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.

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.

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.

The domestication syndrome in vegetatively propagated field crops.

BACKGROUND: Vegetatively propagated crops are globally significant in terms of current agricultural production, as well as for understanding the long-term history of early agriculture and plant domestication. Today, significant field crops include sugarcane (Saccharum officinarum), potato (Solanum tuberosum), manioc (Manihot esculenta), bananas and plantains (Musa cvs), sweet potato (Ipomoea batatas), yams (Dioscorea spp.) and taro (Colocasia esculenta). In comparison with sexually reproduced crops, especially cereals and legumes, the domestication syndrome in vegetatively propagated field crops is poorly defined. AIMS AND SCOPE: Here, a range of phenotypic traits potentially comprising a syndrome associated with early domestication of vegetatively propagated field crops is proposed, including: mode of reproduction, yield of edible portion, ease of harvesting, defensive adaptations, timing of production and plant architecture. The archaeobotanical visibility of these syndrome traits is considered with a view to the reconstruction of the geographical and historical pathways of domestication for vegetatively propagated field crops in the past. CONCLUSIONS: Although convergent phenotypic traits are identified, none of them are ubiquitous and some are divergent. In contrast to cereals and legumes, several traits seem to represent varying degrees of plastic response to growth environment and practices of cultivation, as opposed to solely morphogenetic 'fixation'.

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.

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.

Down-regulation of Glucan, Water-Dikinase activity in wheat endosperm increases vegetative biomass and yield.

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.

A survey of ß-glucan and arabinoxylan content in wheat.

BACKGROUND: Dietary fibre lowers the risk of coronary heart disease and colorectal cancer. This survey quantifies mixed link ß-glucan (MBG) and arabinoxylan (AX) in wheat and investigates relationships between the grain carbohydrates. MBG and AX contents were measured in 500 and 200 wheat accessions respectively, including diploid, tetraploid and hexaploid genotypes comprising primitive, synthetic and elite lines. RESULTS: Overall, MBG contents ranged between 1.8 and 18.0 g kg(-1) grain dry weight. In wheat-barley addition lines and triticale hexaploids the levels were 9.0-11.3 and 3.5-9.6 g kg(-1) respectively. The amounts in synthetic wheats were nearer their tetraploid parents than their diploid parents. AX and total non-starch polysaccharide (NSP) contents ranged from 23.7 to 107.5 g kg(-1) and from 31.7 to 136.7 g kg(-1) respectively. Linear regressions showed that the relationships of starch and grain weight with NSP glucose were stronger than those with AX. CONCLUSION: The results indicated insufficient genetic diversity in the germplasm surveyed to initiate a breeding programme to increase the amount of MBG in wheat grain to 20 g kg(-1) , a level considered high enough to confer a 10-15% reduction in blood cholesterol.

Fructan formation in transgenic white clover expressing a fructosyltransferase from Streptococcus salivarius.

White clover (Trifolium repens L.) is an important pasture legume that does not normally accumulate fructan as a storage carbohydrate. We have generated transgenic white clover plants that accumulate fructan, by expressing the fructosyltransferase (Ftf) enzyme from the bacterium Streptococcus salivarius under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Fructan accumulated in leaves, petioles, stolons, flowers, and roots of transgenic plants. Levels of fructan up to approximately 2% dry weight were measured in leaves. The fructan was of high molecular mass ( > 5000 kDa), typical of bacterial fructans. Ftf enzyme activity up to 120 nmol min-1 g-1 fresh weight was determined in leaf extracts of the transformed plants, and appeared to be stable throughout leaf development. Most transformed lines appeared normal, flowered and produced seed, but the growth rate of some transformed lines decreased. Photosynthetic carbon assimilation and levels of endogenous carbohydrates (hexoses, sucrose and starch) were not substantially changed in a clonal line with relatively low fructan. However, in a clonal line with relatively high fructan accumulation, plant growth was reduced, leaf photosynthesis was decreased by 60%, and carbohydrate contents were reduced. The results are discussed in the context of manipulating soluble carbohydrate composition in pasture species to improve nutritive quality for grazing animals.