The notion that plants use specialized metabolism to protect against environmental stresses needs to be experimentally proven by addressing the question of whether stress tolerance by specialized metabolism is directly due to metabolites such as flavonoids. We report that flavonoids with radical scavenging activity mitigate against oxidative and drought stress in Arabidopsis thaliana. Metabolome and transcriptome profiling and experiments with oxidative and drought stress in wild-type, single overexpressors of MYB12/PFG1 (PRODUCTION OF FLAVONOL GLYCOSIDES1) or MYB75/PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1), double overexpressors of MYB12 and PAP1, transparent testa4 (tt4) as a flavonoid-deficient mutant, and flavonoid-deficient MYB12 or PAP1 overexpressing lines (obtained by crossing tt4 and the individual MYB overexpressor) demonstrated that flavonoid overaccumulation was key to enhanced tolerance to such stresses. Antioxidative activity assays using 2,2-diphenyl-1-picrylhydrazyl, methyl viologen, and 3,3′-diaminobenzidine clearly showed that anthocyanin overaccumulation with strong in vitro antioxidative activity mitigated the accumulation of reactive oxygen species in vivo under oxidative and drought stress. These data confirm the usefulness of flavonoids for enhancing both biotic and abiotic stress tolerance in crops.

The growth of plants depends on continuous function of the meristems. Shoot meristems are responsible for all the post-embryonic aerial organs, such as leaves, stems and flowers. It has been assumed that the phytohormone cytokinin has a positive role in shoot meristem function. A severe reduction in the size of meristems in a mutant that is defective in all of its cytokinin receptors has provided compelling evidence that cytokinin is required for meristem activity. Here, we report a novel regulation of meristem activity, which is executed by the meristem-specific activation of cytokinins. The LONELY GUY (LOG) gene of rice is required to maintain meristem activity and its loss of function causes premature termination of the shoot meristem. LOG encodes a novel cytokinin-activating enzyme that works in the final step of bioactive cytokinin synthesis. Revising the long-held idea of multistep reactions, LOG directly converts inactive cytokinin nucleotides to the free-base forms, which are biologically active, by its cytokinin-specific phosphoribohydrolase activity. LOG messenger RNA is specifically localized in shoot meristem tips, indicating the activation of cytokinins in a specific developmental domain. We propose the fine-tuning of concentrations and the spatial distribution of bioactive cytokinins by a cytokinin-activating enzyme as a mechanism that regulates meristem activity.

Drought, the most prominent threat to agricultural production worldwide, accelerates leaf senescence, leading to a decrease in canopy size, loss in photosynthesis and reduced yields. On the basis of the assumption that senescence is a type of cell death program that could be inappropriately activated during drought, we hypothesized that it may be possible to enhance drought tolerance by delaying droughtinduced leaf senescence. We generated transgenic plants expressing an isopentenyltransferase gene driven by a stress-and maturationinduced promoter. Remarkably, the suppression of drought-induced leaf senescence resulted in outstanding drought tolerance as shown by, among other responses, vigorous growth after a long drought period that killed the control plants. The transgenic plants maintained high water contents and retained photosynthetic activity (albeit at a reduced level) during the drought. Moreover, the transgenic plants displayed minimal yield loss when watered with only 30% of the amount of water used under control conditions. The production of drought-tolerant crops able to grow under restricted water regimes without diminution of yield would minimize drought-related losses and ensure food production in water-limited lands.cytokinins ͉ isopentenyltransferase ͉ water stress ͉ water use efficiency ͉ oxidative stress D rought is the most serious environmental factor limiting the productivity of agricultural crops worldwide, with devastating economical and sociological impact. Climate models have indicated that drought episodes will become more frequent because of the long-term effects of global warming (1, 2), emphasizing the urgent need to develop adaptive agricultural strategies for a changing environment. These range from changes in traditional management and agronomic practices to the use of marker-assisted selection for the improvement of drought-related traits and the development of transgenic crops with enhanced tolerance of drought and improved water use efficiency that would minimize drought-related losses and ensure food production for a growing population.Plants can use a combination of different strategies to avoid or tolerate drought stress (3, 4). In arid regions, for example, winter annuals combine a relatively short life cycle with a high growth rate during the wet season to avoid drought altogether. Other types of avoidance include closing of stomata to minimize water loss, adjusting sink/source allocation by increasing root growth, and decreasing canopy by reducing growth and shedding of older leaves (5). Accelerated leaf senescence and leaf abscission are associated with drought in nature as a means to decrease canopy size. In perennial plants, this strategy contributes to the survival of the plant and the completion of the plant life cycle under drought stress. In contrast, this strategy reduces the yields of annual crops, with concomitant economical loss to farmers. We hypothesized that it is possible to enhance the tolerance of plants of drought stress by delaying the drought-induced...

Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development.

SummaryPlant architecture is mostly determined by shoot branching patterns. Apical dominance is a well-known control mechanism in the development of branching patterns, but little is known regarding its role in monocots such as rice. Here, we show that the concept of apical dominance can be applied to tiller bud outgrowth of rice. In dwarf10 (d10), an enhanced branching mutant of rice, apical dominance can be observed, but the inhibitory effects of the apical meristem was reduced. D10 is a rice ortholog of MAX4/RMS1/DAD1 that encodes a carotenoid cleavage dioxygenase 8 and is supposed to be involved in the synthesis of an unidentified inhibitor of shoot branching. D10 expression predominantly occurs in vascular cells in most organs. Real-time polymerase chain reaction analysis revealed that accumulation of D10 mRNA is induced by exogenous auxin. Moreover, D10 expression is upregulated in six branching mutants, d3, d10, d14, d17, d27 and high tillering dwarf (htd1). No such effects were found for D3 or HTD1, the MAX2 and MAX3 orthologs, respectively, of rice. These findings imply that D10 transcription might be a critical step in the regulation of the branching inhibitor pathway. In addition, we present observations that suggest that FINE CULM1 (FC1), a rice ortholog of teosinte branched 1 (tb1), possibly works independently of the branching inhibitor pathway.

Many multicellular organisms have remarkable capability to regenerate new organs after wounding. As a first step of organ regeneration, adult somatic cells often dedifferentiate to reacquire cell proliferation potential, but mechanisms underlying this process remain unknown in plants. Here we show that an AP2/ERF transcription factor, WOUND INDUCED DEDIFFERENTIATION 1 (WIND1), is involved in the control of cell dedifferentiation in Arabidopsis. WIND1 is rapidly induced at the wound site, and it promotes cell dedifferentiation and subsequent cell proliferation to form a mass of pluripotent cells termed callus. We further demonstrate that ectopic overexpression of WIND1 is sufficient to establish and maintain the dedifferentiated status of somatic cells without exogenous auxin and cytokinin, two plant hormones that are normally required for cell dedifferentiation. In vivo imaging of a synthetic cytokinin reporter reveals that wounding upregulates the B-type ARABIDOPSIS RESPONSE REGULATOR (ARR)-mediated cytokinin response and that WIND1 acts via the ARR-dependent signaling pathway to promote cell dedifferentiation. This study provides novel molecular insights into how plants control cell dedifferentiation in response to wounding.

Cytokinins affect plant immunity to various pathogens; however, the mechanisms coupling plant-derived cytokinins to pathogen responses have been elusive. Here, we found that plant-derived cytokinins promote resistance of Arabidopsis to Pseudomonas syringae pv. tomato DC3000 (Pst). Modulated cytokinin levels or signaling activity in CKX- or IPT-overexpressing plants or in ahk2 ahk3 mutants correlated with altered resistance. In fact, the cytokinin-activated transcription factor ARR2 contributes specifically to Pst resistance. The salicylic acid (SA) response factor TGA3 binds ARR2, and mutation of TGA-binding cis-elements in the Pr1 promoter abolished cytokinin- and ARR2-dependent Pr1 activation. Cytokinin treatment did not increase pathogen resistance in tga3 plants, as the cytokinin-dependent induction of Pr1 was eliminated. Moreover, SA signaling enhanced binding of ARR2/TGA3 to the Pr1 promoter. Taken together, these results show that cytokinins modulate the SA signaling to augment resistance against Pst, a process in which the interaction between TGA3 and ARR2 is important.

Cytokinins play crucial roles in diverse aspects of plant growth and development. Spatiotemporal distribution of bioactive cytokinins is finely regulated by metabolic enzymes. LONELY GUY (LOG) was previously identified as a cytokinin-activating enzyme that works in the direct activation pathway in rice (Oryza sativa) shoot meristems. In this work, nine Arabidopsis thaliana LOG genes (At LOG1 to LOG9) were predicted as homologs of rice LOG. Seven At LOGs, which are localized in the cytosol and nuclei, had enzymatic activities equivalent to that of rice LOG. Conditional overexpression of At LOGs in transgenic Arabidopsis reduced the content of N 6 -(D 2 -isopentenyl)adenine (iP) riboside 59-phosphates and increased the levels of iP and the glucosides. Multiple mutants of At LOGs showed a lower sensitivity to iP riboside in terms of lateral root formation and altered root and shoot morphology. Analyses of At LOG promoter:b-glucuronidase fusion genes revealed differential expression of LOGs in various tissues during plant development. Ectopic overexpression showed pleiotropic phenotypes, such as promotion of cell division in embryos and leaf vascular tissues, reduced apical dominance, and a delay of leaf senescence. Our results strongly suggest that the direct activation pathway via LOGs plays a pivotal role in regulating cytokinin activity during normal growth and development in Arabidopsis.