Sulphur nutrition affects delivery and metabolism of S in developing endosperms of wheat.

Experiments were conducted to investigate the effect of S nutrition and availability on the forms of S and N in the endosperm cavity and endosperm of wheat, and on the capacity of the endosperm to utilize those compounds for the synthesis of proteins. Plants were grown in solution culture with 2 mM N and either 200 microM S (high-S) or 50 microM S (low-S) and all nutrients were withdrawn at various times from booting until 8 d post-anthesis. Sulphate was the major form of soluble S in the endosperm cavity and endosperm of high-S plants during the time of rapid grain development. By contrast, glutathione (GSH) was the major form of soluble S in the endosperm cavity and in the endosperm in low-S plants. Crude extracts of endosperm tissue from both high-S and low-S plants supported (i) the hydrolysis of GSH to gamma-glutamyl cysteine and glycine, and of gamma-glutamyl cysteine to glutamate and cysteine, and (ii) sulphate-dependent PPi-ATP exchange and the sulphydration of O-acetylserine catalysed by ATP sulphurylase and cysteine synthase, respectively. High-S nutrition enhanced the in vitro rates of ATP sulphurylase and cysteine synthase.     

Transport of zinc and manganese to developing wheat grains

An understanding of the transport pathway used by Zn and Mn to enter developing grains may allow measures to increase the Zn and Mn content of wheat grain grown on Zn/Mn deficient soils. For this reason, transport of Zn and Mn into developing grains of wheat ( Triticum aestivum L. cv. Aroona) was investigated. Detached ears (18–22 days post-anthesis) were cultured for 48 h in a solution containing 185 kBq of ⁶⁵Zn and 185 kBq of ⁵⁴Mn. Transport of ⁶⁵Zn to the grain was unaffected by removal of glumes but was slightly reduced after the lemma was removed. Heat girdling the peduncle slightly reduced the amount of ⁶⁵Zn transported to the grain, whilst heat girdling the rachilla reduced transport of ⁶⁵Zn to the grain to a greater degree, suggesting phloem transport to the rachilla. The transport inhibitor CCCP (carbonyl cyanide m -chlorophenyl hydrazone) blocked ⁶⁵Zn transport to grain but not to lemma and glumes. Removing glumes and lemma and heat girdling the peduncle did not affect transport of ⁵⁴Mn, but transport was slightly affected by heat girdling the rachilla, indicating xylem transport. CCCP blocked transport of ⁵⁴Mn into the grain but not to lemma and glumes. It was concluded that xylem-to-phloem transfer of Zn occurs in the rachis and to a lesser extent in peduncle and lemma. The results suggest that the lemma may be an important site for phloem loading when the concentration of Zn within the xylem is high. The data also suggest that Mn was predominantly translocated to the spikelets in the xylem, but that transport to the grain was dependent upon membrane transport before entering the grain. Phloem loading of Mn into the grain vascular system may have occurred at the site of xylem discontinuity in the floral axis.     

Manipulation of xylem transport affects Zn and Mn transport into developing wheat grains of cultured ears

Zinc and manganese loading into developing wheat grain is little understood at present. The objective of this work was to investigate factors that may affect the rate of transport of Zn and Mn into developing wheat grain of cultured ears. Ears 18-22 days post-anthesis were cultured in solutions containing labelled Zn and Mn. The effect of additions of Cu, Fe, citrate, malate and EDTA to the culture solution was observed. The effect of humidity and awn removal as well as the sucrose status of the ears on Zn and Mn transport was also investigated. The effect of high concentration of Zn and Mn on [¹⁴C]-sucrose transport was determined. High humidity almost completely blocked transport of Zn and Mn into the grain. Awn removal reduced the transport of Zn and Mn to the lemma but not the grain. When the ears were depleted of sucrose (by maintaining them in the dark prior to labelling) transport of Zn and Mn to the grain was reduced compared to ears cultured with sucrose. The presence of Cu reduced the loading of Zn into the grain. There was little effect of Cu on Mn transport or Fe on either Zn or Mn transport. High concentrations of Zn and Mn in the culture solution did not affect [¹⁴C]-sucrose loading into the grain but loading of Zn and Mn was limited at high concentrations suggesting membrane saturation. This study demonstrates that sucrose status and humidity clearly influence the transport of Zn and Mn into the grain, and that other ions may influence Zn and Mn transport.     

The induction of sensitivity to gibberellin in aleurone tissue of developing wheat grains

A method for isolating viable protoplasts in high yield from the aleurone layers of developing wheat grains is described, and the techniques for their subsequent culture outlined. Protoplasts from untreated tissue do not produce α-amylase in response to gibberellic acid (GA₃) if the incubation temperature is left at 25°C. However, pre-treatment of the protoplast preparation at temperatures above 27°C for at least 8 h followed by a short incubation at 25°C induces sensitivity to the growth regulator such that α-amylase is produced. The requirements of the sensitisation process are similar to those for intact aleurone tissue although additional adjustment to the calcium ion is beneficial. Pre-treatment of aleurone layers with the sensitising temperature regimes prior to protoplast isolation have the advantage of increasing protoplast viability. Once sensitised, the protoplasts respond to a GA₃ concentration as low as 10^-11 mol dm^-3 with a maximal response at 10^-9 mol dm^-3. The successful isolation of wheat aleurone protoplasts whose sensitivity to GA₃ can be manipulated represents a useful step towards investigating the role of cell membranes in growth-regulator action.     

The induction of sensitivity to gibberellin in aleurone tissue of developing wheat grains

Aleurone tissue from undried immature developing wheat grains (Triticum aestivum L. cv. Sappo), normally insensitive to gibberellic acid, can be made to respond to the hormone by a series of temperature treatments. Incubation of the de-embryoed grains at temperatures above 27° C for at least 8 h causes the tissue to become sensitive. Prolonged incubation at temperatures below 27° C does not effect a change in sensitivity. In addition to the requirement for exposure to an elevated temperature for a period of several hours the tissue must also subsequently be subjected to a period at a lower temperature for just a few seconds for the response to be observed. Once sensitized, the tissue remains responsive to gibberellic acid for substantial periods of time. Exposure of the tissue to temperatures which induce sensitivity to gibberellic acid also results in an increased leakage of amino acids. It is suggested that the increase in sensitivity to gibberellin requires two separate processes to take place. One could be a homeoviscous adaptation of the cell membranes in response to elevated temperature, the other a subsequent, permanent change in conformation of membrane components.     

Properties of NADP-malic enzyme from glumes of developing wheat grains

Properties of partially purified NADP-malic enzyme (EC 1.1.1.40) from glumes of developing wheat grains were examined. The pH optimum for enzyme activity was influenced by malate and shifted from 7.3 to 7.6 when the concentration of malate was increased from 2 to 10 mM. The K_m values, at pH 7.3, for various substrates were: malate, 0.76 mM; NADP, 20 μM and Mn²⁺, 0.06 mM. The requirement of Mn²⁺ cation for enzyme activity could be partially replaced by Mg²⁺ or Co²⁺. Mn²⁺ dependent enzyme activity was inhibited by Pb²⁺, Ni²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Al³⁺ and Fe³⁺. During the reaction, substrate molecules (malate and NADP) reacted with enzyme sequentially. Activity of malic enzyme was inhibited by products of the reaction viz pyruvate, HCO₃^? and NADPH₂. At a limiting fixed concentration of NADP, these products induced a positive cooperative response to increasing concentrations of malate.     

Fluoride-induced alteration of carbon and nitrogen metabolism in developing wheat grains

Effect of fluoride (10 and 50 mM) on the activities of sucrose metabolizing enzymes, alkaline inorganic pyrophosphatase, and transaminases in relation to the accumulation of free sugars, starch, and soluble protein was studied in detached ears of wheat cultured in a liquid medium. Culturing for 5 d in the presence of fluoride reduced the amount of grain starch whereas contents of total free sugars, particularly sucrose, and soluble protein increased. Fluoride inhibited the activities of soluble acid and neutral invertases, as well as sucrose synthase acting in the cleavage direction. Uptake of uniformly labelled ¹⁴C-sucrose or fructose was also drastically reduced by fluoride. Glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities also increased with fluoride addition in correspondence with an increase in soluble protein. Apparently, the wheat grain responds to fluoride-mediated disruption of carbon metabolism by a compensatory effect on nitrogen metabolism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Abscisic acid in developing wheat grains and its relationship to grain growth and maturation

Summary During the later stages of growth of grains of wheat (Triticum aestivum L. cvs. WW15 and Gabo) there is a dramatic increase (up to 40fold) in the content of abscisic acid (ABA) to 4–6 ng per grain. This level remains high from 25 to 40 days after anthesis. Then, in association with natural or forced drying of the grain, there is a rapid drop (5–10 fold) in the ABA content and a brief increase in the content of bound ABA. The bulk of ABA in an ear was in the grain (95%) and although the embryo contributed 19% of this ABA it was less than 5% of the grain by weight. There was no clear relationship between ABA content and the growth of grains in various spikelet or floret positions. Application of (±)-ABA to the ear had no effect on grain growth rate but led to an earlier cessation of grain growth and hastened the drying of the grain. Isolated embryos and whole grains were capable of germinating during the mid grain growth period (15–25 days), but germination capacity declined subsequently as ABA accumulated. Later, still, with grain drying and loss of ABA, embryo and grain became germinable again. At this time there was also a dramatic increase in the ability of the grain to synthesize -amylase. It is suggested that the accumulation of ABA at the later stages of grain growth prevents precocious germination and premature hydrolysis of starch reserves of the morphologically mature but still unripe grain. An inevitable consequence of such action may be in triggering grain maturation.     

Starch synthase activity and heat shock protein in relation to thermal tolerance of developing wheat grains

Wheat (Triticum aestivum L.) cvs. HD 2285 (relatively tolerant) and WH 542 (susceptible) were exposed to ambient and elevated temperature (3–4 °C higher) in open top chambers during post anthesis period. The grain yield components were determined at the time of maturity. In order to elucidate the basis of differential tolerance of these cultivars, the excised developing grains (20 d after anthesis) of ambient grown plants were incubated at 15, 25, 35 and 45 °C for 2 h and then analysed for the activities of soluble starch synthase (SSS), granule bound starch synthase (GBSS), kinetic parameters of SSS and content of heat shock protein (HSP 100). The elevated temperature during grain development significantly decreased grain growth in WH 542 whereas no such decrease was observed in HD 2285. High temperature tolerance of HD 2285 was found to be associated with higher catalytic efficiency (V_max/K_m) of SSS at elevated temperature and higher content of HSP 100.     

Peroxidase and amylase activity in developing grains of triticale,wheat and rye

Changes in the activities of peroxidase and amylase were measured during the development of grain of triticale, wheat and rye. Peroxidase and amylase activities were found to be higher in Triticale-1 which possesses highly shrivelled grains. A direct relationship between the degree to which the grain is shrivelled and the activity of peroxidase and enzymes was observed. During grain development, peroxidase and amylase activity per grain increased in Triticale-1, while it decreased in wheat, rye and well filled triticale grains.     

不同pH模拟酸雨对冬小麦籽粒营养品质的影响

在大田试验条件下研究了不同pH模拟酸雨对冬小麦籽粒氨基酸、蛋白质、可溶性糖、还原糖、淀粉以及总酸度的胁迫效应,以期为防治我国农作物的酸雨灾害提供基础理论依据。结果表明,pH≤4.5酸雨处理会抑制总游离氨基酸的合成,且酸度越强,抑制越明显。蛋白质(可溶性蛋白质、总蛋白)含量随着酸雨酸度的增强逐步下降,且在极酸性条件下降低较明显。pH≤4.5酸雨处理对可溶性糖的合成会有较强的抑制作用。pH≤2.5时还原糖的合成明显受抑。随着酸雨酸度的增强,总酸度和酸糖比均表现出先增加后下降的变化趋势,且在pH=3.5处达到最大值。pH≤3.5酸雨处理显著抑制淀粉的合成,且对支链淀粉的影响较明显,致使淀粉支/直比下降。     

Differential transport of Zn, Me and sucrose along the longitudinal axis of developing wheat grains

The hypothesis that Zn and Mn are transported within the grain in a similar manner to sucrose was investigated in the developing wheat grain. Detached ears were cultured in solution containing ⁶⁵Zn, ⁵⁴Mn and [¹⁴C]-sucrose for 10 to 120 min at 18–22 days post-anthesis. At different times the grain was cut transversely into 1-mm sections and the radioactivity in each section determined The embryo region was damaged in some grains to investigate the effect of reduced accumulation rate on the transport of ⁶⁵Za, ⁵⁴Mn and [¹⁴C]-sucrose to the embryo. The distribution of ⁶⁵Zn. ⁵⁴Mn and [¹⁴C]-sucrose between the endosperm cavity sap. endosperm, embryo and pericarp in grains labelled for 2.5 and 6 h at 18–22 days post-anthesis was also determined. [¹⁴C]-su-crose was initially high in the first, embryo-containing section of the grain but decreased progressively to the distal end of the grain. The amount of ⁶⁵Zn along the longitudinal axis of the grain was distributed evenly in each 1-mm section, whilst ⁵⁴Mn accumulated exponentially in the first proximal 1-mm section of the grain and was distributed evenly in the remaining sections. Damaging the embryo had no effect on ⁶⁵Zn and ⁵⁴Mn transport to the section containing the embryo. The pericarp contained almost all of the grain ⁶⁵Za and ⁵⁴Mn, with small amounts found in the embryo, endosperm and endosperm cavity sap. Increasing amounts of [¹⁴C]-sucrose were found in the endosperm as time progressed. The rate of accumulation of ⁶⁵Zn, ⁵⁴Mn and [¹⁴C]-sucrose was much higher in the embiyo than the endosperm: the difference between the embryo and endosperm was especially large for ⁶⁵Zn and ⁵⁴Mn. It is suggested that ⁶⁵Zn and ⁵⁴Mn are not transported within the grain in the same way as [¹⁴C]-sucrose. [¹⁴C]-sucrose moves laterally out of the vascular system of the crease into the endosperm cavity and is subsequently taken up and stored in the endosperm. In contrast, ⁶⁵Zn and ⁵⁴Mn appear to be retained within the vascular system of the crease and may be transported more slowly to grain parts such as the embryo and pericarp tissue.     

Abscisic acid in developing grains of wheat and barley genotypes differing in grain weight

Two genetically related wheat lines growing in cabinets were given different temperatures during grain filling, and abscisic acid (ABA) was measured in whole grains by gas chromatography with an electron-capture detector. Three genetically related barley lines grown in the field were assayed for ABA content in endosperm and embryo fractions separately by radiommunoassay.Maximum grain growth rate and final weight per grain of the two wheat lines differed by 50–60% at low temperature and 30–40% at high temperature. During grain development two peaks in ABA level were observed at low temperature but only one at high temperature. At times when differences in grain growth rate between genotypes and between temperature treatments were large, the corresponding differences in ABA concentration were small. In barley, one line (Iabo 14) had 30% heavier grains than the other two (Onice and Opale). Endosperm ABA concentrations showed no clear differences between genotypes until grain filling was nearly complete. Embryo ABA levels were up to 10-times greater than those in the endosperm, with Opale having significantly less ABA in the embryo than the other two cultivars.Our experiments did not provide evidence for a causal relationship between ABA levels during grain filling and grain growth rate or final weight.Abbreviations ABA Abscisic acid - DAA days after anthesis - DW dry weight - FW fresh weight     

Environmentally-induced changes in protein composition in developing grains of wheat are related to changes in total protein content

Nitrogen (N) nutrition, post-anthesis temperature and drought-induced changes in the kinetics of accumulation of dry mass, total grain N and protein fractions (albumins-globulins, amphiphils, gliadins, and glutenins) contents were examined for winter wheat (Triticum aestivum L.). Crops were grown in controlled environment tunnels in 1994 and 1998. In 1994, five post-anthesis temperatures averaging from 15-25 degrees C were applied during grain-filling. In 1998 two post-anthesis temperatures averaging 13 degrees C and 20 degrees C were applied and factorized with two post-anthesis water regimes. In 1994 crops also were grown in the field, where different application rates and timing of N nutrition were tested. When expressed in thermal time, the kinetics of accumulation of the protein fractions were not significantly affected by post-anthesis temperature or drought; whereas N nutrition significantly increased the rate and duration of accumulation of storage proteins. Albumin-globulin proteins accumulated during the early stage of grain development. The rate of accumulation of that fraction decreased significantly at c. 250 degrees Cd after anthesis, when the storage proteins (gliadins and glutenins) started to accumulate significantly. Single allometric relationships for the different environmental conditions exist between the quantity of each protein fraction and the total quantity of N per grain. From these results it was concluded that (1) the process of N partitioning is neither significantly affected by post-anthesis temperature or drought nor by the rate and timing of N nutrition and (2) at maturity, variations in protein fraction composition are mainly because of differences in the total quantity of N accumulated during grain-filling.     

Temporal and spatial changes in cell wall composition in developing grains of wheat cv. Hereward

A combination of enzyme mapping, FT-IR microscopy and NMR spectroscopy was used to study temporal and spatial aspects of endosperm cell wall synthesis and deposition in developing grain of bread wheat cv. Hereward. This confirmed previous reports that changes in the proportions of the two major groups of cell wall polysaccharides occur, with β-glucan accumulating earlier in development than arabinoxylan. Changes in the structure of the arabinoxylan occurred, with decreased proportions of disubstituted xylose residues and increased proportions of monosubstituted xylose residues. These are likely to result, at least in part, from arabinoxylan restructuring catalysed by enzymes such as arabinoxylan arabinofurano hydrolase and lead to changes in cell wall mechanical properties which may be required to withstand stresses during grain maturation and desiccation.     

Asparagine metabolism-key to the nitrogen nutrition of developing legume seeds

Asparagine accounted for 50 to 70% of the nitrogen carried in translocatory channels serving fruit and seed of white lupin (Lupinus albus L.). Rates of supply of the amide always greatly exceeded its incorporation as such into protein. An asparaginase (l-asparagine amido hydrolase EC 3.5.1.1) was demonstrated in crude extracts of seeds. In vitro activity was up to 5 mumoles of aspartate formed per hour per gram fresh weight at the apparent Km(Asn) value of 10 mM, and this more than accounted for the estimated rates of asparagine utilization in vivo. Asparaginase activity per seed increased 10-fold in the period 5 to 7 weeks after anthesis, coinciding with early stages of storage protein synthesis in the cotyledons.Double labeled ((14)C (U), (15)N (amide)) asparagine was fed to fruiting shoots through the transpiration steram. Fruit phloem sap analysis indicated that virtually all of the label was translocated to seeds in the form of asparagine. In young seeds (15)N from asparagine breakdown was traced to the ammonia, glutamine, and alanine of endospermic fluid, the (14)C appearing mainly in nonamino compounds. In the cotyledon-filling stage the C and N of asparagine was contributed to a variety of amino acid residues of protein.     

Long-term in vitro culture of wheat grains

Wheat (Triticum aestivum, Triticum durum) grains were excised immediately following fertilization and cultured until maturity. A rachis fragment attached to the grain was required to ensure an increase in grain size for the first 10 days following fertilization. A ¹⁴C-labeling study revealed that 8-day-old grains accumulated more dry matter into the ethanol-insoluble fraction when grown on agar rather than when immersed in liquid medium. Light enhanced the absorption of sucrose from the medium only in the latter case. In agar-based culture, when no contact was made between the grain surface and the medium, peeling off the outer pericarp layers increased sugar absorption, leading to a threefold increase in the amount of accumulated dry matter in the ethanol-insoluble fraction. Culturing of wheat grains with attached rachis fragment and peeled pericarp is recommended for maximum in vitro growth.