Non-TZF Protein AtC3H59/ZFWD3 Is Involved in Seed Germination, Seedling Development, and Seed Development, Interacting with PPPDE Family Protein Desi1 in Arabidopsis.

Despite increasing reports on the function of CCCH zinc finger proteins in plant development and stress response, the functions and molecular aspects of many non-tandem CCCH zinc finger (non-TZF) proteins remain uncharacterized. AtC3H59/ZFWD3 is an Arabidopsis non-TZF protein and belongs to the ZFWD subfamily harboring a CCCH zinc finger motif and a WD40 domain. In this study, we characterized the biological and molecular functions of AtC3H59, which is subcellularly localized in the nucleus. The seeds of AtC3H59-overexpressing transgenic plants (OXs) germinated faster than those of wild type (WT), whereas atc3h59 mutant seeds germinated slower than WT seeds. AtC3H59 OX seedlings were larger and heavier than WT seedlings, whereas atc3h59 mutant seedlings were smaller and lighter than WT seedlings. Moreover, AtC3H59 OX seedlings had longer primary root length than WT seedlings, whereas atc3h59 mutant seedlings had shorter primary root length than WT seedlings, owing to altered cell division activity in the root meristem. During seed development, AtC3H59 OXs formed larger and heavier seeds than WT. Using yeast two-hybrid screening, we isolated Desi1, a PPPDE family protein, as an interacting partner of AtC3H59. AtC3H59 and Desi1 interacted via their WD40 domain and C-terminal region, respectively, in the nucleus. Taken together, our results indicate that AtC3H59 has pleiotropic effects on seed germination, seedling development, and seed development, and interacts with Desi1 in the nucleus via its entire WD40 domain. To our knowledge, this is the first report to describe the biological functions of the ZFWD protein and Desi1 in Arabidopsis.

Investigation of a Novel Salt Stress-Responsive Pathway Mediated by Arabidopsis DEAD-Box RNA Helicase Gene AtRH17 Using RNA-Seq Analysis.

Previously, we reported that overexpression of AtRH17, an Arabidopsis DEAD-box RNA helicase gene, confers salt stress-tolerance via a pathway other than the well-known salt stress-responsive pathways. To decipher the salt stress-responsive pathway in AtRH17-overexpressing transgenic plants (OXs), we performed RNA-Sequencing and identified 397 differentially expressed genes between wild type (WT) and AtRH17 OXs. Among them, 286 genes were upregulated and 111 genes were downregulated in AtRH17 OXs relative to WT. Gene ontology annotation enrichment and KEGG pathway analysis showed that the 397 upregulated and downregulated genes are involved in various biological functions including secretion, signaling, detoxification, metabolic pathways, catabolic pathways, and biosynthesis of secondary metabolites as well as in stress responses. Genevestigator analysis of the upregulated genes showed that nine genes, namely, LEA4-5, GSTF6, DIN2/BGLU30, TSPO, GSTF7, LEA18, HAI1, ABR, and LTI30, were upregulated in Arabidopsis under salt, osmotic, and drought stress conditions. In particular, the expression levels of LEA4-5, TSPO, and ABR were higher in AtRH17 OXs than in WT under salt stress condition. Taken together, our results suggest that a high AtRH17 expression confers salt stress-tolerance through a novel salt stress-responsive pathway involving nine genes, other than the well-known ABA-dependent and ABA-independent pathways.

Overexpression of the DEAD-Box RNA Helicase Gene AtRH17 Confers Tolerance to Salt Stress in Arabidopsis.

Plants adapt to abiotic stresses by complex mechanisms involving various stress-responsive genes. Here, we identified a DEAD-box RNA helicase (RH) gene, AtRH17, in Arabidopsis, involved in salt-stress responses using activation tagging, a useful technique for isolating novel stress-responsive genes. AT895, an activation tagging line, was more tolerant than wild type (WT) under NaCl treatment during germination and seedling development, and AtRH17 was activated in AT895. AtRH17 possesses nine well-conserved motifs of DEAD-box RHs, consisting of motifs Q, I, Ia, Ib, and II-VI. Although at least 12 orthologs of AtRH17 have been found in various plant species, no paralog occurs in Arabidopsis. AtRH17 protein is subcellularily localized in the nucleus. AtRH17-overexpressing transgenic plants (OXs) were more tolerant to high concentrations of NaCl and LiCl compared with WT, but no differences from WT were detected among seedlings exposed to mannitol and freezing treatments. Moreover, in the mature plant stage, AtRH17 OXs were also more tolerant to NaCl than WT, but not to drought, suggesting that AtRH17 is involved specifically in the salt-stress response. Notably, transcriptions of well-known abscisic acid (ABA)-dependent and ABA-independent stress-response genes were similar or lower in AtRH17 OXs than WT under salt-stress treatments. Taken together, our findings suggest that AtRH17, a nuclear DEAD-box RH protein, is involved in salt-stress tolerance, and that its overexpression confers salt-stress tolerance via a pathway other than the well-known ABA-dependent and ABA-independent pathways.

Arabidopsis non-TZF gene AtC3H17 functions as a positive regulator in salt stress response.

Functional studies of CCCH-type zinc finger proteins in abiotic stress responses have largely focused on tandem CCCH-type zinc finger (TZF) genes, whereas the study of functional roles of non-TZF genes in abiotic stress responses has largely been neglected. Here, we investigated the functional roles of AtC3H17, a non-TZF gene of Arabidopsis, in salt stress responses. AtC3H17 expression significantly increased under NaCl, mannitol, and ABA treatments. AtC3H17-overexpressing transgenic plants (OXs) were more tolerant under NaCl and MV treatment conditions than the wild type (WT). atc3h17 mutants were more sensitive under NaCl and MV treatment conditions compared with the WT. The transcription of the salt stress-responsive genes in ABA-dependent pathway, such as RAB18, COR15A, and RD22, was significantly higher in AtC3H17 OXs than in WT both under NaCl-free condition and after NaCl treatment. Our results demonstrate that AtC3H17 functions as a positive regulator in salt stress response, via the up-regulation of ABA-dependent salt stress-response pathway.

Arabidopsis AtNAP functions as a negative regulator via repression of AREB1 in salt stress response.

MAIN CONCLUSION: AtNAP , an Arabidopsis NAC transcription factor family gene, functions as a negative regulator via transcriptional repression of AREB1 in salt stress response. AtNAP is an NAC family transcription factor in Arabidopsis and is known to be a positive regulator of senescence. However, its exact function and underlying molecular mechanism in stress responses are not well known. Here, we investigated functional roles of AtNAP in salt stress response. AtNAP expression significantly increased at the seedling stage, with higher expression in both shoots and roots under NaCl, mannitol, and ABA treatments. T-DNA insertional loss-of-function mutants of AtNAP were more tolerant to salt stress than wild type (WT), whereas AtNAP-overexpressing transgenic plants (OXs) were more sensitive to salt stress than WT during germination, seedling development, and mature plant stage. Transcript levels of stress-responsive genes in the ABA-dependent pathway, such as AREB1, RD20, and RD29B, were significantly higher and lower in atnap mutants and AtNAP OXs, respectively, than in WT under salt stress conditions, suggesting that AtNAP might negatively regulate the expression of those genes under salt stress conditions. Indeed, AtNAP repressed the promoter activity of AREB1 under normal and salt stress conditions. These results indicate that AtNAP functions as a negative regulator in the salt stress response. Our results, together with previous studies, suggest that AtNAP functions as a negative regulator in osmotic stress responses, whereas it functions as a positive regulator in senescence.