Ray Shorter

Molecular Dissection of Variation in Carbohydrate Metabolism Related to Water-Soluble Carbohydrate Accumulation in Stems of Wheat

Water-soluble carbohydrates (WSCs; composed of mainly fructans, sucrose [Suc], glucose [Glc], and fructose) deposited in wheat (Triticum aestivum) stems are important carbon sources for grain filling. Variation in stem WSC concentrations among wheat genotypes is one of the genetic factors influencing grain weight and yield under water-limited environments. Here, we describe the molecular dissection of carbohydrate metabolism in stems, at the WSC accumulation phase, of recombinant inbred Seri/Babax lines of wheat differing in stem WSC concentrations. Affymetrix GeneChip analysis of carbohydrate metabolic enzymes revealed that the mRNA levels of two fructan synthetic enzyme families (Suc:Suc 1-fructosyltransferase and Suc:fructan 6-fructosyltransferase) in the stem were positively correlated with stem WSC and fructan concentrations, whereas the mRNA levels of enzyme families involved in Suc hydrolysis (Suc synthase and soluble acid invertase) were inversely correlated with WSC concentrations. Differential regulation of the mRNA levels of these Suc hydrolytic enzymes in Seri/Babax lines resulted in genotypic differences in these enzyme activities. Down-regulation of Suc synthase and soluble acid invertase in high WSC lines was accompanied by significant decreases in the mRNA levels of enzyme families related to sugar catabolic pathways (fructokinase and mitochondrion pyruvate dehydrogenase complex) and enzyme families involved in diverting UDP-Glc to cell wall synthesis (UDP-Glc 6-dehydrogenase, UDP-glucuronate decarboxylase, and cellulose synthase), resulting in a reduction in cell wall polysaccharide contents (mainly hemicellulose) in the stem of high WSC lines. These data suggest that differential carbon partitioning in the wheat stem is one mechanism that contributes to genotypic variation in WSC accumulation.

Use of expression analysis to dissect alterations in carbohydrate metabolism in wheat leaves during drought stress

Water deficit in plants causes a reduction in photosynthesis and high demands for osmolyte synthesis. To elucidate regulation of carbohydrate metabolic genes in wheat (Triticum aestivum) leaves during drought stress, we performed a systematic expression study using quantitative RT-PCR and cDNA microarray. These analyses revealed that expression levels of most genes encoding chloroplast enzymes involved in carbon fixation (Calvin cycle) were reduced in the leaves during prolonged drought stress. Transcript levels of highly expressed isoenzymes of hexokinase and fructokinase also decreased. Conversely, genes encoding cytoplasmic and vacuolar enzymes in the pathways leading to glucose, fructose and fructan production were up-regulated in the stressed leaves. Systematic expression analysis of an almost complete set of genes involved in conversion of triose phosphates to hexoses and hexose phosphorylation showed that isoenzymes of many enzymes were differentially regulated during drought stress. Correlation analysis indicated that the drought down-regulated Calvin cycle genes were coordinately regulated. This coordinated down-regulation extended to genes encoding major isoenzymes of chloroplast triose-phosphate/phosphate translocator, cytoplasmic fructose-1,6-bisphosphate aldolase and fructose bisphosphatase. Highly correlated expression was also observed between drought up-regulated genes involved in sucrose synthesis and hydrolysis or fructan synthesis. These data dissect coordination in regulation of key enzyme genes involved in carbon fixation and accumulation of hexoses and fructans and provide an insight into molecular mechanisms at the transcript level underlying changes in carbohydrate metabolism in wheat adaptation to drought stress.

TaNAC69 from the NAC superfamily of transcription factors is up-regulated by abiotic stresses in wheat and recognises two consensus DNA-binding sequences

NAC proteins are one of the largest families of plant transcription factors and have recently been implicated in diverse physiological processes. To elucidate their role in gene regulation, we determined the DNA-binding specificity of a drought- and cold-inducible NAC protein, TaNAC69 from wheat, and analysed its homologues from other species. Two consensus DNA-binding sequences (spanning 23-24 bp) of TaNAC69 were identified through binding site selection and both consisted of two half sites. Comprehensive data on the DNA-binding specificity of TaNAC69 were generated through extensive base substitution mutagenesis. TaNAC69 and its homologue in Arabidopsis, NAP, sharing 75% sequence identity in the NAC domain, exhibited similar DNA-binding specificity. TaNAC69 was able to homodimerise through its NAC domain. The NAC domain consists of five conserved subdomains. Subdomain mutation showed that a loss or reduction in TaNAC69 dimerisation capacity was accompanied with abolition or decrease in its DNA-binding activity. These data suggest that all subdomains are necessary to maintain a functional NAC domain structure required for interaction with DNA and dimerisation.

Differential gene expression of wheat progeny with contrasting levels of transpiration efficiency.

High water use efficiency or transpiration efficiency (TE) in wheat is a desirable physiological trait for increasing grain yield under water-limited environments. The identification of genes associated with this trait would facilitate the selection for genotypes with higher TE using molecular markers. We performed an expression profiling (microarray) analysis of approximately 16,000 unique wheat ESTs to identify genes that were differentially expressed between wheat progeny lines with contrasting TE levels from a cross between Quarrion (high TE) and Genaro 81 (low TE). We also conducted a second microarray analysis to identify genes responsive to drought stress in wheat leaves. Ninety-three genes that were differentially expressed between high and low TE progeny lines were identified. One fifth of these genes were markedly responsive to drought stress. Several potential growth-related regulatory genes, which were down-regulated by drought, were expressed at a higher level in the high TE lines than the low TE lines and are potentially associated with a biomass production component of the Quarrion-derived high TE trait. Eighteen of the TE differentially expressed genes were further analysed using quantitative RT-PCR on a separate set of plant samples from those used for microarray analysis. The expression levels of 11 of the 18 genes were positively correlated with the high TE trait, measured as carbon isotope discrimination (Δ13C). These data indicate that some of these TE differentially expressed genes are candidates for investigating processes that underlie the high TE trait or for use as expression quantitative trait loci (eQTLs) for TE.

The Q-type C2H2 zinc finger subfamily of transcription factors in Triticum aestivum is predominantly expressed in roots and enriched with members containing an EAR repressor motif and responsive to drought stress

Q-type C2H2 zinc finger proteins (ZFPs) form a subfamily of transcription factors that contain a plant-specific QALGGH amino acid motif. A total of 47 expressed Q-type C2H2 zinc finger genes in bread wheat (Triticum aestivum) (designated TaZFP) were identified from the current databases. Protein sequence analysis for the presence of ERF-associated amphiphilic repressor (EAR) motif sequences from known transcriptional repressors revealed that 26% of the TaZFP subfamily members contain an EAR motif. Quantitative RT-PCR analysis of the mRNA distribution of 44 TaZFP genes in various organs revealed that 30 genes were predominantly expressed in the roots. The majority of the TaZFP genes showed significant changes in their mRNA levels during leaf development and aging. Expression of 37 TaZFP genes in the leaves and roots responded to drought stress at least in one organ with 74% of the drought-responsive TaZFP genes being down-regulated in the drought-stressed roots. In contrast, only 6 out of the 44 TaZFP genes showed expression changes in the leaves with sucrose treatment. Expression of 50% of the drought-responsive TaZFP genes in the leaves (16 genes analysed) did not respond to ABA treatment, indicating that some TaZFP genes are involved in ABA-independent signalling pathways. These results indicate that the Q-type TaZFP subfamily is likely to have an important role in wheat roots and is enriched with members that are potentially involved in regulating cellular activities during changes of the physiological status of plant cells, as it occurs during drought stress or leaf development/aging.

Use of dry matter content as a rapid and low-cost estimate for ranking genotypic differences in water-soluble carbohydrate concentrations in the stem and leaf sheath of Triticum aestivum

Stem water-soluble carbohydrates (WSC) are an important source of temporary carbohydrate reserve in cool-season cereals. Genotypic variation in stem WSC concentration in wheat at anthesis is often positively associated with grain weight and yield in water-limited environments. In this study we have examined the relationship between dry matter content (DMC, dry weight per unit of fresh weight) and WSC concentration in field-grown bread wheat. Strong correlations (r = 0.92–0.95) were observed between DMC and WSC concentration in the stem and leaf sheath from the top two or three internodes of recombinant inbred lines from a cross between Seri M82 and Babax, at anthesis or 1 week after anthesis, in several field experiments. This strong correlation was also observed in diverse genotypes grown under rainfed or irrigated conditions. DMC and WSC concentration were also positively correlated in the whole above-ground biomass of wheat at anthesis (r = 0.74–0.91). Measurement of stem and leaf sheath DMC and WSC concentration in a small number of samples would allow the rapid prediction of WSC concentrations in a large number of field samples with reasonable accuracy, as demonstrated in a small dataset in this study. These data indicate that DMC can serve cereal breeding as a rapid and low-cost selection tool for genotypic ranking of WSC concentrations in breeding populations.

Genotypic variation in the accumulation of water soluble carbohydrates in wheat

Water-soluble carbohydrates (WSC) stored in the stems and leaf sheaths of winter cereals provide an important source of assimilate for remobilisation during grain-filling. Consequently, WSC are a major contributor to wheat grain yield and grain size in all environments but especially where photosynthesis is compromised as occurs where water is limiting. Breeding programs targeting greater WSC should provide improved varieties with greater and more stable yields in stress environments. To facilitate selection for WSC, genetic and genomic approaches are being used to determine the genetic basis of - and define DNA probes for - marker-aided selection for this important drought-adaptive trait. Empirical studies have identified both WSC concentration and content to be under complex genetic control of many genes. Quantitative trait loci (QTL) for WSC have been identified in several wheat populations with individual QTL explaining small amounts of phenotypic variation, typically of less than 20%. Many of these QTL are common across multiple, genetically-unrelated wheat populations. Evaluation of gene expression in high and low WSC wheat progeny lines from a well characterised wheat population has identified significant differences in expression of genes from different gene categories. For example, high WSC progeny lines have higher levels of expression of genes involved in carbohydrate metabolism and lower levels of expression of genes involved in cell wall and amino acid metabolism than low WSC lines. Genetic mapping reveals several candidate genes co-locating with QTL for WSC. In addition, expression QTL (eQTL) for selected candidate genes co-locate with WSC QTL; co-location of the genes and eQTL with WSC QTL make these genes stronger candidate genes for the WSC trait.

Linked gene networks involved in nitrogen and carbon metabolism and levels of water-soluble carbohydrate accumulation in wheat stems

High levels of water-soluble carbohydrates (WSC) provide an important source of stored assimilate for grain filling in wheat. To better understand the interaction between carbohydrate metabolism and other metabolic processes associated with the WSC trait, a genome-wide expression analysis was performed using eight field-grown lines from the high and low phenotypic tails of a wheat population segregating for WSC and the Affymetrix wheat genome array. The 259 differentially expressed probe sets could be assigned to 26 functional category bins, as defined using MapMan software. There were major differences in the categories to which the differentially expressed probe sets were assigned; for example, probe sets upregulated in high relative to low WSC lines were assigned to category bins such as amino acid metabolism, protein degradation and transport and to be involved in starch synthesis-related processes (carbohydrate metabolism bin), whereas downregulated probe sets were assigned to cell wall-related bins, amino acid synthesis and stress and were involved in sucrose breakdown. Using the set of differentially expressed genes as input, chemical–protein network analyses demonstrated a linkage between starch and N metabolism via pyridoxal phosphate. Twelve C and N metabolism-related genes were selected for analysis of their expression response to varying N and water treatments in the field in the four high and four low WSC progeny lines; the two nitrogen/amino acid metabolism genes demonstrated a consistent negative association between their level of expression and level of WSC. Our results suggest that the assimilation of nitrogen into amino acids is an important factor that influences the levels of WSC in the stems of field-grown wheat.

Preferential retention of chromosome regions in derived synthetic wheat lines: a source of novel alleles for wheat improvement

Abstract. Synthetic hexaploid wheats (SHWs) and their synthetic derivative lines (SDLs) are being used as a means of introducing novel genetic variation into bread wheat (BW). Phenotypic information for days to flowering, height, grain weight and grain yield was collected from multiple environments for three SDL families, each with ∼50 lines, and their elite BW parents. In general, the SDLs were earlier flowering and taller with larger grain size, but similar grain yield to the BWs. The three SDL families and their SHW and BW parents were genotyped using mapped DArT (diversity arrays technology) markers. Within each SDL family, SHW-specific DArT markers were used to identify SHW-derived chromosomal regions that appeared to be preferentially retained in the SDL families, as determined by retention at frequencies >0.25, the expected frequency for Mendelian segregation. Regions on chromosomes 2BS and 7BL appeared to be preferentially retained in all three SDL families, while regions on chromosomes 1AL, 1BS, 3BS, 5AS, 5BL, and 7AS were preferentially retained in two of the three SDL families. Other regions were preferentially retained in single families only, including some regions located on the D genome. Single-marker regression analysis was performed using the preferentially retained markers and identified markers and regions that were significantly associated with one or more of the four traits measured. Comparative mapping also indicates that these preferentially retained markers and chromosome regions may co-locate with previously identified QTLs for anthesis, height, grain weight and/or grain yield. Therefore, SHWs may contain novel alleles at these loci in these regions for these traits, which may provide a selective advantage to the SDLs. This approach could provide a useful method for identifying chromosomal regions of interest with potentially novel alleles for introgression for further BW improvement.