Limits...
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

SBE activity in tubers from a series of lines transformed with a SBEII overexpression construct. (A) Native PAGE activity gel with SBE activity visualized by iodine staining. (B) Mean relative SBEII activity ± SD as determined from the density of the spots. Data are from technical replicate protein preparations from the same pooled tissue as Figure 1 run on different gels on different days (n = 3). Means not sharing a common letter are significantly different between groups at P = 0.05 as determined by LSD after a one-way ANOVA test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: SBE activity in tubers from a series of lines transformed with a SBEII overexpression construct. (A) Native PAGE activity gel with SBE activity visualized by iodine staining. (B) Mean relative SBEII activity ± SD as determined from the density of the spots. Data are from technical replicate protein preparations from the same pooled tissue as Figure 1 run on different gels on different days (n = 3). Means not sharing a common letter are significantly different between groups at P = 0.05 as determined by LSD after a one-way ANOVA test.

Mentions: To assess the effect of overexpression of a SBEII intragene on SBE enzyme activity, SBEII was separated from the major and predominant SBEI by separating proteins using native polyacrylamide gel electrophoresis (PAGE), followed by an in-gel activity reaction and staining for SBE reaction products (Figure 2A). The two isoforms of SBE were identified based on the migration of rice and wheat enzymes [17,22,24] and the relative activities of potato SBEI and SBEII [14]. SBEII activity approximately reflected the abundance of SBEII mRNA, being similar to wild-type in line 1047–3, and with a range of activities greater than wild-type in a selection of other lines (WT < 1047–5 < 1047–23 = 1047–12 ≤ 1047–17 ≤ 1047–15). The greatest enhancement was in lines 1047–15 and 17, which possessed SBEII activity almost 3-fold that of wild-type (Figure 2B).Figure 2


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)

SBE activity in tubers from a series of lines transformed with a SBEII overexpression construct. (A) Native PAGE activity gel with SBE activity visualized by iodine staining. (B) Mean relative SBEII activity ± SD as determined from the density of the spots. Data are from technical replicate protein preparations from the same pooled tissue as Figure 1 run on different gels on different days (n = 3). Means not sharing a common letter are significantly different between groups at P = 0.05 as determined by LSD after a one-way ANOVA test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: SBE activity in tubers from a series of lines transformed with a SBEII overexpression construct. (A) Native PAGE activity gel with SBE activity visualized by iodine staining. (B) Mean relative SBEII activity ± SD as determined from the density of the spots. Data are from technical replicate protein preparations from the same pooled tissue as Figure 1 run on different gels on different days (n = 3). Means not sharing a common letter are significantly different between groups at P = 0.05 as determined by LSD after a one-way ANOVA test.
Mentions: To assess the effect of overexpression of a SBEII intragene on SBE enzyme activity, SBEII was separated from the major and predominant SBEI by separating proteins using native polyacrylamide gel electrophoresis (PAGE), followed by an in-gel activity reaction and staining for SBE reaction products (Figure 2A). The two isoforms of SBE were identified based on the migration of rice and wheat enzymes [17,22,24] and the relative activities of potato SBEI and SBEII [14]. SBEII activity approximately reflected the abundance of SBEII mRNA, being similar to wild-type in line 1047–3, and with a range of activities greater than wild-type in a selection of other lines (WT < 1047–5 < 1047–23 = 1047–12 ≤ 1047–17 ≤ 1047–15). The greatest enhancement was in lines 1047–15 and 17, which possessed SBEII activity almost 3-fold that of wild-type (Figure 2B).Figure 2

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