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.     

Dissecting the molecular basis of the contribution of source strength to high fructan accumulation in wheat

Fructans represent the major component of water soluble carbohydrates (WSCs) in the maturing stem of temperate cereals and are an important temporary carbon reserve for grain filling. To investigate the importance of source carbon availability in fructan accumulation and its molecular basis, we performed comparative analyses of WSC components and the expression profiles of genes involved in major carbohydrate metabolism and photosynthesis in the flag leaves of recombinant inbred lines from wheat cultivars Seri M82 and Babax (SB lines). High sucrose levels in the mature flag leaf (source organ) were found to be positively associated with WSC and fructan concentrations in both the leaf and stem of SB lines in several field trials. Analysis of Affymetrix expression array data revealed that high leaf sucrose lines grown in abiotic-stress-prone environments had high expression levels of a number of genes in the leaf involved in the sucrose synthetic pathway and photosynthesis, such as Calvin cycle genes, antioxidant genes involved in chloroplast H₂O₂ removal and genes involved in energy dissipation. The expression of the majority of genes involved in fructan and starch synthetic pathways were positively correlated with sucrose levels in the leaves of SB lines. The high level of leaf fructans in high leaf sucrose lines is likely attributed to the elevated expression levels of fructan synthetic enzymes, as the mRNA levels of three fructosyltransferase families were consistently correlated with leaf sucrose levels among SB lines. These data suggest that high source strength is one of the important genetic factors determining high levels of WSC in wheat.     

Phosphate transport in plants

Transport of inorganic phosphate (P_i) through plant membranes is mediated by a number of families of transporter proteins. Studies on the topology, function, regulation and sites of expression of the genes that encode the members of these transporter families are enabling roles to be ascribed to each of them. The Pht1 family, of which there are nine members in the Arabidopsis genome, includes proteins involved in the uptake of P_i from the soil solution and the redistribution of P_i within the plant. Members of this family are H₂PO₄ ^–/H⁺ symporters. Most of the genes of the Pht1 family that are expressed in roots are up-regulated in P-stressed plants. Two members of the Pht1 family have been isolated from the cluster roots of white lupin. These same genes are expressed in non-cluster roots. The evidence available to date suggests that there are no major differences between the types of transport systems that cluster roots and non-cluster roots use to acquire P_i. Differences in uptake rates between cluster and non-cluster roots can be ascribed to more high-affinity P_i transporters in the plasma membranes of cluster roots, rather than any difference in the characteristics of the transporters. The efficient acquisition of P_i by cluster roots arises primarily from their capacity to increase the availability of soil P_i immediately adjacent to the rootlets by excretion of carboxylates, protons and phosphatases within the cluster. This paper reviews P_i transport processes, concentrating on those mediated by the Pht1 family of transporters, and attempts to relate those processes involved in P_i acquisition to likely P_i transport processes in cluster roots.     

Co-ordinated synthesis of gentiobiitol and sorbitol, evidence of sorbitol glycosylation in transgenic sugarcane

Sugarcane (a Saccharum spp. interspecific hybrid) was previously engineered to synthesize sorbitol (designated as sorbitolcane). Motivated by the atypical development of the leaves in some sorbitolcane, the polar metabolite profiles in the leaves of those plants were compared against a group of control sugarcane plants. Eighty-six polar metabolites were detected in leaf extracts by GC-MS. Principal component analysis of the metabolites indicated that three compounds were strongly associated with sorbitolcane. Two were identified as sorbitol and gentiobiose and the third was unknown. Gentiobiose and the unknown compound were positively correlated with sorbitol accumulation. The unknown compound was only abundant in sorbitolcane. This compound was structurally characterized and found to be a sorbitol-glucose conjugate. ¹³C NMR analysis indicated that the glucopyranose and glucitol moieties were 1,6-linked. Ligand exchange chromatography confirmed that the compound was a β-anomer, thus identifying the compound as 6-O-β-d-glucopyranosyl-d-glucitol, or gentiobiitol.     

Changes in the sugarcane metabolome with stem development. Are they related to sucrose accumulation?

Sucrose content increases with internode development down the stem of sugarcane. In an attempt to determine which other changes in metabolites may be linked to sucrose accumulation gas chromatography-mass spectrometry was used to obtain metabolic profiles from methanol/water extracts of four samples of different age down the stem of cultivar Q117. Extracts were derivatized with either N-methyl-N-(trimethylsilyl) trifluoracetamide (TMS) or N-methyl N-(tert-butyldimethylsilyl) trifluoroacetamide (TBS) separately in order to increase the number of metabolites that could be detected. This resulted in the measurement of 121 and 71 metabolites from the TMS and TBS derivatization, respectively. Fifty-five metabolites were identified using commercial and publicly available libraries. Statistical analysis of the metabolite profiles resulted in clustering of tissue types. Particular metabolites were correlated with the level of sucrose accumulation, which as expected increased down the stem. Metabolites, such as tricarboxylic acid cycle intermediates and amino acids, were more abundant in the M2 sample (meristem to internode 2) that was actively growing and decreased in an apparently coordinated developmentally programmed manner in more mature internodes down the stem. However, other metabolites such as trehalose and raffinose showed positive correlations with sucrose concentration. Here we discuss the technique used to measure metabolites in sugarcane and the changes in metabolite abundance down the sugarcane stem.     

The Complement of Soluble Sugars in the Saccharum Complex

The use of sugarcane as a biofactory and source of renewable biomass is being investigated increasingly due to its vigorous growth and ability to fix a large amount of carbon dioxide compared to other crops. The high biomass resulting from sugarcane production (up to 80 t/ha) makes it a candidate for genetic manipulation to increase the production of other sugars found in this research that are of commercial interest. Sucrose is the major sugar measured in sugarcane with hexoses glucose and fructose present in lower concentrations; sucrose can make up to 60% of the total dry weight of the culm. Species related to modern sugarcane cultivars were examined for the presence of sugars other than glucose, fructose and sucrose with the potential of this crop as a biofactory in mind. The species examined form part of the Saccharum complex, a closely-related interbreeding group. Extracts of the immature and mature internodes of six different species and a hybrid were analysed with gas chromatography mass spectrometry to identify mono-, di- and tri-saccharides, as well as sugar acids and sugar alcohols. Thirty two sugars were detected, 16 of which have previously not been identified in sugarcane. Apart from glucose, fructose and sucrose the abundance of sugars in all plants was low but the research demonstrated the presence of sugar pathways that could be manipulated. Since species from the Saccharum complex can be interbred, any genes leading to the production of sugars of interest could be introgressed into commercial Saccharum species or manipulated through genetic engineering.     

Growth and metabolism in sugarcane are altered by the creation of a new hexose-phosphate sink

An efficient in planta sugarcane-based production system may be realized by coupling the synthesis of alternative products to the metabolic intermediates of sucrose metabolism, thus taking advantage of the sucrose-producing capability of the plant. This was evaluated by synthesizing sorbitol in sugarcane (Saccharum hybrids) using the Malus domestica sorbitol-6-phosphate dehydrogenase gene (mds6pdh). Mature transgenic sugarcane plants were compared with untransformed sugarcane variety Q117 by evaluation of the growth, metabolite levels and extractable activity of relevant enzymes. The average amounts of sorbitol detected in the most productive line were 120 mg/g dry weight (equivalent to 61% of the soluble sugars) in the leaf lamina and 10 mg/g dry weight in the stalk pith. The levels of enzymes involved in sucrose synthesis and cleavage were elevated in the leaves of plants accumulating sorbitol, but this did not affect sucrose accumulation in the culm. The activity of oxidative reactions in the pentose phosphate pathway and the non-reversible glyceraldehyde-3-phosphate dehydrogenase reaction were elevated to replenish the reducing power consumed by sorbitol synthesis. Sorbitol-producing sugarcane generated 30%-40% less aerial biomass and was 10%-30% shorter than control lines. Leaves developed necrosis in a pattern characteristic of early senescence, and the severity was related to the relative quantity of sorbitol accumulated. When the Zymomonas mobilis glucokinase (zmglk) gene was co-expressed with mds6pdh to increase the production of glucose-6-phosphate, the plants were again smaller, indicating that glucose-6-phosphate deficiency was not responsible for the reduced growth. In summary, sorbitol hyperaccumulation affected sugarcane growth and metabolism, but the outcome was not lethal for the plant. This work also demonstrated that impressive yields of alternative products can be generated from the intermediates of sucrose metabolism in Saccharum spp.