection of transgenic plants
in all,  <dig>  immature embryos of the triticum durum cv.
ofanto were transformed using pgub-g + sbeiia containing also the bar gene cassette in the t-dna.
thirteen t <dig> transgenic lines were obtained with a transformation efficiency of  <dig> %.
the presence of the transgene was verified as described above.
as with transgenic plants generated by biolistic transformation, no morphological differences were observed among the different lines or between transgenics and controls.
molecular characterization of transgenic plants
the absence of sbeiia transcript was investigated by semiquantitive rt-pcr and real time rt-pcr on total rna, extracted from t <dig> immature kernels , using a primer pair specific for sbeiia genes .
we focused on six transgenic lines, three derived from the biolistic method and three from agrobacterium-mediated transformation.
gadph  was used as housekeeping gene.
a complete silencing of sbeiia genes was observed in four lines and partial silencing in two lines .
both the partially silenced lines were obtained by biolistic bombardment while the three lines obtained through agrobacterium-mediated transformation were completely silenced.
in one plant  the level of sbeiia transcript was 30-fold less than in the control line, while in line mj16- <dig> the expression of the target gene appeared to be unaffected at  <dig> dpa .
starch granule-bound proteins, extracted from transgenic seeds, were separated on sds-page gel and compared with those present in the untransformed cultivars.
unexpectedly, two new starch granule-bound proteins were identified in the transformed lines , showing a molecular weight between  <dig> and  <dig> kda.
moreover, the intensity of sgp- <dig>  and to a lesser extent sgp- <dig>  sgp- <dig> and waxy was higher in the transgenic lines.
the two novel sgps  were investigated by mass spectrometry and shown to be waxy-like proteins .
sgp- <dig> and sgp- <dig> were similar to durum wheat genbank accession baa <dig> with a coverage of 41%  and 36% , respectively.
in both cases the probability that the two proteins corresponded to accession baa <dig> was 100%.
determination of total starch, amylose and protein content
the amylose content was strongly increased in sbeiia rnai transgenic lines in comparison to untransformed controls of svevo and ofanto.
amylose values ranged from  <dig> % up to 75% .
although the amylose percentage was more than 70% in endosperm from the mj16- <dig> line, the plant morphology, shape and yield of seeds  was not significantly different from non-transformed controls.
a reduction in the endosperm starch content of transgenic lines obtained by biolistic transformation was observed .
the protein content of the same lines ranged between  <dig> % and  <dig> % and were similar to untransformed control  .
values represent means of three replicates.
standard error is also reported for each value.
nd: not determined.
starch granule analysis
sem analysis showed deep alterations of starch granules in mj16- <dig> line.
the granules with reduced sbeiia expression were deformed with irregular shape, looked deflated and were smaller than wild type .
in particular, a high number of a-type granules had a size of  <dig> μm and b-type granules had lost the spherical shape typical of the controls .
this phenotype was observed both in svevo and ofanto transgenic lines and is clearly visible in figure  <dig>  deformation of starch granules was more evident in line mj16- <dig> compared to a <dig>  this could be associated to the different level of amylose content existing between the two lines.
rva analysis
viscosity properties of whole wheat flours were measured by the rapid visco analyser  on svevo and the silenced lines mj16- <dig> and mj16- <dig>  in the case of the high-amylose lines, an overall decrease in viscosity was observed in comparison with the non-transgenic line .
significant variations were observed for all rva parameters.
mean trough , breakdown , setback  and final viscosities  of sbeiia silenced lines were significantly lower than for the control wheat samples .
there was a clear difference in the retrogradation rates of starches between svevo and the corresponding transgenic lines, mj16- <dig> and mj16- <dig>  even though sbeiia rnai lines had high amylose content, the setback value was smaller than the control, suggesting that a lower retrogradation of these rnai lines is due to their lower viscosity value.
we observed a negative correlation between amylose percentage and viscosity; in particular the line mj16- <dig> had the higher amylose content  and the lower viscosity values.
whereas the line mj16- <dig>  possessing  <dig> % of amylose, had a intermediate rva profile compared to svevo and mj16- <dig>
pv, peak viscosity; bd, breakdown; fv, final viscosity; sb, setback; pt, peak time.
real time rt-pcr on genes involved in starch biosynthesis
to understand if there were pleiotropic effects on the expression of other genes involved in starch biosynthesis, semiquantitative rt-pcr was performed on gbssi, ssi, ssii, ssiii, sbei, sbeiib, isa <dig> and ld genes.
the expression levels of many starch biosynthetic genes were affected by the silencing of sbeiia genes.
compared to the control lines, the transcripts of the genes encoding gbssi, ssiii, ld and isa <dig> were increased in transgenic plants, whereas significant changes were not found in ssi, ssii, sbei and sbeiib genes.
transcript accumulation of gbssi, ssiii, ld and isa <dig> were investigated by quantitative real-time rt-pcr.
as expected, all analyzed genes were up-regulated in comparison with untransformed plants but their level of up-regulation varied .
the expression of gbssi was strongly increased in transgenic seeds with a 12- <dig> fold increase compared to controls).
the expression of ssiii, ld and isa <dig> was up-regulated by 6- <dig>   <dig> - <dig>  and 4- <dig> fold respectively in svevo transgenic plants compared to controls .
discussion
different strategies can be used to modify amylose content in wheat starch.
in the last twenty years the identification of null mutations in starch biosynthetic genes and the crossing of these materials to combine functional mutations have permitted the production of lines with low and high amylose content  <cit> .
unfortunately this strategy is not easy to apply for all starch biosynthetic proteins, due to the difficulty of obtaining the relevant mutants in polyploid species such as wheat.
both transgenic and non-transgenic tools are available, and the latter, along with novel technologies, offers the possibility to manipulate starch composition and develop wheat varieties with new functionalities.
recently a reverse genetic strategy called tilling  has permitted the identification of induced mutations for genes involved in amylose  <cit>  and amylopectin  <cit>  biosynthesis.
the tilling strategy is an efficient, non-transgenic method to identify mutants in genes of interest, but the effect of silencing is often masked by the presence of other functional alleles in polyploid species.
to obtain a null phenotype, it is usually necessary to generate double or triple mutants by crossing single null genotypes  <cit> .
recent studies have demonstrated that transgenic technology is a powerful research tool to modify amylose/amylopectin ratio.
starch composition can be manipulated through genetic engineering, either by overexpression or repression of starch biosynthetic genes.
rnai technology is a specific and powerful mean to silence a target gene.
this technology is particularly useful in polyploid plants such as wheat because it increases the possibility to obtain transgenic plants with a novel phenotype.
in this study, an rnai strategy has been followed to silence sbeiia genes in order to increase the amylose content in two durum wheat cultivars, svevo and ofanto.
although two different methods have been used for genetic transformation , the silencing of sbeiia genes produced similar effects.
the agrobacterium- or the biolistic-mediated genetic transformation showed various levels of silencing between independent transgenic wheat lines.
the abundance of mrnas of sbeiia genes varied in the transgenic lines from nearly undetectable to wild type level confirming that the rnai construct, designed to reduce the expression of sbeiia genes, was effective in inducing gene silencing.
the variation in the levels of gene silencing observed here is in agreement with previously reported results in wheat  <cit>  and other species, such for example tobacco  <cit> , maize  <cit>  and arabidopsis  <cit> .
the silencing of the sbeiia gene caused marked modifications on amylose content, granule morphology and starch composition.
an increase in amylose content was found in all transgenic lines but its percentage varied between lines, presumably due to differences in copy number and/or positional effects.
the line mj16- <dig> showed the exceptionally high value of 75% amylose content, while in the other lines it varied between 31-56%.
a variable level of amylose content between rnai lines was also found in sweetpotato  <cit>  and potato  <cit> .
no significant difference was observed in grain yield between transformed and control lines, although rnai lines showed a lower total starch content.
the reduction of total starch in rnai lines could be due to the difficulty to estimate starch content in high amylose wheats.
mcclearly et al.
<cit>  used different approaches to determine total starch content and found that in high amylose maize the starch content resulted always underestimated.
morphological analysis of starch granules showed a marked difference between sbeiia-silenced lines and control plants.
type-a granules appeared smaller and deflated, whereas type-b granules lost their normal spherical shape and became extended.
similar results were obtained by regina et al.
<cit>  who suppressed the activity of sbeiia through rnai technology in bread wheat.
rva analysis, carried out on whole wheat flour, highlighted large alterations in chemical-physical properties of transgenic starch.
rva parameters, as mean trough, breakdown, setback and final viscosities of sbeiia-silenced lines, were significantly lower than normal wheats.
these results are consistent with those obtained on high amylose flour of wheat  <cit> , corn  <cit>  and barley  <cit> .
the decreased swelling and viscosity affects chemical-physical pasting properties of semolina, with subsequent changes in functional characteristics of derived food products.
although high amylose flour resulted in a smaller bread loaf volume  <cit> , it has a positive impact in the pasta industry, reducing starch loss during the cooking and increasing pasta firmness.
in addition, high amylose foods might have beneficial effects on human diet because of an increased amount of resistant starch, which has a role similar to dietary fibre inside the intestine, protecting against diseases as colon cancer, type ii diabetes, obesity and osteoporosis  <cit> .
sds-page analysis of starch granule-bound proteins allowed the identification of two newly accumulated proteins localized between the sgp- <dig> and sgp- <dig> in the transgenic lines.
these novel proteins are present in all transgenic plants, suggesting that they are a consistent result of sbeiia silencing rather than an unintended effect of random transgene insertions.
the novel proteins show a high similarity to the waxy isoforms.
since the molecular mass of the novel proteins is larger  than that of waxy proteins , one possibility is that the novel proteins are encoded by genes that are poorly or not expressed in wild type cultivar.
an alternative explanation is that a mechanism of post-translation modification has somehow been triggered in these transgenic lines.
the staining intensity of the other starch granule proteins  on sds-page is strongly increased in the silenced lines compared to the control.
real-time rt-pcr analyses on genes involved in starch biosynthesis have confirmed possible interaction and feedback mechanisms between these genes.
in particular, the absence of sbeiia isoforms leads to an up-regulation of wx, ssiii, ld and isa <dig> genes.
pleiotropic effects associated with up-regulation or suppression of specific genes present in the starch biosynthetic pathway have been reported previously.
for example, kosar-hashemi et al.
<cit>  observed a reduction of granule-bound branching enzymes and starch synthases in the starch granule of the ssii mutants and suggested that the lack of ssii would lead to the absence of the complex on starch granule.
similarly, the rice sugary- <dig>  and the maize dull- <dig>  mutants had drastic changes in accumulation of pullulanase and sbeiib isoforms, respectively  <cit> .
in a recent work, the down-regulation of a pullulanase-type dbe inhibitor produced pleiotropic effects on activities of soluble and granule-bound starch synthase, that were significantly reduced  <cit> .
tetlow et al.
<cit>  have discovered the formation of a heterocomplex comprising sbei, sbeiib, and starch phosphorylase in wheat.
more recently, tetlow et al.
<cit>  have identified trimeric assemblages of ssi with ssiia and either sbeiia or sbeiib.
hennen-bierwagen et al.
<cit>  have demonstrated that sss, sbes and dbes associate physically with each other in multisubunit complexes and the lack of one subunit can cause variation on the other isoforms.
in maize the ae- mutation, in the gene coding for sbeiib, was associated with the detection of four novel proteins not observed in wild-type granules.
lc-ms/ms analysis showed that the four bands corresponded to sbei, sbeiia, ssiii and starch phosphorylase  <cit> .
grimaud et al.
<cit>  suggested that structural changes in amylopectin, due to the loss of sbeiib, could offer a more efficient glucan substrate for binding of sbei, sbeiia, starch phosphorylase and ssiii.
the pleiotropic effects observed in our sbeiia-silenced lines represent additional evidence of a strict correlation between all starch enzymes and that the lack of one subunit can cause variation in the other isoforms.
CONCLUSIONS
the rnai-mediated down-regulation of genes coding the sbeiia isoforms produced remarkable changes in starch chemical-physical properties of durum wheat.
no significant differences were identified in grain weight between transgenic plants and wild type controls.
as well as a considerable increase in the amylose content, the silenced sbeiia wheat lines also possessed altered starch granule structure and up-regulated transcriptional profiles of gbssi, ssiii, ld and isa <dig>  the use of two different transformation methods, biolistic and agrobacterium-mediated, and two different durum wheat cultivars, highlights that the effect of sbeiia silencing on starch metabolism is genotype and protocol independent because similar effects have been detected in all the rnai plants obtained.
the variation in amylose content and the different starch physical-chemical properties make these plant materials particularly valuable in a vast range of applications in both food and non food industries.

 cereal grains contain a good balance of proteins, fats, carbohydrate, vitamins and minerals required for human growth and health. unlike other cereals, wheat is rarely consumed in an unprocessed form but prepared into a wide range of end products. common wheat is used in the preparation of bread, noodles, biscuits, and cakes. durum wheat is used mainly for pasta production but also in an array of other regional foods in italy, north africa and west asia. the processing and end-use quality of wheat-based products depends on different factors such as protein content and composition, grain hardness and starch composition. starch, the most important polysaccharide in human diet and is the major component of the wheat kernel, representing more than 70% of its dry weight. as well as its importance in the food industry, starch is also used as a raw material for the production of non-food products in the paper, plastic, adhesive, textile, medical and pharmaceutical industries. reserve starch is accumulated in the amyloplast organelles and is composed of two different glucosidic polymers, amylose and amylopectin. the main differences between these polymers are the degree of polymerization and the number of side branches. amylose is a linear chain of d-glucose molecules with a low degree of polymerization, whereas amylopectin shows a higher degree of polymerization and which has important implications for function. amylopectin is the major constituent of starch in wheat endosperm and comprises about 70-80%; with amylose constituting the remaining 20-30%. amylose and amylopectin are synthesized by two different pathways having a common substrate. a granule bound starch synthase is involved in amylose synthesis, whereas amylopectin is produced by the concerted action of starch synthases, starch branching enzymes and starch debranching enzymes of isoamylase- and limit dextrinase-type. sbes are transglycosylase enzymes that catalyze the formation of α- linkages within the polymer by cleaving an internal alpha- linkage. in monocots, three starch branching isoforms are found: sbei, sbeiia and sbeiib. in maize, rice and pea, suppression of sbeiib leads to amylose-extender phenotype, with a very high amylose content, in contrast suppression of sbei or sbeiia has no effect on the amount of amylose. in wheat sbeiia and sbeiib genes have been characterized and found to be located on the long arm of the homoeologous group chromosomes. regina et al. demonstrated that wheat sbeiia gene is syntenic to the corresponding gene in other cereals, in contrast the sbeiib gene is not in a syntenic position. in wheat, sbeiia is the predominant isoform present in the soluble phase of the endosperm, whereas in maize and rice endosperm sbeiib is the predominant isoform involved in amylopectin biosynthesis. the role of sbeiia and sbeiib isoforms in bread wheat endosperm has been investigated by rna interference technology. in contrast to other cereals, the silencing of sbeiib genes has no effect on amylose content and starch granule shape; whereas silencing of sbeiia genes results in a strong increase in amylose content and granule deformation. there is increasing interest in the manipulation of starch composition in wheat due to the recognition of its important role in food and non food applications and its uses in industry. in addition, the research is also focusing on the production of high amylose starch flours because derived foods have an increased amount of resistant starch which has been shown to have beneficial effects on human health. resistant starch refers to the portion of starch that resists digestion in the stomach and small intestine and it can act as a substrate for microbial fermentation in the large intestine, the end-products being hydrogen, carbon dioxide, methane and short chain fatty acids. the nutritionists believe that the resistant starch has a role similar to dietary fibre inside the intestine, protecting against diseases as colon cancer, type ii diabetes, obesity and osteoporosis. a recent study demonstrated that high-amylose flours might be used to substitute for up to 50% normal wheat flour to make bread with both an acceptable bread quality and a significantly higher amount of resistant starch. furthermore, pasta produced with semolina containing a higher amylose content shows good cooking resistance and firmness, satisfying consumer preferences. in the present study, a transgenic approach was used to increase the amylose content in durum wheat seeds. in particular, the effects of sbeiia silencing were investigated in terms of amylose content, transcript accumulation and protein profile of the enzymes involved in starch biosynthesis.  biolistic transformation and selection of transgenic plants in total, immature embryos obtained from the triticum durum cv. svevo were co-transformed using prdpt + sbeiia and bar selectable marker plasmids. forty eight t independent transgenic lines resistant to bialaphos and containing the rnai cassette were obtained, with a co-transformation efficiency of %. the presence of the transgene was verified by polymerase chain reaction analysis of genomic dna from regenerated plants, by using two primer pairs, one specific for the promoter of the construct and another for the bar gene, to produce amplicons of bp and bp, respectively. no significant differences in morphology and growth were observed between transgenic lines and either the corresponding "null" genotype that had lost the transgene by segregation, or untransformed plants. agrobacterium-mediated transformation and selection of transgenic plants in all, immature embryos of the triticum durum cv. ofanto were transformed using pgub-g + sbeiia containing also the bar gene cassette in the t-dna. thirteen t transgenic lines were obtained with a transformation efficiency of %. the presence of the transgene was verified as described above. as with transgenic plants generated by biolistic transformation, no morphological differences were observed among the different lines or between transgenics and controls. molecular characterization of transgenic plants the absence of sbeiia transcript was investigated by semiquantitive rt-pcr and real time rt-pcr on total rna, extracted from t immature kernels, using a primer pair specific for sbeiia genes. we focused on six transgenic lines, three derived from the biolistic method and three from agrobacterium-mediated transformation. gadph was used as housekeeping gene. a complete silencing of sbeiia genes was observed in four lines and partial silencing in two lines. both the partially silenced lines were obtained by biolistic bombardment while the three lines obtained through agrobacterium-mediated transformation were completely silenced. in one plant the level of sbeiia transcript was 30-fold less than in the control line, while in line mj16- the expression of the target gene appeared to be unaffected at dpa. starch granule-bound proteins, extracted from transgenic seeds, were separated on sds-page gel and compared with those present in the untransformed cultivars. unexpectedly, two new starch granule-bound proteins were identified in the transformed lines, showing a molecular weight between and kda. moreover, the intensity of sgp- and to a lesser extent sgp- sgp- and waxy was higher in the transgenic lines. the two novel sgps were investigated by mass spectrometry and shown to be waxy-like proteins. sgp- and sgp- were similar to durum wheat genbank accession baa with a coverage of 41% and 36%, respectively. in both cases the probability that the two proteins corresponded to accession baa was 100%. determination of total starch, amylose and protein content the amylose content was strongly increased in sbeiia rnai transgenic lines in comparison to untransformed controls of svevo and ofanto. amylose values ranged from % up to 75%. although the amylose percentage was more than 70% in endosperm from the mj16- line, the plant morphology, shape and yield of seeds was not significantly different from non-transformed controls. a reduction in the endosperm starch content of transgenic lines obtained by biolistic transformation was observed. the protein content of the same lines ranged between % and % and were similar to untransformed control. values represent means of three replicates. standard error is also reported for each value. nd: not determined. starch granule analysis sem analysis showed deep alterations of starch granules in mj16- line. the granules with reduced sbeiia expression were deformed with irregular shape, looked deflated and were smaller than wild type. in particular, a high number of a-type granules had a size of μm and b-type granules had lost the spherical shape typical of the controls. this phenotype was observed both in svevo and ofanto transgenic lines and is clearly visible in figure deformation of starch granules was more evident in line mj16- compared to a this could be associated to the different level of amylose content existing between the two lines. rva analysis viscosity properties of whole wheat flours were measured by the rapid visco analyser on svevo and the silenced lines mj16- and mj16- in the case of the high-amylose lines, an overall decrease in viscosity was observed in comparison with the non-transgenic line. significant variations were observed for all rva parameters. mean trough, breakdown, setback and final viscosities of sbeiia silenced lines were significantly lower than for the control wheat samples. there was a clear difference in the retrogradation rates of starches between svevo and the corresponding transgenic lines, mj16- and mj16- even though sbeiia rnai lines had high amylose content, the setback value was smaller than the control, suggesting that a lower retrogradation of these rnai lines is due to their lower viscosity value. we observed a negative correlation between amylose percentage and viscosity; in particular the line mj16- had the higher amylose content and the lower viscosity values. whereas the line mj16- possessing % of amylose, had a intermediate rva profile compared to svevo and mj16- pv, peak viscosity; bd, breakdown; fv, final viscosity; sb, setback; pt, peak time. real time rt-pcr on genes involved in starch biosynthesis to understand if there were pleiotropic effects on the expression of other genes involved in starch biosynthesis, semiquantitative rt-pcr was performed on gbssi, ssi, ssii, ssiii, sbei, sbeiib, isa and ld genes. the expression levels of many starch biosynthetic genes were affected by the silencing of sbeiia genes. compared to the control lines, the transcripts of the genes encoding gbssi, ssiii, ld and isa were increased in transgenic plants, whereas significant changes were not found in ssi, ssii, sbei and sbeiib genes. transcript accumulation of gbssi, ssiii, ld and isa were investigated by quantitative real-time rt-pcr. as expected, all analyzed genes were up-regulated in comparison with untransformed plants but their level of up-regulation varied. the expression of gbssi was strongly increased in transgenic seeds with a 12- fold increase compared to controls). the expression of ssiii, ld and isa was up-regulated by 6- - and 4- fold respectively in svevo transgenic plants compared to controls. discussion different strategies can be used to modify amylose content in wheat starch. in the last twenty years the identification of null mutations in starch biosynthetic genes and the crossing of these materials to combine functional mutations have permitted the production of lines with low and high amylose content. unfortunately this strategy is not easy to apply for all starch biosynthetic proteins, due to the difficulty of obtaining the relevant mutants in polyploid species such as wheat. both transgenic and non-transgenic tools are available, and the latter, along with novel technologies, offers the possibility to manipulate starch composition and develop wheat varieties with new functionalities. recently a reverse genetic strategy called tilling has permitted the identification of induced mutations for genes involved in amylose and amylopectin biosynthesis. the tilling strategy is an efficient, non-transgenic method to identify mutants in genes of interest, but the effect of silencing is often masked by the presence of other functional alleles in polyploid species. to obtain a null phenotype, it is usually necessary to generate double or triple mutants by crossing single null genotypes. recent studies have demonstrated that transgenic technology is a powerful research tool to modify amylose/amylopectin ratio. starch composition can be manipulated through genetic engineering, either by overexpression or repression of starch biosynthetic genes. rnai technology is a specific and powerful mean to silence a target gene. this technology is particularly useful in polyploid plants such as wheat because it increases the possibility to obtain transgenic plants with a novel phenotype. in this study, an rnai strategy has been followed to silence sbeiia genes in order to increase the amylose content in two durum wheat cultivars, svevo and ofanto. although two different methods have been used for genetic transformation, the silencing of sbeiia genes produced similar effects. the agrobacterium- or the biolistic-mediated genetic transformation showed various levels of silencing between independent transgenic wheat lines. the abundance of mrnas of sbeiia genes varied in the transgenic lines from nearly undetectable to wild type level confirming that the rnai construct, designed to reduce the expression of sbeiia genes, was effective in inducing gene silencing. the variation in the levels of gene silencing observed here is in agreement with previously reported results in wheat and other species, such for example tobacco, maize and arabidopsis. the silencing of the sbeiia gene caused marked modifications on amylose content, granule morphology and starch composition. an increase in amylose content was found in all transgenic lines but its percentage varied between lines, presumably due to differences in copy number and/or positional effects. the line mj16- showed the exceptionally high value of 75% amylose content, while in the other lines it varied between 31-56%. a variable level of amylose content between rnai lines was also found in sweetpotato and potato. no significant difference was observed in grain yield between transformed and control lines, although rnai lines showed a lower total starch content. the reduction of total starch in rnai lines could be due to the difficulty to estimate starch content in high amylose wheats. mcclearly et al. used different approaches to determine total starch content and found that in high amylose maize the starch content resulted always underestimated. morphological analysis of starch granules showed a marked difference between sbeiia-silenced lines and control plants. type-a granules appeared smaller and deflated, whereas type-b granules lost their normal spherical shape and became extended. similar results were obtained by regina et al. who suppressed the activity of sbeiia through rnai technology in bread wheat. rva analysis, carried out on whole wheat flour, highlighted large alterations in chemical-physical properties of transgenic starch. rva parameters, as mean trough, breakdown, setback and final viscosities of sbeiia-silenced lines, were significantly lower than normal wheats. these results are consistent with those obtained on high amylose flour of wheat, corn and barley. the decreased swelling and viscosity affects chemical-physical pasting properties of semolina, with subsequent changes in functional characteristics of derived food products. although high amylose flour resulted in a smaller bread loaf volume, it has a positive impact in the pasta industry, reducing starch loss during the cooking and increasing pasta firmness. in addition, high amylose foods might have beneficial effects on human diet because of an increased amount of resistant starch, which has a role similar to dietary fibre inside the intestine, protecting against diseases as colon cancer, type ii diabetes, obesity and osteoporosis. sds-page analysis of starch granule-bound proteins allowed the identification of two newly accumulated proteins localized between the sgp- and sgp- in the transgenic lines. these novel proteins are present in all transgenic plants, suggesting that they are a consistent result of sbeiia silencing rather than an unintended effect of random transgene insertions. the novel proteins show a high similarity to the waxy isoforms. since the molecular mass of the novel proteins is larger than that of waxy proteins, one possibility is that the novel proteins are encoded by genes that are poorly or not expressed in wild type cultivar. an alternative explanation is that a mechanism of post-translation modification has somehow been triggered in these transgenic lines. the staining intensity of the other starch granule proteins on sds-page is strongly increased in the silenced lines compared to the control. real-time rt-pcr analyses on genes involved in starch biosynthesis have confirmed possible interaction and feedback mechanisms between these genes. in particular, the absence of sbeiia isoforms leads to an up-regulation of wx, ssiii, ld and isa genes. pleiotropic effects associated with up-regulation or suppression of specific genes present in the starch biosynthetic pathway have been reported previously. for example, kosar-hashemi et al. observed a reduction of granule-bound branching enzymes and starch synthases in the starch granule of the ssii mutants and suggested that the lack of ssii would lead to the absence of the complex on starch granule. similarly, the rice sugary- and the maize dull- mutants had drastic changes in accumulation of pullulanase and sbeiib isoforms, respectively. in a recent work, the down-regulation of a pullulanase-type dbe inhibitor produced pleiotropic effects on activities of soluble and granule-bound starch synthase, that were significantly reduced. tetlow et al. have discovered the formation of a heterocomplex comprising sbei, sbeiib, and starch phosphorylase in wheat. more recently, tetlow et al. have identified trimeric assemblages of ssi with ssiia and either sbeiia or sbeiib. hennen-bierwagen et al. have demonstrated that sss, sbes and dbes associate physically with each other in multisubunit complexes and the lack of one subunit can cause variation on the other isoforms. in maize the ae- mutation, in the gene coding for sbeiib, was associated with the detection of four novel proteins not observed in wild-type granules. lc-ms/ms analysis showed that the four bands corresponded to sbei, sbeiia, ssiii and starch phosphorylase. grimaud et al. suggested that structural changes in amylopectin, due to the loss of sbeiib, could offer a more efficient glucan substrate for binding of sbei, sbeiia, starch phosphorylase and ssiii. the pleiotropic effects observed in our sbeiia-silenced lines represent additional evidence of a strict correlation between all starch enzymes and that the lack of one subunit can cause variation in the other isoforms.  the rnai-mediated down-regulation of genes coding the sbeiia isoforms produced remarkable changes in starch chemical-physical properties of durum wheat. no significant differences were identified in grain weight between transgenic plants and wild type controls. as well as a considerable increase in the amylose content, the silenced sbeiia wheat lines also possessed altered starch granule structure and up-regulated transcriptional profiles of gbssi, ssiii, ld and isa the use of two different transformation methods, biolistic and agrobacterium-mediated, and two different durum wheat cultivars, highlights that the effect of sbeiia silencing on starch metabolism is genotype and protocol independent because similar effects have been detected in all the rnai plants obtained. the variation in amylose content and the different starch physical-chemical properties make these plant materials particularly valuable in a vast range of applications in both food and non food industries. 
in this work, genes encoding starch branching enzymes of class ii were silenced using the rna interference technique in two cultivars of durum wheat, using two different methods of transformation. results obtained with two different methods of transformation and in two durum wheat cultivars were comparable. high amylose starch has attracted particular interest because of its correlation with the amount of resistant starch in food. two novel granule bound proteins, identified by sds-page in sbeiia rnai lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. we have found that the silencing of sbeiia genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. rs plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. rva analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. amylose content was markedly increased in the durum wheat transgenic lines exhibiting sbeiia gene silencing. in particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content.
 high amylose starch has attracted particular interest because of its correlation with the amount of resistant starch in food. rs plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. in particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. in this work, genes encoding starch branching enzymes of class ii were silenced using the rna interference technique in two cultivars of durum wheat, using two different methods of transformation. expression of rnai transcripts was targeted to the seed endosperm using a tissue-specific promoter.  amylose content was markedly increased in the durum wheat transgenic lines exhibiting sbeiia gene silencing. moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. two novel granule bound proteins, identified by sds-page in sbeiia rnai lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. rva analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. finally, pleiotropic effects on other starch genes were found by semi-quantitative and real-time reverse transcription-polymerase chain reaction.  we have found that the silencing of sbeiia genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. results obtained with two different methods of transformation and in two durum wheat cultivars were comparable. 