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


CONCLUSIONS
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.
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.
David A
DA
Brummell
The New Zealand Institute for Plant & Food Research Limited, Food Industry Science Centre, Private Bag 11600, Palmerston North, 4442, New Zealand. lynwatsonnz@gmail.com.
Lyn M
LM
Watson
The New Zealand Institute for Plant & Food Research Limited, Food Industry Science Centre, Private Bag 11600, Palmerston North, 4442, New Zealand. jun_zcn@hotmail.com.
Jun
J
Zhou
The New Zealand Institute for Plant & Food Research Limited, Food Industry Science Centre, Private Bag 11600, Palmerston North, 4442, New Zealand. marian.mckenzie@plantandfood.co.nz.
Marian J
MJ
McKenzie
The New Zealand Institute for Plant & Food Research Limited, Mount Albert Research Centre, Private Bag 92169, Auckland, 1142, New Zealand. ian.hallett@plantandfood.co.nz.
Ian C
IC
Hallett
The New Zealand Institute for Plant & Food Research Limited, Canterbury Agriculture & Science Centre, Private Bag 4704, Christchurch, 8140, New Zealand. lyall.simmons@plantandfood.co.nz.
Lyall
L
Simmons
The New Zealand Institute for Plant & Food Research Limited, Canterbury Agriculture & Science Centre, Private Bag 4704, Christchurch, 8140, New Zealand. margaret.carpenter@plantandfood.co.nz.
Margaret
M
Carpenter
The New Zealand Institute for Plant & Food Research Limited, Canterbury Agriculture & Science Centre, Private Bag 4704, Christchurch, 8140, New Zealand. gail.timmerman-vaughan@plantandfood.co.nz.
Gail M
GM
Timmerman-Vaughan
D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000687:Amylopectin / Q000737:chemistry* / Q000378:metabolism; D002236:Carbohydrate Conformation; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D011198:Solanum tuberosum / Q000235:genetics / Q000378:metabolism*; D013214:Starch Synthase / Q000235:genetics / Q000378:metabolism
D000687:Amylopectin; D015061:1,4-alpha-Glucan Branching Enzyme; C112590:starch-branching enzyme II; D013214:Starch Synthase