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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.

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 inhibited 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.

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.

Grafting success by cell wall remodeling Plants that produce great fruit may not always have great roots. Grafting of a productive scion onto a resilient rootstock has provided agriculturalists with solutions to this and other challenges. Notaguchi et al. have now studied why some plant grafts work better than others (see the Perspective by McCann). The tobacco relative Nicotiana benthamiana ( Nb ) turns out to be the superhero of grafting, able to form grafts with plants from a wide range of evolutionary families. A bit of Nb , set as a middleman between a tomato scion and an Arabidopsis rootstock, negotiated a successful junction between these two otherwise nonconversant plant species. The expression of β-1,4-glucanases secreted into the extracellular region turns out to be key in facilitating cell wall reconstruction. Science , this issue p. 698 ; see also p. 618

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.

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.

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.

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., Matsuoka et al. study the mechanism by which transcription factors ANAC071 and ANAC096 promotes regeneration of wounded tissue in Arabidopsis by mutagenesis and morphological characterization. They find that these factors are essential for wound-induced cambium formation from dedifferentiated cells before the initiation of cell division.

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.

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.

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, β-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. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-1066-7) contains supplementary material, which is available to authorized users.

In this study the relationship between blue light- and brassinosteroid-enhanced leaf lamina bending and unrolling in rice was investigated. Twenty-four hours (h) irradiation with white or blue light increased endogenous brassinosteroid levels, especially those of typhasterol and castasterone, in aerial tissues of rice seedlings. There was an accompanying up-regulation of transcript levels of CYP85A1/OsDWARF, encoding an enzyme catalyzing C-6 oxidation, after 6h under either white or blue light. These effects were not observed in seedlings placed under far-red or red light regimes. It was concluded that blue light up-regulates the levels of several cytochrome P450 enzymes including CYP85A1, thereby promoting the synthesis of castasterone, a biologically active brassinosteroid in rice. Based on these findings, it is considered that blue light-mediated rice leaf bending and unrolling are consequences of the enhanced biosynthesis of endogenous castasterone. In contrast to aerial tissues, brassinosteroid synthesis in roots appeared to be negatively regulated by white, blue and red light but positively controlled by far-red light.

Small peptides act as local signals during plant development, but few studies have examined their interaction with phytohormone signaling. Here, we show that application of gibberellin (GA) to Arabidopsis shoots induces substantial accumulation of transcripts encoded by CLE6, a member of the CLAVATA/ESR-RELATED (CLE) gene family, in the root stele, followed by promotion of organ growth by CLE6 in GA-deficient plants. The long-distance effect of GA4 was demonstrated by the observation that its application to the shoot apex of the GA-deficient mutant ga3ox1/ga3ox2 rescued the short-root phenotype. Microarray analysis was used to identify root-expressed genes that respond to systemic application of GA, and CLE6 was selected for further analysis. CLE6 was highly expressed in roots at the young seedling stage, and CLE6 promoter activity was strong in hypocotyls and roots, especially in root stele cells at branch points. Application of CLE6 peptide had no obvious effect on the growth and development of GA-deficient mutant plants. Nonetheless, the fact that ectopic over-expression of CLE6 in the GA-deficient mutant promoted root growth and branching, petiole elongation, bolting rate and stem length showed that CLE6 expression partially compensates for the GA deficiency. Reciprocal grafting of GA-deficient mutant plants to 35S::CLE6 transformants complemented the shoot phenotype associated with GA deficiency, demonstrating the systemic effect of CLE6 from root to shoot. These data suggest that root-expressed CLE6 is systemically involved in shoot growth under GA action in Arabidopsis.

In dicots, iron (Fe) is acquired from the soil by IRT1 (IRON-REGULATED TRANSPORTER 1) and FRO2 (FERRIC REDUCTION OXIDASE 2) that are localized at the root epidermis. IRT1 and FRO2 expression is induced by local and systemic signals under Fe-deficient conditions in Arabidopsis thaliana. In this study, the expression of IRT1, FRO2, bHLH038 and bHLH39 (the latter two of which control IRT1 and FRO2 expression) was promoted by GA4 treatment of gibberellin (GA) deficient ga3ox1 ga3ox2 mutants. In contrast, the expression of FIT, which encodes a transcription factor necessary for IRT1 and FRO2 induction under Fe deficiency, was not induced by the application of GA4. The induction of those genes triggered by shoot-applied GA4 was observed, even in the fit-2 mutant which had reduced endogenous GA levels caused by treatment with paclobutrazol (PBZ), a GA biosynthesis inhibitor. These results suggested that FIT was not a key regulator in the GA responses under Fe-sufficient conditions. On the other hand, among Fe uptake-related genes, the expression of IRT1, bHLH038 and bHLH39 was lower in ga3ox1 ga3ox2 compared with the wild type (WT) under Fe-sufficient conditions, but the expression of all Fe uptake-related genes decreased under Fe-deficient conditions. Additionally, the PBZ treatment decreased IRT1 expression in the WT under Fe-deficient conditions, but not in the fit-2 mutant. These data suggest the contribution of GA to the induction of Fe uptake-related genes under Fe-sufficient and Fe-deficient conditions, possibly in FIT-independent and FIT-dependent manners, respectively.

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.

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.

Background Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains β-carotene and xanthophylls (such as zeaxanthin, diatoxanthin, diadinoxanthin and 9′-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated. Results To clarify the carotenoid biosynthetic pathway in E. gracilis, we searched for the putative E. gracilis geranylgeranyl pyrophosphate (GGPP) synthase gene (crtE) and phytoene synthase gene (crtB) by tblastn searches from RNA-seq data and obtained their cDNAs. Complementation experiments in Escherichia coli with carotenoid biosynthetic genes of Pantoea ananatis showed that E. gracilis crtE (EgcrtE) and EgcrtB cDNAs encode GGPP synthase and phytoene synthase, respectively. Phylogenetic analyses indicated that the predicted proteins of EgcrtE and EgcrtB belong to a clade distinct from a group of GGPP synthase and phytoene synthase proteins, respectively, of algae and higher plants. In addition, we investigated the effects of light stress on the expression of crtE and crtB in E. gracilis. Continuous illumination at 460 or 920 μmol m−2 s−1 at 25 °C decreased the E. gracilis cell concentration by 28–40 % and 13–91 %, respectively, relative to the control light intensity (55 μmol m−2 s−1). When grown under continuous light at 920 μmol m−2 s−1, the algal cells turned reddish-orange and showed a 1.3-fold increase in the crtB expression. In contrast, EgcrtE expression was not significantly affected by the light-stress treatments examined. Conclusions We identified genes encoding CrtE and CrtB in E. gracilis and found that their protein products catalyze the early steps of carotenoid biosynthesis. Further, we found that the response of the carotenoid biosynthetic pathway to light stress in E. gracilis is controlled, at least in part, by the level of crtB transcription. This is the first functional analysis of crtE and crtB in Euglena. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0698-8) contains supplementary material, which is available to authorized users.

†These authors contributed equally to this work. SUMMARY The micropylar region of endosperm in a seed, which is adjacent to the radicle tip, is called the ‘endosperm cap’, and is specifically activated before radicle emergence. This activation of the endosperm cap is a widespread phenomenon among species and is a prerequisite for the completion of germination. To understand the mechanisms of endosperm cap-specific gene expression in tomato seeds, GeneChip analysis was performed. The major groups of endosperm cap-enriched genes were pathogenesis-, cell wall-, and hormone-associated genes. The promoter regions of endosperm cap-enriched genes contained DNA motifs recognized by ethylene response factors (ERFs). The tomato ERF1 (TERF1) and its experimentally verified targets were enriched in the endosperm cap, suggesting an involvement of the ethylene response cascade in this process. The known endosperm cap enzyme endo-b-mannanase is induced by gibberellin (GA), which is thought to be the major hormone inducing endosperm cap-specific genes. The mechanism of endo-bmannanase induction by GA was also investigated using isolated, embryoless seeds. Results suggested that GA might act indirectly on the endosperm cap. We propose that endosperm cap activation is caused by the ethylene response of this tissue, as a consequence of mechanosensing of the increase in embryonic growth potential by GA action.

Transgenic potatoes were employed to test whether a diverse range of gene actions can induce different stress tolerance(s). To select available transgenic lines, we measured growth profiles under non-stress conditions in our evaluation of transgenic lines and confirmed productivity under stress(es). We performed microarray analyses to clarify the abiotic stress tolerance mechanism controlled by AtDREB1A. Two transgenic lines out of over 200 independent genotypes displayed stable tuber production under high salinity stress conditions. We identified five significant genes that were induced by abiotic stress with AtDREB1A in potato; the reported homologs in Arabidopsis are downstream of the AtDREB1A gene. Our results suggest that the AtDREB1A gene acts as a transcriptional factor against abiotic stresses in potato, and that potato may have mechanisms in abiotic stress tolerance(s) controlled by a native transcriptional factor similar to AtDREB1A. Besides uses of the exotic genes which can enhance abiotic stress tolerance, exploitation of the native genes in cultivated potatoes is also valuable for elucidating the basic knowledge and for orienting towards uses in crop improvement. Further challenges would be comparing the stress responses between cultivated potatoes and wild species in more detail, in order to examine (1) whether alternative tolerance mechanisms are possible, and (2) if so, whether effective germplasm enhancement could be carried out based on wild species to improve drought and salt stress tolerance of cultivated potato.

When grafting or wounding disconnects stem tissues, new tissues are generated to restore the lost connection. In this study, the molecular mechanism of such healing was elucidated in injured stems of Arabidopsis . Soon after the inflorescence stems were incised, the pith cells started to divide. This process was strongly inhibited by the elimination of cauline leaves, shoot apices, or lateral buds that reduced the indole-3-acetic acid supply. Microarray and quantitative RT-PCR analyses revealed that genes related to cell division, phytohormones, and transcription factors were expressed because of incision. Among them, two plant-specific transcription factor genes, ANAC071 and RAP2 . 6L , were abundantly expressed. ANAC071 was expressed at 1–3 d after cutting exclusively in the upper region of the cut gap, with concomitant accumulation of indole-3-acetic acid. In contrast, RAP2.6L was expressed at 1 d after cutting exclusively in the lower region, with concomitant deprivation of indole-3-acetic acid. The expression of ANAC071 and RAP2.6L were also promoted by ethylene and jasmonic acid, respectively. In transformants suppressing the function of RAP2.6L or ANAC071 , the division of pith cells was inhibited. Furthermore, the ethylene signaling-defective ein2 mutant showed incomplete healing. Hence, plant-specific transcription factors differentially expressed around the cut position were essential for tissue reunion of Arabidopsis wounded flowering stems and were under opposite control by polar-transported auxin, with modification by the ethylene and jasmonic acid wound-inducible hormones.

Germination and early seedling development are critical for successful stand establishment of plants. Following germination, the cotyledons, which are derived from embryonic tissue, emerge from the seed. Arabidopsis seedlings at post-germinative stages are supported mainly by the supply of nutrition from the cotyledons until vegetative leaves emerge and initiate photosynthesis. The switch to autotrophic growth is a significant transition at the post-germinative stage. Here, we provide evidence that down-regulation of SQUAMOSA PROMOTER-BINDING PROTEIN LIKE13 (SPL13) by microRNA156 (miR156) plays an important role in the regulation of the post-germinative switch from the cotyledon stage to the vegetative-leaf stage. Silent mutations created in the SPL13 sequence in the region that is complementary to the miR156 sequence caused the deregulation of the mutant form of SPL13 (mSPL13) mRNA from miR156. Mutant seedlings over-accumulated miRNA-resistant messages and exhibited a delay in the emergence of vegetative leaves compared to wild-type seedlings. The delay was not observed in control transgenic plants expressing non-mutated SPL13, indicating that the phenotype was caused specifically by the silent mutations and deregulation of SPL13 from miR156. Characterization of the SPL13 promoter indicated that this gene is expressed mainly in the hypocotyl and affects leaf primordium development. These results suggest that the repression of SPL13 by miR156 is essential for normal post-germinative growth in Arabidopsis.

MicroRNAs (miRNAs) are involved in developmental programmes of plants, including seed germination and post-germination. Here, we provide evidence that two different miRNA pathways, miR156 and miR172, interact during the post-germination stages inArabidopsis. Mutant seedlings expressing miR156-resistantSQUAMOSA PROMOTER-BINDING PROTEIN-LIKE13(mSPL13), which has silent mutations in the miR156 complementary sequence, over-accumulatedSPL13mRNA and exhibited a delay in seedling development. Microarray analysis indicated thatSCHNARCHZAPFEN(SNZ), anAP2-like gene targeted by miR172, was down-regulated in these mutants. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and miRNA gel blot analyses showed that theMIR172genes were up-regulated inmSPL13mutants. These results suggest that the miRNA regulation cascades (miR156⊣SPL13 → miR172⊣SNZ) play a critical role during the post-germination developmental stages inArabidopsis.

To elucidate the involvement of gibberellin (GA) in the growth regulation of Arabidopsis roots, effects of shoot-applied GA and GA-biosynthesis inhibitors on the root were examined. Applying GA to the shoot of Arabidopsis slightly enhanced the primary root elongation. Treating shoots with uniconazole, a GA biosynthesis inhibitor, also resulted in enhancement of primary root elongation, while shoots treated with uniconazole were stunted and bolting was delayed. Analysis of the expression of GA3ox and GA20ox confirmed the up-regulation of these genes in roots following the inhibitor application to shoots. The results suggest that the inhibition of GA-biosynthesis enhances the production of bioactive GAs in roots and promotes root elongation.

Glycine betaine (GB) is an important compatible solute that protects plants against the damaging effects of abiotic stresses. A number of plants have been engineered to contain genes of the GB biosynthetic pathway, which confers enhanced tolerance to a range of abiotic stresses during various plant developmental stages. Unlike natural accumulators, the transgenic plants accumulate very low GB concentrations, insignificant in terms of coping with osmotic stress. The GB accumulation in these transgenic plants varies depending upon their capacity for endogenous choline uptake, the type of gene that catalyzes the GB biosynthetic pathway, and the localization of the transgene product in a particular cellular compartment. This review focuses on recent progress in studies of abiotic stress tolerance conferred by GB in transgenic plants.

We have previously shown that gibberellin (GA) is required for tissue-reunion in the cortex of cut hypocotyls in cucumber (Cucumis sativus) and tomato (Solanum lycopersicum) seedlings, and that intact cotyledons are also necessary for this process. These results suggested that cotyledons might play an important role in controlling GA levels in the hypocotyl in these plant species. In this study, we found that a local application of a GA biosynthesis inhibitor uniconazole to cotyledons was effective to inhibit hypocotyl elongation, and that simultaneous application of GA canceled this inhibition. To study the role of cotyledons in GA content in the hypocotyl directly, cotyledons were removed from 7-days-old seedlings and endogenous GA levels in the hypocotyl were determined. Our results demonstrated that the cotyledon-removed seedlings contained lower levels of bioactive GAs and their precursors in the hypocotyls than did intact seedlings. Quantitative RT-PCR analysis indicated that transcript levels of LeGA20ox1 and LeGA3ox2 genes were elevated in the hypocotyl after the removal of cotyledons, suggesting that the reduced bioactive GA levels caused upregulation of these genes via the feedback regulation mechanism. Taken together, our results suggest that cotyledons are necessary for maintaining normal GA levels in young cucumber and tomato hypocotyls.

Cucumber (Cucumis sativus) hypocotyls were cut to one-half of their diameter transversely, and morphological and histochemical analyses of the process of tissue reunion in the cortex were performed. Cell division in the cortex commenced 3 d after cutting, and the cortex was nearly fully united within 7 d. 4′,6-Diamidino-2-phenylindole staining and 5-bromo-2′-deoxyuridine labeling experiments indicate that nDNA synthesis occurred during this process. In addition, specific accumulation of pectic substances was observed in the cell wall of attached cells in the reunion region of the cortex. Cell division during tissue reunion was strongly inhibited when the cotyledon was removed. This inhibition was reversed by applying gibberellin (GA, 10−4 mGA3) to the apical tip of the cotyledon-less plant. Supporting this observation, cell division in the cortex was inhibited by treatment of the cotyledon with 10−4 municonazole-P (an inhibitor of GA biosynthesis), and this inhibition was also reversed by simultaneous application of GA. In contrast to the essential role of cotyledon, normal tissue reunion in cut hypocotyls was still observed when the shoot apex was removed. The requirement of GA for tissue reunion in cut hypocotyls was also evident in the GA-deficient gib-1 mutant of tomato (Lycopersicon esculentum). Our results suggest that GA, possibly produced in cotyledons, is essential for cell division in reuniting cortex of cut hypocotyls.

Interactions among the functionally specialized organs of higher plants ensure that the plant body develops and functions properly in response to changing environmental conditions. When an incision or grafting procedure interrupts the original organ or tissue connection, cell division is induced and tissue reunion occurs to restore physiological connections. Such activities have long been observed in grafting techniques, which are advantageous not only for agriculture and horticulture but also for basic research. To understand how this healing process is controlled and how this process is initiated and regulated at the molecular level, physiological and molecular analyses of tissue reunion have been performed using incised hypocotyls of cucumber (Cucumis sativus) and tomato (Solanum lycopersicum) and incised flowering stems of Arabidopsis thaliana. Our results suggest that leaf gibberellin and microelements from the roots are required for tissue reunion in the cortex of the cucumber and tomato incised hypocotyls. In addition, the wound-inducible hormones ethylene and jasmonic acid contribute to the regulation of the tissue reunion process in the upper and lower parts, respectively, of incised Arabidopsis stems. Ethylene and jasmonic acid modulate the expression of ANAC071 and RAP2.6L, respectively, and auxin signaling via ARF6/8 is essential for the expression of these transcription factors. In this report, we discuss recent findings regarding molecular and physiological mechanisms of the graft union and the tissue reunion process in wounded tissues of plants.

One week after partial incision of Arabidopsis inflorescence stems, the repair process in damaged tissue includes pith cell proliferation. Auxin is a key factor driving this process, and ANAC071, a transcription factor gene, is upregulated in the distal region of the incised stem. Here we show that XTH20 and the closely related XTH19, members of xyloglucan endotransglucosylase/hydrolases family catalyzing molecular grafting and/or hydrolysis of cell wall xyloglucans, were also upregulated in the distal part of the incised stem, similar to ANAC071. XTH19 was expressed in the proximal incision region after 3 days or after auxin application to the decapitated stem. Horizontal positioning of the plant with the incised side up resulted in decreased ProDR 5 :GUS, ANAC071, XTH20, and XTH19 expression and reduced pith cell proliferation. In incised stems of Pro35S :ANAC071-SRDX plants, expression of XTH20 and XTH19 was substantially and moderately decreased, respectively. XTH20 and XTH19 expression and pith cell proliferation were suppressed in anac071 plants and were increased in Pro35S :ANAC071 plants. Pith cell proliferation was also inhibited in the xth20xth19 double mutant. Furthermore, ANAC071 bound to the XTH20 and XTH19 promoters to induce their expression. This study revealed XTH20 and XTH19 induction by auxin via ANAC071 in the distal part of an incised stem and their involvement in cell proliferation in the tissue reunion process.

Totipotency is the ability of a cell to regenerate the entire organism, even after previous differentiation as a specific cell. When totipotency is coupled with active cell division, it was presumed that cell division is essential for this expression. Here, using the stress-induction system of somatic embryos in carrots, we show that cell division is not essential for the expression of totipotency in somatic/embryonic conversion. Morphological and histochemical analyses showed that the cell did not divide during embryo induction. Inhibitors of cell division did not affect the rate of somatic embryo formation. Our results indicate that the newly acquired trait of differentiation appears without cell division, but does not arise with cell division as a newborn cell.

The micropylar region of endosperm in a seed, which is adjacent to the radicle tip, is called the 'endosperm cap', and is specifically activated before radicle emergence. This activation of the endosperm cap is a widespread phenomenon among species and is a prerequisite for the completion of germination. To understand the mechanisms of endosperm cap-specific gene expression in tomato seeds, GeneChip analysis was performed. The major groups of endosperm cap-enriched genes were pathogenesis-, cell wall-, and hormone-associated genes. The promoter regions of endosperm cap-enriched genes contained DNA motifs recognized by ethylene response factors (ERFs). The tomato ERF1 (TERF1) and its experimentally verified targets were enriched in the endosperm cap, suggesting an involvement of the ethylene response cascade in this process. The known endosperm cap enzyme endo-β-mannanase is induced by gibberellin (GA), which is thought to be the major hormone inducing endosperm cap-specific genes. The mechanism of endo-β-mannanase induction by GA was also investigated using isolated, embryoless seeds. Results suggested that GA might act indirectly on the endosperm cap. We propose that endosperm cap activation is caused by the ethylene response of this tissue, as a consequence of mechanosensing of the increase in embryonic growth potential by GA action.

Cucumber (Cucumis sativus L.) hypocotyls were transversely cut to half their diameter, and morphological analyses of the tissue-reunion process in the cortex were conducted to elucidate the involvement of root-derived factors. Cell division in the cortex commenced 3 days after cutting, and the cortex was nearly fully united within 7 days. In shoots from which the roots were removed and which were cultured in water, cell division occurred during tissue reunion; however, thick-wall layer formed in the reunion region, and intrusive cell elongation and interdigitation of cortex cells at the cut surface did not occur, even after 7 days. Interdigitation of cells, followed by normal tissue reunion, was observed in shoots from which the roots were removed and which were cultured in squash xylem sap or Murashige and Skoog (MS) medium. The same effect was observed with the simultaneous application of B, Mn, and Zn, which are the major inorganic microelements of MS medium. Our results suggest that application of these inorganic elements, which are taken up from the soil and transferred to the xylem sap, are required for interdigitation of cells during tissue reunion in the cortex of cucumber hypocotyls, possibly because they are required for cell wall function and metabolism.