Advancing Sustainable Malting Practices: Aquaporins as Potential Breeding Targets for Improved Water Uptake during Controlled Germination of Barley (Hordeum vulgare L.).

The conversion of raw barley (Hordeum vulgare L.) to malt requires a process of controlled germination, where the grain is submerged in water to raise the moisture content to >40%. The transmembrane proteins, aquaporins, influence water uptake during the initial stage of controlled germination, yet little is known of their involvement in malting. With the current focus on sustainability, understanding the mechanisms of water uptake and usage during the initial stages of malting has become vital in improving efficient malting practices. In this study, we used quantitative proteomics analysis of two malting barley genotypes demonstrating differing water-uptake phenotypes in the initial stages of malting. Our study quantified 19 transmembrane proteins from nine families, including seven distinct aquaporin isoforms, including the plasma intrinsic proteins (PIPs) PIP1;1, PIP2;1, and PIP2;4 and the tonoplast intrinsic proteins (TIPs) TIP1;1, TIP2;3, TIP3;1, and TIP3;2. Our findings suggest that the presence of TIP1;1, TIP3;1, and TIP3;2 in the mature barley grain proteome is essential for facilitating water uptake, influencing cell turgor and the formation of large central lytic vacuoles aiding storage reserve hydrolysis and endosperm modification efficiency. This study proposes that TIP3s mediate water uptake in malting barley grain, offering potential breeding targets for improving sustainable malting practices.

Determining whether granule structural or surface features govern the wheat starch digestion, a kinetic analysis.

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.

Proteomic exploration reveals a metabolic rerouting due to low oxygen during controlled germination of malting barley (Hordeum vulgare L.).

Barley (Hordeum vulgare L.) is used in malt production for brewing applications. Barley malting involves a process of controlled germination that modifies the grain by activating enzymes to solubilize starch and proteins for brewing. Initially, the grain is submerged in water to raise grain moisture, requiring large volumes of water. Achieving grain modification at reduced moisture levels can contribute to the sustainability of malting practices. This study combined proteomics, bioinformatics, and biochemical phenotypic analysis of two malting barley genotypes with observed differences in water uptake and modification efficiency. We sought to reveal the molecular mechanisms at play during controlled germination and explore the roles of protein groups at 24 h intervals across the first 72 h. Overall, 3,485 protein groups were identified with 793 significant differentially abundant (DAP) within and between genotypes, involved in various biological processes, including protein synthesis, carbohydrate metabolism, and hydrolysis. Functional integration into metabolic pathways, such as glycolysis, pyruvate, starch and sucrose metabolism, revealed a metabolic rerouting due to low oxygen enforced by submergence during controlled germination. This SWATH-MS study provides a comprehensive proteome reference, delivering new insights into the molecular mechanisms underlying the impacts of low oxygen during controlled germination. It is concluded that continued efficient modification of malting barley subjected to submergence is largely due to the capacity to reroute energy to maintain vital processes, particularly protein synthesis.

Beyond amylose content, selecting starch traits impacting in vitro α-amylase degradability in a wheat MAGIC population.

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.

How Does Starch Structure Impact Amylolysis? Review of Current Strategies for Starch Digestibility Study.

In vitro digestibility of starch is a common analysis in human nutrition research, and generally consists of performing the hydrolysis of starch by α-amylase in specific conditions. Similar in vitro assays are also used in other research fields, where different methods can be used. Overall, the in vitro hydrolysis of native starch is a bridge between all of these methods. In this literature review, we examine the use of amylolysis assays in recent publications investigating the complex starch structure-amylolysis relation. This review is divided in two parts: (1) a brief review of the factors influencing the hydrolysis of starch and (2) a systematic review of the experimental designs and methods used in publications for the period 2016-2020. The latter reports on starch materials, factors investigated, characterization of the starch hydrolysis kinetics and data analysis techniques. This review shows that the dominant research strategy favors the comparison between a few starch samples most frequently described through crystallinity, granule type, amylose and chain length distribution with marked characteristics. This strategy aims at circumventing the multifactorial aspect of the starch digestion mechanism by focusing on specific features. An alternative strategy relies on computational approaches such as multivariate statistical analysis and machine learning techniques to decipher the role of each factor on amylolysis. While promising to address complexity, the limited use of a computational approach can be explained by the small size of the experimental datasets in most publications. This review shows that key steps towards the production of larger datasets are already available, in particular the generalization of rapid hydrolysis assays and the development of quantification approaches for most analytical results.

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.

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.

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.

Increasing growth and yield by altering carbon metabolism in a transgenic leaf oil crop.

Engineering high biomass plants that produce oil (triacylglycerol or TAG) in vegetative rather than seed-related tissues could help meet our growing demand for plant oil. Several studies have already demonstrated the potential of this approach by creating transgenic crop and model plants that accumulate TAG in their leaves and stems. However, TAG synthesis may compete with other important carbon and energy reserves, including carbohydrate production, and thereby limit plant growth. The aims of this study were thus: first, to investigate the effect of TAG accumulation on growth and development of previously generated high leaf oil tobacco plants; and second, to increase plant growth and/or oil yields by further altering carbon fixation and partitioning. This study showed that TAG accumulation varied with leaf and plant developmental stage, affected leaf carbon and nitrogen partitioning and reduced the relative growth rate and final biomass of high leaf oil plants. To overcome these growth limitations, four genes related to carbon fixation (encoding CBB cycle enzymes SBPase and chloroplast-targeted FBPase) or carbon partitioning (encoding sucrose biosynthetic enzyme cytosolic FBPase and lipid-related transcription factor DOF4) were overexpressed in high leaf oil plants. In glasshouse conditions, all four constructs increased early growth without affecting TAG accumulation while chloroplast-targeted FBPase and DOF4 also increased final biomass and oil yields. These results highlight the reliance of plant growth on carbon partitioning, in addition to carbon supply, and will guide future attempts to improve biomass and TAG accumulation in transgenic leaf oil crops.

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.

Oil Accumulation in Transgenic Potato Tubers Alters Starch Quality and Nutritional Profile.

Plant storage compounds such as starch and lipids are important for human and animal nutrition as well as industry. There is interest in diverting some of the carbon stored in starch-rich organs (leaves, tubers, and cereal grains) into lipids in order to improve the energy density or nutritional properties of crops as well as providing new sources of feedstocks for food and manufacturing. Previously, we generated transgenic potato plants that accumulate up to 3.3% triacylglycerol (TAG) by dry weight in the tubers, which also led to changes in starch content, starch granule morphology and soluble sugar content. The aim of this study was to investigate how TAG accumulation affects the nutritional and processing properties of high oil potatoes with a particular focus on starch structure, physical and chemical properties. Overall, TAG accumulation was correlated with increased energy density, total nitrogen, amino acids, organic acids and inorganic phosphate, which could be of potential nutritional benefit. However, TAG accumulation had negative effects on starch quality as well as quantity. Starch from high oil potatoes had lower amylose and phosphate content, reduced peak viscosity and higher gelatinization temperature. Interestingly, starch pasting properties were disproportionately affected in lines accumulating the highest levels of TAG (>2.5%) compared to those accumulating only moderate levels (0.2-1.6%). These results indicate that optimized engineering of specialized crops for food, feed, fuel and chemical industries requires careful selection of traits, and an appropriate level of transgene expression, as well as a better understanding of starch structure and carbon partitioning in plant storage organs.

Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy.

Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber-specific (patatin) and constitutive (CaMV-35S) promoters. This coordinated metabolic engineering approach resulted in over a 100-fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild-type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof-of-concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.

Step changes in leaf oil accumulation via iterative metabolic engineering.

Synthesis and accumulation of plant oils in the entire vegetative biomass offers the potential to deliver yields surpassing those of oilseed crops. However, current levels still fall well short of those typically found in oilseeds. Here we show how transcriptome and biochemical analyses pointed to a futile cycle in a previously established Nicotiana tabacum line, accumulating up to 15% (dry weight) of the storage lipid triacylglycerol in leaf tissue. To overcome this metabolic bottleneck, we either silenced the SDP1 lipase or overexpressed the Arabidopsis thaliana LEC2 transcription factor in this transgenic background. Both strategies independently resulted in the accumulation of 30-33% triacylglycerol in leaf tissues. Our results demonstrate that the combined optimization of de novo fatty acid biosynthesis, storage lipid assembly and lipid turnover in leaf tissue results in a major overhaul of the plant central carbon allocation and lipid metabolism. The resulting further step changes in oil accumulation in the entire plant biomass offers the possibility of delivering yields that outperform current oilseed crops.

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.

Engineering high α-amylase levels in wheat grain lowers Falling Number but improves baking properties.

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.

Suppression of glucan, water dikinase in the endosperm alters wheat grain properties, germination and coleoptile growth.

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.

Engineering α-amylase levels in wheat grain suggests a highly sophisticated level of carbohydrate regulation during development.

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.

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.

New perspectives on the role of α- and ß-amylases in transient starch synthesis.

Transient starch in leaves is synthesized by various biosynthetic enzymes in the chloroplasts during the light period. This paper presents the first mathematical model for the (bio)synthesis of the chain-length distribution (CLD) of transient starch to aid the understanding of this synthesis. The model expresses the rate of change of the CLD in terms of the actions of the enzymes involved. Using this to simulate the experimental CLD with different enzyme combinations is a new means to test for enzymes that are significant to the rate of change of the CLD during synthesis. Comparison between the simulated CLD from different enzyme combinations and the experimental CLD in the leaves of the model plant Arabidopsis thaliana indicate α-amylase, in addition to the core starch biosynthetic enzymes, is also involved in the modification of glucans for the synthesis of insoluble starch granules. The simulations suggest involvement of ß-amylase, in the absence of α-amylase in mutants, slows the rate of attaining a crystalline-competent CLD for crystallization of glucans to form insoluble starch. This suggests a minor role of ß-amylase in shaping normal starch synthesis. The model simulation predicts that debranching of glucans is an efficient mechanism for the attainment of crystalline-competent CLD; however, attaining this is still possible, albeit slower, through combinations of α- and ß-amylase in the absence of isoamylase-type debranching enzyme. In Arabidopsis defective in one of the isoamylase-type debranching enzymes, the impact of α-amylase in starch synthesis is reduced, while ß-amylase becomes significantly involved, slowing the rate of synthesis in this mutant. Modeling of transient starch CLD brings to light previously unrecognized but significant effects of α- and ß-amylase on the rate of transient starch synthesis.