We have recently found that, in Arabidopsis, reactive oxygen species levels and flowering negatively correlate with linolenic acid composition of plant lipids (LAC). Here we report that this correlation is the case for boreal conifers. To investigate whether LAC negatively correlates with floral determination in the trees, we monthly harvested the needles from Larix gmelinii, L. kaempferi, Picea glehnii and Abies sachalinensis grown some tree species grown in the field of Hokkaido Forestry Research Institute. Larix LAC in June negatively correlated with corn production, while that in July positively correlated. Abies LAC was higher than that of L. gmelinii and kaempferi and negatively correlated in May, Jun, July. Picea LAC was constantly lower than the tree species and positively correlated with corn production. Based on these results and the fact that one linolenic acid derivatives induces flowering, we will discuss the role of linolenic acid in flowering regulation.

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Linolenic acid composition of lipids (LAC) suppresses flowering in Arabidopsis thaliana. Compared to 22°C, a low temperature (15°C) increases LAC and retards flowering in wild-type plants (Col). Since the floral retardation by low temperatures was reduced by triple mutations in FAD3, FAD7 and FAD8 encoding enzyme synthesizing LA, it is considered to be attributed to LAC. Here we investigated the relationship between LAC-dependent flowering and AP1 function. A decrease in contents followed AP1 expression when Col was grown at 22°C. The triple mutant plants showed earlier expression of AP1, while 35S-FAD3 plants showed delayed expression of AP1 with late flowering. The early-flowering phenotype in 35S-AP1 plants was suppressed by low temperatures or a cross with 35S-FAD3 plants. Taken together, LAC regulates AP1 at the levels of transcription and protein.

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Mammalian protein tyrosine phosphatase (PTPase) is easily inactivated by oxidative modification of the catalytic cysteine residue. And plant PTPase is postulated to be regulated in a similar manner. However, we report that the activity of Arabidopsis PTPase (AtPTP1) is regulated by redox status of the non-catalytic cysteine residue. Wild type AtPTP1 was inactivated by oxidized glutathione (GSSG) or H₂O₂ in a dose-dependent manner. A mutant AtPTP1 where C175 was substituted for serine (C175S) showed more resistance to GSSG and H₂O₂, suggesting that AtPTP1 is regulated by redox status of the non-catalytic C175 rather than catalytic C265. Considering plants expressing the C175S mutant AtPTP1 showed stronger phenotype than those expressing wild-type AtPTP1, C175 is likely to be redox-regulated in vivo.

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In higher plants, ascorbic acid (AsA) is mainly synthesized from hexose phosphates via D-mannose/L-galactose pathway. Phosphomannose isomerase (PMI) catalyses the reversible isomerization of D-fructose 6-phosphate and D-mannose 6-phosphate (M6P), as the first step of AsA biosynthesis. Accordingly, PMI may act as a determinant of the carbon partitioning from major carbon metabolisms to AsA biosynthesis. Two genes of PMI (PMI-1 and PMI-2) existed in Arabidopsis. To analyze the enzymological properties of Arabidopsis PMIs, recombinant proteins were expressed from each cDNA in Escherichia coli and purified. The V_max and K_m value for M6P of the recombinant PMI-2 were 21.3 μmol/min/mg protein and 328 μM, respectively. PMI-2 was inhibited by zinc and cadmium ions. Next, we obtained T-DNA insertion Arabidopsis line in the PMI-2 gene. However, the lack of PMI-2 had no effect on the cellular AsA level. Now, we are analyzing enzymological property of PMI-1 and isolating PMI-1 knockout mutants.

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Thioredoxin (Trx) is a small ubiquitous protein that regulates a number of phenomena through formation or dissociation of a disulphide bridge in the target enzyme. Cytosolic thioredoxin has been reported to regulate several membrane proteins that involve in the self-incompatibility and in the innate immunity of plant so far. However, a membrane protein involved in the redox cascade is still poorly understood.
To uncover a whole picture of membrane proteins involved in the redox cascade, we screened target proteins of thioredoxin in plasma membranes from the suspension culture of Arabidopsis thaliana. The resulting proteins were identified by MALDI-TOF/TOF MS analysis. A list of potential targets for thioredoxin contained some signaling molecules, such as membrane-associated protein kinases. We prepared some recombinant proteins of the molecules and analyzed their redox regulation in vitro. The result allows us to presume a mechanism for redox regulation of the membrane signaling molecule in plant cells.

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We previously reported in this meeting that the rice gene responsible for cpm1 mutation, which results in a defect in phytochrome-mediated inhibition of coleoptile growth, encodes an allene oxide synthase (OsAOS1). Sequence analysis indicated that the cpm1 mutant expresses a full-length OsAOS1 in which one amino acid is substituted. Using recombinant proteins, we confirmed that the wild-type OsAOS1 has AOS activity, but the point-mutated OsAOS1 has little activity. We further showed that both wounding and red light induce an enhancement of jasmonate level in wild-type coleoptiles, but only a slight enhancement in cpm1 coleoptiles. A rice microarray was used to investigate wound- and red light-regulated genes. In wild type coleoptiles, expressions of more than 5000 and 6000 genes were regulated by wounding and red light, respectively. About 2000 genes were regulated by both treatments. The cpm1 mutant was used to show that many of these common genes are jasmonate-dependent.

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We investigated a function of the rice heterotrimeric G protein α subunit (RGA1) in plant hormone responses. We found that a mutant of the RGA1 gene (d1) have a decreased sensitivity to brassinosteroid(BR). d1 also has features commonly found in BR-related mutants, namely short second internodes and an aberrant skotomorphogenesis under the dark condition.
In general, BR is percepted by BRI1 and causes a negative feedback of BR-related gene expression through the action of BZR1. In contrast to an impairment of feedback regulation in Osbri1 (d61), d1 responded normally. Intrinsic BRs levels in d1 were not different from those in wild type rice. The double mutant between d1 and d61 exhibited a more dwarf phenotype due to the additively decreased cell number.
Taken together, we will propose that RGA1 involves in the investment of BR-related features, but BR pathway from BRI1 to BZR1 may be normal in d1.

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Rice heterotrimeric G Protein α, β and γ subunits were localized on plasma membrane fraction. Gel filtration of solubilized plasma membrane proteins showed that all subunits were present in the large protein complexes (about 400kDa). Interestingly, β and γ subunits are present in the 60kDa fraction. As β and γ subunit form a dimmer in mammals and yeast, the 60kDa fraction may be the βγ dimmer in rice. In order to presume the function of the β subunit, we produced the transformants suppressed the expression of β subunit gene by using RNAi methods. First, transformants which the amount of the β subunit was decreased were selected. All transformants showed the abnormal phenotypes, dwarf, lethality and small seeds. These phenotypes were not same as that of the α subunit deficient mutant. These results indicate that the β subunit may be partially separated from the α subunit.

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Chloroplasts possess a system for generating (p)ppGpp, which triggers the stringent response in eubacteria. Here we present the identification and characterization of genes for a novel type of (p)ppGpp synthetase (CRSH) in rice and Arabidopsis. The proteins encoded by these genes contain a putative chloroplast transit peptide at the N-terminus, a central RelA-SpoT like domain, and two EF-hand motifs at the C-terminus. The recombinant OsCRSH1 protein was imported into chloroplasts in vitro. Biochemical analysis showed that the OsCRSH proteins possess (p)ppGpp synthetase activity that is dependent both on Ca2+ and on the EF-hand motifs. CRSH proteins thus likely function as Ca2+-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca2+ and (p)ppGpp signaling in regulation of the genetic system of these organelles.
Tozawa Y. et al. J. Biol. Chem. 282, 35536-35545, 2007.

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Phospholipase D (PLD) is the enzyme that hydrolyzes phosphatidylcholine and generates phosphatidic acid and choline. The Arabidopsis genome encodes 12 PLDs which are classified into 10 plant-specific PLDs and 2 eukaryote-general PLDs by their domain structures.
The promoter activity of PLDz2 is detected in upper region of root-tips and pollen grains, while that of PLDz1 is strongly detected in most of the tissues. Furthermore, expression of PLDz2 is increased under phosphate-starved condition. Mutations in 4 of the PHR1 recognition site upstream of PLDz2 gene reduced responsibility for phosphate starvation on PLDz2 expression. Additionally, PLDz2 expression remained unchanged under phosphate-starved condition in phr1 mutant. These results indicate that the response of PLDz2 to phosphate starvation is mediated through PHR1.
Interestingly PLDz2 mutants do not show phenotypic changes under normal or phosphate-starved conditions. Hypothesis on the role of PLDz2 to retrieve phosphate from plasma membrane under phosphate-starved condition will be discussed.

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Carbon and nitrogen availavility is one of the most important factor regulating plant development. To utilize limited resource of carbon and nitrogen efficiently, plants enable to sense and respond to balance of carbon (C) and nitrogen (N) metabolites, called C/N response.
To clarify mechanisms involved in C/N response in higher plants, we had isolated and characterized a mutant named ssv1-D (super survival 1-D) which was resistant to severe C/N nutrition stress (high-sugar and low-nitrogen) conditions. The mutant was due to overexpression of the SSV1 gene and the expressions of C and/or N-regulated genes were not regulated precisely in the ssv1-D mutant. SSV1 loss of function mutant was hypersensitive to change of C/N conditions. The SSV1 gene encodes RING type ubiquitin ligase (E3) and the translated protein showed E3 activity in vitro. These results suggest that the SSV1 regulates C/N response via ubiquitin-26S proteasome system in higher plants.

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It has been reported that oxalic acid secreted by Sclerotium rolfsii plays a key role in development of pathogenesity by initiating the programmed cell death through activation of anion channels in the host cells such as those of Arabidopsis thaliana. In this study, we observed that oxalic acid and structurally related dicarboxylic acids induce the generation of superoxide anion radicals in the suspension-cultures of tobacco (Nicotiana tabacum L.; cell line, BY-2) and rice (Oryza sativa, cv. Nipponbare). Data suggested that among related chemicals, oxalic acid were shown to be most active in induction of superoxide generation in both plant materials.

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To study signaling mechanisms involved in leaf abscission in Capsicum plants, we developed an in vitro abscission system. Microscopic analysis revealed reactive oxygen species (ROS) were constitutively produced by the abscission zone (AZ) cells. ROS scavengers and diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, suppressed the in vitro abscission and cellulase expression. Conversely, application of ROS promoted in vitro abscission and expression of cellulase. Ethephon-induced abscission accompanied the production of ROS at the AZ and this abscission was suppressed by the inhibitors of ROS. During in vitro abscission, expression of upregulated ethylene-responsive genes was unaffected by the ROS or ROS inhibitor. These indicate that ROS act downstream from ethylene in the in vitro abscission signaling. In planta, salinity induced expression of the ROS-responsive genes and ROS production at the AZ, which preceded leaf abscission, indicating that ROS have roles in leaf abscission.

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Rapid production of nitric oxide (NO) and reactive oxygen species, called NO burst and oxidative burst, respectively, is characterized during diverse physiological processes, such as resistance to biotic and abiotic stress, hormonal signaling and development. MAPK cascade is a major evolutionary conserved signaling pathway used to transduce extracellular stimuli into intracellular responses among eukaryotes. In this study, we investigated the roles of MEK2-WIPK/SIPK and cytokinesis-related NPK1-MEK1-NTF6 cascades in the regulation of NO and oxidative bursts in N. benthamiana. Agrobacterium-mediated transient gene expression and virus-induced gene silencing as gain and loss of function analyses, respectively, showed that MEK2-SIPK cascade controls the NbNOA1-mediated NO burst, and that MEK2-SIPK and NPK1-MEK1-NTF6 cascades regulate the NbRBOHB-dependent oxidative burst.

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Phosphatidylinositol 5-kinase (PIPK) regulates vesicle trafficking and cytoskeletal organization via phospharylation of phosphatidylinositol-monophosphates in animals and yeasts. However, the activation mode and function of PIPKs in plants are largely unknown. We analyzed plasma membrane-localization and activation of PIPKs from Physcomitrella patens (PpPIPKs). Plant PIPKs consist of N-terminal MORN and C-terminal catalytic domains and the former is thought to be responsible for plasma membrane-localization. In contrast, the catalytic domain is important for membrane localization for animal PIPKs. When the catalytic domain of PpPIPKs was fused to GFP, plasma membrane-localization was observed, although a MORN-GFP fusion located in cytoplasm. Thus, the MORM domain is not involved in membrane localization in Physcomitrella. Moreover, amino acids regulating membrane localization and enzymatic activity were identified in the activation loop as is in animal PIPKs. We therefore concluded that the machineries regulating plasma membrane-localization and enzymatic activity of PIPKs are conserved in eukaryotes.

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Plants take many environmental changes around itself and managing to do by expressing some sets of genes. We focused the His-Asp phosphorelay signal transduction system of Oryza sativa and trying to study the functional characteristics of this system. This system was composed from 3 kinds of signaling factors, Histidine Kinase: HK, Response Regulator: RR, Histidine-containing phosphotransfer: HPt, by looking over the Rice genome information as reported in other higher plants, Arabidopsis and Zea mays. And each has some members. Here by using the yeast two-hybrid system, we studied the interaction between 2 OsRRs and 2 OsHPt signaling factors and furthermore which domain of each factor functioned on signaling.

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At various environmental conditions, its signal was accepted by plant cells at first and transduced to the whole plant. His-Asp phosphorelay signaling system was known as one of the outstanding means to recognize stress signals and it has been also reported that one of two kinds of plant RR(For abbreviation, see above summary) called typeB RR had transcriptional activity with B-motif revealed from the 3-D structure analysis, DNA-binding and transcriptional activity analysis etc. But some different functions of these in each plants species, monocot and dicot, has been reported recently. So we tried to see the intracellular characteristics of RR and HPt signaling factors of this system from the point of cellular view, the cellular localization or interaction of these and what kinds of environmental stress signal was recognized using the GFP-RR/-HPt fusion protein with rice culture cells. From these results wed like to discuss about this system of rice.

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Cytokinesis is the final critical step in the generation of two daughter cells that contain a full complement of genomic components and cytoplasmic organelles. Cytokinesis of plant cells occurs in phragmoplast, a cytokinetic machinery, that is mainly consist of bundles of microtubules (MTs) and microfilaments. The NACK-PQR pathway that includes kinesin-like protein NACK and the MAP kinase cascade is a key regulator for such cytokinesis. The pathway is activated after metaphase of M phase, induces dynamic instability of MTs and stimulates phragmoplast expansion (Sasabe et al., Genes & Dev., 2006). The activation is initiated by binding of NACK to NPK1 MAPKKK in the cascade. We here report that phosphorylation of NACK and NPK1 by CDK interferes binding of these two proteins before metaphase and that dephosphoraylation induces the binding and activates the pathway. CDK may be a major regulator that represses functions of factors controlling progressions of cytokinesis after metaphase.

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We have elucidated that the components of MAPK cascade were essential for cytokinesis of plant cell. The NPK1(MAPKKK) binds NACK1(kinesin-like protein ) and is activated during late M phase in tobacco. The NQK1(MAPKK) and NRK1(MAPK) were identified as the downstream components of NPK1. Over expressinon of these, which were dominant-negative form, inhibit the growth of cell plate and result in multinucleate cells with incomplete cell plates. But, the direct evidence, which NRK1 is involved in cytokinesis, has not come. In the present study, we elucidated that AtMPK4 was involved in cytokinesis in Arabidopsis. The ANQ1, which is the homolog of NQK1, activated AtMPK4 specifically in vitro. The avtivity of AtMPK4 was reduced in the anq1mutant . Furthermore, it was observed that multinucleate cells with incomplete cell plates in the root and the leaf in the atmpk4 mutant. These results suggest that MPK4 regulates the growth of cell plate positively.

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Plant cytokinesis is accomplished with the formation of phragmoplast, which is formed between two daughter chromatins during anaphase. The development of phragmoplast is regulated by the MAP kinase cascade. Although the MAP65, one of microtubule binding proteins, has been shown to be phosphorylated by MAPK, most of targets are unidentified. We applied the phosphoprotein purification with immobilized metal ion affinity chromatography and two-dimensional difference in gel electrophoresis for the identification of the phosphorylated targets of MAP kinase cascade in Arabidopsis. We identified PATL2 as one of the targets of MAP kinase and found that recombinant PATL2 protein was phosphorylated by MAPK in vitro. Further analysis identified one serine residue of PATL2 located in SEC14 domain was phosphorylated. Since PATL2 is a member of SEC14 family which were speculated to be involved in membrane metabolism and transport, MAPK cascade might regulate membrane trafficking concomitant with expansion of phragmoplast

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Higher plants are constructed from billions of cells that have variable sizes and shapes. What controls these important cellular features are not well understood, but it is known that the total amount of nuclear DNA influence the final size of cells. In Arabidopsis, an increase in nuclear DNA content is mediated by a process called endoreduplication, however, the molecular mechanisms underlying this process is largely unknown. To gain new insights on the control of endoreduplication, we performed a new genetic screen to identify mutants that have altered plant size and ploidy. To date we have isolated four new mutations which we named high ploidy 1-4 (hip1-4). Endoreduplication in wild type seedlings usually brings the ploidy up to 32C. In contrast, we found that the ploidy levels in hip1-4 go up to 128C or 256C. Our current hypothesis is that HIP1-4 are involved in some pathways that negatively regulate endoreduplication.

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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. An Aurora kinase inhibitor, Hesperadin, can inhibit the kinase activity of AtAUR3. Hesperadin treated tobacco BY-2 cultured cells increased in the ratio of metaphase cells, while the ratio of anaphase/telophase cells decreased, although the mitotic index was not affected. These results suggest that Hesperadin induces delayed transition from metaphase to anaphase, and early exit from mitosis after chromosome segregation. In addition, lagging chromosomes, which caused by the delay and failure of sister chromatid separation, were observed at anaphase and telophase in Hesperadin-treated cells. These data suggest that the plant Aurora kinase plays a role in chromosome alignment and segregation.

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The moss Physcomitrella patens development starts from a germinating spore that gives rise to a filamentous structure called protonema. Bud that is arisen form protonema develops to give rise to a leafy shoot called gametohpore. After dissection of a leaf from a gametophore, leaf cells facing to the dissected cells change to protonemal cells. However, the molecular mechanisms of this process have been unknown. To understand the mechanisms, we focused on the cell cycle activation in leaf cells after leaf dissection. Our analysis with RT-PCR demonstrated that transcripts encoding several cell-cycle regulators, such as CYCLINs, increased in dissected leaves. Furthermore, we found that the CDK activity in dissected leaves increased after leaf dissection. Taken together, our results indicate that the CYCLIN expression and the CDK activation by leaf dissection are associated with cell cycle progression in leaf cells facing to the dissected leaf cells.

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Ubiquitin(Ub)/26S proteasome pathway plays an essential housekeeping role to eliminate the proteins which are damaged or misfolded. It is also essential for aspects of cellular regulation by removing short-lived regulatory proteins as a way to fine-tune homeostasis, adapt to new environments, and redirect growth and development. The 26S proteasome consists of two multisubunit complexes, 20S core particle (CP) and 19S regulatory particle (RP). The RP contains thirteen non-ATPase subunits (RPN) and a ring of six AAA-ATPase subunits (RPT).
We are isolating and characterizing Arabidopsis mutants with defective RPT subunits. Mutations in RPT2a and RPT5a resulted in enlarged leaves, which was caused by increased endoreduplication. Endoreduplication is a type of cell cycle where nuclear chromosomal DNA replication occurs without cell division. The function of RPTs will be discussed in terms of cell size regulation.

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Brca2 is a breast tumor susceptibility factor with functions in maintaining genome stability through ensuring efficient double-strand DNA break (DSB) repair via homologous recombination. To investigate the role of Arabidopsis BRCA2 genes in DNA repair in somatic cells, we identified and characterized transposon insertion mutants of the AtBRCA2a and AtBRCA2b genes. atbrca2a-1 and atbrca2b-1 single mutant, and atbrca2a-1/atbrca2b-1 double mutant showed hypersensitivity to genotoxic stresses compared to wild-type. Interestingly, approximately 13% of double mutant plants showed stem fasciation. In addition, the frequency of the fasciation phenotype in the double mutant increased by up to 46% following g-irradiation. Moreover, we established atbrca2 double mutant line carrying a CYCB1-GUS reporter construct. The GUS fusion gene was expressed in shoot and root apical meristems of the double mutant. These data suggest that inefficient DSB repair in the atbrca2 double mutant leads to disorganization of the programmed cell cycle of apical meristems.

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5-Bromodeoxyuridine (BrdU), a thymidine analog, has been reported to affect various aspects of development and growth of plants. In tissue culture of Arabidopsis, BrdU show different effects on explants, depending on treatment conditions.
We isolated two BrdU-resistant mutants of Arabidopsis, bro1 and bro2, by screening with a focus on the inhibitory effect of BrdU on hypocotyl dedifferentiation. Chromosome mapping and DNA sequencing detected a missense mutation in a gene encoding RNA-binding protein UBA1a in the bro1 genome and a missense mutation in a gene encoding thymidine kinase in the bro2 genome, to which the BrdU-resistant natures of these mutants might be attributable.
We characterized knockout lines of these genes. In the knockout lines of the BRO1 candidate gene, callus induction from hypocotyls of both homozygous and heterozygous mutants appeared partially resistant to BrdU. In the knockout lines of the BRO1 candidate gene, only homozygous mutants were resistant to BrdU.

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For revealing the mechanisms underlying the differentiation of chloroplast, reporter genes were placed under the control of the promoter of photosynthesis gene RBCS-3B, and subjected to the activation tagging to find callus expression of RBCS (CES) genes which promoted the expression of photosynthesis genes in the dedifferentiated calli (Plant Cell Physiol., 47, 319-331, 2006). Contrary to the functions of CES genes, it has been tried to hunt genes for depression of the expression of photosynthesis genes in greened calli.

We have established the culture condition under which calli become green. Mutants called des (depressed expression of RBCS) in which green calli change into white by activation tagging, have been selected. DNA prepared from des mutants was subjected to thermal asymmetric interlaced (TAIL)-PCR, resulting in identification of 4 loci. Gene expression in des mutants has been analyzed by real-time RT-PCR and Arabidopsis ATH1 Genome Array (Affymetrix).

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Stomata are essential for the exchange of gases and water vapor between a plant and its environment. Although previous studies revealed several factors involved in the signaling that creates normal stomatal pattern, less is known about the positive regulators of stomatal differentiation. We are studying FAMA, a bHLH transcription factor, which is highly expressed in plants having excess stomata and repressed in plants without stomata. A T-DNA insertion line of FAMA has no morphologically identifiable stomata in any organ. Instead, fama mutants make tumor-like clusters in normal stomatal positions. The cells in these tumors express markers of developing stomata, but do not express mature stomatal markers. FAMA RNA and protein are expressed in specific cells of the stomatal lineage. FAMA-overexpressing plants make many ectopic unpaired-guard cells. These results suggest that FAMA's function is to promote differentiation of guard cells and to inhibit excess cell divisions in stomatal development.

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In Arabidopsis, leaves have the adaxial (upper side) and abaxial sides (lower side), which differ in shapes of cells and in the number of stomata or trichomes. In order to examine genetic mechanism determining the adaxial/abaxial axis in leaf primordia, we have isolated a series of mutants which show abnormal expression pattern of the abaxial specific gene, FILAMENTOUS FLOWER (FIL). enlarged fil-expression domain1 (enf1), has two types of leaves with abnormal FIL expression pattern: the one with enlarged FIL-expression domain and the other with reduced FIL-expression domain. Map-based cloning reveals that ENF1 gene encodes a metabolic enzyme. We will also report the expression pattern of ENF1 gene.

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Loss-of-function mutations in the ASYMMETRIC LEAVES2 (AS2) gene in Arabidopsis result in various defects in leaf development, such as formation of asymmetrically lobed and downwardly curled leaves, formation of leaflet-like structures from petioles, generation of an abnormal venation system, shorter length of petioles and leaf laminas than WT, and ectopic expression of class1 knox genes in the leaves. To uncover the function of AS2 in the leaf development, we screened enhancer and suppressor mutants of as2. We obtained several enhancer mutants and no suppressor mutant of as2, suggesting several pathways downstream of AS2 in leaf development. The enhancers of as2 could be divided into three types; formation of filamentous and trumpet-like leaves, increased formation of asymmetrically lobed and leaves, and shorter length of petioles and leaf laminas than as2. We will report the phenotype of these enhancer mutants of as2.

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asymmetric leaves1 (as1) and asymmetric leacves2 (as2) mutants of Arabidopsis show similar phenotype, such as the formation of asymmetrically lobed and downwardly curled leaves. It has been reported that as1 and as2 mutants also show the abaxialized leaves. The AS1 gene encodes a protein containing two myb repeats. The AS2 gene encodes a plant-specific protein with a domain containing a cysteine repeat and a leucine-zipper-like sequence. To elucidate the molecular function of AS1 and AS2, we isolated several enhancer mutants that enhance the abnormality in the adaxial-abaxial polarity in the leaves of the as1 mutant. One of the enhancer mutants, enhancer of asymmetric leaves1 (eal1) had abaxialized filamentous leaves. RT-PCR analysis revealed that the transcripts of EAL1 gene were accumulated in leaves and shoot apices.

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