Genome-Wide Analysis of Stress-Responsive Genes and Alternative Splice Variants in Arabidopsis Roots under Osmotic Stresses.

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.

Influence of black rice (Oryza sativa L.) bran derived anthocyanin-extract on growth rate, immunological response, and immune-antioxidant gene expression in Nile tilapia (Oreochromis niloticus) cultivated in a biofloc system.

This study investigated the effects of dietary supplementation with anthocyanin extracted from black rice bran (AR) on the growth rate, immunological response, and expression of immune and antioxidant genes in Nile tilapia raised in an indoor biofloc system. A total of 300 Nile tilapia fingerlings (15.14 ± 0.032 g) were maintained in 150 L tanks and acclimatized for two weeks. Five experimental AR diets (0, 1, 2, 4, and 8 g kg-1) with various anthocyanin doses were used to feed the fish. We observed that the growth and feed utilization of fish fed with different dietary AR levels increased significantly after eight weeks (p < 0.05). In addition, the serum immunity of fish fed AR diets was much greater than that of those fed non-AR diets (p < 0.05). However, there were little or no difference in between fish fed AR enriched diets and the control AR-free diet (p > 0.05). After eight weeks, fish fed AR-supplemented diets had significantly higher mRNA transcript levels in immune (interleukin [IL]-1, IL-8, and liposaccharide-binding protein [LBP]) and antioxidant (glutathione transferase-alpha [GST-α] and glutathione reductase [GSR]) genes compared to control fish fed the AR-free diet, with the greatest enhancement of mRNA transcript levels (in the case of IL-8 by up to about 5.8-fold) in the 4 g kg-1 AR diet. These findings suggest that dietary inclusion of AR extract from black rice bran at 4-8 g kg-1 could function as a herbal immunostimulant to enhance growth performance, feed consumption, and immunity in Nile tilapia.

AtERF71/HRE2, an Arabidopsis AP2/ERF Transcription Factor Gene, Contains Both Positive and Negative Cis-Regulatory Elements in Its Promoter Region Involved in Hypoxia and Salt Stress Responses.

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.

Non-TZF Transcriptional Activator AtC3H12 Negatively Affects Seed Germination and Seedling Development in Arabidopsis.

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 PAtC3H12::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.

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.

Two Alternative Splicing Variants of AtERF73/HRE1, HRE1α and HRE1ß, Have Differential Transactivation Activities in Arabidopsis.

AtERF73/HRE1 is an AP2/ERF transcription factor in Arabidopsis and has two distinct alternative splicing variants, HRE1α and HRE1ß. In this study, we examined the differences between the molecular functions of HRE1α and HRE1ß. We found that HRE1α and HRE1ß are both involved in hypoxia response and root development and have transactivation activity. Two conserved motifs in the C-terminal region of HRE1α and HRE1ß, EELL and LWSY-like, contributed to their transactivation activity, specifically the four E residues in the EELL motif and the MGLWS amino acid sequence at the end of the LWSY-like motif. The N-terminal region of HRE1ß also showed transactivation activity, mediated by the VDDG motif, whereas that of HRE1α did not. The transactivation activity of HRE1ß was stronger than that of HRE1α in Arabidopsis protoplasts. Both transcription factors transactivated downstream genes via the GCC box. RNA-sequencing analysis further supported that both HRE1α and HRE1ß might regulate gene expression associated with the hypoxia stress response, although they may transactivate different subsets of genes in downstream pathways. Our results, together with previous studies, suggested that HRE1α and HRE1ß differentially transactivate downstream genes in hypoxia response and root development in Arabidopsis.

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.

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 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.

AtC3H17, a Non-Tandem CCCH Zinc Finger Protein, Functions as a Nuclear Transcriptional Activator and Has Pleiotropic Effects on Vegetative Development, Flowering and Seed Development in Arabidopsis.

Despite increasing reports that CCCH zinc finger proteins function in plant development and stress responses, the functions and molecular aspects of many CCCH zinc finger proteins remain uncharacterized. Here, we characterized the biological and molecular functions of AtC3H17, a unique Arabidopsis gene encoding a non-tandem CCCH zinc finger protein. AtC3H17 was ubiquitously expressed throughout the life cycle of Arabidopsis plants and their organs. The rate and ratio of seed germination of atc3h17 mutants were slightly slower and lower, respectively, than those of the wild type (WT), whereas AtC3H17-overexpressing transgenic plants (OXs) showed an enhanced germination rate. atc3h17 mutant seedlings were smaller and lighter than WT seedlings while AtC3H17 OX seedlings were larger and heavier. In regulation of flowering time, atc3h17 mutants showed delayed flowering, whereas AtC3H17 OXs showed early flowering compared with the WT. In addition, overexpression of AtC3H17 affected seed development, displaying abnormalities compared with the WT. AtC3H17 protein was localized to the nucleus and showed transcriptional activation activity in yeast and Arabidopsis protoplasts. The N-terminal region of AtC3H17, containing a conserved EELR-like motif, was necessary for transcriptional activation activity, and the two conserved glutamate residues in the EELR-like motif played an important role in transcriptional activation activity. Real-time PCR and transactivation analyses showed that AtC3H17 might be involved in seed development via transcriptional activation of OLEO1, OLEO2 and CRU3. Our results suggest that AtC3H17 has pleiotropic effects on vegetative development such as seed germination and seedling growth, flowering and seed development, and functions as a nuclear transcriptional activator in Arabidopsis.

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.

Production of superoxide from Photosystem II in a rice (Oryza sativa L.) mutant lacking PsbS.

BACKGROUND: PsbS is a 22-kDa Photosystem (PS) II protein involved in non-photochemical quenching (NPQ) of chlorophyll fluorescence. Rice (Oryza sativa L.) has two PsbS genes, PsbS1 and PsbS2. However, only inactivation of PsbS1, through a knockout (PsbS1-KO) or in RNAi transgenic plants, results in plants deficient in qE, the energy-dependent component of NPQ. RESULTS: In studies presented here, under fluctuating high light, growth of young seedlings lacking PsbS is retarded, and PSII in detached leaves of the mutants is more sensitive to photoinhibitory illumination compared with the wild type. Using both histochemical and fluorescent probes, we determined the levels of reactive oxygen species, including singlet oxygen, superoxide, and hydrogen peroxide, in leaves and thylakoids. The PsbS-deficient plants generated more superoxide and hydrogen peroxide in their chloroplasts. PSII complexes isolated from them produced more superoxide compared with the wild type, and PSII-driven superoxide production was higher in the mutants. However, we could not observe such differences either in isolated PSI complexes or through PSI-driven electron transport. Time-course experiments using isolated thylakoids showed that superoxide production was the initial event, and that production of hydrogen peroxide proceeded from that. CONCLUSION: These results indicate that at least some of the photoprotection provided by PsbS and qE is mediated by preventing production of superoxide released from PSII under conditions of excess excitation energy.

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.

Arabidopsis MKKK20 is involved in osmotic stress response via regulation of MPK6 activity.

Plants have developed various regulatory pathways to adapt to environmental stresses. In this study, we identified Arabidopsis MKKK20 as a regulator in the response to osmotic stress. mkkk20 mutants were found to be sensitive to high concentration of salt and showed higher water loss rates than wild-type (WT) plants under dehydration conditions. In addition, mkkk20 mutants showed higher accumulation of superoxide, a reactive oxygen species (ROS), compared to WT plants under high salt condition. In contrast, transgenic plants overexpressing MKKK20 displayed tolerance to salt stress. MKKK20 transcripts were increased by the treatments with NaCl, mannitol, MV, sorbitol, and cold, suggesting that MKKK20 is involved in the response to osmotic, ROS, and cold stresses. In-gel kinase assay showed that MKKK20 regulates the activity of MPK6 under NaCl, cold, and H(2)O(2) treatments. Taken together, our results suggest that MKKK20 might be involved in the response to various abiotic stresses, especially osmotic stress, through its regulation of MPK6 activity.

Identification of a C2H2-type zinc finger transcription factor (ZAT10) from Arabidopsis as a substrate of MAP kinase.

Mitogen-activated protein kinases (MAPKs or MPKs) are one of the most important and conserved signaling molecules in plants. MPKs can directly modulate gene expression by the phosphorylation of transcription factors. However, only a few target substrates of MPKs have been isolated. Here, we identified a C(2)H(2)-type zinc finger transcription factor from Arabidopsis, ZAT10, as a substrate of MPKs. Using in vitro and in vivo protein-protein interaction analyses, we demonstrated that ZAT10 directly interacted with MPK3 and MPK6. ZAT10 was phosphorylated by recombinant Arabidopsis MPK3 and MPK6 in a kinase assay. Furthermore, ZAT10 was also phosphorylated by native MPK3 and MPK6 prepared from Arabidopsis plants in an in-gel kinase assay. Mass spectrometry analysis of phosphopeptides was used to determine two MPK phosphorylation sites in ZAT10. These sites were verified by site-directed mutagenesis and in vitro kinase assays.

EMF1 interacts with EIP1, EIP6 or EIP9 involved in the regulation of flowering time in Arabidopsis.

The EMBRYONIC FLOWER (EMF) 1 gene has been shown to be necessary for maintenance of vegetative development. To investigate the molecular mechanism of EMF1-mediated plant development, we screened EMF1-interacting proteins and identified 11 candidate proteins using the yeast two-hybrid system. Among the candidate genes, three EMF1-Interacting Protein (EIP) genes, EIP1, EIP6 and EIP9, are predicted to encode a WNK (with-no-lysine) kinase, a B-box zinc-finger protein and a DnaJ-domain protein, respectively. The expression patterns of EIP1, EIP6 and EIP9 were similar to that of EMF1, and EMF1-EIP1, EMF1-EIP6 and EMF1-EIP9 heterodimers were localized in the nucleus. In addition, eip1, eip6 and eip9 mutants flowered early and showed increased expression of flowering-time and floral organ identity genes, while EIP1-, EIP6- and EIP9-overexpressing transgenic plants showed late flowering phenotypes. Our results suggest that EMF1 interacts with EIP1, EIP6 and EIP9 during vegetative development to regulate flowering time in Arabidopsis.

Arabidopsis MKK4 mediates osmotic-stress response via its regulation of MPK3 activity.

Plants have developed disparate regulatory pathways to adapt to environmental stresses. In this study, we identified MKK4 as an important mediator of plant response to osmotic stress. mkk4 mutants were more sensitive to high salt concentration than WT plants, exhibiting higher water-loss rates under dehydration conditions and additionally accumulating high levels of ROS. In contrast, MKK4-overexpressing transgenic plants showed tolerance to high salt as well as lower water-loss rates under dehydration conditions. In-gel kinase assays revealed that MKK4 regulates the activity of MPK3 upon NaCl exposure. Semi-quantitative RT-PCR analysis showed that expression of NCED3 and RD29A was lower and higher in mkk4 mutants and MKK4-overexpressing transgenic plants, respectively. Taken together, our results suggest that MKK4 is involved in the osmotic-stress response via its regulation of MPK3 activity.