Masaru Ohta

Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and drought stress responses.

The phytohormone abscisic acid (ABA) modulates the expression of many genes important to plant growth and development and to stress adaptation. In this study, we found that an APETALA2/EREBP-type transcription factor, AtERF7, plays an important role in ABA responses. AtERF7 interacts with the protein kinase PKS3, which has been shown to be a global regulator of ABA responses. AtERF7 binds to the GCC box and acts as a repressor of gene transcription. AtERF7 interacts with the Arabidopsis thaliana homolog of a human global corepressor of transcription, AtSin3, which in turn may interact with HDA19, a histone deacetylase. The transcriptional repression activity of AtERF7 is enhanced by HDA19 and AtSin3. Arabidopsis plants overexpressing AtERF7 show reduced sensitivity of guard cells to ABA and increased transpirational water loss. By contrast, AtERF7 and AtSin3 RNA interference lines show increased sensitivity to ABA during germination. Together, our results suggest that AtERF7 plays an important role in ABA responses and may be part of a transcriptional repressor complex and be regulated by PKS3.

Reconstitution in yeast of the Arabidopsis SOS signaling pathway for Na+ homeostasis

The Arabidopsis thaliana SOS1 protein is a putative Na+/H+ antiporter that functions in Na+ extrusion and is essential for the NaCl tolerance of plants. sos1 mutant plants share phenotypic similarities with mutants lacking the protein kinase SOS2 and the Ca2+ sensor SOS3. To investigate whether the three SOS proteins function in the same response pathway, we have reconstituted the SOS system in yeast cells. Expression of SOS1 improved the Na+ tolerance of yeast mutants lacking endogenous Na+ transporters. Coexpression of SOS2 and SOS3 dramatically increased SOS1-dependent Na+ tolerance, whereas SOS2 or SOS3 individually had no effect. The SOS2/SOS3 kinase complex promoted the phosphorylation of SOS1. A constitutively active form of SOS2 phosphorylated SOS1 in vitro independently of SOS3, but could not fully substitute for the SOS2/SOS3 kinase complex for activation of SOS1 in vivo. Further, we show that SOS3 recruits SOS2 to the plasma membrane. Although sos1 mutant plants display defective K+ uptake at low external concentrations, neither the unmodified nor the SOS2/SOS3-activated SOS1 protein showed K+ transport capacity in vivo, suggesting that the role of SOS1 on K+ uptake is indirect. Our results provide an example of functional reconstitution of a plant response pathway in a heterologous system and demonstrate that the SOS1 ion transporter, the SOS2 protein kinase, and its associated Ca2+ sensor SOS3 constitute a functional module. We propose a model in which SOS3 activates and directs SOS2 to the plasma membrane for the stimulatory phosphorylation of the Na+ transporter SOS1.

LOS2, a genetic locus required for cold-responsive gene transcription encodes a bi-functional enolase

The Arabidopsis mutation, los2, impairs cold‐responsive gene transcription, acquired freezing tolerance and plant resistance to chilling under certain conditions. LOS2 was isolated through positional cloning and shown to encode an enolase in the glycolytic pathway. In animal cells, enolase has also been known to function as a transcription factor that represses the expression of c‐myc by binding to the c‐myc gene promoter. LOS2 fused to green fluorescent protein is targeted to the nucleus as well as to the cytoplasm. LOS2/enolase protein can bind to the cis‐element of the human c‐myc gene promoter and to the gene promoter of STZ/ZAT10, a zinc finger transcriptional repressor from Arabidopsis. STZ/ZAT10 expression is induced rapidly and transiently by cold in the wild type, and this induction is stronger and more sustained in the los2 mutant. Furthermore, the expression of a RD29A‐LUC reporter gene is repressed significantly by STZ/ZAT10 in transient expression assays in Arabidopsis leaves. Our results demonstrate that cold‐responsive gene transcription in plants is controlled by a bi‐functional enolase.

A novel domain in the protein kinase SOS2 mediates interaction with the protein phosphatase 2C ABI2.

SOS2 (salt overly sensitive 2) is a serine/threonine protein kinase required for salt tolerance in Arabidopsis thaliana. In this study, we identified the protein phosphatase 2C ABI2 (abscisic acid-insensitive 2) as a SOS2-interacting protein. Deletion analysis led to the discovery of a novel protein domain of 37 amino acid residues, designated as the protein phosphatase interaction (PPI) motif, of SOS2 that is necessary and sufficient for interaction with ABI2. The PPI motif is conserved in protein kinases of the SOS2 family (i.e., protein kinase S, PKS) and in the DNA damage repair and replication block checkpoint kinase, Chk1, from various organisms including humans. Mutations in the conserved amino acid residues in the PPI motif abolish the interaction of SOS2 with ABI2. We also identified a protein kinase interaction domain in ABI2 and examined the interaction specificity between PKS and the ABI phosphatases. We found that some PKSs interact strongly with ABI2 whereas others interact preferentially with ABI1. The interaction between SOS2 and ABI2 was disrupted by the abi2-1 mutation, which causes increased tolerance to salt shock and abscisic acid insensitivity in plants. Our results establish the PPI motif and the protein kinase interaction domain as novel protein interaction domains that mediate the binding between the SOS2 family of protein kinases and the ABI1/2 family of protein phosphatases.

Transgenic Evaluation of Activated Mutant Alleles of SOS2 Reveals a Critical Requirement for Its Kinase Activity and C-Terminal Regulatory Domain for Salt Tolerance in Arabidopsis thaliana

In Arabidopsis thaliana, the calcium binding protein Salt Overly Sensitive3 (SOS3) interacts with and activates the protein kinase SOS2, which in turn activates the plasma membrane Na+/H+ antiporter SOS1 to bring about sodium ion homeostasis and salt tolerance. Constitutively active alleles of SOS2 can be constructed in vitro by changing Thr168 to Asp in the activation loop of the kinase catalytic domain and/or by removing the autoinhibitory FISL motif from the C-terminal regulatory domain. We expressed various activated forms of SOS2 in Saccharomyces cerevisiae (yeast) and in A. thaliana and evaluated the salt tolerance of the transgenic organisms. Experiments in which the activated SOS2 alleles were coexpressed with SOS1 in S. cerevisiae showed that the kinase activity of SOS2 is partially sufficient for SOS1 activation in vivo, and higher kinase activity leads to greater SOS1 activation. Coexpression of SOS3 with SOS2 forms that retained the FISL motif resulted in more dramatic increases in salt tolerance. In planta assays showed that the Thr168-to-Asp–activated mutant SOS2 partially rescued the salt hypersensitivity in sos2 and sos3 mutant plants. By contrast, SOS2 lacking only the FISL domain suppressed the sos2 but not the sos3 mutation, whereas truncated forms in which the C terminus had been removed could not restore the growth of either sos2 or sos3 plants. Expression of some of the activated SOS2 proteins in wild-type A. thaliana conferred increased salt tolerance. These studies demonstrate that the protein kinase activity of SOS2 is partially sufficient for activation of SOS1 and for salt tolerance in vivo and in planta and that the kinase activity of SOS2 is limiting for plant salt tolerance. The results also reveal an essential in planta role for the SOS2 C-terminal regulatory domain in salt tolerance.

SIZ1, a small ubiquitin-related modifier ligase, controls cold signaling through regulation of salicylic acid accumulation

Low temperature induces several genes to acquire plant cold tolerance. Here, we demonstrate that accumulation of salicylic acid (SA) is involved in the regulation of the DREB1A/CBF3 regulon and plant tolerance to cold stresses. The SA-accumulating mutant siz1 exhibits sensitivity to chilling and freezing conditions and decreased expression of DREB1A/CBF3 and its regulon genes. Reduction of SA levels in siz1 by nahG restored cold sensitivity and down-regulation of these genes. Database analyses and RT-PCR analysis revealed that the ice1 mutation also increased expression of SA-responsive genes. As well as siz1, another SA-accumulating mutant acd6 exhibited freezing sensitivity and the sensitivity was suppressed in acd6 nahG plants. Taken together, these data indicate that SA is involved in regulation of cold signaling.

ICE1 Ser403 is necessary for protein stabilization and regulation of cold signaling and tolerance

ICE1, a MYC-type transcription factor, has an important role in the induction of CBF3/DREB1A for regulation of cold signaling and tolerance. Here we reveal that serine 403 of ICE1 is involved in regulating the transactivation and stability of the ICE1 protein. Substitution of serine 403 by alanine enhanced the transactivational activity of ICE1 in Arabidopsis protoplasts. Over-expression of ICE1(S403A) conferred more freezing tolerance than ICE1(WT) in Arabidopsis, and the expression of cold-regulated genes such as CBF3/DREB1A, COR47 and KIN1 was enhanced in plants over-expressing ICE1(S403A). Furthermore, the ICE1(S403A) protein level was not changed after cold treatment, whereas the ICE1(WT) protein level was reduced. Interestingly, polyubiquitylation of the ICE1(S403A) protein in vivo was apparently blocked. These results demonstrate that serine 403 of ICE1 has roles in both transactivation and cold-induced degradation of ICE1 via the ubiquitin/26S proteasome pathway, suggesting that serine 403 is a key residue for the attenuation of cold-stress responses by HOS1-mediated degradation of ICE1.

A new MADS-box gene (IbMADS10) from sweet potato (Ipomoea batatas (L.) Lam) is involved in the accumulation of anthocyanin

A new MADS-box gene designated as IbMADS10 was cloned and its expression was characterized from sweet potato (Ipomoea batatas (L.) Lam.) cv. Beniazuma. The deduced amino acid sequence of the gene indicated high homology with members of the MADS-box family of transcription factors. IbMADS10 shares high amino acid sequence similarity with the DEFH28 of Antirrhinum majus (64%) and with BpMADS4 of Betula pendula (61%) of the SQUA subfamily. Southern blot analysis revealed that the IbMADS10 is present in one or low copy number in the sweet potato genome. The gene is specifically expressed in the pigmented tissues such as in the flower bud, in the pink and in red roots, and hence, it was speculated that the IbMADS10 gene might be correlated with anthocyanin biosynthesis in sweet potato. RNA blot expression of the anthocyanin biosynthesis genes encoding for CHS, CHI, F3H, DFR, ANS and UFTG carried out in the tissues where the IbMADS10 gene was expressed revealed similar transcript levels in all tissues where the IbMADS10 gene is highly expressed, indicating that the IbMADS10 gene is highly correlated with the anthocyanin biosynthesis genes. Another important aspect is the pigmented phenotypes of transgenic calli that ectopically express the IbMADS10 gene, thereby supporting its involvement in the developmental regulation of pigment formation. Tissue printing result further strengthens the hypothesis that the IbMADS10 gene is indeed involved in anthocyanin pigmentation in sweet potato. As the purpose of the IbMADS10 gene is pigmentation, its function, therefore, resembles that of the transparent testa (tt) genes of Arabidopsis.

Cold-responsive gene regulation during cold acclimation in plants.

Regulation of the transcriptome is necessary for plants to acquire cold tolerance, and cold induces several genes via a cold signaling pathway. The transcription factors CBF/DREB1 (C-repeat binding factor/dehydration responsive element binding1) and ICE1 (inducer of CBF expression1) have important roles in the regulation of cold-responsive gene expression. ICE1 is post-translationally regulated by ubiquitylation-mediated proteolysis and sumoylation. This mini-review highlights some recent studies on plant cold signaling. The relationships among cold signaling, salicylic acid accumulation and stomatal development are also discussed.

Increased tolerance to salt stress in the phosphate-accumulating Arabidopsis mutants siz1 and pho2

High salinity is an environmental factor that inhibits plant growth and development, leading to large losses in crop yields. We report here that mutations in SIZ1 or PHO2, which cause more accumulation of phosphate compared with the wild type, enhance tolerance to salt stress. The siz1 and pho2 mutations reduce the uptake and accumulation of Na+. These mutations are also able to suppress the Na+ hypersensitivity of the sos3-1 mutant, and genetic analyses suggest that SIZ1 and SOS3 or PHO2 and SOS3 have an additive effect on the response to salt stress. Furthermore, the siz1 mutation cannot suppress the Li+ hypersensitivity of the sos3-1 mutant. These results indicate that the phosphate-accumulating mutants siz1 and pho2 reduce the uptake and accumulation of Na+, leading to enhanced salt tolerance, and that, genetically, SIZ1 and PHO2 are likely independent of SOS3-dependent salt signaling.