Carotenoids exhibit qualitative and quantitative differences, depending on the plant organs and species in which they are expressed. The green tissues of most plants exhibit similar carotenoid profiles and accumulate carotenoids essential for photosynthesis. In contrast, the carotenoid composition of different flowers is distinctive and depends on the plant species. In addition, even within the same plant species, flower petals have a wide range of carotenoid content, from little or none (white petals) to large amounts (yellow petals). At present, however, the understanding of the mechanism that causes such differences is limited. Recently, we showed that in white chrysanthemum petals, carotenoids are synthesized but are subsequently cleaved by CmCCD4a into colorless compounds (Ohmiya et al., 2006). In contrast, most of the carotenogenic gene expression was notably suppressed in the white petals of the Japanese morning glory (Yamamizo et al., 2009). These results suggest that the mechanisms for controlling carotenoid accumulation in flower petals differ among plant species.

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The red colors of flowers are mainly produced by two types of pigments: anthocyanins and betacyanins. Although anthocyanins are widely found as flower and fruit pigments in higher plants, betacyanins have largely replaced anthocyanins in the Caryophyllales, except in the families Caryophyllaceae and Molluginaceae. The occurrence of anthocyanins in the betacyanin-producing Caryophyllales has not been reported. Thus, these two red pigments, anthocyanins and betacyanins, have never been demonstrated to co-exist in one plant. Although this curious mutual exclusion has been examined from genetic and evolutionary perspectives, nothing is known about it at the molecular level. Thus, the evolutionary mechanism of the mutual exclusion of these two pathways remains a mystery.
Although the biosynthesis of betacyanins is poorly understood, the biosynthetic pathway of flavonoids is probably one of the best-studied examples of secondary metabolism in higher plants. To gain further insight into the diversification of red pigments in higher plants, we focus on the potential for anthocyanin biosynthesis in the Caryophyllales at the molecular level.

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Hundreds and thousands varieties of anthocyanin molecules are generated by complicated and diverse modification with glycosyl and acyl moieties. It has been reported that glycosyl and acyl modifications to anthocyanidins catalyzed by transferases using UDP-glycoside and acyl-CoA, respectively, as donor molecules in cytosol. Recent researches on the mechanism of acyl modification have revealed that plants have the other type of the aliphatic and aromatic acyltransferases located in vacuoles, which use acyl-glucose as a donor. In case of glucosyltransfers, the enzyme activities has not been detected to 5 and 7 positions of anthocyanins in carnation and delphinium, respectively, using UDP-glucose as a donor. We purified a glucosyl-donor molecule from petals of carnation and succeeded to detect glucosyltransferase activity to 5 position of anthocyanin in the crude extract prepared from petals of carnation. The protein for this glucosyltransferase was purified and its partial amino acid sequences were determined. Using the degenerated primers based on the amino acid sequences, we succeeded to isolate a cDNA corresponding to the amino acid sequences from carnation.

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Plants have species specific wide varieties of flower color by combining anthocyanin structures, oexisting compounds, vacuolar pH and metal ions. A plant species can accumulate limited kinds of anthocyanins due to its genetic constrain. Rose, carnation and chrysanthemum lack violet/blue colour due to the lack of delphinidin and gentian and iris lack blight red due to the lack of pelargonidin.
The number of hydroxyl group on the B-ring greatly affects the color and is regulated by flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. Their heterologous expression and/or suppression of their endogenous expression can confer a new pathway leading to anthocyanins that the target spices does not accumulate by nature. Careful selection of a host cultivar, coexpression of dihydroflavonol 4-reductase having proper substrate specificity and/or down regulation of a competing pathway is also necessary.
Transgenic rose and carnation accumulating delphinidin and wearing blue hue have been commercialized. Intense red petunia and tobacco have been also made. Regulation of anthocyanin modification, vacuolar pH and metal ion uptake would pave a way to generate more colorful flower.

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Photosynthetic plants have developed various mechanisms to cope with oxidative stress, such as the production of antioxidants and enzymes that scavenge reactive oxygen species (ROS). Plants are also equipped with mechanisms for producing ROS in response to internal and external stimuli. ROS production is induced during many physiological processes, including stress responses, cell growth, hormonal responses, stomatal closure, and disease resistance. In this symposium, a brief overview on ROS signaling in mammalian cells and its impact on medical sciences will firstly be presented. We then discuss various aspects on ROS signaling in plants including regulation of the ROS-producing enzymes, physiological significance of ROS production in biotic and abiotic stress responses, development and programmed cell death, as well as signal transduction network downstream of ROS. We look forward to interdisciplinary discussion among all the participants on the current status and future perspectives of ROS signaling research in plants.

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Aerobes conduct high-level vital activities by means of energy metabolism that uses the chemical reactivity of molecular oxygen (oxidation-reduction: redox activity). Reactive oxygen species (ROS), reduced derivatives of molecular oxygen (e.g., superoxide, hydrogen peroxide), are produced during energy metabolism and infection defense processes in cells and tissues. ROS have been thought to be noxious agents that mediate oxygen toxicity (ROS toxicity theory). Several isoforms of ROS-producing enzymes, called Nox, the NADPH oxidase (and Duox), have been recognized. Expressed by many types of cells, these isoforms have not only antimicrobial actions but also other biological functions in various organisms. As a result of evolving ROS toxicology, it is now thought that ROS may play important roles in regulation of physiological cell signal transductions. In fact, in a wide variety of life science fields, the recognition of 'the physiological cell signaling functions of ROS' has greatly advanced. My talk will overview the ROS signaling occurring in mammalian systems, as a new paradigm evolving from the changing concept of ROS toxicity.

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Reactive oxygen species (ROS) are short-lived molecules produced through various cellular mechanisms. In stomatal guard cells, ROS function as second messengers to mediate abscisic acid (ABA) signaling. The phytohormone ABA plays an essential role in protection of plants from environmental stresses. Previously, we showed that two NADPH oxidases AtrbohD and AtrbohF are responsible for ABA-triggered ROS production in guard cells, ABA-activation of plasma membrane calcium channels, and ABA-induced stomatal closure in Arabidopsis. Our recent study unequivocally shows that two Arabidopsis MAPK genes, MPK9 and MPK12, act downstream of ROS in guard cell ABA signaling. MPK9 and MPK12 are highly and preferentially expressed in guard cells, positively regulate ABA- and calcium-activation of anion channels and ABA-induced stomatal closure. Further progress will be discussed.

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We have been studying molecular mechanisms regulating innate immunity in rice. In the previous studies we found that a small GTPase OsRac1 belonging to the Rac(Rop) GTPase family plays a major role in rice innate immunity. By studying a number of proteins directly and indirectly interacting with OsRac1 we have identified a network of proteins which function as a core protein complex which regulates rice innate immunity. We designate the model for this protein network as defensome model and studying various aspects of the model. OsRac1 interacts with Rboh (NADPH oxidase) and an adapter protein RACK1 (RWD) and these three proteins are major regulators of ROS production during innate immune responses in rice. We present results on regulation of ROS production by these proteins in rice.

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Ca²⁺ mobilization by Ca²⁺ channels and production of reactive oxygen species (ROS) are often induced concomitantly during biotic/abiotic stress responses and development in plants. ROS-activated Ca²⁺ channels have also been postulated to play critical roles. Ten respiratory burst oxidase homolog (rboh) genes (AtrbohA-J) have been implicated in ROS production in Arabidopsis. By applying a heterologous expression system using HEK293T cells, we recently showed that AtrbohC and D possess ROS-producing activity synergistically activated by binding of Ca²⁺ to the EF-hand region of the cytosolic domain and phosphorylation. (Ogasawara et al. JBC 2008; Takeda et al. Science 2008). Ca²⁺-triggered activation of rboh-mediated ROS production and Ca²⁺ mobilization activated by ROS could establish a positive feedback loop to amplify intracellular and intercellular signals. To understand the significance and regulation of ROS production, we have been extensively characterizing rboh genes using the heterologous expression system. We will report comparative analyses of ROS-producing activities and regulatory mechanisms of various rboh proteins, and discuss their functional significance.

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Rapid production of reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. We found that two MAPK cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NbRBOHB (Respiratory Burst Oxidase Homolog B), an inducible form of the NADPH oxidase at the transcriptional level in Nicotiana benthamiana. In addition, we have isolated a potato calcium-dependent protein kinase (StCDPK5) that activates an NADPH oxidase StRBOHB by direct phosphorylation of its N-terminal region. The transgenic potato plants that carry a constitutively active StCDPK5 driven by a pathogen-inducible promoter of the potato showed high resistance to late blight pathogen Phytophthora infestans accompanied by HR-like cell death and H₂O₂ accumulation in the attacked cells. In contrast, these plants showed high susceptibility to early blight necrotrophic pathogen Alternaria solani, suggesting that oxidative burst confers high resistance to biotrophic pathogen, but high susceptibility to necrotrophic pathogen. We discuss roles of nitric oxide (NO) and ROS in basal defense against biotrophic and necrotrophic pathogens.

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Plants are continuously exposed to environmental changes in temperature, water and nutrient availability, oxygen and CO₂ levels, and light intensity, in order to make adaptive responses to the changes. Sometimes, herbivory and infection stresses may affect the adaptive mechanism, so that the resultant cellular redox change follows increases in levels of reactive oxygen species (ROS) and redox-buffering compounds. Such a redox unbalance will modify the redox status of the certain cellular components to modulate the function of the components. On the other hand, plants always generate ROS for the use of their growth. Thus, plant growth is strongly affected by biotic and abiotic stresses. Considering ROS signaling for adaptive responses and development, we must take into consideration the role of the tripeptide compound glutathione. Glutathione participates in cellular redox homeostasis, but it seem to have two different roles in ROS signaling. Here I will discuss what is common and different in ROS-dependent regulations for adaptive responses and development in view of glutathione's role.

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Bax inhibitor-1(BI-1), an endoplasmic reticulum membrane protein, is widely conserved cell death suppressor in plants and animals. The overexpression of Arabidopsis BI-1 (AtBI-1) suppresses the oxidative stress-induced plant cell death. To investigate the molecular mechanism of BI-1-medited cell death suppression, we identified BI-1 interactors from Arabidopsis. Calmodulin (CaM), a calcium signaling mediator, is a first identified BI-1 binding protein. The C-terminal region of AtBI-1 interacted with CaM, and such interaction was essential for the cell death suppression activity of AtBI-1. In addition, cytochrome b5 (Cb5), an electron transfer protein localized mainly in the ER membrane, was isolated as an interactor of AtBI-1. On the other hand, functional screening of yeast gene disruptant demonstrated that AtBI-1 required sphingolipid fatty acid 2-hydroxylase (FAH) for its cell death suppression activity. The BiFC and FRET analysis demonstrated interaction of AtCb5 and AtFAHs in plant cells, suggesting thatAtBI-1 interacts with AtFAHs via Cb5 in plant cells. These data indicates that lipid modification by BI-1 may be important to protect plant cells from oxidative stresses.

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Ubiquitin/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. In this paper, we briefly introduce characters of plant ubiquitin proteasome systems. Cell and organ size regulation mediated by ubiquitin proteasome system will be reported.

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The ubiquitin-proteasome system controls almost all cellular processes by degrading regulatory proteins. The 26S proteasome is composed of 70 different subunits arranged in two sub-complexes, a 20S core particle (CP) and one or two 19S regulatory particles (RP). The proteolytic sites are sequestered inside the CP so that substrate proteins must be unfolded to reach the sites. The RP recognizes the polyubiquitin chains, deconjugates ubiquitin chains, unfolds substrate proteins, and translocates into the catalytic CP. To gain insights into the molecular mechanisms how the 26S proteasome degrades the ubiquitinated substrates, we established a novel method that ubiquitinates any favorable protein. By using this method, we found that Lys63-linked polyubiquitin chain, known as non-degrading signal, is efficiently promotes the substate degradation (Saeki et al, 2009, Embo J). Tightly folded proteins such as GFP are degraded slowly, suggesting that physicalproperty of the substrate itself is important for the proteasomal degradation. Recently, we also unravelled the assembly mechanism of the 19S RP, which involves four proteasome-specific chaperones (Saeki et al, 2009, Cell).

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Ubiquitination is one of the most prevalent post-translational modifications. Ubiquitin (Ub) share a common fold, and their transfer to the target protein is accomplished through the sequential action of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (UBE2), and a ubiquitin-protein ligase (E3). UBE2s accept a Ub from the E1 and typically interact with an E3 to promote Ub transfer to the target. All UBE2 share a ~150 residue conserved catalytic core domain. Although their three-dimensional structures have been reported, the recognition mechanisms between UBE2 and E3 are not completely clarified. UBE2 isoform that exceeds 20 kinds exists also in the rice plant. We have determined that the interaction between the RING-H2 finger domain of the EL5 protein, which is rapidly induced by N-acethylchitooligosaccharides, and OsUBE2D, which is one of Rice UBE2 isoform, at an atomic level. From the results, the correlation of the structure and the function of UBE2 will be reported.

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Protein ubiquitination is one of the major posttranslational modifications occurring in eukaryotic cells. To obtain the proteomic dataset related to the ubiquitin (Ub)-dependent regulatory system in Arabidopsis, affinity purification with an anti-Ub antibody under native condition was performed. Using MS/MS analysis, 196 distinct proteins represented by 251 distinct genes were identified. The identified proteins were involved in metabolism (23.0%), stress response (21.4%), translation (16.8%), transport (6.7%), cell morphology (3.6%), and signal transduction (1.5%), in addition to proteolysis (16.8%) to which proteasome subunits (14.3%) is included. On the basis of potential ubiquitination-targeting signal motifs, in-gel mobilities, and previous reports, 78 of the identified proteins were classified as ubiquitinated proteins and the rest were speculated to be associated proteins of ubiquitinated proteins. We also show identified Ub-related proteins from lily anther in this symposium.

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Among E3 F-BOX protein genes make big gene family in plant and it is reported that they are involved in various physiological phenomena. In Arabidopsis about 600 F-BOX protein genes are annotated by us and other groups. We have collected almost all possible F-BOX protein cDNAs to understand the functions of these F-BOX proteins. Currently we collected 454 independent F-BOX protein ORFs and after confirming their sequences we cloned them into mating type Y2H vectors to construct F-BOX protein panel that makes easy for protein interaction analysis.
Using this F-BOX panel we performed interaction with 22 Arabidopsis ASK (Arabidopsis Skp1 homolog) proteins. By examination of around 10,000 combination of interaction analysis we found 130 F-BOX proteins interacted with any of 22 ASK proteins and many of them are ASK1, ASK2, ASK11 and ASK12. There is also some F-BOX protein group that has specificity with ASK3 and ASK4.We examined ASKs and F-BOX protein expression profiles from microarray data to understand the possible interaction in physiological conditions.
This F-BOX panel will be rapid and suitable tool to analyze E3 for any proteins.

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Gravitropism is the directed growth forward or opposite direction of gravity. In higher plants, relative directional change of gravity is suscepted in the specialized cells (statocytes), and then the following processes are initiated. We have studied the molecular genetic mechanism of gravitropism of Arabidopsis inflorescence stems and demonstrated that endodermal cells are most likely to be the statocytes in Arabidopsis shoots. We have also reported that amyloplast sedimentation to the direction of gravity in the endodermal cells is important for gravity perception. The sgr (shoot gravitropism) 9 mutant exhibits greatly reduced shoot gravitropism. In endodermal cells of sgr9, amyloplasts dynamically moved around but did not sediment to the direction of gravity. Interestingly, inhibition of actin polymerization restored gravitropism and amyloplast sedimentation in sgr9. The SGR9 encodes a novel RING finger protein, which is localized to amyloplasts in endodermal cells. SGR9 showed ubiquitin E3 ligase activity in vitro. Together with live cell imaging of amyloplasts and F-actin, our data suggest that SGR9 modulate interaction between amyloplasts and F-actin on amylopalsts.

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SUMO (small ubiquitin-related modifier) conjugation (sumoylation) to protein substrates is a reversible posttranslational modification that regulates signaling by modulating transcription factor activity. The SUMO conjugation cycle involves activation (E1), conjugation (E2), and ligation (E3) steps. SUMO conjugation to transcription factors in animals and yeast facilitates transcription factor activation or repression, chromatin modifications and subnuclear partitioning. Our research established that Arabidopsis SIZ1 is a SUMO E3 ligase, which facilitates ligation of SUMO1/2 to proteins in the final step of the conjugation cycle, and has been implicated in hormonal and environmental stress signaling, including ABA response, phosphate starvation response, and basal thermotolerance. SIZ1-dependent sumoylation controls adaptation to low and high temperature extremes, and ABA response. To control cold signaling and ABA signaling, SIZ1 mediates sumoylation of ICE1 and ABI5, respectively. The function of sumoylation in plant response to abiotic stresses will be discussed.

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MADS-box and FLOWERING LOCOS T (FT) family genes play important roles in floral regulation and morphogenesis of plants. Tomato is a day-neutral plant and insensitive to photoperiod, suggesting that it may have a different mechanism of floral regulation and morphogenesis from those of a long-day plant, Arabidopsis, and a short-day plant, rice. Tomato is one of model crops and genome projects on it have been performed as international collaborations (http://solgenomics.net/ and http://www.kazusa.or.jp/tomato/). In this study, we will show phenotypes of transgenic plants overexpressing genes related to floral regulation and morphogenesis using Micro-Tom. We will discuss possible roles of these genes in Micro-Tom.
This work was supported by the Ministry of Economy, Trade, and Industry of Japan (METI).

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ACC synthase (ACS) is the rate-limiting enzyme of the ethylene biosynthesis pathway. We reported that LeACS2, a wound-inducible ACS in tomato, is phosphorylated in vivo, and suggested the possibility that phosphorylation regulates protein stability. In this presentation, we demonstrate phosphorylation/dephosphorylation of LeACS2 regulates its stability. Pulse-chase experiments coupled with treatment with protein kinase/phosphatase inhibitors demonstrated LeACS2 is stabilized by phosphorylation and immediately degraded after dephosphorylation. LeACS2 amount affected by the protein kinase/phosphatase inhibitors significantly influenced cellular ACS activity, ACC content, and ethylene production levels in fruit, suggesting that post-translational regulation by phosphorylation plays an important role in the control of ethylene production as well as in transcriptional regulation. Furthermore, we isolated LeCDPK2 as the protein kinase that phosphorylates LeACS2 at Ser-460. LeACS2 was immediately phosphorylated after translation by CDPK and MAP kinase at different sites in response to wound signaling and almost all functional LeACS2 molecules are in their phosphorylated form in the cell.

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Root-knot nematodes (RKNs) are a major parasite of plants, causing approximately 5% loss of total agriculture world-wide. RKN are sedentary endoparasites that establish a single permanent feeding site within plant roots as a juvenile, and then spend their whole remaining life-cycle at this single site. Due to the hidden nature of RKN, most current control methods use toxic chemicals to treat soil prior to sowing, which targets RKN during its brief free-moving stage. The development of safe and effective control methods requires understanding of how RKN induces a feeding site within host plants. Tomato is one agricultural crop affected by RKN. Based on increasing resources at the Tomato National BioResource Project, we have selected the tomato variety Micro-Tom as a model plant to study RKN infection. We have shown that RKN readily infects Micro-Tom and are studying early infection events using in vitro cultures. We have also initiated a forward genetics approach to identify host genes involved in RKN infection, and have identified several putative mutants from this screening. Lab home page: http://gotolab.cris.hokudai.ac.jp

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Any individual virus can infect only a limited range of hosts, and most plant species are 'nonhosts' to a given virus, i.e., all members of the species are insusceptible to the virus. In nonhost plants, the factors that control virus resistance are not genetically tractable, and how the host range of a virus is determined remains poorly understood. Tm-1, a resistance gene of tomato to Tomato mosaic virus (ToMV), encodes a protein that binds to ToMV replication proteins and inhibits RNA replication. ToMV-susceptible tomato cultivars have the allelic gene tm-1, whose product can neither bind to ToMV replication proteins nor inhibit ToMV multiplication. Tomato is a nonhost species for Tobacco mild green mosaic virus (TMGMV), another member of the genus Tobamovirus. We found that the tm-1 protein binds to the replication proteins of TMGMV and inhibits RNA replication. These findings suggest that inhibitory interactions between viral and cellular factors exist in non-adapted host cells and restrict host ranges, while viruses must have evolved to escape from such inhibition during adaptation to their hosts.

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To analyze the comprehensive proteome involved in chloroplast to chromoplast differentiation, we established methods to isolate and identify chromoplast proteins of Micro-Tom fruits at four developmental stages (Mature Green, Yellow, Orange and Red). Plastid lipid associated protein, HSP70 and lipocalin accumulated the most in red fruit. We are trying to make these mutants using RNA interference.
In this study, we has have identified approximately 440 proteins using shotgun proteomics. Half of these proteins are classified as "metabolic process", and the majority of the other proteins, as "response to stimulus" and "cellular process", as defined by gene ontology terms.
We are trying to collect natural variations and mutants that have various colors of fruits such as white, black and orange, and then stop their ripening at the intermediate stage. We compare the chromoplast proteome in these fruits with that of Micro-Tom fruits at four developmental stages using two-dimensional gel electrophoresis, and find specific proteins related to chromoplast development, ripening, and the fruit's color.

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It has been generally accepted that the biosynthetic pathway of phytosteroid is only via cycloartenol. However, recently, we identified lanosterol synthase gene (LAS1) from Arabidopsis and discovered that a part of phytosteroid is biosynthesized via lanosterol by the tracer experiment of labeled mevalonate (MVL). Then, why does plant have the biosynthetic pathway of phytosteroid via lanosterol? Since LAS1 expression is induced by methyl jasmonate treatment, the metabolites of lanosterol may be plant defense related compounds. Tomatine, steroidal alkaroid, which seem to be related to defense response, included in Tomato. Although it is thought that tomatine is biosynthesized from phytosterol, the biosynthetic mechanisms are poorly understood. We undertook the present study to identify the genes for tomatine biosynthesis using Micro-Tom, which is a model plant of Tomato. By the tracer experiment of MVL, it was suggested that biosynthetic pathway via lanosterol exists in the plant. Now, we are screening EMS mutation lines for tomatine biosynthesis deficient mutants by TLC analysis. In this symposium, we discuss the possibility of biosynthetic studies using Micro-Tom.

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Certain kinds of plants, such as lupin and Proteaceae, form a unique root structure so-called 'cluster roots'. Cluster root is a bottle-brush like structure with dense rootlets at a certain part of secondary roots and increases the area of root surface and the amount of exudates. P deficiency significantly stimulates cluster root formation, which contributes P accumulation from unavailable forms in the soil. Mature cluster roots of lupin secrete huge amount of citrate and mobilize sparingly soluble phosphate by chelating function. It was suggested that acidification and secretion of antifungal compounds protects citrate from consumption by microbes in the rhizosphere. Secretion of acid phosphatase (APase) is also stimulated at cluster roots. It was indicated that the activity of APase in the rhizosphere of cluster roots was almost root-secreted APase instead of microbial activities. The optimal pH of the APase is 4.3. It is consistent with the citrate protection by acidification in the rhizosphere of cluster roots. Parallel increase of APase and citrate exudation could be important for organic P mobilization, because organic P frequently exists as insoluble forms in the soil.

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Arbuscular mycorrhizal (AM) fungi establish symbiotic associations with the majority of land plant species, allowing improved uptake of phosphate (Pi) and other mineral nutrients from the soil in exchange for plant-assimilated carbohydrates. My group is investigating mechanisms of nutrient absorption through AM fungi, using rice and soybean as plant materials. We prepared transgenic rice plants that express a fusion of rice AM-inducible Pi transporter OsPT11-GFP, and grew them with AM fungi. The fluorescence of the fusion transporter was specifically observed around arbuscule branch domain. We also invented a simple device for in planta observation of fluorescent proteins in living AM roots. In cortical cells, arbuscules seemed to be functional for only a couple of days. Suddenly, the arbuscular branches shrank. At this stage, however, the periarbuscular membranes appeared intact. Then, the fluorescence of the transporter disappeared within only 2.5 h. The collapse of arbuscules occurred in the subsequent several days. In addition, we identified three AM-inducible Pi transporter genes of soybean. Their phylogenetic relationship and expression patterns will be presented.

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Mineral starvation is though to reduce plant growth by reducing metabolic activity, however this might be an oversimplified view. By using the Arabidopsis natural variation we have identified a major QTL (LPR1 = Low Phosphate Response1), and its paralogue LPR2, that reduce the primary growth during phosphate stavation. The molecular origin of the LPR1 QTL is explained by the differential allelic expression of LPR1 in the root tip (1,2,3). Physical contact of the primary root tip with low-Pi medium is required to arrest root growth. These results provide strong evidences for the involvement of the root cap in sensing and/or responding to mineral deficiency. Our results suggest that when the root encounters a mineral-deficient zone, a signaling pathway restraining growth is triggered in the root tip. We are currently dissecting this signalling by various approaches including classical (4) and chemical genetics, microscopy, biochemistry (5,6) and new tools to manipulate phosphate flux in vivo.
1 Reymond et al. 2006. PCE, 2 Svistoonoff et al. 2007. Nature Genet., 3 Ticconi et al. 2009. PNAS, 4 Misson et al. 2004. PMB, 5 Misson et al. 2005. PNAS, 6 Hirsch et al. 2006. Biochimie

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Phosphorus is one of the most essential elements for biological organisms. Free phosphate concentrations in the soil, however, are very low, usually less than 10 micro M. Plants must take up large amounts of phosphorus from this poor supply. To ameliorate such situation, plants have developed various physiological mechanisms, that is, changes in root architecture, release of inorganic phosphates in the soil, activation of membrane transport, long distance transport via xylem and phloem, re-translocation from older tissues and cells to younger ones, changes in phosphate-related metabolism, store in the vacuole for cytoplasmic Pi homeostasis, and synthesis and accumulation of phytate in seeds.
In the present study, we report some of these physiological phenomena based on the phosphate membrane transport system.

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Recently imaging technique using fluorescent probes has developed. However, fluorescent imaging cannot be performed under light condition and it is extremely difficult to carry out numerical treatment. To overcome these problems, we developed real-time imaging systems which can visualize any beta-ray emitters except for tritium. A sample box was prepared to irradiate light at above-ground part of the plant and keep roots in dark. When 32P-phosphate was applied in culture solution, not only the phosphate uptake manner of roots but also translocation of phosphate was able to visualize. In a soybean plant, as soon as phosphate was taken up from the roots, it was selectively moved up to younger tissues in above-ground part. In some leaves, phosphate was accumulated between the leaves vain. In pods, phosphate was first accumulated at the bottom part of the pod and then accumulated equally in each seed. With this system it was able to image the phosphate uptake manner of the root. In a microscopic level of imaging, a fluorescent microscope was remodeled so that a radioisotope image was taken at the same time with that of the fluorescent one. The resolution is now about 0.1mm.

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Phosphorous is the second abundantly required nutrient for most crop plants, and is the most limited nutrient in natural soil. On the other hand, phosphorous in crop fields forms both insoluble organic and inorganic compounds due to its chemical properties. Taken together, P-limitation is one of the most serious factors that reduce crop yield in local farming system of developing countries, while over-input of P-fertilizer would be one of the problems for that of developed countries. In this situation, improvement of tolerance of crops to P-limited environment is one of the most important targets in molecular breeding. In fact, several procedures of molecular breeding to this target were proposed in late 90's, while almost of all attempts were limited in model plant system. In the current talk, I will focus on recent progress in plant molecular physiology that may enable "real-improvement" of tolerance of crop plants to P-limited condition. This will include 1) molecular mechanisms of organic acid excretion from the roots, 2) internal P-recycling system and 3) plant-microbial interaction for efficient P-acquisition.

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We aimed to reveal the mechanisms for equal water supply to all leaves in the branches using mathematical model. The high production requires the large amount of water delivered from roots to leaves. The transport distance of water distinctively differs among the position of the leaves in a large plant. The velocity of water transport decreases with transport distance. Theoretically, therefore, the water supply is much less in the more distal leaves in taller plants.
We analyzed the mechanism for equal water supply to all leaves in the branches using the mathematical model assumed that water moves according to the Fick's law. The model predicted that 1)It is important that the lateral pathways (leaves or twigs) is less conductive than the axial pathway (branch or stunk), 2)larger difference in hydraulic conductance between the lateral and the axial pathways is essential for more branched pathways.
These predictions were tested by the measurements using over 10 m long shoot of kudzu (Pueraria lobata). The results indicated that the high conductance of the stem and the low conductance of the leaf lamina prevent the water flow in a shoot from being favored at the base.

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The axial growth of plant organs changes under genetic and environmental influences, which can be observed as alternations in cell proliferation and volume growth. For understanding growth controls of plant axial organs, we have studied root growth of Arabidopsis by kinematic analysis, which measures cell flux to calculate local rates of cell proliferation and volume growth at various positions. The kinematic data have been further analyzed by a newly developed mathematical model. The model assumes that the biological activity of a given organ is proportional to the cell number of the organ and is allocated into three aspects; cell proliferation, volume growth, and organ maintenance. The model-assisted analysis estimates efficiencies for these three aspects. We have applied this analysis to assess ploidy and temperature effects on the root growth. The analysis suggests that tetraploids are more efficient than diploids in volume growth and organ maintenance but less efficient in cell proliferation, and that the efficiencies of cell proliferation and volume growth decrease at low temperature. Based on the results, we will discuss availability and weakness of the current model.

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In the face of the wide diversity of reproductive tactics, it is conspicuous how many long-lived perennials employ a masting strategy: most individuals in a population reproduce in synchrony, but with large inter-annual variation in effort. The traditional hypothesis attributes the causes of masting to simultaneous induction by environmental cues such as low temperature. However, recent observations show that environmental cues are not sufficient for masting because some individuals do not flower even when they encounter favorable environmental cues. This suggests a potential interplay between resource availability and environmental cues in floral initiation. Recent rapid progress in genetic and molecular analyses has provided the genetic description of well-established flowering pathways in model plants. Proposed in Arabidopsis thaliana, the gene regulatory model of floral transition describes the complex interactions between environmental signals (e.g. photoperiod and cold temperature) and endogenous cues (e.g. size, leaf number or age). In this talk, three mathematical models that integrate regulation of flowering-time genes and stored resource dynamics are presented.

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Nearly all cells in plants show self-sustained circadian oscillation, which is generated through expression of clock genes and interacts each other via the diffusion of materials. Therefore, the plant can be considered as a coupled oscillator system. In general, the coupled oscillator system can regulate itself and produces various spatiotemporal dynamics. Such behavior can be described by use of the coupled oscillator model.
In this presentation, I introduce the experiments of the spatiotemporal dynamics in plant circadian clocks using transgenic Arabidopsis thaliana CCA1::LUC with the clock gene CCA1 fused to a modified firefly luciferase gene. I explain the mathematics of the plant circadian clocks as a coupled oscillator system, and then discuss about the emergence principles of plant systems with regarded to the circadian clock.

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Leaf development can be abstracted as two elements, cell divisions and expansions. Only when we can describe the behaviors of division and expansion, we know how leaf regulates its shape. On actual leaves, there are gradients of cell division and expansion activities that change spaciotemporally with the progress of leaf development. Gradient is not only attractive but also an obstacle for us to grasp the process. For example, although we can experimentally quantify cell number and sizes from micrographs at a particular moment, we cannot estimate the division and expansion activities, because cell size and cell division are not independent. Imaging technology vigorously clarifies problems, however, it is still difficult to observe the process throughout leaf development. We are approaching the problem via analyses on computational simulation. We have constructed a simulation model based on Free Dirichlet model where cell has shape and size, expands, immigrates, and divides. We can operate division and expansion activities, independently, on the simulator. Some results will be introduced on the presentation.

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Pattern formation in biological systems usually involves interaction and diffusion of molecular factors such as genes and proteins. In this presentation, we will introduce analysis of mathematical models of leaf venation and shoot apical meristem (SAM) as examples of plant pattern formation.
Leaf venation pattern shows distinct variation such as pinnate, branching, palmate and parallel patterns. Since feedback regulation between plant hormone auxin and its efflux carrier PIN1 is important for plant vascular formation, a mathematical model based on auxin-PIN1 dynamics was investigated. The model generates diverse leaf venation patterns and provides insight into pattern diversity among plants.
SAM keeps almost constant size despite active cell division. Arabidopsis clv mutant results in enlarged SAM and frequently shows fasciation and dichotomous branching of shoots. On the other hand, wus mutant shows reduced SAM activity but produces many ectopic shoots after prolonged incubation. Because WUS-CLV interaction is essential for the formation and maintenance of SAM, a model including WUS-CLV dynamics was constructed and analyzed to generate SAM patterning of wild type and mutants.

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Stomata regulate carbon dioxide uptake and transpirational water loss rates upon environmental fluctuation. The plant hormone abscisic acid (ABA) and methyljasmonate (MeJA) induce stomatal closure under drought and biological stresses, respectively. In Arabidopsis, several genetic factors involving in ABA and MeJA signaling pathways in guard cells have been identified, such as protein phosphatase components ABI1, ABI2 and RCN1, and protein kinases OST1 and CDPKs. In order to understand the signal integration process in guard cells, signal events (reactive oxygen species generation, Ca²⁺ and anion channel activation) were examined in the mutants defective in such signaling components. Our results depict the outline of early signal integration in ABA and MeJA signaling in guard cells.

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The plant hormone ABA induces rapid stomatal closure and regulates gene expression to orchestrate adaptive response to drought stress. In Arabidopsis thaliana, genetic evidences have revealed that Protein Phosphatase 2C (PP2C) and Snf1-Related Kinases 2 (SnRK2) including OST1 play prominent roles in ABA signaling.
Starting with a protein phosphatase substrate profiling strategy, we have identified the activation loop of the ABA-activated SnRK2 kinase OST1 as a substrate of the PP2Cs HAB1, ABI1 and ABI2 involved in ABA signaling. Together with the recent studies on the ABA receptors of the PYL/RCAR protein family, our results indicate that SnRK2 kinases are activated very early in ABA signaling through the inhibition of PP2Cs by the PYL/RCAR-ABA complex.
To identify downstream elements of ABA signaling, we performed a bioinformatic screen using the substrate preferences of the OST1 kinase. This screen revealed several putative OST1 substrates including the NADPH oxidase AtRBOHF and transcription factors of the ABF/AREB family.
Together, our results suggest a relatively straightforward architecture of ABA signaling to regulate stomatal closure and gene expression in plants.

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The phytohormone abscisic acid (ABA) regulates diverse cellular processes including modulation of seed dormancy, seed maturation, stomatal movements, gene expression, and vegetative growth during plant development. Many studies have shown that protein de/phosphorylation is the major mechanism regulating ABA signaling in guard cells. We have identified two MAP kinase genes, MPK9 and MPK12, which are highly and preferentially expressed in guard cells. Our results show that MPK12 is activated by ABA and H2O2. Furthermore, our molecular genetic and cell biological data demonstrate that MPK9 and MPK12 have overlapping functions and act downstream of ROS to positively regulate guard cell ABA signaling. Using a biochemical approach, we show that the ABA-activated OST1 protein kinase physically interacts with and phosphorylates the NADPH oxidase AtrbohF, which is responsible for the production of reactive oxygen species (ROS) in response to ABA in order to mediate ABA signaling in guard cells. Further progress will be also discussed.

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Ozone is a secondary air pollutant known to induce programmed cell death (PCD) in plant at high concentration. We showed that O₃ induced the activation of plasma membrane anion channel which is an early prerequisite of this PCD in Arabidopsis thaliana cells. Our data further suggest interplay between anion channel activation, Ca²⁺ influx and ROS generation by NADPH-oxidase leading to PCD. This interplay might be fuelled by several ways in addition of direct ROS generation by O₃; namely; increase in anion channel activity by oxalate generated from ascorbate degradation by O₃ and H₂O₂ generation from apoplastic salicylic acid pool. Anion channel activation was also shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases which contribute to the disruption of vacuole integrity during PCD. Collectively, our data indicate that anion efflux is an early key component to morphological and biochemical events involved in O₃-induced PCD.

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As a part of SAKURA program (a Japan-France collaborative research program), we conducted a survey on the historical corpus of sciences (especially that of botanical science) based on the literatures originated from Univ. Paris (Sorbonne), one of the world oldest Universities (establishment in 1211). The Sorbonne-derived botanical literatures re-collected in recent two years include a number of classical books, doctoral theses and journals (dated between 1815 and 1970). Most of them are now preserved in Japan and registered as the open-access sources for future analyses. In this presentation, some of very early works on the plant responses to environmental changes and plant-microbe interactions at the cellular level will be reviewed by showing the original literatures. In addition, recent evidences (obtained through molecular biological, biochemical and electro-physiological experiments) in support of historical views on plant sensing mechanism will be provided for discussion.

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Stomata open in response to blue light, and this response is regulated by stomatal guard cells. The response includes, photoreception of blue light, uptake of K+ in guard cells by inward-rectifying K+ channels, and the generation of driving force of the K+ uptake by the plasma membrane H+-ATPase. These processes are regulated by phosphorylation and dephosphorylation, and the responses are initiated by autophosphorylating protein kinase phototropins and blue light signal is transmitted to the H+-ATPase by phosphorylation. Type1 protein phosphatase mediates the signaling between phototropins and the H+-ATPase. Recent investigations also revealed that phytohormone abscisic acid (ABA) causes the inhibition of phosphorylation of the H+-ATPase, thereby, effectively prevents water loss in the daytime under drought. In this presentation, we will talk about the protein components involved in blue light signaling processes, and the interaction between blue light-induced opening system and closure system induced by ABA.

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Recent identification of the soluble ABA receptors, PYR/PYL/RCAR1,2), made a great progress in our understanding of ABA signaling. The receptor inhibits protein phosphatase 2C (PP2C), a major negative regulator of ABA response, in an ABA-dependent manner. On the other hand, our group have studied in planta or molecular functions of PP2C or the SNF1-related protein kinase 2 (SnRK2) for long years. In this study, we demonstrated that 1) SnRK2 functions as a central hub in ABA signaling network, 2) SnRK2 and PP2C interact in various combinations, and 3) PP2C inactivates SnRK2 by direct dephosphorylation3). In addition, three subclass III SnRK2s phosphorylate and activate bZIP transcription factors AREB/ABF/ABI5, major regulators of ABA-responsive gene expression. Combining these results, the ABA signal transduction pathway can be simplified to just 4 steps of signaling processes from perception to gene expression3). We also showed that abi1-1-type mutation of PP2C constitutively inactivate SnRK2s, and solved a long-standing mystery of ABA insensitivity of abi1-1 mutant3).
References: 1) Ma et al. (2009) Science, 2) Park et al. (2009) Science, 3) Umezawa et al. (2009) PNAS

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By genetical and biochemical analyses using rice mutants, we have elucidated the nature of GA signaling during the past decade. Now, GA perception has been considered as follows; the binding of GA to GID1 induces the formation of a GID1-GA-DELLA complex and, following the degradation of DELLA with aid of F box protein, GID2, via 26S proteasome pathway, results in various GA-triggered actions. Our recent study on the structure of rice GID1 protein revealed that 1) GID1 resembles hormone sensitive lipase (HSL) and interacts with GA in its binding pocket, 2) bioactive GA is held in its pocket by specific polar and non-polar amino acids via hydrogen bonds and hydrophobic interaction, 3) GID1 evolved by replacement of some important amino acids that were refined for high affinity and specificity to the currently active higher plant GAs.
The next question is why the formation of GID1-GA-DELLA complex induces the interaction between DELLA and GID2. We produced various mutated DELLA and GID2 to determine the regions of DELLA essential for GID2 binding and GID2 for DELLA binding by Y3H. We also examined the effect of these mutations in planta using transgenic plants.

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Endoreduplication involves a doubling of chromosomal DNA without corresponding cell division. In plants many cell types transit from the mitotic cycle to the endoreduplication cycle or endocycle, and this transition is often coupled with the initiation of cell expansion and differentiation. In spite of the importance of the cell cycle transition, it is still largely unknown how this process is developmentally regulated. We recently found that a SUMO E3 ligase, HIGH PLOIDY2 (HPY2), functions as a repressor of endocycle onset in Arabidopsis meristems. Loss of HPY2 results in a premature transition from the mitotic cycle to the endocycle, leading to severe dwarfism with defective meristems. HPY2 functions as a SUMO E3 ligase in vivo and in vitro through an SP-RING domain characteristic of MMS21-type SUMO E3 ligases. HPY2 is predominantly expressed in proliferating cells of root meristems and acts downstream of meristem patterning transcription factors PLETHORA1 (PLT1) and PLT2. These results establish that HPY2-mediated sumoylation modulates the cell cycle progression and meristem development in the auxin-PLT-dependent signaling pathway.

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Precise modulation of transcription plays a vital role in both development and the response to environment. Temporal activation or repression of specific genes is accompanied via a plethora of transcriptional regulators. However, relatively little is known about how the activities of these proteins are controlled. Recent findings indicate that post-translational modifications fine-tune the function of transcription regulators by affecting their localization, conformation or stability. The plant immune system lends itself particularly well to studies of transcriptional regulators as activation of the immunity is associated with rapid and dramatic reprogramming of the transcriptome. A case study of the plant immune coactivator NPR1, a key regulator of salicylic acid-mediated gene expression, illustrates that transcription regulator activity may be controlled by redox-based modifications of cysteine thiols, phosphorylation, and ubiquitinylation coupled to protein degradation. Importantly, cross-talk between distinct protein modifications may determine the spatial and temporal activity of transcription regulators that in turn profile the cellular transcriptome.

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Secreted peptide hormones often contain posttranslationally modified amino acid residues by which their biological activities and receptor binding affinities are highly affected. Posttranslational modifications in peptide hormones in plants so far include tyrosine sulfation, proline hydroxylation and arabinosylation. Tyrosine sulfation is found in PSK and PSY1 peptides and is critical for their function. Recently, we identified tyrosylprotein sulfotransferase (TPST), a key enzyme for tyrosine sulfation, and found that a loss-of-function mutant of AtTPST displayed a marked dwarf phenotype accompanied by stunted roots, reduction in higher order veins and early senescence. Arabinosylation has been found in CLV3, a regulator of stem cell fate. We treated clv3-2 mutant seedlings with purified CLV3 glycopeptide and observed that the clv3-2 SAM treated with CLV3 at 30 nM were substantially reduced in size comparable to wild-type levels. In contrast, synthetic peptide devoid of arabinose showed only weak activity, indicating that the arabinose chain of CLV3 is critical for full activity. Recent progress and future perspective in this research area will be discussed.

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Ralstonia solanacearum first invades intercellular spaces of roots where it multiplies before invading xylem vessels and producing exopolysaccharide (EPS), leading to wilt of the infected plant. R. solanacearum strain OE1-1 (OE1-1), which is pathogenic to tobacco, possessed hrp encoding the type III secretion system (T3SS), and its pathogenicity depended on interactions between the host plants and type III effectors. HrpB positively regulated expression of not only hrpbut also genes encoding exoproteins secreted through the type II secretion system (T2SS), such as an exo-polygalacturonase, PehC. A consortium of T2SS-secreted plant cell wall-degrading enzymes contributed to not only invasion of xylem vessels, leading to systemic infection, but also quantitative control of virulence. PhcA activated by quorum sensing in response to the bacterial cell density induces biosynthesis of EPS. Moreover, active PhcA also suppressed expression of the prhR/prhI operon, resulting in suppression of hrp expression. Therefore, R. solanacearum pathogenicity is globally regulated by mutual regulation by pathogenicity factors through multiplication of the bacteria.

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Bacteria of the family Rhizobiaceae and compatible legumes establish symbiosis to exchanges nitrogen and carbon in root nodules where rhizobial cells differentiate to bacteroids in host cytoplasm. Although the interaction is mutualistic under nitrogen limited conditions, it resembles a pathogenic interaction at some stages including invasion of rhizobial cells to the host cytoplasm. It is now known that at least some of host legumes commence defense reactions. These reactions might exist not only to exclude incompatible and parasitic rhizobial cells but also to regulate the excess nodule formation by compatible rhizobial cells. To evade such host reactions, rhizobial cells seem to utilize a number of tools to actively modulate host functions as well as to passively protect themselves. Our molecular genetic investigation on Mesorhizobium loti, the microsymbiont of a model legume Lotus japonicus, indicated difference in the extent of host aggression between Lotus-M. loti system and Medicago-Sinorhizobium meliloti system.

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