Genome-wide analysis of WRKY gene family and the dynamic responses of key WRKY genes involved in cadmium stress in Brassica juncea.

The WRKY transcription factors comprise one of the most extensive gene families and serve as pivotal regulators of plant responses to heavy metal stress. They contribute significantly to maintaining plant growth and development by enhancing plant tolerance. However, research on the role of WRKY genes in response to cadmium (Cd) stress in mustard is minimal. In this study, we conducted a genome-wide analysis of the mustard WRKY gene family using bioinformatics. The results revealed that 291 WRKY putative genes (BjuWRKYs) were identified in the mustard genome. These genes were categorized into seven subgroups (I, IIa-e and III) through phylogenetic analysis, with differences in motif composition between each subgroup. Homology analysis indicated that 31.62% of the genes originated from tandem duplication events. Promoter analysis revealed an abundance of abiotic stress-related elements and hormone-related elements within the BjuWRKY genes. Transcriptome analysis demonstrated that most BjuWRKY genes exhibited differential expression patterns at different Cd treatment stages in mustard. Furthermore, 10 BjuWRKY genes were confirmed to respond to Cd stress through the construction of a BjuWRKY protein interaction network, prediction of hub genes, and real-time fluorescence quantitative PCR analysis, indicating their potential involvement in Cd stress. Our findings provide a comprehensive insight into the WRKY gene family in mustard and establish a foundation for further studies of the functional roles of BjuWRKY genes in Cd stress response.

Genome-Wide Analysis of the Nramp Gene Family in Kenaf (Hibiscus cannabinus): Identification, Expression Analysis, and Response to Cadmium Stress.

Kenaf (Hibiscus cannabinu) is a grass bast fiber crop that has the ability to tolerate and accumulate heavy metals, and it has been considered as a potential heavy metal accumulator and remediation plant. Nramp is a natural resistance-related macrophage, which plays an important role in the transport of divalent metal ions, plant growth and development, and abiotic stress. In this study, the Nramp gene family of kenaf was analyzed at the whole genome level. A total of 15 HcNramp genes were identified. They are distributed unevenly on chromosomes. Phylogenetic analysis classified 15 HcNramp proteins into 3 different subfamilies. All proteins share specific motif 4 and motif 6, and the genes belonging to the same subfamily are similar in structure and motif. The promoters are rich in hormone response, meristem expression, and environmental stress response elements. Under different treatments, the expression levels of HcNramp genes vary in different tissues, and most of them are expressed in roots first. These findings can provide a basis for understanding the potential role of the Nramp gene family in kenaf in response to cadmium (Cd) stress, and are of great significance for screening related Cd tolerance genes in kenaf.

SpCTP3 from the hyperaccumulator Sedum plumbizincicola positively regulates cadmium tolerance by interacting with SpMDH1.

Cadmium (Cd) is a heavy metal pollutant mainly originating from the discharge of industrial sewage, irrigation with contaminated water, and the use of fertilizers. The phytoremediation of Cd polluted soil depends on the identification of the associated genes in hyperaccumulators. Here, a novel Cd tolerance gene (SpCTP3) was identified in hyperaccumulator Sedum plumbizincicola. The results of Cd2+ binding and thermodynamic analyses, revealed the CXXC motif in SpCTP3 functions is a Cd2+ binding site. A mutated CXXC motif decreased binding to Cd by 59.93%. The subcellular localization analysis suggested that SpCTP3 is primarily a cytoplasmic protein. Additionally, the SpCTP3-overexpressing (OE) plants were more tolerant to Cd and accumulated more Cd than wild-type Sedum alfredii (NHE-WT). The Cd concentrations in the cytoplasm of root and leaf cells were significantly higher (53.75% and 71.87%, respectively) in SpCTP3-OE plants than in NHE-WT. Furthermore, malic acid levels increased and decreased in SpCTP3-OE and SpCTP3-RNAi plants, respectively. Moreover, SpCTP3 interacted with malate dehydrogenase 1 (MDH1). Thus, SpCTP3 helps regulate the subcellular distribution of Cd and increases Cd accumulation when it is overexpressed in plants, ultimately Cd tolerance through its interaction with SpMDH1. This study provides new insights relevant to improving the Cd uptake by Sedum plumbizincicola.

Enriched rhizospheric functional microbiome may enhance adaptability of Artemisia lavandulaefolia and Betula luminifera in antimony mining areas.

Dominant native plants are crucial for vegetation reconstruction and ecological restoration of mining areas, though their adaptation mechanisms in stressful environments are unclear. This study focuses on the interactions between dominant indigenous species in antimony (Sb) mining area, Artemisia lavandulaefolia and Betula luminifera, and the microbes in their rhizosphere. The rhizosphere microbial diversity and potential functions of both plants were analyzed through the utilization of 16S, ITS sequencing, and metabarcoding analysis. The results revealed that soil environmental factors, rather than plant species, had a more significant impact on the composition of the rhizosphere microbial community. Soil pH and moisture significantly affected microbial biomarkers and keystone species. Actinobacteria, Proteobacteria and Acidobacteriota, exhibited high resistance to Sb and As, and played a crucial role in the cycling of carbon, nitrogen (N), phosphorus (P), and sulfur (S). The genes participating in N, P, and S cycling exhibited metabolic coupling with those genes associated with Sb and As resistance, which might have enhanced the rhizosphere microbes' capacity to endure environmental stressors. The enrichment of these rhizosphere functional microbes is the combined result of dispersal limitations and deterministic assembly processes. Notably, the genes related to quorum sensing, the type III secretion system, and chemotaxis systems were significantly enriched in the rhizosphere of plants, especially in B. luminifera, in the mining area. The phylogenetic tree derived from the evolutionary relationships among rhizosphere microbial and chloroplast whole-genome resequencing results, infers both species especially B. luminifera, may have undergone co-evolution with rhizosphere microorganisms in mining areas. These findings offer valuable insights into the dominant native rhizosphere microorganisms that facilitate plant adaptation to environmental stress in mining areas, thereby shedding light on potential strategies for ecological restoration in such environments.

A novel gene SpCTP3 from the hyperaccumulator Sedum plumbizincicola redistributes cadmium and increases its accumulation in transgenic Populus × canescens.

A cadmium (Cd) tolerance protein (SpCTP3) involved in the Sedum plumbizincicola response to Cd stress was identified. However, the mechanism underlying the Cd detoxification and accumulation mediated by SpCTP3 in plants remains unclear. We compared wild-type (WT) and SpCTP3-overexpressing transgenic poplars in terms of Cd accumulation, physiological indices, and the expression profiles of transporter genes following with 100 µmol/L CdCl2. Compared with the WT, significantly more Cd accumulated in the above-ground and below-ground parts of the SpCTP3-overexpressing lines after 100 µmol/L CdCl2 treatment. The Cd flow rate was significantly higher in the transgenic roots than in the WT roots. The overexpression of SpCTP3 resulted in the subcellular redistribution of Cd, with decreased and increased Cd proportions in the cell wall and the soluble fraction, respectively, in the roots and leaves. Additionally, the accumulation of Cd increased the reactive oxygen species (ROS) content. The activities of three antioxidant enzymes (peroxidase, catalase, and superoxide dismutase) increased significantly in response to Cd stress. The observed increase in the titratable acid content in the cytoplasm might lead to the enhanced chelation of Cd. The genes encoding several transporters related to Cd2+ transport and detoxification were expressed at higher levels in the transgenic poplars than in the WT plants. Our results suggest that overexpressing SpCTP3 in transgenic poplar plants promotes Cd accumulation, modulates Cd distribution and ROS homeostasis, and decreases Cd toxicity via organic acids. In conclusion, genetically modifying plants to overexpress SpCTP3 may be a viable strategy for improving the phytoremediation of Cd-polluted soil.

Molecular insights into lignin biosynthesis on cadmium tolerance: Morphology, transcriptome and proteome profiling in Salix matsudana.

Soil pollution caused by cadmium (Cd) is a serious concern. Phytoremediation is a popular technology in the remediation of Cd-contaminated soil. Salix matsudana var. matsudana f. umbraculifera Rehd. has been characterized as a high Cd-accumulating and tolerant willow (HCW). Here, transcriptome and proteome profiling, along with morphology analyses were performed to explore molecular cross-talk involved in Cd tolerance. Our results showed that 73%- 83% of the Cd in roots accumulated in the cell walls and root xylem cell walls were significantly thickened. From transcriptome and proteome analysis, a total of 153 up-regulated differentially-expressed genes and 655 up-regulated differentially-expressed proteins were found in common between two comparison groups (1 d and 4 d vs. respective control). Furthermore, phenylpropanoid biosynthesis was identified as a key pathway in response to Cd stress. In this pathway, lignin biosynthesis genes or proteins were significantly up-regulated, and lignin content increased significantly in roots under Cd stress. Two Cd-induced genes cinnamoyl-CoA reductase 1 (SmCCR1) and cinnamyl alcohol dehydrogenase 7 (SmCAD7) from HCW increased the lignin content and enhanced Cd tolerance in transgenic poplar calli. These results lay the foundation for further clarifying the molecular mechanisms of Cd tolerance in woody plants.

MAPK Cascades and Transcriptional Factors: Regulation of Heavy Metal Tolerance in Plants.

In nature, heavy metal (HM) stress is one of the most destructive abiotic stresses for plants. Heavy metals produce toxicity by targeting key molecules and important processes in plant cells. The mitogen-activated protein kinase (MAPK) cascade transfers the signals perceived by cell membrane surface receptors to cells through phosphorylation and dephosphorylation and targets various effector proteins or transcriptional factors so as to result in the stress response. Signal molecules such as plant hormones, reactive oxygen species (ROS), and nitric oxide (NO) can activate the MAPK cascade through differentially expressed genes, the activation of the antioxidant system and synergistic crosstalk between different signal molecules in order to regulate plant responses to HMs. Transcriptional factors, located downstream of MAPK, are key factors in regulating plant responses to heavy metals and improving plant heavy metal tolerance and accumulation. Thus, understanding how HMs activate the expression of the genes related to the MAPK cascade pathway and then phosphorylate those transcriptional factors may allow us to develop a regulation network to increase our knowledge of HMs tolerance and accumulation. This review highlighted MAPK pathway activation and responses under HMs and mainly focused on the specificity of MAPK activation mediated by ROS, NO and plant hormones. Here, we also described the signaling pathways and their interactions under heavy metal stresses. Moreover, the process of MAPK phosphorylation and the response of downstream transcriptional factors exhibited the importance of regulating targets. It was conducive to analyzing the molecular mechanisms underlying heavy metal accumulation and tolerance.

Genome-Wide Characterization of Sedum plumbizincicola HMA Gene Family Provides Functional Implications in Cadmium Response.

Heavy-metal ATPase (HMA), an ancient family of transition metal pumps, plays important roles in the transmembrane transport of transition metals such as Cu, Zn, Cd, and Co. Although characterization of HMAs has been conducted in several plants, scarcely knowledge was revealed in Sedum plumbizincicola, a type of cadmium (Cd) hyperaccumulator found in Zhejiang, China. In this study, we first carried out research on genome-wide analysis of the HMA gene family in S. plumbizincicola and finally identified 8 SpHMA genes and divided them into two subfamilies according to sequence alignment and phylogenetic analysis. In addition, a structural analysis showed that SpHMAs were relatively conserved during evolution. All of the SpHMAs contained the HMA domain and the highly conserved motifs, such as DKTGT, GDGxNDxP, PxxK S/TGE, HP, and CPx/SPC. A promoter analysis showed that the majority of the SpHMA genes had cis-acting elements related to the abiotic stress response. The expression profiles showed that most SpHMAs exhibited tissue expression specificity and their expression can be regulated by different heavy metal stress. The members of Zn/Co/Cd/Pb subgroup (SpHMA1-3) were verified to be upregulated in various tissues when exposed to CdCl2. Here we also found that the expression of SpHMA7, which belonged to the Cu/Ag subgroup, had an upregulated trend in Cd stress. Overexpression of SpHMA7 in transgenic yeast indicated an improved sensitivity to Cd. These results provide insights into the evolutionary processes and potential functions of the HMA gene family in S. plumbizincicola, laying a theoretical basis for further studies on figuring out their roles in regulating plant responses to biotic/abiotic stresses.