Antagonistic regulation of the gibberellic acid response during stem growth in rice.

The size of plants is largely determined by growth of the stem. Stem elongation is stimulated by gibberellic acid1-3. Here we show that internode stem elongation in rice is regulated antagonistically by an 'accelerator' and a 'decelerator' in concert with gibberellic acid. Expression of a gene we name ACCELERATOR OF INTERNODE ELONGATION 1 (ACE1), which encodes a protein of unknown function, confers cells of the intercalary meristematic region with the competence for cell division, leading to internode elongation in the presence of gibberellic acid. By contrast, upregulation of DECELERATOR OF INTERNODE ELONGATION 1 (DEC1), which encodes a zinc-finger transcription factor, suppresses internode elongation, whereas downregulation of DEC1 allows internode elongation. We also show that the mechanism of internode elongation that is mediated by ACE1 and DEC1 is conserved in the Gramineae family. Furthermore, an analysis of genetic diversity suggests that mutations in ACE1 and DEC1 have historically contributed to the selection of shorter plants in domesticated populations of rice to increase their resistance to lodging, and of taller plants in wild species of rice for adaptation to growth in deep water. Our identification of these antagonistic regulatory factors enhances our understanding of the gibberellic acid response as an additional mechanism that regulates internode elongation and environmental fitness, beyond biosynthesis and gibberellic acid signal transduction.

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.

The endophytic fungus Serendipita indica affects auxin distribution in Arabidopsis thaliana roots through alteration of auxin transport and conjugation to promote plant growth.

Plants share their habitats with a multitude of different microbes. This close vicinity promoted the evolution of interorganismic interactions between plants and many different microorganisms that provide mutual growth benefits both to the plant and the microbial partner. The symbiosis of Arabidopsis thaliana with the beneficial root colonizing endophyte Serendipita indica represents a well-studied system. Colonization of Arabidopsis roots with S. indica promotes plant growth and stress tolerance of the host plant. However, until now, the molecular mechanism by which S. indica reprograms plant growth remains largely unknown. This study used comprehensive transcriptomics, metabolomics, reverse genetics, and life cell imaging to reveal the intricacies of auxin-related processes that affect root growth in the symbiosis between A. thaliana and S. indica. Our experiments revealed the sustained stimulation of auxin signalling in fungus infected Arabidopsis roots and disclosed the essential role of tightly controlled auxin conjugation in the plant-fungus interaction. It particularly highlighted the importance of two GRETCHEN HAGEN 3 (GH3) genes, GH3.5 and GH3.17, for the fungus infection-triggered stimulation of biomass production, thus broadening our knowledge about the function of GH3s in plants. Furthermore, we provide evidence for the transcriptional alteration of the PIN2 auxin transporter gene in roots of Arabidopsis seedlings infected with S. indica and demonstrate that this transcriptional adjustment affects auxin signalling in roots, which results in increased plant growth.

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.

Genetic Interaction between Arabidopsis SUR2/CYP83B1 and GNOM Indicates the Importance of Stabilizing Local Auxin Accumulation in Lateral Root Initiation.

Lateral root (LR) formation is an important developmental event for the establishment of the root system in most vascular plants. In Arabidopsis thaliana, the fewer roots (fwr) mutation in the GNOM gene, encoding a guanine nucleotide exchange factor of ADP ribosylation factor that regulates vesicle trafficking, severely inhibits LR formation. Local accumulation of auxin response for LR initiation is severely affected in fwr. To better understand how local accumulation of auxin response for LR initiation is regulated, we identified a mutation, fewer roots suppressor1 (fsp1), that partially restores LR formation in fwr. The gene responsible for fsp1 was identified as SUPERROOT2 (SUR2), encoding CYP83B1 that positions at the metabolic branch point in the biosynthesis of auxin/indole-3-acetic acid (IAA) and indole glucosinolate. The fsp1 mutation increases both endogenous IAA levels and the number of the sites where auxin response locally accumulates prior to LR formation in fwr. SUR2 is expressed in the pericycle of the differentiation zone and in the apical meristem in roots. Time-lapse imaging of the auxin response revealed that local accumulation of auxin response is more stable in fsp1. These results suggest that SUR2/CYP83B1 affects LR founder cell formation at the xylem pole pericycle cells where auxin accumulates. Analysis of the genetic interaction between SUR2 and GNOM indicates the importance of stabilization of local auxin accumulation sites for LR initiation.

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.

Endogenous gibberellins affect root nodule symbiosis via transcriptional regulation of NODULE INCEPTION in Lotus japonicus.

Legumes and nitrogen-fixing rhizobial bacteria establish root nodule symbiosis, which is orchestrated by several plant hormones. Exogenous addition of biologically active gibberellic acid (GA) is known to inhibit root nodule symbiosis. However, the precise role of GA has not been elucidated because of the trace amounts of these hormones in plants and the multiple functions of GAs. Here, we found that GA signaling acts as a key regulator in a long-distance negative-feedback system of root nodule symbiosis called autoregulation of nodulation (AON). GA biosynthesis is activated during nodule formation in and around the nodule vascular bundles, and bioactive GAs accumulate in the nodule. In addition, GA signaling induces expression of the symbiotic transcription factor NODULE INCEPTION (NIN) via a cis-acting region on the NIN promoter. Mutants with deletions of this cis-acting region have increased susceptibility to rhizobial infection and reduced GA-induced CLE-RS1 and CLE-RS2 expression, suggesting that the inhibitory effect of GAs occurs through AON. This is supported by the GA-insensitive phenotypes of an AON-defective mutant of HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) and a reciprocal grafting experiment. Thus, endogenous GAs induce NIN expression via its GA-responsive cis-acting region, and subsequently the GA-induced NIN activates the AON system to regulate nodule formation.

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.

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.

Internalization of FITC-albumin in Human Adipose-derived Stem Cells: Involvement of Clathrin and Caveolin.

Adipose tissue-derived stem cells (AdSCs) are one of the most promising cell types for cell-based therapies. In addition, AdSCs systematically injected into the body have been reported to localize to damaged tissues and certain types of tumor. As an important part of establishing a potent drug delivery system with AdSCs, the mechanism and efficiency of uptake into AdSCs has drawn much research attention. However, this remains to be fully clarified. The aim of this study was to examine the characteristics of endocytosis-mediated uptake in human AdSCs. We used fluorescein isothiocyanate-labeled albumin (FITC-albumin) as a potent marker of endocytosis. FITC-albumin uptake was time- and temperature-dependent. Confocal microscopy showed punctate localization of fluorescence in the cytoplasm. FITC-albumin uptake was inhibited by human serum albumin in a concentration-dependent manner. FITC-albumin uptake was inhibited by a metabolic inhibitor (2,4-dinitrophenol), a microtubule polymerization inhibitor (colchicine), an actin polymerization inhibitor (cytochalasin D), endosomal acidification inhibitors (chloroquine and bafilomycin A1), clathrin-dependent endocytosis inhibitors (chloropromazine, phenylarsine oxide, and Pitstop2), and caveolin-dependent endocytosis inhibitors (nystatin and methyl-ß-cyclodextrin). Furthermore, the knockdown of the clathrin heavy chain and caveolin-1 significantly reduced FITC-albumin uptake. These findings suggest that AdSCs take up albumin via endocytic pathways in which clathrin and caveolin are involved.

WUSCHEL-RELATED HOMEOBOX4 acts as a key regulator in early leaf development in rice.

Rice (Oryza sativa) has long and narrow leaves with parallel veins, similar to other grasses. Relative to Arabidopsis thaliana which has oval-shaped leaves, our understanding of the mechanism of leaf development is insufficient in grasses. In this study, we show that OsWOX4, a member of the WUSCHEL-RELATED HOMEOBOX gene family, plays important roles in early leaf development in rice. Inducible downregulation of OsWOX4 resulted in severe defects in leaf development, such as an arrest of vascular differentiation, a partial defect in the early cell proliferation required for midrib formation, and a failure to maintain cellular activity in general parenchyma cells. In situ analysis showed that knockdown of OsWOX4 reduced the expression of two LONELY GUY genes, which function in the synthesis of active cytokinin, in developing vascular bundles. Consistent with this, cytokinin levels were downregulated by OsWOX4 knockdown. Transcriptome analysis further showed that OsWOX4 regulates multiple genes, including those responsible for cell cycle progression and hormone action, consistent with the effects of OsWOX4 downregulation on leaf phenotypes. Collectively, these results suggest that OsWOX4 acts as a key regulator at an early stage of leaf development. Our previous work revealed that OsWOX4 is involved in the maintenance of shoot apical meristem in rice, whereas AtWOX4 is specifically associated with the maintenance of vascular stem cells in Arabidopsis. Thus, the function of the two orthologous genes seems to be diversified between rice and Arabidopsis.

A gene-stacking approach to overcome the trade-off between drought stress tolerance and growth in Arabidopsis.

The molecular breeding of drought stress-tolerant crops is imperative for stable food and biomass production. However, a trade-off exists between plant growth and drought stress tolerance. Many drought stress-tolerant plants overexpressing stress-inducible genes, such as DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 1A (DREB1A), show severe growth retardation. Here, we demonstrate that the growth of DREB1A-overexpressing Arabidopsis plants could be improved by co-expressing growth-enhancing genes whose expression is repressed under drought stress conditions. We used Arabidopsis GA REQUIRING 5 (GA5), which encodes a rate-limiting gibberellin biosynthetic enzyme, and PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), which encodes a transcription factor regulating cell growth in response to light and temperature, for growth improvement. We observed an enhanced biomass and floral induction in the GA5 DREB1A and PIF4 DREB1A double overexpressors compared with those in the DREB1A overexpressors. Although the GA5 DREB1A double overexpressors continued to show high levels of drought stress tolerance, the PIF4 DREB1A double overexpressors showed lower levels of stress tolerance than the DREB1A overexpressors due to repressed expression of DREB1A. A multiomics analysis of the GA5 DREB1A double overexpressors showed that the co-expression of GA5 and DREB1A additively affected primary metabolism, gene expression and plant hormone profiles in the plants. These multidirectional analyses indicate that the inherent trade-off between growth and drought stress tolerance in plants can be overcome by appropriate gene-stacking approaches. Our study provides a basis for using genetic modification to improve the growth of drought stress-tolerant plants for the stable production of food and biomass.

Molecular Basis for Natural Vegetative Propagation via Regeneration in North American Lake Cress, Rorippa aquatica (Brassicaceae).

Some plant species have a striking capacity for regeneration in nature, including regeneration of the entire individual from explants. However, due to the lack of suitable experimental models, the regulatory mechanisms of spontaneous whole plant regeneration are mostly unknown. In this study, we established a novel model system to study these mechanisms using an amphibious plant within Brassicaceae, Rorippa aquatica, which naturally undergoes vegetative propagation via regeneration from leaf fragments. Morphological and anatomical observation showed that both de novo root and shoot organogenesis occurred from the proximal side of the cut edge transversely with leaf vascular tissue. Time-series RNA-seq analysis revealed that auxin and cytokinin responses were activated after leaf amputation and that regeneration-related genes were upregulated mainly on the proximal side of the leaf explants. Accordingly, we found that both auxin and cytokinin accumulated on the proximal side. Application of a polar auxin transport inhibitor retarded root and shoot regeneration, suggesting that the enhancement of auxin responses caused by polar auxin transport enhanced de novo organogenesis at the proximal wound site. Exogenous phytohormone and inhibitor applications further demonstrated that, in R. aquatica, both auxin and gibberellin are required for root regeneration, whereas cytokinin is important for shoot regeneration. Our results provide a molecular basis for vegetative propagation via de novo organogenesis.

Fatty acids bound to albumin induce prostaglandin E2 production in human renal proximal tubular epithelial cell line HK-2.

Fatty acids bound to albumin have been reported to be involved in various responses in renal proximal tubular cells following albumin overload, leading to progression of tubulointerstitial damage in the kidneys. In addition, it has been reported that prostaglandin E2 (PGE2) plays an important role in nephrotoxicity. The aim of this study was to examine whether albumin-bound fatty acids induce PGE2 production in human renal proximal tubular epithelial cell line HK-2. Fatty acid-bearing human serum albumin increased PGE2 release in the culture medium in concentration-dependent and time-dependent manners, but fatty acid-depleted albumin had no effect on PGE2 production. Next, we investigated the effect of arachidonic acid, a precursor of eicosanoids, on PGE2 production. Arachidonic acid with fatty acid-free albumin significantly enhanced the release of PGE2 into the medium in a concentration-dependent manner. Furthermore, we examined the effect of arachidonic acid on mRNA expression of hypoxia inducible factor-1α (HIF-1α). Arachidonic acid increased HIF-1α mRNA expression in a concentration-dependent manner. These findings suggest that fatty acids, at least in part arachidonic acid, bound to albumin increase PGE2 production and expression of HIF-1α mRNA and protein, possibly resulting in various cell responses induced by albumin overload.

Cytokinin-Mediated Regulation of Reactive Oxygen Species Homeostasis Modulates Stomatal Immunity in Arabidopsis.

Stomata play an important role in preinvasive defense responses by limiting pathogen entry into leaves. Although the stress hormones salicylic acid (SA) and abscisic acid (ABA) are known to regulate stomatal immunity, the role of growth promoting hormones is far from understood. Here, we show that in Arabidopsis thaliana, cytokinins (CKs) function in stomatal defense responses. The cytokinin receptor HISTIDINE KINASE3 (AHK3) and RESPONSE REGULATOR2 (ARR2) promote stomatal closure triggered by pathogen-associated molecular pattern (PAMP) and resistance to Pseudomonas syringae pv tomato bacteria. Importantly, the cytokinin trans-zeatin induces stomatal closure and accumulation of reactive oxygen species (ROS) in guard cells through AHK3 and ARR2 in an SA-dependent and ABA-independent manner. Using pharmacological and reverse genetics approaches, we found that CK-mediated stomatal responses involve the apoplastic peroxidases PRX4, PRX33, PRX34, and PRX71, but not the NADPH oxidases RBOHD and RBOHF. Moreover, ARR2 directly activates the expression of PRX33 and PRX34, which are required for SA- and PAMP-triggered ROS production. Thus, the CK signaling pathway regulates ROS homeostasis in guard cells, which leads to enhanced stomatal immunity and plant resistance to bacteria.

Differences in xylem development between Dutch and Japanese tomato (Solanum lycopersicum) correlate with cytokinin levels in hypocotyls.

BACKGROUND AND AIMS: Dutch tomato cultivars tend to have a greater yield than Japanese cultivars even if they are grown under the same conditions. Factors contributing to the increased yield of the Dutch cultivars were a greater light use efficiency and greater leaf photosynthetic rate. On the other hand, the relationship between tomato yields and anatomical traits is still unclear. The aim of this study is to identify the anatomical traits related to the difference in yield between Dutch and Japanese cultivars. METHODS: Anatomical properties were compared during different growth stages of Dutch and Japanese tomatoes. Hormone profiles and related gene expression in hypocotyls of Dutch and Japanese cultivars were compared in the hypocotyls of 3- and 4-week-old plants. KEY RESULTS: Dutch cultivars have a more developed secondary xylem than Japanese cultivars, which would allow for greater transport of water, mineral nutrients and phytohormones to the shoots. The areas and ratios of the xylem in the hypocotyls of 3- to 6-week-old plants were larger in the Dutch cultivars. In reciprocal grafts of the Japanese and Dutch cultivars, xylem development at the scion and rootstock depended on the scion cultivar, suggesting that some factors in the scion are responsible for the difference in xylem development. The cytokinin content, especially the level of N6-(Δ 2-isopentenyl) adenine (iP)-type cytokinin, was higher in the Dutch cultivars. This result was supported by the greater expression of Sl-IPT3 (a cytokinin biosynthesis gene) and Sl-RR16/17 (a cytokinin-responsive gene) in the Dutch cultivars. CONCLUSIONS: These results suggest that iP-type cytokinins, which are locally synthesized in the hypocotyl, contribute to xylem development. The greater xylem development in Dutch cultivars might contribute to the high yield of the tomato.

WIND1 induces dynamic metabolomic reprogramming during regeneration in Brassica napus.

Plants often display a high competence for regeneration under stress conditions. Signals produced in response to various types of stress serve as critical triggers for de novo organogenesis, but the identity of these signaling molecules underlying cellular reprogramming are largely unknown. We previously identified an AP2/ERF transcription factor, WOUND INDUCED DEDIFFERENTIATION1 (WIND1), as a key regulator involved in wound-induced cellular reprogramming in Arabidopsis. In this study, we found that activation of Arabidopsis WIND1 (AtWIND1) in hypocotyl explants of Brassica napus (B. napus) enhances callus formation and subsequent organ regeneration. Gene expression analyses revealed that AtWIND1 enhances expression of B. napus homologs of ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN (ESR1/DRN), which is a direct target of WIND1 in Arabidopsis. Further, time-course hormonal analyses showed that an altered balance of endogenous auxin/cytokinin exists in AtWIND1-activated B. napus explants. Our mass spectrometry analyses, in addition, uncovered dynamic metabolomic reprogramming in AtWIND1-activated explants, including accumulation of several compounds, e.g. proline, gamma aminobutyric acid (GABA), and putrescine, that have historically been utilized as additives to enhance plant cell reprogramming in tissue culture. Our findings thus provide new insights into how WIND1 functions to promote cell reprogramming.

Suppression of DELLA signaling induces procambial cell formation in culture.

The post-embryonic growth of plants requires the activities of apical meristems and lateral meristems. In the meristems, self-proliferation and differentiation of stem cells is tightly modulated by plant hormone signaling networks and specific transcription factors. Despite extensive studies on stem cell maintenance in plants, the mechanism by which stem cells are initially established is largely unknown. Vascular stem cells consisting of procambial/cambial cells give rise to xylem and phloem cells. In this study, we analyzed the establishment of procambial cells using the in vitro culture system VISUAL, in which mesophyll cells rapidly differentiate into xylem tracheary elements and phloem sieve elements via procambial cells. We found that procambial cell formation in VISUAL is initiated by light, which can be replaced by application of gibberellin (GA). Gibberellin was able to promote procambial cell formation through degradation of DELLA, whereas light did not elevate the endogenous GA content. Indeed, light in combination with bikinin reduced the accumulation of DELLA protein in VISUAL. Consistently, overexpression of a constitutively active DELLA protein repressed vascular cell differentiation even under light. These combined results suggest that DELLA signaling suppresses procambial cell formation during vascular development in VISUAL.

Shotgun Proteomic Analysis Highlights the Roles of Long-Lived mRNAs and De Novo Transcribed mRNAs in Rice Seeds upon Imbibition.

During seed germination, proteins are translated not only from mRNAs newly transcribed upon imbibition but also from long-lived mRNAs that are synthesized during seed maturation and stored in the mature dry seeds. To clarify the distinct roles of proteins translated from long-lived mRNAs and de novo transcribed mRNAs in germinating rice embryos, proteome analysis based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) combining the use of a transcriptional inhibitor was performed. We observed that α-amanitin significantly represses transcription in germinating embryos; nevertheless, the embryos could germinate, albeit slowly. The proteomic analysis revealed that a total of 109 proteins were translated from long-lived mRNAs associated with germination as well as 222 proteins whose expression were dependent on de novo transcription upon imbibition. Transcriptomic datasets available in public databases demonstrated that mRNAs of the 222 proteins notably increased during germination while those of the 109 proteins highly accumulated in dry embryos and constitutively expressed upon imbibition. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that many of the 109 proteins from long-lived mRNAs are implicated in energy production such as glycolysis or annotated as nucleotide binding proteins, while the 222 proteins are involved in pathways such as pyruvate metabolism and TCA cycle following glycolysis, and momilactones biosynthesis. We propose that long-lived mRNAs support initial energy production and activation of translational machinery upon imbibition whereas de novo transcription accelerates the energy production after glycolysis, which enables rice seeds to germinate vigorously.

Aberrant Stamen Development is Associated with Parthenocarpic Fruit Set Through Up-Regulation of Gibberellin Biosynthesis in Tomato.

Parthenocarpy, a process in which fruit set occurs without fertilization, leads to the production of seedless fruit. A number of floral homeotic mutants with abnormal stamen development exhibit parthenocarpic fruit set. Flower development is thought to repress ovary growth before anthesis. However, the mechanism of parthenocarpic fruit development caused by aberrant flower formation is poorly understood. To investigate the molecular mechanism of parthenocarpic fruit development in floral homeotic mutants, we performed functional analysis of Tomato APETALA3 (TAP3) by loss-of-function approaches. Organ-specific promoter was used to induce organ-specific loss of function in stamen and ovary/fruit. We observed increased cell expansion in tap3 mutants and TAP3-RNAi lines during parthenocarpic fruit growth. These were predominantly accompanied by the up-regulation of GA biosynthesis genes, including SlGA20ox1, SlGA20ox2, and SlGA20ox3, as well as reduced expression of the GA-inactivating gene SlGA2ox1 and the auxin signaling gene SlARF7 involved in a crosstalk between GA and auxin. These transcriptional profiles are in agreement with the GA levels in these lines. These results suggest that stamen development negatively regulates fruit set by repressing the GA biosynthesis.