Sucrose metabolism during endosperm development in wheat (Triticum aestivum)

This work reports changes in sucrose synthase and invertase activities throughout endosperm development in wheat, together with the associated substrates and metabolites, sucrose, UDP, glucose, fructose and UDP-glucose. Throughout endosperm development, sucrose synthase had consistently higher activity than invertase and indeed invertase activity did not change appreciably. The observed variation in pattern and amounts of glucose and fructose present during the mid- and late stages of endosperm development confirmed the suggestion that invertase was not the preferred pathway of sucrose catabolism. Kinetic parameters for sucrose synthase were determined in crude extracts. Estimates of UDP and sucrose concentrations suggest that sucrose synthase is unlikely to achieve its potential maximum velocity. This limitation may however be overcome in part by the apparent excess catalytic activity measured during endosperm development.     

Accumulation and conversion of sugars by developing wheat grains. 4. Effects of phosphate and potassium ions in endosperm slices

Abstract. Transverse slices through developing grains of Triticum aestivum cv. SUN 9E 16 d after anthesis were incubated in simple defined media with various radioactive labels. In some enzymic assays slices were pretreated with 2.5% Triton X-100 or with 5% butanol to remove cellular membranes and endogenous substrates.
Endogenous potassium leaked from endosperm slices into 30mol m⁻³ sucrose while sucrose was converted partly into starch. Exogenous alkali-ions, except Li⁺, stimulated conversion of sucrose to insoluble matter, specifically to starch with K⁺. Starch synthetase activity of Triton-pretreated slices was stimulated by K⁺ at both high and low substrate ADPG concentration, but was not affected by phosphate (25 mol m⁻³).
Phosphate in the medium had no effect on incorporation of sucrose or glucose into alcohol-insoluble material or starch in fresh slices (internal inorganic phosphate (P,) concentration was about 11 mol m⁻³). Three- to four-fold contrasts in internal P_i level, achieved by prolonged preincubations in different media, did not show an inhibition of starch synthesis by P_i. However, phosphate (25mol m⁻³) inhibited starch synthesis, that was mediated by ADPG pyrophosphorylase in butanol-pretreated endosperm slices by 15–18%.
It is concluded that starch synthesis in wheat endosperm is not regulated directly by apoplastic P_i; level.     

Expression of fission yeast cdc25 driven by the wheat ADP-glucose pyrophosphorylase large subunit promoter reduces pollen viability and prevents transmission of the transgene in wheat

Cell number was to be measured in wheat (Triticum aestivum) endosperm expressing Spcdc25 (a fission yeast cell-cycle regulator) controlled by a supposedly endosperm-specific promoter, AGP2 (from the large subunit of ADP glucose pyrophosphorylase). Wheat was transformed by biolistics either with AGP2::GUS or AGP2::Spcdc25. PCR and RT-PCR checked integration and expression of the transgene, respectively. In cv. Chinese Spring, AGP2::GUS was unexpectedly expressed in carpels and pollen, as well as endosperm. In cv. Cadenza, three AGP2::Spcdc25 plants, AGP2::Spcdc25.1, .2 and .3, were generated. Spcdc25 expression was detected in mature leaves of AGP2::Spcdc25.1/.3 which exhibited abnormal spikes, 50% pollen viability and low seed set per plant; both were small compared with the nonexpressing and normal AGP2::Spcdc25.2. Spcdc25 was not transmitted to the T(1) in AGP2::Spcdc25.1 or .3, which developed normally. Spcdc25 was PCR-positive in AGP2::Spcdc25.2, using primers for a central portion, but not with primers for the 5' end, of the ORF, indicating a rearrangement; Spcdc25 was not expressed in either T(0) or T(1). The AGP2 promoter is not tissue-specific and Spcdc25 expression disrupted reproduction.     

Two forms of fructose 1,6-bisphosphatase from immature wheat endosperm

Fructose 1,6-bisphosphatase (α-D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase) from immature wheat endosperm has been resolved into two forms, FBPase-I and FBPase-II. Their specific activities over crude homogenate increased 47- and 77-fold, respectively, by using ammonium sulfate fractionation, DEAE-cellulose chromatography and gel filtration through Sephadex G-200. The pH optimum was 7.6 for FBPase-I and 8.4 for FBPase-II. The two forms were highly specific for the substrate FBP with K_m values of 0.17 and 0.08 m M , respectively, for FBPase-I and FBPase-II at their respective pH optimum and saturating Mg²⁺ concentration. pH had no effect on the K_m value for FBPase-I, but that for FBPase-II increased below optimum pH. Neither of the forms had an absolute requirement for Mg²⁺, although it was essential for maximum activity. Mg²⁺ could not be replaced by Cu²⁺, Ca²⁺, Ba²⁺, Co²⁺ or Ni²⁺. Sulfhydryl reagents inactivated both FBPase-I and FBPase-II. Of the metabolites, only 6-phosphogluconate was inhibitory with 50% inhibition at 2 and 4 m M for FBPase-I and FBPase-II, respectively.     

Failure to corroborate claims that the developing endosperm of wheat (Triticum aestivum) contains significant activities of fructose-1,6-bisphosphatase

This work was done to test claims (Sangwan and Singh, Physiol. Plant. 73: 21–26) that the developing endosperm of wheat ( Triticum aestivum L.) contains a cytosolic and a plastidic fructose- 1,6-bisphosphatase (EC 3.1.3.11; FBPase). Repetition of the procedure of Sangwan and Singh with extracts of developing endosperm of Triticum aestivum cv. Mercia produced two peaks of apparent FBPase activity on elution from DEAE-cellulose. Both peaks showed high activity of pyrophosphate:fructose-6-phos-phate 1-phosphotransferase [EC 2.7.1.90; PFK(PP_i)]. The apparent FBPase activity in both peaks was stimulated by 20 μ M fructose-2,6-bisphosphate and inhibited by antibodies to PFK(PP_i). Antibody to plastidic FBPase did not react positively in an immunoblot analysis with any protein of M_r comparable to that of known FBPase in either peak. It is argued that the ability of each peak to convert fructose-1,6-bisphosphate to fructose-6-phosphate was due to PFK(PP_i). and that there remains no substantiated evidence for the presence of a plastidic FBPase in the developing endosperm of wheat.     

Fructan contents and dry matter deposition in different tissues of the wheat grain during development

The role of fructan metabolism in the assimilate relations of the grain of wheat (Triticum aestivum L.) was investigated by determination of the dry matter and fructan content of grain components at short intervals during grain filling. During the initial phase of rapid expansion, most of the assimilates entering the grain were partitioned to the outer pericarp. A large fraction of these assimilates were used for the synthesis of fructan. Dry matter deposition and fructan synthesis in the outer pericarp ceased at about 5d after anthesis. At the same time, the endosperm and the inner pericarp and testa started to accumulate dry matter at a fast rate. This was also associated with significant fructan synthesis in the latter tissues. The outer pericarp lost about 45% of its former maximum dry weight between 9 and 19 d after anthesis. This loss was due almost entirely to the near complete disappearance of water-soluble carbohydrates, most of which was fructan. The inner pericarp and testa accumulated dry matter until about mid-grain filling. The fructan contents of the inner pericarp and testa and the endosperm decreased slowly towards the end of grain filling. Most of the fructans in the inner pericarp and testa and the endosperm had a low molecular weight, whereas higher molecular weight fructans predominated in the outer pericarp. The embryo did not contain fructan. The presence of low molecular weight fructans in the endosperm cavity at mid-grain filling was confirmed. It is suggested that fructan synthesis is closely linked to growth-related water deposition in the different tissues of the wheat grain and serves to sequester the surplus of imported sucrose.     

BiP, HSP70, NDK and PDI in wheat endosperm. I. Accumulation of mRNA and protein during grain development

Biosynthesis and accumulation of seed storage proteins such as the wheat glutens depend on the activity of a variety of other proteins, including chaperones and foldases. cDNA probes and antibodies to two chaperone proteins and a foldase were used to follow mRNA and protein accumulation in developing grains of wheat ( Triticum aestivum , cvs Cheyenne and Butte). Endosperm was separated from other grain components and protein accumulation was analyzed on a per mg fresh weight basis. The ER resident chaperone BiP (binding protein) and foldase PDI (protein disulfide isomerase) accumulated to maximal levels in the middle stage of endosperm development, a period of rapid cell expansion and storage protein accumulation, whereas levels of a cytosolic chaperone, HSP70, remained relatively constant throughout grain development. In contrast, nucleoside diphosphate kinase (NDK), a cytosolic enzyme needed for synthesis of nucleoside triphosphates, accumulated early in endosperm development during the period of nuclear division and cell formation. When analyzed as a fraction of total protein the relative abundance of all four proteins peaked early in grain development and then declined. Accumulation of mRNA for the four proteins also peaked early in grain development. Although BiP and PDI formed a declining percentage of total protein as storage protein accumulated, their pattern of accumulation was compatible with a proposed role as catalysts for storage protein folding and accumulation in the ER.     

Analysis of programmed cell death in wheat endosperm reveals differences in endosperm development between cereals

Although maize endosperm undergoes programmed cell death during its development, it is not known whether this developmental feature is common to cereals or whether it arose inadvertently from the selection process that resulted in the enlarged endosperm of modern maize. Examination of wheat endosperm during its development revealed that this tissue undergoes a programmed cell death that shares features with the maize program but differs in some aspects of its execution. Cell death initiated and progressed stochastically in wheat endosperm in contrast to maize where cell death initiates within the upper central endosperm and expands outward. After a peak of ethylene production during early development, wheat endosperm DNA underwent internucleosomal fragmentation that was detectable from mid to late development. The developmental onset and progression of DNA degradation was regulated by the level of ethylene production and perception. These observations suggest that programmed cell death of the endosperm and regulation of this program by ethylene is not unique to maize but that differences in the execution of the program appear to exist among cereals.     

Assessment of salinity indices to identify Iranian wheat varieties using an artificial neural network

In arid and semi‐arid regions of the world, including Iran, soil salinity is one of the major abiotic stresses. One of the ways to achieve high performance in these areas is to use salt‐tolerant varieties of wheat. Iran is known as one of the places where the D‐genome originated and evolved. In order to evaluate the salt tolerance of Iranian genotypes based on the eight indices using analysis of variance, regression and an artificial neural network (ANN), 41 Iranian wheat varieties (Trticum aestivum L.) were planted in a randomised complete block design with three replications under two saline irrigation conditions, 0.631 and 11.8 dS m^?1, in Kerman, Iran. Significant differences between the varieties were observed, and the significant two‐way interaction of environment × varieties in combined analysis and non‐significant correlation, 0.07, between the yield in two environments (yield in non‐stress conditions, Yp, and yield in stress conditions, Ys) indicates the existence of genetic variation among varieties and the different responses of the varieties in both the environments. The indices of tolerance, geometric mean product (GMP), mean product (MP), harmonic mean (HM) and stress tolerance index (STI) were calculated based on grain yield evidence of positive significant correlation with Yp and Ys. Based on the ANN results, yield stability index (YSI), MP, GMP and STI were the best indices to predict salinity‐tolerant varieties. The varieties selected based on these indices, such as Bolani, Sistan, Ofogh, Pishtaz, Karchia and Arg, produced high yield in both the environments. These results show that bread wheat originating from Iran has salt tolerance potential and can also be used in studies related to salinity tolerance mechanisms.     

Synthesis and degradation of proteins during wheat endosperm development

Synthesis of proteins rich in lysine declines progressively with endosperm development and these proteins appear to be degraded preferentially at later stages. The proteolytic enzymes in extracts of endosperms at a late stage of development release considerably more lysine radioactivity from labelled endosperm proteins as compared with the enzymes in endosperms at an early stage.     

BiP, HSP70, NDK and PDI in wheat endosperm. II. Effects of high temperature on protein and mRNA accumulation

The effects of high temperature on accumulation of the 70‐kDa heat shock protein (HSP70) and nucleoside diphosphate kinase (NDK) as well as two other proteins that have roles in the biosynthesis of storage proteins were examined during grain development. An HSP70 homolog and a 17‐kDa NDK were co‐purified from wheat endosperm, their identity verified, and a cDNA for an HSP70 expressed in endosperm was isolated. Wheat plants ( Triticum aestivum , cvs Butte and Vulcan) were heat shocked at 40°C or exposed to maximum daily temperatures of 37 or 40°C during early or mid‐grain fill. Antibodies and cDNA probes for BiP, HSP70, NDK and PDI were used to examine the effect of high temperatures on the accumulation of protein and mRNA in the endosperm. HSP70 mRNA levels increased substantially when plants were exposed to heat shock or to a 1‐day gradual increase to 40°C. The effects of a 5‐day heat treatment on mRNA levels were more complicated and depended on the developmental stage of the grain. A treatment that began at 7 days post‐anthesis (DPA) decreased the level of mRNA for HSP70, BiP, PDI and NDK, whereas a treatment that began at 14 DPA slightly increased mRNA levels. The same treatments increased the accumulation of HSP70 but did not affect BiP, PDI, or NDK protein levels. This is the first detailed report on the effects of heat on mRNA and protein levels for HSP70 in a developing seed storage tissue.     

The influence of reduced photorespiration on long-term growth and development of wheat

The long-term role of photorespiration was investigated by comparing growth, development, gas exchange characteristics and mineral nutrition of a wheat crop ( Triticum aestivum L. cv. Courtot) cultivated in a culture chamber during a life cycle, either in 4% O₂ or in normal O₂ Low O₂ pressure reduced photorespiration, but CO₂ was controlled so that net photosynthesis remained the same as in the control crop. The growth and development of the low O₂ crop was slowed down. Ear appearance was 16 days late, but the rate of tillering was the same as in the control and was maintained longer so that the final number of tillers was doubled. Pigment, ribulose bisphosphate carboxylase (EC 4.1.1.39) and soluble sugar contents were similar. The response of photosynthesis to CO₂ and O₂ was not appreciably changed by the low O₂ treatment. There was almost no seed formation, and the senescence of the leaves was delayed. It appears that in non-stress conditions most of the photorespiration can be suppressed without damage to the photosynthetic apparatus. Retardation of development and inhibition of reproduction are likely due to other effects of O₂.     

Endogenous abscisic acid and wheat germ agglutinin content in wheat grains during development

Abscisic acid (ABA) and wheat germ agglutinin content of immature wheat grains and embryos was determined by immunoassay throughout the development of a field-grown wheat crop ( Triticum aestivum cv. Timmo). Wheat germ agglutinin accumulation in the embryo was not preceded by an increase in endogenous abscisic acid amount or concentration in either embryos or grains. At a later stage in development the endogenous concentration of abscisic acid in both embryos and grains was found to be two orders of magnitude lower than the endogenous levels required to inhibit precocious germination and promote wheat germ agglutinin accumulation in excised embryos cultured in vitro. These findings are discussed in the context of the control of embryo development in vivo by both ABA and the water status of the grain and embryo.     

Postanthesis temperatures influence cytokinin accumulation and wheat kernel weight

High temperatures during reproductive development alter kernel development and reduce yield and quality in wheat ( Triticum aestivum L.). Understanding how temperature alters kernel formation will help the development of genetic approaches to enhance heat tolerance in this cereal. A relationship between kernel development and postanthesis cytokinin accumulation is well documented, but the effect of temperature on this relationship is not known. This study quantified the effects of a postanthesis temperature treatment (7 d at 35/25 °C day/night) on kernel development and cytokinin accumulation in a soft white winter wheat (c.v. Stephens). Kernels from control plants maintained at 25/15 °C accumulated zeatin, dihydrozeatin and their corresponding 9-ribosides from 1 to 4 d postanthesis. Postanthesis kernel cytokinin accumulation was reduced by 50–80% by the temperature treatment. Kernel cytokinin content in control plants declined to baseline by 5 d postanthesis and remained at that level. Kernels from treated plants had a secondary peak of cytokinin accumulation 6–8 d after anthesis. This treatment significantly reduced kernel weight. The magnitude of the effect on kernel number was smaller than on kernel weight, but was statistically significant. Reduced kernel weight was accompanied by reduced cytokinin accumulation. Exogenous cytokinins did not mitigate the temperature effects on kernel weight, but at the highest concentrations, did reduce the effect on kernel number.