Five recombinant inbred lines (RILs) of Arabidopsis (Arabidopsis thaliana), previously selected from the Bay-0 3 Shahdara RIL population on the basis of differential leaf senescence phenotypes (from early senescing to late senescing) when cultivated under nitrogen (N)-limiting conditions, were analyzed to monitor metabolic markers related to N assimilation and N remobilization pathways. In each RIL, a decrease of total N, free amino acid, and soluble protein contents with leaf aging was observed. In parallel, the expression of markers for N remobilization such as cytosolic glutamine synthetase, glutamate dehydrogenase, and CND41-like protease was increased. This increase occurred earlier and more rapidly in early-senescing lines than in late-senescing lines. We measured the partitioning of 15 N between sink and source leaves during the vegetative stage of development using 15 N tracing and showed that N remobilization from the source leaves to the sink leaves was more efficient in the early-senescing lines. The N remobilization rate was correlated with leaf senescence severity at the vegetative stage. Experiments of 15 N tracing at the reproductive stage showed, however, that the rate of N remobilization from the rosettes to the flowering organs and to the seeds was similar in early-and late-senescing lines. At the reproductive stage, N remobilization efficiency did not depend on senescence phenotypes but was related to the ratio between the biomasses of the sink and the source organs.

Comparison of the extent of leaf senescence depending on the genetic background of different recombinant inbred lines (RILs) of Arabidopsis (Arabidopsis thaliana) is described. Five RILs of the Bay-0 3 Shahdara population showing differential leaf senescence phenotypes (from early senescing to late senescing) were selected to determine metabolic markers to discriminate Arabidopsis lines on the basis of senescence-dependent changes in metabolism. The proportion of g-aminobutyric acid, leucine, isoleucine, aspartate, and glutamate correlated with (1) the age and (2) the senescence phenotype of the RILs. Differences were observed in the glycine/serine ratio even before any senescence symptoms could be detected in the rosettes. This could be used as predictive indicator for plant senescence behavior. Surprisingly, late-senescing lines appeared to mobilize glutamine, asparagine, and sulfate more efficiently than early-senescing lines. The physiological basis of the relationship between leaf senescence and flowering time was analyzed.Leaf senescence is a key developmental step in the life of annual plants. During growth, green leaves accumulate nutrients. The main purpose of senescence is the mobilization and recycling of these nutrients to the developing seeds to prepare the next generation. Developmental signals, aging, or stress can induce leaf senescence. The final stage of this process is death, but cell death is actively delayed until nutrients have been removed (Buchanan-Wollaston et al., 2003).During senescence, cell constituents are dismantled in an ordered progression. Chlorophyll degradation is the first visible symptom of senescence, but by the time yellowing can be seen, some senescence has already occurred. Chlorophyll, protein, and lipid degradation processes have been largely investigated (Hö rtensteiner and Feller, 2002). Protein and mRNA degradation parallels the loss in photosynthetic activity and participates in nutrient mobilization. Nucleic acids, especially RNA, form a valuable source of phosphorus in mature leaves. Total RNA levels fall rapidly during senescence, whereas nuclear DNA is maintained to allow gene expression to continue, until late in the process. Nutrients such as sulfur, phosphorus, metal ions, and minerals are also transferred out of the leaves (Himelblau and Amasino, 2001). Accelerated metabolism of membrane lipids results in a decline in the structural and functional integrity of cellular membranes. Thylakoid membranes provide an abundant source of carbon that can be mobilized for use as an energy source during senescence. Rubisco is one of the major sources of nitrogen for mobilization. A major question in leaf senescence is how leaf proteins, up to 75% of which are located within the chloroplast, are degraded and mobilized (Mae, 2004).The molecular events that induce and contribute to the senescence process have recently been extensively investigated. The genomic resources that are now available for Arabidopsis (Arabidopsis thaliana) have allowed the rapid identification of ...

For the first time in Arabidopsis thaliana, this work proposes the identification of quantitative trait loci (QTLs) associated with leaf senescence and stress response symptoms such as yellowing and anthocyanin-associated redness. When Arabidopsis plants were cultivated under low nitrogen conditions, we observed that both yellowing of the old leaves of the rosette and whole rosette redness were promoted. Leaf yellowing is a senescence symptom related to chlorophyll breakdown. Redness is a symptom of anthocyanin accumulation related to whole plant ageing and nutrient limitation. In this work, Arabidopsis is used as a model system to dissect the genetic variation of these parameters by QTL mapping in the 415 recombinant inbred lines of the Bay-0xShahdara population. Fifteen new QTLs and two epistatic interactions were described in this study. The yellowing of the rosette, estimated by visual notation and image processing, was controlled by four and five QTLs, respectively. The visual estimation of redness allowed us to detect six QTLs among which the major one explained 33% of the total variation. Two main QTLs were confirmed in near-isogenic lines (heterogenous inbred family; HIF), thus confirming the relevance of the visual notation of these traits. Co-localizations between QTLs for leaf yellowing, redness and nitrogen use efficiency described in a previous publication indicate complex interconnected pathways involved in both nitrogen management and senescence- and stress-related processes. No co-localization between QTLs for leaf yellowing and redness has been found, suggesting that the two characters are genetically independent.

A mechanism for integrating light perception and the endogenous circadian clock is central to a plant's capacity to coordinate its growth and development with the prevailing daily light/dark cycles. Under short-day (SD) photocycles, hypocotyl elongation is maximal at dawn, being promoted by the collective activity of a quartet of transcription factors, called PIF1, PIF3, PIF4, and PIF5 (phytochromeinteracting factors). PIF protein abundance in SDs oscillates as a balance between synthesis and photoactivated-phytochrome-imposed degradation, with maximum levels accumulating at the end of the long night. Previous evidence shows that elongation under diurnal conditions (as well as in shade) is also subjected to circadian gating. However, the mechanism underlying these phenomena is incompletely understood. Here we show that the PIFs and the core clock component Timing of CAB expression 1 (TOC1) display coincident cobinding to the promoters of predawn-phased, growthrelated genes under SD conditions. TOC1 interacts with the PIFs and represses their transcriptional activation activity, antagonizing PIF-induced growth. Given the dynamics of TOC1 abundance (displaying high postdusk levels that progressively decline during the long night), our data suggest that TOC1 functions to provide a direct output from the core clock that transiently constrains the growth-promoting activity of the accumulating PIFs early postdusk, thereby gating growth to predawn, when conditions for cell elongation are optimal. These findings unveil a previously unrecognized mechanism whereby a core circadian clock output signal converges immediately with the phytochrome photosensory pathway to coregulate directly the activity of the PIF transcription factors positioned at the apex of a transcriptional network that regulates a diversity of downstream morphogenic responses.PIFs | photoperiod | TOC1 | circadian clock | gating of growth G iven the importance of solar energy to plants, they have evolved sophisticated photosensory-response systems to monitor and adapt to the diurnal photoperiod (1). This environmental parameter provides a precise index of the progression of the earth's seasons and the time of the day and thereby a signal that regulates a spectrum of growth and developmental responses (such as elongation growth, flowering, and dormancy) appropriate to the prevailing conditions.The photoreceptors in the phytochrome family (phyA-E in Arabidopsis) are the primary sensors of this signal (2, 3). These chromoproteins regulate two pathways in parallel that converge to control the morphogenic response: (i) the phytochromeinteracting factor (PIF) pathway, whereby the photoactivatedphytochrome molecules bind to and induce the degradation of the PIF proteins (notably the PIF1, PIF3, PIF4, and PIF5 quartet, a subfamily of basic helix-loop-helix transcription factors), thereby altering the expression of the PIF direct-target genes and the cognate downstream transcriptional network (4, 5), and (ii) the circadian clock, whereby the phytochromes entrain t...

SUMMARYRice plants grown in paddy fields preferentially use ammonium as a source of inorganic nitrogen. Glutamine synthetase (GS) catalyses the conversion of ammonium to glutamine. Of the three genes encoding cytosolic GS in rice, OsGS1;1 is critical for normal growth and grain filling. However, the basis of its physiological function that may alter the rate of nitrogen assimilation and carbon metabolism within the context of metabolic networks remains unclear. To address this issue, we carried out quantitative comparative analyses between the metabolite profiles of a rice mutant lacking OsGS1;1 and its background wild type (WT). The mutant plants exhibited severe retardation of shoot growth in the presence of ammonium compared with the WT. Overaccumulation of free ammonium in the leaf sheath and roots of the mutant indicated the importance of OsGS1;1 for ammonium assimilation in both organs. The metabolite profiles of the mutant line revealed: (i) an imbalance in levels of sugars, amino acids and metabolites in the tricarboxylic acid cycle, and (ii) overaccumulation of secondary metabolites, particularly in the roots under a continuous supply of ammonium. Metabolite-to-metabolite correlation analysis revealed the presence of mutant-specific networks between tryptamine and other primary metabolites in the roots. These results demonstrated a crucial function of OsGS1;1 in coordinating the global metabolic network in rice plants grown using ammonium as the nitrogen source.

Background14-3-3 proteins are considered master regulators of many signal transduction cascades in eukaryotes. In plants, 14-3-3 proteins have major roles as regulators of nitrogen and carbon metabolism, conclusions based on the studies of a few specific 14-3-3 targets.ResultsIn this study, extensive novel roles of 14-3-3 proteins in plant metabolism were determined through combining the parallel analyses of metabolites and enzyme activities in 14-3-3 overexpression and knockout plants with studies of protein-protein interactions. Decreases in the levels of sugars and nitrogen-containing-compounds and in the activities of known 14-3-3-interacting-enzymes were observed in 14-3-3 overexpression plants. Plants overexpressing 14-3-3 proteins also contained decreased levels of malate and citrate, which are intermediate compounds of the tricarboxylic acid (TCA) cycle. These modifications were related to the reduced activities of isocitrate dehydrogenase and malate dehydrogenase, which are key enzymes of TCA cycle. In addition, we demonstrated that 14-3-3 proteins interacted with one isocitrate dehydrogenase and two malate dehydrogenases. There were also changes in the levels of aromatic compounds and the activities of shikimate dehydrogenase, which participates in the biosynthesis of aromatic compounds.ConclusionTaken together, our findings indicate that 14-3-3 proteins play roles as crucial tuners of multiple primary metabolic processes including TCA cycle and the shikimate pathway.

14-3-3 proteins are regulatory proteins found in all eukaryotes and are known to selectively interact with phosphorylated proteins to regulate physiological processes. Through an affinity purification screening, many light-related proteins were recovered as 14-3-3 candidate binding partners. Yeast two-hybrid analysis revealed that the 14-3-3 kappa isoform (14-3-3κ) could bind to PHYTOCHROME INTERACTING FACTOR3 (PIF3) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). Further analysis by in vitro pull-down assay confirmed the interaction between 14-3-3κ and PIF3. Interruption of putative phosphorylation sites on the 14-3-3 binding motifs of PIF3 was not sufficient to inhibit 14-3-3κ from binding or to disturb nuclear localization of PIF3. It was also indicated that 14-3-3κ could bind to other members of the PIF family, such as PIF1 and PIF6, but not to LONG HYPOCOTYL IN FAR-RED1 (HFR1). 14-3-3 mutants, as well as the PIF3 overexpressor, displayed longer hypocotyls, and a pif3 mutant displayed shorter hypocotyls than the wild-type in red light, suggesting that 14-3-3 proteins are positive regulators of photomorphogenesis and function antagonistically with PIF3. Consequently, our results indicate that 14-3-3 proteins bind to PIFs and initiate photomorphogenesis in response to a light signal.

Plants have developed mechanisms to adapt to the potassium deficient conditions over the years. In Arabidopsis thaliana, expression of a potassium transporter HAK5 is induced in low potassium conditions as an adaptive response to nutrient deficiency. In order to understand the mechanism in which HAK5 is regulated, the full-length cDNA overexpressor gene hunting system was employed as a screening method. Of 40 genes recovered, At4g18280 was found to be dramatically induced in response to potassium-deficiency and salt stress. Plants overexpressing this gene showed higher HAK5 expression and enhanced growth. These plants were also less sensitive to potassium-deficiency in terms of primary root growth. Taken together, these data suggest that this novel component, At4g18280, contributes to regulation of HAK5 and, consequently, tolerance to potassium-deficiency in plants.