The establishment of symbiosis between legume and rhizobium results in the formation of nodule. Phytocyanins (PCs) are a class of plant-specific blue copper proteins, playing critical roles in plant development including nodule formation. Although a few PC genes have been isolated from nodules, their functions are still unclear. Here, we performed a genome-wide identification of PC family in seven sequenced legume species (Medicago truncatula, Glycine max, Cicer arietinum, Cajanus cajan, Lotus japonicus, Vigna angularis and Phaseolus vulgaris) and found PCs experienced a remarkable expansion in M. truncatula and G. max. Further, we conducted an in-depth analysis of PC family in the model legume M. truncatula. Briefly, 82 MtPCs were divided into four subfamilies and clustered into seven clades, with a large proportion of tandem duplications and various cross-tissues expression patterns. Importantly, some PCs, such as MtPLC1, MtENODL27 and MtENODL28 were preferentially expressed in nodules. Further, RNA interference (RNAi) experiment revealed the knockdown of MtENDOL27 and MtENDOL28 impaired rhizobia infection, nodule numbers and nitrogenase activity. Moreover, in the MtENODL27-RNAi nodules, the infected cells were reduced and the symbiosomes did not reach the elongated stage, indicating MtENDOL27 is required for rhizobia infection and nodule development. In addition, co-expression analysis showed MtPLC1, MtENODL27 and MtENODL28 were grouped into two different functional modules and co-expressed with the known symbiotic nitrogen fixation-related genes, suggesting that they might participate in nodulation via different ways. In summary, this study provides a useful resource for future researches on the structure and function of PCs in nodulation.

Maize (Zea mays L.) is sensitive to salt stress, especially during seed germination and seedling morphogenesis, which limits maize growth and productivity formation. As a novel recognized plant hormone, melatonin (MT) participates in multiple growth and developmental processes and mediates biotic/abiotic stress responses, yet the effects of salt stress on maize seedlings remain unclear. Herein, we investigated the effects of 150 μM exogenous MT on multiple phenotypes and physiologic metabolisms in three-leaf seedlings across eight maize inbred lines under 180 mM NaCl salt stress, including growth parameters, stomatal morphology, photosynthetic metabolisms, antioxidant enzyme activities, and reactive oxygen species (ROS). Meanwhile, the six gene expression levels controlling antioxidant enzyme activities and photosynthetic pigment biosynthesis in two materials with contrasting salt resistance were examined for all treatments to explore the possible molecular mechanism of exogenous MT alleviating salt injury in maize. The results showed that 150 μM exogenous MT application protected membrane integrity and reduced ROS accumulation by activating the antioxidant system in leaves of maize seedlings under salt stress, their relative conductivity and H₂O₂ level average reduced by 20.91% and 17.22%, while the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) averaged increased by 13.90%, 17.02%, 22.00%, and 14.24% relative to salt stress alone. The improvement of stomatal size and the deposition of photosynthetic pigments were more favorable to enhancing photosynthesis in leaves when these seedlings treated with MT application under salt stress, their stomatal size, chlorophyll content, and net photosynthetic rate averaged increased by 11.60%, 19.64%, and 27.62%. Additionally, Gene expression analysis showed that MT stimulation significantly increased the expression of antioxidant enzyme genes (Zm00001d009990, Zm00001d047479, Zm00001d014848, and Zm00001d007234) and photosynthetic pigment biosynthesis genes (Zm00001d011819 and Zm00001d017766) under salt stress. At the same time, 150 μM MT significantly promoted seedling growth and biomass accumulation. In conclusion, our study may unravel crucial evidence of the role of MT in maize seedlings against salt stress, which can provide a novel strategy for improving maize salt stress resistance.

Chromatin accessibility and post-transcriptional histone modifications play important roles in gene expression regulation. However, little is known about the joint effect of multiple chromatin modifications on the gene expression level in plants, despite that the regulatory roles of individual histone marks such as H3K4me3 in gene expression have been well-documented. By using machine-learning methods, we systematically performed gene expression level prediction based on multiple chromatin modifications data in Arabidopsis and rice. We found that as few as four histone modifications were sufficient to yield good prediction performance, and H3K4me3 and H3K36me3 being the top two predictors with known functions related to transcriptional initiation and elongation, respectively. We demonstrated that the predictive powers differed between protein-coding and non-coding genes as well as between CpG-enriched and CpG-depleted genes. We also showed that the predictive model trained in one tissue or species could be applied to another tissue or species, suggesting shared underlying mechanisms. More interestingly, the gene expression levels of conserved orthologs are easier to predict than the species-specific genes. In addition, chromatin state of distal enhancers was moderately correlated to gene expression but was dispensable if given the chromatin features of the proximal regions of genes. We further extended the analysis to transcription factor (TF) binding data. Strikingly, the combinatorial effects of only a few TFs were roughly fit to gene expression levels in Arabidopsis. Overall, by using quantitative modeling, we provide a comprehensive and unbiased perspective on the epigenetic and TF-mediated regulation of gene expression in plants.

Low-temperature (LT) is one of the major abiotic stresses that restrict the growth and development of maize seedlings. Brassinolides (BRs) have been shown to enhance LT tolerance in several plant species; the physiological and molecular mechanisms by which BRs enhance maize tolerance are still unclear. Here, we characterized changes in the physiology and transcriptome of N192 and Ji853 seedlings at the three-leaf stage with or without 2 μM 2,4-epibrassinolide (EBR) application at 25 and 15 °C environments via high-performance liquid chromatography and RNA-Sequencing. Physiological analyses revealed that EBR increased the antioxidant enzyme activities, enhanced the cell membrane stability, decreased the malondialdehyde formation, and inhibited the reactive oxygen species (ROS) accumulation in maize seedlings under 15 °C stress; meanwhile, EBR also maintained hormone balance by increasing indole-3-acetic acid and gibberellin 3 contents and decreasing the abscisic acid level under stress. Transcriptome analysis revealed 332 differentially expressed genes (DEGs) enriched in ROS homeostasis, plant hormone signal transduction, and the mitogen-activated protein kinase (MAPK) cascade. These DEGs exhibited synergistic and antagonistic interactions, forming a complex LT tolerance network in maize. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that 109 hub genes involved in LT stress regulation pathways were discovered from the four modules with the highest correlation with target traits. In conclusion, our findings provide new insights into the molecular mechanisms of exogenous BRs in enhancing LT tolerance of maize at the seedling stage, thus opening up possibilities for a breeding program of maize tolerance to LT stress.

Cereal/legume intercropping enhances legume nodulation and improves nitrogen use efficiency (NUE) in cereal crops. This facilitation of symbiotic nitrogen fixation (SNF) in intercropped legumes involves a complex eco-physiological mechanism driven by multiple factors. Among them, interspecific root interactions (IRIs) are a key factor influencing SNF in intercropped legumes. Currently, it remains unclear whether and how IRIs modulate SNF to affect NUE and yield formation in legume species. In this study, maize/pea intercropping with different types of root separation [no barrier (NB) and plastic barrier (PB)] and pea monocropping (IP) were simulated in a nitrogen (N)-free nutrient matrix in pots, and the SNF, N metabolism, and N partitioning were investigated. We demonstrated that IRIs optimize SNF performance. N assimilation is positively regulated following increases in enzyme activity and gene expression in intercropped roots and nodules. Furthermore, IRIs facilitate amino acid (AA) export from nodules to roots and shoots, which is followed by an increase in AA levels in leaves (source) and leaf exudates (sink). Overall, intensive SNF drives N metabolism and alters source-to-sink N partitioning, thereby increasing NUE (by 23%) and yield (by 15%) in intercropped pea. This study reveals the positive roles of IRIs to the NUE and yield and provides useful reference material for increasing N contents derived from SNF to maximize NUE and crop yields in intercropped legumes.