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.

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.

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.

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.

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.

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.

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.

Identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene (crtE) and phytoene synthase gene (crtB) for carotenoid biosynthesis in Euglena gracilis.

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 ß-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.

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.

CLE6 expression recovers gibberellin deficiency to promote shoot growth in Arabidopsis.

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.

XTH20 and XTH19 regulated by ANAC071 under auxin flow are involved in cell proliferation in incised Arabidopsis inflorescence stems.

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.

Molecular and physiological mechanisms regulating tissue reunion in incised plant tissues.

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.

Gibberellin-induced expression of Fe uptake-related genes 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.

Spatially selective hormonal control of RAP2.6L and ANAC071 transcription factors involved in tissue reunion in Arabidopsis.

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.

Mechanisms of hormonal regulation of endosperm cap-specific gene expression in tomato seeds.

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-ß-mannanase is induced by gibberellin (GA), which is thought to be the major hormone inducing endosperm cap-specific genes. The mechanism of endo-ß-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.

Acquisition of embryogenic competency does not require cell division in carrot somatic cell.

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.