Determining novel functions of Arabidopsis 14-3-3 proteins in central metabolic processes

<p>Abstract</p> <p>Background</p> <p>14-3-3 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.</p> <p>Results</p> <p>In 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.</p> <p>Conclusion</p> <p>Taken 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.</p

Integrative analyses of genetic variation in enzyme activities of primary carbohydrate metabolism reveal distinct modes of regulation in Arabidopsis thaliana

Multiparallel QTL analysis of 15 Arabidopsis primary carbohydrate metabolism enzymes reveals that traits affecting primary metabolism are often correlated

A long photoperiod relaxes energy management in Arabidopsis leaf six

AbstractPlants adapt to the prevailing photoperiod by adjusting growth and flowering to the availability of energy. To understand the molecular changes involved in adaptation to a long-day condition we comprehensively profiled leaf six at the end of the day and the end of the night at four developmental stages on Arabidopsis thaliana plants grown in a 16h photoperiod, and compared the profiles to those from leaf 6 of plants grown in a 8h photoperiod. When Arabidopsis is grown in a long-day photoperiod individual leaf growth is accelerated but whole plant leaf area is decreased because total number of rosette leaves is restricted by the rapid transition to flowering. Carbohydrate measurements in long- and short-day photoperiods revealed that a long photoperiod decreases the extent of diurnal turnover of carbon reserves at all leaf stages. At the transcript level we found that the long-day condition has significantly reduced diurnal transcript level changes than in short-day condition, and that some transcripts shift their diurnal expression pattern. Functional categorisation of the transcripts with significantly different levels in short and long day conditions revealed photoperiod-dependent differences in RNA processing and light and hormone signalling, increased abundance of transcripts for biotic stress response and flavonoid metabolism in long photoperiods, and for photosynthesis and sugar transport in short photoperiods. Furthermore, we found transcript level changes consistent with an early release of flowering repression in the long-day condition. Differences in protein levels between long and short photoperiods mainly reflect an adjustment to the faster growth in long photoperiods. In summary, the observed differences in the molecular profiles of leaf six grown in long- and short-day photoperiods reveal changes in the regulation of metabolism that allow plants to adjust their metabolism to the available light. The data also suggest that energy management is in the two photoperiods fundamentally different as a consequence of photoperiod-dependent energy constraints

Single feature polymorphism (SFP)-based selective sweep identification and association mapping of growth-related metabolic traits in Arabidopsis thaliana

Background: Natural accessions of Arabidopsis thaliana are characterized by a high level of phenotypic variation that can be used to investigate the extent and mode of selection on the primary metabolic traits. A collection of 54 A. thaliana natural accession-derived lines were subjected to deep genotyping through Single Feature Polymorphism (SFP) detection via genomic DNA hybridization to Arabidopsis Tiling 1.0 Arrays for the detection of selective sweeps, and identification of associations between sweep regions and growth-related metabolic traits. Results: A total of 1,072,557 high-quality SFPs were detected and indications for 3,943 deletions and 1,007 duplications were obtained. A significantly lower than expected SFP frequency was observed in protein-, rRNA-, and tRNA-coding regions and in non-repetitive intergenic regions, while pseudogenes, transposons, and non-coding RNA genes are enriched with SFPs. Gene families involved in plant defence or in signalling were identified as highly polymorphic, while several other families including transcription factors are depleted of SFPs. 198 significant associations between metabolic genes and 9 metabolic and growth-related phenotypic traits were detected with annotation hinting at the nature of the relationship. Five significant selective sweep regions were also detected of which one associated significantly with a metabolic trait. Conclusions: We generated a high density polymorphism map for 54 A. thaliana accessions that highlights the variability of resistance genes across geographic ranges and used it to identify selective sweeps and associations between metabolic genes and metabolic phenotypes. Several associations show a clear biological relationship, while many remain requiring further investigation

Mild Reductions in Mitochondrial NAD- Dependent Isocitrate Dehydrogenase Activity Result in Altered Nitrate Assimilation and Pigmentation But Do Not Impact Growth

ABSTRACT Transgenic tomato (Solanum lycopersicum) plants were generated expressing a fragment of the mitochondrial NAD-dependent isocitrate dehydrogenase gene (SlIDH1) in the antisense orientation. The transgenic plants displayed a mild reduction in the activity of the target enzyme in the leaves but essentially no visible alteration in growth from the wild-type. Fruit size and yield were, however, reduced. These plants were characterized by relatively few changes in photosynthetic parameters, but they displayed a minor decrease in maximum photosynthetic efficiency (Fv/Fm). Furthermore, a clear reduction in flux through the tricarboxylic acid (TCA) cycle was observed in the transformants. Additionally, biochemical analyses revealed that the transgenic lines exhibited considerably altered metabolism, being characterized by slight decreases in the levels of amino acids, intermediates of the TCA cycle, photosynthetic pigments, starch, and NAD(P)H levels, but increased levels of nitrate and protein. Results from these studies show that even small changes in mitochondrial NAD-dependent isocitrate dehydrogenase activity lead to noticeable alterations in nitrate assimilation and suggest the presence of different strategies by which metabolism is reprogrammed to compensate for this deficiency

Defining the robust behaviour of the plant clock gene circuit with absolute RNA timeseries and open infrastructure

Our understanding of the complex, transcriptional feedback loops in the circadian clock mechanism has depended upon quantitative, timeseries data from disparate sources. We measure clock gene RNA profiles in Arabidopsis thaliana seedlings, grown with or without exogenous sucrose, or in soil-grown plants and in wild-type and mutant backgrounds. The RNA profiles were strikingly robust across the experimental conditions, so current mathematical models are likely to be broadly applicable in leaf tissue. In addition to providing reference data, unexpected behaviours included co-expression of PRR9 and ELF4, and regulation of PRR5 by GI. Absolute RNA quantification revealed low levels of PRR9 transcripts (peak approx. 50 copies cell−1) compared with other clock genes, and threefold higher levels of LHY RNA (more than 1500 copies cell−1) than of its close relative CCA1. The data are disseminated from BioDare, an online repository for focused timeseries data, which is expected to benefit mechanistic modelling. One data subset successfully constrained clock gene expression in a complex model, using publicly available software on parallel computers, without expert tuning or programming. We outline the empirical and mathematical justification for data aggregation in understanding highly interconnected, dynamic networks such as the clock, and the observed design constraints on the resources required to make this approach widely accessible

Genetic Analysis of Central Carbon Metabolism Unveils an Amino Acid Substitution That Alters Maize NAD-Dependent Isocitrate Dehydrogenase Activity

Background: Central carbon metabolism (CCM) is a fundamental component of life. The participating genes and enzymes are thought to be structurally and functionally conserved across and within species. Association mapping utilizes a rich history of mutation and recombination to achieve high resolution mapping. Therefore, applying association mapping in maize (Zea mays ssp. mays), the most diverse model crop species, to study the genetics of CCM is a particularly attractive system. Methodology/Principal Findings: We used a maize diversity panel to test the CCM functional conservation. We found heritable variation in enzyme activity for every enzyme tested. One of these enzymes was the NAD-dependent isocitrate dehydrogenase (IDH, E.C. 1.1.1.41), in which we identified a novel amino-acid substitution in a phylogenetically conserved site. Using candidate gene association mapping, we identified that this non-synonymous polymorphism was associated with IDH activity variation. The proposed mechanism for the IDH activity variation includes additional components regulating protein level. With the comparison of sequences from maize and teosinte (Zea mays ssp. Parviglumis), the maize wild ancestor, we found that some CCM genes had also been targeted for selection during maize domestication. Conclusions/Significance: Our results demonstrate the efficacy of association mapping for dissecting natural variation in primary metabolic pathways. The considerable genetic diversity observed in maize CCM genes underlies heritable phenotypic variation in enzyme activities and can be useful to identify putative functional sites