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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

RNA gel blots showing alterations to SBEII and GBSS mRNA abundance in transgenic plants. (A)SBEII mRNA abundance in tubers from plants transformed with a SBEII overexpression construct (1047 lines). (B)GBSS mRNA abundance in tubers from plants transformed with a GBSS RNAi construct (1041 lines). Wild-type (WT) and empty vector transformants (EV) are shown for comparison. Ethidium bromide (EtBr) staining of RNA (with contrast reversed) was used to confirm the equivalency of loading.
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Fig1: RNA gel blots showing alterations to SBEII and GBSS mRNA abundance in transgenic plants. (A)SBEII mRNA abundance in tubers from plants transformed with a SBEII overexpression construct (1047 lines). (B)GBSS mRNA abundance in tubers from plants transformed with a GBSS RNAi construct (1041 lines). Wild-type (WT) and empty vector transformants (EV) are shown for comparison. Ethidium bromide (EtBr) staining of RNA (with contrast reversed) was used to confirm the equivalency of loading.

Mentions: Initial experiments confirmed an earlier report that a complete cDNA of potato SBEII was unable to be propagated in Escherichia coli [14]. Despite the use of several different bacterial strains, very few colonies were produced and all were subsequently discovered to contain frame-shift mutations (data not shown). Therefore, a complete but hybrid SBEII intragene containing a single intron to prevent bacterial translation was assembled from cDNA and genomic DNA fragments (See Methods and Additional file 1: Figure S1). A population of 28 plants transformed with the SBEII-intron intragene under the control of the GBSS promoter was generated (Additional file 1: Figure S2). Of these, 20 plantlets showing good root development were grown to maturity, and tubers exhibited a range of SBEII mRNA abundances (Figure 1A). SBEII mRNA accumulation in these lines ranged from less than wild-type controls (line 1047–3), moderate overexpression (lines 1047–1, 2, 4, 5, 6, 7, 8, 9, 11, 14, 18, 20, 22, 24) to high overexpression (lines 1047–12, 15, 17, 23). Line 1047–19 exhibited approximately wild-type mRNA abundance of SBEII but with an additional anomalous larger SBEII transcript presumably derived from an aberrant insertion event. To confirm that mRNA accumulation of the structurally unrelated starch synthase enzymes SSII and SSIII was not affected by manipulation of SBEII expression, the same gel blot was stripped and re-hybridised. Sequential re-hybridisation with labelled probes for SSII and SSIII showed that the mRNA abundances of these two genes were essentially indistinguishable from wild-type and empty vector controls (Additional file 1: Figure S3).Figure 1


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)

RNA gel blots showing alterations to SBEII and GBSS mRNA abundance in transgenic plants. (A)SBEII mRNA abundance in tubers from plants transformed with a SBEII overexpression construct (1047 lines). (B)GBSS mRNA abundance in tubers from plants transformed with a GBSS RNAi construct (1041 lines). Wild-type (WT) and empty vector transformants (EV) are shown for comparison. Ethidium bromide (EtBr) staining of RNA (with contrast reversed) was used to confirm the equivalency of loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: RNA gel blots showing alterations to SBEII and GBSS mRNA abundance in transgenic plants. (A)SBEII mRNA abundance in tubers from plants transformed with a SBEII overexpression construct (1047 lines). (B)GBSS mRNA abundance in tubers from plants transformed with a GBSS RNAi construct (1041 lines). Wild-type (WT) and empty vector transformants (EV) are shown for comparison. Ethidium bromide (EtBr) staining of RNA (with contrast reversed) was used to confirm the equivalency of loading.
Mentions: Initial experiments confirmed an earlier report that a complete cDNA of potato SBEII was unable to be propagated in Escherichia coli [14]. Despite the use of several different bacterial strains, very few colonies were produced and all were subsequently discovered to contain frame-shift mutations (data not shown). Therefore, a complete but hybrid SBEII intragene containing a single intron to prevent bacterial translation was assembled from cDNA and genomic DNA fragments (See Methods and Additional file 1: Figure S1). A population of 28 plants transformed with the SBEII-intron intragene under the control of the GBSS promoter was generated (Additional file 1: Figure S2). Of these, 20 plantlets showing good root development were grown to maturity, and tubers exhibited a range of SBEII mRNA abundances (Figure 1A). SBEII mRNA accumulation in these lines ranged from less than wild-type controls (line 1047–3), moderate overexpression (lines 1047–1, 2, 4, 5, 6, 7, 8, 9, 11, 14, 18, 20, 22, 24) to high overexpression (lines 1047–12, 15, 17, 23). Line 1047–19 exhibited approximately wild-type mRNA abundance of SBEII but with an additional anomalous larger SBEII transcript presumably derived from an aberrant insertion event. To confirm that mRNA accumulation of the structurally unrelated starch synthase enzymes SSII and SSIII was not affected by manipulation of SBEII expression, the same gel blot was stripped and re-hybridised. Sequential re-hybridisation with labelled probes for SSII and SSIII showed that the mRNA abundances of these two genes were essentially indistinguishable from wild-type and empty vector controls (Additional file 1: Figure S3).Figure 1

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