Overexpression of a wheat α-amylase type 2 impact on starch metabolism and abscisic acid sensitivity during grain germination.

Despite being of vital importance for seed establishment and grain quality, starch degradation remains poorly understood in organs such as cereal or legume seeds. In cereals, starch degradation requires the synergetic action of different isoforms of α-amylases. Ubiquitous overexpression of TaAmy2 resulted in a 2.0-437.6-fold increase of total α-amylase activity in developing leaf and harvested grains. These increases led to dramatic alterations of starch visco-properties and augmentation of soluble carbohydrate levels (mainly sucrose and α-gluco-oligosaccharide) in grain. Interestingly, the overexpression of TaAMY2 led to an absence of dormancy in ripened grain due to abscisic acid (ABA) insensitivity. Using an allosteric α-amylase inhibitor (acarbose), we demonstrated that ABA insensitivity was due to the increased soluble carbohydrate generated by the α-amylase excess. Independent from the TaAMY2 overexpression, inhibition of α-amylase during germination led to the accumulation of soluble α-gluco-oligosaccharides without affecting the first stage of germination. These findings support the hypotheses that (i) endosperm sugar may overcome ABA signalling and promote sprouting, and (ii) α-amylase may not be required for the initial stage of grain germination, an observation that questions the function of the amylolytic enzyme in the starch degradation process during germination.

Over-Expression of a Wheat Late Maturity Alpha-Amylase Type 1 Impact on Starch Properties During Grain Development and Germination.

The hydrolysis of starch is a complex process that requires synergistic action of multiple hydrolytic enzymes, including α-amylases. Wheat over-expression of TaAmy1, driven by seed specific promoter, resulted in a 20- to 230-fold total α-amylase activity in mature grains. Ectopic expression of TaAmy1 showed a significant elevated α-amylase activity in stem and leaf without consequences on transitory starch. In mature grain, overexpressed TaAMY1 was mainly located in the endosperm with high expression of TaAmy1. This is due to early developing grains having effect on starch granules from 18 days post-anthesis (DPA) and on soluble sugar accumulation from 30 DPA. While accumulation of TaAMY1 led to a high degree of damaged starch in grain, the dramatic alterations of starch visco-properties caused by the elevated levels of α-amylase essentially occurred during processing, thus suggesting a very small impact of related starch damage on grain properties. Abnormal accumulation of soluble sugar (α-gluco-oligosaccharide and sucrose) by TaAMY1 over-expression reduced the grain dormancy and enhanced abscisic acid (ABA) resistance. Germination study in the presence of α-amylase inhibitor suggested a very limited role of TaAMY1 in the early germination process and starch conversion into soluble sugars.

Evaluation of the impact of heat on wheat dormancy, late maturity α-amylase and grain size under controlled conditions in diverse germplasm.

In the Australian wheat belts, short episodes of high temperatures or hot spells during grain filling are becoming increasingly common and have an enormous impact on yield and quality, bringing multi-billion losses annually. This problem will become recurrent under the climate change scenario that forecast increasing extreme temperatures, but so far, no systematic analysis of the resistance to hot spells has yet been performed in a diverse genetic background. We developed a protocol to study the effects of heat on three important traits: grain size, grain dormancy and the presence of Late Maturity α-Amylase (LMA), and we validated it by analysing the phenotypes of 28 genetically diverse wheat landraces and exploring the potential variability existing in the responses to hot spells. Using controlled growth environments, the different genotypes were grown in our standard conditions until 20 days after anthesis, and then moved for 10 days into a heat chamber. Our study showed that our elevated temperature treatment during mid-late filling triggered multiple detrimental effects on yield and quality. We observed a reduction in grain size, a reduction in grain dormancy and increased LMA expression in most of the tested genotypes, but potential resistant lines were identified for each analyzed trait opening new perspectives for future genetic studies and breeding for heat-insensitive commercial lines.

Does Late Maturity Alpha-Amylase Impact Wheat Baking Quality?

Late maturity α-amylase (LMA) and pre-harvest sprouting (PHS) are both recognized as environmentally induced grain quality defects resulting from abnormally high levels of α-amylase. LMA is a more recently identified quality issue that is now receiving increasing attention worldwide and whose prevalence is now seen as impeding the development of superior quality wheat varieties. LMA is a genetic defect present in specific wheat genotypes and is characterized by elevated levels of the high pI TaAMY1 α-amylase, triggered by environmental stress during wheat grain development. TaAMY1 remains present in the aleurone through the harvest, lowering Falling Number (FN) at receival, causing a down-grading of the grain, often to feed grade, thus reducing the farmers' income. This downgrading is based on the assumption within the grain industry that, as for PHS, a low FN represents poor quality grain. Consequently any wheat line possessing low FN or high α-amylase levels is automatically considered a poor bread wheat despite there being no published evidence to date, to show that LMA is detrimental to end product quality. To evaluate the validity of this assumption a comprehensive evaluation of baking properties was performed from LMA prone lines using a subset of tall non-Rht lines from a multi-parent advanced generation inter-cross (MAGIC) wheat population grown at three different sites. LMA levels were determined along with quality parameters including end product functionality such as oven spring, bread loaf volume and weight, slice area and brightness, gas cell number and crumb firmness. No consistent or significant phenotypic correlation was found between LMA related FN and any of the quality traits. This manuscript provides for the first time, compelling evidence that LMA has limited impact on bread baking end product functionality.

Fast and Efficient Screening for Wheat Loss-of-Gene Mutants Using Multiplexed Melt Curve Analyses.

This study describes a new approach in the screening for loss-of-gene mutants in Heavy Ion Bombardment (HIB) mutant populations of genetically complex organisms such as hexaploid bread wheat using multiplexed single-color (SYBR Green) melt curve analyses. The assay was set up for three target genes to test its validity and applicability. For each gene, three genome-specific primer pairs (one for each genome) with distinct melt curves were developed and multiplexed. This allowed screening for "single null mutants" (plants with the target gene deleted in one of the three genomes) for all three genomes in a single reaction. The first two genes (α-Amylase 3 and Epsilon Cyclase) were used to test the approach as HIB null lines for all three genomes were already available for these. The third assay was successfully applied to identify new single null lines of the target gene α-Amylase 2 in an in-house HIB wheat collection. The use of SYBR Green greatly reduced the time and/or cost investment compared to other techniques and the approach proved highly suitable for high-throughput applications.

GrainScan: a low cost, fast method for grain size and colour measurements.

BACKGROUND: Measuring grain characteristics is an integral component of cereal breeding and research into genetic control of seed development. Measures such as thousand grain weight are fast, but do not give an indication of variation within a sample. Other methods exist for detailed analysis of grain size, but are generally costly and very low throughput. Grain colour analysis is generally difficult to perform with accuracy, and existing methods are expensive and involved. RESULTS: We have developed a software method to measure grain size and colour from images captured with consumer level flatbed scanners, in a robust, standardised way. The accuracy and precision of the method have been demonstrated through screening wheat and Brachypodium distachyon populations for variation in size and colour. CONCLUSION: By using GrainScan, cheap and fast measurement of grain colour and size will enable plant research programs to gain deeper understanding of material, where limited or no information is currently available.