The wheat (Triticum aestivum L.) leaf proteome

The wheat leaf proteome was mapped and partially characterized to function as a comparative template for future wheat research. In total, 404 proteins were visualized, and 277 of these were selected for analysis based on reproducibility and relative quantity. Using a combination of protein and expressed sequence tag database searching, 142 proteins were putatively identified with an identification success rate of 51%. The identified proteins were grouped according to their functional annotations with the majority (40%) being involved in energy production, primary, or secondary metabolism. Only 8% of the protein identifications lacked ascertainable functional annotation. The 51% ratio of successful identification and the 8% unclear functional annotation rate are major improvements over most previous plant proteomic studies. This clearly indicates the advancement of the plant protein and nucleic acid sequence and annotation data available in the databases, and shows the enhanced feasibility of future wheat leaf proteome research.     

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.     

Contribution of the wheat (Triticum aestivum L.) flag leaf to grain yield in response to plant growth regulators

In West-Europe, intensive cereal management uses plant growth regulators (PGRs) especially for wheat. A green-house experiment compared the effects of two PGRs on flag leaf characteristics and yield of winter wheat. Chlormequat chloride + choline chloride (CCC) and chlormequat chloride + choline chloride + imazaquin (CCC+I) were applied to winter wheat at growth stage 5 (Feekes Large scale). CCC and CCC+I significantly increased flag leaf surface area at anthesis. Both treatments also enhanced chlorophyll content of the main stem flag leaf. The grain filling period was extended with PGR application by 2 days. CCC and CCC+I significantly increased net CO₂ assimilation rates during the flag leaf life. No effects of PGR spraying were observed on the pattern of ¹⁴C labelled assimilate distribution. Increased grain yield was due to the increase in average grain weight. The results indicate that PGR treatments increased flag leaf contribution to grain filling. The addition of imazaquin (I) to chlormequat (CCC) improved the effects of CCC.     

Comparison of the photosynthetic characteristics in the pericarp and flag leaves during wheat (Triticum aestivum L.) caryopsis development

The pericarp of cereal crops is considered a photosynthetically active tissue. Although extensive studies have been performed on green leaves, the photosynthetic role of the pericarp in cereal caryopsis development has not been well investigated. In the present study, we investigated the anatomy, ultrastructure, chlorophyll (Chl) fluorescence, and oxygen evolution of the pericarp during caryopsis ontogenesis in field wheat (Triticum aestivum L.). The results showed that wheat pericarp cross-cells contained Chl; the grana stacks and thylakoid membranes in the cross-cells were more distinct in the pericarp than those in the flag leaves as shown by transmission electron microscopy. Chl fluorescence revealed that the photosynthetic efficiency, which was indicated by values of maximum efficiency of PSII photochemistry and effective PSII quantum yield, was lower in the pericarp compared to that of the flag leaf eight days after anthesis (DAA), whereas similar values were subsequently observed. The nonphotochemical quenching values were lower from 8–16 DAA but significantly increased in the pericarp from 24–32 DAA compared to the flag leaf. The oxygen evolution rate of the flag leaves was consistently higher than that of pericarp; notably, isolated pericarps released more oxygen than intact pericarps during caryopsis development. These results suggest that the pericarp plays a key role in caryopsis development by performing photosynthesis as well as by supplying oxygen to the endosperm and dissipating excessive energy during the grain-filling stages.     

Relative sensitivity of various spectral forms of photosynthetic pigments to leaf senescence in wheat (Triticum aestivum L.)

The change in the characteristics of the absorption spectrum of chloroplasts which were isolated from the mature and senescing primary wheat leaves, was examined at various wavelengths in which the photosynthetic pigments mostly absorb. Chlorophyll (Chl) a was observed to be relatively more sensitive to leaf senescence than Chl b and carotenoids. Furthermore, the various spectral in vivo forms of Chl a, did not degrade to a similar extent; the far red absorbing forms of Chl a including species that absorb maximally at 692 nm (Chl a-692), 700 nm (Chl a-700) and 708 nm (Chl a 708) were found to be extremely sensitive to senescence induced losses. Both attached and detached senscing primary wheat leaves exhibited nearly similar pattern in the loss of photosynthetic pigments which suggests that the loss in long wavelength absorbing forms of Chl a is a selective indicator of leaf senescence.     

Fine mapping TaFLW1, a major QTL controlling flag leaf width in bread wheat (Triticum aestivum L.)

Introduction

Flag leaf width (FLW) is directly related to photosynthetic capacity and yield potential in wheat. In a previous study, Qflw.nau-5A controlling FLW was detected on chromosome 5A in the interval possessing Fhb5 for type I Fusarium head blight (FHB) resistance using a recombinant inbred line population derived from Nanda2419 × Wangshuibai.

Materials and methods

Qflw.nau-5A near-isogenic line (NIL) with the background of Mianyang 99-323 and PH691 was developed and evaluated. FLW inheritance was investigated using two F₂ populations developed from crossing the Qflw.nau-5A NILs with their recurrent parents. One hundred ten and 28 recombinants, which included 10 and 5 types of recombinants, were identified from 2816 F₂ plants with Mianyang 99-323 background and 1277 F₂ plants with PH691 background, respectively, and phenotyped in field trials for FLW and type I FHB resistance. Deletion bin mapping was applied to physically map Qflw.nau-5A.

Results and conclusions

The introduction of Wangshuibai Qflw.nau-5A allele reduced the FLW up to 3 mm. In the F₂ populations, Qflw.nau-5A was inherited like a semi-dominant gene, and was therefore designated as TaFLW1. The FLW of the recombinant lines displayed a distinct two-peak distribution. Recombinants with wider leaves commonly have Mianyang 99-323 or PH691 chromatin in the 0.2 cM Xwmc492-Xwmc752 interval that resided in the 5AL12-0.35–0.57 deletion bin, and recombinants with narrow leaves were Wangshuibai genotype in this interval. Phenotypic recombination between FLW and type I FHB resistance was identified, implying TaFLW1 was in close linkage with Fhb5. These results should aid wheat breeders to break the linkage drag through marker-assisted selection and assist in the map-based cloning of TaFLW1.     

Vacuolar cysteine proteases of wheat (Triticum aestivum L.) are common to leaf senescence induced by different factors

Cellular proteins are extensively degraded during leaf senescence, and this correlates with an up-regulation of protease gene expression, particularly cysteine proteases. The objectives of this work were (i) to detect cysteine proteases associated with senescence of wheat leaves under different conditions and (ii) to find out their subcellular location. Activity labelling of cysteine proteases with the biotinylated inhibitor DCG-04 detected five bands at 27, 36, 39, 42, and 46 kDa in leaves of wheat senescing under continuous darkness. In-gel activity assays showed that these proteases are only active in an acid milieu (pH 4), and their activity increased several-fold in senescing leaves. Fractionation experiments showed that the senescence-associated cysteine proteases of 36, 39, 42, and 46 kDa localize to a vacuolar-enriched fraction. The vacuolar cysteine proteases of 36, 39, and 42 kDa increased in activity in attached flag leaves senescing naturally during post-anthesis, and in attached leaves of plants subjected to a period of water deficit. Thus, the activity of these vacuolar cysteine proteases is associated with developmental (post-anthesis) senescence and with senescence induced by stress factors (i.e. protracted darkness or drought). This suggests that vacuoles are involved in senescence-associated cellular degradation, and that different senescence-inducing factors may converge on a single degradation pathway.     

Comparative changes in the antioxidant system in the flag leaf of early and normally senescing near-isogenic lines of wheat (Triticum aestivum L.)

Key message

The antioxidant system was significantly inhibited in the early aging line than the near-isogenic normal aging line during senescence.

Abstract

The antioxidant system plays pivotal roles in removal of reactive oxygen species (ROS) produced during leaf senescence. To explore its roles in leaf senescence of wheat (Triticum aestivum L.), the concentrations of antioxidants, activities, and gene expression of antioxidant enzymes were evaluated in flag leaves of the early aging line (EAL) and the near-isogenic normal aging line (NL) during senescence. The results showed that the total chlorophyll and soluble protein in the EAL declined earlier and faster, while more malondialdehyde and ROS accumulated compared with the NL. The activities of superoxide dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase were lower in the EAL than in the NL across multiple measuring dates. Additionally, the EAL had less amounts of reduced ascorbate and glutathione as well as lower reduction state with the progression of senescence. Concomitantly, the gene expression of antioxidant enzymes in the EAL was also significantly repressed relative to those in the NL during natural senescence. Taken together, the earlier onset and faster rate of senescence in the EAL could be a result of an imbalance of ROS production and ROS-scavenging antioxidant system, which provided valuable hints toward understanding leaf senescence of wheat.     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The flag leaf of wheat was examined for changes in quantity and activity of ribulose-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39), in the proteolytic degradation of RuBPCase and other native proteins, and in the ultrastructure of the leaf cells during grain development. Proteolytic degradation of RuBPCase at pH 4.8 increased until 8–10 d after anthesis, then declined, and increased again 16–18 d after anthesis. The second peak coincided with the onset of a preferential loss of immunologically recognizable RuBPCase. The specific activity and number of active sites per molecule of RuBPCase did not change during senescence. Examination of ultrastructure with the electron microscope showed little change in the appearance of the mitochondria as the flag leaf aged. Prominent cristae were still evident 35 d after anthesis. In contrast, the chloroplasts showed a progressive disruption of the thylakoid structure and an increasing number of osmiophilic glubules. The double membrane envelope surrounding the chloroplast appeared intact until at least 20 d after anthesis. The tonoplast also appeared intact up to 20 d. At later stages of senescence of the leaf the outer membrane of the chloroplast adjacent to the tonoplast appeared to break but the inner membrane of the envelope appeared intact until at least 35 d after anthesis.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase (EC. 4.1.1.39) I=Waters et al. 1980     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The technique of EDTA-enhanced phloem exudation (King and Zeevaart, 1974: Plant Physiol. 53, 96–103) was evaluated with respect to the collection and identification of amino acids exported from senescing wheat leaves. Whilst the characteristics of the exudate collected conform with many of the accepted properties of phloem exudate, unexpectedly high molar proportions of phenylalanine and tyrosine were observed. By comparing exudation into a range chelator solutions with exudation into water, the increased exudation of phenylalanine and tyrosine relative to the other amino acids occurring when ethylene-diaminetetracetic acid was used, was considered to an artefact.In plants thought to be relying heavily on mobilisation of protein reserves to satisfy the nitrogen requirements of the grain, the major amino acids present in flag-leaf phloem exudate were glutamate, aspartate, serine, alanine and glycine. Only small proportions of amides were present until late in senescence when glutamine became the major amino acid in phloem exudate (25 molar-%). Glutamine was always the major amino acid in xylem sap (50 molar-%).The activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), glutamate dehydrogenase (EC 1.4.1.3) and asparagine synthetase (EC 5.3.5.4) were measured in the flag leaf throughout the grain-filling period. Glutamine synthetase and glutamate-synthase activities declined during this period. Glutamate-dehydrogenase activity was markedly unchanged despite variation in the number of multiple forms visualised after gel electrophoresis. The activity of the enzyme reached a peak only very late in the course of senescence of the flag leaf. No asparagine-synthetase activity could be detected in the flag leaf during the grain-filling period.II. Peoples et al. (1980)     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The activity of a range of endo- and exopeptidase enzymes have been measured in the glumes, flag leaf and stem during the period of grain development in wheat. The enzymes show a sequential pattern of appearance with activity peaks occurring at a number of intervals from anthesis until just prior to the cessation of grain growth. Of the enzymes studied only the haemoglobin- and casein-degrading activity and alanylglycine-dipeptidase activity increased during the period of rapid protein loss, while aminopeptidase, carboxypeptidase and leucyltyrosine dipeptidase reached maximum activity prior to this period.     

Subcellular localization of mineral deposits in the roots of wheat (Triticum aestivum L.)

Summary Mineral distribution in the roots of wheat (Triticum aestivum L. cv. Wheaton) was investigated using X-ray microanalysis of bulk frozen hydrated roots in SEM and of freeze substituted sections in TEM. Results obtained using the two methods agreed reasonably well. A total often elements were detected: Na, Mg, Si, P, S, Cl, K, Ca, Mn, and Fe. Of these Si, P, Ca, and Mn were incorporated into biomineralized structures. Silica was deposited in the endodermal walls in the older parts of the root. Silicon was also detected in the large central metaxylem lumina in the basal zone of the root, and in the smaller peripheral metaxylem and the immediately contiguous pericycle and outer parenchyma cells bridging the small metaxylem vessels to the endodermal layer. In the basal zone of the root some of the inner cortical cells contained intracellular electron opaque deposits. These were associated with the cell walls, had non-opaque inclusions and microanalysis revealed that they consisted of calcium, phosphorus and manganese.Abbreviations A apical zone of root - M midzone of root - B basal zone of root - SEM scanning electron microscope - TEM transmission electron microscope     

Silicon absorption by wheat (Triticum aestivum L.)

Although silicon (Si) is a quantitatively major inorganic constituent of higher plants the element is not considered generally essential for them. Therefore it is not included in the formulation of any of the solution cultures widely used in plant physiological research. One consequence of this state of affairs is that the absorption and transport of Si have not been investigated nearly as much as those of the elements accorded 'essential' status. In this paper we report experiments showing that Si is rapidly absorbed by wheat (Triticum aestivum L.) plants from solution cultures initially containing Si at 0.5 mM, a concentration realistic in terms of the concentrations of the element in soil solutions. Nearly mature plants (headed out) 'preloaded' with Si absorbed it at virtually the same rate as did plants grown previously in solutions to which Si had not been added. The rate of Si absorption increased by more than an order of magnitude between the 2-leaf and the 7-8 leaf stage, with little change thereafter. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro enzyme activities and products of 14CO2 assimilation in flag leaf and ear parts of wheat (Triticum aestivum L.)

Activities of key enzymes of Calvin cycle and C₄ metabolism, rate of ¹⁴CO₂ fixation in light and dark and the initial products of photosynthetic ¹⁴CO₂ fixation were determined in flag leaf and different ear parts of wheat viz. pericarp, awn and glumes. Compared to the activities of RuBP carboxylase and other Calvin cycle enzymes viz. NADP-glyceraldehyde-3-phosphate dehydrogenase, NAD-glyceraldehyde-3-phosphate dehydrogenase and ribulose-5-phosphate kinase, the levels of PEP carboxylase and other enzymes of C₄ metabolism viz. NADP-malate dehydrogenase, NAD-malate dehydrogenase, NADP-malic enzyme, NAD-malic enzyme, glutamate oxaloacetate transaminase genase, NADP-malic enzyme, NAD-malic enzyme, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase, were generally greater in ear parts than in the flag leaf. In contrast to CO₂ fixation in light, the various ear parts incorporated CO₂ in darkness at much higher rates than flag leaf. In short term assimilation of ¹⁴CO₂ by illuminated ear parts, most of the ¹⁴C was in malate with less in 3-phosphoglyceric acid, whereas flag leaves incorporated most into 3-phosphoglyceric acid. It seems likely that ear parts have the capability of assimilating CO₂ by the C₄ pathway of photosynthesis and utilise PEP carboxylase for recapturing the respired CO₂.     

Agrobacterium-mediated In-planta transformation of bread wheat (Triticum aestivum L.)

Journal of Plant Biochemistry and Biotechnology - Agrobacterium-mediated in-planta transformation method allows efficient plant transformation without tissue culture. In the present study, a tissue...     

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.