Osmotic Stress in Coleoptiles and Primary Leaves of Wheat. II. Main Phosphorylated Fractions

Seedlings of wheat (Triticum durum, cv. Balcarceno-INTA) werewater-stressed in darkness with 20% polyethylene glycol (PEG)6000 or 0.3 M mannitol added to the root medium. At differenttimes and up to a total of 36 h of treatment the coleoptilesand primary leaves were cut and analysed for acid soluble-P,lipid-P, protein-P, alkali-stable organic-P, and nucleic acid-P.All the phosphorylated fractions were expressed on 100 mg oftotal coleoptile and primary leaves dry wt. Acid soluble-P (14.6µmol) accounted for most of the phosphorus. The lipid-Pfraction remained at an unchanged level (6.2 µmol/100mg dry wt) in control or in mildly osmotic stressed shoots ofseedlings over the period of treatment. The protein-P representedonly 2.4% (0.8 µmol) of the total phosphorus found incoleoptiles and primary leaves. The alkali-stable organic-Pand nucleic acid-P fractions represented 22.2% (7.2µ mol)and 11.1% (3.6 µmol) of the total phosphorus content ofshoots, respectively. All the phosphorylated fractions, exceptingthe lipid-P fraction, underwent a significant (P < 0.025)fall over periods of up to 36 h of wheat seedlings growth indarkness. A common characteristic found in all the phosphorylatedfractions was the fact that there were not significant differencesbetween control and 20% PEG 6000 or 0.3 M mannitol treated seedlingsduring 36 h of treatment and growth in darkness. However, whenseedlings were pulse-labelled with 32P during imbibition, someeffects of the osmotic stressants on several fractions couldbe seen. Specific radioactivity fell in acid soluble-P of controlshoots, but increased in 20% PEG 6000 and in 0.3 M mannitoltreated seedlings. Radioactive phosphorus was not found in thealkali-stable organic-P fraction. Lipid-P, nucleic acid-P, andprotein-P fractions increased their specific activities during36 h of shoot growth in control plants. Such 20% PEG 6000 and0.3 M mannitol restricted this increase during the same periodof time, the former being more active. Possible implicationsof phospholipid and phosphoprotein turnover in relation to waterstress are discussed.     

Mild phosphorus stress in barley and a related low-phosphorus-adapted barleygrass: Phosphorus fractions and phosphate absorption in relation to growth

Barleygrass ( Hordeum leporinum ) from Australian low-P (phosphorus) soils and commercial barley ( H. vulgare ) with high fertilizer requirements were grown in solution culture at 3 levels of P supply. The high-P-adapted barley produced more biomass at all levels of P supply and was more responsive to added P in terms of rate of tillering, rate of leaf production, final leaf size, and therefore total shoot weight compared to barleygrass. In both species root: shoot ratio decreased in response to improved tissue P status, even at P levels where total biomass did not respond to P supply. Removal of endosperm reserves of barley reduced total biomass to a greater extent than it altered phosphate absorption rate, thus increasing tissue P status and making plants less responsive to added P. Similarly, barleygrass had a slower growth rate but a comparable P absorption rate to that of barley. Thus barleygrass also accumulated tissue P and was unresponsive to added P. All phosphorus chemical fractions increased in response to improved tissue P status, but to differing extents (inorganic-P > nucleic acid-P > lipid-P > ester-P), suggesting that all P fractions (particularly inorganic P) serve, in part, a storage function. Both barleygrass and barley without endosperm had higher concentrations of all P fractions (particularly inorganic P) than did unaltered barley, but this was due entirely to their higher P status (due to slow growth) rather than to any major difference in P metabolism between species. We conclude that slow growth is more important than interspecific differences in P metabolism, P absorption, or efficiency of P utilization in explaining the success of barleygrass and other low-P-adapted species on infertile soils.     

Reduced phytic Acid content does not have an adverse effect on germination of soybean seeds

Altering the level of phytic acid phosphorus by nutritional means had no effect on the ability of soybean (Glycine max L. [Merr.], cv `Williams 79') seeds to germinate under laboratory or greenhouse conditions. Dry matter moved out of the cotyledons at similar rates whether the germinating seeds initially contained low (0.19), medium (0.59), or high (1.00 milligram per seed) phytic acid phosphorus. Growth of roots and shoots from 3 to 9 days after planting was similar for seeds containing low and medium levels of phytic acid phosphorus. The medium level of phytic acid resembles that found in field-grown seed, so it is clear that soybean seeds normally contain a phosphorus reserve far above that needed for germination and early seedling growth.     

The role of phytic Acid in the wheat grain

The concentrations of adenosine triphosphate and phytic acid in testa, embryo plus scutellum, aleurone, and endosperm fractions from grain of Triticum vulgare cv. Insignia have been determined during development under both normal conditions and those of water stress. Phytic acid was not detected in the endosperm. In the embryo plus scutellum and aleurone fractions there was a rapid build-up of phytic acid, but the adenosine triphosphate level did not change markedly at this time. These results are not consistent with physiological roles previously suggested for phytic acid other than the role of phytin as a phosphorus and cation store for the germinating seed.     

Free and Bound Amino Acids during Germination of Maize Seed with Preincorporated Foreign Prolamines

Wheat prolamines were preincorporated into the endosperm of maize seed, and concentrations of free and bound amino acids were measured in the endosperm and seedlings during seed germination. Incorporation of foreign prolamines lowered the rate of decrease of free proline concentration in both the endosperm (sprouting and shoots) and the embryo (sprouting). The seedlings at the stage of shoots were characterized by higher concentrations of aspartic acid, glutamic acid, and tyrosine. At the stage of transition to autotrophic nutrition, the pool of amino acids that are physiologically important for plant growth was greater in maize seedlings with incorporated wheat prolamines.     

Free and bound amino acids during germination of maize seeds with implemented foreign prolamines

Wheat prolamines were preincorporated into the endosperm of maize seed, and concentrations of free and bound amino acids were measured in the endosperm and seedlings during seed germination. Incorporation of foreign prolamines lowered the decrease rate of free proline concentration in both the endosperm (sprouting and shoots) and the embryo (sprouting). The seedlings at the stage of shoots were characterized by higher concentrations of aspartic acid, glutamic acid, and tyrosine. At the stage of transition to autotrophic nutrition, the pool of amino acids that are physiologically important for the plant growth was greater in maize seedlings with incorporated wheat prolamines.     

Dynamics of nutrient remobilisation from seed of wheat genotypes during imbibition,germination and early seedling growth

The changes in nutrient content of grain tissues and seedling parts of two wheat genotypes (Triticum aestivum L., Excalibur and Janz) with low or high seed Zn content were followed from imbibition to early seedling development (12 days). The grains were separated into seed coat, endosperm and embryo, while the seedlings were separated into roots and shoots. The dry weight of the seed coat did not change throughout the experimental period, whereas the endosperm weight declined rapidly from day 4 onward. The weight of embryo did not show any difference between and within cultivars. About a half of seed Zn was remobilised into shoot and roots during 12 days of growth, regardless of the initial seed Zn content in both genotypes. The seed coat contained 55–77% of the total seed nutrients in the two wheat genotypes, except in the case of S (around 40%). Manganese, Fe, Ca, K, and P were remobilised effectively from the seed coat as well as from the endosperm, while remobilisation of Zn and Cu was relatively less from the seed coat than from the endosperm. After 10 days of growth, all nutrients monitored were completely remobilised from the endosperm. Remobilised K was directed primarily into shoots; an increase in K content in shoots was relatively higher than the accumulation of dry matter, with a consequent increase in K concentration in shoot tissue. The remobilisation of some nutrients (eg. Fe, Ca and Zn) from various grain tissues during inbibition, germination and early growth is different from the remobilisation in more mature plants.     

Mobilization of Phosphorus in the Cotyledons of Young Seedlings of the Garden Pea (Pisum sativum L.)

Changes in the levels of various phosphorus fractions and ofphytase activity in the cotyledons of young pea seedlings grownin the light have been studied. It is shown that from the onsetof germination there is a lag of several days in the hydrolysisof phytic acid and that this is associated with a low levelof phytase activity in cotyledon extracts. Rapid developmentof phytase during the next few days is accompanied by a rapidincrease in the rate of phytic acid break-down and both reachmaximum levels after 6–7 days from soaking the seed. Theamount of phytic acid in the cotyledons becomes negligible afterabout 15 days and at the same time phytase activity declinesmarkedly. At this point protease activity is at a maximum andthe water content of the cotyledons begins to fall. Removal of the shoot 4 days after soaking the seed caused animmediate decrease in export of phosphorus from the cotyledonsbut did not affect the level of phytic acid for several days.Subsequently there was a small, but significant reduction inthe rate of phytic acid hydrolysis in de-shooted seedlings ascompared with intact plants in spite of the fact that phytaseactivity was not affected for several days. Similar effectswere observed when excised cotyledons were cultured on moistfilter-paper. Control mechanisms for phytic acid hydrolysis are discussedand it is concluded that regulation by the axis of the inorganicphosphate concentration at the sites of phytase activity maybe a means of controlling phytic acid hydrolysis.     

Localization of myo-inositol-1-phosphate synthase to the endosperm in developing seeds of Arabidopsis

Expression and localization of myo-inositol-1-phosphate synthase (MIPS) in developing seeds of Arabidopsis thaliana was investigated. MIPS is an essential enzyme for production of inositol and inositol phosphates via its circularization of glucose-6-phosphate as the initial step. myo-inositol-6-phosphate (InsP(6) or phytic acid) is the predominant form of phosphorus found in seeds and accumulates as a consequence of MIPS action. Three MIPS genes have been identified in Arabidopsis, all of which were expressed not only in siliques but in both leaves and roots. Immunoelectron microscopy using a MIPS antibody showed that MIPS localizes to the cytosol primarily in the endosperm during seed development and not in the embryo. This is consistent with results obtained using fluorescent microscopy and western blot analysis that showed a similar pattern of localization. However, InsP(6), which is the final product of inositol phosphate metabolism, was present mainly in the embryo. This suggests that a complex interaction between the endosperm and embryo occurs during the synthesis and subsequent accumulation of InsP(6) in developing seeds of Arabidopsis.     

植酸对杂交水稻V20B种子萌发及生理特性的影响

以杂交水稻V20B 为材料,研究外源植酸对种子萌发的影响。结果表明,外源植酸浓度为10 mmol/L 时能明显抑制种子萌发。生理生化检测表明,植酸对杂交水稻种子萌发过程中α-淀粉酶活性、可溶性糖和内源植酸含量变化有不同程度的影响。     

Biochemical changes during germination and seedling growth in Cuscuta campestris

Changes in the contents of starch, protein, DNA, RNA, total phosphorus, acid soluble phosphorus and inorganic phosphorus, and in the activities of some enzymes of carbohydrate, amino acid, nucleic acid and phosphate metabolism were studied during the germination of Cuscuta campestris seeds. The results are expressed on per seed basis.
Starch content in Cuscuta seeds showed a steady decline with most of it depleted by the end of the eighth day of germination. Protein content increased with germination up to 48 h and then decreased. RNA and DNA contents increased to a maximal level on the fourth day of germination and then decreased. Total phosphorus in the seeds remained almost unchanged during the period of study. Both trichloroacetic acid soluble and inorganic phosphorus increased until the third day and then decreased. Phytin was rapidly hydrolyzed with little being detectable by the seventh day of germination. Glucose-6-phosphate dehydrogenase increased with germination, while fructose bisphosphate aldolase which is indispensable for glycolysis, decreased with germination. Ribonuclease and deoxyribonuclease increased till the third and fourth day, respectively, and then decreased. Aspartate and alanine aminotransferases showed a maximum on the second day and then decreased. Activities of alkaline fructose-1,6-bisphosphatase and phytase were absent in the dry seeds and appeared only on the second day of germination. Both α- and β-amylase activities were present in the dry seed.     

Application of 31P NMR to Monitor Phosphorus Compounds and their Changes During Germination of Legume Seeds

Extracts of embryonic axes and cotyledons of 19 species of legumeswere analysed by ³¹P nuclear magnetic resonance; the effectof different extraction procedures on the solubilization ofthe several phosphocompounds was investigated. The contentsof phytic acid, inorganic phosphate, phosphomonoesters and phosphodiesterswere determined. The relative contents of these phosphocompoundswere very different when embryonic axes and cotyledons werecompared, and were greatly influenced by the taxonomic affiliationof the corresponding species. Phytic acid and inorganic phosphatewere by far the major phosphocompounds found in cotyledon extracts;an unusual phosphodiester component was detected only in embryonicaxes and was particularly abundant in species belonging to theLotoideae sub-family. The changes in the amount of the phosphodiestercompound were followed during germination and the results suggestthat it is not a phosphorus reserve. Furthermore, this componentwas preferentially present in the roots of the new plants. Itwas partially hydrolysed by RNAse and to a smaller extent byproteases, but not affected by DNAse. The hypothesis that thisunusual component might be a ribonucleoprotein is discussed. Key words: ³¹P NMR, legumes, seed components, phosphorus compounds, solubilization     

Polyamines and Protein Metabolism in Maize Inbreds Differing in Seed Protein Content

Polyamine metabolism was evaluated in the embryo and the endosperm,during the early stages of seed germination, of two maize inbreds(Lo5 and B73) differing in the protein nitrogen content of thecaryopscs. On germination, the concentration of buffer-extractableproteins and of polyamines increases more quickly and to greatervalues in Lo5 than in B73. In the caryopses, the embryos havea higher polyamine content than the endosperms and in the seedlings,after three days of growth, the shoots show a higher polyaminecontent than in the case of the scutellum and the roots. Duringseed germination, spermidine is the main polyamine and its contentvaries while the spermine remains virtually constant. The polyaminesand protein pattern in the embryo and the endosperm of the twoinbreds are discussed in relation to the differences in theirgermination energy and early seedling growth.     

Changes in the patterns of phosphatase isozymes during the germination of rice seed

Crude phosphatase preparations from both the embryo and endosperm of germinating rice seeds were resolved into several components by Sephadex G-100 column chromatography. The substrate specificity of each isozyme was broad, but their spectra were more or less different. One of these iso-phosphatases in the embryo increased prominently during seed germination and decreased at the stage of senescence. Phosphatase activities for several phosphate esters were increased during germination, but to different extents. The rate increased more in the embryo than in the endosperm. Especially in the embryo, gibberellin A₃ promoted an increase in the activity of some phosphatases as well as an increase in the content of free phosphate.     

A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings

Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. Due to the lack of phytases in the non-ruminant digestive tract, monogastric animals cannot utilize dietary phytic acid and it is excreted into manure. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The use of phytases to degrade seed phytic acid has potential for reducing the negative environmental impact of livestock production. A phytase was purified to electrophoretic homogeneity from cotyledons of germinated soybeans (Glycine max L. Merr.). Peptide sequence data generated from the purified enzyme facilitated the cloning of the phytase sequence (GmPhy) employing a polymerase chain reaction strategy. The introduction of GmPhy into soybean tissue culture resulted in increased phytase activity in transformed cells, which confirmed the identity of the phytase gene. It is surprising that the soybean phytase was unrelated to previously characterized microbial or maize (Zea mays) phytases, which were classified as histidine acid phosphatases. The soybean phytase sequence exhibited a high degree of similarity to purple acid phosphatases, a class of metallophosphoesterases.     

Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study

Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.     

Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds

Phytic acid in cereal grains and oilseeds is poorly digested by monogastric animals and negatively affects animal nutrition and the environment. However, breeding programs involving mutants with less phytic acid and more inorganic phosphate (P(i)) have been frustrated by undesirable agronomic characteristics associated with the phytic acid-reducing mutations. We show that maize lpa1 mutants are defective in a multidrug resistance-associated protein (MRP) ATP-binding cassette (ABC) transporter that is expressed most highly in embryos, but also in immature endosperm, germinating seed and vegetative tissues. Silencing expression of this transporter in an embryo-specific manner produced low-phytic-acid, high-Pi transgenic maize seeds that germinate normally and do not show any significant reduction in seed dry weight. This dominant transgenic approach obviates the need for incorporating recessive lpa1 mutations to create maize hybrids with reduced phytic acid. Suppressing the homologous soybean MRP gene also generated low-phytic-acid seed, suggesting that the strategy might be feasible for many crops.