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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. [ABSTRACT FROM AUTHOR]

Summary: 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. Significance Statement: This study described the application of plant biotechnology to provide evidence as to whether elevated levels of α‐amylase in the mature grain can affect germination positively or negatively. These findings support the hypotheses that (i) endosperm sugar may overcome abscisic acid signalling and promote sprouting, and (ii) α‐amylase may not be required for the initial stage of grain germination. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In glasshouse studies we have previously shown that endosperm-specific RNAi suppression of the primary starch phosphorylation enzyme, Glucan,Water Dikinase (GWD) leads to enhanced early vigor, greater leaf biomass, and increases in both head size and yield. To confirm these affects in a field setting, trials were conducted in three Australian environments. Field results were consistent with those in the glasshouse for increased flag leaf area and rachis nodes. However, there was also a decrease in tiller number and consequently a decrease in yield for one event at two sites. These findings provide potentially important information on plant vigor enhancement and highlight the challenges of transferring the modification of complex traits from single plants in controlled environments to the field. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Late maturity α-amylase (LMA) and preharvest sprouting (PHS) are genetic defects in wheat. They are both characterized by the expression of specific isoforms of α-amylase in particular genotypes in the grain prior to harvest. The enhanced expression of α-amylase in both LMA and PHS results in a reduction in Falling Number (FN), a test of gel viscosity, and subsequent downgrading of the grain, along with a reduced price for growers. The FN test is unable to distinguish between LMA and PHS; thus, both defects are treated similarly when grain is traded. However, in PHS-affected grains, proteases and other degradative process are activated, and this has been shown to have a negative impact on end product quality. No studies have been conducted to determine whether LMA is detrimental to end product quality. This work demonstrated that wheat in which an isoform α-amylase (TaAmy3) was overexpressed in the endosperm of developing grain to levels of up to 100-fold higher than the wild-type resulted in low FN similar to those seen in LMA- or PHS-affected grains. This increase had no detrimental effect on starch structure, flour composition and enhanced baking quality, in small-scale 10-g baking tests. In these small-scale tests, overexpression of TaAmy3 led to increased loaf volume and Maillard-related browning to levels higher than those in control flours when baking improver was added. These findings raise questions as to the validity of the assumption that (i) LMA is detrimental to end product quality and (ii) a low FN is always indicative of a reduction in quality. This work suggests the need for a better understanding of the impact of elevated expression of specific α-amylase on end product quality. [ABSTRACT FROM AUTHOR]

Starch phosphate ester content is known to alter the physicochemical properties of starch, including its susceptibility to degradation. Previous work producing wheat (Triticum aestivum) with down-regulated glucan, water dikinase, the primary gene responsible for addition of phosphate groups to starch, in a grain-specific manner found unexpected phenotypic alteration in grain and growth. Here, we report on further characterization of these lines focussing on mature grain and early growth. We find that coleoptile length has been increased in these transgenic lines independently of grain size increases. No changes in starch degradation rates during germination could be identified, or any major alteration in soluble sugar levels that may explain the coleoptile growth modification. We identify some alteration in hormones in the tissues in question. Mature grain size is examined, as is Hardness Index and starch conformation. We find no evidence that the increased growth of coleoptiles in these lines is connected to starch conformation or degradation or soluble sugar content and suggest these findings provide a novel means of increasing coleoptile growth and early seedling establishment in cereal crop species. [ABSTRACT FROM AUTHOR]

Endosperm-specific over-expression of wheat α-amylase TaAMY3 during grain development revealed unexpected effects on grain composition, carbon partitioning, fatty acid accumulation, and germination.Wheat starch degradation requires the synergistic action of different amylolytic enzymes. Our spatio-temporal study of wheat α-amylases throughout grain development shows that AMY3 is the most abundant isoform compared with the other known α-amylases. Endosperm-specific over-expression of AMY3 resulted in an increase of total α-amylase activity in harvested grains. Unexpectedly, increased activity did not have a significant impact on starch content or composition but led to an increase of soluble carbohydrate (mainly sucrose) in dry grain. In AMY3 overexpression lines (A3OE), germination was slightly delayed and triacylglycerol (TAG) content was increased in the endosperm of mature grain. Despite increased AMY3 transcript and protein content throughout grain development, alterations of α-amylase activity and starch granule degradation were not detected until grain maturation, suggesting a post-translational inhibition of α-amylase activity in the endosperm during the starch filling period. These findings show unexpected effects of a high level of α-amylase on grain development and composition, notably in carbon partitioning and TAG accumulation, and suggest the presence of a hitherto unknown regulatory pathway during grain filling. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

Background: A crucial step in the evaluation of newly produced transgenic plants is the selection of homozygous plants. Here we describe an efficient and highly flexible real-time PCR-based method for the development of homozygous lines in plant models with complex (multiple) genomes and/or relatively long generation times (>3 months) using direct copy number determinations. Results: An existing DNA extraction method was converted into a high-throughput plant leaf DNA extraction procedure yielding DNA suitable for real-time PCR analyses. Highly specific and efficient primer pairs were developed for a bread wheat reference gene (Epsilon Cyclase) and for standard sequence elements in the gene cassette routinely used for cereal transformations (an intron bridge and the Nopaline Synthase terminator). The real-time PCR assay reliably distinguished wheat plants with a single copy of the transgene from individuals with multiple copies or those lacking the transgene. To obtain homozygous lines carrying a unique insertion event as efficiently as possible, T0 plants (plants raised from transformed callus) with a single copy of the transgene were selected and their progeny screened for homozygous plants. Finally, the assay was adapted to work on rice. Conclusions: The ability to quickly, easily and accurately quantify the construct copy numbers, as provided by the real-time PCR assay, greatly improved the efficiency and reliability of the selection of homozygous transgenic plants in our case study. We were able to select homozygous plants in early generations, avoiding time-consuming methods such as large scale analysis of segregation patterns of descendants and/or Southern blotting. Additionally, the ability to specifically develop homozygous lines carrying a unique insertion event could be important in avoiding gene silencing due to co-suppression, and if needed assist in the selection of lines suitable for future deregulation. The same primer pairs can be used to quantify many different wheat transgenic events because the construct-specific primer pairs are targeted to standard sequence elements of the cereal gene cassettes, making the method widely applicable in wheat GM research. Moreover, because all procedures described here are standardized, the method may easily be adapted to vectors lacking the target regions used here and/or to other plant models. [ABSTRACT FROM AUTHOR]

A novel mechanism for increasing vegetative biomass and grain yield has been identified in wheat ( Triticum aestivum). RNAi-mediated down-regulation of Glucan, Water-Dikinase (GWD), the primary enzyme required for starch phosphorylation, under the control of an endosperm-specific promoter, resulted in a decrease in starch phosphate content and an increase in grain size. Unexpectedly, consistent increases in vegetative biomass and grain yield were observed in subsequent generations. In lines where GWD expression was decreased, germination rate was slightly reduced. However, significant increases in vegetative growth from the two leaf stage were observed. In glasshouse pot trials, down-regulation of GWD led to a 29% increase in grain yield while in glasshouse tub trials simulating field row spacing and canopy development, GWD down-regulation resulted in a grain yield increase of 26%. The enhanced yield resulted from a combination of increases in seed weight, tiller number, spikelets per head and seed number per spike. In field trials, all vegetative phenotypes were reproduced with the exception of increased tiller number. The expression of the transgene and suppression of endogenous GWD RNA levels were demonstrated to be grain specific. In addition to the direct effects of GWD down-regulation, an increased level of α-amylase activity was present in the aleurone layer during grain maturation. These findings provide a potentially important novel mechanism to increase biomass and grain yield in crop improvement programmes. [ABSTRACT FROM AUTHOR]

Deciphering the determinants of starch digestion from multiple interrelated properties is a challenge that can benefit from multifactorial data analysis. The present study investigated the digestion kinetic parameters (rate, final extent) of size-fractions from four commercial wheat starches with different amylose contents. Each size-fraction was isolated and characterized comprehensively using a large range of analytic techniques (FACE, XRD, CP-MAS NMR, time-domain NMR, DSC...). A statistical clustering analysis applied on the results revealed that the mobility of water and starch protons measured by time-domain NMR was consistently related to the macromolecular composition of the glucan chains and to the ultrastructure of the granule. The final extent of starch digestion was determined by the granule structural features. The digestion rate coefficient dependencies, on the other hand, changed significantly with the range of granule size, i.e. the accessible surface for initial binding of α-amylase. The study particularly showed the molecular order and the chains mobility predominantly limiting or accelerating the digestion rate depending on the accessible surface. This result confirmed the need to differentiate between the surface and the inner-granule related mechanisms in starch digestion studies. [Display omitted] [ABSTRACT FROM AUTHOR]

A major challenge faced when studying the "structure-degradability" interaction of native starch is deciphering the interdependency between different structural levels, especially when experimental conditions limit the number of samples. To tackle this challenge, 224 wheat starches from a 4-way multiparent advanced generation inter-cross population were screened for structural features and degradation profiles by porcine pancreatic α-amylase. A hierarchical clustering on principal components (HCPC) were used as multifactorial analysis to explore the data structure. The degradation procedure was proved to be robust and sensible enough to screen a large collection of starches. The HCPC highlighted the combined effects of granule size distribution (GSD), amylopectin chain length distribution (CLD), amylose content and endogenous α-amylase activity on degradation kinetics. Especially the GSD and amylopectin CLD showed high co-occurrences with specific hydrolysis profiles. These findings provide an innovative screening method and structural factors to be primarily considered for wheat starch selection in breeding programs. [Display omitted] [ABSTRACT FROM AUTHOR]