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Wheat (Triticum aestivum L.) is a major food crop worldwide whose complex and very large genome hinders stable genetic transformation. An optimized method for transformation by particle bombardment is therefore greatly needed, both in general and for Korean commercial wheat varieties, which have not yet been successfully transformed. In this study, we describe the stable transformation of the Korean commercial wheat variety Keumkang and the DH20, which lacks the Glu-B3 and Gli-B1 loci (both related to gluten production), via particle bombardment of a vector harboring the enhanced green fluorescent protein (eGFP) gene. We confirmed the insertion of the eGFP transgene into the genome of primary (T0) transformants by genomic PCR, RT-qPCR, and fluorescence imaging. Our estimated average transformation efficiencies were 4.4% for Keumkang and 3.5% for DH20 in the T0 generation. We also validated the stable expression of the transgene in the T1 generation. We hypothesize that donor plant health, centrifugation, and high-sucrose pre-treatment increased the transformation efficiency reported here. Taken together, these results describe a useful method for transforming Korean commercial wheat varieties, including Keumkang and DH20, that will allow the manipulation of gene expression via such techniques as RNA interference, overexpression, and genome editing.

The stress hormone abscisic acid (ABA) helps plants to survive under abiotic stresses; however, its use as an agrochemical is limited by its chemical instability and expense. Here, we report the development of an in vivo screening system to isolate chemicals able to induce ABA signalling responses in rice (Oryza sativa) protoplasts. This system consists of an ABA-hypersensitive synthetic promoter containing ABRE and DRE motifs driving a luciferase reporter gene. After efficiently transfecting rice protoplasts with this construct, we screened chemicals library with a similar molecular weight and chemical structure to ABA and identified one chemical, S7, that induced ABA signalling by mediating interactions between the group I and II OsPYL receptors and certain OsPP2CAs in a yeast two-hybrid assay. In an in vitro pulldown assay, S7 was found to mediate a weak interaction between OsPYL5/8 and various OsPP2CAs. S7 treatments did not affect seedling growth or seed germination, but could reduce water loss. Rice seedlings treated with S7 exhibited transcriptome profiles that partially overlapped those treated with ABA. Taken together, we concluded that S7 is a new partial ABA agonist, which has potential use in future dissections of ABA signalling and as an agrochemical.

Transient expression of genes in protoplasts has been used widely for purposes ranging from subcellular localization to promoter activity analyses. Here, we describe methods for reconstituting the abscisic acid (ABA) signaling pathway using a transient expression system in rice protoplasts. ABA signaling is monitored via reporter systems consisting of synthetic promoters and luciferase. Thus, the effects of each signaling component as well as complexes involved in ABA signaling can be characterized in rice protoplasts, overcoming many of the limitations that hamper efforts to identify biological functions of effector genes in whole plants. This protoplast-based transient assay system for ABA signaling thus provides valuable tools and knowledge for understanding complicated ABA signaling networks.

Flavonols and anthocyanins are the two major classes of flavonoids in Brassica rapa. To elucidate the flavonoid biosynthetic pathway in Chinese cabbage (B. rapa L. subsp. pekinensis), we analyzed flavonoid contents in two varieties of Chinese cabbage with normal green (5546) and purple (8267) leaves. The 8267 variety accumulates significantly higher levels of quercetin, isorhamnetin, and cyanidin than the 5546 variety, indicating that 3′-dihydroxylated flavonoids are more prevalent in the purple than in the green variety. Gene expression analysis showed that the expression patterns of most phenylpropanoid pathway genes did not correspond to the flavonoid accumulation patterns in 5546 and 8267 varieties, except for BrPAL1.2 while most early and late flavonoid biosynthetic genes are highly expressed in 8267 variety. In particular, the flavanone 3′-hydroxylase BrF3′H (Bra009312) is expressed almost exclusively in 8267. We isolated the coding sequences of BrF3′H from the two varieties and found that both sequences encode identical amino acid sequences and are highly conserved with F3'H genes from other species. An in vitro enzymatic assay demonstrated that the recombinant BrF3′H protein catalyzes the 3′-hydroxylation of a wide range of 4′-hydroxylated flavonoid substrates. Kinetic analysis showed that kaempferol is the most preferred substrate and dihydrokaempferol (DHK) is the poorest substrate for recombinant BrF3′H among those tested. Transient expression of BrF3′H in Nicotiana benthamiana followed by infiltration of naringenin and DHK as substrates resulted in eriodictyol and quercetin production in the infiltrated leaves, demonstrating the functionality of BrF3′H in planta. As the first functional characterization of BrF3′H, our study provides insight into the molecular mechanism underlying purple coloration in Chinese cabbage.

Leaf angle and grain size are important agronomic traits affecting rice productivity directly and/or indirectly through modulating crop architecture. OsBC1, as a typical bHLH transcription factor, is one of the components comprising a complex formed with LO9-177 and OsBUL1 contributing to modulation of rice leaf inclination and grain size. In the current study, two homologues of OsBC1, OsBCL1 and OsBCL2 were functionally characterized by expressing them under the control of OsBUL1 promoter, which is preferentially expressed in the lamina joint and the spikelet of rice. Increased leaf angle and grain length with elongated cells in the lamina joint and the grain hull were observed in transgenic rice containing much greater gibberellin A3&nbsp, (GA3) levels than WT, demonstrating that both OsBCL1 and OsBCL2 are positive regulators of cell elongation at least partially through increased GA biosynthesis. Moreover, the cell elongation was likely due to cell expansion rather than cell division based on the related gene expression and, the cell elongation-promoting activities of OsBCL1 and OsBCL2 were functional in a dicot species, Arabidopsis.

DNA methylation plays an important role in diverse developmental processes in many eukaryotes, including the response to environmental stress. Abscisic acid (ABA) is a plant hormone that is up-regulated under stress. The involvement of DNA methylation in the ABA response has been reported but is poorly understood. DNA demethylation is a reverse process of DNA methylation and often induces structural changes of chromatin leading to transcriptional activation. In Arabidopsis (Arabidopsis thaliana), active DNA demethylation depends on the activity of REPRESSOR OF SILENCING 1 (ROS1), which directly excises 5-methylcytosine from DNA. Here we showed that ros1 mutants were hypersensitive to ABA during early seedling development and root elongation. Expression levels of some ABA-inducible genes were decreased in ros1 mutants, and more than 60% of their proximal regions became hypermethylated, indicating that a subset of ABA-inducible genes are under the regulation of ROS1-dependent DNA demethylation. Notable among them is NICOTINAMIDASE 3 (NIC3) that encodes an enzyme that converts nicotinamide to nicotinic acid in the NAD+ salvage pathway. Many enzymes in this pathway are known to be involved in stress responses. The nic3 mutants display hypersensitivity to ABA, whereas overexpression of NIC3 restores normal ABA responses. Our data suggest that NIC3 is responsive to ABA but requires ROS1-mediated DNA demethylation at the promoter as a prerequisite to transcriptional activation. These findings suggest that ROS1-induced active DNA demethylation maintains the active state of NIC3 transcription in response to ABA.

The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.

Clade A Type 2C protein phosphatases (PP2CAs) negatively regulate abscisic acid (ABA) signaling and have diverse functions in plant development and in response to various stresses. In this study, we showed that overexpression of the rice ABA receptor OsPYL/RCAR3 reduces the growth retardation observed in plants exposed to osmotic stress. By contrast, overexpression of the OsPYL/RCAR3-interacting protein OsPP2C09 rendered plant growth more sensitive to osmotic stress. We tested whether OsPP2CAs activate an ABA-independent signaling cascade by transfecting rice protoplasts with luciferase reporters containing the drought-responsive element (DRE) or ABA-responsive element (ABRE). We observed that OsPP2CAs activated gene expression via the cis-acting drought-responsive element. In agreement with this observation, transcriptome analysis of plants overexpressing OsPP2C09 indicated that OsPP2C09 induces the expression of genes whose promoters contain DREs. Further analysis showed that OsPP2C09 interacts with DRE-binding (DREB) transcription factors and activates reporters containing DRE. We conclude that, through activating DRE-containing promoters, OsPP2C09 positively regulates the drought response regulon and activates an ABA-independent signaling pathway.

The wheat gliadins are a complex group of flour proteins that can trigger celiac disease and serious food allergies. As a result, mutation breeding and biotechnology approaches are being used to develop new wheat lines with reduced immunogenic potential. Key to these efforts is the development of rapid, high-throughput methods that can be used as a first step in selecting lines with altered gliadin contents. In this paper, we optimized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and reversed-phase high-performance liquid chromatography (RP-HPLC) methods for the separation of gliadins from Triticum aestivum cv. Chinese Spring (CS). We evaluated the quality of the resulting profiles using the complete set of gliadin gene sequences recently obtained from this cultivar as well as a set of aneuploid lines in CS. The gliadins were resolved into 13 peaks by MALDI-TOF-MS. α- or γ-gliadins that contain abundant celiac disease epitopes and are likely targets for efforts to reduce the immunogenicity of flour were found in several peaks. However, other peaks contained multiple α- and γ-gliadins, including one peak with as many as 12 different gliadins. In comparison, separation of proteins by RP-HPLC yielded 28 gliadin peaks, including 13 peaks containing α-gliadins and eight peaks containing γ-gliadins. While the separation of α- and γ-gliadins gliadins achieved by RP-HPLC was better than that achieved by MALDI-TOF-MS, it was not possible to link peaks with individual protein sequences. Both MALDI-TOF-MS and RP-HPLC provided adequate separation of ω-gliadins. While MALDI-TOF-MS is faster and could prove useful in studies that target specific gliadins, RP-HPLC is an effective method that can be applied more broadly to detect changes in gliadin composition.

Overexpression of abscisic acid (ABA) receptors has been reported to enhance drought tolerance, but also to cause stunted growth and decreased crop yield. Here, we constructed transgenic rice for all monomeric ABA receptors and observed that only transgenic rice over-expressing OsPYL/RCAR7 showed similar phenotype with wild type, without total yield loss when grown under normal growth condition in a paddy field. Even though transgenic rice over-expressing OsPYL/RCAR7 showed neither an ABA-sensitivity nor an osmotic stress tolerance in plate assay, it showed drought tolerance. We investigated the ABA-dependent interaction with OsPP2CAs and ABA signaling induction by OsPYL/RCAR7. In yeast two hybrid assay, OsPYL/RCAR7 required critically higher ABA concentrations to interact with OsPP2CAs than other ABA receptors, and co-immunoprecipitation assay showed strong interaction under ABA treatment. When ABA-responsive signaling activity was monitored using a transient expression system in rice protoplasts, OsPYL/RCAR7 had the lowest ABA-responsive signaling activity as compared with other ABA receptors. OsPYL/RCAR7 also showed weak suppression of phosphatase activity as compared with other ABA receptors in vitro. Transcriptome analysis of transgenic rice over-expressing OsPYL/RCAR7 suggested that only a few genes were induced similar to control under without exogenous ABA, but a large number of genes was induced under ABA treatment compared with control. We conclude that OsPYL/RCAR7 is a novel functional ABA receptor that has low ABA signaling activity and exhibits high ABA dependence. These results lay the foundation for a new strategy to improve drought stress tolerance without compromising crop growth.

The AP2/EREBP family transcription factors play important roles in a wide range of stress tolerance and hormone signaling. In this study, a heat-inducible rice ERF gene was isolated and functionally characterized. The OsERF115/AP2EREBP110 was categorized to Group-IIIc of the rice AP2/EREBP family and strongly induced by heat and drought treatment. The OsERF115/AP2EREBP110 protein targeted to nuclei and suppressed the ABA-induced transcriptional activation of Rab16A promoter in rice protoplasts. Overexpression of OsERF115/AP2EREBP110 enhanced thermotolerance of seeds and vegetative growth stage plants. The OsERF115/AP2EREBP110 overexpressing (OE) plants exhibited higher proline level and increased expression of a proline biosynthesis P5CS1 gene. Phenotyping of water use dynamics of the individual plant indicates that the OsERF115/AP2EREBP110-OE plant exhibited better water saving traits under heat and drought combined stress. Our combined results suggest the potential use of OsERF115/AP2EREBP110 as a candidate gene for genetic engineering approaches to develop heat and drought stress-tolerant crops.

This work suggests 2020 potential candidates in rice for the functional annotation of unannotated genes using meta-analysis of anatomical samples derived from microarray and RNA-seq technologies and this information will be useful to identify novel morphological agronomic traits. Although the genome of rice (Oryza sativa) has been sequenced, 14,365 genes are considered unannotated because they lack putative annotation information. According to the Rice Genome Annotation Project Database ( http://rice.plantbiology.msu.edu/ ), the proportion of functionally characterized unannotated genes (0.35%) is quite limited when compared with the approximately 3.9% of annotated genes with assigned putative functions. Researchers require additional information to help them investigate the molecular mechanisms associated with those unannotated genes. To determine which of them might regulate morphological or physiological traits in the rice genome, we conducted a meta-analysis of expression data that covered a wide range of tissue/organ samples. Overall, 2020 genes showed cultivar-, tissue-, or organ-preferential patterns of expression. Representative candidates from featured groups were validated by RT-PCR, and the GUS reporter system was used to validate the expression of genes that were clustered according to their leaf or root preference. Taking a molecular and genetics approach, we examined meta-expression data and found that 127 genes were differentially expressed between japonica and indica rice cultivars. This is potentially significant for future agronomic applications. We also used a T-DNA insertional mutant and performed a co-expression network analysis of Sword shape dwarf1 (SSD1), a gene that regulates cell division. This network was refined via RT-PCR analysis. Our results suggested that SSD1 represses the expression of four genes related to the processes of DNA replication or cell division and provides insight into possible molecular mechanisms. Together, these strategies present a valuable tool for in-depth characterization of currently unannotated genes.

Drought, the most devastating abiotic stress, has detrimental effects on crop production. To minimize such adverse effects, it is crucial to improve drought tolerance in crops. Several members of the plant Nuclear Factor Y (NF-Y) gene family are involved in drought tolerance. In this study, we isolated the potato NF-Y subunit A 7 (StNF-YA7) gene from Solanum tuberosum ‘Superior’ and analyzed its relationship to abiotic stress tolerance. StNF-YA7 was induced by drought, salt stress, and abscisic acid (ABA), and a transient expression assay using 35S:StNF-YA7-smGFP showed that StNF-YA7 is localized to the nucleus, as reported for other plant NF-Ys. To examine the involvement of StNF-YA7 in drought tolerance, we generated 35S:StNF-YA7 transgenic potato plants. The transgenic potato plants exhibited enhanced drought tolerance, likely due to reduced leaf water loss. Analysis of the StNF-YA7 promoter (pStNF-YA7) fused to GUS revealed that StNF-YA7 is strongly expressed in vascular tissue. Taken together, these results suggest that enhancing drought tolerance via StNF-YA7 overexpression is associated with reduced leaf water loss, likely through mechanism(s) involving the vascular system.

Plants adapt to adverse environmental conditions through physiological responses, such as induction of the abscisic acid signaling pathway, stomatal regulation, and root elongation. Altered gene expression is a major molecular response to adverse environmental conditions in plants. Several transcription factors function as master switches to induce the expression of stress-tolerance genes. To find out a master regulator for the cold stress tolerance in rice, we focused on functionally identifying DREB subfamily which plays important roles in cold stress tolerance of plants. Here, we characterized OsDREB1G (LOC_Os02g45450), a functionally unidentified member of the DREB1 subgroup. OsDREB1G is specifically induced under cold stress conditions among several abiotic stresses examined. This gene is dominantly expressed in leaf sheath, blade, node, and root. Transgenic rice overexpressing this gene exhibited strong cold tolerance and growth retardation, like transgenic rice overexpressing other OsDREB1 genes. However, unlike these rice lines, transgenic rice overexpressing OsDREB1G did not exhibit significant increases in drought or salt tolerance. Cold-responsive genes were highly induced in transgenic rice overexpressing DREB1G compared to wild type. In addition, OsDREB1G overexpression directly induced the expression of a reporter gene fused to the promoters of cold-induced genes in rice protoplasts. Therefore, OsDREB1G is a typical CBF/DREB1 transcription factor that specifically functions in the cold stress response. Therefore, OsDREB1G could be useful for developing transgenic rice with enhanced cold-stress tolerance.

We determined the structure of OsPYL/RCAR3:OsPP2C50 complex with pyrabactin. Our results suggest that a less-conserved phenylalanine of OsPYL/RCAR subfamily I is one of considerations of ABA agonist development for Oryza sativa. Pyrabactin is a synthetic chemical mimicking abscisic acid (ABA), a naturally occurring phytohormone orchestrating abiotic stress responses. ABA and pyrabactin share the same pocket in the ABA receptors but pyrabactin modulates ABA signaling differently, exhibiting both agonistic and antagonistic effects. To explore structural determinants of differential functionality of pyrabactin, we determined the crystal structure of OsPYL/RCAR3:pyrabactin:OsPP2C50, the first rice ABA receptor:co-receptor complex structure with a synthetic ABA mimicry. The water-mediated interaction between the wedging Trp-259 of OsPP2C50 and pyrabactin is lost, undermining the structural integrity of the ABA receptor:co-receptor. The loss of the interaction of the wedging tryptophan of OsPP2C with pyrabactin appears to contribute to the weaker functionality of pyrabactin. Pyrabactin in the OsPYL/RCAR3:OsPP2C50 complex adopts a conformation different from that in ABA receptors from Arabidopsis. Phe125, specific to the subfamily I of OsPYL/RCARs in the ABA binding pocket, appears to be the culprit for the differential conformation of pyrabactin. Although the gate closure essential for the integrity of ABA receptor:co-receptor is preserved in the presence of pyrabactin, Phe125 apparently restricts accessibility of pyrabactin, leading to decreased affinity for OsPYL/RCAR3 evidenced by phosphatase assay. However, Phe125 does not affect conformation and accessibility of ABA. Yeast two-hybrid, germination and gene transcription analyses in rice also support that pyrabactin imposes a weak effect on the control of ABA signaling. Taken together, our results suggest that phenylalanine substitution of OsPYL/RCARs subfamily I may be one of considerations for ABA synthetic agonist development.

Supplementary methods. Purification of GST-N75-OsSLAC1. To obtain the GST-N75-OsSLAC1, we constructed pGEX-5x-N75-OsSLAC1. N-terminal region (N75) of OsSLAC1 was amplified by PCR reaction with specific primers (Additional file 8: Table S1) and harbored in pGEX-5x-1 vector with digestion by restriction enzymes (BamH1 and EcoR1) and ligation. Expressed GST-N75-OsSLAC1 in E. coli (BL21 pLysS) was purified using Glutathione Sepharose high performance (GE healthcare). (DOCX 13 kb)

DNA methylation plays an important role in diverse developmental processes in many eukaryotes, including the response to environmental stress. Abscisic acid (ABA) is a plant hormone that is up-regulated under stress. The involvement of DNA methylation in the ABA response has been reported but is poorly understood. DNA demethylation is a reverse process of DNA methylation and often induces structural changes of chromatin leading to transcriptional activation. In Arabidopsis (Arabidopsis thaliana), active DNA demethylation depends on the activity of REPRESSOR OF SILENCING 1 (ROS1), which directly excises 5-methylcytosine from DNA. Here we showed that ros1 mutants were hypersensitive to ABA during early seedling development and root elongation. Expression levels of some ABA-inducible genes were decreased in ros1 mutants, and more than 60% of their proximal regions became hypermethylated, indicating that a subset of ABA-inducible genes are under the regulation of ROS1-dependent DNA demethylation. Notable among them is NICOTINAMIDASE 3 (NIC3) that encodes an enzyme that converts nicotinamide to nicotinic acid in the NAD(+) salvage pathway. Many enzymes in this pathway are known to be involved in stress responses. The nic3 mutants display hypersensitivity to ABA, whereas overexpression of NIC3 restores normal ABA responses. Our data suggest that NIC3 is responsive to ABA but requires ROS1-mediated DNA demethylation at the promoter as a prerequisite to transcriptional activation. These findings suggest that ROS1-induced active DNA demethylation maintains the active state of NIC3 transcription in response to ABA.

Plants adapt to adverse environmental conditions through physiological responses, such as induction of the abscisic acid signaling pathway, stomatal regulation, and root elongation. Altered gene expression is a major molecular response to adverse environmental conditions in plants. Several transcription factors function as master switches to induce the expression of stress-tolerance genes. To find out a master regulator for the cold stress tolerance in rice, we focused on functionally identifying DREB subfamily which plays important roles in cold stress tolerance of plants. Here, we characterized OsDREB1G (LOC_Os02g45450), a functionally unidentified member of the DREB1 subgroup. OsDREB1G is specifically induced under cold stress conditions among several abiotic stresses examined. This gene is dominantly expressed in leaf sheath, blade, node, and root. Transgenic rice overexpressing this gene exhibited strong cold tolerance and growth retardation, like transgenic rice overexpressing other OsDREB1 genes. However, unlike these rice lines, transgenic rice overexpressing OsDREB1G did not exhibit significant increases in drought or salt tolerance. Cold-responsive genes were highly induced in transgenic rice overexpressing DREB1G compared to wild type. In addition, OsDREB1G overexpression directly induced the expression of a reporter gene fused to the promoters of cold-induced genes in rice protoplasts. Therefore, OsDREB1G is a typical CBF/DREB1 transcription factor that specifically functions in the cold stress response. Therefore, OsDREB1G could be useful for developing transgenic rice with enhanced cold-stress tolerance.

The phytohormone abscisic acid (ABA) enables plants to adapt to adverse environmental conditions through the modulation of metabolic pathways and of growth and developmental programs. We used comparative microarray analysis to identify genes exhibiting ABA-dependent expression and other hormone-dependent expression among them in Oryza sativa shoot and root. We identified 854 genes as significantly up- or down-regulated in root or shoot under ABA treatment condition. Most of these genes had similar expression profiles in root and shoot under ABA treatment condition, whereas 86 genes displayed opposite expression responses in root and shoot. To examine the crosstalk between ABA and other hormones, we compared the expression profiles of the ABA-dependently regulated genes under several different hormone treatment conditions. Interestingly, around half of the ABA-dependently expressed genes were also regulated by jasmonic acid based on microarray data analysis. We searched the promoter regions of these genes for cis-elements that could be responsible for their responsiveness to both hormones, and found that ABRE and MYC2 elements, among others, were common to the promoters of genes that were regulated by both ABA and JA. These results show that ABA and JA might have common gene expression regulation system and might explain why the JA could function for both abiotic and biotic stress tolerance.

Highlight OsCYP19-4 is a novel apoplastic immunophilin that plays a role in developmental acclimation and cold stress, probably via regulation of auxin transport., AtCYP19-4 (also known as CYP5) was previously identified as interacting in vitro with GNOM, a member of a large family of ARF guanine nucleotide exchange factors that is required for proper polar localization of the auxin efflux carrier PIN1. The present study demonstrated that OsCYP19-4, a gene encoding a putative homologue of AtCYP19-4, was up-regulated by several stresses and showed over 10-fold up-regulation in response to cold. The study further demonstrated that the promoter of OsCYP19-4 was activated in response to cold stress. An OsCYP19-4-GFP fusion protein was targeted to the outside of the plasma membrane via the endoplasmic reticulum as determined using brefeldin A, a vesicle trafficking inhibitor. An in vitro assay with a synthetic substrate oligomer confirmed that OsCYP19-4 had peptidyl-prolyl cis-trans isomerase activity, as was previously reported for AtCYP19-4. Rice plants overexpressing OsCYP19-4 showed cold-resistance phenotypes with significantly increased tiller and spike numbers, and consequently enhanced grain weight, compared with wild-type plants. Based on these results, the authors suggest that OsCYP19-4 is required for developmental acclimation to environmental stresses, especially cold. Furthermore, the results point to the potential of manipulating OsCYP19-4 expression to enhance cold tolerance or to increase biomass.

The sensitivity of rice to salt stress greatly depends on growth stages, organ types and cultivars. Especially, the roots of young rice seedlings are highly salt-sensitive organs that limit plant growth, even under mild soil salinity conditions. In an attempt to identify metabolic markers of rice roots responding to salt stress, metabolite profiling was performed by H-1-NMR spectroscopy in 38 rice genotypes that varied in biomass accumulation under long-term mild salinity condition. Multivariate statistical analysis showed separation of the control and salt-treated rice roots and rice genotypes with differential growth potential. By quantitative analyses of H-1-NMR data, five conserved salt-responsive metabolic markers of rice roots were identified. Sucrose, allantoin and glutamate accumulated by salt stress, whereas the levels of glutamine and alanine decreased. A positive correlation of metabolite changes with growth potential and salt tolerance of rice genotypes was observed for allantoin and glutamine. Adjustment of nitrogen metabolism in rice roots is likely to be closely related to maintain the growth potential and increase the stress tolerance of rice.

Shaker-like potassium channels are highly conserved voltage-dependent ion channels in plants. Rice, a monocot model plant, has similar numbers of shaker-like potassium channels as Arabidopsis. Although several inward-rectifying potassium channels have been identified, no outward-rectifying potassium channels have yet been reported for rice. Here, we identified two outward-rectifying shaker-like potassium channels in rice, with high amino acid sequence similarities to Arabidopsis stelar K+ outward rectifier (SKOR) and guard cell outward rectifying K+ channel (GORK). To characterize these channels we monitored their expression patterns in several tissues. qRTPCR and promoter-GUS analysis showed that OsSKOR is expressed in root vascular tissues, flower, and seed scutellum. OsGORK was expressed to some degree in most tissues, such as leaf blade, node, leaf sheath and root, but at high levels in flowers. This is the first report characterizing rice outward-rectifying potassium channels and contributes to understanding the regulation of potassium homeostasis in monocot plants.

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.

Cyclophilin 18-2 (CYP18-2) genes, homologues of human peptidyl-prolyl isomerase-like 1 (PPiL1), are conserved across multicellular organisms and Schizosaccharomyces pombe. Although PPiL1 is known to interact with ski-interacting protein (SKIP), a transcriptional co-regulator and spliceosomal component, there have been no functional analyses of PPiL1 homologues in plants. Rice cyclophilin 18-2 (OsCYP18-2) bound directly to amino acids 56-95 of OsSKIP and its binding was independent of cyclosporin A, a cyclophilin-binding drug. Moreover, OsCYP18-2 exhibited PPIase activity regardless of its interaction with OsSKIP. Therefore, the binding site for OsCYP18-2's interaction with SKIP was distinct from the PPIase active site. OsCYP18-2's interaction with SKIP full-length protein enabled OsCYP18-2's translocation from the cytoplasm into the nucleus and AtSKIP interacted in planta with both AtCYP18-2 and OsCYP18-2. Drought and salt stress induced similar expression of OsCYP18-2 and OsSKIP. Overexpression of OsCYP18-2 in transgenic rice and Arabidopsis thaliana plants enhanced drought tolerance and altered expression and pre-mRNA splicing patterns of stress-related genes in Arabidopsis under drought conditions. Furthermore, OsCYP18-2 caused transcriptional activation with/without OsSKIP in the GAL4 system of yeast; thus the OsSKIP-OsCYP18-2 interaction has an important role in the transcriptional and post-transcriptional regulation of stress-related genes and increases tolerance to drought stress.

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.