Autonomous differentiation of transgenic cells requiring no external hormone application: the endogenous gene expression and phytohormone behaviors.

The ectopic overexpression of developmental regulator (DR) genes has been reported to improve the transformation in recalcitrant plant species because of the promotion of cellular differentiation during cell culture processes. In other words, the external plant growth regulator (PGR) application during the tissue and cell culture process is still required in cases utilizing DR genes for plant regeneration. Here, the effect of Arabidopsis BABY BOOM (BBM) and WUSCHEL (WUS) on the differentiation of tobacco transgenic cells was examined. We found that the SRDX fusion to WUS, when co-expressed with the BBM-VP16 fusion gene, significantly influenced the induction of autonomous differentiation under PGR-free culture conditions, with similar effects in some other plant species. Furthermore, to understand the endogenous background underlying cell differentiation toward regeneration, phytohormone and RNA-seq analyses were performed using tobacco leaf explants in which transgenic cells were autonomously differentiating. The levels of active auxins, cytokinins, abscisic acid, and inactive gibberellins increased as cell differentiation proceeded toward organogenesis. Gene Ontology terms related to phytohormones and organogenesis were identified as differentially expressed genes, in addition to those related to polysaccharide and nitrate metabolism. The qRT-PCR four selected genes as DEGs supported the RNA-seq data. This differentiation induction system and the reported phytohormone and transcript profiles provide a foundation for the development of PGR-free tissue cultures of various plant species, facilitating future biotechnological breeding.

Plant hormone profiling of scion and rootstock incision sites and intra- and inter-family graft junctions in Nicotiana benthamiana.

Many previous studies have suggested that various plant hormones play essential roles in the grafting process. In this study, to understand the plant hormones that accumulate in the graft junctions, whether these are supplied from the scion or rootstock, and how these hormones play a role in the grafting process, we performed a hormonome analysis that accumulated in the incision site of the upper plants from the incision as "ungrafted scion" and lower plants from the incision as "ungrafted rootstock" in Nicotiana benthamiana. The results revealed that indole-3-acetic acid (IAA) and gibberellic acid (GA), which regulate cell division; abscisic acid (ABA) and jasmonic acid (JA), which regulate xylem formation; cytokinin (CK), which regulates callus formation, show different accumulation patterns in the incision sites of the ungrafted scion and rootstock. In addition, to try discussing the differences in the degree and speed of each event during the grafting process between intra- and inter-family grafting by determining the concentration and accumulation timing of plant hormones in the graft junctions, we performed hormonome analysis of graft junctions of intra-family grafted plants with N. benthamiana as scion and Solanum lycopersicum as rootstock (Nb/Sl) and inter-family grafted plants with N. benthamiana as scion and Arabidopsis thaliana as rootstock (Nb/At), using the ability of Nicotiana species to graft with many plant species. The results revealed that ABA and CK showed different accumulation timings; IAA, JA, and salicylic acid (SA) showed similar accumulation timings, while different accumulated concentrations in the graft junctions of Nb/Sl and Nb/At. This information is important for understanding the molecular mechanisms of plant hormones in the grafting process and the differences in molecular mechanisms between intra- and inter-family grafting.

Large-scale single-cell profiling of stem cells uncovers redundant regulators of shoot development and yield trait variation.

Stem cells in plant shoots are a rare population of cells that produce leaves, fruits and seeds, vital sources for food and bioethanol. Uncovering regulators expressed in these stem cells will inform crop engineering to boost productivity. Single-cell analysis is a powerful tool for identifying regulators expressed in specific groups of cells. However, accessing plant shoot stem cells is challenging. Recent single-cell analyses of plant shoots have not captured these cells, and failed to detect stem cell regulators like CLAVATA3 and WUSCHEL . In this study, we finely dissected stem cell-enriched shoot tissues from both maize and arabidopsis for single-cell RNA-seq profiling. We optimized protocols to efficiently recover thousands of CLAVATA3 and WUSCHEL expressed cells. A cross-species comparison identified conserved stem cell regulators between maize and arabidopsis. We also performed single-cell RNA-seq on maize stem cell overproliferation mutants to find additional candidate regulators. Expression of candidate stem cell genes was validated using spatial transcriptomics, and we functionally confirmed roles in shoot development. These candidates include a family of ribosome-associated RNA-binding proteins, and two families of sugar kinase genes related to hypoxia signaling and cytokinin hormone homeostasis. These large-scale single-cell profiling of stem cells provide a resource for mining stem cell regulators, which show significant association with yield traits. Overall, our discoveries advance the understanding of shoot development and open avenues for manipulating diverse crops to enhance food and energy security.

Phosphate starvation response precedes abscisic acid response under progressive mild drought in plants.

Drought severely damages crop production, even under conditions so mild that the leaves show no signs of wilting. However, it is unclear how field-grown plants respond to mild drought. Here, we show through six years of field trials that ridges are a useful experimental tool to mimic mild drought stress in the field. Mild drought reduces inorganic phosphate levels in the leaves to activate the phosphate starvation response (PSR) in soybean plants in the field. Using Arabidopsis thaliana and its mutant plants grown in pots under controlled environments, we demonstrate that PSR occurs before abscisic acid response under progressive mild drought and that PSR plays a crucial role in plant growth under mild drought. Our observations in the field and laboratory using model crop and experimental plants provide insight into the molecular response to mild drought in field-grown plants and the relationship between nutrition and drought stress response.

A cell wall-localized cytokinin/purine riboside nucleosidase is involved in apoplastic cytokinin metabolism in Oryza sativa.

In the final step of cytokinin biosynthesis, the main pathway is the elimination of a ribose-phosphate moiety from the cytokinin nucleotide precursor by phosphoribohydrolase, an enzyme encoded by a gene named LONELY GUY (LOG). This reaction accounts for most of the cytokinin supply needed for regulating plant growth and development. In contrast, the LOG-independent pathway, in which dephosphorylation and deribosylation sequentially occur, is also thought to play a role in cytokinin biosynthesis, but the gene entity and physiological contribution have been elusive. In this study, we profiled the phytohormone content of chromosome segment substitution lines of Oryza sativa and searched for genes affecting the endogenous levels of cytokinin ribosides by quantitative trait loci analysis. Our approach identified a gene encoding an enzyme that catalyzes the deribosylation of cytokinin nucleoside precursors and other purine nucleosides. The cytokinin/purine riboside nucleosidase 1 (CPN1) we identified is a cell wall-localized protein. Loss-of-function mutations (cpn1) were created by inserting a Tos17-retrotransposon that altered the cytokinin composition in seedling shoots and leaf apoplastic fluid. The cpn1 mutation also abolished cytokinin riboside nucleosidase activity in leaf extracts and attenuated the trans-zeatin riboside-responsive expression of cytokinin marker genes. Grain yield of the mutants declined due to altered panicle morphology under field-grown conditions. These results suggest that the cell wall-localized LOG-independent cytokinin activating pathway catalyzed by CPN1 plays a role in cytokinin control of rice growth. Our finding broadens our spatial perspective of the cytokinin metabolic system.

Identification of a pluripotency-inducing small compound, PLU, that induces callus formation via Heat Shock Protein 90-mediated activation of auxin signaling.

Plants retain the ability to generate a pluripotent tissue called callus by dedifferentiating somatic cells. A pluripotent callus can also be artificially induced by culturing explants with hormone mixtures of auxin and cytokinin, and an entire body can then be regenerated from the callus. Here we identified a pluripotency-inducing small compound, PLU, that induces the formation of callus with tissue regeneration potency without the external application of either auxin or cytokinin. The PLU-induced callus expressed several marker genes related to pluripotency acquisition via lateral root initiation processes. PLU-induced callus formation required activation of the auxin signaling pathway though the amount of active auxin was reduced by PLU treatment. RNA-seq analysis and subsequent experiments revealed that Heat Shock Protein 90 (HSP90) mediates a significant part of the PLU-initiated early events. We also showed that HSP90-dependent induction of TRANSPORT INHIBITOR RESPONSE 1, an auxin receptor gene, is required for the callus formation by PLU. Collectively, this study provides a new tool for manipulating and investigating the induction of plant pluripotency from a different angle from the conventional method with the external application of hormone mixtures.

The trans-zeatin-type side-chain modification of cytokinins controls rice growth.

Cytokinins (CKs), a class of phytohormones with vital roles in growth and development, occur naturally with various side-chain structures, including N6-(Δ2-isopentenyl)adenine-, cis-zeatin- and trans-zeatin (tZ)-types. Recent studies in the model dicot plant Arabidopsis (Arabidopsis thaliana) have demonstrated that tZ-type CKs are biosynthesized via cytochrome P450 monooxygenase (P450) CYP735A and have a specific function in shoot growth promotion. Although the function of some of these CKs has been demonstrated in a few dicotyledonous plant species, the importance of these variations and their biosynthetic mechanism and function in monocots and in plants with distinctive side-chain profiles other than Arabidopsis, such as rice (Oryza sativa), remain elusive. In this study, we characterized CYP735A3 and CYP735A4 to investigate the role of tZ-type CKs in rice. Complementation test of the Arabidopsis CYP735A-deficient mutant and CK profiling of loss-of-function rice mutant cyp735a3 cyp735a4 demonstrated that CYP735A3 and CYP735A4 encode P450s required for tZ-type side-chain modification in rice. CYP735As are expressed in both roots and shoots. The cyp735a3 cyp735a4 mutants exhibited growth retardation concomitant with reduction in CK activity in both roots and shoots, indicating that tZ-type CKs function in growth promotion of both organs. Expression analysis revealed that tZ-type CK biosynthesis is negatively regulated by auxin, abscisic acid, and CK and positively by dual nitrogen nutrient signals, namely glutamine-related and nitrate-specific signals. These results suggest that tZ-type CKs control the growth of both roots and shoots in response to internal and environmental cues in rice.

Comprehensive analyses of plant hormones in etiolated pea and maize seedlings grown under microgravity conditions in space: Relevance to the International Space Station experiment "Auxin Transport".

Functional relationships between endogenous levels of plant hormones in the growth and development of shoots in etiolated Alaska pea and etiolated Golden Cross Bantam maize seedlings under different gravities were investigated in the "Auxin Transport" experiment aboard the International Space Station (ISS). Comprehensive analyses of 31 species of plant hormones of pea and maize seedlings grown under microgravity (µg) in space and 1 g conditions were conducted. Principal component analysis (PCA) and a multiple regression analysis with the dataset from the plant hormone analysis of the etiolated pea seedlings grown under µg and 1 g conditions in the presence and absence of 2,3,5-triiodobenzoic acid (TIBA) revealed endogenous levels of auxin correlated positively with bending and length of epicotyls. Endogenous cytokinins correlated negatively with them. These results suggest an interaction of auxin and cytokinins in automorphogenesis and growth inhibition of etiolated Alaska pea epicotyls grown under µg conditions in space. Less polar auxin transport with reduced endogenous levels of auxin increased endogenous levels of cytokinins, resulting in changing the growth direction of epicotyls and inhibiting growth. On the other hand, almost no close relationship between endogenous plant hormone levels and growth and development in etiolated maize seedlings grown was observed under µg conditions in space, as per Schulze et al. (1992). However, endogenous levels of IAA in the seedlings grown under µg conditions in space were significantly higher than those grown on Earth, similar to the cases of polar auxin transport already reported.

Plant Hormone and Inorganic Ion Concentrations in the Xylem Exudate of Grafted Plants Depend on the Scion-Rootstock Combination.

In grafted plants, inorganic ions and plant hormones in the xylem exudate transported from the rootstock to the scion directly or indirectly affect the scion, thereby improving the traits. Therefore, the concentration of these components in the xylem exudate of grafted plants may be an indicator for rootstock selection. On the other hand, few reports have presented a comprehensive analysis of substances transferred from the rootstock to the scion in plants grafted onto different rootstocks, primarily commercial cultivars. In this study, we measured inorganic ions and plant hormones in the xylem exudate from the rootstock to the scion in various grafted plants of tomato and eggplant. The results revealed that the concentrations of inorganic ions and plant hormones in the xylem exudate significantly differed depending on the type of rootstock. In addition, we confirmed the concentration of the inorganic ions and plant hormones in the xylem exudate of plants grafted onto the same tomato rootstock cultivars as rootstock with tomato or eggplant as the scions. As a result, the concentrations of inorganic ions and plant hormones in the xylem exudate were significantly different in the grafted plants with eggplant compared with tomato as the scion. These results suggest that signals from the scion (shoot) control the inorganic ions and plant hormones transported from the rootstock (root).

ABCG11 modulates cytokinin responses in Arabidopsis thaliana.

The Arabidopsis ABC transporter ABCG11 transports lipidic precursors of surface coating polymers at the plasma membrane of epidermal cells. Mutants in ABCG11 exhibit severe developmental defects, suggesting that ABCG11 might also participate in phytohormone-mediated development. Here, we report that ABCG11 is involved in cytokinin-mediated development. The roots of abcg11 mutant seedlings failed to respond to cytokinins and accumulated more cytokinins than wild-type roots. When grown under short-day conditions, abcg11 exhibited longer roots and shorter hypocotyls compared to wild type, similar to abcg14, a knockout mutant in a cytokinin transporter. Treatment with exogenous trans-zeatin, which inhibits primary root elongation in the wild type, enhanced abcg11 primary root elongation. It also increased the expression of cytokinin-responsive Arabidopsis response regulator (ARR) genes, and the signal of the TCS::GFP reporter in abcg11 roots compared to wild-type roots, suggesting that cytokinin signaling was enhanced in abcg11 roots. When we treated only the roots of abcg11 with trans-zeatin, their shoots showed lower ARR induction than the wild type. The abcg14 abcg11 double mutant did not have additional root phenotypes compared to abcg11. Together, these results suggest that ABCG11 is necessary for normal cytokinin-mediated root development, likely because it contributes to cytokinin transport, either directly or indirectly.

Transcriptomic, Hormonomic and Metabolomic Analyses Highlighted the Common Modules Related to Photosynthesis, Sugar Metabolism and Cell Division in Parthenocarpic Tomato Fruits during Early Fruit Set.

Parthenocarpy, the pollination-independent fruit set, can raise the productivity of the fruit set even under adverse factors during the reproductive phase. The application of plant hormones stimulates parthenocarpy, but artificial hormones incur extra financial and labour costs to farmers and can induce the formation of deformed fruit. This study examines the performance of parthenocarpic mutants having no transcription factors of SlIAA9 and SlTAP3 and sldella that do not have the protein-coding gene, SlDELLA, in tomato (cv. Micro-Tom). At 0 day after the flowering (DAF) stage and DAFs after pollination, the sliaa9 mutant demonstrated increased pistil development compared to the other two mutants and wild type (WT). In contrast to WT and the other mutants, the sliaa9 mutant with pollination efficiently stimulated the build-up of auxin and GAs after flowering. Alterations in both transcript and metabolite profiles existed for WT with and without pollination, while the three mutants without pollination demonstrated the comparable metabolomic status of pollinated WT. Network analysis showed key modules linked to photosynthesis, sugar metabolism and cell proliferation. Equivalent modules were noticed in the famous parthenocarpic cultivars 'Severianin', particularly for emasculated samples. Our discovery indicates that controlling the genes and metabolites proffers future breeding policies for tomatoes.

Cytosolic Glutamine Synthetase GS1;3 Is Involved in Rice Grain Ripening and Germination.

Ammonium is combined with glutamate to form glutamine. This reaction is catalyzed by glutamine synthetase (GS or GLN). Plants harbor several isoforms of cytosolic GS (GS1). Rice GS1;3 is highly expressed in seeds during grain filling and germination, suggesting a unique role in these processes. This study aimed to investigate the role of GS1;3 for rice growth and yield. Tos17 insertion lines for GS1;3 were isolated, and the nitrogen (N), amino acid, and ammonium contents of GS1;3 mutant grains were compared to wild-type grains. The spatiotemporal expression of GS1;3 and the growth and yield of rice plants were evaluated in hydroponic culture and the paddy field. Additionally, the stable isotope of N was used to trace the foliar N flux during grain filling. Results showed that the loss of GS1;3 retarded seed germination. Seeds of GS1;3 mutants accumulated glutamate but did not show a marked change in the level of phytohormones. The expression of GS1;3 was detected at the beginning of germination, with limited promoter activity in seeds. GS1;3 mutants showed a considerably decreased ripening ratio and decreased N efflux in the 12th leaf blade under N deficient conditions. The ß-glucuronidase gene expression under control of the GS1;3 promoter was detected in the vascular tissue and aleurone cell layer of developing grains. These data suggest unique physiological roles of GS1;3 in the early stage of seed germination and grain filling under N deficient conditions in rice.

Regulation of ammonium acquisition and use in Oryza longistaminata ramets under nitrogen source heterogeneity.

Oryza longistaminata, a wild rice, vegetatively reproduces and forms a networked clonal colony consisting of ramets connected by rhizomes. Although water, nutrients, and other molecules can be transferred between ramets via the rhizomes, inter-ramet communication in response to spatially heterogeneous nitrogen availability is not well understood. We studied the response of ramet pairs to heterogeneous nitrogen availability using a split hydroponic system that allowed each ramet root to be exposed to different conditions. Ammonium uptake was compensatively enhanced in the sufficient-side root when roots of the ramet pairs were exposed to ammonium-sufficient and ammonium-deficient conditions. Comparative transcriptome analysis revealed that a gene regulatory network for effective ammonium assimilation and amino acid biosynthesis was activated in the sufficient-side roots. Allocation of absorbed nitrogen from the nitrogen-sufficient to the nitrogen-deficient ramets was rather limited. Nitrogen was preferentially used for newly growing axillary buds on the sufficient-side ramets. Biosynthesis of trans-zeatin (tZ), a cytokinin, was upregulated in response to the nitrogen supply, but tZ appeared not to target the compensatory regulation. Our results also implied that the O. longistaminata putative ortholog of rice (Oryza sativa) C-terminally encoded peptide1 plays a role as a nitrogen-deficient signal in inter-ramet communication, providing compensatory upregulation of nitrogen assimilatory genes. These results provide insights into the molecular basis for efficient growth strategies of asexually proliferating plants growing in areas where the distribution of ammonium ions is spatially heterogeneous.

Root-specific Reduction of Cytokinin Perception Enhances Shoot Growth in Arabidopsis thaliana.

Previous studies suggest that root-derived cytokinins (CKs) contribute to shoot growth via long-distance transport; therefore, we hypothesized that an increase in root-derived CKs enhances shoot growth. To verify this, we grafted Arabidopsis Col-0 (wild type, WT) scion onto rootstock originated from WT or a double-knockout mutant of CK receptors Arabidopsis histidine kinase 2 (AHK2) and AHK3 (ahk2-5 ahk3-7; ahk23) because this mutant overaccumulates CKs in the body probably due to feedback homeostasis regulation. The grafted plants (scion/rootstock: WT/WT and WT/ahk23) were grown in vermiculite pots or solid media for vegetative growth and biochemical analysis. The root-specific deficiency of AHK2 and AHK3 increased root concentrations of trans-zeatin (tZ)-type and N6-(Δ2-isopentenyl) adenine (iP)-type CKs, induced CK biosynthesis genes and repressed CK degradation genes in the root. The WT/ahk23 plants had significantly larger shoot weight, rosette diameter and leaves area than did the WT/WT plants. Shoot concentrations of tZ-type CKs showed increasing trends in the WT/ahk23 plants. Moreover, the root-specific deficiency of AHK2 and AHK3 enhanced shoot growth in the WT scion more strongly than in the ahk23 scion, suggesting that shoot growth enhancement could occur through increased shoot perception of CKs. In the WT/ahk23 shoots compared with the WT/WT shoots, however, induction of most of CK-inducible response regulator genes was not statistically significant. Thus we suggest that the root-specific reduction of CK perception enhances shoot growth only partly by increasing the amount of root-derived tZ-type CKs and their perception by shoots. The unknown mechanism(s) distinct from CK signaling would also be involved in the shoot growth enhancement.

Evolutionary alterations in gene expression and enzymatic activities of gibberellin 3-oxidase 1 in Oryza.

Proper anther and pollen development are important for plant reproduction. The plant hormone gibberellin is important for anther development in rice, but its gametophytic functions remain largely unknown. Here, we report the functional and evolutionary analyses of rice gibberellin 3-oxidase 1 (OsGA3ox1), a gibberellin synthetic enzyme specifically expressed in the late developmental stages of anthers. Enzymatic and X-ray crystallography analyses reveal that OsGA3ox1 has a higher GA7 synthesis ratio than OsGA3ox2. In addition, we generate an osga3ox1 knockout mutant by genome editing and demonstrate the bioactive gibberellic acid synthesis by the OsGA3ox1 action during starch accumulation in pollen via invertase regulation. Furthermore, we analyze the evolution of Oryza GA3ox1s and reveal that their enzyme activity and gene expression have evolved in a way that is characteristic of the Oryza genus and contribute to their male reproduction ability.

Diminished Auxin Signaling Triggers Cellular Reprogramming by Inducing a Regeneration Factor in the Liverwort Marchantia polymorpha.

Regeneration in land plants is accompanied by the establishment of new stem cells, which often involves reactivation of the cell division potential in differentiated cells. The phytohormone auxin plays pivotal roles in this process. In bryophytes, regeneration is enhanced by the removal of the apex and repressed by exogenously applied auxin, which has long been proposed as a form of apical dominance. However, the molecular basis behind these observations remains unexplored. Here, we demonstrate that in the liverwort Marchantia polymorpha, the level of endogenous auxin is transiently decreased in the cut surface of decapitated explants, and identify by transcriptome analysis a key transcription factor gene, LOW-AUXIN RESPONSIVE (MpLAXR), which is induced upon auxin reduction. Loss of MpLAXR function resulted in delayed cell cycle reactivation, and transient expression of MpLAXR was sufficient to overcome the inhibition of regeneration by exogenously applied auxin. Furthermore, ectopic expression of MpLAXR caused cell proliferation in normally quiescent tissues. Together, these data indicate that decapitation causes a reduction of auxin level at the cut surface, where, in response, MpLAXR is up-regulated to trigger cellular reprogramming. MpLAXR is an ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION 1/DORNRÖSCHEN, which has dual functions as a shoot regeneration factor and a regulator of axillary meristem initiation, the latter of which requires a low auxin level. Thus, our findings provide insights into stem cell regulation as well as apical dominance establishment in land plants.

Integrative omics approaches revealed a crosstalk among phytohormones during tuberous root development in cassava.

KEY MESSAGE: Integrative omics approaches revealed a crosstalk among phytohormones during tuberous root development in cassava. Tuberous root formation is a complex process consisting of phase changes as well as cell division and elongation for radial growth. We performed an integrated analysis to clarify the relationships among metabolites, phytohormones, and gene transcription during tuberous root formation in cassava (Manihot esculenta Crantz). We also confirmed the effects of the auxin (AUX), cytokinin (CK), abscisic acid (ABA), jasmonic acid (JA), gibberellin (GA), brassinosteroid (BR), salicylic acid, and indole-3-acetic acid conjugated with aspartic acid on tuberous root development. An integrated analysis of metabolites and gene expression indicated the expression levels of several genes encoding enzymes involved in starch biosynthesis and sucrose metabolism are up-regulated during tuberous root development, which is consistent with the accumulation of starch, sugar phosphates, and nucleotides. An integrated analysis of phytohormones and gene transcripts revealed a relationship among AUX signaling, CK signaling, and BR signaling, with AUX, CK, and BR inducing tuberous root development. In contrast, ABA and JA inhibited tuberous root development. These phenomena might represent the differences between stem tubers (e.g., potato) and root tubers (e.g., cassava). On the basis of these results, a phytohormonal regulatory model for tuberous root development was constructed. This model may be useful for future phytohormonal studies involving cassava.

Identification of the unique molecular framework of heterophylly in the amphibious plant Callitriche palustris L.

Heterophylly is the development of different leaf forms in a single plant depending on the environmental conditions. It is often observed in amphibious aquatic plants that can grow under both aerial and submerged conditions. Although heterophylly is well recognized in aquatic plants, the associated developmental mechanisms and the molecular basis remain unclear. To clarify these underlying developmental and molecular mechanisms, we analyzed heterophyllous leaf formation in an aquatic plant, Callitriche palustris. Morphological analyses revealed extensive cell elongation and the rearrangement of cortical microtubules in the elongated submerged leaves of C. palustris. Our observations also suggested that gibberellin, ethylene, and abscisic acid all regulate the formation of submerged leaves. However, the perturbation of one or more of the hormones was insufficient to induce the formation of submerged leaves under aerial conditions. Finally, we analyzed gene expression changes during aerial and submerged leaf development and narrowed down the candidate genes controlling heterophylly via transcriptomic comparisons, including a comparison with a closely related terrestrial species. We discovered that the molecular mechanism regulating heterophylly in C. palustris is associated with hormonal changes and diverse transcription factor gene expression profiles, suggesting differences from the corresponding mechanisms in previously investigated amphibious plants.

Nitrogen Nutrition Promotes Rhizome Bud Outgrowth via Regulation of Cytokinin Biosynthesis Genes and an Oryza longistaminata Ortholog of FINE CULM 1.

Oryza longistaminata, a wild rice, can propagate vegetatively via rhizome formation and, thereby, expand its territory through horizontal growth of branched rhizomes. The structural features of rhizomes are similar to those of aerial stems; however, the physiological roles of the two organs are different. Nitrogen nutrition is presumed to be linked to the vegetative propagation activity of rhizomes, but the regulation of rhizome growth in response to nitrogen nutrition and the underlying biological processes have not been well characterized. In this study, we analyzed rhizome axillary bud growth in response to nitrogen nutrition and examined the involvement of cytokinin-mediated regulation in the promotion of bud outgrowth in O. longistaminata. Our results showed that nitrogen nutrition sufficiency promoted rhizome bud outgrowth to form secondary rhizomes. In early stages of the response to nitrogen application, glutamine accumulated rapidly, two cytokinin biosynthesis genes, isopentenyltransferase, and CYP735A, were up-regulated with accompanying cytokinin accumulation, and expression of an ortholog of FINE CULM1, a negative regulator of axillary bud outgrowth, was severely repressed in rhizomes. These results suggest that, despite differences in physiological roles of these organs, the nitrogen-dependent outgrowth of rhizome axillary buds in O. longistaminata is regulated by a mechanism similar to that of shoot axillary buds in O. sativa. Our findings provide a clue for understanding how branched rhizome growth is regulated to enhance nutrient acquisition strategies.

Acetic-acid-induced jasmonate signaling in root enhances drought avoidance in rice.

Conferring drought resistant traits to crops is one of the major aims of current breeding programs in response to global climate changes. We previously showed that exogenous application of acetic acid to roots of various plants could induce increased survivability under subsequent drought stress conditions, but details of the metabolism of exogenously applied acetic acid, and the nature of signals induced by its application, have not been unveiled. In this study, we show that rice rapidly induces jasmonate signaling upon application of acetic acid, resulting in physiological changes similar to those seen under drought. The major metabolite of the exogenously applied acetic acid in xylem sap was determined as glutamine-a common and abundant component of xylem sap-indicating that acetic acid is not the direct agent inducing the observed physiological responses in shoots. Expression of drought-responsive genes in shoot under subsequent drought conditions was attenuated by acetic acid treatment. These data suggest that acetic acid activates root-to-shoot jasmonate signals that partially overlap with those induced by drought, thereby conferring an acclimated state on shoots prior to subsequent drought.