Salt stress is a major abiotic stress factor affecting crop production, and understanding of the response mechanisms of seed germination to salt stress can help to improve crop tolerance and yield. The differences in regulatory pathways during germination in different salt-tolerant barley seeds are not clear. Therefore, this study investigated the responses of different salt-tolerant barley seeds during germination to salt stress at the proteomic and metabolic levels. To do so, the proteomics and metabolomics of two barley seeds with different salt tolerances were comprehensively examined. Through comparative proteomic analysis, 778 differentially expressed proteins were identified, of which 335 were upregulated and 443 were downregulated. These proteins, were mainly involved in signal transduction, propanoate metabolism, phenylpropanoid biosynthesis, plant hormones and cell wall stress. In addition, a total of 187 salt-regulated metabolites were identified in this research, which were mainly related to ABC transporters, amino acid metabolism, carbohydrate metabolism and lipid metabolism; 72 were increased and 112 were decreased. Compared with salt-sensitive materials, salt-tolerant materials responded more positively to salt stress at the protein and metabolic levels. Taken together, these results suggest that salt-tolerant germplasm may enhance resilience by repairing intracellular structures, promoting lipid metabolism and increasing osmotic metabolites. These data not only provide new ideas for how seeds respond to salt stress but also provide new directions for studying the molecular mechanisms and the metabolic homeostasis of seeds in the early stages of germination under abiotic stresses.

Low phosphorus (LP) stress leads to a significant reduction in wheat yield, primarily in the reduction of biomass, the number of tillers and spike grains, the delay in heading and flowering, and the inhibition of starch synthesis and grouting. However, the differences in regulatory pathway responses to low phosphorus stress among different wheat genotypes are still largely unknown. In this study, metabolome and transcriptome analyses of G28 (LP-tolerant) and L143 (LP-sensitive) wheat varieties after 72 h of normal phosphorus (CK) and LP stress were performed. A total of 181 and 163 differentially accumulated metabolites (DAMs) were detected for G28CK vs. G28LP and L143CK vs. L143LP, respectively. Notably, the expression of pilocarpine (C07474) in G28CK vs. G28LP was significantly downregulated 4.77-fold, while the expression of neochlorogenic acid (C17147) in L143CK vs. L143LP was significantly upregulated 2.34-fold. A total of 4023 differentially expressed genes (DEGs) were acquired between G28 and L143, of which 1120 DEGs were considered as the core DEGs of LP tolerance of wheat after LP treatment. The integration of metabolomics and transcriptomic data further revealed that the LP tolerance of wheat was closely related to 15 metabolites and 18 key genes in the sugar and amino acid metabolism pathway. The oxidative phosphorylation pathway was enriched to four ATPases, two cytochrome c reductase genes, and fumaric acid under LP treatment. Moreover, PHT1;1, TFs (ARFA, WRKY40, MYB4, MYB85), and IAA20 genes were related to the Pi starvation stress of wheat roots. Therefore, the differences in LP tolerance of different wheat varieties were related to energy metabolism, amino acid metabolism, phytohormones, and PHT proteins, and precisely regulated by the levels of various molecular pathways to adapt to Pi starvation stress. Taken together, this study may help to reveal the complex regulatory process of wheat adaptation to Pi starvation and provide new genetic clues for further study on improving plant Pi utilization efficiency.

Drought stress is a major meteorological threat to crop growth and yield. Barley (Hordeum vulgare L.) is a vital cereal crop with strong drought tolerance worldwide. However, the underlying growth properties and metabolomic regulatory module of drought tolerance remains less known. Here, we investigated the plant height, spike length, effective tiller, biomass, average spikelets, 1000-grain weight, number of seeds per plant, grain weight per plant, ash content, protein content, starch content, cellulose content, and metabolomic regulation mechanisms of drought stress in barley. Our results revealed that the growth properties were different between ZDM5430 and IL-12 under drought stress at different growth stages. We found that a total of 12,235 metabolites were identified in two barley genotype root samples with drought treatment. More than 50% of these metabolites showed significant differences between the ZDM5430 and IL-12 roots. The Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 368 differential metabolites mainly involved in starch and sucrose metabolism, the pentose phosphate pathway, pyrimidine metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis in ZDM5430 under drought stress, whereas the different metabolites of IL-12 under drought stress related to starch and sucrose metabolism, the pentose phosphate pathway, 2-oxocarboxylic acid metabolism, cutin, suberine and wax biosynthesis, carbon metabolism, fatty acid biosynthesis, and C5-branched dibasic acid metabolism. These metabolites have application in the tricarboxylic cycle, the urea cycle, the met salvage pathway, amino acid metabolism, unsaturated fatty acid biosynthesis, phenolic metabolism, and glycolysis. On the other hand, the expression patterns of 13 genes related to the abovementioned bioprocesses in different barley genotypes roots were proposed. These findings afford an overview for the understanding of barley roots’ metabolic changes in the drought defense mechanism by revealing the differently accumulated compounds.

Melatonin (N-acetyl-5-methoxytryptamine) plays an important role in plant growth and development, and in the response to various abiotic stresses. However, its role in the responses of barley to low phosphorus (LP) stress remains largely unknown. In the present study, we investigated the root phenotypes and metabolic patterns of LP-tolerant (GN121) and LP-sensitive (GN42) barley genotypes under normal P, LP, and LP with exogenous melatonin (30 μM) conditions. We found that melatonin improved barley tolerance to LP mainly by increasing root length. Untargeted metabolomic analysis showed that metabolites such as carboxylic acids and derivatives, fatty acyls, organooxygen compounds, benzene and substituted derivatives were involved in the LP stress response of barley roots, while melatonin mainly regulated indoles and derivatives, organooxygen compounds, and glycerophospholipids to alleviate LP stress. Interestingly, exogenous melatonin showed different metabolic patterns in different genotypes of barley in response to LP stress. In GN42, exogenous melatonin mainly promotes hormone-mediated root growth and increases antioxidant capacity to cope with LP damage, while in GN121, it mainly promotes the P remobilization to supplement phosphate in roots. Our study revealed the protective mechanisms of exogenous MT in alleviating LP stress of different genotypes of barley, which can be used in the production of phosphorus-deficient crops.

Nitrogen is one of the most important mineral elements for plant growth and development. Excessive nitrogen application not only pollutes the environment, but also reduces the quality of crops. However, are few studies on the mechanism of barley tolerance to low nitrogen at both the transcriptome and metabolomics levels. In this study, the nitrogen-efficient genotype (W26) and the nitrogen-sensitive genotype (W20) of barley were treated with low nitrogen (LN) for 3 days and 18 days, then treated with resupplied nitrogen (RN) from 18 to 21 days. Later, the biomass and the nitrogen content were measured, and RNA-seq and metabolites were analyzed. The nitrogen use efficiency (NUE) of W26 and W20 treated with LN for 21 days was estimated by nitrogen content and dry weight, and the values were 87.54% and 61.74%, respectively. It turned out to have a significant difference in the two genotypes under the LN condition. According to the transcriptome analysis, 7926 differentially expressed genes (DEGs) and 7537 DEGs were identified in the leaves of W26 and W20, respectively, and 6579 DEGs and 7128 DEGs were found in the roots of W26 and W20, respectively. After analysis of the metabolites, 458 differentially expressed metabolites (DAMs) and 425 DAMs were found in the leaves of W26 and W20, respectively, and 486 DAMs and 368 DAMs were found in the roots of W26 and W20, respectively. According to the KEGG joint analysis of DEGs and DAMs, it was discovered that glutathione (GSH) metabolism was the pathway of significant enrichment in the leaves of both W26 and W20. In this study, the metabolic pathways of nitrogen metabolism and GSH metabolism of barley under nitrogen were constructed based on the related DAMs and DEGs. In leaves, GSH, amino acids, and amides were the main identified DAMs, while in roots, GSH, amino acids, and phenylpropanes were mainly found DAMs. Finally, some nitrogen-efficient candidate genes and metabolites were selected based on the results of this study. The responses of W26 and W20 to low nitrogen stress were significantly different at the transcriptional and metabolic levels. The candidate genes that have been screened will be verified in future. These data not only provide new insights into how barley responds to LN, but also provide new directions for studying the molecular mechanisms of barley under abiotic stress.

Cuticular waxes comprise the hydrophobic layer that protects crops against nonstomatal water loss and biotic and abiotic stresses. Expanding on our current knowledge of the genes that are involved in cuticular wax biosynthesis and regulation plays an important role in dissecting the processes of cuticular wax metabolism. In this study, we identified the Cer-GN1 barley (Hordeum vulgare L.) mutant that is generated by ethyl methanesulfonate mutagenesis with a glossy spike phenotype that is controlled by a single recessive nuclear gene. A physiological analysis showed that the total cuticular wax loads of Cer-GN1 were one-third that of the progenitor wild-type (WT), and its water loss rate was significantly accelerated (p < 0.05). In addition, Cer-GN1 was defective in the glume’s cuticle according to the toluidine blue dye test, and it was deficient in the tubule-shaped crystals which were observed on the glume surfaces by scanning electron microscopy. Using metabolomics and transcriptomics, we investigated the impacts of cuticular wax composition and waxy regulatory genes on the loss of the glaucous wax in the spikes of Cer-GN1. Among the differential metabolites, we found that 16-hydroxyhexadecanoic acid, which is one of the predominant C16 and C18 fatty acid-derived cutin monomers, was significantly downregulated in Cer-GN1 when it was compared to that of WT. We identified two novel genes that are located on chromosome 4H and are downregulated in Cer-GN1 (HvMSTRG.29184 and HvMSTRG.29185) that encode long-chain fatty acid omega-monooxygenase CYP704B1, which regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid. A quantitative real-time PCR revealed that the expression levels of HvMSTRG.29184 and HvMSTRG.29185 were downregulated at 1, 4, 8, 12, and 16 days after the heading stage in Cer-GN1 when it was compared to those of WT. These results suggested that HvMSTRG.29184 and HvMSTRG.29185 have CYP704B1 activity, which could regulate the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley. Their downregulation in Cer-GN1 reduced the synthesis of the cuticular wax components and ultimately caused the loss of the glaucous wax in the spikes. It is necessary to verify whether HvMSTRG.29184 and HvMSTRG.29185 truly encode a CYP704B1 that regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley.