Proteomic analysis of the similarities and differences of soil drought and polyethylene glycol stress responses in wheat (Triticum aestivum L.).

KEY MESSAGE: Our results reveal both soil drought and PEG can enhance malate, glutathione and ascorbate metabolism, and proline biosynthesis, whereas soil drought induced these metabolic pathways to a greater degree than PEG. Polyethylene glycol (PEG) is widely used to simulate osmotic stress, but little is known about the different responses of wheat to PEG stress and soil drought. In this study, isobaric tags for relative quantification (iTRAQ)-based proteomic techniques were used to determine both the proteomic and physiological responses of wheat seedlings to soil drought and PEG. The results showed that photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, maximum potential efficiency of PS II, leaf water content, relative electrolyte leakage, MDA content, and free proline content exhibited similar responses to soil drought and PEG. Approximately 15.8% of differential proteins were induced both by soil drought and PEG. Moreover, both soil drought and PEG inhibited carbon metabolism and the biosynthesis of some amino acids by altering the accumulation of glyceraldehyde-3-phosphate dehydrogenase, ribulose-bisphosphate carboxylase, and phosphoglycerate kinase, but they both enhanced the metabolism of malate, proline, glutathione, and ascorbate by increasing the accumulation of key enzymes including malate dehydrogenase, monodehydroascorbate reductase, pyrroline-5-carboxylate dehydrogenase, pyrroline-5-carboxylate synthetase, ascorbate peroxidase, glutathione peroxidase, and glutathione S-transferase. Notably, the latter five of these enzymes were found to be more sensitive to soil drought. In addition, polyamine biosynthesis was specifically induced by increased gene expression and protein accumulation of polyamine oxidase and spermidine synthase under PEG stress, whereas fructose-bisphosphate aldolase and arginase were induced by soil drought. Therefore, present results suggest that PEG is an effective method to simulate drought stress, but the key proteins related to the metabolism of malate, glutathione, ascorbate, proline, and polyamine need to be confirmed under soil drought.

Proteomic analysis of melatonin-mediated osmotic tolerance by improving energy metabolism and autophagy in wheat (Triticum aestivum L.).

MAIN CONCLUSION: Melatonin-mediated osmotic tolerance was attributed to increased antioxidant capacity, energy metabolism, osmoregulation and autophagy in wheat (Triticum aestivum L.). Melatonin is known to play multiple roles in plant abiotic stress tolerance. However, its role in wheat has been rarely investigated. In this study, 25% polyethylene glycol 6000 (PEG 6000) was used to simulate osmotic stress, and wheat seeds and seedlings were treated with different concentrations of melatonin under PEG stress. Isobaric tag for relative and absolute quantification (iTRAQ)-based proteomic techniques were used to identify the differentially accumulated proteins from melatonin-treated and non-treated seedlings. Seeding priming with melatonin significantly increased the germination rate, coleoptile length, and primary root number of wheat under PEG stress, as well as the fresh weight, dry weight, and water content of wheat seedlings. Under PEG stress, melatonin significantly improved reactive oxygen species homeostasis, as revealed by lower H2O2 and O 2· content; and the expression of antioxidant enzymes at the transcription and translation levels was increased. Melatonin maintained seedling growth by improving photosynthetic rates and instantaneous and intrinsic water use efficiencies, as well as carbon fixation and starch synthesis at the protein level. Melatonin treatment significantly affected the expression of glycolytic proteins, including fructose-1,6-bisphosphate aldolase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and enolase, and remarkably increased the expression of the nicotinamide adenine dinucleotide transporter and nicotinamide adenine dinucleotide binding protein, thereby indirectly modulating electron transport in the respiratory chain. This indicated that melatonin improved energy production in PEG-stressed seedlings. Further, melatonin played a regulatory role in autophagy, protease expression, and ubiquitin-mediated protein degradation by significantly upregulating rab-related protein, fused signal recognition particle receptor, aspartyl protease, serine protease, ubiquitin-fold modifier 1, and ubiquitin at the mRNA or protein level. These findings suggested that melatonin might activate a metabolic cascade related to autophagy under PEG stress in wheat seedlings.

Regulatory Network of Preharvest Sprouting Resistance Revealed by Integrative Analysis of mRNA, Noncoding RNA, and DNA Methylation in Wheat.

Preharvest sprouting (PHS) of grain occurs universally and sharply decreases grain quality and yield, but the mechanism remains unclear. MingXian169, a breeding inducer wheat for stripe rust, is widely used in the Huanghuai wheat-producing region, China. In this study, we found that MingXian169 could be considered an ideal material for PHS research because of its high PHS resistance. To further analyze the network of PHS, transcriptome sequencing of mRNA, noncoding RNA (ncRNA), and DNA methylome data were used to comparison germination seeds (GS) and dormant seeds (DS); 3027, 1516, and 22 genes and 95 103 methylation regions were identified as differentially expressed mRNA, DE-microRNAs (DE-miRNA), DE-long noncoding RNAs (DE-lncRNA), and differentially methylated regions (DMRs). Pathway enrichment tests highlighted plant hormone biosynthesis and signal transduction, glutathione-ascorbate metabolism, and starch and sucrose metabolism processes related to PHS mechanisms. Further analysis demonstrated that long noncoding RNA, miRNA, and DNA methylation played critical roles in transcriptional regulation of critical pathways during PHS by modifying and interacting with target genes. Quantitative real-time polymerase chain reaction (PCR) analyses of mRNA and miRNA confirmed the sequencing results. In the phytohormone content assay, abscisic acid (ABA) and jasmonic acid (JA) increased significantly in DS, and GA19 increased in GS. The ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and ß-d-glucosidase (BGLU) enzyme activities and the substance content of glutathione and sucrose were significantly higher in GS than in DS, implying that they were responsible for increasing PHS in MingXian169. Our results provide new insights into wheat PHS resistance at mRNA, ncRNA, and DNA methylation levels, with suggestions for crop breeding and production.

RNA Sequencing Reveals Dynamic Carbohydrate Metabolism and Phytohormone Signaling Accompanying Post-mowing Regeneration of Forage Winter Wheat (Triticum aestivum L.).

Winter wheat (Triticum aestivum L.) is used as fresh green winter forage worldwide, and its ability to regenerate after mowing determines whether it can be used for forage production; however, the molecular mechanism of regeneration is poorly understood. This study identified long-chain coding and non-coding RNAs in the wheat cultivar "XN9106," which is cultivated for forage and grain production separately in winter and summer, and analyzed their function during post-mowing regeneration. The results showed that the degradation of carbohydrate plays an important role in regeneration, as demonstrated by decreased carbohydrate content. The increased gene expression of enzymes including ß-amylase, ß-fructofuranosidase, sucrose synthase, sucrose-6-phosphate synthase, trehalose-6-phosphate synthase, and trehalose-6-phosphate phosphatase in mowed seedlings suggests regeneration is fueled by degraded carbohydrates that provide energy and carbon skeletons for the Krebs cycle and amino acid synthesis. The decreased auxin content relieved the inhibition of cytokinin synthesis, that controls the transition from cell division to cell expansion and stimulates cell expansion and differentiation during the cell expansion phase, and eventually accelerate post-mowing regeneration of seedlings. Additionally, differentially expressed long-chain non-coding RNAs (lncRNAs) might participate in the regulation of gene expression related to carbohydrate metabolism and hormone signal transduction. This study demonstrated the responses of key mRNAs and lncRNAs during post-mowing regeneration of winter wheat and revealed the importance of carbohydrate and hormone during regeneration, providing valuable information for genetic improvement of forage wheat.

MicroRNA and regulation of auxin and cytokinin signalling during post-mowing regeneration of winter wheat (Triticum aestivum L.).

Winter wheat not only provides adequate fresh forage grass in winter, but also ensures ample grain production in summer. The mechanisms underlying the regeneration of winter wheat after mowing or grazing are not well understood. In this study, the miRNA expression profile of winter wheat was determined using RNA sequencing and the endogenous auxin and cis-zeatin concentrations, as well as the expression of related miRNA-targeted genes, were measured. During the post-mowing regeneration of winter wheat, the concentrations of endogenous indole-3-acetic acid (IAA), methyl indole-3-acetate (ME-IAA), and indole-3-carboxaldehyde (ICA) decreased, while those of cis-zeatin (cZ) increased. Moreover, 15 novel miRNAs and three known miRNAs were found to be involved in the synthesis and signalling transduction of auxins and cytokinins (CKs). Among these miRNAs, miR1153-y, miR5059-x, miR2916-x, novel-miR1532-3p, novel-miR1060-3p, and novel-miR0890-3p, were found to be negatively correlated with the expression of their target genes including auxin response GH3.7, auxin response factor (ARF), type-A two-component response regulator (A-ARR), aldehyde dehydrogenase (ALDH), and O-glucosyltransferase (CISZOG). Furthermore, miR1153-y was identified as mediating the cleavage of GH3.7 by RACE assay. In turn, these genes inhibited the biosynthesis and signalling of IAA and activated CK signal transduction, resulting in the rapid regeneration of mowed winter wheat. This study revealed that some miRNAs exert a positive regulatory effect on the post-mowing regeneration of winter wheat by controlling the synthesis and signal transduction of IAA and CK, and our founding will aid developments in biotechnology aimed at improving the post-mowing regeneration ability of winter wheat.

Beneficial effects of melatonin in overcoming drought stress in wheat seedlings.

Melatonin plays an important role in abiotic stress in plant, but its role in wheat drought tolerance is less known. To verify its role, wheat seedlings (Triticum aestivum L. 'Yan 995') at 60% and 40% of field capacity were treated with 500 µM melatonin in this study. Melatonin treatment significantly enhanced the drought tolerance of wheat seedlings, as demonstrated by decreased membrane damage, more intact grana lamella of chloroplast, higher photosynthetic rate, and maximum efficiency of photosystem II, as well as higher cell turgor and water holding capacity in melatonin-treated seedlings. Besides, melatonin markedly decreased the content of hydrogen peroxide and superoxide anion in melatonin-treated seedlings, which is attributed to the increased total antioxidant capacity, GSH and AsA contents, as well as enzyme activity including ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX), and glutathione transferase (GST). The GSH-AsA related genes including APX, MDHAR, and DHAR were commonly upregulated by melatonin and correlated to the antioxidant enzyme activity as well as the content of GSH and AsA, indicating that the increase of GSH and AsA was attributed to the expression of these genes. Our result confirmed the mitigation potential of melatonin in drought stress and certain mechanisms of melatonin-induced GSH and AsA accumulation, which could deepen our understanding of melatonin-induced drought tolerance in wheat.