The transition from juvenility through maturation to senescence is a complex process that involves the regulation of longevity. Here, we identify JUNGBRUNNEN1 (JUB1), a hydrogen peroxide (H 2 O 2 )-induced NAC transcription factor, as a central longevity regulator in Arabidopsis thaliana. JUB1 overexpression strongly delays senescence, dampens intracellular H 2 O 2 levels, and enhances tolerance to various abiotic stresses, whereas in jub1-1 knockdown plants, precocious senescence and lowered abiotic stress tolerance are observed. A JUB1 binding site containing a RRYGCCGT core sequence is present in the promoter of DREB2A, which plays an important role in abiotic stress responses. JUB1 transactivates DREB2A expression in mesophyll cell protoplasts and transgenic plants and binds directly to the DREB2A promoter. Transcriptome profiling of JUB1 overexpressors revealed elevated expression of several reactive oxygen species-responsive genes, including heat shock protein and glutathione S-transferase genes, whose expression is further induced by H 2 O 2 treatment. Metabolite profiling identified elevated Pro and trehalose levels in JUB1 overexpressors, in accordance with their enhanced abiotic stress tolerance. We suggest that JUB1 constitutes a central regulator of a finely tuned control system that modulates cellular H 2 O 2 level and primes the plants for upcoming stress through a gene regulatory network that involves DREB2A.

). † Both authors contributed equally to this work and should jointly be considered second authors. SUMMARYThe onset and progression of senescence are under genetic and environmental control. The Arabidopsis thaliana NAC transcription factor ANAC092 (also called AtNAC2 and ORE1) has recently been shown to control age-dependent senescence, but its mode of action has not been analysed yet. To explore the regulatory network administered by ANAC092 we performed microarray-based expression profiling using estradiolinducible ANAC092 overexpression lines. Approximately 46% of the 170 genes up-regulated upon ANAC092 induction are known senescence-associated genes, suggesting that the NAC factor exerts its role in senescence through a regulatory network that includes many of the genes previously reported to be senescence regulated. We selected 39 candidate genes and confirmed their time-dependent response to enhanced ANAC092 expression by quantitative RT-PCR. We also found that the majority of them (24 genes) are up-regulated by salt stress, a major promoter of plant senescence, in a manner similar to that of ANAC092, which itself is salt responsive. Furthermore, 24 genes like ANAC092 turned out to be stage-dependently expressed during seed growth with low expression at early and elevated expression at late stages of seed development. Disruption of ANAC092 increased the rate of seed germination under saline conditions, whereas the opposite occurred in respective overexpression plants. We also detected a delay of salinity-induced chlorophyll loss in detached anac092-1 mutant leaves. Promoter-reporter (GUS) studies revealed transcriptional control of ANAC092 expression during leaf and flower ageing and in response to salt stress. We conclude that ANAC092 exerts its functions during senescence and seed germination through partly overlapping target gene sets.

Tomato (Solanum lycopersicum) is a well-studied model of fleshy fruit development and ripening. Tomato fruit development is well understood from a hormonal-regulatory perspective, and developmental changes in pigment and cell wall metabolism are also well characterized. However, more general aspects of metabolic change during fruit development have not been studied despite the importance of metabolism in the context of final composition of the ripe fruit. In this study, we quantified the abundance of a broad range of metabolites by gas chromatography-mass spectrometry, analyzed a number of the principal metabolic fluxes, and in parallel analyzed transcriptomic changes during tomato fruit development. Metabolic profiling revealed pronounced shifts in the abundance of metabolites of both primary and secondary metabolism during development. The metabolite changes were reflected in the flux analysis that revealed a general decrease in metabolic activity during ripening. However, there were several distinct patterns of metabolite profile, and statistical analysis demonstrated that metabolites in the same (or closely related) pathways changed in abundance in a coordinated manner, indicating a tight regulation of metabolic activity. The metabolite data alone allowed investigations of likely routes through the metabolic network, and, as an example, we analyze the operational feasibility of different pathways of ascorbate synthesis. When combined with the transcriptomic data, several aspects of the regulation of metabolism during fruit ripening were revealed. First, it was apparent that transcript abundance was less strictly coordinated by functional group than metabolite abundance, suggesting that posttranslational mechanisms dominate metabolic regulation. Nevertheless, there were some correlations between specific transcripts and metabolites, and several novel associations were identified that could provide potential targets for manipulation of fruit compositional traits. Finally, there was a strong relationship between ripening-associated transcripts and specific metabolite groups, such as TCA-cycle organic acids and sugar phosphates, underlining the importance of the respective metabolic pathways during fruit development.

Tomato (Solanum lycopersicum) is an established model to study fleshy fruit development and ripening. Tomato ripening is regulated independently and cooperatively by ethylene and transcription factors, including nonripening (NOR) and ripeninginhibitor (RIN). Mutations of NOR, RIN, and the ethylene receptor Never-ripe (Nr), which block ethylene perception and inhibit ripening, have proven to be great tools for advancing our understanding of the developmental programs regulating ripening. In this study, we present systems analysis of nor, rin, and Nr at the transcriptomic, proteomic, and metabolomic levels during development and ripening. Metabolic profiling marked shifts in the abundance of metabolites of primary metabolism, which lead to decreases in metabolic activity during ripening. When combined with transcriptomic and proteomic data, several aspects of the regulation of metabolism during ripening were revealed. First, correlations between the expression levels of a transcript and the abundance of its corresponding protein were infrequently observed during early ripening, suggesting that posttranscriptional regulatory mechanisms play an important role in these stages; however, this correlation was much greater in later stages. Second, we observed very strong correlation between ripening-associated transcripts and specific metabolite groups, such as organic acids, sugars, and cell wall-related metabolites, underlining the importance of these metabolic pathways during fruit ripening. These results further revealed multiple ethylene-associated events during tomato ripening, providing new insights into the molecular biology of ethylene-mediated ripening regulatory networks.

We report here that ORS1, a previously uncharacterized member of the NAC transcription factor family, controls leaf senescence in Arabidopsis thaliana. Overexpression of ORS1 accelerates senescence in transgenic plants, whereas its inhibition delays it. Genes acting downstream of ORS1 were identified by global expression analysis using transgenic plants producing dexamethasone-inducible ORS1–GR fusion protein. Of the 42 up-regulated genes, 30 (∼70%) were previously shown to be up-regulated during age-dependent senescence. We also observed that 32 (∼76%) of the ORS1-dependent genes were induced by long-term (4 d), but not short-term (6 h) salinity stress (150 mM NaCl). Furthermore, expression of 16 and 24 genes, respectively, was induced after 1 and 5 h of treatment with hydrogen peroxide (H2O2), a reactive oxygen species known to accumulate during salinity stress. ORS1 itself was found to be rapidly and strongly induced by H2O2 treatment in both leaves and roots. Using in vitro binding site selection, we determined the preferred binding motif of ORS1 and found it to be present in half of the ORS1-dependent genes. ORS1 is a paralog of ORE1/ANAC092/AtNAC2, a previously reported regulator of leaf senescence. Phylogenetic footprinting revealed evolutionary conservation of the ORS1 and ORE1 promoter sequences in different Brassicaceae species, indicating strong positive selection acting on both genes. We conclude that ORS1, similarly to ORE1, triggers expression of senescence-associated genes through a regulatory network that may involve cross-talk with salt- and H2O2-dependent signaling pathways.

It has been previously demonstrated, utilizing intraspecific introgression lines, that Lycopersicum Invertase5 (LIN5), which encodes a cell wall invertase, controls total soluble solids content in tomato (Solanum lycopersicum). The physiological role of this protein, however, has not yet been directly studied, since evaluation of data obtained from the introgression lines is complicated by the fact that they additionally harbor many other wild species alleles. To allow a more precise comparison, we generated transgenic tomato in which we silenced the expression of LIN5 using the RNA interference approach. The transformants were characterized by an altered flower and fruit morphology, displaying increased numbers of petals and sepals per flower, an increased rate of fruit abortion, and a reduction in fruit size. Evaluation of the mature fruit revealed that the transformants were characterized by a reduction of seed number per plant. Furthermore, detailed physiological analysis revealed that the transformants displayed aberrant pollen morphology and a reduction in the rate of pollen tube elongation. Metabolite profiling of ovaries and green and red fruit revealed that metabolic changes in the transformants were largely confined to sugar metabolism, whereas transcript and hormone profiling revealed broad changes both in the hormones themselves and in transcripts encoding their biosynthetic enzymes and response elements. These results are discussed in the context of current understanding of the role of sugar during the development of tomato fruit, with particular focus given to its impact on hormone levels and organ morphology.The importance of the supply to, and the subsequent mobilization of Suc in, plant heterotrophic organs has been the subject of intensive research effort over many

SummaryGlucosinolates are a group of secondary metabolites that function as defense substances against herbivores and micro-organisms in the plant order Capparales. Indole glucosinolates (IGS), derivatives of tryptophan, may also influence plant growth and development. In Arabidopsis thaliana, indole-3-acetaldoxime (IAOx) produced from tryptophan by the activity of two cytochrome P450 enzymes, CYP79B2 and CYP79B3, serves as a precursor for IGS biosynthesis but is also an intermediate in the biosynthetic pathway of indole-3-acetic acid (IAA). Another cytochrome P450 enzyme, CYP83B1, funnels IAOx into IGS. Although there is increasing information about the genes involved in this biochemical pathway, their regulation is not fully understood. OBP2 has recently been identified as a member of the DNA-binding-with-one-finger (DOF) transcription factors, but its function has not been studied in detail so far. Here we report that OBP2 is expressed in the vasculature of all Arabidopsis organs, including leaves, roots, flower stalks and petals. OBP2 expression is induced in response to a generalist herbivore, Spodoptera littoralis, and by treatment with the plant signalling molecule methyl jasmonate, both of which also trigger IGS accumulation. Constitutive and inducible overexpression of OBP2 activates expression of CYP83B1. In addition, auxin concentration is increased in leaves and seedlings of OBP2 over-expression lines relative to wild-type, and plant size is diminished due to a reduction in cell size. RNA interference-mediated OBP2 blockade leads to reduced expression of CYP83B1. Collectively, these data provide evidence that OBP2 is part of a regulatory network that regulates glucosinolate biosynthesis in Arabidopsis.

The antioxidant defense system involves complex functional coordination of multiple components in different organelles within the plant cell. Here, we have studied the Arabidopsis thaliana early response to the generation of superoxide anion in chloroplasts during active photosynthesis. We exposed plants to methyl viologen (MV), a superoxide anion propagator in the light, and performed biochemical and expression profiling experiments using Affymetrix ATH1 GeneChip Ò microarrays under conditions in which photosynthesis and antioxidant enzymes were active. Data analysis identified superoxideresponsive genes that were compared with available microarray results. Examples include genes encoding proteins with unknown function, transcription factors and signal transduction components. A common GAA AAGTCAAAC motif containing the W-box consensus sequence of WRKY transcription factors, was found in the promoters of genes highly up-regulated by superoxide. Band shift assays showed that oxidative treatments enhanced the specific binding of leaf protein extracts to this motif. In addition, GUS reporter gene fused to WRKY30 promoter, which contains this binding motif, was induced by MV and H 2 O 2 . Overall, our study suggests that genes involved in signalling pathways and with unknown functions are rapidly activated by superoxide anion generated in photosynthetically active chloroplasts, as part of the early antioxidant response of Arabidopsis leaves.