Enzymatic and metabolic diagnostic of nitrogen deficiency in Arabidopsis thaliana Wassileskija accession

Adaptation to steady-state low-nutrient availability was investigated by comparing the Wassileskija (WS) accession of Arabidopsis thaliana grown on 2 or 10 mM nitrate. Low nitrogen conditions led to a limited rosette biomass and seed yield. The latter was mainly due to reduced seed number, while seed weight was less affected. However, harvest index was lower in high nitrate compared with limited nitrate conditions. Under nitrogen-limiting conditions, nitrate reductase activity was decreased while glutamine synthetase activity was increased due to a higher accumulation of the cytosolic enzyme. The level of nitrogen remobilization to the seeds was higher under low nitrogen, and the vegetative parts of the plants remaining after seed production stored very low residual nitrogen. Through promoting nitrogen remobilization and recycling pathways, nitrogen limitation modified plant and seed compositions. Rosette leaves contained more sugars and less free amino acids when grown under nitrogen-limiting conditions. Compared with high nitrogen, the levels of proline, asparagine and glutamine were decreased. The seed amino acid composition reflected that of the rosette leaves, thus suggesting that phloem loading for seed filling was poorly selective. The major finding of this report was that together with decreasing biomass and yield, nitrogen limitation triggers large modifications in vegetative products and seed quality.     

Level of Abscisic Acid in Integuments, Nucellus, Endosperm, and Embryo of Peach Seeds (Prunus persica L. cv Springcrest) during Development

Free abscisic acid (ABA) in integuments, nucellus, endosperm, and embryo was determined throughout seed development of peach (Prunus persica L. cv Springcrest). Quantification of ABA was performed using combined high performance liquid chromatography-radioimmunoassay based on a monoclonal antibody raised against free (S)-ABA. In the integuments and endosperm, ABA concentration remained constant during the first 100 days after anthesis and rose in the following days when fresh weight was rapidly decreasing. In the nucellus, the ABA concentration variation pattern paralleled that of tissue growth. ABA concentration in the embryo increased constantly with the growth of the tissues to reach a maximum at the last growth stage. The role of ABA in peach seeds is discussed in relation to the development of the different seed tissues.     

Changes in the Contents of Free and Protein Amino Acids in Tobacco Seeds and Placentae during Maturation

Changes in the levels of protein and free amino acids in theseeds and placentae of Nicotiana tabacum were studied duringseed development. Seed maturation was completed 24 days afteranthesis. During maturation, protein rapidly accumulated inthe seeds between the 6th and 18th day, along with an appreciablecompositional change in the protein amino acids as the proportionsof glutamic acid and arginine increased. The amount of freeamino acids in the seeds gradually decreased throughout maturation.The major free amino acid on the 6th day after anthesis wasglutamine, which then drastically decreased between the 6thand 12th day with increases of glutamic acid, proline, arginineand alanine. The latter amino acids decreased thereafter untilthe 24th day. On the other hand, the amount and composition of the proteinsin the placentae did not change significantly throughout seedmaturation. In the early stage of development, the major freeamino acids in the placentae were glutamine, asparagine andglutamic acid, while in the later stage asparagine was mostabundant. (Received March 12, 1982; Accepted August 16, 1982)     

Intensification of the initial stage in amino acid metabolism in germinating cereal seeds with exogenous glutamine and proline

The initial stage of amino acid metabolism was intensified in germinating wheat seeds with exogenous glutamine and proline. Exogenous glutamine and proline accumulated over 2 h at 4°C in swelling seeds were spent at different rates over the following 2 h at 20°C, thus compensating for insufficiency of these amino acids during the initial stage of development. Creation of additional pools of glutamine and proline during the initial stage of amino acid metabolism had positive effects on the seed germination and vital activity of the plants.     

4-methyleneglutamine in peanut plants: dynamics of formation, levels, and turnover in relation to other free amino acids

Neither 4-methyleneglutamine nor 4-methyleneglutamic acid were found in free or bound form in ungerminated peanut seeds (Arachis hypogaea L.). Both, however, were formed soon after germination; whereas, 4-methyleneglutamic acid appeared slightly before 4-methyleneglutamine, the former remained at a low concentration while the level of 4-methyleneglutamine rose rapidly between 2 and 10 days of germination and declined slowly thereafter. Free proline and glutamine followed a pattern similar to 4-methyleneglutamine; on the other hand, asparagine increased for at least 20 days but other free amino acids remained at relatively low, constant levels. In mature peanut plants, 4-methyleneglutamine occurred in all parts except developing pods, was virtually the only free amino acid in xylem sap, and constituted about 70% of the total soluble nitrogen of sap. In contrast, 4-methyleneglutamic acid was found only in leaves and stems in highly variable amounts.     

Changes in carbohydrates,amino acids and proteins in developing seed of chickpea

Developing seeds of chickpea cultivars G-130, L-550 and 850-3/27 grown under field conditions were sampled at different stages of maturity and analysed for soluble sugars, starch, soluble nitrogen, protein nitrogen and amino acids. Fr. wt of seeds of all three cultivars decreased after 28 days of flowering while the dry wt continued to increase. Rapid starch accumulation was observed between 14 and 28 days after flowering. Starch as per cent of seed dry wt started to decrease after 28 days, while starch per seed increased till maturity. The percentage of salt-soluble proteins decreased with maturation of seed. The electrophoretic pattern revealed that deposition of seed storage protein in cotyledons occurred 14 days after flowering. Most of the biochemical activity apparently occurred between 14 and 28 days after flowering.     

Nitrate, a signal relieving seed dormancy in Arabidopsis

Nitrate is an important nitrogen source for plants, but also a signal molecule that controls various aspects of plant development. In the present study the role of nitrate on seed dormancy in Arabidopsis was investigated. The effects of either mutations affecting the Arabidopsis nitrate reductase genes or of different nitrate regimes of mother plants on the dormancy of the seeds produced were analysed. Altogether, data show that conditions favouring nitrate accumulation in mother plants and in seeds lead to a lower dormancy of seeds with little other morphological or biochemical differences. Analysis of germination during seed development indicated that nitrate does not prevent the onset of dormancy but rather its maintenance. The effect of an exogenous supply of nitrate on seed germination was tested: nitrate in contrast to glutamine or potassium chloride clearly stimulated the germination of dormant seeds. Data show, moreover, that the Arabidopsis dual affinity nitrate transporter NRT1.1 (CHL1) may be involved in conveying the nitrate signal into seeds. Thus, nitrate provided exogenously or by mother plants to the produced seeds, acts as a signal molecule favouring germination in Arabidopsis. This signalling may involve interaction with the abscisic acid or gibberellin pathway.     

Intensification of the initial stage of amino acid metabolism in germinating cereal seeds by exogenous glutamine and proline

The initial stage of amino acid metabolism was intensified in germinating wheat seeds with exogenous glutamine and proline. Exogenous glutamine and proline accumulated over 2 h at 4 degrees C in swelling seeds were spent at different rates over the following 2 h at 20 degrees C, thus compensating for insufficiency of these amino acids during the initial stage of development. Creation of an additional store of glutamine and proline during the initial stage of amino acid metabolism had positive effects on the seed germination and vital activity of the plants.     

Development of seed coat-imposed dormancy during seed maturation in Cynoglossum officinale

The relationship between seed phenolics and appearance of seed coat–imposed dormancy during seed development in Cynoglossum officinale L. was studied. Up to 24 days after anthesis, seeds failed to germinate upon imbibition in Petri dishes at 25°C. At 44 days after anthesis, seeds were fully germinable; removal of seed coats did not improve their germination or O₂ uptake. At 72 days after anthesis, mature seeds at the base of the cyme did not germinate unless their coats were removed. Removal of seed coat also stimulated O₂ uptake at this harvest date. The methanol-soluble phenolic content of the seeds increased during the early stages of seed development, in both the seed coat and the embryo. As seed development continued, the methanol-soluble phenolic content of the embryo stabilized, but that of the seed coat declined. This decline was associated with an increase in the thioglycolic acid–soluble phenolics, presumably lignins, in the seed coat. These results suggest that polymerization of methanol–soluble phenolics into lignins in the seed coat during later stages of seed development renders the seed coat of C. officinale impermeable to 03, and thus keeps the seed dormant.     

Structural and expression analysis of prolamin genes in <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

Rice is a staple crop with a small genome of 389 Mb. Rice grain is a source of carbohydrates and proteins and has a relatively low protein content compared to other legume seeds. Glutelin and prolamin are the major storage proteins in rice. Prolamins are characterized by high glutamine and proline content and are generally soluble only in strong alcohol solutions. In this study, we obtained a total of 51,383 expressed sequence tags (ESTs) from Ilpumbyeo (Oryza sativa L.), of which 33,201 and 18,182 clones were obtained from immature and germinating seeds, respectively. From the EST clones, 15,148 unigenes were identified, and 2,590 genes were expressed in both immature and germinating seeds. Gene expression profiling of rice prolamins indicated that prolamin gene expression increased 5 days after heading and reached maximal expression after 30 days, suggesting a high demand for prolamins during seed development and germination. Phylogenetic analysis grouped 33 prolamin genes based on the abundance of sulfur-containing amino acids methionine and cysteine according to the deduced amino acid sequences. Our results enhance the understanding of the regulation of seed maturation and germination, which can result in improved agricultural traits for the seed industry.     

Ozone Alteration of Nitrate Reduction in Soybean

Soybeans [Glycine max (L.) Merr.] were harvested at various periods after a 2 h exposure to determine the effects of ozone on nitrate reductase and selected metabolites related to nitrate reduction. Ozone initially depressed the concentrations of reducing sugars, amino acids, and nitrate reductase activity. On subsequent days following exposure, amino acid and protein levels were higher than the respective controls while the levels of reducing sugars and nitrate reductase activity returned to control levels. The ozone depression of nitrate reduction was not the result of a direct ozone reaction with the nitrate reductase protein but rather an interference with reactions that supplied the NAD(P)H needed for nitrate reduction.     

Influence of Water Stress on Water Relations, Photosynthetic Parameters and Nitrogen Metabolism of Moth Bean Genotypes

Effects of water stress at pre-flowering stage were studied in three genotypes (RMO-40, Maru moth and CZM-32 E) of moth bean [Vigna aconitifolia (Jacq.) Marechal]. Increasing water stress progressively decreased plant water potential, leaf area, net photosynthetic rate, starch and soluble protein contents and nitrate reductase activity while contents of reducing sugars, total soluble sugar, free amino acids and free proline progressively increased. Significant genotypic differences were observed and genotype CZM-32-E displayed a better drought tolerance than other genotypes.     

Drought tolerance of Periploca sepium during seed germination: antioxidant defense and compatible solutes accumulation

Periploca sepium Bunge is a native and widespread shrub on the Loess Plateau, an arid and semi-arid region in China. To understand the adaptability of its seed germination to dry environments, we investigated the germination rate, water relations, lipid peroxidation, antioxidant capacity and accumulation of major organic solutes during seed germination under water deficit conditions. Results showed that seeds pre-treated by hydration–dehydration or ?0.9 MPa PEG germinated faster than control seeds, indicating strong resistance of P. sepium to drought condition. The re-dried seeds showed higher proline, total free amino acids (TFAA) and soluble proteins (SP) contents than control dry seeds, indicating the maintenance of physiological advancement when dehydrated. Osmotic stress made seed germination stay on the plateau phase (phase II). However, germinating seeds moved into phase III immediately once transferred into distilled water. Large increases in SP and soluble sugars (SS) of both re-dried and osmotic stressed seeds help themselves to resist drought stress. The re-hydrated seeds showed significantly higher levels of proline, TFAA, SP and SS than control seeds. The largely accumulated SS during osmotic stress declined sharply when transferred into distilled water. Our data demonstrate that P. sepium’s tolerance to drought stresses during germination is associated with enhanced activity of antioxidant enzymes and accumulation of some compatible solutes. Seed physiological advancement progressed slowly under low water conditions and it was maintained when seeds were air dried. This strategy ensures high and more rapid seed germination of P. sepium under drying and wetting conditions in drought-prone regions.     

Nitrate Nutrition and Temperature Effects on Wheat: Enzyme Composition, Nitrate and Total Amino Acid Content of Leaves

Lawlor, D. W., Boyle, F. A., Kendall, A. C. and Keys, A. J.1987. Nitrate nutrition and temperature effects on wheat: Enzymecomposition, nitrate and total amino acid content of leaves.—J.exp. Bot. 38: 378–392. Wheat plants were grown in controlled environments in two temperatureregimes with two rates of nitrate fertilization. In some experimentstwo light intensities were combined with the nitrogen and temperaturetreatments. The composition of the third leaf was studied fromsoon after emergence until early senescence. The amounts ofchlorophyll, soluble protein, ribulose bisphosphate carboxylase-oxygenase(RuBPc-o) protein, nitrate, and total amino acids were measuredtogether with the activities of RuBPc-o, fructose- 1,6-bisphosphatase,glycolate oxidase, carbonic anhydrase, nitrate reductase, glutaminesynthetase and serine- and glutamate-glyoxylate aminotransferases.Additional nitrate supply increased the amounts, per unit leafarea, of chlorophyll, total soluble protein and RuBPc-o proteinand the activities of all the enzymes. The ratio of RuBP carboxylaseto RuBP oxygenase activity, when measured at constant CO²/O²ratio and temperature, was unaffected by growth conditions orleaf age. Leaves grown at the lower temperature, especiallywith more nitrate, contained much more soluble protein, nitratereductase, fructose bisphosphatase and free amino acids perunit area than the plants grown in the warmer conditions. However,young leaves grown in the warm contained more nitrate than thosegrown in the cool. Amounts of protein, amino acids and chlorophylland most enzyme activities reached maxima near full leaf expansionand decreased with age; additional nitrate slowed the decreaseand senescence was delayed. Nitrate content and nitrate reductaseactivities were highest in leaves before full expansion andthen fell rapidly after full expansion. Increased light intensityincreased the content of RuBPc-o protein at the higher rateof nitrate supply. Chloroplast components and, to a lesser extent,peroxisomal enzymes associated with photosynthetic nitrogenassimilation changed in proportion with different treatmentsbut nitrate reductase activity was not closely related to chloroplastenzymes. Control of tissue composition in relation to environmentalconditions is discussed. Key words: Nitrate nutrition, temperature, wheat, enzyme, amino acid, leaves, ribulose bisphosphate carboxylase oxygenase, nitrate reductase     

Biochemical Characteristics Associated with the Development of the Desiccation-sensitive Seeds of Machilus thunbergii Sieb. & Zucc.

Biochemical properties, i.e. endogenous abscisic acid, proline,sugars, respiration, adenosine phosphates and adenylate energycharge, and growth and moisture content were measured duringthe development of seeds of Machilus thunbergii. As dry matteraccumulated in the embryo during development, moisture content,ABA, proline, respiration and sugars all declined. At maturity,the dry mass of the seeds failed to attain a plateau beforethe period of natural seed shedding; the axis and cotyledonsreached moisture contents of 58 and 45%, respectively. Dryingof immature seeds at 73% relative humidity and 25 °C for30 d resulted in a complete loss of viability at all developmentalstages tested with the exception of mature seeds that were ableto tolerate a 5% decrease in moisture content before germinationdeclined. ABA was detected in all embryos tested, with a maximum value16.·16 µg g^-1 d. wt about midway through development.Although the presence of ABA induced no tolerance to desiccationof mature seeds, it did coincide with decreased content of waterin the developing seeds and decreased respiration. Desiccationdamage of M. thunbergii seeds occurred when moisture contentwas still high (45%) and this damage was not related to theabsence of oligosaccharides in the mature seeds. We concludethat developing embryos and mature seeds of M. thunbergii haveproperties common to many recalcitrant seeds, with seeds beingsensitive to desiccation at all stages, having a prominent ABApeak, little proline, lacking oligosaccharides, and specifically,little dormancy and a moderate rate of respiration of matureseeds (0·9 µmol O₂ min^-1 g^-1 f. wt). Adenosinetriphosphate content and energy charge decreased from stagefour to stage eight of seed development, then increased againto 103 nmol g^-1 d. wt and 0·73, respectively, in matureseeds. The moderate energy charge observed in mature seeds indicatesthat continuous metabolism is also a characteristic of recalcitrantseeds.Copyright 1995, 1999 Academic Press Machilus thunbergii, seed development, recalcitrant seed, abscisic acid, energy charge     

Changes in growth,proteins and free amino acids of developing seed and pod of fenugreek

The growth and development under field conditions of the pods and seeds of two cvs of Trigonella foenum graecum are described. Samples were harvested at different stages of ripeness for the determination of dry matter, protein and free amino acid content. During maturation, reserves of solutes are established in the pod wall before the seeds begin their exponential phase of growth. Later, these reserves disappear, providing about 20% of the seed's requirements for nitrogen. SDS-electrophoresis was used to follow the formation of proteins and it was shown that the synthesis of storage proteins takes place prior to dehydration of the seed. Production soluble nitrogenous compounds precedes protein accumulation. Free amino acids follow the same pattern. 4-Hydroxyisoleucine represents nearly 80% of free amino acid of dry seeds. The concentration does not decrease in the later stages of maturation of the seed but this unusual amino acid is absent in the storage proteins of the seeds.     

板栗种子发育期间ABA等生理指标与脱水耐性的相关性研究

对板栗种子发育期间胚轴和子叶中部分生理指标的变化以及它们与板栗种子脱水耐性的相关性进行研究.结果表明:随着板栗种子不断发育,在花后80 d胚轴和子叶中ABA、淀粉、可溶性蛋白质量分数同步达到最大值,可溶性糖质量分数达到最低,此时板栗种子的脱水耐性最强,可确定为板栗的最佳采收期.另外,通过相关分析可知,板栗种子在发育期间...     

Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): study of enzymes and nitrogen-containing compounds.

Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-l-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound.     

Starch synthases and starch branching enzymes from Pisum sativum

Concentrations of ADPglucose:α-1,4-glucan-4-glucosyltransferase (starch synthase) and α-1,4 glucan: α-1,4-glucan-6-glycosyltransferase (branching enzyme) from developing seeds of Pisum sativum were measured. Primed starch synthase activity increased from 8 to 14 days after anthesis and decreased by 50 % at 26 days. Citrate-stimulated starch synthase activity was highest at 10 days after anthesis decreasing to low levels by 22 days. Branching enzyme activity increased from 8 to 18 days after anthesis and decreased little by 26 days. Two fractions of starch synthase were recovered by gradient elution from DEAE-cellulose of extracts from 12- and 18-day-old seeds. The two fractions differed in primer specificity, K_m for ADPG and relative amounts of citrate-stimulated activity. A major and minor fraction of branching enzyme were observed in extracts from both 12- and 18-day-old seeds. Marked differences in the relative abilities ofthe two branching enzyme fractions to stimulate phosphorylase and to branch amylose as well as pH optima were found. Although the content of the starch synthase and branching enzyme fractions varied with seed age, little difference was seen in the properties of chromatographically similar fractions. Therefore, the changes in starch synthase and branching enzyme activity during pea seed development resulted from changes in the concentrations of a few enzyme forms, but not the appearance of different enzyme forms.