In the signal transduction network, from the perception of stress signals to stress-responsive gene expression, various transcription factors and cis-regulatory elements in stress-responsive promoters coordinate plant adaptation to abiotic stresses. Among the AP2/ERF transcription factor family, group VII ERF (ERF-VII) genes, such as RAP2.12, RAP2.2, RAP2.3, AtERF73/HRE1, and AtERF71/HRE2, are known to be involved in the response to hypoxia in Arabidopsis. Notably, HRE2 has been reported to be involved in responses to hypoxia and osmotic stress. In this study, we dissected HRE2 promoter to identify hypoxia- and salt stress-responsive region(s). The analysis of the promoter deletion series of HRE2 using firefly luciferase and GUS as reporter genes indicated that the −116 to −2 region is responsible for both hypoxia and salt stress responses. Using yeast one-hybrid screening, we isolated HAT22/ABIG1, a member of the HD-Zip II subfamily, which binds to the −116 to −2 region of HRE2 promoter. Interestingly, HAT22/ABIG1 repressed the transcription of HRE2 via the EAR motif located in the N-terminal region of HAT22/ABIG1. HAT22/ABIG1 bound to the 5′-AATGATA-3′ sequence, HD-Zip II-binding-like cis-regulatory element, in the −116 to −2 region of HRE2 promoter. Our findings demonstrate that the −116 to −2 region of HRE2 promoter contains both positive and negative cis-regulatory elements, which may regulate the expression of HRE2 in responses to hypoxia and salt stress and that HAT22/ABIG1 negatively regulates HRE2 transcription by binding to the HD-Zip II-binding-like element in the promoter region.

Plant roots show distinct gene-expression profiles from those of shoots under abiotic stress conditions. In this study, we performed mRNA sequencing (mRNA-Seq) to analyze the transcriptional profiling of Arabidopsis roots under osmotic stress conditions—high salinity (NaCl) and drought (mannitol). The roots demonstrated significantly distinct gene-expression changes from those of the aerial parts under both the NaCl and the mannitol treatment. We identified 68 closely connected transcription-factor genes involved in osmotic stress-signal transduction in roots. Well-known abscisic acid (ABA)-dependent and/or ABA-independent osmotic stress-responsive genes were not considerably upregulated in the roots compared to those in the aerial parts, indicating that the osmotic stress response in the roots may be regulated by other uncharacterized stress pathways. Moreover, we identified 26 osmotic-stress-responsive genes with distinct expressions of alternative splice variants in the roots. The quantitative reverse-transcription polymerase chain reaction further confirmed that alternative splice variants, such as those for ANNAT4, MAGL6, TRM19, and CAD9, were differentially expressed in the roots, suggesting that alternative splicing is an important regulatory mechanism in the osmotic stress response in roots. Altogether, our results suggest that tightly connected transcription-factor families, as well as alternative splicing and the resulting splice variants, are involved in the osmotic stress response in roots.

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.

CCCH zinc finger proteins are a large protein family and are classified as either tandem CCCH zinc finger (TZF) or non-TZF proteins. The roles of TZF genes in several plants have been well determined, whereas the functions of many non-TZF genes in plants remain uncharacterized. Herein, we describe biological and molecular functions of AtC3H12, an Arabidopsis non-TZF protein containing three CCCH zinc finger motifs. AtC3H12 has orthologs in several plant species but has no paralog in Arabidopsis. AtC3H12-overexpressing transgenic plants (OXs) germinated slower than wild-type (WT) plants, whereas atc3h12 mutants germinated faster than WT plants. The fresh weight (FW) and primary root lengths of AtC3H12 OX seedlings were lighter and shorter than those of WT seedlings, respectively. In contrast, FW and primary root lengths of atc3h12 seedlings were heavier and longer than those of WT seedlings, respectively. AtC3H12 was localized in the nucleus and displayed transactivation activity in both yeast and Arabidopsis. We found that the 97–197 aa region of AtC3H12 is an important part for its transactivation activity. Detection of expression levels and analysis of Arabidopsis transgenic plants harboring a P_AtC3H12::GUS construct showed that AtC3H12 expression increases as the Arabidopsis seedlings develop. Taken together, our results demonstrate that AtC3H12 negatively affects seed germination and seedling development as a nuclear transcriptional activator in Arabidopsis. To our knowledge, this is the first report to show that non-TZF proteins negatively affect plant development as nuclear transcriptional activators.

Salinity causes widespread crop loss and prompts plants to adapt through changes in gene expression. In this study, we aimed to investigate the function of the non-tandem CCCH zinc-finger (non-TZF) protein gene AtC3H3 in response to salt stress in Arabidopsis. AtC3H3, a gene from the non-TZF gene family known for its RNA-binding and RNase activities, was up-regulated under osmotic stress, such as high salt and drought. When overexpressed in Arabidopsis, AtC3H3 improved tolerance to salt stress, but not drought stress. The expression of well-known abscisic acid (ABA)-dependent salt stress-responsive genes, namely Responsive to Desiccation 29B (RD29B), RD22, and Responsive to ABA 18 (RAB18), and representative ABA-independent salt stress-responsive genes, namely Dehydration-Responsive Element Binding protein 2A (DREB2A) and DREB2B, was significantly higher in AtC3H3-overexpressing transgenic plants (AtC3H3 OXs) than in wild-type plants (WT) under NaCl treatment, indicating its significance in both ABA-dependent and -independent signal transduction pathways. mRNA-sequencing (mRNA-Seq) analysis using NaCl-treated WT and AtC3H3 OXs revealed no potential target mRNAs for the RNase function of AtC3H3, suggesting that the potential targets of AtC3H3 might be noncoding RNAs and not mRNAs. Through this study, we conclusively demonstrated that AtC3H3 plays a crucial role in salt stress tolerance by influencing the expression of salt stress-responsive genes. These findings offer new insights into plant stress response mechanisms and suggest potential strategies for improving crop resilience to salinity stress.

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.