Ammonium and amino acid metabolism in excised leaves of wheat (Triticum aestivum) senescing in the dark

Several parameters of amino acid metabolism were studied in detached primary leaves of wheat (Triticum aestivum L. cv. Castell) during a 14 day incubation period in the dark. Protein loss was accompanied by a 5-fold increase in the total amount of free amino acids during the first 4 days of the incubation period with asparagine being the most important. Beyond this stage a pronounced intracellular accumulation of ammonium occured. A gradual decrease in the levels of free amino acids and ammonium at the later stages of senescence could in part be accounted for by leakage from the leaves. Additionally, some nitrogen was lost due to ammonia volatilization. The rapid decay of the glutamine synthetase (GS; EC 6.3.1.2)-glutamate synthase (Fd-GOGAT; EC 1.4.7.1) system and the fast decline of glutamate-pyruvate transaminase (GPT; EC 2.6.1.2) activity appear to be predominant features of senescence in the dark. Decreasing Fd-GOGAT activity was slightly compensated by a small and temporary increase in the activity of NADH-GOGAT (EC 1.4.1.14). Glutamateoxalocetate transaminase (GOT: EC 2.6.1.1) activity, although declining continuously, proved to be much more persistent. Changes in glutamate dehydrogenase (GDH; EC 1.4.1.3) activity closely resembled the profile of ammonium evolution in the leaves and NADP-isocitrate dehydrogenase (IDH; EC 1.1.1.42) activity revealed a temporary maximum during the period of rapid increase in GDH activity. Increased activity of GDH could also be induced by exogenous ammonium. Ammonium accumulation could, at least partly, be caused by increased asparaginase (EC 3.5.1.1) activity which accompanied the rapid conversion of asparagine to aspartic acid. Asparagine aminotransferase (EC 2.6.1.14) activity declined sharply from the beginning of the senescence period. Although the activity profile of glutaminase (EC 3.5.1.2) was similar to that of asparaginase, glutamine was of little importance quantitatively and an analogous relationship between glutamine and glutamic acid could not be detected.     

Distribution of gluten proteins in bread wheat (Triticum aestivum) grain

Background and Aims

Gluten proteins are the major storage protein fraction in the mature wheat grain. They are restricted to the starchy endosperm, which forms white flour on milling, and interact during grain development to form large polymers which form a continuous proteinaceous network when flour is mixed with water to give dough. This network confers viscosity and elasticity to the dough, enabling the production of leavened products. The starchy endosperm is not a homogeneous tissue and quantitative and qualitative gradients exist for the major components: protein, starch and cell wall polysaccharides. Gradients in protein content and composition are the most evident and are of particular interest because of the major role played by the gluten proteins in determining grain processing quality.

Methods

Protein gradients in the starchy endosperm were investigated using antibodies for specific gluten protein types for immunolocalization in developing grains and for western blot analysis of protein extracts from flour fractions obtained by sequential abrasion (pearling) to prepare tissue layers.

Key Results

Differential patterns of distribution were found for the high-molecular-weight subunits of glutenin (HMW-GS) and γ-gliadins when compared with the low-molecular-weight subunits of glutenin (LMW-GS), ω- and α-gliadins. The first two types of gluten protein are more abundant in the inner endosperm layers and the latter more abundant in the subaleurone. Immunolocalization also showed that segregation of gluten proteins occurs both between and within protein bodies during protein deposition and may still be retained in the mature grain.

Conclusions

Quantitative and qualitative gradients in gluten protein composition are established during grain development. These gradients may be due to the origin of subaleurone cells, which unlike other starchy endosperm cells derive from the re-differentiation of aleurone cells, but could also result from the action of specific regulatory signals produced by the maternal tissue on specific domains of the gluten protein gene promoters.     

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.     

Effects of foliar applied benzyladenine on grain yield and grain protein in wheat (Triticum aestivum L.)

The effects of foliar applications of nitrogen and benzyladenine (BA) on grain yield and grain protein of wheat grown under field conditions were studied over 2 years with 5 cultivars at 2 locations. Nitrogen (N) at 20 kg.ha^–1, and BA at 100 or 800 mg.l^–1 were applied alone or combined at pre and post-anthesis; applications of BA at 8 mg.l^–1 were also made on individual ears in order to study the effect on cell number. Weekly determinations of the chlorophyll content of the flag leaf were conducted after anthesis to study leaf senescence. At harvest, yield, yield components and grain protein percentage were determined. N and BA applications delayed chlorophyll loss in the flag leaf, but modified neither yield nor yield components. Foliarly applied BA increased grain protein in four of the five cultivars tested. It is concluded that delay of the senescence induced by BA might allow more energy to be available for N uptake by the crop leading to an increase in grain protein.Research supported by a CAFPTA grant 1656/86 and by CONICET, PID 30017700/85.CONICETComisión de Investigaciones Cientificas de la Provincia de Buenos AiresInstituto de Fisiologia Vegetal     

Antioxidative enzymes and the Russian wheat aphid (Diuraphis noxia) resistance response in wheat (Triticum aestivum)

A crucial function of antioxidative enzymes is to remove excess reactive oxygen species (ROS), which can be toxic to plant cells. The effect of Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), infestation on the activities of antioxidative enzymes was investigated in the resistant (cv. Tugela DN) and the near-isogenic susceptible (cv. Tugela) wheat (Triticum aestivum L.). RWA infestation significantly induced the activity of superoxide dismutase, glutathione reductase and ascorbate peroxidase to higher levels in the resistant than in susceptible plants. These findings suggest the involvement of antioxidative enzymes in the RWA-wheat resistance response, which was accompanied by an early oxidative burst. The results are consistent with the role of ROS in the resistance response and the control of their levels to minimise toxic effects.     

Lignin deposition induced by aluminum in wheat (Triticum aestivum) roots

We investigated the relation between the toxic effect of aluminum (Al) on root growth and the lignin deposition in wheat ( Triticum aestivum L. cvs Atlas 66 and Scout 66). In the Al-tolerant cultivar Atlas 66, control treatment without AlCl₃ at pH 4.75, cell length increased dramatically in the portion of the root that was 0.6 to 3.2 mm from the root cap junction (approximately 1.0 to 3.6 mm from the root tip). However, treatment with 20 μ M AlCl₃ for 24 and 48 h completely inhibited root elongation and markedly decreased the length and increased the diameter of the cells in the same portion of the root. Moreover, marked deposition of lignin was observed in the cells that corresponded to the portion 1.5 to 4.5 mm from the root tip in Atlas 66 roots treated with 20 μ M AlCl₃, while no deposition of lignin was detected in control roots. Treatment with 5 μ M AlCl₃ slightly inhibited root growth and there was no deposition of lignin in the root. On the other hand, in roots of the Al-sensitive cultivar Scout 66, treatment with 5 μ M AlCl₃ completely inhibited root growth and markedly induced deposition of lignin. These results suggest that lignification in the elongating region coincided with the extent of inhibition of root growth by Al in two wheat cultivars that differed in their sensitivity to Al.     

Amino acid metabolism associated with N-mobilization from the flag leaf of wheat (Triticum aestivum L.) during grain development

Field-grown winter wheat (Triticum aestivum L. cv. Castell) was used to study changes in the free amino acid pools of different plant parts and related enzyme activities in the flag leaf throughout the grain-filling period in three consecutive growing seasons. Amino acid analysis data indicated that, during senescence, the nitrogen flow in the flag leaf was directed towards the synthesis of glutamine as a specific nitrogen transport form. Of the enzymes involved, total glutamine synthetase (GS; EC 6.3.1.2) and especially ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) activities declined continuously as senescence progressed. Unlike (chloroplastic) GS₂, (cytosolic) GS₁ was shown to be very persistent suggesting a special role for this isoenzyme in the N-reallocation process. Glutamate-oxaloacetate transaminase (GOT; EC 2.6.1.1), glutamate-pyruvate transaminase (GPT; EC 2.6.1.2) and isocitrate dehydrogenase (IDH; EC 1.1.1.42) showed a characteristic biphasic activity profile after anthesis. It is proposed that these enzymes, for each of which at least two isoenzymes were demonstrated, are involved in glutamate synthesis at the later stages of leaf senescence. Ammonium levels were fairly constant throughout the flag leafs life span, an ultimate rise often following peak values of glutamate dehydrogenase (GDH; EC 1.4.1.4) activity. The enzymology of flag leaf amino acid metabolism during grain development is further discussed in relation to observations of NH₃-volatilization from naturally senescing wheat plants.     

Translocation and cycling through roots of recently absorbed nitrogen and sulphur in wheat (Triticum aestivum) during vegetative and generative growth

¹⁵N-Nitrate and ³⁵S-sulphate labelling experiments were performed with spring wheat ( Triticum aestivum L. cv. Timmo) 44. 64, 79, 95 and 115 days after sowing (growth stages arbitrarily denoted I to V). Label was fed to the plants via a fraction of the root system, termed donor root, whereas the rest of the root (receiver root) was fed non-labelled nutrient solution. Net uptake rates for both nitrate and sulphate per unit root weight changed little from growth stage I to IV, but were considerably lower at stage V. On a whole-plant weight basis, uptake declined from stage I to IV, because root contribution to total plant weight declined. Between 80 and 95% of absorbed label was translocated to the shoot at all growth stages. At stage V, up to 30% of absorbed label was recovered in the ears. Labelling of the receiver root indicated that, at all growth stages, 10 to 17% of N and 12 to 32% of S translocated to the shoot was retranslocated to the root. This corresponds to between 35 and 85% of the label actually recovered in the roots. Analysis of ¹⁵N-labelling of xylem sap collected from receiver roots at growth stages I to IV indicated that about half of the reduced N in the sap is derived from cycling through roots of recently assimilated N. Evidence of cycling was also obtained at stage V. Labelled sulphate was the only form of S cycled in the plant, but it accounted for only 1 to 7% of the sulphate in the xylem sap.     

Nitrate accumulation by wheat (Triticum aestivum) in relation to growth and tissue N concentrations

The accumulation of nitrate in relation to total N concentrations ([N]_i) in tissues of wheat (Triticum aestivum L., cv Sicco) grown in solution culture was investigated. Root, shoot and leaf tissues showed qualitatively similar relationships between internal nitrate concentrations and [N]_i, both expressed on a tissue water basis. At low [N]_i, no nitrate was detectable but once a particular [N]_i was exceeded, nitrate accumulated as a linear function of [N]_i. The threshold [N]_i values for nitrate accumulation were 110, 450, and 550 mM for roots, total shoot and leaf 4, respectively. The slope of the relationship between nitrate and [N]_i indicated that in all tissues nitrate accounted for 50–55% of the extra N accumulated above the threshold [N]_i. All growth requirements for N were satisfied before nitrate accumulated.     

Endogenous gibberellins in young seedlings of wheat (Triticum aestivum) cultivars

Gibberellins A₁, A₃, A₄ and A₇ were identified by combined gas chromatography mass spectrometry (GC-MS) in leaf and stem tissues of 17-day-old seedlings of wheat ( Triticum aestivum L. ), cvs Siete Cerros (semi-dwarf, Rht1) and Møystad (tall), of F₁, hybrids from the cross Møystad × Siete Cerros and of 2 selected lines from the cross Møystad x Sonora 64 (Rht1 and Rht2). GA, and GA, were identified by full scan mass spectra separately in all 5 extracts, GA₄ and GA₇, were identified by selected ion monitoring in a bulked fraction. About 90% of the biological activity (Tan-ginbozu dwarf rice bioassay) in all 5 extracts was due to the GA₁/GA₃-fraction.     

Accelerated production of transgenic wheat (Triticum aestivum L.) plants

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 g of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.     

Effect of source-sink relations and nitrogen nutrition on senescence and N remobilization in the flag leaf of wheat

The relation between the source-sink ratio and nitrogen nutrition on grain yield of wheat ( Triticum aestivum L. cv. Klein Chamaco) was studied in a greenhouse experiment. Plants were grown until anthesis in pots with soil fertilized with 0.16 mmol N per plant twice a week. At anthesis, all leaves but the flag leaf were excised in a group of plants. In another group the treatment consisted in a similar defoliation plus the longitudinal excision of half the ear, while a third group was left untouched as a control. At the same time, the N supply to half of the plants in each group was interrupted, while the other half continued receiving 16 m M N. The defoliated plants showed a longer functional life of the flag leaf than the control, retaining the chlorophyll, soluble proteins and total reduced nitrogen for a longer time. The ear-excised plants showed an intermediate behavior. The plants with the interrupted N supply showed a faster leaf senescence than the N supplied ones, and this coincided with an increase in the proteolytic activity and nitrogen transport to the ear. However there were no differences in ear weight between the two nitrogen treatments. It is concluded that leaves and ear compete for the nitrogen, and that a low level of carbohydrates in the flag leaf, due to a low source-sink ratio, delays leaf senescence.     

Gibberellic acid (GA) increases fibre cell differentiation and secondary cell-wall deposition in spring wheat (Triticum aestivum L.) culms

The role of gibberellic acid (GA) in differentiation and secondary cell-wall deposition of fibre cells of spring wheat (Triticum aestivum) culms was studied using applications of GA and chlormequat (a GA biosynthesis inhibitor). In certain genotypes, higher GA levels may increase the number of cortical fibre cell files by changing cell fate from parenchyma to fibre, and induce thicker secondary cell-walls.     

Transport and Partitioning of Lead in Indian Mustard (Brassica juncea) and Wheat (Triticum aestivum)

Lead (Pb) contamination in soils is a serious concern because it can be taken up by crops and then transferred through the food chain, posing a potential risk to human health. Indian mustard (Brassica juncea) and wheat (Triticum aestivum) are important crop species known to accumulate heavy metals in their tissues. This study aimed at understanding the transport and accumulation of Pb in these two species and the risk associated with consumption of these foods, which would help us in mitigating accumulation of Pb in edible tissues. The plants were grown at different Pb concentrations for the entire life cycle, and the partitioning of the metal to different tissues was examined. The results showed that plant species differ widely in their ability to transport and accumulate Pb in different tissues. In B. juncea, there was significant accumulation of Pb in both siliques and seeds, whereas most of the Pb in wheat was concentrated in the vegetative tissues and less to the flag leaf and reproductive tissues. In both species, although seed Pb concentrations exceeded acceptable limits, dietary intake did not exceed acceptable limits in most treatments, indicating that more studies on Pb transport and redistribution in crop species is necessary.     

Wheat (Triticum aestivum) seedlings secrete proteases from the roots and, after protein addition, grow well on medium without inorganic nitrogen

This paper reports on the role of proteases secreted by roots in nitrogen capture by plants. The study was conducted on aseptically cultivated wheat seedlings (Triticum aestivum cv. Tacher) obtained from embryos isolated from grains. Seedlings were cultivated for 21 days on deionised water, Murashige Skoog medium (MS), MS without inorganic nitrogen (IN), and MS without IN, in which IN was replaced by casein (0.01%, 0.1% or 1%). Comparison of seedlings grown on these media showed that casein entirely compensated for the lack of inorganic nitrogen in the medium. Shoots and roots of seedlings cultivated on MS medium with this protein had higher fresh weight than those cultivated on MS medium without casein. The increase in fresh weight of seedlings was correlated with casein concentration and proteolytic activity in the medium. In conclusion, wheat that uses proteases secreted by the roots can directly utilise proteins in the medium as a source of nitrogen without prior digestion by microbial proteases and without protein mineralisation. These results suggest the important role of organic nitrogen fertilisers in increasing wheat yield.     

High frequency regeneration of plantlets from the leaf-bases via somatic embryogenesis and comparison of polypeptide profiles from morphogenic and non-morphogenic calli in wheat (Triticum aestivum)

High frequency regeneration via somatic embryogenesis was achieved in the leaf-base cultures of wheat ( Triticum aestivum L. cv Sonalika) by optimizing the concentration of the hormone, 2,4-dichlorophenoxy acetic acid, and selecting for the appropriate part of the leaf base as explant. It was possible to distinguish compact (morphogenic) and friable (non-morphogenic) calli by the naked eye, after about 60 days of culture on medium enriched with 2,4-dichlorophenoxy acetic acid. The fact that the compact calli are morphogenic, while the friable ones are not, was evident from the observation that only the former formed plantlets after transfer to the basal medium. The morphogenic and non-morphogenic cultures showed substantial difference in soluble protein content on a fresh weight basis. A comparison of silver-stained profiles of soluble polypeptides from morphogenic and non-morphogenic calli revealed many polypeptides specifically associated with either type of calli.     

Identification of quantitative trait locus of zinc and phosphorus density in wheat (Triticum aestivum L.) grain

Zinc (Zn) is an essential micronutrient for human beings. However, Zn malnutrition has become a major problem throughout the world. Wheat is the most important food crop in the world, therefore, developing Zn-enriched wheat varieties provides an effective approach to reduce Zn malnutrition in human beings. The aim of this study was to understand the genetic control of grain Zn density in wheat and hence, to provide genetic basis for breeding wheat with high grain Zn density using molecular approach. A doubled haploid (DH) population developed from a cross between winter wheat varieties Hanxuan10 and Lumai 14 was used to identify quantitative trait loci (QTLs) for Zn concentration and content in wheat grains. In addition, phosphorus (P) concentration and content in wheat grain were also investigated to examine possible interactions between these two nutrients. The wheat grains used in this study were harvested from the plants grown under normal condition in a field trial. We found the grain Zn concentrations of the DH population varied from 25.9 to 50.5 mg/kg and the Zn content varied from 0.90 to 2.21 μg/seed. The grain P concentrations of the DH population varied from 0.258 to 0.429 mg/kg, and the P contents varied from 0.083 to 0.186 mg/seed. A significant positive correlation was observed between Zn and P density in this experiment. The results showed that both grain Zn and P densities were controlled by polygenes. Four and seven QTLs for Zn concentration and Zn content were detected, respectively. All the four QTLs for Zn concentration co-located with the QTLs for Zn content, suggesting a possibility to improve both grain Zn concentration and content simultaneously. Four and six QTLs for P concentration and P content were detected, respectively. The two QTLs for grain Zn concentration on chromosomes 4A and 4D co-located with the QTLs for P concentration. The four QTLs for grain Zn content on chromosome 2D, 3A and 4A co-located with the QTLs for P contents, reflecting the positive correlations between the grain Zn and P density, and providing possibility of improving grain micro- and macronutrient density simultaneously in wheat. In order to improve human health, the effect of P–Zn relation in grain on the Zn bioavailability should also be considered in the future work.