The transcription factor ThDOF8 binds to a novel cis-element and mediates molecular responses to salt stress in Tamarix hispida.

Salt stress is a common abiotic factor that restricts plant growth and development. As a halophyte, Tamarix hispida is a good model plant for exploring salt-tolerance genes and regulatory mechanisms. DNA-binding with one finger (DOF) is an important transcription factor (TF) that influences and controls various signaling substances involved in diverse biological processes related to plant growth and development, but the regulatory mechanisms of DOF TFs in response to salt stress are largely unknown in T. hispida. In the present study, a newly identified Dof gene, ThDOF8, was cloned from T. hispida, and its expression was found to be induced by salt stress. Transient overexpression of ThDOF8 enhanced T. hispida salt tolerance by enhancing proline levels, and increasing the activities of the antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD). These results were also verified in stably transformed Arabidopsis. Results from TF-centered yeast one-hybrid (Y1H) assays and EMSAs showed that ThDOF8 binds to a newly identified cis-element (TGCG). Expression profiling by gene chip analysis identified four potential direct targets of ThDOF8, namely the cysteine-rich receptor-like kinases genes, CRK10 and CRK26, and two glutamate decarboxylase genes, GAD41, and GAD42, and these were further verified by ChIP-quantitative-PCR, EMSAs, Y1H assays, and ß-glucuronidase enzyme activity assays. ThDOF8 can bind to the TGCG element in the promoter regions of its target genes, and transient overexpression of ThCRK10 also enhanced T. hispida salt tolerance. On the basis of our results, we propose a new regulatory mechanism model, in which ThDOF8 binds to the TGCG cis-element in the promoter of the target gene CRK10 to regulate its expression and improve salt tolerance in T. hispida. This study provides a basis for furthering our understanding the role of DOF TFs and identifying other downstream candidate genes that have the potential for improving plant salt tolerance via molecular breeding.

BpGRP1 acts downstream of BpmiR396c/BpGRF3 to confer salt tolerance in Betula platyphylla.

Glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, but the function of GRP genes involved in salt stress and the underlying mechanism remain unclear. In this study, we identified BpGRP1 (glycine-rich RNA-binding protein), a Betula platyphylla gene that is induced under salt stress. The physiological and molecular responses to salt tolerance were investigated in both BpGRP1-overexpressing and suppressed conditions. BpGRF3 (growth-regulating factor 3) was identified as a regulatory factor upstream of BpGRP1. We demonstrated that overexpression of BpGRF3 significantly increased the salt tolerance of birch, whereas the grf3-1 mutant exhibited the opposite effect. Further analysis revealed that BpGRF3 and its interaction partner, BpSHMT, function upstream of BpGRP1. We demonstrated that BpmiR396c, as an upstream regulator of BpGRF3, could negatively regulate salt tolerance in birch. Furthermore, we uncovered evidence showing that the BpmiR396c/BpGRF3 regulatory module functions in mediating the salt response by regulating the associated physiological pathways. Our results indicate that BpmiR396c regulates the expression of BpGRF3, which plays a role in salt tolerance by targeting BpGRP1.

Bp-miR408a participates in osmotic and salt stress responses by regulating BpBCP1 in Betula platyphylla.

The microRNAs, which are small RNAs of 18-25 nt in length, act as key regulatory factors in posttranscriptional gene expression during plant growth and development. However, little is known about their regulatory roles in response to stressful environments in birch (Betula platyphylla). Here, we characterized and further explored miRNAs from osmotic- and salt-stressed birch. Our analysis revealed a total of 190 microRNA (miRNA) sequences, which were classified into 180 conserved miRNAs and 10 predicted novel miRNAs based on sequence homology. Furthermore, we identified Bp-miR408a under osmotic and salt stress and elucidated its role in osmotic and salt stress responses in birch. Notably, under osmotic and salt stress, Bp-miR408a contributed to osmotic and salt tolerance sensitivity by mediating various physiological changes, such as increases in reactive oxygen species accumulation, osmoregulatory substance contents and Na+ accumulation. Additionally, molecular analysis provided evidence of the in vivo targeting of BpBCP1 (blue copper protein) transcripts by Bp-miR408a. The overexpression of BpBCP1 in birch enhanced osmotic and salt tolerance by increasing the antioxidant enzyme activity, maintaining cellular ion homeostasis and decreasing lipid peroxidation and cell death. Thus, we reveal a Bp-miR408a-BpBCP1 regulatory module that mediates osmotic and salt stress responses in birch.

Comparative transcriptomic and metabolomic analyses provide insights into the responses to NaCl and Cd stress in Tamarix hispida.

Salinity and heavy metal pollution seriously affect plant growth. Tamarix hispida (T. hispida) has the potential to remediate soil saline-alkali and heavy metal pollution. In this study, the response mechanisms of T. hispida under NaCl, CdCl2 (Cd) and combined CdCl2 and NaCl (Cd-NaCl) stresses were explored. Overall, the antioxidant system showed changes under the three stresses. The addition of NaCl inhibited the absorption of Cd2+. However, there were obvious differences in the transcripts and metabolites identified among the three stress responses. Interestingly, the number of DEGs was greatest under NaCl stress (929), but the number of differentially expressed metabolites (DEMs) was lowest (48), with 143 and 187 DEMs identified under Cd and Cd-NaCl stress, respectively. It is worth noting that both DEGs and DEMs were enriched in the linoleic acid metabolism pathway under Cd stress. In particular, the content of lipids changed significantly under Cd and Cd-NaCl stress, suggesting that maintaining normal lipid synthesis and metabolism may be an important way to improve the Cd tolerance of T. hispida. Flavonoids may also play an important role in the response to NaCl and Cd stress. These results provide a theoretical basis for cultivating plants with improved salt and cadmium repair abilities.

The R2R3-MYB transcription factor ThRAX2 recognized a new element MYB-T (CTTCCA) to enhance cadmium tolerance in Tamarix hispida.

R2R3-MYB transcription factors play an important role in plant development and response to various environmental stresses. In this study, a new R2R3-MYB gene, named ThRAX2, was isolated from T. hispida. ThRAX2 has an open reading frame (ORF) of 1191 bp and encodes a protein of 396 amino acids. ThRAX2 was localized in the nucleus. The overexpression of ThRAX2 in Arabidopsis and T. hispida significantly increased Cadmium (Cd) tolerance. Moreover, the accumulation of cadmium in roots and leaves was significantly reduced. The TF-centred Y1H and Y1H results showed that ThRAX2 was able to specifically bind a new cis-element (MYB-T, CTTCCA). The promoters of some Cd-responsive genes, such as ThSOS1, ThCKX3, ThCAX3A, ThMYB78, ThMIP2, ThTPS4, and ThSOD2, all contained 1-3 MYB-T sequences. Furthermore, chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) and ChIPquantitative (q)PCR showed that the ThRAX2 gene can bind to ThSOS1, ThCKX3, ThCAX3A and ThMYB78 promoter fragments, including the MYB-T motif. Meanwhile, the qRTPCR results also showed that the expression trends of ThSOS1, ThCKX3, ThCAX3A and ThMYB78 were similar to that of ThRAX2. This finding suggests that Cd tolerance of the ThRAX2 gene may regulate the expression of some downstream genes through specific recognition of the MYB-T motif and participate in regulating intracellular ion homeostasis, transport, and protein activity or enhance antioxidant enzyme activity. This study found a novel cis-acting element that binds ThRAX2 to regulate Cd tolerance, which lays the foundation for the ThRAX2 regulatory mechanism of Cd stress. This study provides a genetic and theoretical basis for the bioremediation of Cd-contaminated land by cultivating transgenic plants in the future.

The multilayered hierarchical gene regulatory network reveals interaction of transcription factors in response to cadmium in Tamarix hispida roots.

Cadmium (Cd) is a toxic metal that affects the normal growth and development of plants. Roots may directly contact Cd and thus serve as the first barrier in the defense responses of plants. In this study, Tamarix hispida (T. hispida) roots treated with 150 µM CdCl2 were collected for RNA-seq. A total of 2004 differentially expressed genes (DEGs) were identified at different time points. Kyoto Encyclopedia of Genes and Genomes enrichment revealed that the DEGs were significantly enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis and other metabolic pathways. To explore the regulatory role of transcription factors (TFs) involved in the Cd stress response, a multilayer hierarchical gene regulatory network (ML-hGRN) was constructed, including 53 TFs and 54 structural genes in ML-hGRN, with 341 predicted regulatory relationships. Binding of DRE1A, MYC1, FEZ, ERF4 and ERF17 to predicted target genes was detected by ChIP-PCR, and DRE1A, MYC1 and FEZ were transiently overexpressed in T. hispida. The results suggest that these TFs play a key role in the Cd stress response by scavenging reactive oxygen species. In conclusion, this study predicts some Cd-responsive TFs that may have an important function under Cd stress and provides useful information for molecular breeding.

Transcriptomic Analysis of Cadmium Stressed Tamarix hispida Revealed Novel Transcripts and the Importance of Abscisic Acid Network.

Cadmium (Cd) pollution is widely detected in soil and has been recognized as a major environmental problem. Tamarix hispida is a woody halophyte, which can form natural forest on the desert and soil with 0.5 to 1% salt content, making it an ideal plant for the research on response to abiotic stresses. However, no systematic study has investigated the molecular mechanism of Cd tolerance in T. hispida. In the study, RNA-seq technique was applied to analyze the transcriptomic changes in T. hispida treated with 150 µmol L-1 CdCl2 for 24, 48, and 72 h compared with control. In total, 72,764 unigenes exhibited similar sequences in the Non-redundant nucleic acid database (NR database), while 36.3% of all these unigenes may be new transcripts. In addition, 6,778, 8,282, and 8,601 DEGs were detected at 24, 48, and 72 h, respectively. Functional annotation analysis indicated that many genes may be involved in Cd stress response, including ion bonding, signal transduction, stress sensing, hormone responses and ROS metabolism. A ThUGT gene from the abscisic acid (ABA) signaling pathway can enhance Cd resistance ability of T. hispida by regulating the production of ROS under Cd stress and inhibit absorption of Cd. The new transcriptome resources and data that we present in this study for T. hispida may facilitate investigation of molecular mechanisms governing Cd resistance.