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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

Images of tuber starch granules from wild-type and transgenically modified potato tubers. (A) Brightfield. (B) Brightfield, starch stained with I2/KI. (C) Polarised light. (D) Differential interference contrast. (E) Variable pressure scanning electron microscopy. (F) Optical section of starch fluorescently labelled with APTS taken using confocal scanning laser microscopy. Lines used were WT898, 1041–3 and 1047–17. The scale bar (in panel A) for light microscopy pictures (A–D) represents 100 μm, and for the scanning electron microscopy (E) represents 40 μm.
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Fig5: Images of tuber starch granules from wild-type and transgenically modified potato tubers. (A) Brightfield. (B) Brightfield, starch stained with I2/KI. (C) Polarised light. (D) Differential interference contrast. (E) Variable pressure scanning electron microscopy. (F) Optical section of starch fluorescently labelled with APTS taken using confocal scanning laser microscopy. Lines used were WT898, 1041–3 and 1047–17. The scale bar (in panel A) for light microscopy pictures (A–D) represents 100 μm, and for the scanning electron microscopy (E) represents 40 μm.

Mentions: To test whether the morphology of the starch granules was altered by the transgenic modifications, a range of microscopy techniques was employed (Figure 5). Brightfield microscopy showed a wide range of granule sizes in wild-type starch (Figure 5A), and neither knockdown of GBSS nor overexpression of SBEII had any obvious effect on the size range distribution of the granules. Granules from wild-type and SBEII overexpressors stained dark blue with iodine (Figure 5B), whereas granules from GBSS knockdown plants showed the pale purple colour typical of a low amylose content but with visible dark blue staining at the hilum core. Examination under polarised light detected birefringence visible as a ‘Maltese cross’ shape and blue and yellow sectors (Figure 5C). Birefringence was higher in the GBSS knockdown line, presumably since these lines are higher in amylopectin content, which has a more ordered orientation (crystallinity) than amylose. An increased birefringence in the GBSS knockdown line was also observed using differential interference contrast microscopy (Figure 5D). Variable pressure scanning electron microscopy showed that the external morphology of the starch granules was not affected by GBSS knockdown or by SBEII overexpression (Figure 5E).Figure 5


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)

Images of tuber starch granules from wild-type and transgenically modified potato tubers. (A) Brightfield. (B) Brightfield, starch stained with I2/KI. (C) Polarised light. (D) Differential interference contrast. (E) Variable pressure scanning electron microscopy. (F) Optical section of starch fluorescently labelled with APTS taken using confocal scanning laser microscopy. Lines used were WT898, 1041–3 and 1047–17. The scale bar (in panel A) for light microscopy pictures (A–D) represents 100 μm, and for the scanning electron microscopy (E) represents 40 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Images of tuber starch granules from wild-type and transgenically modified potato tubers. (A) Brightfield. (B) Brightfield, starch stained with I2/KI. (C) Polarised light. (D) Differential interference contrast. (E) Variable pressure scanning electron microscopy. (F) Optical section of starch fluorescently labelled with APTS taken using confocal scanning laser microscopy. Lines used were WT898, 1041–3 and 1047–17. The scale bar (in panel A) for light microscopy pictures (A–D) represents 100 μm, and for the scanning electron microscopy (E) represents 40 μm.
Mentions: To test whether the morphology of the starch granules was altered by the transgenic modifications, a range of microscopy techniques was employed (Figure 5). Brightfield microscopy showed a wide range of granule sizes in wild-type starch (Figure 5A), and neither knockdown of GBSS nor overexpression of SBEII had any obvious effect on the size range distribution of the granules. Granules from wild-type and SBEII overexpressors stained dark blue with iodine (Figure 5B), whereas granules from GBSS knockdown plants showed the pale purple colour typical of a low amylose content but with visible dark blue staining at the hilum core. Examination under polarised light detected birefringence visible as a ‘Maltese cross’ shape and blue and yellow sectors (Figure 5C). Birefringence was higher in the GBSS knockdown line, presumably since these lines are higher in amylopectin content, which has a more ordered orientation (crystallinity) than amylose. An increased birefringence in the GBSS knockdown line was also observed using differential interference contrast microscopy (Figure 5D). Variable pressure scanning electron microscopy showed that the external morphology of the starch granules was not affected by GBSS knockdown or by SBEII overexpression (Figure 5E).Figure 5

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