Soil salinity is a main abiotic stress in agriculture worldwide. The Na⁺/H⁺ antiporters (NHXs) play pivotal roles in intracellular Na⁺ excretion and vacuolar Na⁺ compartmentalization, which are important for plant salt stress resistance (SSR). However, few systematic analyses of NHXs has been reported in allotetraploid rapeseed so far. Here, a total of 18 full-length NHX homologs, representing seven subgroups (NHX1-NHX8 without NHX5), were identified in the rapeseed genome (A_nA_nC_nC_n). Number variations of BnaNHXs might indicate their significantly differential roles in the regulation of rapeseed SSR. BnaNHXs were phylogenetically divided into three evolutionary clades, and the members in the same subgroups had similar physiochemical characteristics, gene/protein structures, and conserved Na⁺ transport motifs. Darwin´s evolutionary pressure analysis suggested that BnaNHXs suffered from strong purifying selection. The cis-element analysis revealed the differential transcriptional regulation of NHXs between the model Arabidopsis and B. napus. Differential expression of BnaNHXs under salt stress, different nitrogen forms (ammonium and nitrate), and low phosphate indicated their potential involvement in the regulation of rapeseed SSR. Global landscapes of BnaNHXs will give an integrated understanding of their family evolution and molecular features, which will provide elite gene resources for the genetic improvement of plant SSR through regulating the NHX-mediated Na⁺ transport.

Background: Cadmium (Cd) is a heavy metal with high toxicity that severely inhibits wheat growth and development. Cd easily accumulates in wheat kernels and enters the human food chain. Genetic variation in the resistance to Cd toxicity found in wheat genotypes emphasizes the complex response architecture. Understanding the Cd resistance mechanisms is crucial for combating Cd phytotoxicity and meeting the increasing daily food demand.Results: Using two wheat genotypes (Cd resistant and sensitive genotypes T207 and S276, respectively) with differing root growth responses to Cd, we conducted comparative physiological and transcriptomic analyses and exogenous application tests to evaluate Cd detoxification mechanisms. S276 accumulated more H₂O₂, O₂⁻, and MDA than T207 under Cd toxicity. Catalase activity and levels of ascorbic acid (AsA) and glutathione (GSH) were greater, whereas superoxide dismutase (SOD) and peroxidase (POD) activities were lower in T207 than in S276. Transcriptomic analysis showed that the expression ofRBOHA,RBOHC, andRBOHEwas significantly increased under Cd toxicity, and two-thirds (22 genes) of the differentially expressedRBOHgenes had higher expression levels in S276 than inT207. Cd toxicity reshaped the transcriptional profiling of the genes involving the AsA-GSH cycle, and a larger proportion (74.25%) of the corresponding differentially expressed genes showed higher expression in T207 than S276. The combined exogenous application of AsA and GSH alleviated Cd toxicity by scavenging excess ROS and coordinately promoting root length and branching, especially in S276.Conclusions: The results indicated that the ROS homeostasis plays a key role in differential Cd resistance in wheat genotypes, and the AsA-GSH cycle fundamentally and vigorously influences wheat defense against Cd toxicity, providing insight into the physiological and transcriptional mechanisms underlying Cd detoxification.

Background: Nitrogen (N), referred to as a “life element”, is a macronutrient essential for optimal plant growth and yield production. Amino acid (AA) permease (AAP) genes play pivotal roles in root import, long-distance translocation, remobilization of organic amide-N from source organs to sinks, and other environmental stress responses. However, few systematic analyses of AAPs have been reported in Brassica napus so far. Results: In this study, we identified a total of 34 full-length AAP genes representing eight subgroups (AAP1–8) from the allotetraploid rapeseed genome (A_nA_nC_nC_n, 2n = 4x = 38). Great differences in the homolog number among the BnaAAP subgroups might indicate their significant differential roles in the growth and development of rapeseed plants. The BnaAAPs were phylogenetically divided into three evolutionary clades, and the members in the same subgroups had similar physiochemical characteristics, gene/protein structures, and conserved AA transport motifs. Darwin’s evolutionary analysis suggested that BnaAAPs were subjected to strong purifying selection pressure. Cis-element analysis showed potential differential transcriptional regulation of AAPs between the model Arabidopsis and B. napus. Differential expression of BnaAAPs under nitrate limitation, ammonium excess, phosphate shortage, boron deficiency, cadmium toxicity, and salt stress conditions indicated their potential involvement in diverse nutrient stress responses. Conclusions: The genome-wide identification of BnaAAPs will provide a comprehensive insight into their family evolution and AAP-mediated AA transport under diverse abiotic stresses. The molecular characterization of core AAPs can provide elite gene resources and contribute to the genetic improvement of crop stress resistance through the modulation of AA transport.

Improving crop nitrogen (N) limitation adaptation (NLA) is a core approach to enhance N use efficiency (NUE) and reduce N fertilizer application. Rapeseed has a high demand for N nutrients for optimal plant growth and seed production, but it exhibits low NUE. Epigenetic modification, such as DNA methylation and modification from small RNAs, is key to plant adaptive responses to various stresses. However, epigenetic regulatory mechanisms underlying NLA and NUE remain elusive in allotetraploid B. napus. In this study, we identified overaccumulated carbohydrate, and improved primary and lateral roots in rapeseed plants under N limitation, which resulted in decreased plant nitrate concentrations, enhanced root-to-shoot N translocation, and increased NUE. Transcriptomics and RT-qPCR assays revealed that N limitation induced the expression of NRT1.1, NRT1.5, NRT1.7, NRT2.1/NAR2.1, and Gln1;1, and repressed the transcriptional levels of CLCa, NRT1.8, and NIA1. High-resolution whole genome bisulfite sequencing characterized 5094 differentially methylated genes involving ubiquitin-mediated proteolysis, N recycling, and phytohormone metabolism under N limitation. Hypermethylation/hypomethylation in promoter regions or gene bodies of some key N-metabolism genes might be involved in their transcriptional regulation by N limitation. Genome-wide miRNA sequencing identified 224 N limitation-responsive differentially expressed miRNAs regulating leaf development, amino acid metabolism, and plant hormone signal transduction. Furthermore, degradome sequencing and RT-qPCR assays revealed the miR827-NLA pathway regulating limited N-induced leaf senescence as well as the miR171-SCL6 and miR160-ARF17 pathways regulating root growth under N deficiency. Our study provides a comprehensive insight into the epigenetic regulatory mechanisms underlying rapeseed NLA, and it will be helpful for genetic engineering of NUE in crop species through epigenetic modification of some N metabolism-associated genes.

Background: Salinity severely inhibit crop growth, yield, and quality worldwide. Allotetraploid rapeseed (Brassica napus L.), a major glycophyte oil crop, is susceptible to salinity. Understanding the physiological and molecular strategies of rapeseed salinity resistance is a promising and cost-effective strategy for developing highly resistant cultivars. Results: First, early leaf senescence was identified and root system growth was inhibited in rapeseed plants under severe salinity conditions. Electron microscopic analysis revealed that 200 mM NaCl induced fewer leaf trichomes and stoma, cell plasmolysis, and chloroplast degradation. Primary and secondary metabolite assays showed that salinity led to an obviously increased anthocyanin, osmoregulatory substances, abscisic acid, jasmonic acid, pectin, cellulose, reactive oxygen species, and antioxidant activity, and resulted in markedly decreased photosynthetic pigments, indoleacetic acid, cytokinin, gibberellin, and lignin. ICP-MS assisted ionomics showed that salinity significantly constrained the absorption of essential elements, including the nitrogen, phosphorus, potassium, calcium, magnesium, iron, mangnese, copper, zinc, and boron nutrients, and induced the increase in the sodium/potassium ratio. Genome-wide transcriptomics revealed that the differentially expressed genes were involved mainly in photosynthesis, stimulus response, hormone signal biosynthesis/transduction, and nutrient transport under salinity. Conclusions: The high-resolution salt-responsive gene expression profiling helped the efficient characterization of central members regulating plant salinity resistance. These findings might enhance integrated comprehensive understanding of the morpho-physiologic and molecular responses to salinity and provide elite genetic resources for the genetic modification of salinity-resistant crop species.

Background: Plants worldwide are often stressed by low Fe availability around the world, especially in aerobic soils. Therefore, the plant growth, seed yield, and quality of crop species are severely inhibited under Fe deficiency. Fe metabolism in plants is controlled by a series of complex transport, storage, and regulatory mechanisms in cells. Allohexaploid wheat (Triticum aestivum L.) is a staple upland crop species that is highly sensitive to low Fe stresses. Although some studies have been previously conducted on the responses of wheat plants to Fe deficiency, the key mechanisms underlying adaptive responses are still unclear in wheat due to its large and complex genome. Results: Transmission electron microscopy showed that the chloroplast structure was severely damaged under Fe deficiency. Paraffin sectioning revealed that the division rates of meristematic cells were reduced, and the sizes of elongated cells were diminished. ICP-MS-assisted ionmics analysis showed that low-Fe stress significantly limited the absorption of nutrients, including N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, and B nutrients. High-throughput transcriptome sequencing identified 378 and 2,619 genome-wide differentially expressed genes (DEGs) were identified in the shoots and roots between high-Fe and low-Fe conditions, respectively. These DEGs were mainly involved in the Fe chelator biosynthesis, ion transport, photosynthesis, amino acid metabolism, and protein synthesis. Gene coexpression network diagrams indicated that TaIRT1b-4A, TaNAS2-6D, TaNAS1a-6A, TaNAS1-6B, and TaNAAT1b-1D might function as key regulators in the adaptive responses of wheat plants to Fe deficiency. Conclusions: These results might help us fully understand the morpho-physiological and molecular responses of wheat plants to low-Fe stress, and provide elite genetic resources for the genetic modification of efficient Fe use.