Overexpression of AtBBD1, Arabidopsis Bifunctional Nuclease, Confers Drought Tolerance by Enhancing the Expression of Regulatory Genes in ABA-Mediated Drought Stress Signaling.

Drought is the most serious abiotic stress, which significantly reduces crop productivity. The phytohormone ABA plays a pivotal role in regulating stomatal closing upon drought stress. Here, we characterized the physiological function of AtBBD1, which has bifunctional nuclease activity, on drought stress. We found that AtBBD1 localized to the nucleus and cytoplasm, and was expressed strongly in trichomes and stomatal guard cells of leaves, based on promoter:GUS constructs. Expression analyses revealed that AtBBD1 and AtBBD2 are induced early and strongly by ABA and drought, and that AtBBD1 is also strongly responsive to JA. We then compared phenotypes of two AtBBD1-overexpression lines (AtBBD1-OX), single knockout atbbd1, and double knockout atbbd1/atbbd2 plants under drought conditions. We did not observe any phenotypic difference among them under normal growth conditions, while OX lines had greatly enhanced drought tolerance, lower transpirational water loss, and higher proline content than the WT and KOs. Moreover, by measuring seed germination rate and the stomatal aperture after ABA treatment, we found that AtBBD1-OX and atbbd1 plants showed significantly higher and lower ABA-sensitivity, respectively, than the WT. RNA sequencing analysis of AtBBD1-OX and atbbd1 plants under PEG-induced drought stress showed that overexpression of AtBBD1 enhances the expression of key regulatory genes in the ABA-mediated drought signaling cascade, particularly by inducing genes related to ABA biosynthesis, downstream transcription factors, and other regulatory proteins, conferring AtBBD1-OXs with drought tolerance. Taken together, we suggest that AtBBD1 functions as a novel positive regulator of drought responses by enhancing the expression of ABA- and drought stress-responsive genes as well as by increasing proline content.

Arabidopsis TCX8 functions as a senescence modulator by regulating LOX2 expression.

KEY MESSAGE: TCX8 localizes to nucleus and has transcriptional repression activity. TCX8 binds to the promoter region of LOX2 encoding lipoxygenase, causing JA biosynthesis suppression, and thereby delays plant senescence. Conserved CXC domain-containing proteins are found in most eukaryotes. Eight TCX proteins, which are homologs of animal CXC-Hinge-CXC (CHC) proteins, were identified in Arabidopsis, and three of them, TSO1, TCX2/SOL2 and TCX3/SOL1, have been reported to affect cell-cycle control. TCX8, one of the TCX family proteins, was believed to be a TF but its precise function has not been reported. Yeast two-hybrid screening revealed TCP20, a TF that binds to the promoter of LOX2 encoding lipoxygenase, as a strong candidate for interaction with TCX8. We confirmed that TCX8 directly interacts with TCP20 using in vitro pull-down assay and in vivo BiFC and observed that TCX8, as a TF, localizes to nucleus. Using EMSA and by analyzing phenotypes of TCX8-overexpression lines, we demonstrated that TCX8 regulates the expression of LOX2 by binding to either cis-element of LOX2 promoter to which TCP20 or TCP4 binds, affecting JA biosynthesis, and thereby delaying plant senescence. Our study provides new information about the role of TCX8 in modulating plant senescence through regulating LOX2 expression.

AtMYB109 negatively regulates stomatal closure under osmotic stress in Arabidopsis thaliana.

Osmotic stress, caused by drought, salinity, or PEG (polyethylene glycol), is one of the most important abiotic factors that hinder plant growth and development. In Arabidopsis, more than 100 R2R3-MYB transcription factors (TFs) have been identified, and many of them are involved in the transcriptional regulation of a variety of biological processes related to growth and development, as well as responses to biotic and abiotic stresses. However, the MYB TF involving in both plant development and stress response has rarely been reported. We report here that Arabidopsis AtMYB109, a R2R3-MYB TF, functions as a negative regulator of stomatal closure under osmotic stress as well as of pollen tube elongation. Under PEG-induced osmotic stress, whole leaves of AtMYB109-OXs were intensely wilted, while leaves of the wild-type (WT) and myb109 were weakly affected. Moreover, we confirmed that the wilting in AtMYB109-OXs was more severe than in WT and myb109 under drought conditions, and that after re-watering, WT and myb109 plants promptly recovered, while AtMYB109-OXs failed to survive. In addition, stomatal closure was delayed in the AtMYB109-OXs compared to the WT and myb109. However, proline content and the expression of stress-induced and proline synthesis genes were higher in the overexpression lines than in WT and myb109. Then, we observed that the expression of ICS1, a key gene in SA biosynthesis, was greatly suppressed in AtMYB109-OXs. In addition, we found that AtMYB109 expression gradually increased until the flowers were fully opened and thereafter dramatically decreased during silique development. The pollen tube growth was significantly suppressed in AtMYB109-OXs compared to the WT and myb109. Using EMSA and ChIP-qPCR, we confirmed that AtMYB109 bound to the promoter of RABA4D, a gene encoding a pollen development regulator. Taken together, we suggest the delayed stomatal closing and vulnerable phenotypes in the AtMYB109-OXs under osmotic stress are possibly directly or indirectly associated with a SA-mediated mechanism, and that AtMYB109 suppresses RABA4D that modulates pollen tube growth.

Phylogenetic Analysis and In Vitro Bifunctional Nuclease Assay of Arabidopsis BBD1 and BBD2.

Nucleases are a very diverse group of enzymes that play important roles in many crucial physiological processes in plants. We previously reported that the highly conserved region (HCR), domain of unknown function 151 (DUF151) and UV responsive (UVR) domain-containing OmBBD is a novel nuclease that does not share homology with other well-studied plant nucleases. Here, we report that DUF151 domain-containing proteins are present in bacteria, archaea and only Viridiplantae kingdom of eukarya, but not in any other eukaryotes. Two Arabidopsis homologs of OmBBD, AtBBD1 and AtBBD2, shared 43.69% and 44.38% sequence identity and contained all three distinct domains of OmBBD. We confirmed that the recombinant MBP-AtBBD1 and MBP-AtBBD2 exhibited non-substrate-specific DNase and RNase activity, like OmBBD. We also found that a metal cofactor is not necessarily required for DNase activity of AtBBD1 and AtBBD2, but their activities were much enhanced in the presence of Mg2+ or Mn2+. Using a yeast two-hybrid assay, we found that AtBBD1 and AtBBD2 each form a homodimer but not a heterodimer and that the HCR domain is possibly crucial for dimerization.

CBSX3-Trxo-2 regulates ROS generation of mitochondrial complex II (succinate dehydrogenase) in Arabidopsis.

Despite being toxic at a high concentrations, reactive oxygen species (ROS) play a pivotal role as signaling molecules in responses to stress and regulation of plant development. The mitochondrial electron transport chain (ETC) is the major source of ROS in cells. Although the regulation of ROS in mitochondria has been well elucidated, the protein-protein interaction-based regulation of ETC members has not been well elucidated. In this study, we identified a CBS domain-containing protein, CBSX3, and found that CBSX3 activates o-type thioredoxin (Trx-o2) in mitochondria. In addition, we found that Trx-o2 interacts with SDH1, a subunit of ETC complex II. Knockdown (KD) of CBSX3 revealed anther indehiscence due to deficient lignin deposition caused by insufficient ROS accumulation, and increased expression of genes related to cell cycle and accelerated plant growth. However, in the CBSX3-overexpression plants, ROS accumulation increased, and cell cycle-related gene expression decreased, and thereby plant growth was retarded and leaf size decreased. Moreover, KD of CBSX3 and Trx-o2 conferred resistance to mitochondria ETC inhibitors in terms of ROS release. Taken together, we suggest that CBSX3-Trx-o2 is a ROS generation regulator of mitochondria in plants and plays an important role in regulating plant development and the redox system.

The novel protein CSAP accelerates leaf senescence and is negatively regulated by SAUL1 in the dark.

KEY MESSAGE: The chloroplast-localized protein CSAP is an ABA-responsive factor and positively regulates dark-induced senescence. This phenomenon is controlled by SAUL1 in Arabidopsis. We report here that CSAP (Chloroplast-localized Senescence-Associated Protein, AT5G39520) functions as a positive regulator of senescence and is controlled by SAUL1 (Senescence Associated E3 Ubiquitin Ligase 1) in Arabidopsis. CSAP transcript level was gradually increased when senescence was progressed. Under dark conditions, the csap mutant showed delayed leaf senescence and reduced chlorophyll breakdown, but overexpression of CSAP accelerated leaf senescence and expressions of chlorophyll catabolic genes were up-regulated compared to the wild-type (WT). NCED3 and AAO3, which are involved in ABA biosynthesis, also showed higher expression in the overexpression lines than the WT. It is known that the CSAP transcript is increased in the saul1 mutant that shows precocious senescence. In our experiments, we confirmed that CSAP interacts with SAUL1 by the yeast two-hybrid and pull-down assays. In addition, we found that SAUL1 decreases the stability of CSAP in the presence of ABA. Taken together, we suggest that CSAP accelerates leaf senescence in the dark and this process is controlled by SAUL1.

Occurrence of land-plant-specific glycerol-3-phosphate acyltransferases is essential for cuticle formation and gametophore development in Physcomitrella patens.

During the evolution of land plants from aquatic to terrestrial environments, their aerial surfaces were surrounded by cuticle composed of cutin and cuticular waxes to protect them from environmental stresses. Glycerol-3-phosphate acyltransferase (GPAT) harboring bifunctional sn-2 acyltransferase/phosphatase activity produces 2-monoacylglycerol, a precursor for cutin synthesis. Here, we report that bifunctional sn-2 GPATs play roles in cuticle biosynthesis and gametophore development of Physcomitrella patens. Land plant-type cuticle was observed in gametophores but not in protonema. The expression of endoplasmic reticulum-localized PpGPATs was significantly upregulated in gametophores compared with protonema. Floral organ fusion and permeable cuticle phenotypes of Arabidopsis gpat6-2 petals were rescued to the wild type (WT) by the expression of PpGPAT2 or PpGPAT4. Disruption of PpGPAT2 and PpGPAT4 caused a significant reduction of total cutin loads, and a prominent decrease in the levels of palmitic and 10,16-dihydroxydecanoic acids, which are major cutin monomers in gametophores. Δppgpat2 mutants displayed growth retardation, delayed gametophore development, increased cuticular permeability, and reduced tolerance to drought, osmotic and salt stresses compared to the WT. Genome-wide analysis of genes encoding acyltransferase or phosphatase domains suggested that the occurrence of sn-2 GPATs with both domains may be a key event in cuticle biogenesis of land plants.

Cytokinin oxidase PpCKX1 plays regulatory roles in development and enhances dehydration and salt tolerance in Physcomitrella patens.

KEY MESSAGE: PpCKX1 localizes to vacuoles and is dominantly expressed in the stem cells. PpCKX1 regulates developmental changes with increased growth of the rhizoid and enhances dehydration and salt tolerance. Cytokinins (CKs) are plant hormones that regulate plant development as well as many physiological processes, such as cell division, leaf senescence, control of shoot/root ratio, and reproductive competence. Cytokinin oxidases/dehydrogenases (CKXs) control CK concentrations by degradation, and thereby influence plant growth and development. In the moss Physcomitrella patens, an evolutionarily early divergent plant, we identified six putative CKXs that, by phylogenetic analysis, form a monophyletic clade. We also observed that ProPpCKX1:GUS is expressed specifically in the stem cells and surrounding cells and that CKX1 localizes to vacuoles, as indicated by Pro35S:PpCKX1-smGFP. Under normal growth conditions, overexpression of PpCKX1 caused many phenotypic changes at different developmental stages, and we suspected that increased growth of the rhizoid could affect those changes. In addition, we present evidence that the PpCKX1-overexpressor plants show enhanced dehydration and salt stress tolerance. Taken together, we suggest that PpCKX1 plays regulatory roles in development and adaptation to abiotic stresses in this evolutionarily early land plant species.

Lon domain-containing protein 1 represses thioredoxin y2 and regulates ROS levels in Arabidopsis chloroplasts.

Plant thioredoxins (Trxs) act as antioxidants and function as redox regulators in the chloroplast. Although the regulation of ROS in chloroplasts is well elucidated, the precise regulation mechanism of Trx remains unknown. Here, we characterize a novel chloroplast protein, Lon domain-containing protein 1 (LCP1), which contains only a Lon domain, the precise function of which is not known. We find that LCP1 interacts with Trx-y2 and represses its activity, and that knockdown (KD) of LCP1 causes anther indehiscence due to deficient lignin deposition. In addition, LCP1 KD plants show less ROS accumulation and lower expression of ROS-responsive marker genes than the wild-type plant. Taken together, we suggest that LCP1 directly regulates Trx-y2 and controls H2 O2 levels and, thereby, regulates lignin polymerization in the anther endothecium.

The transcription factor γMYB2 acts as a negative regulator of secondary cell wall thickening in anther and stem.

Secondary cell wall (SCW) thickening provides the mechanical force for anther dehiscence and plays an important role in the formation of xylem structure. We have previously reported that γMYB2, a MYB coiled-coil protein, directly binds to the P1BS cis-element of the PLA2-γ promoter and acts as a co-activator of γMYB1 in controlling the expression of PLA2-γ. In this study, we analyzed morphological phenotypes of the constitutive overexpression (γMYB2-OE) and knock-down (γMYB2-KD) lines of γMYB2. We found that γMYB2 overexpression caused the collapse of the endothecium layer, thereby suppressing anther dehiscence and forming short infertile siliques. The γMYB2-OE also showed less cellulose deposition in the xylem and had a longer primary stem than the wild-type, while γMYB2-KD had greater cellulose accumulation and a shorter primary stem than the wild-type. We demonstrated that the male sterility and the longer primary stem in γMYB2-OE were caused by reduced expression of SCW thickening-related genes. Our results suggest that γMYB2 acts as a negative regulator in controlling the SCW thickening in Arabidopsis.

The novel transcription factor TRP interacts with ZFP5, a trichome initiation-related transcription factor, and negatively regulates trichome initiation through gibberellic acid signaling.

KEY MESSAGE: The trichome-related protein (TRP) is a novel transcription factor (TF) that negatively regulates trichome initiation-related TFs through gibberellin (GA) signaling. Trichomes, which are outgrowths of leaf epidermal cells, provide the plant with a first line of defense against damage from herbivores and reduce transpiration. The initiation and development of trichomes are regulated by a network of positively or negatively regulating transcription factors (TFs). However, little information is currently available on transcriptional regulation related to trichome formation. Here, we report a novel TF Trichome-Related Protein (TRP) that was observed to negatively regulate the trichome initiation-related TFs through gibberellic acid (GA) signaling. ProTRP:GUS revealed that TRP was only expressed in the trichome. The TRP loss-of-function mutant (trp) had an increased number of trichomes on the flower, cauline leaves, and main inflorescence stems compared to the wild-type. In contrast, TRP overexpression lines (TRP-Ox) exhibited a decreased number of trichomes on cauline leaves and main inflorescence stem following treatment with exogenous GA. Moreover, the expressions of trichome initiation regulators (GIS, GIS2, ZFP8, GL1, and GL3) increased in trp plants but decreased in TRP-Ox lines after GA treatment. TRP was observed to physically interact with ZFP5, a C2H2 TF that controls trichome cell development through GA signaling, both in vivo and in vitro. Based on these results, we suggest that TRP functions upstream of the trichome initiation regulators and represses the binding of ZFP5 to the ZFP8 promoter.

The FBA motif-containing protein AFBA1 acts as a novel positive regulator of ABA response in Arabidopsis.

ABA plays a critical role in regulating seed germination and stomatal movement in response to drought stress. Screening ABA-responsive genes led to the identification of a novel Arabidopsis gene encoding a protein which contained a conserved F-box-associated (FBA) domain, subsequently named ABA-responsive FBA domain-containing protein 1 (AFBA1). Expression of ProAFBA1:GUS revealed that this gene was mainly expressed in guard cells. Expression of AFBA1 increased following the application of exogenous ABA and exposure to salt (NaCl) and drought stresses. Seed germination of the loss-of-function mutant (afba1) was insensitive to ABA, salt or mannitol, whereas AFBA1-overexpressing (Ox) seeds were more sensitive to these stresses than the wild-type seeds. The afba1 plants showed decreased drought tolerance, increased water loss rate and ABA-insensitive stomatal movement compared with the wild-type. In contrast, AFBA1-Ox plants exhibited enhanced drought tolerance and a rapid ABA-induced stomatal closure response. The expression of genes encoding serine/threonine protein phosphatases that are known negative regulators of ABA signaling increased in afba1 plants but decreased in AFBA1-Ox plants. AFBA1 was also found to be localized in the nucleus and to interact with an R2R3-type transcription factor, MYB44, leading to the suggestion that it functions in the stabilization of MYB44. Based on these results, we suggest that AFBA1 functions as a novel positive regulator of ABA responses, regulating the expression of genes involved in ABA signal transduction in Arabidopsis through its interaction with positive regulators of ABA signaling including MYB44, and increasing their stability during ABA-mediated responses.

A Transcription Factor γMYB1 Binds to the P1BS cis-Element and Activates PLA2-γ Expression with its Co-Activator γMYB2.

Phospholipase A2(PLA2) hydrolyzes phospholipid molecules to produce two products that are both precursors of second messengers of signaling pathways and signaling molecules per se.Arabidopsis thaliana PLA2 paralogs (-ß,-γ and -δ) play critical roles during pollen development, pollen germination and tube growth. In this study, analysis of the PLA2-γ promoter using a deletion series revealed that the promoter region -153 to -1 is crucial for its pollen specificity. Using a yeast one-hybrid screening assay with the PLA2-γ promoter and an Arabidopsis transcription factor (TF)-only library, we isolated two novel MYB-like TFs belonging to the MYB-CC family, denoted here as γMYB1 and γMYB2. By electrophoretic mobility shift assay, we found that these two TFs bind directly to the P1BS (phosphate starvation response 1-binding sequence)cis-element of the PLA2-γ promoter. γMYB1 alone functioned as a transcriptional activator for PLA2-γ expression, whereas γMYB2 directly interacted with γMYB1 and enhanced its activation. Overexpression of γMYB1 in the mature pollen grain led to increased expression of not only the PLA2-γ gene but also of several genes whose promoters contain the P1BS cis-element and which are involved in the Pi starvation response, phospholipid biosynthesis and sugar synthesis. Based on these results, we suggest that the TF γMYB1 binds to the P1BS cis-element, activates the expression of PLA2-γ with the assistance of its co-activator, γMYB2, and regulates the expression of several target genes involved in many plant metabolic reactions.

CML10, a variant of calmodulin, modulates ascorbic acid synthesis.

Calmodulins (CaMs) regulate numerous Ca(2+) -mediated cellular processes in plants by interacting with their respective downstream effectors. Due to the limited number of CaMs, other calcium sensors modulate the regulation of Ca(2+) -mediated cellular processes that are not managed by CaMs. Of 50 CaM-like (CML) proteins identified in Arabidopsis thaliana, we characterized the function of CML10. Yeast two-hybrid screening revealed phosphomannomutase (PMM) as a putative interaction partner of CML10. In vitro and in vivo interaction assays were performed to analyze the interaction mechanisms of CML10 and PMM. PMM activity and the phenotypes of cml10 knock-down mutants were studied to elucidate the role(s) of the CML10-PMM interaction. PMM interacted specifically with CML10 in the presence of Ca(2+) through its multiple interaction motifs. This interaction promoted the activity of PMM. The phenotypes of cml10 knock-down mutants were more sensitive to stress conditions than wild-type plants, corresponding with the fact that PMM is an enzyme which modulates the biosynthesis of ascorbic acid, an antioxidant. The results of this research demonstrate that a calcium sensor, CML10, which is an evolutionary variant of CaM, modulates the stress responses in Arabidopsis by regulating ascorbic acid production.

Heterologous expression of chloroplast-localized geranylgeranyl pyrophosphate synthase confers fast plant growth, early flowering and increased seed yield.

Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast-targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS-transgenic lines (control) or wild-type plants. The gibberellin levels in HaGGPS-transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS-transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS-expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy.

Calcineurin B-like 3 calcium sensor associates with and inhibits 5'-methylthioadenosine nucleosidase 2 in Arabidopsis.

Calcineurin B-like (CBL) proteins constitute a unique family of calcium sensor relays in plants. It is well known that CBLs detect the calcium signals elicited by a variety of abiotic stresses and relay the information to a group of serine/threonine protein kinases called CBL-interacting protein kinases (CIPKs). In this study, we found that a few CBL members can also target another group of enzymes 5'-methylthioadenosine nucleosidases (MTANs), which are encoded by two genes in Arabidopsis, AtMTAN1 and AtMTAN2. In the yeast two-hybrid system, AtMTAN1 interacted with multiple CBL members such as CBL2, CBL3 and CBL6, whereas AtMTAN2 associated exclusively with CBL3. We further demonstrated that the CBL3-AtMTAN2 association occurs in a calcium-dependent manner, which results in a significant decrease in the enzyme activity of the AtMTAN2 protein. Taken together, these results clearly indicate that the CBL family can target at least two distinct groups of enzymes (CIPKs and MTANs), conferring an additional level of complexity on the CBL-mediated signaling networks. In addition, our finding also provides a novel molecular mechanism by which calcium signals are transduced to alter metabolite profiles in plants.

Seed-specific expression of seven Arabidopsis promoters.

Seeds contain storage compounds, from various carbohydrates to proteins and lipids, which are synthesized during seed development. For the purposes of many plant researches or commercial applications, developing promoter systems expressing specifically in seeds or in particular constituents or tissues/compartments of seeds are indispensable. To screen genes dominantly or specifically expressed in seed tissues, we analyzed Arabidopsis ATH1 microarray data open to the public. Thirty-two candidate genes were selected and their expressions in seed tissues were confirmed by RT-PCR. Finally, seven genes were selected for promoter analysis. The promoters of seven genes were cloned into pBI101 vector and transformed into Arabidopsis to assay histochemical ß-glucuronidase (GUS) activity. We found that Pro-at3g03230 promoter drove GUS expression in a chalazal endosperm, Pro-at4g27530:GUS expressed in both chalazal endosperm and embryo, Pro-at4g31830 accelerated GUS expression both in radicle and procambium, Pro-at5g10120 and Pro-at5g16460 drove GUS expression uniquely in embryo, Pro-at5g53100:GUS expressed only in endosperm, and Pro-at5g54000 promoted GUS expression in both embryo and inner integument. These promoters can be used for expressing any genes in specific seed tissues for practical application.

Regulation of jasmonic acid biosynthesis by silicon application during physical injury to Oryza sativa L.

We investigated the effects of silicon (Si) application on rice plants (Oryza sativa L.) and its responses in the regulation of jasmonic acid (JA) during wounding stress. Endogenous JA was significantly higher in wounded rice plants than in non-wounded. In contrast, Si treatment significantly reduced JA synthesis as compared to non-Si applications under wounding stress. mRNA expression of O. sativa genes showed down-regulation of lipoxygenase, allene oxide synthase 1, allene oxide synthase 2, 12-oxophytodienoate reductase 3, and allene oxide cyclase upon Si application and wounding stress as compared to non-Si-treated wounded rice plants. The physical injury-induced-oxidative stress was modulated by Si treatments, which resulted in higher catalase, peroxidase, and polyphenol oxidase activities as compared with non-Si-treated plants under wounding stress. The higher Si accumulation in rice plants also reduced the level of lipid peroxidation, which helped the rice plants to protect it from wounding stress. In conclusion, Si accumulation in rice plants mitigated the adverse effects of wounding through regulation of antioxidants and JA.

Overexpression of sweetpotato expansin cDNA (IbEXP1) increases seed yield in Arabidopsis.

Results of transcriptome analyses suggest that expansin genes play an active role in seed development and yield, but gain- or loss-of-function studies have not yet elucidated the functional role(s) of the expansin gene(s) in these processes. We have overexpressed a sweetpotato expansin gene (IbEXP1) in Arabidopsis under the control of cauliflower mosaic 35S promoter in an attempt to determine the effect of the expansin gene in seed development and yield in heterologous plants. The growth rate was enhanced in IbEXP1-overexpressing (ox) plants relative to wild-type Col-0 plants during early vegetative growth stage. At the reproductive stage, the number of rosette leaves was higher in IbEXP1-ox plants than that in Col-0 plants, and siliques were thicker. IbEXP1-ox plants produced larger seeds, accumulated more protein and starch in each seed, and produced more inflorescence stems and siliques than Col-0 plants, leading to a 2.1-2.5-fold increase in total seed yield per plant. The transcript level of IbEXP1 was up-regulated in response to brassinosteroid (BR) treatment in sweetpotato, and the transcript levels of three BR-responsive genes, fatty acid elongase 3-ketoacyl-CoA synthase 1, HAIKU1 and MINISEED3, were also increased in IbEXP1-ox Arabidopsis plants, suggesting a possible involvement of IbEXP1 in at least one of the BR signaling pathways. Based on these results, we suggest that overexpression of IbEXP1 gene in heterologous plants is effective in increasing seed size and number and, consequently, seed yield.

OsMPK3 is a TEY-type rice MAPK in Group C and phosphorylates OsbHLH65, a transcription factor binding to the E-box element.

KEY MESSAGE: OsMPK3 is a TEY-type rice MAPK belonging to Group C and directly phosphorylates OsbHLH65 in the nucleus. OsMPK3 and OsbHLH65 are induced by biotic stress and defense-related hormones. Mitogen-activated protein kinases (MAPKs) are involved in the majority of signaling pathways that regulate plant development and stress tolerance via the phosphorylation of target molecules. Plant MAPKs are classified into two subtypes, TEY and TDY, according to the TxY (x = E or D) motif in their activation loop, and the TDY motif is unique to plant MAPKs. In rice, 17 MAPKs have been classified into six groups. To date, the functions of many TDY-type rice MAPKs have been characterized, but little is known of the TEY-type MAPKs in Group C and their possible target substrates. In the study reported here, we determined that a TEY-type rice MAPK belonging to subgroup C, named OsMPK3, phosphorylates its substrate OsbHLH65 in the nucleus. Our electrophoresis mobility shift assay results revealed that OsbHLH65 specifically binds to the E-box cis-element, but not to the G-box. Both OsMPK3 and OsbHLH65 were induced by treatments with rice blast (Magnaporthe grisea), brown planthopper (Nilaparvata lugens), and defense-related hormones, such as methyl jasmonic acid and salicylic acid. Our results suggest the possibility that OsMPK3 contributes to the defense signal transduction by phosphorylating the basic helix-loop-helix transcription factor.