DNA-Binding Activity of CAMTA3 Is Essential for Its Function: Identification of Critical Amino Acids for Its Transcriptional Activity.

Calmodulin-binding transcription activators (CAMTAs), a small family of highly conserved transcription factors, function in calcium-mediated signaling pathways. Of the six CAMTAs in Arabidopsis, CAMTA3 regulates diverse biotic and abiotic stress responses. A recent study has shown that CAMTA3 is a guardee of NLRs (Nucleotide-binding, Leucine-rich repeat Receptors) in modulating plant immunity, raising the possibility that CAMTA3 transcriptional activity is dispensable for its function. Here, we show that the DNA-binding activity of CAMTA3 is essential for its role in mediating plant immune responses. Analysis of the DNA-binding (CG-1) domain of CAMTAs in plants and animals showed strong conservation of several amino acids. We mutated six conserved amino acids in the CG-1 domain to investigate their role in CAMTA3 function. Electrophoretic mobility shift assays using these mutants with a promoter of its target gene identified critical amino acid residues necessary for DNA-binding activity. In addition, transient assays showed that these residues are essential for the CAMTA3 function in activating the Rapid Stress Response Element (RSRE)-driven reporter gene expression. In line with this, transgenic lines expressing the CG-1 mutants of CAMTA3 in the camta3 mutant failed to rescue the mutant phenotype and restore the expression of CAMTA3 downstream target genes. Collectively, our results provide biochemical and genetic evidence that the transcriptional activity of CAMTA3 is indispensable for its function.

The amino acid region from 448-517 of CAMTA3 transcription factor containing a part of the TIG domain represses the N-terminal repression module function.

CAMTA3, a Ca2+-regulated transcription factor, is a repressor of plant immune responses. A truncated version of CAMTA3; CAMTA3334 called N-terminal repression module (NRM), and its extended version (CAMTA447), which include the DNA binding domain, were previously reported to complement the camta3/2 mutant phenotype. Here, we generated a series of CAMTA3 truncated versions [the N-terminus (aa 1-517), C-terminus (aa 517-1032), R1 (aa 1-173), R2 (aa 174-345), R3 (aa 346-517), R4 (aa 517-689), R5 (aa 690-861) and R6 (aa 862-1032)], expressed in camta3 mutant and analyzed the phenotypes of the transgenic lines. Interestingly, unlike CAMTA447, extending the N-terminal region to 517 aa did not complement the camta3 phenotype, suggesting that the amino acid region from 448-517 (70 aa), which includes a part of the TIG domain suppresses the NRM activity. The C-terminus and other truncated versions (R1-R6) also failed to complement the camta3 mutant. Expressing the full length or NRM of CAMTA3 in camta3 plants suppressed the activation of immune-responsive genes and increased the expression of cold-induced genes. In contrast, the transgenic lines expressing the N- or C-terminus or R1-R6 of CAMTA3 showed expression patterns like those of the camta3 with enhanced expression of the defense genes and suppressed expression of the cold response genes. Furthermore, like camta3, the transgenic lines expressing the N- or C-terminus, or R1-R6 of CAMTA3 exhibited higher levels of H2O2 and increased resistance to a Pst DC3000 as compared to WT, NRM, or FL-CAMTA3 transgenic plants. Our studies identified a novel regulatory region in CAMTA3 that suppresses the NRM activity. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01401-w.

Salt-Induced Stability of SR1/CAMTA3 mRNA Is Mediated by Reactive Oxygen Species and Requires the 3' End of Its Open Reading Frame.

Soil salinity, a prevalent abiotic stress, causes enormous losses in global crop yields annually. Previous studies have shown that salt stress-induced reprogramming of gene expression contributes to the survival of plants under this stress. However, mechanisms regulating gene expression in response to salt stress at the posttranscriptional level are not well understood. In this study, we show that salt stress increases the level of Signal Responsive 1 (SR1) mRNA, a member of signal-responsive Ca2+/calmodulin-regulated transcription factors, by enhancing its stability. We present multiple lines of evidence indicating that reactive oxygen species generated by NADPH oxidase activity mediate salt-induced SR1 transcript stability. Using mutants impaired in either nonsense-mediated decay, XRN4 or mRNA decapping pathways, we show that neither the nonsense-mediated mRNA decay pathway, XRN4 nor the decapping of SR1 mRNA is required for its decay. We analyzed the salt-induced accumulation of eight truncated versions of the SR1 coding region (∼3 kb) in the sr1 mutant background. This analysis identified a 500-nt region at the 3' end of the SR1 coding region to be required for the salt-induced stability of SR1 mRNA. Potential mechanisms by which this region confers SR1 transcript stability in response to salt are discussed.

Global gene expression analysis using RNA-seq uncovered a new role for SR1/CAMTA3 transcription factor in salt stress.

Abiotic and biotic stresses cause significant yield losses in all crops. Acquisition of stress tolerance in plants requires rapid reprogramming of gene expression. SR1/CAMTA3, a member of signal responsive transcription factors (TFs), functions both as a positive and a negative regulator of biotic stress responses and as a positive regulator of cold stress-induced gene expression. Using high throughput RNA-seq, we identified ~3000 SR1-regulated genes. Promoters of about 60% of the differentially expressed genes have a known DNA binding site for SR1, suggesting that they are likely direct targets. Gene ontology analysis of SR1-regulated genes confirmed previously known functions of SR1 and uncovered a potential role for this TF in salt stress. Our results showed that SR1 mutant is more tolerant to salt stress than the wild type and complemented line. Improved tolerance of sr1 seedlings to salt is accompanied with the induction of salt-responsive genes. Furthermore, ChIP-PCR results showed that SR1 binds to promoters of several salt-responsive genes. These results suggest that SR1 acts as a negative regulator of salt tolerance by directly repressing the expression of salt-responsive genes. Overall, this study identified SR1-regulated genes globally and uncovered a previously uncharacterized role for SR1 in salt stress response.