Plasmodesmata callose binding protein 2 contributes to the regulation of cambium/phloem formation and auxin response during the tissue reunion process in incised Arabidopsis stem.

Plants are exposed to a variety of biotic and abiotic stresses, including wounding at the stem. The healing process (tissue reunion) begins immediately after stem wounding. The plant hormone auxin plays an important role during tissue reunion. In decapitated stems, auxin transport from the shoot apex is reduced and tissue reunion does not occur but is restored by application of indole-3-acetic acid (IAA). In this study, we found that plasmodesmata callose binding protein 2 (PDCB2) affects the expansion of the cambium/phloem region via changes in auxin response during the process of tissue reunion. PDCB2 was expressed in the cortex and endodermis on the incised side of stems 1-3 days after incision. PDCB2-knockout plants showed reduced callose deposition at plasmodesmata and DR5::GUS activity in the endodermis/cortex in the upper region of the incision accompanied by an increase in size of the cambium/phloem region during tissue reunion. In addition, PIN(PIN-FORMED)3, which is involved in lateral auxin transport, was induced by auxin in the cambium/phloem and endodermis/cortex in the upper part of the incision in wild type, but its expression of PIN3 was decreased in pdcb2 mutant. Our results suggest that PDCB2 contributes to the regulation of cambium/phloem development via auxin response.

Propiconazole-induced brassinosteroid deficiency reduces female fertility by inhibiting female gametophyte development in woodland strawberry.

KEY MESSAGE: In woodland strawberry, a brassinosteroid biosynthesis inhibitor propiconazole induced typical brassinosteroid-deficient phenotypes and decreased female fertility due to attenuated female gametophyte development. Brassinosteroids (BRs) play roles in various aspects of plant development. We investigated the physiological roles of BRs in the woodland strawberry, Fragaria vesca. BR-level-dependent phenotypes were observed using a BR biosynthetic inhibitor, propiconazole (PCZ), and the most active natural BR, brassinolide (BL). Endogenous BL and castasterone, the active BRs, were below detectable levels in PCZ-treated woodland strawberry. The plants were typical BR-deficient phenotypes, and all phenotypes were restored by treatment with BL. These observations indicate that PCZ is an effective inhibitor of BR in woodland strawberry. Only one gene for each major step of BR biosynthesis in Arabidopsis is encoded in the woodland strawberry genome. BR biosynthetic genes are highly expressed during the early stage of fruit development. Emasculated flowers treated with BL failed to develop fruit, implying that BR is not involved in parthenocarpic fruit development. Similar to BR-deficient and BR-insensitive Arabidopsis mutants, female fertility was lower in PCZ-treated plants than in mock-treated plants due to failed attraction of the pollen tube to the ovule. In PCZ-treated plants, expression of FveMYB98, the homologous gene for Arabidopsis MYB98 (a marker for synergid cells), was downregulated. Ovules were smaller in PCZ-treated plants than in mock-treated plants, and histological analysis implied that the development of more than half of female gametophytes was arrested at the early stage in PCZ-treated plants. Our findings explain how BRs function during female gametophyte development in woodland strawberry.

Auxin-Induced WUSCHEL-RELATED HOMEOBOX13 Mediates Asymmetric Activity of Callus Formation upon Cutting.

Plants have the regenerative ability to reconnect cut organs, which is physiologically important to survive severe tissue damage. The ability to reconnect organs is utilized as grafting to combine two different individuals. Callus formation at the graft junction facilitates organ attachment and vascular reconnection. While it is well documented that local wounding signals provoke callus formation, how callus formation is differentially regulated at each cut end remains elusive. Here, we report that callus formation activity is asymmetrical between the top and bottom cut ends and is regulated by differential auxin accumulation. Gene expression analyses revealed that cellular auxin response is preferentially upregulated in the top part of the graft. Disruption of polar auxin transport inhibited callus formation from the top, while external application of auxin was sufficient to induce callus formation from the bottom, suggesting that asymmetric auxin accumulation is responsible for active callus formation from the top end. We further found that the expression of a key regulator of callus formation, WUSCHEL-RELATED HOMEOBOX 13 (WOX13), is induced by auxin. The ectopic callus formation from the bottom end, which is triggered by locally supplemented auxin, requires WOX13 function, demonstrating that WOX13 plays a pivotal role in auxin-dependent callus formation. The asymmetric WOX13 expression is observed both in grafted petioles and incised inflorescence stems, underscoring the generality of our findings. We propose that efficient organ reconnection is achieved by a combination of local wounding stimuli and disrupted long-distance signaling.

Genome Editing Reveals Both the Crucial Role of OsCOI2 in Jasmonate Signaling and the Functional Diversity of COI1 Homologs in Rice.

Jasmonic acid (JA) regulates plant growth, development and stress responses. Coronatine insensitive 1 (COI1) and jasmonate zinc-finger inflorescence meristem-domain (JAZ) proteins form a receptor complex for jasmonoyl-l-isoleucine, a biologically active form of JA. Three COIs (OsCOI1a, OsCOI1b and OsCOI2) are encoded in the rice genome. In the present study, we generated mutants for each rice COI gene using genome editing to reveal the physiological functions of the three rice COIs. The oscoi2 mutants, but not the oscoi1a and oscoi1b mutants, exhibited severely low fertility, indicating the crucial role of OsCOI2 in rice fertility. Transcriptomic analysis revealed that the transcriptional changes after methyl jasmonate (MeJA) treatment were moderate in the leaves of oscoi2 mutants compared to those in the wild type or oscoi1a and oscoi1b mutants. MeJA-induced chlorophyll degradation and accumulation of antimicrobial secondary metabolites were suppressed in oscoi2 mutants. These results indicate that OsCOI2 plays a central role in JA response in rice leaves. In contrast, the assessment of growth inhibition upon exogenous application of JA to seedlings of each mutant revealed that rice COIs are redundantly involved in shoot growth, whereas OsCOI2 plays a primary role in root growth. In addition, a co-immunoprecipitation assay showed that OsJAZ2 and OsJAZ5 containing divergent Jas motifs physically interacted only with OsCOI2, whereas OsJAZ4 with a canonical Jas motif interacts with all three rice COIs. The present study demonstrated the functional diversity of rice COIs, thereby providing clues to the mechanisms regulating the various physiological functions of JA.

The simple and rapid quantification method for L-3,4-dihydroxyphenylalanine (L-DOPA) from plant sprout using liquid chromatography-mass spectrometry.

L-3,4-dihydroxyphenylalanine (L-DOPA) is one of the important secondary metabolites of plants and has been used for various purposes, such as in clinical treatment for Parkinson's disease and dopamine-responsive dystonia. In plants, L-DOPA is a precursor of many alkaloids, catecholamines, and melanin; the L-DOPA synthesis pathway is similar to that in mammals. L-DOPA acts as an allelochemical, has an important role in several biological processes, such as stress response and metabolism, in plants. L-DOPA is widely used in the clinical treatment as well as a dietary supplement or psychotropic drug, understanding of biosynthesis of L-DOPA in plant could lead to a stable supply of L-DOPA. This paper describes an improved method for simple and rapid quantification of L-DOPA content using liquid chromatography-tandem mass spectrometry. The standard quantitative methods for L-DOPA require multiple purification steps or relatively large amounts of plant material. In our improved method, quantification of L-DOPA was possible with extract of one-two pieces of cotyledon without any partitioning or column for purification. The endogenous L-DOPA (approximately 4,000 µg g-1 FW (fresh weight)) could be detected from the one pieces of cotyledon of the faba bean sprout using this method. This method was also effective for samples with low endogenous amounts of L-DOPA such as broccoli, Japanese white radish, pea, and red cabbage sprouts. Therefore, this improved method will allow to measurement of L-DOPA content easily and accurately from a small amount of plant tissue and contribute to understanding biosynthesis, catabolism, and transport of L-DOPA.

Cell-wall damage activates DOF transcription factors to promote wound healing and tissue regeneration in Arabidopsis thaliana.

Wound healing is a fundamental property of plants and animals that requires recognition of cellular damage to initiate regeneration. In plants, wounding activates a defense response via the production of jasmonic acid and a regeneration response via the hormone auxin and several ethylene response factor (ERF) and NAC domain-containing protein (ANAC) transcription factors. To better understand how plants recognize damage and initiate healing, we searched for factors upregulated during the horticulturally relevant process of plant grafting and found four related DNA binding with one finger (DOF) transcription factors, HIGH CAMBIAL ACTIVITY2 (HCA2), TARGET OF MONOPTEROS6 (TMO6), DOF2.1, and DOF6, whose expression rapidly activated at the Arabidopsis graft junction. Grafting or wounding a quadruple hca2, tmo6, dof2.1, dof6 mutant inhibited vascular and cell-wall-related gene expression. Furthermore, the quadruple dof mutant reduced callus formation, tissue attachment, vascular regeneration, and pectin methylesterification in response to wounding. We also found that activation of DOF gene expression after wounding required auxin, but hormone treatment alone was insufficient for their induction. However, modifying cell walls by enzymatic digestion of cellulose or pectin greatly enhanced TMO6 and HCA2 expression, whereas genetic modifications to the pectin or cellulose matrix using the PECTIN METHYLESTERASE INHIBITOR5 overexpression line or korrigan1 mutant altered TMO6 and HCA2 expression. Changes to the cellulose or pectin matrix were also sufficient to activate the wound-associated ERF115 and ANAC096 transcription factors, suggesting that cell-wall damage represents a common mechanism for wound perception and the promotion of tissue regeneration.

Spatiotemporal plant hormone analysis from cryosections using laser microdissection-liquid chromatography-mass spectrometry.

Laser microdissection (LMD) is used for isolating specific regions or single cells from a wide variety of tissue samples under direct microscopic observation. The LMD method enables the harvest of the cells of interest in a region or specific cells for several analyses, such as DNA/RNA analysis, proteomics, metabolomics, and other molecular analyses. Currently, LMD is used to study various biological events at the tissue or cellular level; it has been used in a wide range of research fields. In this report, we describe techniques for isolating different tissues/specific cells from cryosections of incised Arabidopsis flowering stems by LMD for spatiotemporal quantitative plant hormone analysis. The endogenous indole-3-acetic acid levels in the epidermis/cortex, vascular bundles, and pith of Arabidopsis flowering stems were approximately 19.0 pg mm-3, 33.5 pg mm-3, and 3.32 pg mm-3, respectively, and these endogenous levels were altered spatiotemporally after incision. We also analyzed jasmonic acid from LMD-isolated cells and showed that the endogenous levels increased in the range of approximately 200-3,500 pg mm-3 depending on the tissue and region at 1 h after incision and then decreased to less than 100 pg mm-3 or undetectable levels at 24 h after incision. Quantitative analyses of phytohormones, including jasmonic acid-related molecules, gibberellin, abscisic acid, and cytokinins, could also be performed using the same cell samples. These results showed that spatiotemporal changes in plant hormones could be quantitatively and simultaneously analyzed by LMD-isolated cells from cryosections with positional information. The combination of quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) and sampling by the LMD method provides a comprehensive and quantitative understanding of spatiotemporal changes in plant hormones in a region- and tissue-specific manner. Therefore, LMD-LC-MS methods will contribute to our understanding of the physiological events that control the process of plant growth and development.

Suppression of the Lycopene Cyclase Gene Causes Downregulation of Ascorbate Peroxidase Activity and Decreased Glutathione Pool Size, Leading to H2O2 Accumulation in Euglena gracilis.

Carotenoids are photosynthetic pigments and hydrophobic antioxidants that are necessary for the survival of photosynthetic organisms, including the microalga Euglena gracilis. In the present study, we identified an uncharacterized gene encoding the E. gracilis ß-carotene synthetic enzyme lycopene cyclase (EgLCY) and discovered a relationship between EgLCY-mediated carotenoid synthesis and the reactive oxygen species (ROS) scavenging system ascorbate-glutathione cycle. The EgLCY cDNA sequence was obtained via homology searching E. gracilis transcriptome data. An enzyme assay using Escherichia coli demonstrated that EgLCY converts lycopene to ß-carotene. E. gracilis treated with EgLCY double-stranded RNA (dsRNA) produced colorless cells with hypertrophic appearance, inhibited growth, and marked decrease in carotenoid and chlorophyll content, suggesting that EgLCY is essential for the synthesis of ß-carotene and downstream carotenoids, which are abundant and physiologically functional. In EgLCY dsRNA-treated cells, the ascorbate-glutathione cycle, composed of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR), was unusually modulated; APX and GR activities significantly decreased, whereas DHAR and MDAR activities increased. Ascorbate content was significantly increased and glutathione content significantly decreased in EgLCY dsRNA-treated cells and was correlated with their recycling enzyme activities. Fluorescent imaging demonstrated that EgLCY dsRNA-treated cells accumulated higher levels of H2O2 compared to wild-type cells. Taken together, this study revealed that EgLCY-mediated synthesis of ß-carotene and downstream carotenoid species upregulates APX activity and increases glutathione pool size for H2O2 scavenging. Our study suggests a possible relationship between carotenoid synthesis and the ascorbate-glutathione cycle for ROS scavenging in E. gracilis.

Involvement of Auxin Biosynthesis and Transport in the Antheridium and Prothalli Formation in Lygodium japonicum.

The spores of Lygodium japonicum, cultured in the dark, form a filamentous structure called protonema. Earlier studies have shown that gibberellin (GA) induces protonema elongation, along with antheridium formation, on the protonema. In this study, we have performed detailed morphological analyses to investigate the roles of multiple phytohormones in antheridium formation, protonema elongation, and prothallus formation in L. japonicum. GA4 methyl ester is a potent GA that stimulates both protonema elongation and antheridium formation. We found that these effects were inhibited by simultaneous application of abscisic acid (ABA). On the other hand, IAA (indole-3-acetic acid) promoted protonema elongation but reduced antheridium formation, while these effects were partially recovered by transferring to an IAA-free medium. An auxin biosynthesis inhibitor, PPBo (4-phenoxyphenylboronic acid), and a transport inhibitor, TIBA (2,3,5-triiodobenzoic acid), both inhibited protonema elongation and antheridium formation. L. japonicum prothalli are induced from germinating spores under continuous white light. Such development was negatively affected by PPBo, which induced smaller-sized prothalli, and TIBA, which induced aberrantly shaped prothalli. The evidence suggests that the crosstalk between these plant hormones might regulate protonema elongation and antheridium formation in L. japonicum. Furthermore, the possible involvement of auxin in the prothalli development of L. japonicum is suggested.

WIND transcription factors orchestrate wound-induced callus formation, vascular reconnection and defense response in Arabidopsis.

Wounding triggers de novo organogenesis, vascular reconnection and defense response but how wound stress evoke such a diverse array of physiological responses remains unknown. We previously identified AP2/ERF transcription factors, WOUND INDUCED DEDIFFERENTIATION1 (WIND1) and its homologs, WIND2, WIND3 and WIND4, as key regulators of wound-induced cellular reprogramming in Arabidopsis. To understand how WIND transcription factors promote downstream events, we performed time-course transcriptome analyses after WIND1 induction. We observed a significant overlap between WIND1-induced genes and genes implicated in cellular reprogramming, vascular formation and pathogen response. We demonstrated that WIND transcription factors induce several reprogramming genes to promote callus formation at wound sites. We, in addition, showed that WIND transcription factors promote tracheary element formation, vascular reconnection and resistance to Pseudomonas syringae pv. tomato DC3000. These results indicate that WIND transcription factors function as key regulators of wound-induced responses by promoting dynamic transcriptional alterations. This study provides deeper mechanistic insights into how plants control multiple physiological responses after wounding.

Wound-inducible ANAC071 and ANAC096 transcription factors promote cambial cell formation in incised Arabidopsis flowering stems.

ANAC071 and its homolog ANAC096 are plant-specific transcription factors required for the initiation of cell division during wound healing in incised Arabidopsis flowering stems and Arabidopsis hypocotyl grafts; however, the mechanism remains mostly unknown. In this study, we showed that wound-induced cambium formation involved cell proliferation and the promoter activity of TDR/PXY (cambium-related gene) in the incised stem. Prior to the wound-induced cambium formation, both ANAC071 and ANAC096 were expressed at these sites. anac-multiple mutants significantly decreased wound-induced cambium formation in the incised stems and suppressed the conversion from mesophyll cells to cambial cells in an ectopic vascular cell induction culture system (VISUAL). Our results suggest that ANAC071 and ANAC096 are redundantly involved in the process of "cambialization", the conversion from differentiated cells to cambial cells, and these cambium-like cells proliferate and provide cells in wound tissue during the tissue-reunion process.

Cell-cell adhesion in plant grafting is facilitated by ß-1,4-glucanases.

Plant grafting is conducted for fruit and vegetable propagation, whereby a piece of living tissue is attached to another through cell-cell adhesion. However, graft compatibility limits combinations to closely related species, and the mechanism is poorly understood. We found that Nicotiana is capable of graft adhesion with a diverse range of angiosperms. Comparative transcriptomic analyses on graft combinations indicated that a subclade of ß-1,4-glucanases secreted into the extracellular region facilitates cell wall reconstruction near the graft interface. Grafting was promoted by overexpression of the ß-1,4-glucanase. Using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. These findings demonstrate that the process of cell-cell adhesion is a potential target to enhance plant grafting techniques.

RAP2.6L and jasmonic acid-responsive genes are expressed upon Arabidopsis hypocotyl grafting but are not needed for cell proliferation related to healing.

KEY MESSAGE: Jasmonic acid and RAP2.6L are induced upon wounding but are not involved in cell proliferation during healing in Arabidopsis hypocotyls. Plants produce jasmonic acid in response to wounding, but its role in healing, if any, has not been determined. Previously, the jasmonic acid-induced transcription factor, RAP2.6L, related to APETALA 2.6-like, was identified as a spatially expressed factor involved in tissue reunion in partially incised flowering stems of Arabidopsis. In the present study, we investigated the function of JA and RAP2.6L on wound healing using an Arabidopsis hypocotyl-grafting system, in which separated tissues are reattached by vascular tissue cell proliferation. The jasmonic acid-responsive genes AOS and JAZ10 were transiently expressed immediately after grafting. We confirmed that the endogenous content of jasmonic acid-Ile, which is the bioactive form of jasmonic acid, increased in hypocotyls 1 h after grafting. Morphological analysis of the grafted tissue revealed that vascular tissue cell proliferation occurred in a similar manner in wild-type Arabidopsis, the jasmonic acid-deficient mutant aos, the jasmonic acid-insensitive mutant coi1, and in Arabidopsis that had been exogenously treated with jasmonic acid. RAP2.6L expression was also induced during graft healing. Because RAP2.6L expression occurred during graft healing in aos and coi1, its expression must be regulated via a jasmonic acid-independent pathway. The rap2.6L mutant and dominant repressor transformants for RAP2.6L showed normal cell proliferation during graft healing. Taken together, our results suggest that JA and RAP2.6L, induced by grafting, are not necessary for cell proliferation process in healing.

YUCCA9-Mediated Auxin Biosynthesis and Polar Auxin Transport Synergistically Regulate Regeneration of Root Systems Following Root Cutting.

Recovery of the root system following physical damage is an essential issue for plant survival. An injured root system is able to regenerate by increases in lateral root (LR) number and acceleration of root growth. The horticultural technique of root pruning (root cutting) is an application of this response and is a common garden technique for controlling plant growth. Although root pruning is widely used, the molecular mechanisms underlying the subsequent changes in the root system are poorly understood. In this study, root pruning was employed as a model system to study the molecular mechanisms of root system regeneration. Notably, LR defects in wild-type plants treated with inhibitors of polar auxin transport (PAT) or in the auxin signaling mutant auxin/indole-3-acetic acid19/massugu2 were recovered by root pruning. Induction of IAA19 following root pruning indicates an enhancement of auxin signaling by root pruning. Endogenous levels of IAA increased after root pruning, and YUCCA9 was identified as the primary gene responsible. PAT-related genes were induced after root pruning, and the YUCCA inhibitor yucasin suppressed root regeneration in PAT-related mutants. Therefore, we demonstrate the crucial role of YUCCA9, along with other redundant YUCCA family genes, in the enhancement of auxin biosynthesis following root pruning. This further enhances auxin transport and activates downstream auxin signaling genes, and thus increases LR number.

Suppression of the phytoene synthase gene (EgcrtB) alters carotenoid content and intracellular structure of Euglena gracilis.

BACKGROUND: Photosynthetic organisms utilize carotenoids for photoprotection as well as light harvesting. Our previous study revealed that high-intensity light increases the expression of the gene for phytoene synthase (EgcrtB) in Euglena gracilis (a unicellular phytoflagellate), the encoded enzyme catalyzes the first committed step of the carotenoid biosynthesis pathway. To examine carotenoid synthesis of E. gracilis in response to light stress, we analyzed carotenoid species and content in cells grown under various light intensities. In addition, we investigated the effect of suppressing EgcrtB with RNA interference (RNAi) on growth and carotenoid content. RESULTS: After cultivation for 7 days under continuous light at 920 µmol m-2 s-1, ß-carotene, diadinoxanthin (Ddx), and diatoxanthin (Dtx) content in cells was significantly increased compared with standard light intensity (55 µmol m-2 s-1). The high-intensity light (920 µmol m-2 s-1) increased the pool size of diadinoxanthin cycle pigments (i.e., Ddx + Dtx) by 1.2-fold and the Dtx/Ddx ratio from 0.05 (control) to 0.09. In contrast, the higher-intensity light treatment caused a 58% decrease in chlorophyll (a + b) content and diminished the number of thylakoid membranes in chloroplasts by approximately half compared with control cells, suggesting that the high-intensity light-induced accumulation of carotenoids is associated with an increase in both the number and size of lipid globules in chloroplasts and the cytoplasm. Transient suppression of EgcrtB in this alga by RNAi resulted in significant decreases in cell number, chlorophyll, and total major carotenoid content by 82, 82 and 86%, respectively, relative to non-electroporated cells. Furthermore, suppression of EgcrtB decreased the number of chloroplasts and thylakoid membranes and increased the Dtx/Ddx ratio by 1.6-fold under continuous illumination even at the standard light intensity, indicating that blocking carotenoid synthesis increased the susceptibility of cells to light stress. CONCLUSIONS: Our results indicate that suppression of EgcrtB causes a significant decrease in carotenoid and chlorophyll content in E. gracilis accompanied by changes in intracellular structures, suggesting that Dtx (de-epoxidized form of diadinoxanthin cycle pigments) contributes to photoprotection of this alga during the long-term acclimation to light-induced stress.

Visualisation of abscisic acid and 12-oxo-phytodienoic acid in immature Phaseolus vulgaris L. seeds using desorption electrospray ionisation-imaging mass spectrometry.

The plant hormone abscisic acid (ABA) and the jasmonic acid related-compound 12-oxo-phytodienoic acid (OPDA) play crucial roles in seed development, dormancy, and germination. However, a lack of suitable techniques for visualising plant hormones has restricted the investigation of their biological mechanisms. In the present study, desorption electrospray ionisation-imaging mass spectrometry (DESI-IMS), a powerful tool for visualising metabolites in biological tissues, was used to visualise ABA and OPDA in immature Phaseolus vulgaris L. seed sections. The mass spectra, peak values and chemical formulae obtained from the analysis of seed sections were consistent with those determined for ABA and OPDA standards, as were the precursor and major fragment ions observed in tandem mass spectrometry (MS/MS) imaging. Furthermore, the precursor and fragment ion images showed similar distribution patterns. In addition, the localisation of ABA and OPDA using DESI-IMS was confirmed using liquid chromatography-MS/MS (LC-MS/MS). The results indicated that ABA was mainly distributed in the radical and cotyledon of the embryo, whereas OPDA was distributed exclusively in external structures, such as the hilum and seed coat. The present study is the first to report the visualisation of plant hormones using IMS, and demonstrates that DESI-IMS is a promising technique for future plant hormone research.

Occurrence of brassinosteroids in non-flowering land plants, liverwort, moss, lycophyte and fern.

Endogenous brassinosteroids (BRs) in non-flowering land plants were analyzed. BRs were found in a liverwort (Marchantia polymorpha), a moss (Physcomitrella patens), lycophytes (Selaginella moellendorffii and S. uncinata) and 13 fern species. A biologically active BR, castasterone (CS), was identified in most of these non-flowering plants but another biologically active BR, brassinolide, was not. It may be distinctive that levels of CS in non-flowering plants were orders of magnitude lower than those in flowering plants. 22-Hydroxycampesterol and its metabolites were identified in most of the non-flowering plants suggesting that the biosynthesis of BRs via 22-hydroxylation of campesterol occurs as in flowering plants. Phylogenetic analyses indicated that M. polymorpha, P. patens and S. moellendorffii have cytochrome P450s in the CYP85 clans which harbors BR biosynthesis enzymes, although the P450 profiles are simpler as compared with Arabidopsis and rice. Furthermore, these basal land plants were found to have multiple P450s in the CYP72 clan which harbors enzymes to catabolize BRs. These findings indicate that green plants were able to synthesize and inactivate BRs from the land-transition stage.

Differential Cellular Control by Cotyledon-Derived Phytohormones Involved in Graft Reunion of Arabidopsis Hypocotyls.

When wounding or grafting interrupts the original connection of plant tissue, cell proliferation is induced and the divided tissue is reunited. Previous studies suggested that gibberellin derived from the cotyledon is required for tissue reunion in cucumber and tomato incised hypocotyls, and tissue reunion of Arabidopsis incised flowering stems is controlled by auxin. Differences in the hormone requirements of the tissue reunion process between Arabidopsis and cucumber might be due to differences in organs or species. In this study, we performed morphological and gene expression analyses of graft union in Arabidopsis hypocotyl. We found that removal of the cotyledon and treatment of the cotyledon with the auxin transport inhibitor triiodobenzoic acid (TIBA) suppressed cell proliferation of vascular tissue during graft union formation. These treatments also suppressed expression of IAA5, ANAC071, ANAC096 and CYCB1;1. ANAC071 is involved in the tissue reunion process. The anac071 anac096 double mutant suppressed cell proliferation more so than either of the single mutants. On the other hand, paclobutrazol treatment or deficiency of gibberellin biosynthesis genes suppressed expansion of cortex cells, and exogenous gibberellin treatment or rga/gai mutations that lack the negative regulator of gibberellin reversed this inhibition. The up-regulation of the key gibberellin biosynthesis gene GA20ox1 during graft union formation was prevented by cotyledon removal or TIBA treatment. These data suggest that auxin regulates cell proliferation of vascular tissue and expansion of cortex cells by promoting gibberellin biosynthesis during graft attachment. We hypothesize that the cotyledon-derived phytohormones are essential for graft reunion of the hypocotyl, processed in a cell type-specific manner, in Arabidopsis.

Jasmonoyl-l-isoleucine is required for the production of a flavonoid phytoalexin but not diterpenoid phytoalexins in ultraviolet-irradiated rice leaves.

Rice produces low-molecular-weight antimicrobial compounds known as phytoalexins, in response to not only pathogen attack but also abiotic stresses including ultraviolet (UV) irradiation. Rice phytoalexins are composed of diterpenoids and a flavonoid. Recent studies have indicated that endogenous jasmonyl-l-isoleucine (JA-Ile) is not necessarily required for the production of diterpenoid phytoalexins in blast-infected or CuCl2-treated rice leaves. However, JA-Ile is required for the accumulation of the flavonoid phytoalexin, sakuranetin. Here, we investigated the roles of JA-Ile in UV-induced phytoalexin production. We showed that UV-irradiation induces the biosynthesis of JA-Ile and its precursor jasmonic acid. We also showed that rice jasmonate biosynthesis mutants produced diterpenoid phytoalexins but not sakuranetin in response to UV, indicating that JA-Ile is required for the production of sakuranetin but not diterpenoid phytoalexins in UV-irradiated rice leaves.