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Overexpression of STARCH BRANCHING ENZYME II increases short-chain branching of amylopectin and alters the physicochemical properties of starch from potato tuber.

Brummell DA, Watson LM, Zhou J, McKenzie MJ, Hallett IC, Simmons L, Carpenter M, Timmerman-Vaughan GM - BMC Biotechnol. (2015)

Bottom Line: Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity.In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern.The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.

View Article: PubMed Central - PubMed

Affiliation: The New Zealand Institute for Plant & Food Research Limited, Food Industry Science Centre, Private Bag 11600, Palmerston North, 4442, New Zealand. david.brummell@plantandfood.co.nz.

ABSTRACT

Background: Starch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers.

Results: A hybrid SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6-12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern.

Conclusion: This work reports the first time that overexpression of SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.

No MeSH data available.


Related in: MedlinePlus

Comparison of pasting properties of tuber starch from wild-type and transgenic lines. Pasting properties were assessed in starch from lines exhibiting GBSS knockdown (1041–3) or SBEII overexpression (1047–17). Changes in viscosity of 7% (w/w) starch suspensions during heating and subsequent cooling were measured by RVA. Curves are the means of measurements on three independent biological replicates per line. The thin dotted line shows the change in temperature during measurement.
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Fig6: Comparison of pasting properties of tuber starch from wild-type and transgenic lines. Pasting properties were assessed in starch from lines exhibiting GBSS knockdown (1041–3) or SBEII overexpression (1047–17). Changes in viscosity of 7% (w/w) starch suspensions during heating and subsequent cooling were measured by RVA. Curves are the means of measurements on three independent biological replicates per line. The thin dotted line shows the change in temperature during measurement.

Mentions: An examination of starch pasting properties using a rapid visco analyser (RVA) detected differences between the lines (Figure 6), including the lowered To of starch from line 1047–17 and the increased To of starch from line 1041–3. Other than the onset temperature, pasting profiles were similar in shape, but with a reduction in peak viscosity of ~10% for line 1047–17 and ~24% for line 1041–3. Final viscosities were also reduced in both transgenic lines.Figure 6


Overexpression of STARCH BRANCHING ENZYME II increases short-chain branching of amylopectin and alters the physicochemical properties of starch from potato tuber.

Brummell DA, Watson LM, Zhou J, McKenzie MJ, Hallett IC, Simmons L, Carpenter M, Timmerman-Vaughan GM - BMC Biotechnol. (2015)

Comparison of pasting properties of tuber starch from wild-type and transgenic lines. Pasting properties were assessed in starch from lines exhibiting GBSS knockdown (1041–3) or SBEII overexpression (1047–17). Changes in viscosity of 7% (w/w) starch suspensions during heating and subsequent cooling were measured by RVA. Curves are the means of measurements on three independent biological replicates per line. The thin dotted line shows the change in temperature during measurement.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4414359&req=5

Fig6: Comparison of pasting properties of tuber starch from wild-type and transgenic lines. Pasting properties were assessed in starch from lines exhibiting GBSS knockdown (1041–3) or SBEII overexpression (1047–17). Changes in viscosity of 7% (w/w) starch suspensions during heating and subsequent cooling were measured by RVA. Curves are the means of measurements on three independent biological replicates per line. The thin dotted line shows the change in temperature during measurement.
Mentions: An examination of starch pasting properties using a rapid visco analyser (RVA) detected differences between the lines (Figure 6), including the lowered To of starch from line 1047–17 and the increased To of starch from line 1041–3. Other than the onset temperature, pasting profiles were similar in shape, but with a reduction in peak viscosity of ~10% for line 1047–17 and ~24% for line 1041–3. Final viscosities were also reduced in both transgenic lines.Figure 6

Bottom Line: Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity.In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern.The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.

View Article: PubMed Central - PubMed

Affiliation: The New Zealand Institute for Plant & Food Research Limited, Food Industry Science Centre, Private Bag 11600, Palmerston North, 4442, New Zealand. david.brummell@plantandfood.co.nz.

ABSTRACT

Background: Starch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers.

Results: A hybrid SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6-12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern.

Conclusion: This work reports the first time that overexpression of SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.

No MeSH data available.


Related in: MedlinePlus