Abscisic acid （ABA） has critical roles in many physiological processes including seed dormancy, stomatal closure and adaptive responses to abiotic stress.　Synthetic positive or negative regulators of ABA function are expected to use as not only chemical tools for research in plant biology but also plant growth regulators for agriculture application.　We developed inhibitors of ABA-inactivating enzymes as the positive regulators and antagonists of ABA receptors as the negative regulators.　（1） Specific inhibitors of ABA-inactivating enzymes: Key enzymes for ABA inactivation are ABA 8′-hydroxylases, cytochrome P450 enzymes classified into the CYP707A subfamily.　Because the tertiary structure of CYP707A has never been clarified, we developed the CYP707A inhibitors as ABA analogs （AHI） or azoles （abscinazoles） by lead optimization based on structure-activity relationship.　These inhibitors conferred drought tolerance in plants.　（2） Antagonists of ABA receptors: ABA signaling is repressed by PP2Cs, but stress-induced ABA binds PYL, which then bind and inhibit PP2Cs.　X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA’s 3′-CH and 4′-CO that opens at the PP2C binding interface.　Because ABA analogs with sufficiently long 3′ or 4′ chains were predicted to traverse this tunnel and block PYL-PP2C interactions, we designed and synthesized AS6 and PAN.　These compounds functioned as a potent ABA antagonist to block multiple stress-induced ABA responses in vivo.

Diterpenoids are biosynthesized from geranylgeranyl diphosphate （GGDP） through cyclization and successive chemical modification. Various physiologically active diterpenoids have been isolated and identified from higher plants and fungi. Rice （Oryza sativa） produces various labdane-related diterpenoids, phytohormones gibberellins and phytoalexins including phytocassanes, momilactones and oryzalexins. We have identified diterpene cyclase genes responsible for the rice diterpenoids: OsCPS1, CPS2, CPS4, KS1, KSL2, KSL4, KSL5, KSL6, KSL7, KSL8 and KSL10. The identification led to finding biosynthetic gene clusters of phytocassanes and momilactones, respectively, and enabled us to perform reverse-genetic studies. We showed that OsCPS1 for gibberellin biosynthesis expresses at different tissues from its isoform gene OsCSP2 for phytoalexin biosynthesis according to their distinct biological roles in rice. Furthermore, we identified not only fungal labdane-related diterpene cyclases but also unusual fungal chimera diterpene synthases, possessing both GGDP synthase and GGDP cyclase activities.　Identification of chimera diterpene synthase genes PaFS responsible for biosynthesis of fusicoccins, diterpene glucosides produced by Phomopsis amygdali, led to finding its biosynthetic gene cluster, and manipulation of a P450 gene in the cluster helped us to effectively produce a fusicoccin-related anticancer drug.

Abscisic acid （ABA） is one of the classical phytohormones and regulates stomatal closure, stress adaptation, seed maturation and seed dormancy. To elucidate molecular mechanism underlying ABA response, I have been conducting chemical genetic studies on ABA metabolism and reception. In studies on ABA metabolic regulations, integration analyses of ABA measurement and molecular genetic approaches have revealed physiological function of CYP707A, which is key enzyme for ABA catabolism. Prior to germination, endogenous ABA levels in seeds are drastically reduced by activation of CYP707A, and this ABA reduction is essential for germination. In the vegetative tissues, stomatal opening occurs under the high humidity condition, and this physiological action is caused by activation of CYP707A. In studies on ABA reception, I have found chemical probes, which are able to bind to ABA receptors, and those have been utilized for elucidation of receptor functions. Although synthetic ABA agonist named quinabactin selectively activates low-affinity receptors, quinabactin can induce ABA action in both seeds and vegetative tissues in similar concentration to ABA. This result indicates that low-affinity subfamily among receptors has a key function in major ABA response in plant.

Plants maximize fitness by coordinating growth and development of cells as well as organs in response to environmental changes. For the coordination, cell-to-cell and long-distance communication among cells and organs is necessary. Cytokinins, a class of phytohormones that play key roles in various processes of plant growth and development, have been implicated in the communication. It has been shown that cytokinin action is regulated at various steps, including de novo biosynthesis, degradation, transport, perception, and signaling, to play roles in the communication. This review summarizes and provides updates on the regulatory mechanisms of cytokinin action with major emphasis on cytokinin de novo biosynthesis and transport. We also introduce the mechanisms of cytokinin biosynthesis by phytopathogens that exploit cytokinin action for their infection.

Fleshy fruits are one of important human diets and ripening is a critical step for the fruits to be tasty and higher-quality foods. During ripening, a variety of physiological changes, such as pigmentation, softening, and aroma and flavor development, occurs in a highly synchronized manner. These physiological changes at the onset of the ripening are caused by a synchronized and drastic transition of gene expression patterns in the fruit. Tomato has been used as an advantageous model plant for studies on fleshy fruits, and many advances in our understanding of ripening mechanism have been achieved by the studies of mutations suppressing the ripening phenomena entirely, such as ripening inhibitor （rin）, non-ripening （nor） and Colorless non-ripening （Cnr）. These three mutation loci have been identified to encode transcription factors, indicating that these transcription factors play important roles in the drastic and synchronized changes at the onset of ripening. In this review, I will overview the function of ripening regulators including RIN, CNR, NOR and other transcription factors identified recently, and also discuss about the interaction between these transcription factors.

The ability to set fruit without pollination and/or fertilization is one way to improve the quantity and quality of fruits throughout the year dramatically. In tomato, the parthenocarpic fruit 2 （pat-2） gene is a recessive mutation conferring parthenocarpy, which is the capability to set seedless fruits in the absence of pollination and fertilization. To confirm the chromosomal location of the pat-2 locus, F₂ population segregating from a cross between parthenocarpic line and non-parthenocarpic tomato cultivar were grown and classified as parthenocarpic or wild-type. Through genetic analysis, we constructed linkage map based on SSR markers and the pat-2 locus was assigned to chromosome 4, located on 6.4 cM region between 2 SSR markers. Using F₃ individuals, we refined the candidate region to a ~300 kb interval. Comparing the sequence of this region between parental lines, a deletion was found in parthenocarpic line in a predicted gene. It is estimated that the pat-2 is deficient functionally and translated into a truncated protein in parthenocarpic line. Independent RNAi transgenic plants of the gene showed parthenocarpy. Consequently, PAT-2 encodes zinc-finger homeodomain protein that is predicted as a transcription factor.

Flowering plants have developed self-incompatibility （SI） as a genetic system to prevent inbreeding and thus promote outcrossing to generate genetic diversity. SI is based on the self/non-self discrimination between male and female. In many plants, SI is controlled by a single locus, designated S, with multiple haplotypes. Each S-haplotype encodes both male-specificity and female-specificity determinants （S-determinants）, and the self/non-self discrimination is accomplished by the S-haplotype-specific interaction between these S-determinants. Recent studies have revealed that plants utilize diverse self/non-self discrimination systems, which can be classified into two fundamentally different systems, self-recognition and non-self-recognition systems. The self-recognition system, adopted by Brassicaceae and Papaveraceae, depends on a specific interaction between male and female S-determinants derived from the same S-haplotype, and their interaction triggers SI responses to prevent the fertilization. The non-self-recognition system, found in Petunia （Solanaceae）, depends on non-self （different S-haplotype）-specific interaction between male and female S-determinants, and their interaction positively supports the fertilization process. The male S-determinant genes in Solanaceae family were highly duplicated to recognize diverse non-self female S-determinants. In this review, we introduce our recent findings on the molecular mechanism and the evolution of this non-self-recognition system in the Solanaceae.

Protein-protein interaction plays a fundamental role to maintain biological process. Bimolecular fluorescence complementation （BiFC） assay is an imaging technique to visualize protein-protein interaction in living cells. BiFC assay is based on structural complementation between two non-fluorescent N-terminal and C-terminal fragments derived from a fluorescent protein. Over the past decade, BiFC assay has been widely used in plant science fields, due to its technical simplicity. However, designing appropriate control experiment is quite important for BiFC assay, because non-specific self-assembly of the non-fluorescent fragments induces background fluorescence, which may lead to misinterpretation of BiFC results. In this technical note, we describe information regarding fluorescent protein and BiFC assay in plants, and introduce “BiFC competition assay” as a control experiment.

A novel assay system, based on the bi-molecular fluorescence complementation （BiFC） technique in Escherichia coli, was developed for detecting transient interactions such as those between kinases and their substrates. BiFC is an alternative technique for detecting protein-protein associations in live cells. This system detected, the interaction between OsMEK1 and its direct target, OsMAP1. By contrast, BiFC fluorescence was not observed when OsMAP2 or OsMAP3, which are not substrates of OsMEK1, were used as prey proteins. We also screened for interacting proteins of Ca²⁺-dependent protein kinase 8 （OsCPK8）, a regulator of plant immune responses, and identified three proteins as interacting molecules of OsCPK8. The interaction between OsCPK8 and two of these proteins （ARF-GEF and peptidyl prolyl isomerase） was confirmed in rice cells by means of BiFC technology. These results indicate that our new assay system has the potential to screen for protein kinase target molecules.

Sugarcane was brought into Brazil in early 16th century and is currently planted more than 10 mm ha. Sugarcane harvest starts mainly from March and mostly finish in November. Ripeners were registered in Brazil from 1990s and being used in order to increase sugar content to desirable level and/or suppress flowering, which enables to extend harvesting period. Several field research results of bispyribac sodium were shown in comparison with other ripeners.

Glyphosate is the most widely used herbicide in the world.　Resistance to the herbicide in weeds leads to severe yield losses for crop.　The resistance to glyphosate of amaranth is due to two mechanisms: target site resistance conferred by amino acid change in a target enzyme or overexpression of a target enzyme, and non-target site resistance conferred by changes in sequestration and/or translocation of the herbicide.　Various kinds of weed seeds have been introduced as contaminants in imported grains and some of them are resistant to herbicide.　The monitoring of the spread of resistant genes is necessary.　Plant hormones that control sexual reproduction will be one of the powerful candidates to control weeds.

The Convention on Biological Diversity and its Nagoya Protocol have been in force and effective. Researchers dealing with natural products chemistry have to comply with these conventions when conducting research and development activities using genetic resources. To raise awareness among researchers about the conventions, this article explains basic principles and key regimes of compliance regarding access and benefit-sharing on genetic resources. Researchers clearly understand and follow the regulations for legally appropriate research and development.