Barley lines producing grains with either amylopectin-free or hyper-phosphorylated starches were engineered by transgenic methods. Amylopectin-free barley was generated by simultaneously silencing the three genes encoding the starch branching enzymes (SBEIIa, SBEIIb and SBEI) by a chimeric hairpin. This construct was inherited as a single locus with a 3:1 segregation, which makes the method useful for breeding as compared to combing alleles of the three different SBE genes segregating independently. Transgenic grains were wrinkled. This is a phenocopy of Mendel’s wrinkled peas, which were also based on a non-functional allele of an SBE gene. Amylopectin content was below detection level (< 1%) measured by both size exclusion chromatography (SEC) and differential scanning calorimetry (DSC). Scanning electron microscopy (SEM) showed that this amylopectin-free starch is organized into amorphic granules. Plants were propagated for three generations. Yield and grain phenotypes were determined in grains grown under semi-field conditions. Loss of SBE activity was compensated by the cereal grain by up-regulated gene expression of starch synthases. As an effect, yield loss is limited to 20%. This demonstrates for the first time a way for production of pure amylose in plants with limited yield loss. In a different barley line endosperm specific overexpression of glucan water dikinase from potato (StGWD) was conducted. The content of phosphate esters in starch from consecutive generations (T0 and T1) of transgenic grains was tenfold higher than from vector control and wild type grains. Amylose content was not affected in hyperphosphorylated grains. Hyper-phosphorylated starch granules had several pores on the surfaces, similar to pores seen on enzymatically semi-degraded granules. This provides support for the presence of a general mechanism in starch degradation in the plant kingdom where phosphorylation carried out by ectopic expression of StGWD tags barley starch granules for degradation by endogenous enzymes. Together this work shows two new strategies for in planta starch bioengineering of cereals. It demonstrates that bioengineering may be used to obtain novel and technologically interesting cereal starches, and to elucidate the complex pathways of starch biosynthesis and the roles of individual starch biosynthetic enzymes.
6th European Symposium on Enzymes in Grain Processing. Program and Abstract Book, 2011
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6th European Symposium on Enzymes in Grain Processing, 2011