Two aquaporins, SIP1;1 and PIP1;2, mediate water transport for pollen hydration in the Arabidopsis pistil.

Pollination is the crucial initial step that brings together the male and female gametophytes, and occurs at the surface of the stigmatic papilla cell in Arabidopsis thaliana. After pollen recognition, pollen hydration is initiated as a second critical step to activate desiccated mature pollen grains for germination, and thus water transport from pistil to pollen is essential for this process. In this study, we report a novel aquaporin-mediated water transport process in the papilla cell as a control mechanism for pollen hydration. Coupled with a time-series imaging analysis of pollination and a reverse genetic analysis using T-DNA insertion Arabidopsis mutants, we found that two aquaporins, the ER-bound SIP1;1 and the plasma membrane-bound PIP1;2, are key players in water transport from papilla cell to pollen during pollination. In wild type plant, hydration speed reached its maximal value within 5 min after pollination, remained high until 10-15 min. In contrast, sip1;1 and pip1;2 mutants showed no rapid increase of hydration speed, but instead a moderate increase during ∼25 min after pollination. Pollen of sip1;1 and pip1;2 mutants had normal viability without any functional defects for pollination, indicating that decelerated pollen hydration is due to a functional defect on the female side in sip1;1 and pip1;2 mutants. In addition, sip1;1 pip1;2 double knockout mutant showed a similar impairment of pollen hydration to individual single mutants, suggesting that their coordinated regulation is critical for proper water transport, in terms of speed and amount, in the pistil to accomplish successful pollen hydration.

Double-Locking Mechanism of Self-Compatibility in Arabidopsis thaliana: The Synergistic Effect of Transcriptional Depression and Disruption of Coding Region in the Male Specificity Gene.

Self-compatibility in Arabidopsis thaliana represents the relatively recent disruption of ancestral obligate cross pollination, recognized as one of the prevalent evolutionary pathways in flowering plants, as noted by Darwin. Our previous study found that inversion of the male specificity gene (SP11/SCR) disrupted self-incompatibility, which was restored by overexpressing the SCR with the reversed inversion. However, SCR in A. thaliana has other mutations aside from the pivotal inversion, in both promoter and coding regions, with probable effects on transcriptional regulation. To examine the functional consequences of these mutations, we conducted reciprocal introductions of native promoters and downstream sequences from orthologous loci of self-compatible A. thaliana and self-incompatible A. halleri. Use of this inter-species pair enabled us to expand the scope of the analysis to transcriptional regulation and deletion in the intron, in addition to inversion in the native genomic background. Initial analysis revealed that A. thaliana has a significantly lower basal expression level of SCR transcripts in the critical reproductive stage compared to that of A. halleri, suggesting that the promoter was attenuated in inducing transcription in A. thaliana. However, in reciprocal transgenic experiments, this A. thaliana promoter was able to restore partial function if coupled with the functional A. halleri coding sequence, despite extensive alterations due to the self-compatible mode of reproduction in A. thaliana. This represents a synergistic effect of the promoter and the inversion resulting in fixation of self-compatibility, primarily enforced by disruption of SCR. Our findings elucidate the functional and evolutionary context of the historical transition in A. thaliana thus contributing to the understanding of the molecular events leading to development of self-compatibility.

Abscisic acid-mediated developmental flexibility of stigmatic papillae in response to ambient humidity in Arabidopsis thaliana.

Stigmatic papillae develop at the apex of the gynoecium and play an important role as a site of pollination. The papillae in Brassicaceae are of the dry and unicellular type, and more than 15,000 genes are expressed in the papillae; however, the molecular and physiological mechanisms of their development remain unknown. We found that the papillae in Arabidopsis thaliana change their length in response to altered ambient humidity: papillae of flowers incubated under high humidity elongated more than those under normal humidity conditions. Genetic analysis and transcriptome data suggest that an abscisic acid-mediated abiotic stress response mechanism regulates papilla length. Our data suggest a flexible regulation of papilla elongation at the post-anthesis stage, in response to abiotic stress, as an adaptation to environmental conditions.

The Arabidopsis transcription factor NAI1 is required for enhancing the active histone mark but not for removing the repressive mark on PYK10, a seedling-specific gene upon embryonic-to-postgerminative developmental phase transition.

We have recently shown that the expression onset of a seedling-specific gene, PYK10, occurs in a cell-by-cell manner upon the transition from the embryonic to the postgerminative phase and during embryogenesis in seed maturation regulator mutants such as lec1, and implicated epigenetic mechanisms in the process. Here, the role of the NAI1 transcription factor required for PYK10 expression in the developmental switching of PYK10 was investigated. The cell-by-cell onset of PYK10-EGFP in lec1 embryo was still observed in the nai1 background, but at greatly reduced levels. Decreases in the level of the repressive histone mark, H3K27 trimethylation observed upon the transition to the postgeminative phase normally occurred in nai1. However, concomitant increases in the level of the active mark, H3K4 trimethylation observed in wild type was significantly compromised in nai1. These results indicate that the switching of PYK10 upon developmental phase transition involves 2 separable steps of chromatin state change.

Rice SNF2 family helicase ENL1 is essential for syncytial endosperm development.

The endosperm of cereal grains represents the most important source of human nutrition. In addition, the endosperm provides many investigatory opportunities for biologists because of the unique processes that occur during its ontogeny, including syncytial development at early stages. Rice endospermless 1 (enl1) develops seeds lacking an endosperm but carrying a functional embryo. The enl1 endosperm produces strikingly enlarged amoeboid nuclei. These abnormal nuclei result from a malfunction in mitotic chromosomal segregation during syncytial endosperm development. The molecular identification of the causal gene revealed that ENL1 encodes an SNF2 helicase family protein that is orthologous to human Plk1-Interacting Checkpoint Helicase (PICH), which has been implicated in the resolution of persistent DNA catenation during anaphase. ENL1-Venus (enhanced yellow fluorescent protein (YFP)) localizes to the cytoplasm during interphase but moves to the chromosome arms during mitosis. ENL1-Venus is also detected on a thread-like structure that connects separating sister chromosomes. These observations indicate the functional conservation between PICH and ENL1 and confirm the proposed role of PICH. Although ENL1 dysfunction also affects karyokinesis in the root meristem, enl1 plants can grow in a field and set seeds, indicating that its indispensability is tissue-dependent. Notably, despite the wide conservation of ENL1/PICH among eukaryotes, the loss of function of the ENL1 ortholog in Arabidopsis (CHR24) has only marginal effects on endosperm nuclei and results in normal plant development. Our results suggest that ENL1 is endowed with an indispensable role to secure the extremely rapid nuclear cycle during syncytial endosperm development in rice.

Cell-by-cell developmental transition from embryo to post-germination phase revealed by heterochronic gene expression and ER-body formation in Arabidopsis leafy cotyledon mutants.

LEC1, LEC2, FUS3 and ABI3 (collectively abbreviated LEC/ABI3 here) are required for embryo maturation and have apparent roles in repressing post-germinative development. lec mutant embryos exhibit some heterochronic characteristics, as exemplified by the development of true leaf-like cotyledons during embryogenesis. Although the roles of LEC/ABI3 as positive regulators of embryo maturation have been extensively studied, their roles in the negative regulation of post-germinative development have not been explored in detail. Based on microarray analyses, we chose PYK10, which encodes an endoplasmic reticulum (ER)-body-localized protein, as a molecular marker of post-germinative development. lec/abi3 embryos exhibited PYK10 misexpression and the formation of 'constitutive' ER-bodies, which develop specifically during the seedling stage, confirming the heterochronic nature of these mutants at both the gene expression and cellular levels. The PYK10 reporter expression in lec1 embryos started as early as the globular-heart transition stage. The onset of PYK10 promoter-enhanced green fluorescent protein (EGFP) reporter expression occurred in a stochastic, cell-by-cell manner in both developing lec/abi3 embryos and germinating wild-type seedlings. Additionally, clustered EGFP-positive cells were frequently found along cell files, probably representing the transmission of the expression state via cell division. These observations, together with the results of the experiments using PYK10-EGFP/PYK10-CFP double reporter transgenic lines and the analyses of H3K27me3 levels in the PYK10 chromatin, suggested the involvement of epigenetic mechanisms in repressing post-germinative genes during embryogenesis and derepressing these genes upon the transition to post-germinative development.

Ectopic gene expression and organogenesis in Arabidopsis mutants missing BRU1 required for genome maintenance.

Chromatin reconstitution after DNA replication and repair is essential for the inheritance of epigenetic information, but mechanisms underlying such a process are still poorly understood. Previously, we proposed that Arabidopsis BRU1 functions to ensure the chromatin reconstitution. Loss-of-function mutants of BRU1 are hypersensitive to genotoxic stresses and cause release of transcriptional gene silencing of heterochromatic genes. In this study, we show that BRU1 also plays roles in gene regulation in euchromatic regions. bru1 mutations caused sporadic ectopic expression of genes, including those that encode master regulators of developmental programs such as stem cell maintenance and embryogenesis. bru1 mutants exhibited adventitious organogenesis, probably due to the misexpression of such developmental regulators. The key regulatory genes misregulated in bru1 alleles were often targets of PcG SET-domain proteins, although the overlap between the bru1-misregulated and PcG SET-domain-regulated genes was limited at a genome-wide level. Surprisingly, a considerable fraction of the genes activated in bru1 were located in several subchromosomal regions ranging from 174 to 944 kb in size. Our results suggest that BRU1 has a function related to the stability of subchromosomal gene regulation in the euchromatic regions, in addition to the maintenance of chromatin states coupled with heritable epigenetic marks.

Diverse roles and mechanisms of gene regulation by the Arabidopsis seed maturation master regulator FUS3 revealed by microarray analysis.

The FUSCA3 (FUS3) transcription factor is considered a master regulator of seed maturation because a wide range of seed maturation events are impaired in its defective mutant. To identify comprehensively genes under the control of FUS3, two types of microarray experiments were performed. First, transgenic plants in which FUS3 expression could be induced by the application of estrogen (ESTR) were used to identify any genes up-regulated in young seedlings of Arabidopsis in response to the ectopic expression of FUS3. Secondly, the transcriptomes of the fus3 mutant and wild-type developing seeds were compared. The combined results of these experiments identified genes under the relatively immediate and robust control of FUS3 during seed development. The analysis has extended the range of identified gene types under the control of FUS3. The genes positively controlled by FUS3 are not confined to previously known seed maturation-related genes and include those involved in the production of secondary metabolites, such as glucosinolates, phenylpropanoids and flavonoids, and those involved in primary metabolism, such as photosynthesis and fatty acid biosynthesis. Furthermore, several different patterns were identified in the manner of ectopic activation by FUS3 with respect to the induction kinetics and ABA requirement of downstream gene induction depending on the nature of developmental regulation, suggesting mechanistic diversity of gene regulation by FUS3.

Arabidopsis transcription factors, RAV1 and RAV2, are regulated by touch-related stimuli in a dose-dependent and biphasic manner.

Transcription factors, RAV1 and RAV2 from Arabidopsis thaliana, contain two distinct DNA-binding domains, AP2/EREBP and B3, both of which are uniquely found in plants. We found that transcripts of RAV1 and RAV2 were upregulated transiently by touch-related mechanical stimuli. However, the temporal expression patterns of RAV1 and RAV2 differed from those of known touch-induced genes. A striking feature of mechanical stimulus-induced expression of RAV1 and RAV2 was that it was biphasic; the RAV1 and RAV2 expression was reinduced and sustained after a rapid and transient induction. The extent of both transient and subsequent upregulation by touch-stimuli depended on the dose of the initial stimulus. Analysis of transgenic A. thaliana plants carrying a RAV2 promoter-GUS fusion gene indicated that the transient mechanical stimulus-induced RAV2 expression was primarily controlled by its promoter. Histochemical analysis of the transgenic plants revealed that GUS expression was strongly induced in the petioles and primordia of true leaves and shoot apical meristems, which may be related to the alteration in plant growth pattern caused by touch-stimuli. Because RAV1 has been suggested to be a negative regulator of growth and development, the dose-dependent biphasic upregulation of RAV1 and RAV2 may serve not only for immediate physiological responses and but also for developmental adaptation in response to the environmental stimuli.

Arabidopsis NF-YB subunits LEC1 and LEC1-LIKE activate transcription by interacting with seed-specific ABRE-binding factors.

LEAFY COTYLEDON 1 (LEC1) plays vital roles in the regulation of seed maturation in Arabidopsis. LEC1 encodes a homolog of yeast HAP3 or mammalian NF-YB/CBF-A subunit of trimeric CCAAT binding factor (CBF). Among the nine paralogs of NF-YB in Arabidopsis, LEC1-LIKE (L1L) is most closely related to LEC1, and can complement the lec1 mutation when expressed under the control of the LEC1 promoter. Although the nature of the B3-type seed maturation regulators as transcription factors have been investigated, knowledge of the molecular action of LEC1 is limited. When co-expressed with NF-YC2 in the presence of ABA, we found that LEC1 or L1L, but not other NF-YBs, activated the promoter of CRUCIFERIN C (CRC), which encodes a seed storage protein. However, additional expression of an NF-YA subunit interfered with the activation. The LEC1/L1L-[NF-YC2] activation depended on ABA-response elements present in the promoter, which led to the finding that LEC1/L1L-[NF-YC2] can strongly activate the CRC promoter in the absence of ABA when co-expressed with a seed-specific ABA-response element (ABRE)-binding factor, bZIP67. Functional coupling of LEC1/L1L-[AtNF-YC2] and bZIP67 was also observed in the regulation of sucrose synthase 2 (SUS2). Immunoprecipitation experiments revealed that L1L and bZIP67 formed a protein complex in vivo. These results demonstrate a novel plant-specific mechanism for NF-Y subunit function that enables LEC1 and L1L to regulate a defined developmental network.

Abscisic acid-activated SNRK2 protein kinases function in the gene-regulation pathway of ABA signal transduction by phosphorylating ABA response element-binding factors.

The plant hormone abscisic acid (ABA) induces gene expression via the ABA-response element (ABRE) present in the promoters of ABA-regulated genes. A group of bZIP proteins have been identified as ABRE-binding factors (ABFs) that activate transcription through this cis element. A rice ABF, TRAB1, has been shown to be activated via ABA-dependent phosphorylation. While a large number of signalling factors have been identified that are involved in stomatal regulation by ABA, relatively less is known about the ABA-signalling pathway that leads to gene expression. We have shown recently that three members of the rice SnRK2 protein kinase family, SAPK8, SAPK9 and SAPK10, are activated by ABA signal as well as by hyperosmotic stress. Here we show that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases leads to the activation of an ABRE-regulated promoter, suggesting that these kinases are involved in the gene-regulation pathway of ABA signalling. We further show several lines of evidence that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA, but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and is critical for the activation function.

LEAFY COTYLEDON1 controls seed storage protein genes through its regulation of FUSCA3 and ABSCISIC ACID INSENSITIVE3.

Arabidopsis ABSCISIC ACID INSENSEITIVE3 (ABI3), FUSCA3 (FUS3) and LEAFY COTYLEDON1 (LEC1) encode key transcription factors that control seed maturation events, including seed storage protein (SSP) accumulation. Although lec1 mutations are known to down-regulate SSP gene expression as the fus3 or abi3 mutation does, the mechanisms by which LEC1 regulates SSP gene expression are largely unknown compared with the mechanisms utilized by FUS3 or ABI3. We expressed LEC1 ectopically in transgenic plants using an artificial dexamethasone (Dex) induction system. The ectopic expression of LEC1 also resulted in the induction of FUS3 and ABI3 expression, which preceded the induction of SSP expression. The expression of FUS3 and ABI3 was found to be down-regulated in developing siliques of the lec1 mutant. Furthermore, the levels of ectopic SSP induction by LEC1 were greatly or moderately reduced in transgenic plants with an abi3 or fus3 mutant background, respectively. LEC1-induced ectopic expression of the At1g62290 aspartic protease gene, which was identified to be regulated preferentially by FUS3, was more severely affected in the fus3 mutant than in the abi3 mutant. From these data, we suggest that LEC1 controls the expression of the SSP genes in a hierarchical manner, which involves ABI3 and FUS3.

Indirect ABA-dependent regulation of seed storage protein genes by FUSCA3 transcription factor in Arabidopsis.

The key transcription factors that control seed maturation, ABSCISIC ACID INSENSITIVE3 (ABI3) and FUSCA3 (FUS3), share homologous DNA-binding domains. Regulation of seed storage protein genes At2S3 and CRC by ABI3 and FUS3 was investigated using transgenic plants in which ABI3 and FUS3 could be ectopically induced by steroid hormones. Like ABI3, the presence of FUS3 led to expression of At2S3 and CRC in vegetative tissues. FUS3-mediated induction of CRC was completely dependent on exogenous abscisic acid (ABA), while At2S3 was weakly induced without ABA but strongly enhanced with ABA. This ABA dependency of FUS3-induced CRC and At2S3 expression was similar to that observed for ABI3. However, kinetic analysis revealed distinctions between the mechanisms of ABA-dependent CRC regulation by FUS3 or ABI3, and between target genes. While At2S3 activation by FUS3 was rapid, CRC induction by FUS3 in the presence of ABA, and by ABA followed by the presence of FUS3, took a significantly longer time (24-36 h). This suggested the involvement of an indirect mechanism requiring the ABA- and FUS3-dependent synthesis of intermediate regulatory factor(s). A chimeric protein composed of the FUS3 B3 domain, and a heterologous activation domain and nuclear localization signal exhibited a tight coupling with ABA regulation as observed for wild-type FUS3. Simultaneous induction of FUS3 and ABI3 did not result in the synergistic activation of CRC and At2S3. Based on these results, similarities and differences in the mechanisms of seed storage protein gene regulation by FUS3 and ABI3 are discussed.

Differential activation of the rice sucrose nonfermenting1-related protein kinase2 family by hyperosmotic stress and abscisic acid.

To date, a large number of sequences of protein kinases that belong to the sucrose nonfermenting1-related protein kinase2 (SnRK2) family are found in databases. However, only limited numbers of the family members have been characterized and implicated in abscisic acid (ABA) and hyperosmotic stress signaling. We identified 10 SnRK2 protein kinases encoded by the rice (Oryza sativa) genome. Each of the 10 members was expressed in cultured cell protoplasts, and its regulation was analyzed. Here, we demonstrate that all family members are activated by hyperosmotic stress and that three of them are also activated by ABA. Surprisingly, there were no members that were activated only by ABA. The activation was found to be regulated via phosphorylation. In addition to the functional distinction with respect to ABA regulation, dependence of activation on the hyperosmotic strength was different among the members. We show that the relatively diverged C-terminal domain is mainly responsible for this functional distinction, although the kinase domain also contributes to these differences. The results indicated that the SnRK2 protein kinase family has evolved specifically for hyperosmotic stress signaling and that individual members have acquired distinct regulatory properties, including ABA responsiveness by modifying the C-terminal domain.

Abscisic acid-induced transcription is mediated by phosphorylation of an abscisic acid response element binding factor, TRAB1.

The rice basic domain/Leu zipper factor TRAB1 binds to abscisic acid (ABA) response elements and mediates ABA signals to activate transcription. We show that TRAB1 is phosphorylated rapidly in an in vivo labeling experiment and by phosphatase-sensitive mobility shifts on SDS-polyacrylamide gels. We had shown previously that a chimeric promoter containing GAL4 binding sites became ABA inducible when a GAL4 binding domain-TRAB1 fusion protein was present. This expression system allowed us to assay the ABA response function of TRAB1. Using this system, we show that Ser-102 of TRAB1 is critical for this function. Because no ABA-induced mobility shift was observed when Ser-102 was replaced by Ala, we suggest that this Ser residue is phosphorylated in response to ABA. Cell fractionation experiments, as well as fluorescence microscopy observations of transiently expressed green fluorescent protein-TRAB1 fusion protein, indicated that TRAB1 was localized in the nucleus independently of ABA. Our results suggest that the terminal or nearly terminal event of the primary ABA signal transduction pathway is the phosphorylation in the nucleus of preexisting TRAB1.

Temporal and spatial expression pattern of the OSVP1 and OSEM genes during seed development in rice.

The spatial and temporal expression patterns of the rice VP1 (OSVP1) gene, as well as the OSEM gene which it controls, were studied during seed development by in situ hybridization and immuno-localization techniques. The expression of OSVP1 could be detected in embryos as early as 2-3 d after pollination (DAP) and thereafter became preferentially localized to shoot, radicle and vascular tissues during the embryo development at both the mRNA and protein levels. In the aleurone layers, OSVP1 mRNA and protein were detected after 6 DAP. OSEM mRNA was detectable after 6 DAP in the embryo and aleurone tissue. The spatial distribution within the embryo of OSEM mRNA and OSVP1 mRNA/protein was very similar after 6 DAP. Transgenic rice carrying a beta-glucuronidase (GUS) gene transcribed from a chimeric promoter consisting of the CaMV 35S minimal promoter (-46) and the 55-bp promoter fragment of OSEM, minimally required for ABA and VP1 regulation, also exhibited a spatial pattern of GUS expression similar to that of OSEM and OSVP1. These results suggest that (OS)VP1 is a major determinant not only of the seed specificity but also of the spatial pattern of OSEM expression in the developing seed.

Experimentally determined sequence requirement of ACGT-containing abscisic acid response element.

The sequence requirement of the ACGT-containing abscisic acid response element (ABRE) was analyzed by systematically substituting the bases surrounding the ACGT-core of motif A, the principal ABRE of the rice gene, OSEM: This was done within the context of a 55-bp promoter fragment that minimally confers ABA-responsiveness to a heterologous promoter. Based on this analysis, the sequence requirement of the ACGT-containing ABRE was determined as ACGTG G/T C, which matched very well with the consensus derived from sequence comparison of ABA-responsive promoters.