Abstract
Starch represents the most important carbohydrate used for food and feed purposes. Increasingly, it is also used as a renewable raw material, as a source of biofuel, and for many different industrial applications. Progress in understanding starch biosynthesis, and investigations of the genes involved in this process, has enabled the genetic modification f crops in a rational manner to produce novel designer starches with improved functionality.
The hypothesis of the present study is that the hyper-phosphorylation of cereal endosperm starch makes it easily accessible and degradable by the amylolytic enzymes while the amylose-only endosperm starch exhibits high resistance to degradation and hence less available for degradation. With the aim to investigate the hypothesis, starch molecular structures were modulated with the above mentioned modifications and were studied for the effects of modified starches (in comparison
to wild-type) during grain germination, development and on its pathogen resistance during Fusarium Head blight (FHB). The targets were the phosphate content, which has potential industrial applications and amylose content having valuable food and feed applications. This was achieved by endosperm-specific overexpression of Solanum tuberosum GWD to generate hyper-phosphorylated (HP) starch and endosperm-specific RNAi suppression of all three starch branching enzyme (SBE)
isoforms to generate amylose-only (AO) starch in barley (cv. Golden Promise).
In first and second study, the effects of engineering high levels of phosphate and amylose content on starch physico-chemical properties were evaluated by various biochemical and morphological studies. As a result, a substantial increase of 10-fold phosphate content and ~99% amylose content with high-resistant starch was observed in our transgenic barley lines. The results of scanning electron microscope suggested that the HP starch appear to promote premature starch degradation in cereal endosperm. The studies of in-vitro enzymatic starch hydrolysis demonstrated that the AO starch had 2.2-fold higher resistant starch than the wild-type cultivar. These exciting results may provide a potential clean technological approach to starch modification by in-planta bioengineering and avoid environmental hazards resulting from post-harvest treatments by chemical modifications.
The third study was to investigate the effects of these modified starches and the modifications on germination and it was observed that HP did not show higher starch mobilization or degradation (despite of lower hydrolase activities), while AO showed significantly lower starch mobilization compared to wild-type (WT). This study oncluded that the inadequate starch deposition in these lines in combination with suppressed hydrolase activities led to temporal and compensating redirection
of starch, sugar and protein metabolism in order to maintain the metabolic dynamics
during germination. Further investigations on grain development revealed the differences in storage reserve accumulation, metabolite accumulation in AO but no significant differences were observed in HP compared to WT. Scanning electron microscopy and confocal microscopy revealed the details in topography and internal structures of the starch granules in these lines. The results demonstrated for the first time, the grain compositional, starch structural and metabolite level effects of the specific suppression of all of the three SBE isoforms and overexpression of GWD in
cereal endosperm over development. The investigations on the effect of these starch types on fusarium growth and secondary metabolite production revealed few differences, which need further investigations. Overall, this study contributes to the research knowledge of starch bioengineering in cereals.
The hypothesis of the present study is that the hyper-phosphorylation of cereal endosperm starch makes it easily accessible and degradable by the amylolytic enzymes while the amylose-only endosperm starch exhibits high resistance to degradation and hence less available for degradation. With the aim to investigate the hypothesis, starch molecular structures were modulated with the above mentioned modifications and were studied for the effects of modified starches (in comparison
to wild-type) during grain germination, development and on its pathogen resistance during Fusarium Head blight (FHB). The targets were the phosphate content, which has potential industrial applications and amylose content having valuable food and feed applications. This was achieved by endosperm-specific overexpression of Solanum tuberosum GWD to generate hyper-phosphorylated (HP) starch and endosperm-specific RNAi suppression of all three starch branching enzyme (SBE)
isoforms to generate amylose-only (AO) starch in barley (cv. Golden Promise).
In first and second study, the effects of engineering high levels of phosphate and amylose content on starch physico-chemical properties were evaluated by various biochemical and morphological studies. As a result, a substantial increase of 10-fold phosphate content and ~99% amylose content with high-resistant starch was observed in our transgenic barley lines. The results of scanning electron microscope suggested that the HP starch appear to promote premature starch degradation in cereal endosperm. The studies of in-vitro enzymatic starch hydrolysis demonstrated that the AO starch had 2.2-fold higher resistant starch than the wild-type cultivar. These exciting results may provide a potential clean technological approach to starch modification by in-planta bioengineering and avoid environmental hazards resulting from post-harvest treatments by chemical modifications.
The third study was to investigate the effects of these modified starches and the modifications on germination and it was observed that HP did not show higher starch mobilization or degradation (despite of lower hydrolase activities), while AO showed significantly lower starch mobilization compared to wild-type (WT). This study oncluded that the inadequate starch deposition in these lines in combination with suppressed hydrolase activities led to temporal and compensating redirection
of starch, sugar and protein metabolism in order to maintain the metabolic dynamics
during germination. Further investigations on grain development revealed the differences in storage reserve accumulation, metabolite accumulation in AO but no significant differences were observed in HP compared to WT. Scanning electron microscopy and confocal microscopy revealed the details in topography and internal structures of the starch granules in these lines. The results demonstrated for the first time, the grain compositional, starch structural and metabolite level effects of the specific suppression of all of the three SBE isoforms and overexpression of GWD in
cereal endosperm over development. The investigations on the effect of these starch types on fusarium growth and secondary metabolite production revealed few differences, which need further investigations. Overall, this study contributes to the research knowledge of starch bioengineering in cereals.
| Originalsprog | Engelsk |
|---|
| Forlag | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
|---|---|
| Antal sider | 275 |
| Status | Udgivet - 2013 |