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An altitudinal gradient of leaf water repellency is often observed between and within species. In a previous study of Arabidopsis halleri, cauline leaves (stem leaves that wrap flowering buds) showed higher water repellency in exposed semi-alpine plants than in understory low-elevation plants. Here, we examined altitudinal variations in the cuticular wax content of the leaf surface and experimentally evaluated the role of high water repellency of cauline leaves. Leaf cuticular wax was analysed using comprehensive two-dimensional gas chromatography (GC)-mass spectrometry and a GC-flame ionisation detector. Young flowering buds wrapped by cauline leaves were exposed to freezing temperatures with or without water, and frost damage to the flowering buds was compared between plants from semi-alpine and low-elevation habitats. Higher amounts of C29, C31, and C33 alkanes were observed in the cauline leaves of semi-alpine plants than in those of low-elevation plants. In the freezing experiment, water application increased damage to the flowering buds of low-elevation plants, and the extent of damage to the flowering buds was lower in semi-alpine plants than in low-elevation plants when water was applied to the plant surface. Genetic variations in the amounts of alkanes on the leaf surface depending on the altitude occurred specifically in cauline leaves. Our results indicate that the water repellency of cauline leaves presumably minimises frost damage to flowering buds at high altitudes.

An altitudinal gradient of leaf water repellency is often observed between and within species. In a previous study of Arabidopsis halleri, cauline leaves (stem leaves that wrap flowering buds) showed higher water repellency in exposed semi-alpine plants than in understory low-elevation plants. Here, we examined altitudinal variations in the cuticular wax content of the leaf surface and experimentally evaluated the role of high water repellency of cauline leaves. Leaf cuticular wax was analysed using comprehensive two-dimensional gas chromatography (GC)-mass spectrometry and a GC-flame ionisation detector. Young flowering buds wrapped by cauline leaves were exposed to freezing temperatures with or without water, and frost damage to the flowering buds was compared between plants from semi-alpine and low-elevation habitats. Higher amounts of C29, C31, and C33 alkanes were observed in the cauline leaves of semi-alpine plants than in those of low-elevation plants. In the freezing experiment, water application increased damage to the flowering buds of low-elevation plants, and the extent of damage to the flowering buds was lower in semi-alpine plants than in low-elevation plants when water was applied to the plant surface. Genetic variations in the amounts of alkanes on the leaf surface depending on the altitude occurred specifically in cauline leaves. Our results indicate that the water repellency of cauline leaves presumably minimises frost damage to flowering buds at high altitudes.

MIKC classic (MIKCC)-type MADS-box genes encode transcription factors that function in various developmental processes, including angiosperm floral organ identity. Phylogenetic analyses of the MIKCC-type MADS-box family, including genes from non-flowering plants, suggest that the increased numbers of these genes in flowering plants is related to their functional divergence; however, their precise functions in non-flowering plants and their evolution throughout land plant diversification are unknown. Here, we show that MIKCC-type MADS-box genes in the moss Physcomitrella patens function in two ways to enable fertilization. Analyses of protein localization, deletion mutants and overexpression lines of all six genes indicate that three MIKCC-type MADS-box genes redundantly regulate cell division and growth in the stems for appropriate external water conduction, as well as the formation of sperm with motile flagella. The former function appears to be maintained in the flowering plant lineage, while the latter was lost in accordance with the loss of sperm.

Parasitic plants in the genus Striga, commonly known as witchweeds, cause major crop losses in sub-Saharan Africa and pose a threat to agriculture worldwide. An understanding of Striga parasite biology, which could lead to agricultural solutions, has been hampered by the lack of genome information. Here, we report the draft genome sequence of Striga asiatica with 34,577 predicted protein-coding genes, which reflects gene family contractions and expansions that are consistent with a three-phase model of parasitic plant genome evolution. Striga seeds germinate in response to host-derived strigolactones (SLs) and then develop a specialized penetration structure, the haustorium, to invade the host root. A family of SL receptors has undergone a striking expansion, suggesting a molecular basis for the evolution of broad host range among Striga spp. We found that genes involved in lateral root development in non-parasitic model species are coordinately induced during haustorium development in Striga, suggesting a pathway that was partly co-opted during the evolution of the haustorium. In addition, we found evidence for horizontal transfer of host genes as well as retrotransposons, indicating gene flow to S. asiatica from hosts. Our results provide valuable insights into the evolution of parasitism and a key resource for the future development of Striga control strategies.

Floods impede gas (O2and CO2) exchange between plants and the environment. A mechanism to enhance plant gas exchange under water comprises gas films on hydrophobic leaves, but the genetic regulation of this mechanism is unknown. We used a rice mutant (dripping wet leaf 7, drp7) which does not retain gas films on leaves, and its wild-type (Kinmaze), in gene discovery for this trait. Gene complementation was tested in transgenic lines. Functional properties of leaves as related to gas film retention and underwater photosynthesis were evaluated. Leaf Gas Film 1 (LGF1) was identified as the gene determining leaf gas films. LGF1 regulates C30 primary alcohol synthesis, which is necessary for abundant epicuticular wax platelets, leaf hydrophobicity and gas films on submerged leaves. This trait enhanced underwater photosynthesis 8.2-fold and contributes to submergence tolerance. Gene function was verified by a complementation test of LGF1 expressed in the drp7 mutant background, which restored C30 primary alcohol synthesis, wax platelet abundance, leaf hydrophobicity, gas film retention, and underwater photosynthesis. The discovery of LGF1 provides an opportunity to better understand variation amongst rice genotypes for gas film retention ability and to target various alleles in breeding for improved submergence tolerance for yield stability in flood-prone areas.

Allene oxide synthases (AOSs) were isolated from liverworts and charophytes. These AOSs exhibited enzymatic properties similar to those of angiosperms but formed a distinct phylogenetic clade. Allene oxide synthase (AOS) and hydroperoxide lyase (HPL) mediate the formation of precursors of jasmonates and carbon-six volatiles, respectively. AOS and HPL utilize fatty acid hydroperoxides and belong to the plant cytochrome P450 74 (CYP74) family that mediates plant defense against herbivores, pathogens, or abiotic stresses. Although members of the CYP74 family have been reported in mosses and other species, the evolution and function of multiple CYP74 genes in plants remain elusive. Here, we show that the liverwort Marchantia polymorpha belongs to a basal group in the evolution of land plants; has two closely related proteins (59 % identity), MpAOS1 and MpAOS2, that are similar to moss PpAOS1 (49 and 47 % identity, respectively); and exhibits AOS activity but not HPL activity. We also found that the green microalgae Klebsormidium flaccidum, consist of multicellular and non-branching filaments, contains an enzyme, KfAOS, that is similar to PpAOS1 (37 % identity), and converts 13-hydroperoxide of linolenic acid to 12-oxo-phytodienoic acid in a coupled reaction with allene oxide cyclase. Phylogenetic analysis showed two evolutionarily distinct clusters. One cluster comprised AOS and HPL from charophytic algae, liverworts, and mosses, including MpAOSs and KfAOS. The other cluster was formed by angiosperm CYP74. Our results suggest that plant CYP74 enzymes with AOS, HPL, and divinyl ether synthase activities have arisen multiple times and in the two different clades, which occurred prior to the divergence of the flowering plant lineage.

Klebsormidium flaccidum is a charophytic alga living in terrestrial and semiaquatic environments. K. flaccidum grows in various habitats, such as low-temperature areas and under desiccated conditions, because of its ability to tolerate harsh environments. Wax and cuticle polymers that contribute to the cuticle layer of plants are important for the survival of land plants, as they protect against those harsh environmental conditions and were probably critical for the transition from aquatic microorganism to land plants. Bryophytes, non-vascular land plants, have similar, but simpler, extracellular waxes and polyester backbones than those of vascular plants. The presence of waxes in terrestrial algae, especially in charophytes, which are the closest algae to land plants, could provide clues in elucidating the mechanism of land colonization by plants. Here, we compared genes involved in the lipid biosynthetic pathways of Arabidopsis thaliana to the K. flaccidum and the Chlamydomonas reinhardtii genomes, and identified wax-related genes in both algae. A simple and easy extraction method was developed for the recovery of the surface lipids from K. flaccidum and C. reinhardtii. Although these algae have wax components, their surface lipids were largely different from those of land plants. We also investigated aliphatic substances in the cell wall fraction of K. flaccidum and C. reinhardtii. Many of the fatty acids were determined to be lipophilic monomers in K. flaccidum, and a Fourier transform infrared spectroscopic analysis revealed that their possible binding mode was distinct from that of A. thaliana. Thus, we propose that K. flaccidum has a cuticle-like hydrophobic layer composed of lipids and glycoproteins, with a different composition from the cutin polymer typically found in land plant cuticles.

Plant hormones are transported across cell membranes during various physiological events. Recent identification of abscisic acid and strigolactone transporters suggests that transport of various plant hormones across membranes does not occur by simple diffusion but requires transporter proteins that are strictly regulated during development. Here, we report that a major glucosinolate transporter, GTR1/NPF2.10, is multifunctional and may be involved in hormone transport in Arabidopsis thaliana. When heterologously expressed in oocytes, GTR1 transports jasmonoyl-isoleucine and gibberellin in addition to glucosinolates. gtr1 mutants are severely impaired in filament elongation and anther dehiscence resulting in reduced fertility, but these phenotypes can be rescued by gibberellin treatment. These results suggest that GTR1 may be a multifunctional transporter for the structurally distinct compounds glucosinolates, jasmonoyl-isoleucine and gibberellin, and may positively regulate stamen development by mediating gibberellin supply., GTR1 is known to transport glucosinolates in Arabidopsis. Here, Saito et al. show that GTR1 also transports the plant hormones jasmonate and gibberellin when heterologously expressed in Xenopus oocytes, and that gtr1 mutant plants show a gibberellin-related fertility phenotype.

Summary Jasmonic acid (JA) and methyl jasmonate (MeJA), collectively termed jasmonates, are ubiquitous plant signalling compounds. Several types of stress conditions, such as wounding and pathogen infection, cause endogenous JA accumulation and the expression of jasmonate-responsive genes. Although jasmonates are important signalling components for the stress response in plants, the mechanism by which jasmonate signalling contributes to stress tolerance has not been clearly defined. A comprehensive analysis of jasmonate-regulated metabolic pathways in Arabidopsis was performed using cDNA macroarrays containing 13516 expressed sequence tags (ESTs) covering 8384 loci. The results showed that jasmonates activate the coordinated gene expression of factors involved in nine metabolic pathways belonging to two functionally related groups: (i) ascorbate and glutathione metabolic pathways, which are important in defence responses to oxidative stress, and (ii) biosynthesis of indole glucosinolate, which is a defence compound occurring in the Brassicaceae family. We confirmed that JA induces the accumulation of ascorbate, glutathione and cysteine and increases the activity of dehydroascorbate reductase, an enzyme in the ascorbate recycling pathway. These antioxidant metabolic pathways are known to be activated under oxidative stress conditions. Ozone (O3) exposure, a representative oxidative stress, is known to cause activation of antioxidant metabolism. We showed that O3 exposure caused the induction of several genes involved in antioxidant metabolism in the wild type. However, in jasmonate-deficient Arabidopsis 12-oxophytodienoate reductase 3 (opr3) mutants, the induction of antioxidant genes was abolished. Compared with the wild type, opr3 mutants were more sensitive to O3 exposure. These results suggest that the coordinated activation of the metabolic pathways mediated by jasmonates provides resistance to environmental stresses.

Jasmonic acid (JA) and methyl jasmonate (MeJA), collectively known as JAs, regulate diverse physiological processes in plants, including the response to wounding. Recent reports suggest that a cyclopentenone precursor of JA, 12-oxo-phytodienoic acid (OPDA), can also induce gene expression. However, little is known about the physiological significance of OPDA-dependent gene expression. We used microarray analysis of approximately 21,500 Arabidopsis (Arabidopsis thaliana) genes to compare responses to JA, MeJA, and OPDA treatment. Although many genes responded identically to both OPDA and JAs, we identified a set of genes (OPDA-specific response genes [ORGs]) that specifically responded to OPDA but not to JAs. ORGs primarily encoded signaling components, transcription factors, and stress response-related genes. One-half of the ORGs were induced by wounding. Analysis using mutants deficient in the biosynthesis of JAs revealed that OPDA functions as a signaling molecule in the wounding response. Unlike signaling via JAs, OPDA signaling was CORONATINE INSENSITIVE 1 independent. These results indicate that an OPDA signaling pathway functions independently of JA/MeJA signaling and is required for the wounding response in Arabidopsis.

Tetrapyrrole compounds, such as chlorophylls, hemes, and phycobilins, are synthesized in many enzymatic steps. For regulation of the tetrapyrrole metabolic pathway, it is generally considered that several specific isoforms catalyzing particular enzymatic steps control the flow of tetrapyrrole intermediates by differential regulation of gene expression depending on environmental and developmental factors. However, the coordination of such regulatory steps and orchestration of the overall tetrapyrrole metabolic pathway are still poorly understood. In this study, we developed an original mini-array system, which enables the expression profiling of each gene involved in tetrapyrrole biosynthesis simultaneously with high sensitivity. With this system, we performed a transcriptome analysis of Arabidopsis seedlings in terms of the onset of greening, endogenous rhythm, and developmental control. Data presented here clearly showed that based on their expression profiles at the onset of greening, genes involved in tetrapyrrole biosynthesis can be classified into four categories, in which genes are coordinately regulated to control the biosynthesis. Moreover, genes in the same group were similarly controlled in an endogenous rhythmic manner but also by a developmental program. The physiological significance of these gene clusters is discussed.

The distinctive features of plant organs are primarily determined by organ-specific gene expression. We analyzed the expression specificity of 8809 genes in 7 organs of Arabidopsis using a cDNA macroarray system. Using relative expression (RE) values between organs, many known and unknown genes specifically expressed in each organ were identified. We also analyzed the organ specificity of various gene groups using the GRE (group relative expression) value, the average of the REs of all genes in a group. Consequently, we found that many gene groups even ribosomal protein genes, have strong organ-specific expression. Clustering of the expression profiles revealed that the 8809 genes were classified into 9 major categories. Although 3451 genes were clustered into the largest category, which showed constitutive gene expression, 266 and 1005 genes were found to be root- and silique-specific genes, respectively. By this clustering, particular gene groups which showed multi-organ-specific expression profiles, such as bud-flower-specific, stem-silique-specific or bud-flower-root-specific profiles, could be effectively identified. From these results, major features of plant organs could be characterized by their distinct profiles of global gene expression. These data of organ-specific gene expression are available at our web site: Arabidopsis thaliana Tissue-Specific Expression Database, ATTED (http://www.atted.bio.titech.ac.jp/).

The colonization of land by plants was a key event in the evolution of life. Here we report the draft genome sequence of the filamentous terrestrial alga Klebsormidium flaccidum (Division Charophyta, Order Klebsormidiales) to elucidate the early transition step from aquatic algae to land plants. Comparison of the genome sequence with that of other algae and land plants demonstrate that K. flaccidum acquired many genes specific to land plants. We demonstrate that K. flaccidum indeed produces several plant hormones and homologues of some of the signalling intermediates required for hormone actions in higher plants. The K. flaccidum genome also encodes a primitive system to protect against the harmful effects of high-intensity light. The presence of these plant-related systems in K. flaccidum suggests that, during evolution, this alga acquired the fundamental machinery required for adaptation to terrestrial environments., Plant colonization of land is an important evolutionary event. Here, the authors sequence the genome of a filamentous terrestrial alga and, through a comparative analysis with related algae and land plant species, provide insight into how aquatic algae adapted to terrestrial environments.

Jasmonates have crucial roles in plant responses to biotic and abiotic stresses. Given the importance of transcriptional regulation in jasmonate-mediated stress responses, transcription factors are key regulators of jasmonate signaling. The transcription factors JASMONATE-ASSOCIATED MYC2-LIKE 1 (JAM1), JAM2, and JAM3 are negative regulators of jasmonate signaling, although the mechanisms that control the activities of these transcription factors remain unclear. To understand the regulatory mechanisms of JAM proteins, we used a yeast two-hybrid assay to screen for protein interaction partners of JAM1 and found that JAM1 interacted with JAZ proteins.

Jasmonates regulate transcriptional reprogramming during growth, development, and defense responses. Jasmonoyl-isoleucine, an amino acid conjugate of jasmonic acid (JA), is perceived by the protein complex composed of the F-box protein CORONATINE INSENSITIVE1 (COI1) and JASMONATE ZIM DOMAIN (JAZ) proteins, leading to the ubiquitin-dependent degradation of JAZ proteins. This activates basic helix-loop-helix-type MYC transcription factors to regulate JA-responsive genes. Here, we show that the expression of genes encoding other basic helix-loop-helix transcription factors, JASMONATE ASSOCIATED MYC2-LIKE1 (JAM1), JAM2, and JAM3, is positively regulated in a COI1- and MYC2-dependent manner in Arabidopsis (Arabidopsis thaliana). However, contrary to myc2, the jam1jam2jam3 triple mutant exhibited shorter roots when treated with methyl jasmonate (MJ), indicating enhanced responsiveness to JA. Our genome-wide expression analyses revealed that key jasmonate metabolic genes as well as a set of genes encoding transcription factors that regulate the JA-responsive metabolic genes are negatively regulated by JAMs after MJ treatment. Consistently, loss of JAM genes resulted in higher accumulation of anthocyanin in MJ-treated plants as well as higher accumulation of JA and 12-hydroxyjasmonic acid in wounded plants. These results show that JAMs negatively regulate the JA responses in a manner that is mostly antagonistic to MYC2.

While exogenous toxic compounds such as herbicides are thought to be sequestered into vacuoles in the form of glutathione (GSH) conjugates, little is understood about natural plant products conjugated with GSH. To identify natural products conjugated with GSH in plants, metabolites in the Arabidopsis γ-glutamyl transpeptidase (ggt) 4 knockout mutants that are blocked in the degradation of GSH conjugates in the vacuole were compared with those in wild-type plants. Among the metabolites identified, one was confirmed to be the 12-oxo-phytodienoic acid (OPDA)-GSH conjugate, indicating that OPDA, a precursor of jasmonic acid (JA), is transported into the vacuole as a GSH conjugate.

Jasmonates are known as growth regulators, which have cyclopentanone or cyclopentenone ring, and synthesized through lipoxygenase pathway. Jasmonates are widely distributed in the plant kingdom and modulates wounding responses, disease responses, and anther development. Some kinds of jasmonates were shown to have specific effects on plants. 12-oxo-phytodienoic acid (OPDA), a precursor for jasmonic acid (JA) biosynthesis, promotes tendril coiling, and this effect is stronger than MeJA (Falkenstein E., et al., 1991). However, except for the fact, little is known about the OPDA specific function in various physiological events.