Plant disease resistance (R) proteins act as intracellular receptors for sensing pathogen invasion, and trigger a variety of immune responses. We have recently reported that the small GTPase OsRac1 is activated by R protein Pit at the plasma membrane, and this activation plays a critical role in R protein-mediated immunity in rice. However, mechanisms underlying localization of R protein are largely unknown. To identify molecules which are involved in localization of R proteins, we screened inhibitors which make a difference to localization of Pit. Pit was mainly localized at the plasma membrane in rice protoplasts and this membrane localization was compromised in the cells treated with the molecular chaperone Hsp90 inhibitor. Pit formed a complex with Hsp90 in rice suspension cells. Agroinfiltration of the active form of Pit induced the hypersensitive response in Nicotiana benthamiana, whereas the treatment of Hsp90 inhibitor suppressed Pit-induced hypersensitive response. These results suggest that Hsp90 plays a key role in the localization and function of R protein. In addition, we found that Palmitoylation is also a critical role of the localization of Pit.

View full abstract

Plants have the ability to detect invading pathogens through the perception of microbe-associated molecular patterns (MAMPs). Nevertheless, some pathogens are able to inhibit the MAMPs perception/signaling as a part of their pathogenic strategy. It is becoming evident that several pathogenic fungi such as Cladosporium fulvum and Magnaporthe grisea secrete LysM effectors that structurally resemble the extracellular part of plant chitin receptors, CEBiP and CERK1, and inhibit chitin perception by these receptors [1].
We observed that these LysM effectors can bind chitin oligosaccharides with high affinity by BIACORE analysis. We also found that these molecules inhibit the detection of chitin oligosaccharides by plant receptors by competing for the binding of ligand in affinity labeling experiments. Finally, we found that the addition of LysM effectors to rice cells inhibited the chitin induced of defense responses. These observations suggest that competition for the detection of chitin oligosaccharides between plant receptors and fungal effectors is an important strategy in the battle between plants and pathogens.
[1]de Jonge et al., Science, 329, 953 (2010).

View full abstract

Proteomics is one of the best available tools for studying posttranslational modifications (PTMs), and it has no limitation like those encountered with forward genetics. Therefore, it is well suited for the analysis of unknown signaling pathways. Among the several PTMs described thus far, phosphorylation is the most extensively studied, and they have been shown to play a role in plant immune signaling. We have developed a phosphoproteomics platform, which enables monitoring phosphorylation events in plant cells at the cellular level.
To reveal novel players involved in plant immunity, we are analyzing phosphoproteome dynamics upon MAMP (microbe-associated molecular pattern) treatments. Furthermore, we are developing a novel method, which utilizes the phosphoproteomics technique, to identify substrates of protein kinases involved in plant immunity.

View full abstract

Plant bacterial pathogens equipped with the type III secretion system (TTSS) generally deliver different TTSS effector proteins into plant cells. These TTSS effector proteins modulate the function of crucial host regulatory molecules and allow bacteria to invade plant cells. To identify Xoo TTSS effectors that inhibit host immune responses, we generated transgenic rice plants expressing each of Xoo effectors. Transgenic plants expressing some of Xoo effectors developed severe disease lesions by infection with the hrpX mutant of Xoo, suggesting that these effectors may block the important steps in PAMPs-triggered immunity (PTI). We identified two rice receptor like cytoplasmic kinase (OsRLCKs) as potential interacting proteins for one of the effectors using yeast two-hybrid (Y2H) screen. Interaction between the effector and OsRLCKs were analyzed by using Y2H and BiFC. We also found that OsRLCKs interacted with some of PAMP receptors. It was recently reported that RLCKs play important roles in signal transduction involved in plant immunity and development. We will discuss the role of OsRLCKs in PTI.

View full abstract

Rice has perception systems of flagellin from rice-avirulent strain of phytopathogenic bateria Acidovorax avenae. To clarify the flagellin perception mechanism in rice, several domains of the flagellin were prepared and induction activity of each domain was examined. The flagellin C-terminal domain was induced the immune responses in rice, whereas the flagellin N-teminal domain, containing flg22, did not cause any immune responses. Gene expression profiling during flagellin treatment showed that expression of several genes encoding the receptor kinase were increased. Among the identified genes, Flagellin-induced Receptor Kinase 1 (FliRK1) complemented the flagellin recognition ability in fls2 mutant. Two flirk1 disruption mutants in rice lost the ability of the flagellin perception. Immuno precipitation and immuno detection using anti-flagellin antibody, showed that FliRK1 specifically interacted with flagellin. Our results suggest that FliRK1 is specific receptor of the flagellin in rice.

View full abstract

Adenosine 5'-triphosphate (ATP) involved in many cellular biological processes. It has been known that concentration of intracellular ATP ([ATP]_i) is decreased with time after induction of cell death in plant, suggesting that the reduction of [ATP]_i might lead to activation of cell death pathway in plants. To further investigate possible involvement of ATP in the plant cell death, we tried visualization of [ATP]_i dynamics in live plant at single cell level by using a FRET-based ATP indicator, ATeam, which has been recently developed by us. We examined whether pathogen-induced hypersensitive cell death (HCD) is associated with reduction of [ATP]_i in Arabidopsis plants. When the plant was inoculated with virulent bacteria, the [ATP]_i did not changed. On the other hand, inoculation of avirulent bacteria induced small [ATP]_i reduction, which preceded the changes in morphological features of HCD, and then the reduction of [ATP]_i still continued after the morphological changing. This result raised two opposing possibilities, i.e., small reduction of [ATP]_i is essential to activate cell death pathway in HCD, or ATP is not involved in HCD.

View full abstract

The shoot apical meristem (SAM) is an indeterminate structure that maintains itself, and initiates all above ground organs throughout the life cycle. Class I Knotted1-like homeobox (KNOX) genes are specifically expressed in the SAM, and maintain its indeterminacy in flowering plants. Despite the indispensable roles of KNOX genes for SAM formation and maintenance, virtually nothing is known about the mechanisms how positively regulate their expression. By genetic analyses, we found that the expression of KNOX genes depends on the function of themselves. OSH1 positively regulates the expression of all KNOX genes in a direct manner through evolutionarily conserved cis-elements. Furthermore, we show that these cis elements on OSH1 loci are essential for its expression and SAM maintenance. Taking together, the maintenance mechanism of the indeterminate state mediated by positive autoregulation of a KNOX gene emerges as a novel mechanism of self-maintenance system of the SAM that ensures the unique body plan of flowering plants.

View full abstract

Whereas most plant cells differentiate to generate the specialized cell types within the different organs, stem cells remain undifferentiated and retain the potential to divide and generate new cells for sustained growth.
We have previously shown that two members of plant-specific gene families, double AP2-domain PLETHORA (PLT) transcription factors and GRAS family transcription factor SCARECROW (SCR ), are key players in root growth and maintenance of the stem cell niche in Arabidopsis.
However, the molecular mechanisms by which those factors may interact were largely unknown.
To address this question, we screened interactors of both PLT and SCR proteins, and identified several candidate regulatory factors of PLTs and SCR. A third family of plant-specific transcription factor is associated with both SCR and PLT proteins in vitro and in vivo. Genetic and histological analyses support the idea that these factors together with PLT and/or SCR proteins fine tune the progression of differentiation in plants. Progress towards characterizing the interacting proteins and their role(s) in regulating PLT and SCR networks will be presented.

View full abstract

During the development of multicellular organisms, several distinct cell fates arise at defined positions. Cell type specific transcription factors play key roles in determining cell fates through the regulation of gene expression. ATML1, an Arabidopsis homeobox gene, is expressed in the outermost cell layer from the early stage of development. Many epidermis-specific genes including ATML1 contain an ATML1-binding site in their promoters, suggesting ATML1-mediated regulation of epidermal cell fate. However, it still remains unknown whether ATML1 is sufficient for the activation of these epidermis-specific genes. To further assess the role of ATML1 in epidermis-specific gene expression, we examined the effect of overexpression of ATML1 in the postembryonic development. Constitutive expression of ATML1 fused with a transcriptional repressor sequence decreased epidermis-specific gene expression and affected epidermal cell morphology. Conversely, constitutive expression of ATML1 was able to activate epidermis-specific genes in the inner cells of the seedling. These results suggested that ATML1 acts on the promoters of epidermis-specific genes and positively regulate their expressions.

View full abstract

Lysine is the most deficient essential amino acid in cereal grains. A bifunctional lysine degrading enzyme, lysine ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH), is one of the key regulators determining free lysine content in plants. In rice (Oryza sativa. L), a bifunctional OsLKR/SDH is predominantly present in seeds. Here, we show that OsLKR/SDH is directly regulated by RISBZ1 and RPBF, major transcriptional regulators of seed storage protein (SSP) genes. OsLKR/SDH was highly expressed in the aleurone and subaleurone layers of the endosperm. RPBF-recognizing prolamin box and the RISBZ1-recognizing GCN4 motif act as important cis-elements for proper expression of OsLKR/SDH, like SSP genes. Reducing either RISBZ1 or RPBF decreased OsLKR/SDH levels, resulting in an increase in free Lys content in rice grain. This result was in contrast with the fact that a significant reduction of SSP was observed only when these transcription factors were simultaneously reduced. The same combinations of TF and cis-elements are involved in the regulation of OsLKR/SDH and SSP genes, but there is a distinct difference in their regulation mechanisms.

View full abstract

ABA is an important phytohormone regulating various plant processes, including stress tolerance, seed development and germination. SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3 are redundant ABA-activated SNF1-related protein kinases 2 (SnRK2s) in Arabidopsis thaliana. We examined the role of SRK2D, SRK2E and SRK2I in seeds. The triple mutant (srk2d srk2e srk2i) was sensitive to desiccation, showed severe growth defects during seed development, exhibited a loss of dormancy and vivipary, and showed highly enhanced insensitivity to ABA (Nakashima et al., 2009, Fujita et al., 2009). It also showed insensitivity to sugars and paclobutrazol (PAC), a gibberellin biosynthesis inhibitor. Disruption of the three protein kinases induced global changes in the expression of ABA/stress-related genes including genes encoding LEA proteins and HSPs in seeds. Stress tolerant assay revealed that the triple mutant seeds were sensitive to cold and heat stresses. These results indicate that these protein kinases are essential for the control of seed development, dormancy and abiotic stress tolerance through the extensive regulation of global gene expression.

View full abstract

In developing Arabidopsis seeds, assembly of triacylglycelols (TAGs) from fatty acids synthesized in chloroplasts takes place in the endoplasmic reticulumn, and expression of genes involved in TAG assembly such as DGAT1 coding for diacylglycerol acyltransferase follows the expression of genes involved in fatty acid synthesis. A DREB subfamily transcription factor A2, with expression patterns during seed maturation similar to that of DGAT1, trans-activated transient expression of DGAT1:LUC reporter gene in protoplasts. The DGAT1 promoter contained a DRE sequence, and mutation in the DRE site of DGAT1:LUC abolished the trans-activation by A2. Recombinant A2 showed binding to the DRE sequence in vitro. A T-DNA insertion line of A2 showed reduced levels of DGAT1 mRNA in developing fruits compared to the wild type, but seed oil content was not significantly affected. A T-DNA insertion line of a gene for a close homolog of A2 also showed reduced DGAT1 mRNA in fruits, suggesting that they may share overlapping roles. Since the 35S:DGAT1 plants showed reduced DGAT1 mRNA probably due to co-suppression, we are preparing plants expressing A2 under the control of a seed-specific promoter.

View full abstract

HSI2-subfamily B3 domain factors of Arabidopsis are required for repression of genes for embryogenesis and seed maturation after germination. Seeds of hsi2 hsl1 double knockouts express seed maturation genes after germination, accumulate seed storage proteins and oil in hypocotyls, and stop growth 7 days after germination¹⁾. Analyses of DNA-binding of HSI2 and transcripts in various mutants suggest that genes for seed storage proteins and seed oleosins are among the direct targets of HSI2. Chromatin of these genes is enriched with H3K27me3 inactive markers in mature plants. During germination of Col-0 seeds, mRNAs of these genes were lost within several days after germination, while H3K27me3 modification became prominent two weeks after germination. In hsi2 seedlings, these genes did not receive H3K27me3 modification even two weeks after germination. OleS3p::LUC showed transient LUC luminescence 36 hrs after germination in Col-0 seeds on medium containing luciferin. On the other hand, it showed continued expression after germination of hsi2 hsl1 seeds, suggesting that OleS3p::LUC enables us to monitor repression of seed maturation genes. ¹⁾Tsukagoshi et al., PNAS 104: 2543 (2007).

View full abstract

During the maturation of Arabidopsis seeds, a large portion of sugars imported from the source are converted into oil for storage. A double-AP2 domain transcription factor ASML1/WRI1 directly activates genes for fatty acid synthesis in the plastid, while the activation of genes for the synthesis of triacylglycerol (TAG) in the ER requires additional factors. To search for such factors, a luciferase (LUC) reporter gene with the promoter of a gene for diacylglycerol acyltransferase (DGAT1) was introduced into Arabidopsis. In addition to strong expression in maturing seeds, the DGAT1p::LUC plants showed LUC expression in leaves of young seedlings, similar to the expression of DGAT1 mRNA. EMS-treated seeds of the DGAT1p::LUC plants screened for mutants with reduced LUC expression in seedlings by using the real-time bioluminescence monitoring system. Most of the mutants that consistently exhibit low LUC expression in the M₃ generation showed reduced bioluminescence in developing seeds and reduced seed oil content. We are screening for more mutants and trying to identify genes responsible for these mutants by next-generation sequencer.

View full abstract

In Oryza sativa (rice), floral organs such as stamens and carpels are enclosed by the lemma and palea. On the top of the lemma, a needle-like structure, called awn, is formed in O. sativa L. ssp. indica and wild rice species. The awn sharpens at the tip and connects to the midrib of the lemma at the base. Molecular mechanisms underlying awn development are not known. To understand the mechanisms regulating development of the awn, we focused on two genes, DROOPING LEAF (DL) and SHOOTLESS2 (SHL2). The DL gene, a member of the YABBY gene family, encodes a plant specific transcriptional factor and is known to have a crucial function in midrib formation and carpel specification. The SHL2 gene encodes an enzyme responsible for trans-acting siRNA synthesis.
Our genetic analyses revealed that DL had a role to promote awn formation, in addition to the well-known functions of DL. Although DL was specifically expressed in the midrib in the lemma, as it is in the leaves, its expression was not detected in the developing awn itself, suggesting that DL acts to form the awn through a non-cell autonomous mechanism. By contrast, the SHL2 gene negatively regulated awn formation.

View full abstract

The timing of meristem transition from the panicle branch meristem to the spikelet meristem is crucial in the control of inflorescence architecture in rice. Molecular mechanisms of this transition have begun to be understood. Here, we report a new gene, TAWAWA1 (TAW1) that negatively controls meristem transition in inflorescence development. The taw1-D mutation inherited in a semi-dominant manner. The taw1-D allele contained an insertion of nDart1 transposon. It was presented in the 0.7kb downstream of the 3 ^,UTR of TAW1 . This insertion caused an increase of TAW1 mRNA level. TAW1 mRNA accumulation was decreased from shoot apical meristem to branch meristem. These suggest that the change to spikelet meristem identity in taw1-D is delayed depending on the TAW1 expression level. It is interesting to know how complexes consisting of TAW1 regulate meristem transition in rice inflorescence.

View full abstract

The inflorescence of rice consists of several rachis branches. Meristems produced on the branch acquire the identity as a spikelet meristem (SM). SM differentiates a flower and terminates its growth. The timing of the phase transition to the SM is a major determinant of the rice inflorescence structure. It was reported that PAP2 accelerates the acquisition of the identity as a SM. On the other hand, APO1 and RFL suppress the SM identity. The purpose of this study is to clarify the mechanism regulating meristem phase change through the analysis of molecular functions of APO1 and RFL.
APO1 physically binds with RFL and functions interdependently. The expression of APO1 initiates in the inflorescence meristem (IM) after the transition to the reproductive phase and activates cell proliferation in IM. We carried out transcriptome analysis of genes specifically expressed in IM at early stages of inflorescence development by using LMD-microarray. It was shown that RFL and MADS-box genes related to floral transition were identified in this analysis, indicating the validity of this method. Isolation of genes under the control of APO1 by using same LMD-microarray system is in progress.

View full abstract

The control of meristem identity is crucial for plants to determine their body plan. In particular, the transition from vegetative meristem to reproductive meristem and the transition from inflorescence meristem to floral meristem are essential for successful reproduction.
We searched for genes whose expression is induced in the rice SAM by the transition to the reproductive phase. It was found that OsMADS34 was most highly up-regulated and expression of OsMADS14 and OsMADS15 was also enhanced. We already reported that OsMADS34 is one of grass species specific SEP genes and is necessary to determine the spikelet identity. Both OsMADS14 and OsMADS15 are orthologs of Arabidopsis AP1 that determines floral identity. Based on this information, we hypothesize that these three MADS-box genes work together as positive controllers of meristem fate transition. To test this idea, we examined the genetic and molecular interaction among them.
We will discuss the role of OsMADS34 in the transition of meristem identity in grass species.

View full abstract

Lateral root (LR) formation is initiated from the asymmetric cell division of the pericycle cells adjacent to xylem poles in the primary root in Arabidopsis thaliana. It has been shown that the auxin signaling mediated by the auxin response factors ARF7, ARF19, their repressor protein SLR/IAA14, and the direct targets of ARF7/19 including LBD16/ASL18, promotes LR formation in Arabidopsis. ARF7/19 are mainly expressed in root stele tissues including xylem-pole pericycle cells. However, it is unknown exactly when and where ARF7/19-dependent auxin response is required for LR formation. In this study, we analyzed the transgenic Arabidopsis plants expressing ARF7/19 in the spatio-temporal specific manner. 1) We examined the timing when ARF7/19 activity is required for LR formation by using ARF7/19-inducible lines. 2) We also examined whether targeted expression of ARF7/19 or LBD16 in the protoxylem pericycle restores LR formation in the arf7 arf19 mutant background. Based on these results, we will discuss the spatio-temporal regulation of ARF7/19-dependent auxin response in LR formation.

View full abstract

In vascular plants, lateral roots (LRs) contribute to supporting aerial parts and absorbing water and nutrients in the soil. Previous studies have shown that the control of gene expression via auxin-responsive transcriptional factors ARF7, ARF19, and their repressor SOLITARY-ROOT(SLR)/IAA14 is important for LR initiation in Arabidopsis. However, it remains unknown how SLR/IAA14 represses the ARF7/19 activity. To identify the novel factors involved in this process, we screened for suppressor mutants of the lateral rootless mutant slr and isolated the suppressor of slr1 (ssl1) mutant that partially rescued the LR phenotype in slr. The ssl1 is a recessive mutation and the map-based cloning has shown that SSL1 encodes HASTY (HST), which is similar to the mammalian exportin-5 and is involved in miRNA processing and nuclear export in Arabidopsis.
We observed that ssl1 slr arf7 arf19 quadruple mutant formed no LRs, indicating that LR formation in ssl1 slr depends on ARF7/19 activity. In addition, the mutations in the miRNA biosynthesis partially rescued the LR phenotype in slr/+ heterozygous plants. These results indicate that miRNA regulation is involved in auxin-mediated LR formation.

View full abstract

Lateral root (LR) formation in Arabidopsis is regulated by auxin-responsive transcription through ARF7, ARF19 and Aux/IAAs. ARF7/19 directly activate the transcription of Lateral Organ Boundaries domain 16/Asymmetric Leaves2-like 18 (LBD16/ASL18) gene, and overexpression of LBD16/ASL18 induces LR formation in arf7 arf19 mutant, strongly suggesting that LBD16/ASL18 positively regulates LR formation downstream of ARF7/19 (Okushima et al., 2007, Plant Cell). As LBD16 was thought to be a transcriptional activator, we identified the downstream genes of LBD16 using DNA microarray. One of these downstream genes, LLPL2, was specifically expressed in LR primordia. LLPL2 encodes an unknown protein with a conserved amino acid sequence motif (LLPL). LLPL2-GFP fusion protein expressed under the control of the native promoter was also specifically observed in the dividing protoxylem pericycle cells during LR initiation. In this poster, we will discuss a possible role of LLPL2 in LR formation.

View full abstract

ASYMMETRIC LEAVES1 (AS1) and AS2 are involved in the axes-dependent leaf development in Arabidopsis. AS1 and AS2 proteins contain Myb- and AS2/LOB-domains, respectively. These proteins interact each other in vitro. However, the interaction in vivo remains elusive, since the accumulation patterns of each transcripts are not same. To assess it, we attempted to narrow down the regions of AS1 and AS2 proteins for the interaction. And the results were that the essential region of AS1 for the interaction with AS2 was not found and that the essential region of AS2 for the specific interaction with AS1 roughly agreed with the conserved AS2/LOB-domain. We also found the single amino acid replacements, which confer the deficiency in the interaction with AS1, in the essential region. One of them, the LP mutant failed to reverse the phenotype of as2 mutant plants. However, the artificial fusion protein of AS2 and AS1 carrying the LP mutation could reverse and overcome the phenotype. These results suggest that the LP mutant AS2 still has the functions other than the interaction with AS1 and that the interaction with AS1 is essential for the function of AS2 protein.

View full abstract

The ASYMMETRIC LEAVES1 (AS1) and AS2 genes in Arabidopsis thaliana are involved in the establishment of the determinate state of cells in leaves by negatively regulating the expression of both class 1 KNOX genes and abaxial determinants, such as ETT, KAN2, and YAB5 genes in the leaf primordia. When leaf sections of as1 and as2 mutants are incubated on MS medium without exogenous phytohormones, the frequency of shoot regeneration increased as compared to leaf sections of wild-type. We found that mutations in class 1 KNOX genes have no effect on the ability to regenerate shoots in as2 and as1 mutants. To examine whether ett/arf3 and arf4 mutation affect on the regeneration of shoots from leaf sections of as1 and as2 mutants. Frequencies of shoot regeneration from leaf section of the as1 ett arf4 and as2 ett arf4 triple mutants were reduced to those of the wild-type plants, indicating that the potential for formation of shoots in the as1 and as2 mutant was caused by the increased expression of ETT/ARF3 and ARF4 genes. These data suggested that ETT and ARF4 may be involved in shoot regeneration from leaf section in vitro in as2 or as1 mutant background in these mutants.

View full abstract

The ASYMMETRIC LEAVES1 (AS1) and AS2 genes of Arabidopsis thaliana regulate formation of a symmetrical, expanded lamina. AS1 and AS2 are thought to act as a transcriptional regulator of certain genes including class 1 KNOX genes. To identify genes regulated by AS1 and AS2, we performed DNA microarray and clustering analysis by Knowledge-based Fuzzy Adaptive Resonance Theory. We found that AS1 and AS2 repressed abaxial determinants, ETT, KAN2 and YAB5 in addition to class 1 KNOX genes. To know how AS1 and AS2 regulate these genes, we performed ChIP assay and chip-on chip analysis. Our results suggested that AS1 may directly regulate ETT by binding to ETT promoter region. Our genetic analysis of AS1, AS2, ETT, and ARF4 suggested that AS1 and AS2 repress the expression of both ETT and ARF4, which are closely related members of ARF family, and known to be targets of tasiR-ARF. We will discuss the significance of repression of the ETT gene directly and the ARF4 gene indirectly by AS1 and AS2 for the establishment of adxial-abaxial and medio-lateral polarity of leaves in Arabidopsis thaliana.

View full abstract

In Arabidopsis thaliana asymmetric leaves2 (as2) and as1 mutants, asymmetrically lobed, downwardly curled and shorten leaves are observed. The mutants also show defects in the establishment of leaf adaxial-abaxial polarity in certain mutant backgrounds. We screened mutants that show defects in the establishment of the polarity in as1 or as2 backgrounds to identify novel factors genetically interacting with AS1 and AS2. We used formation of filamentous leaves as a marker of defects in the leaf adaxial-abaxial polarity. We identified several mutants and designated one of the mutants as enhancer of asymmetric leaves1 and asymmetric leaves2 (eal). In as1 eal and as2 eal double mutants, abaxialized filamentous leaves were observed. The eal mutation causes early transition from mitotic cell cycle to endocycle. EAL gene encodes a protein well conserved from fungi to human. In fungi Aspergillus nidulans, it has been reported that the function of EAL homolog is required for the nuclear movement. We induced A. thaliana EAL to a A. nidulans mutant. The A. thaliana EAL complemented defects of the A. nidulans mutant. This result indicates that the EAL homologs are also functionally conserved.

View full abstract

In dicot plants, leaf primodia are derived from the shoot apical meristem and develop flat and symmetrical leaf blade. The establishment of adaxial-abaxial polarity is required for lateral expansion of leaf blade. In Arabidopsis thaliana, class III HD-ZIP genes, YABBY and KANADI genes, microRNAs and trans-acting siRNAs regulate leaf development through distinct pathways. The as2-1 mutation causes pleiotropic phenotypes in leaves. Our previous study suggest that AS2, directly or indirectly, represses the expressions of class 1 KNOX genes and abaxial genes, ETTIN, KANADI2 and YABBY5. To identify new components that function together with AS2 in leaf development, we analyzed enhancers of the as2-1 mutant. east1-1 as2-1 and east1-2 as2-1 mutants formed filament-like leaves associated with defect in the adaxial-abaxial leaf polarity. east1-1 also showed weak defects in leaf venation patterns. Expression analysis revealed that mRNA levels of class1 KNOX genes and abaxial genes were increased in east1-1 as2-1. We will also present genetic analysis using as2-1 and other elongata or other enhancer mutants to discuss genetic relationship among them.

View full abstract

Leaves are flat and lateral organs and developed from shoot apical meristem along three axes: the proximal-distal, medial-lateral and adaxial-abaxial axes. Especially, the establishment of adaxial-abaxial axis in leaf primordia is important for flat leaf and the defects of this step result in abnormal filamentous and/or trumpet-like leaves. ASYMMETRIC LEAVES2 (AS2) gene encodes a plant specific protein and is involved in the establishment of all three axes. To understand the molecular function of the AS2 and obtain novel factors involved in the establishment of adaxial-abaxial axis, we performed screening of as2 modifiers and obtained several mutants that generate filamentous and trumpet-like leaves in as2 background. A double mutant of the as2 and a mutant of a DEAD box RNA helicase that is involved in the processing of pre-rRNA frequently generated the abaxialized filamentous leaves at high temperature. . A double mutant of as2 and another pre-rRNA processing factor also showed similar morphological abnormality. These results suggest the importance of the precise pre-rRNA processing in the establishment of adaxial-abaxial axis in leaf primordia.

View full abstract

Leaves develop as flat lateral organs from a shoot apical meristem. The establishment of polarity along three-dimensional axes, namely, proximal-distal, medial-lateral and adaxial-abaxial, is crucial for the growth of normal leaves. ASYMMETRIC LEAVES2 (AS2) and AS1 are involved in repression of both the class 1 KNOX genes and leaf abaxial determinants (ETTIN/ARF3, KANADI2 and YABBY5). AS1 and AS2 may function as key regulatory genes for the establishment of adaxial cell fate in leaves. In addition, it has been reported that several ribosomal protein genes and chromatin remodeling factor genes are also required for the establishment of adaxial-abaxial polarity of leaves. However, the roles of these genes in leaf development have still unknown. We have taken a chemical and genetic approach with the goal of understanding the establishment of adaxial-abaxial polarity of leaves. We identified several small molecules that inhibited the formation of the leaves in as2-1 and as1-1 plants. We will discuss modes of action of these small molecules.

View full abstract

ASYMMETRIC LEAVES2 (AS2) gene is involved in morphogenesis of leaves in Arabidopsis thaliana. Molecular analysis demonstrated that the AS2 represses the expression of class I KNOX genes and some abaxial identity genes. To reveal how AS2 suppresses the expression of these genes and functions in leaf development, we studied the subcellular localization of AS2 protein. The AS2 localizes to a sub-nuclear body around the nucleolus and we designated this body the AS2 body. The AS2 protein contains a plant-specific AS2/LOB domain that consists of the C motif containing four cysteine residues, a single conserved glycine residue and the leucine-zipper-like sequence. The AS2/LOB domain includes a short stretch of basic residues (RRK) in the C-motif. In order to identify the signal in AS2 that is required for its localization to the AS2 body, we made DNA constructs that encoded mutant AS2 proteins with various deletions or amino acid substitutions and investigated sub-nuclear localization of these mutant proteins. We also examined whether the localization to the AS2 body is required for the function of AS2 in morphogenesis of leaves. We will present latest results of these experiments.

View full abstract

In our previous study, we showed that 4 isoenzymes of cytosolic glutamine synthetase (GS1) are expressed in Arabidopsis roots, and that GS1;2 is the only isoenzyme whose transcript accumulates in response to high NH₄⁺. These results suggest significance of GS1;2 in acclimation to high NH₄⁺. In this study, we examined the roles of GS1;2 in root development of Arabidopsis in response to nitrogen sources.
When plants were grown on medium containing NO₃^- as the sole nitrogen source, the wild-type plants (WT) and gs1;2 mutants showed no differences in root development. By contrast, gs1;2 mutants developed shorter and smaller number of lateral roots (LRs) compared to WT plants when plants were grown on medium containing NH₄⁺.
LR development is known to be regulated by auxin. To examine the effects of auxin on NH₄⁺-induced LR development, roots of WT plants and gs1;2 mutants were treated with IAA. The number of LRs was significantly increased in WT plants, but not in gs1;2 mutants, when plants were grown on medium containing NH₄⁺ and 10 nM IAA. The results suggested that gs1;2 mutants are desensitized to auxin and that the response of LRs to auxin is partly dependent on NH₄⁺.

View full abstract

Auxin is known to play a critical role in various aspects of development in plants. To better understand the mechanisms of these auxin responses in plants, we focused on this response in the liverwort M. polymorpha. Application of naphthaleneacetic acid (NAA), an artificial auxin, over 1 μM to gemma confers growth inhibition in M. polymorpha. From γ-ray irradiated spores grown on 10 μM NAA-containing media, we isolated 7 auxin-resistant mutants. Interestingly, these mutants showed abnormal development of gemma, but no obvious phenotype in thallus development from established meristems. Spores of female auxin-resistant mutants did not properly develop, but the male auxin-resistant mutant formed motile sperm and successfully crossed with a wild type female to form spores. We also investigated auxin distribution during gametophyte and sporophyte development using transgenic M. polymorpha expressing the GUS gene driven by a soybean auxin-inducible GH3 promoter. Based on these results, we will discuss the roles of auxin in the development of M. polymorpha.

View full abstract

The phytohormone auxin regulates most aspects of plant growth and development. To understand the origin and evolution of auxin signaling in land plants, we utilized the basal land plant Marchantia polymorpha. We have previously isolated three ARFs (MpARFs), one AUX/IAA (MpIAA), and one TIR1/AFB (MpTIR1) gene from the M. polymorpha genome, implying that M. polymorpha and flowering plants share common auxin-signaling factors. To investigate the role of auxin in the development of M. polymorpha, we generated transgenic plants expressing an mDII-GR fusion protein consisting of degron-modified MpIAA, which dominantly reduces auxin sensitivity, and a C-terminally fused glucocorticoid receptor domain. Dexamethasone (DEX) treatment successfully reduced sensitivity to auxin in these mDII-GR plants. In the presence of DEX, mDII-GR gemmae developed into clumps of cells without forming any organs, such as rhizoids or air chambers, suggesting that auxin signaling mediated by MpIAA contributes to organogenesis from gemma. Based on the detailed observation of mDII-GR plants, we will discuss the role of auxin in the development of M. polymorpha.

View full abstract

The biosynthesis pathway of natural auxin IAA (indole-3-acetic acid) has been studied intensively. However, since the biosynthesis system consists of several pathways and genes playing a role in those pathways are not fully identified, the biosynthesis mechanism of IAA remains to be elucidated more precisely. Chemical genetics approach with biosynthesis inhibitors is a reliable means for studies of hormone biosynthesis. However, biosynthesis inhibitors for IAA had not been available. Recently, we reported the inhibitory activity of L-2-aminooxy-3-phenylpropionic acid (L-AOPP) to IAA biosynthesis in Arabidopsis thaliana, although its specificity to the activity remained to be shown. In this study, we demonstrated biochemical study for the inhibitory activity of L-AOPP to TAA1 (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1), which has been reported to be an IAA biosynthesis enzyme. Furthermore, transcriptome and physiological analysis were carried out and the activity of L-AOPP was multidirectionally examined.

View full abstract

Endoreduplication is one of the special cell cycle which is defined as successive chromosomal DNA replication without cell division and is correlated with cell size. To examin its, segregation distortion 3 (sd3) was isolated from RIKEN Ds insertional mutants collection. sd3 showed short hypocotyls and decreased polyploidy under the dark. Corresponding gene of SD3 encodes a protein with high similarity to yeast Translocase on the Inner Mitochondrial membrane 21 (TIM21), which is a component of the TIM23 complex. Indeed, SD3 protein fused to GFP was localized in mitochondria. SD3 over-expression plants (SD3-ox) showed increased polyploidy and hypocotyl size. Additionaly, expression levels of some subunits of the respiratory-chain complexes and ATP synthase were up-regulated in SD3-ox. Furthermore, SD3-ox showed high ATP levels whereas sd3 showed low ATP levels. Moreover, treatment of seedling with mitochondrial electron transport inhibitor antimycin A resulted in short hypocotyls, decrease in polyploidy levels and ATP levels. Here we speculate that mitochondrial protein regulates endocycle through cellular ATP levels in dark-grown seedlings.

View full abstract

Endoreduplication is a kind of cell cycle that increases nuclear DNA content(ploidy)without cell and nuclear division and is important for plant development.We screened mutants showing increased polyploidy from RIKEN Arabidipsis full length cDNA Overexpressor lines(FOX line).We found that one of FOX line F07144 showed increase in polyploidy in darkness and the corresponding gene encoded a homologue of mitochondrial translocator subunit TIM50.We checked sub-cellular localization of F07144 protein fusing GFP reporter and found that this protein was localized in mitochondria, so this F07144 protein can be designated as AtTIM50. Loss of function mutant showed decrease in polyploidy, reduced plant growth and low ATP levels compared to wild type plants. Transmission electron microscopic analysis showed appearance of large vacuolar space in loss of function mutant,while in overexpressor cristae was significantly thicker. Histochemical analysis of promoter using GUS reporter showed AtTIM50 was expressed in cotyledons and hypocotyls. Here, we demonstrate that AtTIM50 regulate endoreduplication through cellular ATP-levels.

View full abstract

Post-embryonic growth in plants largely depends on activity of meristems in shoots and roots. For proper tissue organisation, it is essential that cell division be coordinated with cell differentiation and that it be in synchrony with the cycles of neighbouring cells. However, it is unknown how the proliferative activities of the meristems and coordination between cell division and differentiation are maintained under stressful conditions. We have shown that a novel rice protein RSS1 contributes to the vigour of meristematic cells and the viability of the entire plant under salinity conditions. This effect is achieved by maintenance of the G1-S transition and the activity of the phytohormone cytokinin in shoots. Recently, we found that RSS1 stability is controlled by cell cycle phases, and that RSS1 interacts with protein phosphatase 1 (PP1). PP1 is known to activate Retinoblastoma protein, a regulator of G1-S transition. A possible role of RSS1 to regulate cell cycle under stress conditions will be discussed.

View full abstract

Circadian rhythms, endogenous oscillations of physiological activities with a period of ~24 h, are found in a wide spectrum of organisms and enhance their fitness in a day/night alternating environment. Cyanobacteria are the simplest organisms that exhibit circadian rhythms. In cyanobacterium Synechococcus elongatus PCC 7942, KaiC is an essential protein for circadian rhythm generation. We screened KaiC-association protein and isolated DnaA, which is known as DNA replication initiation factor in bacteria. Disruption of dnaA gene in wild-type cell shortened the period of gene expression in Synechococcus elongatus PCC 7942, and mutants with inactivated dnaA were viable. We also found that there is relationship between DnaA and cell division and circadian clock. Thus, we think that that DnaA modifies the circadian rhythm and cell cycle by interacting with KaiC.

View full abstract

Aurora kinases are serine/threonine protein kinases with essential roles in cell division through eukaryotes. Although functions of animal and yeast Aurora kinases have been analyzed in detail, those of plant Aurora kinases are unknown. Our live cell imaging analyses using cultured cells indicated that the plant Aurora kinase has dual roles; correction of aberrant kinetochore-microtubule attachment and dissociation of cohesin during chromosome alignment and segregation. We found three homologs and named AtAUR1, 2 and 3 in Arabidopsis thaliana.We also analyzed transgenic plants of Arabidopsis thaliana to express AtAUR3 with tdTomato. AtAUR3 localized around kinetochores of mitotic chromosomes in A. thaliana. We constructed the knockdown plant of AtAUR3. AtAUR3 knockdown induced the abnormality of root growth and leaf extension. An Aurora kinase inhibitor, Hesperadin, can inhibit the kinase activity of AtAUR3 and suppressed cell division in organs. These data suggest that the plant Aurora kinase plays a role in chromosome alignment and segregation in A. thaliana.

View full abstract

Asymmetric cell division, generating two cell types from one, is important to understand mechanisms of ontogenesis in animals and in plants. The various events of asymmetric cell division should be tightly coupled to a cell-cycle regulation. One of the important links between cell-cycle regulation and asymmetric cell division has been reported in cyclin-dependent kinase Cdc2 of Drosophila melanogaster. Attenuation of its Cdc2 function resulted in a symmetric cell division in the neural precursor cell, which normally divides asymmetrically. While homozygous mutant of Arabidopsis thaliana CDKA;1, a homologue of Cdc2, is lethal, little is known about how CDKA functions in a process of asymmetric cell division in plants.
A protonemal apical cell of the moss, Physcomitrella patens provides a good system for the study of asymmetric cell division at a single cell level. P. patens has two copies of CDKA in the genome, and to investigate the function of CDKA in the process of asymmetric cell division, we made double knock-out lines of CDKA by homologous recombination. Thus we will report their phenotypes and discuss the function.

View full abstract

Multicellular organisms efficiently use symmetric cell division and asymmetric cell division, and control the number and the type of cells. In protonemata of Physcomitrella patens, they normally grow by asymmetric cell division and elongation of the apical cells. When treated with abscisic acid (ABA), protonemata divide symmetrically to produce numerous spherical cells, called brood cells. It is largely unknown how ABA regulates such alternation of cell division mode and change of cell fate.
We transiently over-expressed about 3000 full-length cDNAs in protoplasts, and identified three genes which induced brood cell-like cells. One of them encoded ABI3/VP1 orthologue, a positive regulator of ABA signaling pathway, and the other two were presumed to encode a proline-rich cell wall protein and a glycosyltransferase, respectively. Conditionally over-expressed these two genes induced rounded, brood cell-like cells, in which a degree of the angle of the septa was disarranged, indicating that cell polarity was destroyed and that these two genes were in fact involved in ABA responses. Further functional analyses to study knock-out phenotype and protein localization are under progress.

View full abstract

Genome integrity is continuously threatened by external stress or by errors in DNA replication, thus a response to DNA damage is essential for survival and continuous growth of plants. To prevent DNA-damaged cells from proliferating, plants provoke the DNA damage checkpoint, but its regulatory mechanisms remain unknown.
Recently, we found that DNA double-strand breaks (DSBs) trigger transition to endocycle, leading to endoreduplication, in Arabidopsis. In plants, exit from mitotic cell cycle is often associated with endocycle, in which cell increases their DNA content by repeating DNA replication without mitosis. We also found that the protein degradation of CDKB2 was enhanced in response to DSBs. This result suggests that CDKB2 degradation may be involved in switching from the mitotic cell cycle to the endocycle in response to DSBs.
To verify this hypothesis, we are identifying cis regulatory motifs important for DSB-induced CDKB2 degradation. For this purpose, we generated plants expressing several truncated versions of CDKB2 or CDKB2 with several combinations of amino acid substitutions, which are fused with GUS reporter gene, and found some possible important elements.

View full abstract

Upstream open reading frames (uORF) are small ORFs located in 5' leader region of certain eukaryotic genes. The presence of uORF can negatively modulate translational efficiency of the downstream main ORF. Although the effects of most uORFs are independent of their encoded peptide sequences, several uORFs have been shown to control translation of the main ORF in an amino acid sequence-dependent manner. In plants, so far only two genes regulated by such a uORF-encoded bioactive peptide have been reported. In order to further identify uORF peptides that control expression of the main ORF, we searched for Arabidopsis uORFs whose amino acid sequences are conserved beyond rosids group by using a comparative genomic approach. We found 14 novel uORFs conserved in a wide range of dicot plants. Next, we tested whether these uORFs affect expression of the downstream ORF in a uORF peptide sequence-dependent manner, and identified 5 novel genes whose expression is controlled by a uORF-encoded peptide.

View full abstract

Plant organelles have their own genomes and gene expression machinery. Plant organelle gene expression is controlled by various post-transcriptional processes, including site-specific RNA cleavage, RNA splicing and RNA editing. It is known that pentatricopeptide repeat (PPR) proteins have an important role in post-transcriptional regulations. Recently, we have shown that PPR-DYW proteins function as a site-specific recognition factor for RNA editing in the mitochondrial transcript. The moss Physcomitrella patens, a basal land plant, has 10 PPR-DYW proteins. To elucidate the function of mitochondrial-localized PpPPR_43 protein, belonging to the DYW-subclass PPR protein, we constructed the knockout mutants of PpPPR_43 gene and analyzed on the effect of RNA editing. As a result, RNA editing normally occurred at the mitochondrial 11 editing site in the mutants. In contrast, we found that splicing efficiency of cox1 pre-mRNA significantly reduced. In addition, the transcripts of ORF622 gene present in the third intron of cox1 accumulated at the same level of wild type. Based on these results, we discuss on the involvement of PpPPR_43 in the splicing of mitochondrial cox1 pre-mRNA.

View full abstract

One of the regulation factors of mitochondrial gene expression is pentatricopeptide repeat (PPR) protein which binds RNA. PPR motif is composed of 35 amino acids tandem repeat and PPR proteins bind specific RNA. Consequently, PPR proteins are involved in post transcriptional process. In this study, we analyzed rice PPR gene mutant, named Mitochondrial PPR 25 (MPR25) caused by Tos17 insertio. mpr25 mutants exhibited pale green leaves and growth retardation. MPR25 was localized in mitochondria and was involved in C-U RNA editing of nad5 encoding a subunit of complex I (NADH dehydrogenase) respiratory chain in mitochondria. Respiration activity was not significantly lower in the mpr25 mutants but photosynthesis rate was lower in the mpr25 mutants than that of WT at juvenile stage. Internal/external NADH dehydrogenase genes were up-regulated in mpr25 mutant leaves than those of WT at juvenile stage. MPR25 have a function of C-U RNA editing of nad5 and affects photosynthesis.

View full abstract

After the endosymbiosis, a lot of genes encoded by the chloroplast genome have been transferred to the nuclear genome. This gene transfer event has been occurred by following steps. (1) Endosymbiosis, (2) Chloroplast DNA was transferred to the host nucleus and integrated into the nuclear genome, (3) Transferred genes were obtained the expression system in the nucleus, and also obtained the transit peptide to chloroplasts, and (4) Orthologus genes remained in the chloroplast genome were removed. As far as we know, there are no plants that orthologous genes encoded by both chloroplast- and nuclear-genome are functional simultaneously in the chloroplast. In many higher plants, the gene for 30S ribosomal subunit S16, rps16, is encoded not only by the chloroplast genome but also by the nuclear genome, and nuclear-encoded S16 protein is targeted to chloroplasts. Here, we analyzed whether the chloroplast-encoded rps16 gene is functional in chloroplasts using tobacco as a model.

View full abstract

Chloroplast psbD and psbC genes encode a D2 and a CP43 protein of photosystem II complex, respectively. These genes are members of psbD operon with psbZ and are co-transcribed from a promoter upstream of psbD. The psbC is also transcribed from its own promoter. A striking feature of this operon is that psbD and psbC coding regions partially overlap, implying translation of downstream psbC from the polycistronic mRNA is dependent on that of upstream psbD. To examine this possibility, we carried out tobacco chloroplast in vitro translation assays. Our result indicates that translation of psbC from a dicistronic psbD-psbC mRNA was much more efficient than that from monocistronic psbC mRNA by chloroplast in vitro translation assays. In addition, translation of the psbC from the dicistronic mRNA was strongly dependent on that of the first cistron. Mechanism of translational coupling between psbD and psbC will be discussed.

View full abstract

The chloroplast atpB and atpE genes code for subunits beta and epsilon, respectively, of the chloroplast ATP synthase. The atpB stop codon (UGA) overlaps one nucleotide with the atpE start codon (AUG) in most flowering plants. The two genes are co-transcribed as a dicistronic atpB-atpE mRNA. Therefore, it was long thought that translation of the downstream atpE cistron depends on that of the upstream atpB cistron (i.e. translational coupling). Using two different fluorescent proteins, we devised an in vitro assay to measure differentially translation of the atpB and atpE cistrons. We first showed that both atpB and atpE cistrons are translated from the tobacco dicistronic mRNA by using our efficient in vitro translation system from tobacco chloroplasts. When the atpB 5-UTR was replaced with a lower efficient 5-UTR, atpE translation was higher than atpB translation. This result strongly suggests that atpE translation is independent of atpB translation (no translational coupling).

View full abstract

Coexpression analysis data sets generated from comprehensively collected transcriptome data sets have become an efficient resource capable of facilitating the discovery of genes closely correlated in their expression patterns. In order to construct coexpression network of barley, we analyzed publicly available 45 experimental series, which are composed of 1347 GeneChip data in barley. On the basis of gene-to-gene weighted correlation coefficient, we constructed global barley coexpression network. Then, we classified the global co-expression network into clusters of sub-network modules by using the MCODE algorithm. The resulting clusters are candidate for functional regulatory modules in the barley transcriptome. To annotate each of the clusters, we applied functional annotation of genes in Arabidopsis and in rice as well as in Brachypodium distachyon (Brachypodium). On the basis of comparative analysis between barley and these model species, we investigated functional properties from the representative distributions of genes encoding transcription factors and of the Gene Ontology(GO) terms.

View full abstract

Flooding injury is one of the major constraints for cultivation of soybean. A proteomic approach was used to clarify mechanisms of the flooding injury. Two-day-old seedlings were flooded for 1 day, and proteins were extracted from root tips of the seedlings. Comparative analyses on quantity and phosphorylation states of proteins between untreated and flooded samples were performed. For quantitative analysis, tryptic peptides were analyzed using mass spectrometry. For phosphoproteomics, phospho-proteins and -peptides were enriched using phosphoprotein purification column and titanium oxide beads, respectively, and were analyzed using mass spectrometry. As results, proteins related to energy generation and signalling were increased, and protein modification and cell structure were decreased by flooding. Alternatively, phosphoproteomics showed that proteins related to energy generation, cell structure, protein modification and transcription were dephosphorylated, and primary metabolism, protein degradation and translation were phosphorylated by flooding. It is suggested that response to flooding stress is regulated by modulation of protein quantities and protein phosphorylations.

View full abstract

A unicellular green alga Chlamydomonas reinhardtii can acclimate to wide range of CO₂ concentrations. It can grow well even at atmospheric level of CO₂ by inducing the CO₂-concentrating mechanism (CCM). Nine isozymes of extracellular and intracellular carbonic anhydrases (CAs) specifically induced in low-CO₂ acclimated cells play key roles in CCM. When CO₂ concentration is increased, CCM and CAs is known to disappear. We previously found that the external CA was replaced by an extracellular protein H43/Fea1, indicating cellular high-CO₂ response. [Hanawa et al. (2007) Plant Cell Physiol. 48: 299-309]. In this study, we focused to study on high-CO₂-inducible extracellular proteins by proteomic analysis using a wall-deficient strain of C. reinhardtii which releases extracellular matrixes to the culture medium. The mass spectrometric analysis on extracellular proteins showed that 22 proteins, including H43/Fea1, among 129 proteins increased their amounts synthesized during acclimation to high-CO₂ conditions for 1 d and 3 d. Eleven of those proteins were classified into hydroxyproline-rich glycoprotein groups.

View full abstract