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.     

Effects of drought, transgenic expression of a fructan synthesizing enzyme and of mycorrhizal symbiosis on growth and soluble carbohydrate pools in tobacco plants

The effects of three conditions likely to affect soluble carbohydrate pools, namely drought, expression of barley sucrose: fructan 6-fructosyl transferase (6-SFT, EC 2.4.1.10) and the establishment of the arbuscular mycorrhizal symbiosis with Glomus mosseae were studied in a multifactorial experiment using tobacco ( Nicotiana tabacum ). Tobacco, a plant naturally unable to form fructan, accumulated fructan in leaves, and to a larger extent in the roots, when transformed with 6-SFT. Under drought conditions, growth was considerably reduced, but neither expression of 6-SFT nor mycorrhiza formation had an effect on growth rate. However, in response to drought, carbon partitioning was significantly altered towards accumulation of soluble sugars. In plants exposed to drought, pools of sucrose were greater than those of unstressed plants, particularly in their roots. In the transgenic plants expressing 6-SFT, there were also increased contents of the products of 6-SFT, namely fructan, most probably because of the increased availability of the substrate, sucrose. These effects were the same in the presence or absence of mycorrhiza. Hexoses (glucose and fructose) also increased in response to drought, primarily in the leaves. This effect of drought was little affected by the expression of 6-SFT, except that it slightly enhanced drought-induced glucose accumulation in roots. However, the presence of mycorrhiza led to a considerable reduction in drought-induced accumulation of hexoses in the leaves. The content of the fungal disaccharide trehalose was greatly increased in the roots of all mycorrhizal plants upon exposure to drought, particularly in some of the transgenic plants expressing 6-SFT.     

Importance of pre‐anthesis anther sink strength for maintenance of grain number during reproductive stage water stress in wheat

Reproductive stage water stress leads to spikelet sterility in wheat. Whereas drought stress at anthesis affects mainly grain size, stress at the young microspore stage of pollen development is characterized by abortion of pollen development and reduction in grain number. We identified genetic variability for drought tolerance at the reproductive stage. Drought‐tolerant wheat germplasm is able to maintain carbohydrate accumulation in the reproductive organs throughout the stress treatment. Starch depletion in the ovary of drought‐sensitive wheat is reversible upon re‐watering and cross‐pollination experiments indicate that the ovary is more resilient than the anther. The effect on anthers and pollen fertility is irreversible, suggesting that pollen sterility is the main cause of grain loss during drought conditions in wheat. The difference in storage carbohydrate accumulation in drought‐sensitive and drought‐tolerant wheat is correlated with differences in sugar profiles, cell wall invertase gene expression and expression of fructan biosynthesis genes in anther and ovary (sucrose : sucrose 1‐fructosyl‐transferase, 1‐SST; sucrose : fructan 6‐fructosyl‐transferase, 6‐SFT). Our results indicate that the ability to control and maintain sink strength and carbohydrate supply to anthers may be the key to maintaining pollen fertility and grain number in wheat and this mechanism may also provide protection against other abiotic stresses.     

Regulation of photosynthetic carbon assimilation at the cellular level: a review

In green leaves and a number of algae, photosynthetically derived carbon is ultimately converted into two carbohydrate end-products, sucrose and starch. Drainage of carbon from the Calvin cycle proceeds via triose phosphate, fructose 6-phosphate and glycollate. Gluconeogenesis in photosynthetic cells is controlled by light, inorganic phosphate and phosphorylated sugars. Light stimulates the production of dihydroxyacetone phosphate, the initial substrate for sucrose and starch synthesis, and inhibits the degradative pathways in the chloroplast. Phosphate inactivates reactions of synthesis and activates reactions of degradation. Among the phosphorylated sugars a special role is allocated to fructose 2,6-bisphosphate, which is present in the cytoplasm at very low concentrations and inhibits sucrose synthesis directly by inactivating pyrophosphatedependent phosphofructokinase. The synthesis of sucrose plays a central role in the partitioning of photosynthetic carbon. The cytoplasmic enzymes, fructose bisphosphate phosphatase and sucrose phosphate synthase are likely key points of regulation. The regulation is carried out by several effector metabolites. Fructose 2,6-bisphosphate is likely to be the main coordinator of the rate of sucrose synthesis, hence of photosynthetic carbon partitioning between sucrose and starch.Paper presented at the FESP meeting (Strasbourg, 1984)     

NADK2, an <Emphasis Type="Italic">Arabidopsis</Emphasis> Chloroplastic NAD Kinase,Plays a Vital Role in Both Chlorophyll Synthesis and Chloroplast Protection

As one of terminal electron acceptors in photosynthetic electron transport chain, NADP receives electron and H⁺ to synthesize NADPH, an important reducing energy in chlorophyll synthesis and Calvin cycle. NAD kinase (NADK), the catalyzing enzyme for the de novo synthesis of NADP from substrates NAD and ATP, may play an important role in the synthesis of NADPH. NADK activity has been observed in different sub-cellular fractions of mitochondria, chloroplast, and cytoplasm. Recently, two distinct NADK isoforms (NADK1 and NADK2) have been identified in Arabidopsis. However, the physiological roles of NADKs remain unclear. In present study, we investigated the physiological role of Arabidiposis NADK2. Sub-cellular localization of the NADK2–GFP fusion protein indicated that the NADK2 protein was localized in the chloroplast. The NADK2 knock out mutant (nadk2) showed obvious growth inhibition and smaller rosette leaves with a pale yellow color. Parallel to the reduced chlorophyll content, the expression levels of two POR genes, encoding key enzymes in chlorophyll synthesis, were down regulated in the nadk2 plants. The nadk2 plants also displayed hypersensitivity to environmental stresses provoking oxidative stress, such as UVB, drought, heat shock and salinity. These results suggest that NADK2 may be a chloroplast NAD kinase and play a vital role in chlorophyll synthesis and chloroplast protection against oxidative damage.     

Cloning and expression of genes related to the sucrose-metabolizing enzymes and carbohydrate changes in peach

To shed light on the relationship between sucrose metabolism and expression of genes related to sucrose-metabolizing enzymes, six genes encoding sucrose-metabolizing enzymes were isolated, and the levels of four main carbohydrates and related enzyme activities as well as the expression of these six genes were determined in fruits, leaves and phloem-enriched fraction throughout peach fruit development. Sucrose content in mature fruit ranked first followed by glucose, fructose and sorbitol in that order, while sorbitol was the highest and sucrose lowest in phloem-enriched fraction and leaves. Glucose and fructose had similar change patterns throughout fruit development. Cloning results reveal that the nucleotide sequences of the six genes have high similarity to corresponding genes isolated from other plants. In addition, the expression of these genes and the levels of related enzyme activities varied with tissue and stage of fruit development, suggesting a complexity in relationships between carbohydrates, enzymes activities and related gene expression. Sucrose phosphate synthase maybe a key enzyme involved in sucrose synthesis while sucrose synthase may mainly be responsible for sucrose synthesis in peach fruits at later stages of development. Further studies are needed to genetically and physiologically characterize these genes and enzymes in peach and to gain a better understanding of their functions and relationship with carbohydrate metabolism.     

The influence of altered gravity on carbohydrate metabolism in excised wheat leaves

We developed a system to study the influence of altered gravity on carbohydrate metabolism in excised wheat leaves by means of clinorotation. The use of excised leaves in our clinostat studies offered a number of advantages over the use of whole plants, most important of which were minimization of exogenous mechanical stress and a greater amount of carbohydrate accumulation during the time of treatment. We found that horizontal clinorotation of excised wheat leaves resulted in significant reductions in the accumulation of fructose, sucrose, starch and fructan relative to control, vertically clinorotated leaves. Photosynthesis, dark respiration and the extractable activities of ADP glucose pyrophosphorylase (EC 2.7.7.27), sucrose phosphate synthase (EC 2.4.4.14), sucrose sucrose fructosyltransferase (EC 2.4.1.99), and fructan hydrolase (EC 3.2.1.80) were unchanged due to altered gravity treatment.     

The glycolate pathway and photosynthetic competence in euglena

The development of glycolate pathway enzymes has been determined in relation to photosynthetic competence during the regreening of Euglena cultures. Phosphoglycolate phosphatase and glycolate dehydrogenase rapidly reached maximal levels of activity but the complete development of ribulose 1,5-diphosphate carboxylase and concomitant photosynthetic carbon dioxide fixation were not attained until 72 hours of illumination. Specific inhibitors of protein synthesis showed that the formation of ribulose 1,5-diphosphate carboxylase in both division-synchronized and regreening cultures was prevented by both cycloheximide and d-threo-chloramphenicol, whereas phosphoglycolate phosphatase formation was only inhibited by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide. Since cycloheximide prevented ribulose diphosphate carboxylase synthesis and photosynthetic carbon dioxide fixation without affecting phosphoglycolate phosphatase synthesis during regreening, it was concluded that photosynthetic competence was not necessary for the development of the glycolate pathway enzymes. The inhibition of phosphoglycolate phosphatase synthesis by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide shows that the enzyme was synthesized exclusively on chloroplast ribosomes, whereas protein synthesis on both chloroplast and cytoplasmic ribosomes was required for the formation of ribulose 1,5-diphosphate carboxylase. Although light is required for the development of both Calvin cycle and glycolate pathway enzymes during regreening it is concluded that the two pathways are not coordinately regulated.     

硫酸锌添加对酿酒酵母乙酸胁迫条件下全局基因转录的影响

【目的】利用转录组测序研究硫酸锌添加提高絮凝酿酒酵母SPSC01乙酸胁迫耐性的分子机理。【方法】在10.0 g/L乙酸胁迫条件下,添加0.03 g/L硫酸锌,取对数期酿酒酵母细胞,与不添加硫酸锌的对照组细胞进行比较转录组分析。【结果】添加硫酸锌的实验组与对照组相比较,50个基因转录水平上调,162个基因转录水平下调,这些转录水平变化明显的基因涉及糖代谢、甲硫氨酸合成、维生素合成等多条代谢途径,此外,转录水平变化的基因还包括抗氧化酶基因等关键胁迫响应基因。【结论】硫酸锌添加可改变酿酒酵母全局基因转录水平,提高抗氧化酶及其他胁迫耐性相关基因的表达,影响细胞氧化还原平衡和能量代谢,通过对多基因转录的调控提高酿酒酵母乙酸耐受性。     

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.     

Comparison of fructan dynamics in two wheat cultivars with different capacities of accumulation and remobilization under drought stress

Remobilization of stored carbohydrates in the stem of wheat plants is an important contributor to grain filling under drought stress (DS) conditions. A massive screening on Iranian wheat cultivars was performed based on stem dry weight changes under well-watered and DS conditions. Two cultivars, Shole and Crossed Falat Hamun (CFH), with different fructan accumulation and remobilization behavior were selected for further studies. Water-soluble carbohydrates (WSCs) and fructan metabolizing enzymes were studied both in the stem penultimate and in sucrose (Suc) treated, excised leaves. Under drought, CFH produced higher grain yields than Shole (412 vs 220 g m(-2)). Also, grain yield loss under drought was more limited in CFH than in Shole (17 vs 54%). Under drought, CFH accumulated more graminan-type fructo-oligosaccharides than Shole. After anthesis, fructan 6-exohydrolase (6-FEH; EC 3.2.1.154) activities increased more prominently than fructan 1-exohydrolase (EC 3.2.1.153) activities during carbon remobilization. Interestingly, CFH showed higher 6-FEH activities in the penultimate than Shole. The field experiment results suggest that the combined higher remobilization efficiency and high 6-FEH activities in stems of wheat could contribute to grain yield under terminal drought. Similar to the penultimate, fructan metabolism differed strongly in Suc-treated detached leaves of selected cultivars. This suggests that variation in the stem fructan among wheat cultivars grown in the field could be traced by leaf blade induction experiments.     

Relationships between some transcription factors and concordantly expressed drought stress-related genes in bread wheat

The challenge of climate change makes it mandatory to improve tolerance to drought stress in bread wheat (Triticum aestivum) via biotechnological approaches. Drought stress experiment was conducted followed by RNA-Seq analysis for leaves of two wheat cultivars namely Giza 168 and Gemmiza 10 with contrasting genotypes. Expression patterns of the regulated stress-related genes and concordantly expressed TFs were detected, then, validated via qPCR for two loss-of-function mutants in Arabidopsis background harboring mutated genes analogue to those in wheat. Drought-stress related genes were searched for concordantly expressed TFs and a total of eight TFs were shown to coexpress with 14 stress-related genes. Among these genes, one TF belongs to the zinc finger protein CONSTANS family and proved via qPCR to drive expression of a gene encoding a speculative TF namely zinc transporter 3-like and two other stress related genes encoding tryptophan synthase alpha chain and asparagine synthetase. Known functions of the two TFs under drought stress complement those of the two concordantly expressed stress-related genes, thus, it is likely that they are related. This study highlights the possibility to utilize metabolic engineering approaches to decipher and incorporate existing regulatory frameworks under drought stress in future breeding programs of bread wheat.     

干旱与氧化胁迫对小麦根氧化还原状态和叶片ABA积累的影响

通过分析一氧化氮(nitric oxide,NO)、活性氧(reactive oxygen species,ROS)和干旱胁迫对小麦根氧化还原状态和叶片脱落酸(abscisic acid,ABA)积累的影响,探讨了干旱胁迫下NO和H2O2调节ABA合成的可能机制。结果表明:干旱胁迫处理初期小麦根还原型谷胱甘肽含量降低、抗氧化酶活性发生振荡变化,细胞氧化还原状态向氧化型转变。NO和H2O2能模拟干旱胁迫的作用使细胞状态向氧化型转变,还可以使小麦叶片ABA积累量上升。干旱胁迫下NO和H2O2对ABA合成的调节作用可能是通过调节细胞氧化还原状态进行。     

Appearance of Three Chloroplast Isoenzymes in Dark-grown Pea Plants and Pea Seeds

Activity peaks characteristic of the chloroplastic Calvin cycle enzymes triose-phosphate isomerase, ribose 5-phosphate isomerase, and fructose 1,6-diphosphate aldolase are found in isoelectric focusing patterns of dark-grown pea (Pisum sativum) seedlings and seeds. Apparently, in this higher plant these three chloroplastic isoenzymes can be formed in the absence of light and of chloroplast formation.     

Differential expression of alkaline and neutral invertases in response to environmental stresses: characterization of an alkaline isoform as a stress-response enzyme in wheat leaves

It is well accepted that sucrose (Suc) metabolism is involved in responses to environmental stresses in many plant species. In the present study we showed that alkaline invertase (A-Inv) expression is up-regulated in wheat leaves after an osmotic stress or a low-temperature treatment. We demonstrated that the increase of total alkaline/neutral Inv activity in wheat leaves after a stress could be due to the induction of an A-Inv isoform. Also, we identified and functionally characterized the first wheat cDNA sequence that codes for an A-Inv. The wheat leaf full-length sequence encoded a protein 70% similar to a neutral Inv of Lolium temulentum; however, after functional characterization, it resulted to encode a protein that hydrolyzed Suc to hexoses with an optimum pH of 8, and, consequently, the encoding sequence was named Ta-A-Inv. By RT-PCR assays we demonstrated that Ta-A-Inv expression is induced in response to osmotic and cold stress in mature primary wheat leaves. We propose that Ta-A-Inv activity could play an important role associated with a more efficient cytosolic Suc hydrolysis during environmental stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Exogenous nitric oxide effect on fructan accumulation and FBEs expression in chilling-sensitive and chilling-resistant wheat

This study was to investigate the effect of exogenous nitric oxide (NO) on fructan accumulation and fructan biosynthesic enzymes (FBEs) expression in seedlings leaves of two wheat (Triticum aestivum L.) cultivars, winter wheat (Zhoumai18, ZM) and spring wheat (Yanzhan4110, YZ), under 4 °C. The seedlings of two wheat cultivars were subjected to different concentrations of sodium nitroprussiate (SNP) for 0, 24, 48, and 96 h. Relative water content (RWC) was increased by exogenous NO in YZ, but decreased in ZM. Except for glucose, fructose and fructans of degree of polymerization (DP) 3 in YZ, other soluble carbohydrates contents in the two wheat cultivars all increased to different degrees. The activities of FS (including sucrose: sucrose 1-fructosyltransferase (1-SST, EC: 2.4.1.99) and sucrose: fructan 6-fructosyltransferase (6-SFT, EC: 2.4.1.10)) were significantly higher than fructan: fructan 1-fructosyltransferase (1-FFT, EC: 2.4.1.100) in the seedlings of two wheat cultivars. The same phenomenon occurred to FBEs expression. In addition, sucrose content decreased while fructans content increased under low temperature, which was in accordance with the improved 1-FFT activity in ZM. Moreover, fructans content increased to a high level under high concentration of NO in ZM while kept at a constant low level in YZ. The expression levels of FBEs were universally higher in ZM than in YZ, which identified with the high frost resistance of the winter cultivar. It is concluded that exogenous NO treatment on wheat may be a good option to reduce chilling injury by regulating fructan accumulation in leaves. This is the first report owing that exogenous NO alleviated the negative effects of chilling stress by accumulating fructans in wheat.