TaWRKY74 participates copper tolerance through regulation of TaGST1 expression and GSH content in wheat.

Glutathione S-transferase (GST) is the key enzyme in glutathione (GSH) synthesis, and plays a crucial role in copper (Cu) detoxification. Nonetheless, its regulatory mechanisms remain largely unclear. In this study, we identified a Cu-induced glutathione S-transferase 1 (TaGST1) gene in wheat. Yeast one-hybrid (Y1H) screened out TaWRKY74, which was one member from the WRKY transcription factor family. The bindings between TaGST1 promoter and TaWRKY74 were further verified by using another Y1H and luciferase assays. Expression of TaWRKY74 was induced more than 30-folds by Cu stress. Functions of TaWRKY74 were tested by using transiently silence methods. In transiently TaWRKY74-silenced wheat plants, TaWRKY74 and TaGST1 expression, GST activity, and GSH content was significantly inhibited by 25.68%, 19.88%, 27.66%, and 12.68% in shoots, and 53.81%, 52.11%, 23.47%, and 17.11% in roots, respectively. However, contents of hydrogen peroxide, malondialdehyde, or Cu were significantly increased by 2.58%, 12.45%, or 37.74% in shoots, and 25.24%, 53.84%, and 103.99% in roots, respectively. Notably, exogenous application of GSH reversed the adverse effects of transiently TaWRKY74-silenced wheat plants during Cu stress. Taken together, our results suggesting that TaWRKY74 regulated TaGST1 expression and affected GSH accumulation under Cu stress, and could be useful to ameliorate Cu toxicity for crop food safety.

Melatonin promotes potassium deficiency tolerance by regulating HAK1 transporter and its upstream transcription factor NAC71 in wheat.

Melatonin (MT) is involved in various physiological processes and stress responses in animals and plants. However, little is known about the molecular mechanisms by which MT regulates potassium deficiency (DK) tolerance in crops. In this study, an appropriate concentration (50 µmol/L) was found to enhance the tolerance of wheat plants against DK. RNA-seq analysis showed that a total of 6253 and 5873 differentially expressed genes (DEGs) were separately identified in root and leaf tissues of the DK + MT-treated wheat plants. They functionally involved biological processes of secondary metabolite, signal transduction, and transport or catabolism. Of these, an upregulated high-affinity K transporter 1 (TaHAK1) gene was next characterized. TaHAK1 overexpression markedly enhanced the K absorption, while its transient silencing exhibited the opposite effect, suggesting its important role in MT-mediated DK tolerance. Moreover, yeast one-hybrid (Y1H) was used to screen the upstream regulators of TaHAK1 gene and the transcription factor TaNAC71 was identified. The binding between TaNAC71 and TaHAK1 promoter was evidenced by using Y1H, LUC, and EMSA assays. Transient overexpression of TaNAC71 in wheat protoplasts activated the TaHAK1 expression, whereas its transient silencing inhibited the TaHAK1 expression and aggravated the sensitivity to DK. Exogenous MT application greatly upregulated the expression of TaHAK1 in both transient overexpression and silencing systems. Our findings revealed some molecular mechanisms underlying MT-mediated DK tolerance and helped broaden its practical application in agriculture.

Root and nitrate-N distribution and optimization of N input in winter wheat.

Scientific management of nitrogen (N) fertilizer has a significant effect on yield while also reducing the environmental risks. In this study, we conducted field experiments over three years at two different sites (Zhengzhou and Shangshui) in Henan Province, China, using different N application rates (0, 90,180, 270, and 360 kg ha-1) to determine the relationships between soil N supply and N demand in winter wheat (Triticum aestivum L.). Optimal N input was then determined. Both sites showed the same trend. Namely, aboveground N uptake and soil nitrate N (NO3--N) increased with increasing N, while NO3--N decreased with increasing soil depth, gradually moving downwards with growth. A significant correlation (p < 0.001) between increasing aboveground N uptake and increasing NO3--N was also observed under N application, with the best relationships occurring in the 20-60 cm layer during jointing-anthesis (R2 = 0.402-0.431) and the 20-80 cm layer at maturity (R2 = 0.474). Root weight density showed the same spatial-temporal characteristics as NO3--N, following a unimodal trend with increasing N, and peaking at 90 kg ha-1. The root weight density was mainly distributed in the 0-60 cm layer (above 80%), with the 20-60 cm layer accounting for 30% of the total root system. In this layer, the root weight density was also significantly positively correlated with aboveground N uptake. Wheat yield reached saturation under high N (>270 kg ha-1), with a sharp decrease in N use efficiency (NUE) and linear increase in residual NO3--N. To balance yield and the risk of environmental pollution in the experimental area, an N application rate of 180-270 kg ha-1 is recommended under sufficient irrigation, thereby supporting a well-developed root system while ensuring balance between N supply and demand.

Comprehensive analysis of the transcription of starch synthesis genes and the transcription factor RSR1 in wheat (Triticum aestivum) endosperm.

The cDNA sequences of 26 starch synthesis genes were identified in common wheat (Triticum aestivum L.), and their transcript levels were measured using quantitative real-time RT-PCR to assess the function of individual genes and the regulatory mechanism in wheat endosperm. The expression patterns of 26 genes in wheat endosperm were classified into three groups. The genes in group 1 were richly expressed in the early stage of grain development and may be involved in the construction of fundamental cell machinery, synthesis of glucan primers, and initiation of starch granules. The genes in group 2 were highly expressed during the middle and late stages of grain development, and their expression profiles were similar to the accumulation rate of endosperm starch; these genes are presumed to play a crucial role in starch production. The genes in group 3 were scantily expressed throughout the grain development period and might be associated with transitory starch synthesis. Transcripts of the negative transcription factor TaRSR1 were high at the early and late stages of grain development but low during the middle stage. The expression pattern of TaRSR1 was almost opposite to those of the group 2 starch synthesis genes, indicating that TaRSR1 might negatively regulate the expression of many endosperm starch synthesis genes during grain development.

Identification of the TaBTF3 gene in wheat (Triticum aestivum L.) and the effect of its silencing on wheat chloroplast, mitochondria and mesophyll cell development.

The full-length cDNA (882bp) and DNA (1742bp) sequences encoding a basic transcription factor 3, designated as TaBTF3, were first isolated from common wheat (Triticum aestivum L.). Subcellular localization studies revealed that the TaBTF3 protein was mainly located in the cytoplasm and nucleus. In TaBTF3-silenced transgenic wheat seedlings obtained using the Virus-induced gene silencing (VIGS) method, the chlorophyll pigment content was markedly reduced. However, the malonaldehyde (MDA) and H(2)O(2) contents were enhanced, and the structure of the wheat mesophyll cell was seriously damaged. Furthermore, transcripts of the chloroplast- and mitochondrial-encoded genes were significantly reduced in TaBTF3-silenced transgenic wheat plants. These results suggest that the TaBTF3 gene might function in the development of the wheat chloroplast, mitochondria and mesophyll cell. This paper is the first report to describe the involvement of TaBTF3 in maintaining the normal plant mesophyll cell structure.

Identification and expression pattern of ribosomal L5 gene in common wheat (Triticum aestivum L.).

The full-length cDNA sequence (1158 bp) encoding a ribosomal L5 protein, designated as TaL5, was firstly isolated from common wheat (Triticum aestivum L.) using the rapid amplification of cDNA ends method (RACE). The open reading frame (ORF) of TaL5 gene was 906 bp, and its deduced amino acid sequence (301 residues) shared high similarity to those of other higher plant L5 proteins. TaL5 protein contained a putative 5S binding region (74 amino acids). TaL5 DNA sequence was further cloned, and sequence analysis showed that it contained 7 introns and 8 exons. Predicated using TargetP software, TaL5 protein was putatively located in mitochondria and contains a transit peptide of 12 amino acids. During grain filling period, temporal expression pattern of TaL5 gene was approximately consistent with the rates of starch accumulation in grains. Additionally, TaL5 gene was dramatically induced by salt, drought and freezing stresses, exogenous abscisic acid (ABA) and salicylic acid (SA) in wheat seedlings. These implied that TaL5 gene could function in growth, development and abiotic stresses in wheat plants.

[Effects of combined application of biogas slurry and chemical fertilizer on winter wheat rhizosphere soil microorganisms and enzyme activities].

This paper studied the effects of combined application of biogas slurry and chemical fertilizer under same N application rate on the quantities of bacteria, actinomycetes and fungi as well as the activities of urease, protease and catalase in winter wheat rhizosphere soil. With the growth of winter wheat, the quantities of test microorganisms and the activities of urease and catalase showed a trend of increasing after an initial decrease, while the protease activity showed an S-type change. Combined application of biogas slurry and chemical fertilizer increased the quantities of test microorganisms significantly, and improved the activities of soil urease and protease. Applying 50% biogas slurry N as basal plus 50% chemical N as topdressing and applying 25% biogas slurry N as basal plus 75% chemical N as topdressing had the best effect, while applying single conventional urea or biogas slurry had the worst effect. At all growth stages, the activity of soil catalase was the highest in treatments 25% biogas slurry N as basal plus 75% chemical N as topdressing and single biogas slurry, but had greater differences in other treatments among the growth stages. The results suggested that proper biogas slurry application combined with chemical fertilization could increase the microbial quantity and enzyme activities in winter wheat rhizosphere soil.

A novel Ta.AGP.S.1b transcript in Chinese common wheat (Triticum aestivum L.).

ADP-glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis in plants, is composed of two small and two large subunits, and has plastidial and cytosolic isoforms. In kernels of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), transcripts for cytosolic (Ta.AGP.S1a) and plastidial (Ta.AGP.S1b) small subunits of AGPase were encoded by the same gene (Ta.AGP.S.1) by use of the alternative first exons. In this study, a cDNA sequence (1631 bp) [NCBI: EU586278] encoding a novel Ta.AGP.S1b transcript was isolated in kernels of Chinese common wheat cultivars. Compared with another Ta.AGP.S1b transcript [NCBI: FJ643609] isolated in kernels of non-Chinese wheat cultivars, EU586278 lacked a long fragment (117 bp) at its 5'terminal, resulting in a shorten transit peptide. The lacked fragments of Ta.AGP.S1b (EU586278) were universally found in surveyed 22 Chinese common wheat cultivars. Partial genomic DNA sequence [NCBI: FJ907395] of Ta.AGP.S.1 gene, which was corresponded to 5'terminal of EU586278 transcript, was also isolated in Chinese common cultivars and sequencing indicated that FJ907395 contained the corresponding lacked fragment of EU586278 transcript, inferring the lacked fragment in EU586278 transcript was not present in the genome, but possibly occurred at transcription level. Using TargetP software, the predicated transit peptide of putative plastidial SSU encoded by EU586278 contained merely 25 amino acids, considerably shorter than those of other plant AGP. S.1bs (54-70 amino acids). Phylogenetic tree analysis indicated that the amino acid sequence of EU586278 transit peptide was not clustered together with those of other wheat Ta.AGP.S1bs [NCBI: AF536819 and FJ643609] and barley AGP.S1b [NCBI: Z48563]. These implied that EU586278 could be a novel Ta.AGP.S1b transcript. Semi-quantitative PCR analysis indicated that transcripts of EU586278 were abundantly expressed in leaf, moderately in endosperm and stem, and weakly in root.

[Effects of ecological factors on the dough extensograph parameters of different winter wheat cultivars].

In 2000-2001 and 2001-2002, six representative winter wheat cultivars Yumai 34, Gaomai 8901, Yumai 49, Yumai 70, Luoyang 8716, and Yumai 50 were consecutively grown at five locations (Xinyang, Zhumadian, Xuchang, Wuzhi, and Tangyin) with latitudes varying from 32 degrees N to 36 degrees N in Henan Province, aimed to understand the relationships of winter wheat dough extensograph parameters with genetic and ecological factors. The dough extensograph parameters were more affected by genetic factors than by ecological factors. Cultivars Yumai 34 and Gaomai 8901 had significantly higher maximum resistance and extension area than the other four test cultivars, and significant differences in the dough extensograph parameters were observed between the cultivars grown in the south region (Xinyang and Zhumadian) and in the north region (Wuzhi and Tangyin) of the Province. The change patterns of dough extensograph parameters with latitude differed in 2000-2001 and in 2001-2002, and the effects of climatic factors on the dough extensograph parameters varied with year. In 2001-2002, the precipitation at the stage from grain-filling to maturing affected the dough extensograph parameters significantly. Our results suggested that in order to improve the dough extensograph parameters of winter wheat, local meteorological conditions should be taken into full consideration in the soil water management at late-maturing stage.

[Effects of latitude on grain's protein components of winter wheat cultivars].

Six representative winter wheat cultivars were planted at five locations with the latitude varied from 32 degrees N to 36 degrees N in Henan Province to study the effects of latitude on their grain's protein components. The results showed that with increasing latitude, the contents of albumin and gliadin decreased, while glutenin content, glutenin/gliadin ratio, sum of protein components, and grain yield increased. The grain's albumin content of test cultivars at Xinyang, gliadin content at Zhumadian and Xuchang, and glutenin content and glutenin/gliadin ratio at Wuzhi and Tangyin were higher than those at other locations. The average air temperature, total sunlight hours, and precipitation in May had close relations with the grain' s protein components of test winter wheat cultivars at different locations. In order to improve wheat grain quality, suitable cultivars should be chosen to match the local ecological conditions, and, management practices for improving photosynthesis and extending grain-filling period should be performed in May.

[Effects of water-nitrogen interaction on the contents and components of protein and starch in wheat grains].

With wheat cultivars Yumai 34 (strong-gluten wheat) and Yumai 50 (weak-gluten wheat) as test materials, a field experiment was conducted to study the effects of three irrigation treatments (irrigation at jointing stage, at jointing and grain-filling stages, and at jointing, grain-filling, and pre-maturing stages), three nitrogen application rates (0, 150, and 270 kg x hm(-2)), and their combinations on the contents and components of protein and starch in wheat grains. The results showed that for strong-gluten wheat cultivar Yumai 34, applying 270 kg x hm(-2) of N increased the total content of protein and the contents of albumin, gliadin and glutelin, and enhanced the glutelin/gliadin ratio. This application rate of nitrogen also increased the total content of starch and the content of amylopectin, and decreased the amylose/amylopetin ratio. For weak-gluten wheat cultivar Yumai 50, applying 150 kg x hm(-2) of N increased the contents of albumin and gliadin, and decreased the contents of globulin and glutelin and the glutelin/gliadin ratio. The amylopectin and starch contents also increased when the N application rate was 150 kg x hm(-2). Non-N fertilization or applying 270 kg x hm(-2) of N decreased the accumulation of protein and starch, and resulted in a decrease of grain yield. Among the irrigation treatments, irrigation at jointing and grain-filling stages promoted the accumulation of protein and starch in grains and increased the grain yield, while the other two treatments were unbeneficial to the accumulation of protein and starch and decreased the grain yield. Applying 270 kg x hm(-2) and 150 kg x hm(-2) of N combined with irrigation at jointing and grain-filling stages was the ideal management regime for the high yield and good quality of strong- and weak-gluten wheat cultivars, respectively.

[Effects of nitrogen application rate on soil enzyme activities in wheat rhizosphere].

This paper studied the effects of nitrogen application rate on the soil enzyme activities in the rhizosphere of wheat cultivars Lankaoaizao 8, a large spike genotype, and Yumai 49-198, a small spike genotype, under high yield condition. The results showed that the enzyme activities in rhizosphere soil had similar changing trends with wheat growth. The protease, urease and dehydrogenase activities in rhizosphere soil increased with wheat growth, maximized at heading stage, jointing stage, and heading stage, respectively, and decreased thereafter. Catalase activity increased with wheat growth, and peaked at maturing stage. At the same growth stage, the protease, catalase and dehydrogenase activities in rhizosphere soil of the two cultivars increased with increasing nitrogen application rate and peaked at 180 kg N x hm(-2). Urease activity also increased with increasing nitrogen application rate, and the maximum activity was observed at 360 kg N x hm(-2).

[Key enzymes in starch synthesis in plants].

Starch, the most common form of stored carbon in plants, is both the major food source for mankind and important raw material for many industries. It is composed of two types of alpha-1,4-linked glucan polymer: essentially unbranched amylose and regularly branched amylopectin, and synthesized in photosynthetic and non-photosynthetic organs. Starch is synthesized via four committed enzyme steps: ADP-Glc pyrophosphorylase, which synthesizes sugar nucleotide precursors; starch synthase, which extends the alpha-1,4-linked glucan chains using ADP-Glc; starch-branching enzymes, which introduce alpha-1,6 branch points to form amylopectin; and starch debranching enzymes, which hydrolyze alpha-1,6 branches in glucans. In this paper, recent advances in biochemical characterizations and gene engineering concerning these enzymes were reviewed, and the achievements in gene engineering involved in manipulation of starch amount and quality were also cited.