Accumulation and conversion of sugars by developing wheat grains. 3. Non-diffusional uptake of sucrose, the substrate preferred by endosperm slices

Abstract. Starch synthesis by developing wheat endosperm slices incubated in liquid media was more rapid, at optimum concentration, from sucrose as external substrate than from glucose and/or fructose. Fructose inhibited conversion of sucrose or glucose. The results are consistent with the hypothesis that sucrose is not hydrolysed in the apoplast before uptake.
Besides a diffusional influx and efflux of labelled sucrose there was a non-diffusional influx; it was inhibited by dinitrophenol, potassium arsenate, potassium iodide, and parachloromercuribenzene sulphonate (PCMBS). PCMBS inhibited both uptake and conversion of label from 150 molm⁻³¹⁴C-sucrose by 75%. Uptake and conversion of sucrose were stimulated by lowering pH and by fusicoccin, a promoter of proton extrusion.
Extracellular solutes like raffinosc and polyethylene glycol stimulated net uptake of label from ¹⁴C-sucrose — the larger molecule being more effective — this being due to a non-specific inhibition of diffusional efflux. At too high an osmotic concentration such solutes reduced net uptake; the larger the molecule the lower this transitional concentration.
In conclusion, wheat endosperm is better equipped to convert apoplastic sucrose rather than the hydrolysis products to starch; active loading of sucrose possibly involves proton co-transport; and large molecules in the extracellular solution reduce the diffusional elllux of loaded substrate.     

Accumulation of label from radioactive precursors into dry matter by wheat endosperm cultured in vitro

The capacity of in vitro cultured common wheat ( Triticum aestivum L.) endosperms to incorporate starch and protein precursors was investigated. Isolated 2–3 week-old endosperms were cultured up to 2 weeks in a liquid medium containing labelled (¹⁴C)-sucrose and (³H)-glutamine. Cultured endosperms were separated into ethanolsoluble, starch and protein fractions and the incorporation of the label into each of these fractions was assessed at different times after commencement of culture. The same medium was introduced through the peduncle into normally-developing grains, which were then similarly analyzed. Accumulation of both ¹⁴C and ³H in the ethanol-soluble fraction occurred, at a decreasing rate, only during the first 3 days, and then ceased. The accumulated label in the starch fraction, which originated mainly as ¹⁴C sucrose, proceeded at a relatively constant rate for one week and reached only about 1/5 of the expected in vivo starch production. Incorporation of both isotopes into the protein fraction reflected similar utilization of sucrose and glutamine from the medium (molar base), decreasing in rate with time. Culturing beyond one week produced deteriorated endosperms. Compared to cultured endosperms, normally-developing grains incorporated proportionally less precursors into the ethanol-soluble and more into the insoluble fraction. It is suggested that the reduced starch and protein synthesis in cultured grains stems from impaired capacity of the biosynthetic machinery rather than from low availability of precursors.     

Abscisic acid induced stress-like polyamine pattern in wheat seedlings, and its reversal by potassium ions

In 3-day-old wheat ( Triticum aestivum L. cv. Marinat) seedlings, 100 μ M ABA blocked the growth and altered the level of K⁺ in both the shoot and root. The presence of ABA increased the putrescine titer during a 24-h treatment. Increasing the endogenous level of K⁺ by the addition of 10 m M KCl to the ABA-treated seedlings, inhibited the effect of ABA on growth and putrescine level. In both tissues, ABA increased putrescine content at low concentrations (1 μ M ), reaching the maximal effect at 100 μ M . Putrescine increase induced by ABA was inhibited by both α-difluoromethylarginine (DFMA) and α-difluoromethylornithine (DFMO) in shoots while only the inhibitor of arginine decarboxylase was effective in the root. The presence of ABA modulated, in opposite ways, ornithine and arginine decarboxylase activities. These results are discussed in relation to ion balance under stress.     

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.     

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.     

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.     

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.     

两个小麦磷转运蛋白基因的分离、功能鉴定和表达研究

磷是能量代谢、核酸以及许多生物膜合成的重要底物。在光合作用、呼吸作用等过程中发挥了重要作用。中国大多数小麦产区的土壤存在着缺磷的问题。磷饥饿给小麦生产造成了很大损失。培育耐低磷小麦是解决这一问题的一个重要途径。在磷饥饿的过程中,哪些基因的表达发生了变化．它们是如何变化的,弄清楚这些问题对于培育转基因耐低磷小麦具有重要的意义。磷转运蛋白基因在植物吸收磷的过程中发挥着重要作用。利用RT—PCR的方法,我们从普通小麦“小偃54”中分离了两个磷转运蛋白基因TaPT8和TaPHT2;1。通过与酵母突变体互补分析表明这两个基因都能够与磷吸收功能存在缺陷的酵母突变体实现功能互补,在低磷条件下有促进酵母突变体吸收磷的作用。进一步分析表明TaPT8属于Pht1家族。TaPHT2;1属于Pht2家族。运用RQRT—PCR的方法进行分析后发现TaPT8在根中表达,受磷饥饿的诱导;TaPHT2;1主要在绿色组织中表达,受磷饥饿的抑制,受光的诱导。TaPT8可能主要参与了小麦的根从土壤中吸收磷的过程。TaPHT2;1可能在磷从细胞质向叶绿体内转运的过程中发挥了重要作用。     

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.     

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.     

Enzymes of glutamine metabolism in testa-pericarp and endosperm of developing wheat grain

Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate puruvate transaminase and glutamate oxaloacetate transaminase have been assayed in developing testa-pericarp and endosperm of two wheat varieties, namely Shera (11.6% protein) and C-306 (9.8% protein). On per organ basis, activities of all the enzymes studied, except glutamine synthetase, increased during development. Glutamine synthetase activity decreased during development in the testa-pericarp, whereas, no glutamine synthetase activity could be detected in endosperm of either variety at any stage of development. Compared to testa-pericarp, endosperm had higher activities of glutamate synthase and glutamate pyruvate transaminase. On the whole, enzyme activities in Shera were higher, as compared to C-306. Developmental patterns and relative levels of enzyme activities in the two varieties were more or less the same, when expressed on dry weight basis or as specific activities. The results suggest that ammonia assimilation in developing wheat grain takes place by the glutamate dehydrogenase pathway in the endosperm; and both by the glutamate dehydrogenase and glutamine synthetase—glutamate synthase pathways in the testa-pericarp.     

不同供水条件对小麦强、弱势籽粒中淀粉粒度分布的影响

以3个淀粉含量不同的冬小麦品种山农12、鲁麦21和济南17为材料,设灌溉和旱作2种栽培处理,对不同水分条件下小麦强、弱势籽粒中淀粉粒的体积、数目和表面积的分布特征进行了研究.结果表明,小麦强、弱势籽粒均含有A(＞9.8 μm)、B(2.0～9.8 μm)、C(＜2.0 μm)3种类型的淀粉粒,但不同类型淀粉粒的分布状况存在明显差异.在强势籽粒中,淀粉粒的体积和表面积分布均表现为三峰分布,而弱势籽粒中淀粉粒的体积和表面积分布则表现为双峰分布.与弱势粒相比较,强势粒中C型淀粉粒(＜2.0 μm)的体积百分比为7.25%～9.31%,表面积百分比为34.88%～41.51%,而弱势粒的体积和表面积百分比分别为5.33%～6.40%和26.31%～33.54%.强、弱势籽粒中＜0.6 μm和0.6～2.0 μm范围内的淀粉粒数目存在明显差异,强势粒为1.86%～6.13%和83.77%～87.77%,而弱势粒为25.72%～37.42%和52.77%～58.48%.与灌溉栽培相比较,旱作栽培条件下籽粒中B、C型淀粉粒体积和表面积百分比显著增加,而A型淀粉粒体积和表面积显著减少;弱势粒中＜0.6 μm的淀粉粒数目显著增加,强势籽粒中淀粉粒的数目无显著变化.与弱势粒相比较,强势粒中的蛋白质含量较高,C型淀粉粒的体积和表面积所占比例较大,而强势粒中的淀粉含量较低,且A、B型淀粉粒比例也较小.与灌溉栽培相比较,旱作栽培条件下强、弱势籽粒中B、C型淀粉粒体积和表面积百分比增加,蛋白质含量也显著增加,淀粉含量降低.表明水分亏缺能提高籽粒中B、C型淀粉粒体积和表面积百分比及蛋白质含量.     

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.     

The cellular pathway of photosynthate transfer in the developing wheat grain. III. A structural analysis and physiological studies of the pathway from the endosperm cavity to the starchy endosperm

The cellular pathway of sucrose transfer from the endosperm cavity to the starchy endosperm of developing grains of wheat (Triticum turgidum) has been elucidated. The modified aleurone and sub-aleurone cells exhibit a dense cytoplasm enriched in mitochondria and endoplasmic relicilium. Significantly, the sub-aleurone cells are characterized by secondary wall ingrowths. Numerous plasmodesmata interconnect all cells between the modified aleurone and starchy endosperm. The pro-tonophore carbonylcyanide-m-chlorophenyl hydrazone (CCCP) slowed [¹⁴C]sucrose uptake by grain tissue slices enriched in modified aleurone and sub-aleurone cells but had no effect on uptake by the starchy endosperm. The fluorescent weak acid sulphorhodamine G (SRG) was preferentially accumulated by the modified aleurone and sub-aleurone cells, and this uptake was sensitive to CCCP. The combined plasma membrane surface areas of the modified aleurone and sub-aleurone cells appeared to be sufficient to support the in vivo rates of sucrose transfer to the starchy endosperm. Plasmolysis of intact excised grain inhibited [¹⁴C]sucrose transfer from the endosperm cavity to the starchy endosperm. The sulphydryl group modifier p-chloromercuribenzenesulphonie acid (PCMBS) decreased [¹⁴C]sucrose uptake by the modified aleurone and sub-aleurone cells but had little effect on uptake by the starchy endosperm. In contrast, when PCMBS and [¹⁴C]sucrose were supplied to the endosperm cavity of intact excised grain, PCMBS slowed accumulation by all tissues equally. Estimates of potential sucrose fluxes through the interconnecting plasmodesmata were found to be within the published range. It is concluded that the bulk of sucrose is accumulated from the endosperm cavity by the modified aleurone and sub-aleurone cells and subsequently transferred through the symplast to the starchy endosperm.     

Regulation of starch biosynthesis in normal and opaque-2 maize during endosperm development

The absolute activities of sucrose-UDP glucosyltransferase, glucose-6-phosphate ketoisomerase and soluble and bound ADPG-starch glucosyltransferase have been studied in normal and Opaque-2 maize endosperms during development. In general, the activities of these enzymes except sucrose-UDP glucosyltransferase were higher up to 20 days post-pollination and lower at the 30 day stage in Opaque-2 than in normal maize endosperms. However, sucrose-UDP glucosyltransferase activity was higher in normal maize endosperm up to the 20 day stage while it was lower at subsequent stages than in Opaque-2. It is suggested that the lower level of these enzymes, except sucrose-UDP glucosyltransferase, might be responsible for the reduced accumulation of starch in Opaque-2 endosperm during later stages of endosperm development.     

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.     

Protein accumulation in developing endosperm of a high-protein line of Triticum dicoccoides

Abstract. Endosperm tissue from developing grains of a line of wheat ( Triticum dicoccoides ) which accumulates up to 30% protein in the mature grain, was examined by electron microscopy to establish the ontogeny of the storage protein bodies. Ultrastructural evidence suggests that storage proteins of wheat may be transported from their site of synthesis on the rough endoplasmic reticulum (ER) to protein bodies by two different routes within the endomembrane system. The first route, which probably functions throughout protein deposition, involves the transport of protein from the cisternal rough ER to the protein vacuoles via the Golgi apparatus. The second route, observed 20 d after anthesis, appears to lead directly from dilated regions of the rough ER to protein vacuoles, bypassing the dictyosomes. Phytin inclusions are found in protein vacuoles of starchy endosperm cells adjacent to the aleurone layer of developing grain.     

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.