[Show abstract][Hide abstract] ABSTRACT: A collection of 175 melon (Cucumis melo L.) accessions (including wild relatives, feral types, landraces, breeding lines and commercial cultivars) from 50 countries was selected to study the phenotypic variability for ripening behavior and sugar accumulation. The variability of single nucleotide polymorphisms (SNPs) at 53 selected candidate genes involved in sugar accumulation and fruit ripening processes was studied, as well as their association with phenotypic variation of related traits.
The collection showed a strong genetic structure, defining seven groups plus a number of accessions that could not be associated to any of the groups (admixture), which fitted well with the botanical classification of melon varieties. The variability in candidate genes for ethylene, cell wall and sugar-related traits was high and similar to SNPs located in reference genes. Variability at ripening candidate genes had an important weight on the genetic stratification of melon germplasm, indicating that traditional farmers might have selected for ripening traits during cultivar diversification. A strong relationship was also found between the genetic structure and phenotypic diversity, which could hamper genetic association studies. Accessions belonging to the ameri group are the most appropriate for association analysis given the high phenotypic and molecular diversity within the group, and lack of genetic structure.
The most remarkable association was found between sugar content and SNPs in LG III, where a hotspot of sugar content QTLs has previously been defined. By studying the differences in allelic variation of SNPs within horticultural groups with specific phenotypic features, we also detected differential variation in sugar-related candidates located in LGIX and LGX, and in ripening-related candidates located in LGII and X, all in regions with previously mapped QTLs for the corresponding traits.
In the current study we have found an important variability at both the phenotypic and candidate gene levels for ripening behavior and sugar accumulation in melon fruit. By combination of differences in allelic diversity and association analysis, we have identified several candidate genes that may be involved in the melon phenotypic diversity.
[Show abstract][Hide abstract] ABSTRACT: In climacteric fruit-bearing species, the onset of fruit ripening is marked by a transient rise in respiration rate and autocatalytic ethylene production, followed by rapid deterioration in fruit quality. In non-climacteric species, there is no increase in respiration or ethylene production at the beginning or during fruit ripening. Melon is unusual in having climacteric and non-climacteric varieties, providing an interesting model system to compare both ripening types. Transcriptomic analysis of developing melon fruits from Védrantais and Dulce (climacteric) and Piel de sapo and PI 161375 (non-climacteric) varieties was performed to understand the molecular mechanisms that differentiate the two fruit ripening types.
Fruits were harvested at 15, 25, 35 days after pollination and at fruit maturity. Transcript profiling was performed using an oligo-based microarray with 75 K probes. Genes linked to characteristic traits of fruit ripening were differentially expressed between climacteric and non-climacteric types, as well as several transcription factor genes and genes encoding enzymes involved in sucrose catabolism. The expression patterns of some genes in PI 161375 fruits were either intermediate between. Piel de sapo and the climacteric varieties, or more similar to the latter. PI 161375 fruits also accumulated some carotenoids, a characteristic trait of climacteric varieties.
Simultaneous changes in transcript abundance indicate that there is coordinated reprogramming of gene expression during fruit development and at the onset of ripening in both climacteric and non-climacteric fruits. The expression patterns of genes related to ethylene metabolism, carotenoid accumulation, cell wall integrity and transcriptional regulation varied between genotypes and was consistent with the differences in their fruit ripening characteristics. There were differences between climacteric and non-climacteric varieties in the expression of genes related to sugar metabolism suggesting that they may be potential determinants of sucrose content and post-harvest stability of sucrose levels in fruit. Several transcription factor genes were also identified that were differentially expressed in both types, implicating them in regulation of ripening behaviour. The intermediate nature of PI 161375 suggested that classification of melon fruit ripening behaviour into just two distinct types is an over-simplification, and that in reality there is a continuous spectrum of fruit ripening behaviour.
[Show abstract][Hide abstract] ABSTRACT: Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a highly specific and sensitive technique for measuring metabolites. However, co-eluting components in tissue extracts can mutually interfere with ionization at the interface of the LC and MS/MS phases, potentially causing under- or over-estimation of metabolite concentrations. Spiking of samples with known amounts of stable isotope labelled internal standards (SIL-IS) allows measurements of the corresponding metabolites to be corrected for such matrix effects. We describe criteria for selection of suitable SIL-IS, and report the enzymatic synthesis and purification of nine SIL-IS for hexose-, pentose- and triose-phosphates, UDP-glucose and adenosine monophosphate (AMP). Along with commercially available SIL-IS for seven other metabolites, these were these were validated by LC-MS/MS analyses of extracts from leaves, non-photosynthetic plant tissues, mouse liver, and cells of Chlamydomonas reinhardtii, Escherichia coli and baker's yeast (Saccharomyces cerevisiae). With only a few exceptions, spiking with SIL-IS significantly improved the reproducibility of LC-MS/MS-based metabolite measurements across a wide range of extract dilutions, indicating effective correction for matrix effects by this approach. With a suitable tissue sampling procedure and use of SIL-IS to correct for matrix effects, LC-MS/MS offers unprecedented scope for reliable determination of photosynthetic and respiratory intermediates in a diverse range of organisms.
[Show abstract][Hide abstract] ABSTRACT: Trehalose metabolism is essential for normal growth and development in higher plants. It is synthesized in a two-step pathway catalyzed by trehalose-6-phosphate (Tre6P) synthase (TPS) and trehalose phosphatase. Arabidopsis thaliana has 11 TPS or TPS-like proteins, which belong to two distinct clades: class I (AtTPS1-AtTPS4) and class II (AtTPS5-AtTPS11). Only AtTPS1 has previously been shown to have TPS activity. Null A. thaliana tps1 mutants fail to complete embryogenesis and rescued lines have stunted growth and delayed flowering, indicating that AtTPS1 is important throughout the life cycle. Here we show that expression of AtTPS2 or AtTPS4 enables the yeast tps1∆ tps2∆ mutant to grow on glucose and accumulate Tre6P and trehalose. Class II TPS genes did not complement the yeast mutant. Thus, A. thaliana has at least three catalytically active TPS isoforms, suggesting that loss of Tre6P production might not be the only reason for the growth defects of A. thaliana tps1 mutants.
[Show abstract][Hide abstract] ABSTRACT: Mature leaves of plants transferred from low to high light typically increase their photosynthetic capacity. In Arabidopsis thaliana, this dynamic acclimation requires expression of GPT2, a glucose 6-phosphate/phosphate translocator. Here, we examine the impact of GPT2 on leaf metabolism and photosynthesis. Plants of wild-type and of a GPT2 knockout (gpt2.2) grown under low light achieved the same photosynthetic rate, despite having different metabolic and transcriptomic strategies. Immediately upon transfer to high light, gpt2.2 plants showed a higher rate of photosynthesis than wild-type (35%) however, over subsequent days, wild-type plants acclimated photosynthetic capacity, increasing photosynthesis 100 after 7 days. Wild-type plants accumulated more starch than gpt2.2 plants throughout acclimation. We suggest that GPT2 activity results in the net import of glucose 6-phosphate from cytosol to chloroplast, increasing starch synthesis. There was clear acclimation of metabolism, with short terms changes typically being reversed as plants acclimated. Distinct responses to light were seen in wild-type and gpt2.2 leaves. Significantly higher levels of sugar-phosphates were seen in gpt2.2. We suggest that GPT2 alters the distribution of metabolites between compartments and that this plays an essential role in allowing the cell to interpret environmental signals.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: The major component of starch is the branched glucan amylopectin. Structural features of amylopectin - the branching pattern and the chain length distribution - are thought to be key factors that enable it to form semi-crystalline starch granules. We varied both structural parameters by creating Arabidopsis mutants lacking combinations of starch synthases 1, 2 and 3 (to vary chain lengths) and the isoamylase 1 debranching enzyme (isa; to alter branching pattern). The isa mutant accumulates primarily phytoglycogen in leaf mesophyll cells, with only small amounts of starch in other cell types (epidermis and bundle sheath cells). This balance can be significantly shifted by mutating different starch synthases. Mutation of SS1 promoted starch synthesis, restoring granules in mesophyll cell plastids. Mutation of SS2 decreased starch synthesis, abolishing granules in epidermal and bundle sheath cells. Thus, the types of starch synthases present affect the crystallinity - and thus solubility - of the glucans made, compensating for or compounding the effects of an aberrant branching pattern. Interestingly, ss2 mutant plants contained small amounts of phytoglycogen in addition to aberrant starch. Likewise, ss2ss3 plants contained phytoglycogen, but were almost devoid of glucan despite retaining other SS isoforms. Surprisingly, in the ss2ss3isa triple mutants, glucan production was restored, indicating that in ss2ss3, starch synthase activity per se is not limiting but that the isoamylase suppresses glucan accumulation. We conclude that loss of only starch synthases can cause phytoglycogen production. This readily degraded by isoamylase and other enzymes so does not accumulate and was previously unnoticed.
[Show abstract][Hide abstract] ABSTRACT: Non-aqueous fractionation is a technique for enrichment of different subcellular compartments derived from lyophilised material. It was developed to study the subcellular distribution of metabolites. Here we analyse the distribution of about 1000 proteins and 70 metabolites, including 22 phosphorylated intermediates in wild-type Arabidopsis rosette leaves, using non-aqueous gradients divided into 12 fractions. Good separation of plastidial, cytosolic and vacuolar metabolites and proteins was achieved but cytosolic, mitochondrial and peroxisomal proteins clustered together. There was considerable heterogeneity in the fractional distribution of transcription factors, ribosomal proteins and subunits of the vacuolar-ATPase, indicating diverse compartmental location. Within the plastid, sub-organellar separation of thylakoids and stromal proteins was observed. Metabolites from the Calvin-Benson cycle, photorespiration, starch and sucrose synthesis, glycolysis and tricarboxylic acid cycle grouped with their associated proteins of the respective compartment. Non-aqueous fractionation thus proved a powerful method for the study of the organellar, and in some cases sub-organellar, distribution of proteins and their association with metabolites. It remains the technique of choice for assignment of subcellular location to metabolites in intact plant tissues and thus the technique of choice for doing combined metabolite-protein analysis in the same tissue sample.
[Show abstract][Hide abstract] ABSTRACT: Trehalose is a quantitatively important compatible solute and stress protectant in many organisms, including green algae and primitive plants. These functions have largely been replaced by sucrose in vascular plants, and trehalose metabolism has taken on new roles. Trehalose is a potential signal metabolite in plant interactions with pathogenic or symbiotic microorganisms, and herbivorous insects. It is also implicated in responses to cold and salinity, and in regulation of stomatal conductance and water use efficiency. In plants, as in other eukaryotes and many prokaryotes, trehalose is synthesised via a phosphorylated intermediate, trehalose 6-phosphate (Tre6P). A meta-analysis reveals that the levels of Tre6P change in parallel with sucrose, which is the major product of photosynthesis and the main transport sugar in plants. We propose a bi-directional network, in which Tre6P is a signal of sucrose availability, and acts to maintain sucrose concentrations within an appropriate range. Tre6P influences the relative amounts of sucrose and starch accumulated in leaves during the day, and regulates the rate of starch degradation at night to match demand for sucrose. Mutants in Tre6P metabolism have highly pleiotropic phenotypes, showing defects in embryogenesis, leaf growth, flowering, inflorescence branching and seed set. It has been proposed that Tre6P influences plant growth and development via inhibition of the SNF1-related protein kinase (SnRK1). However, current models conflict with some experimental data and do not completely explain the pleiotropic phenotypes exhibited by mutants in Tre6P metabolism. Additional explanations for the diverse effects of altering Tre6P metabolism are discussed. This article is protected by copyright. All rights reserved.
The Plant Journal 03/2014; 79(4). DOI:10.1111/tpj.12509 · 6.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, has a profound influence on plant metabolism, growth, and development. It has been proposed that Tre6P acts as a signal of sugar availability and is possibly specific for sucrose status. Short-term sugar-feeding experiments were carried out with carbon-starved Arabidopsis thaliana seedlings grown in axenic shaking liquid cultures. Tre6P increased when seedlings were exogenously supplied with sucrose, or with hexoses that can be metabolized to sucrose, such as glucose and fructose. Conditional correlation analysis and inhibitor experiments indicated that the hexose-induced increase in Tre6P was an indirect response dependent on conversion of the hexose sugars to sucrose. Tre6P content was affected by changes in nitrogen status, but this response was also attributable to parallel changes in sucrose. The sucrose-induced rise in Tre6P was unaffected by cordycepin but almost completely blocked by cycloheximide, indicating that de novo protein synthesis is necessary for the response. There was a strong correlation between Tre6P and sucrose even in lines that constitutively express heterologous trehalose-phosphate synthase or trehalose-phosphate phosphatase, although the Tre6P:sucrose ratio was shifted higher or lower, respectively. It is proposed that the Tre6P:sucrose ratio is a critical parameter for the plant and forms part of a homeostatic mechanism to maintain sucrose levels within a range that is appropriate for the cell type and developmental stage of the plant.
[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: Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants.
Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume.
We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape.
SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules.
New Phytologist 08/2013; 200(4). DOI:10.1111/nph.12455 · 7.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Starch synthase 4 (SS4) is required for proper starch granule initiation in Arabidopsis thaliana, although starch synthase 3 (SS3) can partially replace its function. Unlike other starch deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than wild type (WT) and lower maximal rates of photosynthesis. There is evidence of photo-oxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose than WT plants. Restricting ADP-glucose synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-glucose pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-glucose in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation leading to photo-oxidative stress, causing the chlorotic and stunted growth phenotypes of the plants.
[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.
The Plant Cell 02/2013; 25(2). DOI:10.1105/tpc.112.106989 · 9.58 Impact Factor