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.

Arabidopsis AtERF71/HRE2 functions as transcriptional activator via cis-acting GCC box or DRE/CRT element and is involved in root development through regulation of root cell expansion.

KEY MESSAGE: AtERF71/HRE2 binds to GCC box or DRE/CRT as transcription activator and plays an important role in root development via root cell expansion regulation. AtERF71/HRE2 transcription factor, a member of the AP2/ERF family, plays a key role in the stress response. GCC box and DRE/CRT, both essential cis-acting elements, have been shown to be recognized by AP2/ERF family transcription factors. However, it remains unclear whether or not AtERF71/HRE2 directly interacts with GCC box and/or DRE/CRT. Here, we showed that AtERF71/HRE2 binds to GCC box and DRE/CRT by electrophoretic mobility shift assay (EMSA). Binding of AtERF71/HRE2 to GCC box and DRE/CRT was also detected by fluorescence measurement and surface plasmon resonance spectroscopy (BIAcore) experiments. Folding properties of AtERF71/HRE2 proteins were characterized by CD spectroscopy, and AtERF71/HRE2 showed thermal stability as evidenced by two endothermic peaks (T d) at 53 and 65 °C. In addition, AtERF71/HRE2 showed transcriptional activation activity via GCC box and DRE/CRT in Arabidopsis protoplasts. Interestingly, AtERF71/HRE2 OXs showed increased primary root length due to elevated root cell expansion. Our data indicate that AtERF71/HRE2 binds to both GCC box and DRE/CRT, transactivates expression of genes downstream via GCC box or DRE/CRT, and plays an important role in root development through regulation of root cell expansion.

Arabidopsis Qc-SNARE gene AtSFT12 is involved in salt and osmotic stress responses and Na(+) accumulation in vacuoles.

KEY MESSAGE: AtSFT12, an Arabidopsis Qc-SNARE protein, is localized to Golgi organelles and is involved in salt and osmotic stress responses via accumulation of Na (+) in vacuoles. To reduce the detrimental effects of environmental stresses, plants have evolved many defense mechanisms. Here, we identified an Arabidopsis Qc-SNARE gene, AtSFT12, involved in salt and osmotic stress responses using an activation-tagging method. Both activation-tagged plants and overexpressing transgenic plants (OXs) of the AtSFT12 gene were tolerant to high concentrations of NaCl, LiCl, and mannitol, whereas loss-of-function mutants were sensitive to NaCl, LiCl, and mannitol. AtSFT12 transcription increased under NaCl, ABA, cold, and mannitol stresses but not MV treatment. GFP-fusion AtSFT12 protein was juxtaposed with Golgi marker, implying that its function is associated with Golgi-mediated transport. Quantitative measurement of Na(+) using induced coupled plasma atomic emission spectroscopy revealed that AtSFT12 OXs accumulated significantly more Na(+) than WT plants. In addition, Na(+)-dependent fluorescence analysis of Sodium Green showed comparatively higher Na(+) accumulation in vacuoles of AtSFT12 OX cells than in those of WT plant cells after salt treatments. Taken together, our findings suggest that AtSTF12, a Golgi Qc-SNARE protein, plays an important role in salt and osmotic stress responses and functions in the salt stress response via sequestration of Na(+) in vacuoles.

Arabidopsis HRE1α, a splicing variant of AtERF73/HRE1, functions as a nuclear transcription activator in hypoxia response and root development.

KEY MESSAGE: HRE1α shows transcriptional activation activity in its C-terminal region via GCC box but not DRE/CRT and plays an important role in root development via root meristem cell division regulation. AtERF73/HRE1 protein, a member of the Arabidopsis AP2/ERF family, contains a conserved AP2/ERF DNA-binding domain. Here, we studied the molecular function of HRE1α, a splicing variant of AtERF73/HRE1, as well as its role in root development. HRE1α-overexpressing transgenic plants (OXs) showed tolerance to submergence. HRE1α showed transcriptional activation activity via GCC box but not DRE/CRT. The 121-211 aa region of HRE1α was responsible for the transcriptional activation activity, and the region was conserved among homologs of other species but was not found in other Arabidopsis proteins. HRE1α OXs showed increased primary root length due to elevated root cell division. Our results suggest that HRE1α functions as a transcription activator in the nucleus, and plays an important role in root development through regulation of root meristem cell division.

The Arabidopsis chloroplast protein S-RBP11 is involved in oxidative and salt stress responses.

S-RBP11, a chloroplast protein, which was isolated using activation tagging system, is shown to be the first Arabidopsis small RNA-binding group protein involved in oxidative and salt stress responses. Activation tagging is one of the most powerful tools in reverse genetics. In this study, we isolated S-RBP11, encoding a small RNA-binding protein in Arabidopsis, by salt-resistant activation tagging line screen and then characterized its function in the abiotic stress response. The isolated activation tagging line of S-RBP11 as well as transgenic plants overexpressing S-RBP11 showed increased tolerance to salt and MV stresses compared to WT plants, whereas s-rbp11 mutants were more sensitive to salt stresses. Transcription of S-RBP11 was elevated upon MV treatment but not NaCl or cold treatment. Interestingly, S-RBP11 protein was localized in the chloroplast and the N-terminal 34 amino acid region of S-RBP11 was necessary for its chloroplast targeting. Our results suggest that S-RBP11 is a chloroplast protein involved in the responses to salt and oxidative stresses.

AtERF71/HRE2 transcription factor mediates osmotic stress response as well as hypoxia response in Arabidopsis.

Various transcription factors are involved in the response to environmental stresses in plants. In this study, we characterized AtERF71/HRE2, a member of the Arabidopsis AP2/ERF family, as an important regulator of the osmotic and hypoxic stress responses in plants. Transcript level of AtERF71/HRE2 was highly increased by anoxia, NaCl, mannitol, ABA, and MV treatments. aterf71/hre2 loss-of-function mutants displayed higher sensitivity to osmotic stress such as high salt and mannitol, accumulating higher levels of ROS under high salt treatment. In contrast, AtERF71/HRE2-overexpressing transgenic plants showed tolerance to salt and mannitol as well as flooding and MV stresses, exhibiting lower levels of ROS under high salt treatment. AtERF71/HRE2 protein was localized in the nucleus, and the C-terminal region of AtERF71/HRE2 was required for transcription activation activity. Taken together, our results suggest that AtERF71/HRE2 might function as a transcription factor involved in the response to osmotic stress as well as hypoxia.