Alfalfa is the most extensively cultivated forage legume worldwide, and salinity constitutes the main environmental scourge limiting its growth and productivity. To unravel the potential molecular mechanism involved in salt tolerance in alfalfa, we accomplished a combined analysis of parallel reaction monitoring-based proteomic technique and targeted metabolism. Based on proteomic analysis, salt stress induced 226 differentially abundant proteins (DAPs). Among them, 118 DAPs related to the antioxidant system, including glutathione metabolism and oxidation-reduction pathways, were significantly up-regulated. Data are available via ProteomeXchange with identifier PXD017166. Overall, 107 determined metabolites revealed that the tricarboxylic acid (TCA) cycle, especially the malate to oxaloacetate conversion step, was strongly stimulated by salt stress. This leads to an up-regulation by about 5 times the ratio of NADPH/NADP⁺, as well as about 3 to 5 times in the antioxidant enzymes activities, including those of catalase and peroxidase and proline contents. However, the expression levels of DAPs related to the Calvin–Benson–Bassham (CBB) cycle and photorespiration pathway were dramatically inhibited following salt treatment. Consistently, metabolic analysis showed that the metabolite amounts related to carbon assimilation and photorespiration decreased by about 40% after exposure to 200 mM NaCl for 14 d, leading ultimately to a reduction in net photosynthesis by around 30%. Our findings highlighted also the importance of the supplied extra reducing power, thanks to the TCA cycle, in the well-functioning of glutathione to remove and scavenge the reactive oxygen species (ROS) and mitigate subsequently the oxidative deleterious effect of salt on carbon metabolism including the CBB cycle.

Ethylene promotes ripening in fruits as well as the biosynthesis of anthocyanins in plants. However, the question of which ethylene response factors (ERFs) interact with the genes along the anthocyanin biosynthesis pathway is yet to be answered. Herein, we conduct an integrated analysis of transcriptomes and metabolome on fruits of two mulberry genotypes (‘Zijin’, ZJ, and ‘Dashi’, DS, with high and low anthocyanin abundance, respectively) at different post-flowering stages. In total, 1035 upregulated genes were identified in ZJ and DS, including MYBA in the MBW complex and anthocyanin related genes such as F3H. A KEGG analysis suggested that flavonoid biosynthesis and plant hormone signaling transduction pathways were significantly enriched in the upregulated gene list. In particular, among 103 ERF genes, the expression of ERF5 showed the most positive correlation with the anthocyanin change pattern across both genotypes and in the post-flowering stages, with a Pearson correlation coefficient (PCC) of 0.93. Electrophoresis mobility shift assay (EMSA) and luciferase assay suggested that ERF5 binds to the promoter regions of MYBA and F3H and transcriptionally activates their gene expression. We elucidated a potential mechanism by which ethylene enhances anthocyanin accumulation in mulberry fruits and highlighted the importance of the ERF5 gene in controlling the anthocyanin content in mulberry species. This knowledge could be used for engineering purposes in future mulberry breeding programs.

Clematis florida Thun (CfT) is an ornamental and medicinal plant. It is a cold resistant but heat sensitive species and deserves to be further investigated to improve its adaptability to heat stress. Exploring the molecular mechanism potential via an omic-analysis constitutes a promising approach towards improving heat tolerance of CfT. Two CfT lines, heat resistance (HR) and heat sensitive (HS), with differential thermotolerance capacities were used for the integrative analyses of proteomics and transcriptomes. Transcriptomes analysis showed that various pathways were significantly enriched including plant hormone signal transduction and carbon fixation pathways in prokaryotes. Proteomics study revealed the enrichment of some other pathways comprising antioxidant activity and carbohydrates metabolism. Based on combined transcriptomes and proteomics analyses and following heat stress treatment, a total of 1724 annotated genes were overlapped between both CfT lines. Particularly, 84 differential expressed genes (DEGs) were overlapped in both CfT lines. Fifteen out of these 84 genes were up-regulated solely for HR line (PS) but not for HS one (SG). This strongly suggests a potential prominent role for these genes in the thermotolerance process in PS line. We corroborate that two Hsps (Hsp18 and Hsp70) out of 20 detected proteins with higher expression levels in PS than in SG based on either global transcripts or proteins levels. According to the transcriptomes and proteomics analyses, 6 proteins and their corresponding genes were found to be significantly abundant in HR line (PS). Data are available via ProteomeXchange with identifier PXD018192. The expressions levels of these 6 genes were checked also for both CfT lines to evaluate their potential contributions in the heat tolerance process. Thus, their expression levels were approximately 2~4 times higher in HR than in HS line. We provided as well a representative schematic model to highlight the key genes involved in ROS scavenging and photorespiratory pathway in CfT. This model could be helpful also in understanding the mechanism of heat tolerance in CfT.

Respiration is a major plant physiological process that generates adenosine triphosphate (ATP) to support the various pathways involved in the plant growth and development. After decades of focused research on basic mechanisms of respiration, the processes and major proteins involved in respiration are well elucidated. However, much less is known about the natural variation of respiration. Here we conducted a survey on the natural variation of leaf dark respiration (R_d) in a global rice minicore diversity panel and applied a genome-wide association study (GWAS) in rice (Oryza sativa L.) to determine candidate loci associated with R_d. This rice minicore diversity panel consists of 206 accessions, which were grown under both growth room (GR) and field conditions. We found that R_d shows high single-nucleotide polymorphism (SNP) heritability under GR and it is significantly affected by genotype-environment interactions. R_d also exhibits strong positive correlation to the leaf thickness and chlorophyll content. GWAS results of R_d collected under GR and field show an overlapped genomic region in the chromosome 3 (Chr.3), which contains a lead SNP (3m29440628). There are 12 candidate genes within this region; among them, three genes show significantly higher expression levels in accessions with high R_d. Particularly, we observed that the LRK1 gene, annotated as leucine rich repeat receptor kinase, was up-regulated four times. We further found that a single significantly associated SNPs at the promoter region of LRK1, was strongly correlated with the mean annual temperature of the regions from where minicore accessions were collected. A rice lrk1 mutant shows only ~37% R_d of that of WT and retarded growth following exposure to 35 °C for 30 days, but only 24% reduction in growth was recorded under normal temperature (25 °C). This study demonstrates a substantial natural variation of R_d in rice and that the LRK1 gene can regulate leaf dark respiratory fluxes, especially under high temperature.

The present study reveals contrasting responses of photosynthesis to salt stress in two C₄ species: a glycophyte Setaria viridis (SV) and a halophyte Spartina alterniflora (SA). Specifically, the effect of short-term salt stress treatment on the photosynthetic CO₂ uptake and electron transport were investigated in SV and its salt-tolerant close relative SA. In this experiment, at the beginning, plants were grown in soil then were exposed to salt stress under hydroponic conditions for two weeks. SV demonstrated a much higher susceptibility to salt stress than SA; while, SV was incapable to survive subjected to about 100 mM, SA can tolerate salt concentrations up to 550 mM with slight effect on photosynthetic CO₂ uptake rates and electrons transport chain conductance (g_ETC). Regardless the oxygen concentration used, our results show an enhancement in the P₇₀₀ oxidation with increasing O₂ concentration for SV following NaCl treatment and almost no change for SA. We also observed an activation of the cyclic NDH-dependent pathway in SV by about 2.36 times upon exposure to 50 mM NaCl for 12 days (d); however, its activity in SA drops by about 25% compared to the control without salt treatment. Using PTOX inhibitor (n-PG) and that of the Q_o-binding site of Cytb₆/f (DBMIB), at two O₂ levels (2 and 21%), to restrict electrons flow towards PSI, we successfully revealed the presence of a possible PTOX activity under salt stress for SA but not for SV. However, by q-PCR and western-blot analysis, we showed an increase in PTOX amount by about 3–4 times for SA under salt stress but not or very less for SV. Overall, this study provides strong proof for the existence of PTOX as an alternative electron pathway in C₄ species (SA), which might play more than a photoprotective role under salt stress.

Quinoa is an annual pseudocereal highly adapted to extreme environments and has become, at this point in time, an extremely popular food due to its exceptional and high nutritional quality. This study aims to investigate the association of quinoa salt tolerance at an early developmental stage with its grain nutritional value under the effect of severe climatic hurdles. The current findings revealed a significant variability between genotypes in salt response attributes at the first development stage, where genotypes Amarilla Sacaca (thereafter, A. Sacaca) and QQ57 exhibited high salt tolerance thresholds with a low salt sensitivity index (SI), and a high capacity for Na⁺ sequestration into vacuoles. A significant positive association was detected between salt tolerance degree and yield parameters, saponins (SAPs), and minerals contents, where genotype A. Sacaca exhibited the highest SAP content with 3.84 mg.g⁻¹ and the highest amounts of K, Ca, P, and Fe. The analysis of fatty acid composition demonstrated a high significant negative correlation between crude fat content and salt SI, and between yield parameters. Despite its low harvest index (HI) and low seed oil content, the salt-tolerant genotype A. Sacaca showed a high nutritional quality for seed oil according to its lowest ω6/ω3 ratio (5.6/1) and lowest level of atherogenicity index (AI). The genotype 115R, defined as the most sensitive to salt stress, exhibited a high seed oil quality due to its low lipid peroxidation susceptibility as reflected by its oxidative susceptibility and peroxidizability indexes. The significance of this study includes the identification of valuable quinoa genotypes showing high efficiency in growth and yield under severe stress accompanied by a high nutritional value satisfying the market requirements for healthy, nutritious, and safe food products.