[show abstract][hide abstract] ABSTRACT: To understand the molecular basis underlying increased triacylglycerol (TAG) accumulation in starchless (sta) Chlamydomonas reinhardtii mutants, we undertook comparative time-course transcriptomics of strains CC-4348 (sta6 mutant), CC-4349, a cell wall-deficient (cw) strain purported to represent the parental STA6 strain, and three independent STA6 strains generated by complementation of sta6 (CC-4565/STA6-C2, CC-4566/STA6-C4, and CC-4567/STA6-C6) in the context of N deprivation. Despite N starvation-induced dramatic remodeling of the transcriptome, there were relatively few differences (5 × 10(2)) observed between sta6 and STA6, the most dramatic of which were increased abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically isocitrate lyase, malate synthase, transaldolase, fructose bisphosphatase and phosphoenolpyruvate carboxykinase (encoded by ICL1, MAS1, TAL1, FBP1, and PCK1 respectively), suggestive of increased carbon movement toward hexose-phosphate in sta6 by upregulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities. Targeted metabolite analysis indicated increased succinate, malate, and Glc-6-P and decreased Fru-1,6-bisphosphate, illustrating the effect of these changes. Comparisons of independent data sets in multiple strains allowed the delineation of a sequence of events in the global N starvation response in C. reinhardtii, starting within minutes with the upregulation of alternative N assimilation routes and carbohydrate synthesis and subsequently a more gradual upregulation of genes encoding enzymes of TAG synthesis. Finally, genome resequencing analysis indicated that (1) the deletion in sta6 extends into the neighboring gene encoding respiratory burst oxidase, and (2) a commonly used STA6 strain (CC-4349) as well as the sequenced reference (CC-503) are not congenic with respect to sta6 (CC-4348), underscoring the importance of using complemented strains for more rigorous assignment of phenotype to genotype.
[show abstract][hide abstract] ABSTRACT: Next-generation technologies generate an overwhelming amount of gene sequence data. Efficient annotation tools are required, to make this data amenable to functional genomics analyses. The Mercator pipeline automatically assigns functional terms to protein or nucleotide sequences. It uses the MapMan "BIN" ontology, which is tailored for functional annotation of plant "omics" data. The classification procedure performs parallel sequence searches against reference databases, compiles the results, and computes the most likely MapMan BINs for each query. In the current version, the pipeline relies on manually curated reference classifications originating from the three reference organisms (Arabidopsis, Chlamydomonas, rice), various other plant species that have a reviewed SwissProt annotation, and more than 2000 protein domain and family profiles at InterPro, CDD and KOG. Functional annotations predicted by Mercator achieve accuracies above 90% when benchmarked against manual annotation. In addition to mapping files for direct use in the visualization software MapMan, Mercator provides graphical overview charts, detailed annotation information in a convenient web browser interface and a MapMan-to-GO translation Table to export results as GO terms. Mercator is available free of charge via http://mapman.gabipd.org/web/guest/app/Mercator.
Plant Cell and Environment 11/2013; · 5.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: In short photoperiods plants accumulate starch more rapidly in the light and degrade it more slowly at night, ensuring that their starch reserves last until dawn. To investigate the accompanying changes in the timing of growth, Arabidopsis was grown in a range of photoperiods and analysed for rosette biomass, photosynthesis, respiration, ribosome abundance, polysome loading, starch, and over 40 metabolites at dawn and dusk. The dataset was used to model growth rates in the daytime and night, and to identify metabolites that correlate with growth. Modelled growth rates and polysome loading were high in the daytime and at night in long photoperiods, but decreased at night in short photoperiods. Ribosome abundance was similar in all photoperiods. It is discussed how the amount of starch accumulated in the light period, the length of the night and maintenance costs interact to constrain growth at night in short photoperiods, and alter the strategy for optimising ribosome use. Significant correlations were found in the daytime and the night between growth rates and the levels of the sugar-signal trehalose 6-phosphate and the amino acid biosynthesis intermediate shikimate, identifying these metabolites as hubs in a network that coordinates growth with diurnal changes in the carbon supply.
[show abstract][hide abstract] ABSTRACT: Many plants accumulate substantial starch reserves in their leaves during the day, and remobilise them at night to provide carbon and energy for maintenance and growth. In this paper we explore the role of a sugar signalling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis leaves. Ethanol-induced overexpression of trehalose-phosphate synthase (TPS) during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2 to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher Pi content than granules from non-induced control plants, consistent with either disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown, or with inhibition of starch hydrolysis by β-amylase. Non-aqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4-7 µM in the cytosol, 0.2-0.5 µM in the chloroplasts and 0.05 µM in the vacuole. It is proposed that Tre6P is a component in a signalling pathway that mediates feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night.
[show abstract][hide abstract] ABSTRACT: In plants, sugars such as glucose act as signalling molecules that promote changes in gene expression programmes that impact on growth and development. Recent evidence has revealed the potential importance of controlling mRNA decay in some aspects of glucose-mediated regulatory responses suggesting a role of microRNAs (miRNAs) in these responses. In order to get a better understanding of glucose-mediated development modulation involving miRNA-related regulatory pathways, early seedling development of mutants impaired in miRNA biogenesis (hyl1-2 and dcl1-11) and miRNA activity (ago1-25) was evaluated. All mutants exhibited a glucose hyposensitive phenotype from germination up to seedling establishment, indicating that miRNA regulatory pathways are involved in the glucose-mediated delay of early seedling development. The expression profile of 200 miRNA primary transcripts (pri-miRs) was evaluated by large-scale quantitative real-time PCR profiling, which revealed that 38 pri-miRs were regulated by glucose. For several of them, the corresponding mature miRNAs are known to participate directly or indirectly in plant development, and their accumulation was shown to be co-regulated with the pri-miR by glucose. Furthermore, the expression of several miRNA target genes was found to be deregulated in response to glucose in the miRNA machinery mutants ago1-25, dcl1-11, and hyl1-2. Also, in these mutants, glucose promoted misexpression of genes for the three abscisic acid signalling elements ABI3, ABI4, and ABI5. Thus, miRNA regulatory pathways play a role in the adjustments of growth and development triggered by glucose signalling.
Journal of Experimental Botany 08/2013; · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plants respond to environmental changes often by reprogramming metabolic and stress-associated pathways. Homeostatic integration of signaling is a central requirement of life to ensure metabolic stability. Under diurnal conditions, properly timed rhythmic metabolism provides fitness benefits to plants. TIME FOR COFFEE is a circadian regulator known to be involved in clock resetting at dawn. Here we explored the mechanism of influence of TIC in plant growth and development, as initiated by a microarray analysis. This global profiling showed that a loss of TIC function causes a major reprogramming of gene expression that predicting numerous developmental, metabolic, and stress-related phenotypes. This led us to demonstrate that this mutant exhibits late flowering, a plastochron defect, and diverse anatomical phenotypes. We further observed a starch-excess phenotype and altered soluble carbohydrate levels. tic exhibited hypersensitivity to oxidative stress and abscisic acid, and this was associated to a striking resistance to drought. These phenotypes were connected to an increase in total glutathione levels that correlated with a readjustment of amino acids and polyamine pools. By comparatively analyzing our transcriptomic and metabolomic data, we concluded that TIC is a central element of plant homeostasis that integrates and coordinates developmental, metabolic, and environmental signals. This article is protected by copyright. All rights reserved.
[show abstract][hide abstract] ABSTRACT: Full text URL: http://www.plantmethods.com/content/9/1/21
Trehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar, carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic plant-microbe interactions, in responses to abiotic stress and in developmental signalling, but its precise functions are unknown. A major obstacle to investigating its function is the technical difficulty of measuring the very low levels of trehalose usually found in plant tissues, as most of the established trehalose assays lack sufficient specificity and/or sensitivity.
A kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose. Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose content of the tissue extract is determined from an internal calibration curve. The assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were >=88%. A. thaliana Col-0 rosettes contain about 20--30 nmol g-1FW of trehalose, increasing to about 50--60 nmol g-1FW in plants grown at 8[degree sign]C. Trehalose is not correlated with sucrose content, whereas a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6+/-2.6 nmol g-1FW and 19.0+/-12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43+/-0.85 and 6.16+/-3.45, respectively.
The fluorometric assay is highly specific for trehalose and sensitive enough to measure the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status. The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia or a downward shift in the trehalose:Tre6P ratio.
[show abstract][hide abstract] ABSTRACT: Growth is driven by newly fixed carbon in the light, but depends at night on reserves, like starch, that are laid down in the light. Unless plants coordinate their growth with diurnal changes in the carbon supply, they will experience acute carbon starvation during the night. Protein synthesis represents a major component of cellular growth. Polysome loading was investigated during the diurnal cycle, an extended night and low CO2 in Arabidopsis Col-0 and in the starchless pgm mutant. In Col-0, polysome loading was 60-70% in the light, 40-45% for much of the night and <20% in an extended night, whilst in pgm it fell to <25% early in the night. Quantification of rRNA species using qRT-PCR revealed that polysome loading remained high for much of the night in the cytosol, was strongly light-dependent in the plastid, and was always high in mitochondria. The rosette sucrose content correlated with overall and with cytosolic polysome loading. Ribosome abundance did not show significant diurnal changes. However, compared to Col-0, pgm had decreased and increased abundance of plastidic and mitochondrial ribosomes, respectively. Incorporation of label from 13CO2 into protein confirmed that protein synthesis continues at a diminished rate in the dark. Modelling revealed that a decrease in polysome loading at night is required to balance protein synthesis with the availability of carbon from starch breakdown. Costs are also reduced by using amino acids that accumulated in previous light period. These results uncover a tight coordination of protein synthesis with the momentary supply of carbon.
[show abstract][hide abstract] ABSTRACT: Photosynthesis is the basis for life, and its optimization is a key biotechnological aim given the problems of population explosion and environmental deterioration. We describe a method to resolve intracellular fluxes in intact Arabidopsis thaliana rosettes based on time-dependent labeling patterns in the metabolome. Plants photosynthesizing under limiting irradiance and ambient CO(2) in a custom-built chamber were transferred into a (13)CO(2)-enriched environment. The isotope labeling patterns of 40 metabolites were obtained using liquid or gas chromatography coupled to mass spectrometry. Labeling kinetics revealed striking differences between metabolites. At a qualitative level, they matched expectations in terms of pathway topology and stoichiometry, but some unexpected features point to the complexity of subcellular and cellular compartmentation. To achieve quantitative insights, the data set was used for estimating fluxes in the framework of kinetic flux profiling. We benchmarked flux estimates to four classically determined flux signatures of photosynthesis and assessed the robustness of the estimates with respect to different features of the underlying metabolic model and the time-resolved data set.
[show abstract][hide abstract] ABSTRACT: The timing of the induction of flowering determines to a large extent the reproductive success of plants. Plants integrate diverse environmental and endogenous signals to ensure the timely transition from vegetative growth to flowering. Carbohydrates are thought to play a crucial role in the regulation of flowering, and trehalose-6-phosphate (T6P) has been suggested to function as a proxy for carbohydrate status in plants. The loss of TREHALOSE-6-PHOSPHATE SYNTHASE 1 (TPS1) causes Arabidopsis thaliana to flower extremely late, even under otherwise inductive environmental conditions. This suggests that TPS1 is required for the timely initiation of flowering. We show that the T6P pathway affects flowering both in the leaves and at the shoot meristem, and integrate TPS1 into the existing genetic framework of flowering-time control.
[show abstract][hide abstract] ABSTRACT: Understanding molecular factors determining local adaptation is a key challenge, particularly relevant for plants, which are sessile organisms coping with a continuously fluctuating environment. Here we introduce a rigorous network-based approach for investigating the relation between geographic location of accessions and heterogeneous molecular phenotypes. We demonstrate for Arabidopsis accessions that not only genotypic variability but also flowering and metabolic phenotypes show a robust pattern of isolation-by-distance. Our approach opens new avenues to investigate relations between geographic origin and heterogeneous molecular phenotypes, like metabolite profiles, which can easily be obtained in species where genome data is not yet available.
[show abstract][hide abstract] ABSTRACT: Photorespiration makes oxygenic photosynthesis possible by scavenging 2-phosphoglycolate. Hence, compromising photorespiration impairs photosynthesis. We examined whether facilitating photorespiratory carbon flow in turn accelerates photosynthesis and found that overexpression of the H-protein of glycine decarboxylase indeed considerably enhanced net-photosynthesis and growth of Arabidopsis thaliana. At the molecular level, lower glycine levels confirmed elevated GDC activity in vivo, and lower levels of the CO(2) acceptor ribulose 1,5-bisphosphate indicated higher drain from CO(2) fixation. Thus, the photorespiratory enzyme glycine decarboxylase appears as an important feed-back signaller that contributes to the control of the Calvin-Benson cycle and hence carbon flow through both photosynthesis and photorespiration.
[show abstract][hide abstract] ABSTRACT: Diurnal cycles provide a tractable system to study the response of metabolism and growth to fluctuating temperatures. We reasoned that the response to daytime and night temperature may vary; while daytime temperature affects photosynthesis, night temperature affects use of carbon that was accumulated in the light. Three Arabidopsis thaliana accessions were grown in thermocycles under carbon-limiting conditions with different daytime or night temperatures (12 to 24 °C) and analyzed for biomass, photosynthesis, respiration, enzyme activities, protein levels, and metabolite levels. The data were used to model carbon allocation and growth rates in the light and dark. Low daytime temperature led to an inhibition of photosynthesis and an even larger inhibition of growth. The inhibition of photosynthesis was partly ameliorated by a general increase in protein content. Low night temperature had no effect on protein content, starch turnover, or growth. In a warm night, there is excess capacity for carbon use. We propose that use of this capacity is restricted by feedback inhibition, which is relaxed at lower night temperature, thus buffering growth against fluctuations in night temperature. As examples, the rate of starch degradation is completely temperature compensated against even sudden changes in temperature, and polysome loading increases when the night temperature is decreased.
The Plant Cell 06/2012; 24(6):2443-69. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent rapid advances in next generation RNA sequencing (RNA-Seq)-based provide researchers with unprecedentedly large data sets and open new perspectives in transcriptomics. Furthermore, RNA-Seq-based transcript profiling can be applied to non-model and newly discovered organisms because it does not require a predefined measuring platform (like e.g. microarrays). However, these novel technologies pose new challenges: the raw data need to be rigorously quality checked and filtered prior to analysis, and proper statistical methods have to be applied to extract biologically relevant information. Given the sheer volume of data, this is no trivial task and requires a combination of considerable technical resources along with bioinformatics expertise. To aid the individual researcher, we have developed RobiNA as an integrated solution that consolidates all steps of RNA-Seq-based differential gene-expression analysis in one user-friendly cross-platform application featuring a rich graphical user interface. RobiNA accepts raw FastQ files, SAM/BAM alignment files and counts tables as input. It supports quality checking, flexible filtering and statistical analysis of differential gene expression based on state-of-the art biostatistical methods developed in the R/Bioconductor projects. In-line help and a step-by-step manual guide users through the analysis. Installer packages for Mac OS X, Windows and Linux are available under the LGPL licence from http://mapman.gabipd.org/web/guest/robin.
Nucleic Acids Research 06/2012; 40(Web Server issue):W622-7. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The diversity of metabolites found in plants is by far greater than in most other organisms. Metabolic profiling techniques, which measure many of these compounds simultaneously, enabled investigating the regulation of metabolic networks and proved to be useful for predicting important agronomic traits. However, little is known about the genetic basis of metabolites in crops such as maize. Here, a set of 289 diverse maize inbred lines was genotyped with 56,110 SNPs and assayed for 118 biochemical compounds in the leaves of young plants, as well as for agronomic traits of mature plants in field trials. Metabolite concentrations had on average a repeatability of 0.73 and showed a correlation pattern that largely reflected their functional grouping. Genome-wide association mapping with correction for population structure and cryptic relatedness identified for 26 distinct metabolites strong associations with SNPs, explaining up to 32.0% of the observed genetic variance. On nine chromosomes, we detected 15 distinct SNP-metabolite associations, each of which explained more then 15% of the genetic variance. For lignin precursors, including p-coumaric acid and caffeic acid, we found strong associations (P values to ) with a region on chromosome 9 harboring cinnamoyl-CoA reductase, a key enzyme in monolignol synthesis and a target for improving the quality of lignocellulosic biomass by genetic engineering approaches. Moreover, lignin precursors correlated significantly with lignin content, plant height, and dry matter yield, suggesting that metabolites represent promising connecting links for narrowing the genotype-phenotype gap of complex agronomic traits.
Proceedings of the National Academy of Sciences 05/2012; 109(23):8872-7. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Many plants store part of their photosynthate as starch during the day and remobilise it to support metabolism and growth at night. Mutants unable to synthesize or degrade starch show strongly impaired growth except in long day conditions. In rapidly growing plants, starch turnover is regulated such that it is almost, but not completely, exhausted at dawn. There is increasing evidence that premature or incomplete exhaustion of starch turnover results in lower rates of plant growth. This review provides an update on the pathways for starch synthesis and degradation. We discuss recent advances in understanding how starch turnover and the use of carbon for growth is regulated during diurnal cycles, with special emphasis on the role of the biological clock. Much of the molecular and genetic research on starch turnover has been performed in the reference system Arabidopsis. This review considers to what extent information gained in this weed species maybe applicable to annual crops and perennial species.
Current opinion in plant biology 04/2012; 15(3):282-92. · 10.33 Impact Factor
[show abstract][hide abstract] ABSTRACT: Maize is both an exciting model organism in plant genetics and also the most important crop worldwide for food, animal feed and bioenergy production. Recent genome-wide association and metabolic profiling studies aimed to resolve quantitative traits to their causal genetic loci and key metabolic regulators. Here we present a complementary approach that exploits large-scale genomic and metabolic information to predict complex, highly polygenic traits in hybrid testcrosses. We crossed 285 diverse Dent inbred lines from worldwide sources with two testers and predicted their combining abilities for seven biomass- and bioenergy-related traits using 56,110 SNPs and 130 metabolites. Whole-genome and metabolic prediction models were built by fitting effects for all SNPs or metabolites. Prediction accuracies ranged from 0.72 to 0.81 for SNPs and from 0.60 to 0.80 for metabolites, allowing a reliable screening of large collections of diverse inbred lines for their potential to create superior hybrids.
[show abstract][hide abstract] ABSTRACT: Sucrose is the main transported form of carbon in several plant species, including Populus species. Sucrose metabolism in developing wood has therefore a central role in carbon partitioning to stem biomass. Half of the sucrose-derived carbon is in the form of fructose, but metabolism of fructose has received little attention as a factor in carbon partitioning to walls of wood cells. We show that RNAi-mediated reduction of FRK2 activity in developing wood of hybrid aspen (Populus tremula × tremuloides) led to the accumulation of soluble neutral sugars and a decrease in hexose phosphates and UDP-glucose, indicating that carbon flux to cell-wall polysaccharide precursors is decreased. Reduced FRK2 activity also led to thinner fiber cell walls with a reduction in the proportion of cellulose. No pleiotropic effects on stem height or diameter were observed. The results establish a central role for FRK2 activity in carbon flux to wood cellulose.
The Plant Journal 01/2012; 70(6):967-77. · 6.58 Impact Factor