Abstract
Rice is a very important cereal crop. It serves as a major source of dietary energy for more than half of the world’s population. Rice grain is usually polished to make it more acceptable to consumers. Consequently, commercially available rice grains have high glycaemic index (GI) and trace amounts of resistant starch (RS). These traits are undesirable considering the alarming global increase in the incidence of ailments like diabetes, cardiovascular disease, colorectal cancer and obesity. The development of rice grains with reduced digestibility can be an effective strategy to manage these nutrition-associated conditions. This can be done by developing rice grains with high amylose because it is associated with elevated RS and reduced GI. Alterations to the amylopectin structure can also be investigated to determine its effect on digestibility. In this study, more than 2,500 wild and cultivated rice accessions from the International Rice Research Institute were screened for amylose content, and the digestibility of a high amylose subset was characterised. Results revealed that the in vitro RS content of the selected grains was around 1%, compared with trace amounts found in the glutinous varieties, where amylose is absent. Furthermore, the selected grains also demonstrated significant reductions in GI estimates compared to glutinous rice grains. However, the apparent amylose content (AAC) in the rice grains tested did not exceed 30%. Hence, to further investigate the structural basis for reduced digestibility and to identify potential novel approaches to increase amylose content, selected mutants were characterised. Of the mutants available for this study, Goami 2 presented the most interesting properties. It has high AAC and RS contents and the genetic basis of its mutation is still unknown. Proteins associated with the starch granule that are involved in starch biosynthesis were characterised. It was found that Goami 2 had elevated levels of an unusual isoform of starch branching enzyme IIb (SBEIIb). The genetic or structural basis of this isoform could not be uncovered in this study despite exhaustive characterisation but it may reside in an interacting gene. Interestingly, Goami 2 had elevated RS despite lack of observable elevations in true amylose chains. In contrast, the well-characterised mutant IR36ae had elevations in both true amylose and intermediate material that were accompanied by an increase in RS, but not to the extent of Goami 2. Furthermore, characterisation of grain digestibility of WAB3-5, an amylose producer in a glutinous rice background, revealed that it also had elevated RS and reduced GI. These findings suggest that aside from amylose, the intermediate chains in rice may play an important role in impacting RS contents and GI estimate. To further increase the variability of starch structure and to obtain more insights into its relationship with digestibility, transgenic experiments using hairpin RNA (hp-RNA) and artificial microRNA (amiRNA) were conducted to modify the expression of selected enzymes involved in starch biosynthesis. Previous studies of down regulating the expression of starch branching enzyme IIa (SBEIIa), SBEIIb and starch synthase IIa (SSIIa) in wheat, maize and barley, respectively, had been found to increase the amylose contents up to 50-75%. However, in this study, only minor alterations in the distribution of debranched amylopectin chains were observed when SBEIIa and SSIIa were down regulated, and it did not affect the amounts of RS. In contrast, down regulating the expression of SBEIIb led to an increase in apparent amylose (35%) and resistant starch (4.8%) contents, as well as a much lower GI estimate (44). Similar to what was observed in IR36ae and Goami 2, down regulating the expression of SBEIIb resulted in significantly elevated intermediate chains. This further strengthened the hypothesis that intermediate chains are associated with an increase in the levels of RS content and reductions in GI. These intermediate chains may be responsible for increasing the AAC values, probably because they are lightly branched and therefore can bind iodine to form a complex that absorbs in the region of 600-700 nm. Despite only subtle alterations in the debranched amylopectin structure in the lines where the expression of SSIIa was down regulated, there was still a slight reduction in glycaemic index estimate compared to its parental line Nipponbare. The rice lines had no increase in amylose and intermediate chains, and their RS content was not elevated. Interestingly, it has relatively lower proportion of debranched short amylopectin chains compared to Nipponbare, which may explain why its digestion was slowed. Decreasing the amount of short chain amylopectin could be an important target in decreasing the glycaemic index of rice, especially if it is not accompanied by any increase in amylose and intermediate chains. This is of practical importance because elevated amylose is associated with a decrease in grain and cooking qualities for many consumers, which reduces consumer acceptance. Since individually down regulating the expression of SSIIa, SBEIIa, and SBEIIb was not effective in Nipponbare in elevating the proportion of true amylose chains, the three genes were simultaneously targeted in various combinations. Simultaneous down regulation of the expression of starch enzymes have been demonstrated to be effective in producing extreme starch phenotypes in wheat, barley, maize and potato. In this study, simultaneously targeting SBEIIa and SBEIIb did not significantly improve the phenotypes compared with lines with the expression of only SBEIIb was reduced. However, the chain length distribution profile of the lines developed showed a more extensive alteration than that observed when SBEIIb alone was down regulated. Since SBEIIa was previously hypothesised to act on longer chains up to degree of polymerisation (DP) 15, it is possible that the role of SBEIIa inside the rice grain is so minor that its phenotype can only be observed when SBEIIb is also down regulated. Simultaneously targeting SSIIa and SBEIIb led to elevations in the proportion of intermediate chains and true amylose. This suggests that when glucan chain elongation (SSIIa effect) and branching activity (SBEIIb effect) are both reduced, amylose biosynthesis is favoured over amylopectin, even in a background where granule bound starch synthase I (GBSSI), critical for amylose synthesis, is only partially active, as is the case for Nipponbare. In these lines, chains up to DP17 were reduced. Furthermore, when a selected line was crossed with IR36 where GBSSI and SSIIa are more abundant and catalytically active, shrivelled grains were produced with a 50% loss of birefringent starch granules in the F1 generation. The lines from this cross are expected to produce more elevated amounts of true amylose chains at the succeeding homozygous generations. Simultaneous targeting of SSIIa, SBEIIa and SBEIIb also increased the proportion of true amylose but unexpectedly there appeared to be only minor changes in digestibility in these lines. Aside from establishing potential functional and nutritional associations with specific starch alterations, this study was also able to demonstrate for the first time that gene silencing using artificial microRNA is possible in the rice endosperm and that this technique can be more effective than the hairpin RNA technique. It also highlighted the importance of tissue-specific down regulation of gene expression in altering starch structure of rice grains. Furthermore, it also established that even minor perturbations in starch structure could be associated with differences in starch crystallinity as well as grain and starch granule morphology. Lines with partially down regulated SBEIIb using hairpin RNA appeared chalky and demonstrated the intermediate C-type crystallinity. Down regulating SBEIIa produced ‘white core’ grains in which starch granules had lost the compound structure and were severely altered in the middle of the grain. In summary, rice grains with altered starch structure and reduced digestibility have been produced using transgenic approaches. Associations between amylopectin structure and resistant starch and glycaemic index have been demonstrated, and the previously known relationships with true amylose have been confirmed. This study has also demonstrated that in Goami, an unknown gene modification has altered the starch structure that resulted in a phenotype identical those with a mutation in SBEIIb, including elevations in RS. In addition, existing germplasm has been shown not to display the variability required for dramatic alterations in digestibility. Novel starch structures for tailored digestibility may be anticipated in the future that can be tapped to alleviate nutrition-related preventable disease.
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Claims 20
Claim · model-written summary
Selected high-amylose grains had around 1% in vitro resistant starch versus trace amounts in glutinous varieties.
- Taxon
- Rice
Oryza sativat0 - Material
- selected grainse0
- Outcome
- in vitro RS contente0
- Value
- 1%e0
- Comparator
- glutinous varietiese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“in vitro RS content of the selected grains was around 1%, compared with trace amounts found in the glutinous varieties”
t0“Rice”
Claim · model-written summary
Selected grains had significantly lower GI estimates than glutinous rice.
- Taxon
- RiceOryza sativat0
- Material
- selected grainse0
- Outcome
- GI estimatese0
- Direction
- reductionse0
- Comparator
- glutinous rice grainse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“selected grains also demonstrated significant reductions in GI estimates compared to glutinous rice grains”
t0“Rice”
Claim · model-written summary
Apparent amylose content did not exceed 30% in tested rice grains.
- Taxon
- riceOryza sativae0
- Outcome
- apparent amylose content (AAC)e0
- Value
- 30%e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“apparent amylose content (AAC) in the rice grains tested did not exceed 30%”
Claim · model-written summary
Goami 2 had high AAC and RS contents.
- Taxon
- RiceOryza sativat0
- Material
- Goami 2e0
- Outcome
- high AAC and RS contentse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Goami 2 presented the most interesting properties. It has high AAC and RS contents”
t0“Rice”
Claim · model-written summary
Goami 2 had elevated levels of an unusual SBEIIb isoform.
- Taxon
- RiceOryza sativat0
- Material
- Goami 2e0
- Gene
- starch branching enzyme IIb (SBEIIb)e0
- Outcome
- elevated levelse0
- Direction
- elevatede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Goami 2 had elevated levels of an unusual isoform of starch branching enzyme IIb (SBEIIb)”
t0“Rice”
Claim · model-written summary
Goami 2 had elevated RS without elevated true amylose chains.
- Taxon
- RiceOryza sativat0
- Material
- Goami 2e0
- Outcome
- RSe0
- Direction
- elevatede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Goami 2 had elevated RS despite lack of observable elevations in true amylose chains”
t0“Rice”
Claim · model-written summary
IR36ae had elevated true amylose and intermediate material with increased RS.
- Taxon
- RiceOryza sativat0
- Material
- IR36aee0
- Outcome
- true amylose and intermediate materiale0
- Direction
- elevationse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“IR36ae had elevations in both true amylose and intermediate material that were accompanied by an increase in RS”
t0“Rice”
Claim · model-written summary
WAB3-5 had elevated RS and reduced GI.
- Taxon
- RiceOryza sativat0
- Material
- WAB3-5e0
- Outcome
- RSe0
- Direction
- elevatede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“WAB3-5, an amylose producer in a glutinous rice background, revealed that it also had elevated RS and reduced GI”
t0“Rice”
Claim · model-written summary
SBEIIa and SSIIa down regulation made minor amylopectin-chain changes and did not affect RS.
- Taxon
- RiceOryza sativat0
- Treatment
- SBEIIa and SSIIa were down regulatede0
- Outcome
- distribution of debranched amylopectin chainse0
- Direction
- minor alterationse0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“only minor alterations in the distribution of debranched amylopectin chains were observed when SBEIIa and SSIIa were down regulated, and it did not affect the amounts of RS”
t0“Rice”
Claim · model-written summary
SBEIIb down regulation raised apparent amylose to 35%.
- Taxon
- RiceOryza sativat0
- Treatment
- down regulating the expression of SBEIIbe0
- Gene
- SBEIIbe0
- Outcome
- apparent amylosee0
- Direction
- increasee0
- Value
- 35%e0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“down regulating the expression of SBEIIb led to an increase in apparent amylose (35%) and resistant starch (4.8%) contents”
t0“Rice”
Claim · model-written summary
SBEIIb down regulation raised resistant starch to 4.8%.
- Taxon
- RiceOryza sativat0
- Treatment
- down regulating the expression of SBEIIbe0
- Gene
- SBEIIbe0
- Outcome
- resistant starche0
- Direction
- increasee0
- Value
- 4.8%e0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“down regulating the expression of SBEIIb led to an increase in apparent amylose (35%) and resistant starch (4.8%) contents”
t0“Rice”
Claim · model-written summary
SBEIIb down regulation produced a GI estimate of 44.
- Taxon
- RiceOryza sativat0
- Treatment
- down regulating the expression of SBEIIbe1
- Outcome
- GI estimatee0
- Direction
- lowere0
- Value
- 44e0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“In contrast, down regulating the expression of SBEIIb”
e0“a much lower GI estimate (44)”
t0“Rice”
Claim · model-written summary
SBEIIb down regulation elevated intermediate chains significantly.
- Taxon
- RiceOryza sativat0
- Treatment
- down regulating the expression of SBEIIbe0
- Outcome
- intermediate chainse0
- Direction
- elevatede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“down regulating the expression of SBEIIb resulted in significantly elevated intermediate chains”
t0“Rice”
Claim · model-written summary
SSIIa down regulation slightly reduced GI versus Nipponbare.
- Taxon
- RiceOryza sativat0
- Treatment
- SSIIa was down regulatede0
- Outcome
- glycaemic index estimatee0
- Direction
- reductione0
- Comparator
- Nipponbaree0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“expression of SSIIa was down regulated, there was still a slight reduction in glycaemic index estimate compared to its parental line Nipponbare”
t0“Rice”
Claim · model-written summary
Joint SBEIIa and SBEIIb targeting did not significantly improve phenotypes over SBEIIb reduction alone.
- Taxon
- RiceOryza sativat0
- Treatment
- simultaneously targeting SBEIIa and SBEIIbe0
- Outcome
- phenotypese0
- Direction
- did not significantly improvee0
- Comparator
- lines with the expression of only SBEIIb was reducede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“simultaneously targeting SBEIIa and SBEIIb did not significantly improve the phenotypes compared with lines with the expression of only SBEIIb was reduced”
t0“Rice”
Claim · model-written summary
Joint SSIIa and SBEIIb targeting increased intermediate chains and true amylose.
- Taxon
- RiceOryza sativat0
- Treatment
- targeting SSIIa and SBEIIbe0
- Outcome
- proportion of intermediate chains and true amylosee0
- Direction
- elevationse0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Simultaneously targeting SSIIa and SBEIIb led to elevations in the proportion of intermediate chains and true amylose”
t0“Rice”
Claim · model-written summary
Crossing a selected line with IR36 produced shrivelled F1 grains with 50% loss of birefringent starch granules.
- Taxon
- RiceOryza sativat0
- Treatment
- crossed with IR36e0
- Outcome
- loss of birefringent starch granulese0
- Direction
- losse0
- Value
- 50%e0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“when a selected line was crossed with IR36 where GBSSI and SSIIa are more abundant and catalytically active, shrivelled grains were produced with a 50% loss of birefringent starch granules in the F1 generation”
t0“Rice”
Claim · model-written summary
Joint SSIIa, SBEIIa and SBEIIb targeting increased true amylose with minor digestibility changes.
- Taxon
- RiceOryza sativat0
- Treatment
- targeting of SSIIa, SBEIIa and SBEIIbe0
- Outcome
- proportion of true amylosee0
- Direction
- increasede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Simultaneous targeting of SSIIa, SBEIIa and SBEIIb also increased the proportion of true amylose but unexpectedly there appeared to be only minor changes in digestibility in these lines”
t0“Rice”
Claim · model-written summary
Artificial microRNA silenced genes in rice endosperm and could outperform hairpin RNA.
- Taxon
- riceOryza sativae0
- Outcome
- gene silencinge0
- Comparator
- hairpin RNA techniquee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“gene silencing using artificial microRNA is possible in the rice endosperm and that this technique can be more effective than the hairpin RNA technique”
Claim · model-written summary
Partial SBEIIb down regulation gave chalky grains with intermediate C-type crystallinity.
- Taxon
- RiceOryza sativat0
- Treatment
- partially down regulated SBEIIb using hairpin RNAe0
- Outcome
- chalky and demonstrated the intermediate C-type crystallinitye0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Lines with partially down regulated SBEIIb using hairpin RNA appeared chalky and demonstrated the intermediate C-type crystallinity”
t0“Rice”
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See all 40 papers →Claims are grouped by organism, treatment and what was measured or targeted. A paper counts as positive when any of its claims in the group is positive.