Auxin, a plant growth regulator, promotes lateral root (LR) formation in many plant species but the regulatory mechanism is not fully understood. We have studied molecular cascade regulating auxin-mediated LR formation using several Arabidopsis mutants defective in LR formation. We have shown that Auxin Response Factor 7 (ARF7) and ARF19 regulate LR formation via activation of Lateral Organ Boundaries domain 16/Asymmetric Leaves2-like 18 (LBD16/ASL18) and LBD29/ASL16, which are nuclear proteins (Okushima et al., 2007). Because these LBD/ASLs are thought to be transcriptional regulators, we tried to identify the downstream genes of LBD16/ASL18 using DNA microarray. Among 11 candidate genes, auxin-induced expression of two genes was repressed in arf7 arf19, slr-1 and lbd16-1 mutants. One of these genes was expressed specifically in LR primordia. In this poster, we will present the results of expression analysis and discuss the functions of several candidate genes.

View full abstract

In most dicots, lateral roots (LRs) are initiated from the root pericycle cells and their formation is regulated by intrinsic hormones and environmental factors. In Arabidopsis, several Aux/IAAs and two Auxin Response Factors (ARF7 and ARF19) regulate LR formation through controlling auxin signaling. We have shown that Lateral Organ Boundaries-domain 16/Asymmetric Leaves2-like 18 (LBD16/ASL18) and LBD29/ASL16 genes act for LR formation as direct targets of ARF7/19 (Okushima et al., 2007). Arabidopsis genome has 42 LBD/ASL family members that encode putative transcription factors. No obvious phenotypes of the knockout mutant of LBD16, lbd16-1, suggested functional redundancy of auxin-inducible LBD/ASL members for LR formation. In this study, we focused on the auxin-inducible LBD/ASL proteins and analyzed multiple mutants of them to reveal the molecular mechanisms of auxin-regulated LR formation.

View full abstract

Monocot plants produce numerous adventitious (crown) roots that are dominant in root system of cereals. We previously reported CRL1 that encodes AS2/LOB, a positive regulator for crown root formation, and is directly regulated by ARF in the auxin signaling pathway. Recently, we isolated CRL5 involved in crown root initiation that encodes the protein highly homologous with AINTEGUMENTA in Arabidopsis and found its expression is induced by auxin. However, it is suggested these genes do not work in the same regulatory pathway of crown root formation by the phenotype of crl1 crl5 double mutants and their expression in each mutant back ground. Then, we tried microarray analysis using crl1 and crl5 mutant to find the candidate genes working in downstream of each CRL. Here, we report the functional analysis of CRL5 through phenotyping of its over expresser and the expression analysis of the candidates working in downstream of each CRL.

View full abstract

Cuscuta japonica is a holoparasitic plant which develops a haustorium, a unique organ for absorption of nutrients and water from the host plant. The haustorium formation is initiated by emergence of meristematic cells in the cortex of Cuscuta stem. Then, the meristematic cells differentiate into haustorium. During such process, several genes related to shoot apical meristem formation are expressed. However, the mechanism involved in de novo formation and maintenance of haustorium meristematic cells has been poorly understood in Cuscuta. In this study, we isolated Cuscuta homologues of the genes for adventitious bud differentiation and shoot meristem maintenance in Arabidopsis thaliana, e.g. CUP-SHAPED COTYLEDON2, WUSCHEL and CLAVATA1. Their expression patterns during haustorium development were analyzed by RT-PCR. The role of their genes in haustorium formation in Cuscuta will be discussed.

View full abstract

The genus Eucalypts includes the most widely used tree species, such as E.globulus, for industrial plantation, mainly for making pulp and paper. However, E.globulus is difficult to vegetatively propagate.
In this study, we investigated the effect of CO₂ on adventitious root formation. Eleavated CO₂ concentration (1000 ppm) increased rooting rate, root length and number of roots compared with atmospheric CO₂ (380 ppm). We examined the expression of small subunit of ribulose 1,5-bisphosphate carboxylase (RBCS ) after elevated CO₂ concentration. The expression of RBCS was induced after 2 d in high CO₂ treatment. These results suggest that increased rate of photosynthesis is responsible for the increase in root growth. Furthermore, adventitious root emergence and development were significantly inhibited in NPA (N-1-naphthylphalamic acid, an auxin transport inhibitor)-treated E.globulus. This data supports the hypothesis that a polar transport of the auxin is critical for the induction of adventitious roots.

View full abstract

A low pH (pH 4) medium induced root hair formation in lettuce seedlings. Decapitation of shoots inhibited root hair formation and the accumulation of chlorogenic acid (CGA). Root hair formation in the isolated roots was recovered by exogenous CGA. Thus CGA should be essential for root hair formation in lettuce seedlings.
Dark condition suppressed root hair formation at pH 4 in intact seedlings. Application of exogenous CGA could recover that. Moreover, dark condition reduced CGA content of the roots to one third. Therefore, CGA synthesis by light facilitates for root hair formation. On the other hand, application of CGA on the isolated roots at pH 4 in dark condition could not induce root hair formation. Addition of sugar with CGA on the isolated roots at pH 4 was restored root hair formation in dark condition.
These results suggest that light controls root hair formation through sugar supplement and CGA synthesis.

View full abstract

Plants have developed various mechanisms to grow even in the presence of pathogens. Our previous research using uni-1D mutant, a constitutively active mutant of a CC-NB-LRR gene, suggests a unique possibility that plants change morphology for protection against pathogens. However molecular mechanisms for this phenomenon are unknown.
uni-1D mutant has 3 major phenotypes; up-regulation of PR1/5 expression (an aspect of pathogenesis responses), ectopic axillary meristem formation and defect of meristem maintenance. We first analyzed uni-1D morphological phenotypes in meristem-related mutants and also promoter-GUS activities of meristem-related genes in uni-1D mutant. Further we isolated suppressor mutants of uni-1D morphological abnormalities and analyzed them. From these experiments, distinct mechanisms regulating each uni-1D phenotype were implied. Interestingly, some suppressor mutants have meristem-related phenotypes without uni-1D mutant allele, suggesting that the mechanisms which are affected by uni-1D have roles in meristem regulation even in wild-type background.

View full abstract

The vascular system formation in higher plants requires cell-cell communication. Xylogen, an arabinogalactan protein (AGP), was isolated from Zinnia cell culture as a tracheary element differentiation-inducing factor. In the Arabidopsis genome, there are at least 14 xylogen homologs with the signal peptide and the well-conserved nsLTP domain, and occasionally, with AGP-like sequence. Expression analysis using promoter::GUS reporter transgenic plants of these 14 homologues showed that AtXYP2 is expressed preferentially in vascular tissues among these transgenic lines. On the other hand, AtXYP1 and AtXYP2 had xylogen activity. Therefore, AtXYP2 may play a major role as xylogen in Arabidopsis. Since xylogen is predicted to be a GPI-anchored and to be secreted in a directional manner, we analyzed subcellular localization of AtXYP2 in transgenic plants expressing a GFP-translational fusion of AtXYP2. Intracellular localization pattern of AtXYP2-GFP varied through developmental stages. Our data also suggested that AtXYP2 is secreted via the vesicle transport system.

View full abstract

To reveal the mechanisms underlying the differentiation of chloroplasts, activation tagging was applied to dedifferentiated calli to screen callus expression of RBCS (ces) , in which the expression of photosynthesis genes was elevated (Plant Cell Physiol. , 47, 319-331, 2006). Contrary to the functions of CES genes, we have 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 named sug (suppressed greening of calli) in which green calli change into white by activation tagging, have been selected. DNA prepared from sug mutants was subjected to thermal asymmetric interlaced (TAIL)-PCR, resulting in identification of 4 loci for sug mutations. Expression of genes around sug loci has been analyzed by real-time RT-PCR. Phenotypes of plants and calli in which candidate genes were over-expressed have been analyzed.

View full abstract

In cereal breeding, semi-dwarfness is a favorable character since it decreases the risk that panicles fall down to the ground. We show that rice dense panicle 1 (dn1 ) plant is semi-dwarf, and that it increases its potential grain weight per plant. The dn1 plant decreased weight of each grain, whereas number of spikelets (grains) per plant increased. This gene has been utilized in breeding of japonica high-yield varieties in China in last 50 years. Cells and various organs were shorter and wider in dn1 plant. Response of dn1 plant to brassinosteroid, gibberellin, cytokinin or auxin was not different from that of wild type. Cloned DN1 was mainly expressed in primordia of leafy organs. DN1 protein fused to sGFP localized to the cell membrane, and that intracellular region localized not only to the membrane but nuclear. DN1 lacking the extracellular region, which caused the dn1 phenotypes, localized only to the membrane.

View full abstract

We isolated the orthologues of WUS, STM and FUL (PnWUS1, PnSTM1, PnFUL1, respectively) in Pharbitis nil., a very sensitive plant for short-day treatment, aiming to reveal the control mechanism of early stage of flowering. The analysis by in situ hybridization in the shoot apical meristem showed that expression patterns of 3 genes were reminiscent of those observed for each orthologues in other plants. The quantitative analysis using RT-PCR showed that PnFUL1 mRNA was already accumulated in the flower induced SAM 10 times higher than that of the SAM without flower induction at the end of the 16 hours inductive dark span. In contrast, accumulations of PnWUS1 mRNA and PnSTM1 mRNA in the SAM were not significantly influenced by the dark treatment, at least within the 48 hours after the end of the dark span. The expected flowering mechanism of Pharbitis nil. will be discussed.

View full abstract

Arabidopsis TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) are phosphatidylethanolamine-binding protein homologues and play important roles in flowering, determining the timing and influorescence rchitecture, with antagonistic functions: While FT promotes flowering, TFL1 represses it. These proteins are believed to be regulators of bZIP transcription factors, FD and FD PALALOG (FDP). It is supposed that TFL1 represses the FD-dependent transcription: In turn, FT activates it. However, molecular mechanisms underlying the FD-dependent transcription controlled by TFL1 or FT are still unclear. To investigate the mechanisms, we made transgenic plants over-expressing TFL1/FT proteins fused with an activator or a repressor domain. Genetic analyses between tfl1, ft, fd and fdp are in progress. Here, we will report the phenotypes of these plants and expressions of target genes.

View full abstract

In Arabidopsis thaliana, inflorescence-stem begins to elongate toward proper direction. We found that misexpression of LEAFY ( LFY) in cormbosa1/big and induction of LFY led to defects in orientation of pedicel and fruit. Further analyses revealed that ectopic LFY activation displayed defects in differential growth of pedicel and overproliferation of xylem elements. Correlated well with these phenotypes, ectopic expression of REVOLUTA, which is a regulator of polarity and xylem development, was observed when LFY was activated.
To understand gene cascade, we focused on APETALA1 ( AP1), a direct target of LFY and revealed that the defects in orientation in plant activating AP1 in ap1 were weaker than those in LFY in lfy or ap1. Our results show that the proper control of LFY expression not only through AP1 but more importantly through other components affects polarity and xylem development and thus ensures the appropriate orientation of pedicel and fruit.

View full abstract

Shoot branching is a major determinant of the aerial architecture of plants. In rice, three types of branches develop through out the life time (i.e. tillers in the vegetative phase, panicle branches and spikelets in the reproductive phase). We previously identified LAX1 gene as a main regulator of axillary meristem (AM) formation in the reproductive phase of rice. The mRNAs locate in the boundary between SAM and the region of new AM formation suggests that LAX1 acts non-cell autonomously.
Here we demonstrate that LAX1 protein trafficked from boundary region to the AM cells at P4 stage, and this protein trafficking is required for LAX1 full function. Detailed analysis of lax1 mutants indicates that LAX1 is required for AM formation in both vegetative and reproductive phases. Together with results of in situ experiments, we propose that LAX1 regulates the activity of cell proliferation in the AM at P4 stage.

View full abstract

Plants produce organs from meristem throughout their life. One of the important events during organ formation is to generate boundary that separates meristem and organs. CUC1, CUC2 and CUC3 encode transcription factors with NAC domain and are involved in organ boundary formation. To understand how boundary formation is regulated, we identified CUC1 direct target genes by microarray and in situ expression analysis. One target, LIGHT-DEPENDENT SHORT HYPOCOTYLS 4 (LSH4), encodes a nuclear-localized protein. LSH4 is expressed at boundary region in a similar pattern with CUC1. Interestingly, overexpression of LSH4 results in formation of aberrant leave shape and production of extra floral organs. LSH3, a closest homolog of LSH4 in Arabidopsis, is also expressed at boundary region under the control of CUC1. We will present functional analysis of LSH3 and LSH4 genes.

View full abstract

We have applied heavy-ion beam irradiation to torenia for providing useful floral traits. Among them, we found a mutant (no. 252) that exhibited a B-function-deficient phenotype in which the organs in the second whorl were changed into sepals. RT-PCR analysis of the torenia class B genes revealed the TfGLO expression was abolished in the mutant. Because no effective mutation was found in the genomic sequence of TfGLO, we supposed the repression of TfGLO was caused by a defection of the upstream signaling. Since SQUA, LFY and UFO have been reported to participate in the expression of class B genes in Arabidopsis, we examined expressions of the torenia ortholog genes in the mutant. RT-PCR analysis revealed only TfUFO expression was diminished, and we found a mutation on TfUFO genome in the mutant.

This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry.

View full abstract

Molecular breeding enables us to produce novel floral traits of horticultural plants which could not be obtained by traditional breeding. However, it is difficult to find out useful genes for valuable floral traits without regenerating transgenic plants. To obtain new floral traits efficiently, we simultaneously introduced 42 chimeric repressors for Arabidopsis transcription factors which highly expressed in flowers into torenia (Torenia fournieri) and screened phenotypically-altered 193 lines of 348 transgenic plants. We found that 82.4% of them had single transgene, and 39 of 42 constructs were introduced independently. One third of transgenic torenias with single transgene induced recognizable phenotypes in floral color and/or shape as expected. These results indicate that bulk introduction of Arabidopsis genes can efficiently produce novel horticultural plants with valuable traits. Now we are transforming another bulk set of 50 genes selected with different approach to explore further variation of floral traits.

View full abstract

The flower of spirally arranged numerous stamens and carpels is found in polycarpicae within ancestral taxa. Such flower is considered a primitive flower by the euanthium theory. In contrast, the flower of core eudicots mostly have fixed number of organs arranged in whorls. Thus, the organization of flower is differ between core eudicots and polycarpicae. Phylogenetic analyses indicates that gene duplications followed by functionalizations might have occurred in the AP1/FUL, AP3 and AG gene lineages at the base of the core eudicots. It is suggest that mechanism of flower development and gene function may differ between core eudicots and polycarpicae. Here, FUL-like, PI and AG-like gene of R.screlatus, belong the basal eudicots and have the flower of polycarpicae, was isolated and examine gene expression patterns. To consider a role of FUL-like genes in floral development in polycarpicae, we frame a hypothesis based on our results and BC model.

View full abstract

Most chrysanthemum flowers have comprehensive structures, an aggregated flower, composed by two components of petals, many disc florets at center and surrounded ray petals. Some wild species have only disc florets but no ray petal. The ray petal specific expressed gene, CmCCD4a, was identified from the cultivated chrysanthemum, and already suggested that CmCCD4a keeps white color ray petals by degradation of carotenoids. The both existences and functions of CmCCD4a in petal less species were never disclosed. Here we show that this gene homolog was existent in ray petal less wild species, inherited next generation and certainly functioned in ray petals of progenies. The CmCCD4a was never the lost of function in also the petal less wild chrysanthemums, independent to ray petal construction.

View full abstract

The male gametophyte and tapetum play different roles during anther development although they are differentiated from the same cell lineage, the L2 layer. Until now, it has not been possible to delineate their transcriptomes due to technical difficulties in separating the two cell types. In the present study, we characterized the separated transcriptomes of the rice microspore/pollen and tapetum using laser microdissection (LM)-mediated microarray. Spatiotemporal expression patterns of 28,141 anther-expressed genes were classified into 20 clusters, which contained 3,468 (12.3%) anther-enriched genes. In some clusters, synchronous gene expression in the microspore and tapetum at the same developmental stage was observed as a novel characteristic of the anther transcriptome. Noteworthy expression patterns are discussed in connection with gene ontology (GO) categories and gene annotations, which are related to important biological events in anther development, such as pollen maturation, pollen germination, pollen tube elongation and pollen wall formation.

View full abstract

To investigate the involvement of phytohormones during rice microspore/pollen (MS/POL) development, endogenous levels of several phytohormones in the mature anther were analyzed. We also analyzed the global expression profiles of genes related to seven phytohormones: auxin, gibberellins (GAs), cytokinins (CKs), brassinosteroids, ethylene, ABA, and jasmonic acids, in MS/POL and tapetum (TAP) using a 44K microarray combined with a laser-microdissection technique (LM-array analysis). IAA and GA₄ accumulated at an extremely higher amount in the mature anther compared to the other tissues and organs, while CKs and ABA did not. LM-array analysis revealed that sets of genes required for IAA and GA synthesis were expressed coordinately during the later stages of MS/POL development. In contrast, genes for GA signaling were preferentially expressed during the early developmental stages of MS/POL and throughout TAP development, while their expression was clearly down-regulated at the later stages of MS/POL development.

View full abstract

Sugi (Cryptomeria japonica) is one of the most commercially important conifers but its pollinosis is the most serious allergic disease in Japan. Recently, male-sterile mutants of C. japonica whose pollen is hardly released have been selected. To clarify the molecular mechanisms of male sterility in C. japonica, we have constructed a DNA microarray using clustered ESTs. This microarray was used to analyze gene expression in male strobili of the male-sterile mutant and wild type. There were about 20 ESTs exhibiting differential expression (4-fold changes) at stages before collapsed microspores were observed in the male-sterile mutant. In contrast, over 100 ESTs showed differential expression in male strobili at the stages of microspore, which were collapsed in the male-sterile mutant.

View full abstract

Protein ubiquitination is one of a major posttranslational modification presented in all eukaryote cells. Ubiqtuitn is attached to a substrate protein as a signal molecule to lead various outcomes, such as degradation by 26S proteasome, DNA repair and endocytosis. Ubiquitination of target proteins requires sequential actions of three enzymes; E1, E2 and E3.
To date, it has been considered that protein ubiquitination is active in male tissue although there have been quite a few information. In this study, we identified ubiquitin-related proteins from lily anther followed by determination of ortholog proteins in Arabidopsis. Now we are analyzing one protein, which is expressed in pollen.

View full abstract

MALE STERILITY1 (MS1) gene of Arabidopsis encodes a PHD-type transcription factor and regulates microspore maturation after meiosis. The ms1 mutant did not develop a characteristic exine structure, suggesting that MS1 controls genes necessary for exine formation. On the other hand, downstream genes regulated by MS1 contain several genes for lipid and phenylpropanoid biosynthetic and modification enzymes. Since exine mainly consists of sporopollenin polymer which is thought to be made from lipids and phenylpropanoids, these genes may be involved in sporopollenin biosynthetic genes. Among these, we report here a functional analysis of an acyltransferase family gene which is homologous to acyltransferases involved in lignin monomer biosynthesis. We will discuss function of the gene by metabolomic, genetic and molecular biological approaches.

View full abstract

The surface of mature pollen grains of many higher plants is covered with a layer of lipophilic materials called pollen coat. The pollen coat consists mainly of lipids and proteins and is necessary for successful pollination. However, little is still known about its molecular composition as well as the biosynthesis of each component. In this study, we are aimed at elucidating the lipid and protein components of Arabidopsis pollen coat exhaustively.
GC-MS analysis showed that the lipid of Arabidopsis pollen coat consists of C24-C31 normal saturated hydrocarbons, isohydrocarbons, and various esterified sterols. Some putative polar lipids were also detected by thin-layer chromatography. SDS-PAGE and TOF-MS analysis revealed that the pollen coat contains only small number of proteins larger than MW 15,000, whereas it contains large numbers of smaller proteins and peptides.

View full abstract

We have previously showed that each component of Arabidopsis phosphatidylinositol 3-kinase (PI3K) complex, AtVps30, AtVps34, and AtVps15, is required for pollen germination. Transmission electron microscope showed that pollen grains of atvps30 and atvps15 were normal in appearance, suggesting that PI3K complex is not essential for early pollen development. However, it should be noted that several atvps34 pollen grains exhibited abnormal morphology, in accordance with the recent report by Lee et al. (2008). We suggest that reduced PI3K activity in atvps30 and atvps15 disturbs pollen germination signaling, which might include ROS production. By contrast, complete loss of PI3K activity in atvps34 may affect pollen development as well as pollen germination.

View full abstract

Boron is an essential micronutrient for plants. Boron is important for pollen tube elongation. Boron transporters BOR6, BOR7 are expressed in pollen grains and tubes. We studied possible roles in pollen tube elongation and fertilization.
T-DNA insertion lines for BOR6, BOR7 were obtained and lines heterozygous for T-DNA insertion were grown under limited supply of B. T-DNA segregation among the seeds from self-pollinated heterozygous plants was examined. It was found that homozygous lines in terms of T-DNA insertion was underrepresented while lines without T-DNA was overrepresented, suggesting the roles of these gene in efficient fertilization under low B conditions. Pollens carrying T-DNAs in both BOR6 and BOR7 were found to elongate shorter pollen tubes when germinated in vitro with normal media. Abnormal pollen tubes elongation was also observed in vivo in pistils of selfed double homozygous lines for BOR6 and BOR7 grown under low B conditions.

View full abstract

Parthenocarpy is the natural or artificially induced production of fruit without fertilization of ovules. It has been revealed that parthenocarpy is regulated in part by the plant hormones, auxin and gibberellin however, the detailed mechanism is still unknown. Since some cultivars of Chinese and European pears were thought to be parthenocarpic, we investigated which cultivars exhibit parthenocarpy, in detail. As a result, 9 out of 27 Chinese pears and 5 out of 8 European pears showed parthenocarpic fruit development. Out of 257 F2 individuals derived from a cross between self-compatible Japanese pear,"Osa-nijisseiki" and a Chinese pear,"Tsu Li", we found that three individuals were parthenocarpic. One of the three individuals showed male sterility, and in the others, seed development was interrupted. We conducted micro array analysis with them, and found that 39 genes were commonly up- or down-regulated as compared to those of the non-parthenocarpic individuals.

View full abstract

The flo2 mutants which have a mutation in a functionally unknown OsCEO1 gene develop floury and chalky endosperm as well as a defect of rice grain developed in high temperature. We found gene expression alteration related to fundamental cell function and storage of starch and protein in both defect. It making us expects the extensive reconstruction of cellular function including ATP starvation. In fact, we found ATP shortage at grain maturation stage, and the alteration of protein storage or synthesis, which was greater in immature stage but shorter in mature grain, supporting the hypothesis. Interestingly, the flo2 alleles which have nucleotide alteration in front half of OsCEO1 coding region showed severe phenotype, and the mild phenotypes were observed in alleles of mutants which have the mutation in later half. This observation supposed existence of a complex mechanism for the functional expression of OsCEO1.

View full abstract

The flo2 mutant shows decreased expression of many genes involved in starch synthesis and storage proteins, suggesting that the Flo2 gene may encode a multi-functional regulatory factor for biosynthesis of storage substances in rice grains. Fine mapping determined the responsible gene for flo2 locating at 110 cM of chromosome 4 and following DNA sequencing including other 7 flo2 alleles revealed point mutations in this region causing stop codon or splicing error. The corresponding genomic fragment from the wild-type plant recovered the flo2 phenotype in a flo2 mutant. RBE1 and RA16 expressions were also recovered, which greatly decreased in flo2 mutant. Taken together, this gene was identified to be the origin of flo2 mutation and termed OsCEO1 (Conductor of Endosperm Organogenesis 1). OsCEO1 expressed higher in leaves and immature seeds. Yeast 2-hybrid analysis detected 14 candidates that may have direct interaction with OsCEO1.

View full abstract

In cultivation of tomato, unfavorable conditions prevent pollination and fruit set. So parthenocarpic trait is very useful for tomato fruits production, but little is known about its mechanism. In this study, we analyze the function of cytochrome P450 (CYP78A subfamily) that may play important roles in fruit development, and try to make parthenocarpic tomato plants by transgenic approach.
Previous study, we isolated four CYP78A cDNAs from tomato (LeCYP78A1-4) and found that the expression of LeCYP78A2 was induced by pollination in ovaries. In this study, we introduced this gene into tomato, and analyzed its function. The transgenic tomato plants overexpressing LeCYP78A2 changed fruit size and seed formation. And the LeCYP78A2 RNAi plants showed pleiotropic developmental phenotypes, including short internode, abnormal flower morphology and small leaf size. These results suggest that LeCYP78A2 may play important roles in morphogenesis and fruit development.

View full abstract

A cultured cell line, GTH4 (N. gossei x N. tabacum), which exhibits hybrid lethality, dies at 26C, but not at 37C. However, GTH4S, a variant cell line derived from GTH4 does not die at 26C. Both gene expression and protein synthesis at 37C are prerequisite for cell death of GTH4, suggesting that those are absent in GHT4S. We compared protein profiles of GTH4 to GTH4S by ion-exchange chromatography/SDS-PAGE and MALDI-TOF MS, found that Annexin (AY14973) involved in cell cycle was present in GTH4 but absent in GTH4S. The RT-PCR analysis showed that transcript of this Annexin gene was totally missing in GTH4S, supporting the results of protein analysis. Transcription of other Annexin genes (AY965682, AY965683) that respond to biotic/abiotic stresses was reduced in GTH4S cells. In relationship to those results, profiles of the genes preferentially expressed in GTH4 will be also discussed.

View full abstract

We have been identified novel putative programmed cell death (PCD) regulators, Arabidopsis thaliana IAP like Protein (AtILP) 1 and AtILP2, which contain two conserved BIR like domains that show weak homology to baculoviral IAP repeat of Inhibitor of Apoptosis Protein in animals (Higashi et al.,(2005) Apoptosis 10:471-480). We previously reported that heterologously expressed AtILP1 or AtILP2 affected PCD in tobacco BY-2 (Kobayashi et al., the 47th Annual Meeting of JSPP in 2006).
To further analyses for ILP function, we have isolated tobacco ILP homologs. We found a tobacco EST, AJ718124 from BY-2 using tblastn, then carried out RACE and successfully obtained cDNA sequences including ORF. The cDNA are highly homologous to AtILP1, thus we designate the cDNA as NtILP1. We made two constructs for overexpression and RNAi of NtILP1, respectively, and introduced them into tobacco BY-2 cells. Using these transformants, we are analyzing the function of NtILP1 for plant PCD.

View full abstract

AtLSD1, a negative regulator of programmed cell death (PCD) in Arabidopsis, is a cytosolic retention protein that indirectly participates in transcriptional regulation by direct interaction with transcription factors. Oppositely AtLOL1 is a positive regulator of PCD although its structure is similar to AtLSD1, which has three LSD1-type zinc-finger motifs (zf-LSD1). Our survey for plant genes with three zf-LSD1s using published papers and databases demonstrated that higher plants has two or three, but Physcomitrella patens and Chlamydomonas reinhardtii have only one. Two-hybrid assay in yeast showed that these plant LSD1-like proteins also interact with Arabidopsis transcription factors, which interacts with AtLSD1, via "GxP" motif. Interestingly Physcomitrella patens and Chlamydomonas reinhardtii have only AtLSD1-type gene, which encodes LSD1-like proteins with conserved C-terminal domain. Taken all, it's supposed that plants have acquired AtLOL1-type gene and PCD regulation mechanism controlled by several LSD1-like proteins on evolutional process.

View full abstract

Among Arabidopsis genes, CLAVATA2 (CLV2) harbors exceptionally many strain-specific polymorphisms accompanying amino-acid substitutions. However, it has not been elucidated the mechanism how such highly polymorphic genes are allowed to be exist. We have shown that CLV2^Ws, a CLV2 in Ws strain, requires for its function the SHEPHERD (SHD), which is an HSP90-like molecular chaperone resident in the endoplasmic reticulum. Our aim is to verify the hypothesis that the chaperone activity of SHD dissolves harmful misfolding of mutated CLV2, allows such mutated genes to behave as functional alleles, and functions as a capacitor of genetic variation.
Recently, we determined a single amino-acid substitution in CLV2^Ws as the caused mutation for SHD requirement. Then we identified 32 types of CLV2 amino-acid sequences among 93 Arabidopsis strains. SHD dependency of these CLV2 variants will be reported.

View full abstract

Lateral root elongation is an important process that contributes to the establishment of root architecture. RCN1/OsABCG5 is firstly reported as an important protein for shoot branching in rice. In the present study, we addressed the alternative role of RCN1/OsABCG5 in root development using two reduced culm number 1 (rcn1) mutants, namely rcn1-1 and rcn1-2. Remarkable inhibition of lateral root elongation was identified in rcn1. In addition, we identified that remarkable inhibition of lateral root emergence on seminal root surface. The mutant specific phenotype was recovered by high temperature treatment suggesting that inhibition effect of rcn1 mutation was temperature sensitive. The present data represented that RCN1/OsABCG5 plays major role in root branching, involving two processes of elongation and emergence on seminal root surface.

View full abstract

Both root meristem size and root growth are regulated systematically as the rates of cell division and elongation are synchronized. However the molecular details of the transcription network regulating rapid root cell elongation are poorly understood. To identify the transcriptional networks regulating rapid root cell elongation, we used our RootMap, which comprises high-resolution gene expression profiles obtained from fine sections along the developmental axis of the Arabidopsis root. We chose several transcription factors show a peak of gene expression at the boundary between the root meristem zone and elongation zone. We then screened Arabidopsis T-DNA insertion mutants for these transcription factors. Preliminary characterization of one of these, UPBEAT1 has shown that downregulation results in a larger plant while upregulation results in a smaller plant.
The molecular and cytological characterization of the upbeat1 mutant should provide new insights into the molecular interactions controlling rapid cell expansion.

View full abstract

EL5 in rice encodes membrane-localized E3 ubiquitin ligase with a RING-H2 domain. We have demonstrated that nitrate causes necrosis in transgenic rice roots that overexpress EL5V162A with impaired E3 activity (mEL5-5). We report here that compared to non-transgenic rice plant (NT), elongation of crown roots in mEL5-5 was normal, but the number of lateral root increased when cultured in N-source-free medium. Cells around the lateral root emergence sites in mEL5-5 became necrotic when treated with nitrite. This treatment enhanced NO production, but its level and location in mEL5-5 and NT were the same. On the other hand, ROS was increased only in mEL5-5 treated with nitrite. The ROS production was at the same site where NO production and cell death occurred. These results suggest that EL5 is involved in the lateral root formation and cell death by controlling the ROS production downstream of NO signaling.

View full abstract

During cell migration, F-actin is asymmetrically accumulated at the front side of monospores from the marine red alga Porphyra yezoensis. To elucidate how the establishment of cell polarity responsible for the F-actin asymmetry is regulated in monospores, we pharmacologically tested the roles of Ca²⁺ and phosphatidylinositol pathway in this event. When extracellular Ca²⁺ was removed by EGTA or Ca²⁺ influx was blocked by LaCl₃, asymmetrical distribution of F-actin was completely inhibited. The same result was observed by the treatment of monospores with phospholipase C (PLC) inhibitor U73122. In contrast, phospholipase D (PLD) inhibitor, 1-butanol, had no effect on the formation of F-actin asymmetry, although migration of monospores was completely inhibited. Therefore, we proposed that in the early development of monospores, Ca²⁺ influx and PLC activity are participated in the establishment of cell polarity and PLD plays a role in the maintenance of cell polarity.

View full abstract

Calcium regulates diverse physiological functions in organisms. Influx of Ca²⁺ is evoked by internal or external stimulus. Alterations of cytoplasmic Ca²⁺ concentration due to Ca²⁺ influx are detected by calcium-binding proteins, ex. calmodulin, calcium dependent protein kinases, which convert Ca²⁺ signals into cellular events.
To analyze Ca²⁺ kinetics in plant cell, we established Ca imaging system in Lotus japonicus. In the root hair cells of leguminous plants, Calcium spiking, a periodical increase of Ca²⁺ concentration, is induced by perception of infection signals released from symbiotic partners.Our system clearly detected the symbiotic Ca spiking in response to root nodule symbiosis signal, Nod factor. In addition, another type of Ca oscillation was detected during elongation of the root hair cells. This 'non-symbiotic' Ca oscillation had different wave profile from that of the symbiotic Ca spiking, suggesting that different Ca signatures trigger different cellular events in the root hair cells.

View full abstract

In plants, the apparent active uptake of water during
elongation growth is thought to depend on local osmotic
gradients in the apoplast canal surrounding the symplast
around xylem vessel. Plenty of simulation and analytical
results have been hitherto obtained on above water uptake
model. However, net solute efflux into symplast and that
of influx from vessels have not been precisely calculated.
Therefore, we analytically calculated and compared both
quantity. It was found that in the steady state, net solute
efflux into symplast is equal to that of influx from xylem
vessels, and in the state for response to a step change of
solute efflux into the symplast, net solute efflux into the
symplast is greater than that of influx from xylem
vessels.

View full abstract

Peptidomics is a challenging field to create a link between genomic information and biological function through biochemical analysis of expressed peptides, including precise identification of post-translational modifications and proteolytic processing. Small peptide hormones are usually encoded by multiple paralogous genes whose primary products are approximately 70- to 110-amino-acid cysteine-poor secreted peptides that exhibit sequence similarity within the C-terminal short conserved domain that correspond to the mature peptide sequences. By analogy with these examples, we speculated that if Arabidopsis contains genes for paralogous secreted peptides with similar features, they may encode small peptide signals generated by proteolytic processing. Using an in silico approach coupled with nano LC-MS/MS analysis of apoplastic peptides, we identified a novel 14-amino-acid peptide generated through post-translational modification and proteolytic processing. We will discuss possible functions of this peptide based on expression pattern analysis and gain-of-function phenotypes.

View full abstract

Oxylipin, 9-hydroxy-10-oxo-(Z), 15(Z)- octadecadienoic acid (KODA) is one of signal compounds, which is synthesized from the α- linolenic acid. It has been suspected that has a function not only to enhance stress tolerance but also to activate inflorescence development. In biosynthesis in KODA and Jasmonate (JA) it has been suspected that two kinds of enzymes (LOX, AOS) were shared. And some differences in substrate specificity of enzyme activity (9 or 13 oxidation) has also suggested. In Arabidopsis thaliana the expression of one AOS and 6 LOXs were detected, but their involvement in KODA biosynthesis and enzyme activity remained to be elucidated. To clear the correlation between the phenotype of the mutants of LOXs and the levels of endogenous KODA, we established GC/MS analysis method. The differences in these mutants were indicated in phenotype of leaf or inflorescence and the levels of endogenous oxylipines.

View full abstract

The maize orange leafhopper Cicadulina bipunctata is a serious pest of second crops of forage maize in Kyushu, Japan. The insect induces galls on various plants of Poaceae, which are characterized by stunted growth and severe swelling of leaf veins. Previous studies indicate that some chemicals injected by the insect affect the shoot apical meristem and induce galls on the leaves that have extended during the feeding. To clarify the mechanism underlying the gall induction, we performed high-throughput and comprehensive plant hormone analyses by LC-ESI-MS/MS, in leaves infested and galled by C. bipunctata. As a result, galled rice leaves contained higher and lower contents abscisic acid (ABA) and salicylic acid (SA) than ungalled leaves, respectively. Galled maize leaves had much more contents of ABA, especially around shoot apical meristems than ungalled leaves. We discuss possible gall-inducing processes by C. bipunctata.

View full abstract

Plant hormones play an important role as signaling molecules in the regulation of almost all phases of plant development, from seed germination to senescence. They regulate expression of genes via each signal transduction systems, and in most cases they mutually regulate the signaling and metabolic systems between the hormones. To understand the fine regulatory systems of hormone levels, we have established a method for high-sensitive and high-throughput analysis of major plant hormones, namely, cytokinins (23 species), auxins (7 species), abscisic acid, and gibberellins (12 species). Our method needs 10 to 100 mg fresh weight of plant tissues for determination and enables us to analyze 180 samples at a time. We are now trying to identify key genes regulating hormone levels in rice using QTL analysis. We have measured hormone contents of Sasanishiki X Habataki BILs. The data will be presented.

View full abstract

The plant-oncogenic 6b gene from Agrobacterium tumefaciens causes auxin localization in tobacco. To study further the auxin localization, we applied 1-naphthoxyacetic acid (NOA, a specific auxin influx carrier inhibitor) to the tobacco seedlings transgenic for the dexamethasone (Dex)-inducible 6b gene. In the presence of Dex alone, tumor developed only abaxial side of cotyledons and auxin (IAA) was localized to the periphery of tumor as well as vascular bundles. In contrast, the presence of both Dex and NOA resulted in the reduction of the abaxial tumor, and the newly development tumors growth hypocotyls. The seedlings became shooty calli at high concentration of NOA. Seedlings containing both 6b gene and IAA responsive reporter gene construct DR5::GUS indicated that IAA was localized to the junction of normal tissue and tumor. Thus, NOA induced additional redistribution of IAA and morphological alterations which had been caused by the 6b gene in tobacco.

View full abstract

In several angiosperm species, excised plant tissues give rise to the entire organism through transdifferentiation from differentiated cells to stem cells when cultured with phytohormones including auxin. However, the action mechanism of auxin on this process has remained elusive. In our current study with Physcomitrella, which has a high-capacity for transdifferentiation, we show that the anti-auxin probe BH-IAA suppresses transdifferentiation from leaf cells to stem cells. Because BH-IAA blocks auxin response through the suppression of the SCF^TIR1-Aux/IAA pathway and the transcriptional regulation by auxin response factors (ARFs), the transcriptional network of ARFs is likely to be important in transdifferentiation of leaf cells. As a first step to elucidate this network, we isolated 13 ARF genes from Physcomitrella, and found 2 to 3 splice variants of 4 genes. In addition, we will show recent studies on the expression of ARFs during transdifferentiation and the phenotypes of transgenic lines expressing high levels of ARFs.

View full abstract

Auxin-induced elongation is thought to be caused by acid-growth and wall modifying proteins, particularly in Graminiae by an expression of endo-1,3:1,4-β-glucanase (EI). However, expression of EIwas detected only after 4-hr treatment with auxin. When the segments were treated with IAA in the presence or absence of 2% of sucrose, sucrose slightly enhanced IAA-induced elongation. Analysis of the level of glucose in hemicellulosic fraction of treated segments revealed that IAA alone reduced the glucose level, but sucrose suppressed this IAA effect, suggesting that exogenously supplied sucrose inhibited the degradation of 1,3:1,4-β-glucan in the cell walls. Next we examined the expression of EI gene by RT-PCR . IAA alone promoted the expression of EI, but this effect was cancelled by the presence of sucrose. These results indicated that the degradation of hemicellulosic 1,3:1,4-β-glucan in barley coleoptile cell walls was not a cause of IAA-induced elongation growth.

View full abstract

Auxin induces cell wall loosening, leading to elongation growth by water absorption. The molecular mechanism of the wall loosening has been speculated that some enzymes modified hemicellulosic polysaccharides. Xyloglucan endotransglycosylase/hydrolases (XTHs) are thought to participate in xyloglucan degradation and/or reconstruction, leading to wall loosening, particularly in dicot. However, the involvement of XTHs in auxin-induced elongation has not been extensively studied, partly because of the little effect of auxin on elongation of Arabidopsis (Columbia) hypocotyls. We examined the effect of auxin on the elongation growth of etiolated hypocotyls and expression of XTHs. IAA stimulated the elongation of hypocotyls within 2 hr either in the absence or presence of sucrose. Expressions of known 33 XTH genes were examined by RT-PCR. XTH 4, 15, 17, 18, 24, 27 and 30 were highly expressed in the etiolated hypocotyls. The early effects of IAA treatment and presence of sucrose on the expressions are reported.

View full abstract

The metabolism of source organs in plants change during the development of the sink organs. The regulation of this metabolism is important in the control of crop productivity. To elucidate these regulation systems, we constructed model experiments using Arabidopsis to analyze metabolic and gene expression changes during leaf-stage progression and after removal of the reproductive organs. Leaf gene expression levels and content of major amino acids, both of which decreased during leaf-stage progression, increased after removal of the reproductive organs. In particular, the levels of expression of cytokinin-biosynthesis genes and cytokinin-responsive genes and the cytokinin content increased after removal of the reproductive organs. Analysis of plants with knockout of a cytokinin-biosynthetic gene (AtIPT3) and a cytokinin-receptor gene (AHK3) suggest that cytokinins regulate communication between reproductive and vegetative organs.

View full abstract