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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

Changes in gelatinisation of tuber starch granules from transgenic potato lines. (A) Effect of GBSS knockdown (1041–3) or SBEII overexpression (1047–17) on starch granule swelling in 4 M urea. (B) Comparison of swelling of starch granules in a range of urea concentrations. Gelatinisation was assessed by measuring the volume of the swollen starch pellet. Data are mean pellet volumes ± SD (n = 4 biological replicates).
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Fig7: Changes in gelatinisation of tuber starch granules from transgenic potato lines. (A) Effect of GBSS knockdown (1041–3) or SBEII overexpression (1047–17) on starch granule swelling in 4 M urea. (B) Comparison of swelling of starch granules in a range of urea concentrations. Gelatinisation was assessed by measuring the volume of the swollen starch pellet. Data are mean pellet volumes ± SD (n = 4 biological replicates).

Mentions: The greatest difference in physicochemical properties was seen in the gelatinisation of starch granules in urea. In 4 M urea, starch granules from line 1041–3 showed very little gelatinisation, with a swollen pellet volume only one-third that of wild-type starch granules (Figure 7A). Starch from lines 1041–1 and 1041–16 produced similar results (data not shown). In contrast, starch granules from line 1047–17 exhibited a huge amount of gelatinisation, producing a large and diffuse pellet with a swollen volume 3-fold greater than that of wild-type. Starch from line 1047–15 produced similar results (data not shown). Examination of a range of urea concentrations showed that gelatinisation was initiated at a lower urea concentration in starch granules from 1047–17, but followed a similar curve to wild-type and achieved a similar maximum swollen volume (Figure 7B). Gelatinisation followed a very different pattern in starch granules from 1041–3, beginning at a higher urea concentration and showing a remarkably steep increase in swollen pellet volume over a change in urea concentration from 4 M to 6 M. The maximum swelling volume achieved by 1041–3 was up to 25% greater than wild-type, and at urea concentrations above 7 M occupied the entire 1 mL volume of the original liquid provided.Figure 7


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)

Changes in gelatinisation of tuber starch granules from transgenic potato lines. (A) Effect of GBSS knockdown (1041–3) or SBEII overexpression (1047–17) on starch granule swelling in 4 M urea. (B) Comparison of swelling of starch granules in a range of urea concentrations. Gelatinisation was assessed by measuring the volume of the swollen starch pellet. Data are mean pellet volumes ± SD (n = 4 biological replicates).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Changes in gelatinisation of tuber starch granules from transgenic potato lines. (A) Effect of GBSS knockdown (1041–3) or SBEII overexpression (1047–17) on starch granule swelling in 4 M urea. (B) Comparison of swelling of starch granules in a range of urea concentrations. Gelatinisation was assessed by measuring the volume of the swollen starch pellet. Data are mean pellet volumes ± SD (n = 4 biological replicates).
Mentions: The greatest difference in physicochemical properties was seen in the gelatinisation of starch granules in urea. In 4 M urea, starch granules from line 1041–3 showed very little gelatinisation, with a swollen pellet volume only one-third that of wild-type starch granules (Figure 7A). Starch from lines 1041–1 and 1041–16 produced similar results (data not shown). In contrast, starch granules from line 1047–17 exhibited a huge amount of gelatinisation, producing a large and diffuse pellet with a swollen volume 3-fold greater than that of wild-type. Starch from line 1047–15 produced similar results (data not shown). Examination of a range of urea concentrations showed that gelatinisation was initiated at a lower urea concentration in starch granules from 1047–17, but followed a similar curve to wild-type and achieved a similar maximum swollen volume (Figure 7B). Gelatinisation followed a very different pattern in starch granules from 1041–3, beginning at a higher urea concentration and showing a remarkably steep increase in swollen pellet volume over a change in urea concentration from 4 M to 6 M. The maximum swelling volume achieved by 1041–3 was up to 25% greater than wild-type, and at urea concentrations above 7 M occupied the entire 1 mL volume of the original liquid provided.Figure 7

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