Albert Batushansky

Metabolite and transcript profiling of berry skin during fruit development elucidates differential regulation between Cabernet Sauvignon and Shiraz cultivars at branching points in the polyphenol pathway

BackgroundGrapevine berries undergo complex biochemical changes during fruit maturation, many of which are dependent upon the variety and its environment. In order to elucidate the varietal dependent developmental regulation of primary and specialized metabolism, berry skins of Cabernet Sauvignon and Shiraz were subjected to gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) based metabolite profiling from pre-veraison to harvest. The generated dataset was augmented with transcript profiling using RNAseq.ResultsThe analysis of the metabolite data revealed similar developmental patterns of change in primary metabolites between the two cultivars. Nevertheless, towards maturity the extent of change in the major organic acid and sugars (i.e. sucrose, trehalose, malate) and precursors of aromatic and phenolic compounds such as quinate and shikimate was greater in Shiraz compared to Cabernet Sauvignon. In contrast, distinct directional projections on the PCA plot of the two cultivars samples towards maturation when using the specialized metabolite profiles were apparent, suggesting a cultivar-dependent regulation of the specialized metabolism. Generally, Shiraz displayed greater upregulation of the entire polyphenol pathway and specifically higher accumulation of piceid and coumaroyl anthocyanin forms than Cabernet Sauvignon from veraison onwards. Transcript profiling revealed coordinated increased transcript abundance for genes encoding enzymes of committing steps in the phenylpropanoid pathway. The anthocyanin metabolite profile showed F3′5′H-mediated delphinidin-type anthocyanin enrichment in both varieties towards maturation, consistent with the transcript data, indicating that the F3′5′H-governed branching step dominates the anthocyanin profile at late berry development. Correlation analysis confirmed the tightly coordinated metabolic changes during development, and suggested a source-sink relation between the central and specialized metabolism, stronger in Shiraz than Cabernet Sauvignon. RNAseq analysis also revealed that the two cultivars exhibited distinct pattern of changes in genes related to abscisic acid (ABA) biosynthesis enzymes.ConclusionsCompared with CS, Shiraz showed higher number of significant correlations between metabolites, which together with the relatively higher expression of flavonoid genes supports the evidence of increased accumulation of coumaroyl anthocyanins in that cultivar. Enhanced stress related metabolism, e.g. trehalose, stilbene and ABA in Shiraz berry-skin are consistent with its relatively higher susceptibility to environmental cues.

Metabolite Profiling and Integrative Modeling Reveal Metabolic Constraints for Carbon Partitioning under Nitrogen Starvation in the Green Algae Haematococcus pluvialis

Background: The microalga H. pluvialis shows a distinct pattern of carbon repartitioning upon nitrogen starvation. Results: Data-driven integrative modeling pinpoints metabolic adjustments underlying carbon repartition. Conclusion: In vitro and in silico experiments can dissect the systemic response to nitrogen starvation. Significance: Experimental data in conjunction with integrative modeling enables model-driven hypothesis testing. The green alga Hematococcus pluvialis accumulates large amounts of the antioxidant astaxanthin under inductive stress conditions, such as nitrogen starvation. The response to nitrogen starvation and high light leads to the accumulation of carbohydrates and fatty acids as well as increased activity of the tricarboxylic acid cycle. Although the behavior of individual pathways has been well investigated, little is known about the systemic effects of the stress response mechanism. Here we present time-resolved metabolite, enzyme activity, and physiological data that capture the metabolic response of H. pluvialis under nitrogen starvation and high light. The data were integrated into a putative genome-scale model of the green alga to in silico test hypotheses of underlying carbon partitioning. The model-based hypothesis testing reinforces the involvement of starch degradation to support fatty acid synthesis in the later stages of the stress response. In addition, our findings support a possible mechanism for the involvement of the increased activity of the tricarboxylic acid cycle in carbon repartitioning. Finally, the in vitro experiments and the in silico modeling presented here emphasize the predictive power of large scale integrative approaches to pinpoint metabolic adjustment to changing environments.

Growth Platform-Dependent and -Independent Phenotypic and Metabolic Responses of Arabidopsis and Its Halophytic Relative, Eutrema salsugineum , to Salt Stress

Eutrema salsugineum maintains its salt tolerance under very different growth conditions even though its development and metabolism show substantial growth condition-dependent differences. Comparative studies of the stress-tolerant Arabidopsis (Arabidopsis thaliana) halophytic relative, Eutrema salsugineum, have proven a fruitful approach to understanding natural stress tolerance. Here, we performed comparative phenotyping of Arabidopsis and E. salsugineum vegetative development under control and salt-stress conditions, and then compared the metabolic responses of the two species on different growth platforms in a defined leaf developmental stage. Our results reveal both growth platform-dependent and -independent phenotypes and metabolic responses. Leaf emergence was affected in a similar way in both species grown in vitro but the effects observed in Arabidopsis occurred at higher salt concentrations in E. salsugineum. No differences in leaf emergence were observed on soil. A new effect of a salt-mediated reduction in E. salsugineum leaf area was unmasked. On soil, leaf area reduction in E. salsugineum was mainly due to a fall in cell number, whereas both cell number and cell size contributed to the decrease in Arabidopsis leaf area. Common growth platform-independent leaf metabolic signatures such as high raffinose and malate, and low fumarate contents that could reflect core stress tolerance mechanisms, as well as growth platform-dependent metabolic responses were identified. In particular, the in vitro growth platform led to repression of accumulation of many metabolites including sugars, sugar phosphates, and amino acids in E. salsugineum compared with the soil system where these same metabolites accumulated to higher levels in E. salsugineum than in Arabidopsis. The observation that E. salsugineum maintains salt tolerance despite growth platform-specific phenotypes and metabolic responses suggests a considerable degree of phenotypic and metabolic adaptive plasticity in this extremophile.

Effects of Parental Temperature and Nitrate on Seed Performance are Reflected by Partly Overlapping Genetic and Metabolic Pathways

Seed performance is affected by the seed maturation environment, and previously we have shown that temperature, nitrate and light intensity were the most influential environmental factors affecting seed performance. Seeds developed in these environments were selected to assess the underlying metabolic pathways, using a combination of transcriptomics and metabolomics. These analyses revealed that the effects of the parental temperature and nitrate environments were reflected by partly overlapping genetic and metabolic networks, as indicated by similar changes in the expression levels of metabolites and transcripts. Nitrogen metabolism-related metabolites (asparagine, γ-aminobutyric acid and allantoin) were significantly decreased in both low temperature (15 °C) and low nitrate (N0) maturation environments. Correspondingly, nitrogen metabolism genes (ALLANTOINASE, NITRATE REDUCTASE 1, NITRITE REDUCTASE 1 and NITRILASE 4) were differentially regulated in the low temperature and nitrate maturation environments, as compared with control conditions. High light intensity during seed maturation increased galactinol content, and displayed a high correlation with seed longevity. Low light had a genotype-specific effect on cell surface-encoding genes in the DELAY OF GERMINATION 6-near isogenic line (NILDOG6). Overall, the integration of phenotypes, metabolites and transcripts led to new insights into the regulation of seed performance.

Paclobutrazol induces tolerance in tomato to deficit irrigation through diversified effects on plant morphology, physiology and metabolism

Dwindling water resources combined with meeting the demands for food security require maximizing water use efficiency (WUE) both in rainfed and irrigated agriculture. In this regard, deficit irrigation (DI), defined as the administration of water below full crop-water requirements (evapotranspiration), is a valuable practice to contain irrigation water use. In this study, the mechanism of paclobutrazol (Pbz)-mediated improvement in tolerance to water deficit in tomato was thoroughly investigated. Tomato plants were subjected to normal irrigated and deficit irrigated conditions plus Pbz application (0.8 and 1.6 ppm). A comprehensive morpho-physiological, metabolomics and molecular analysis was undertaken. Findings revealed that Pbz application reduced plant height, improved stem diameter and leaf number, altered root architecture, enhanced photosynthetic rates and WUE of tomato plants under deficit irrigation. Pbz differentially induced expression of genes and accumulation of metabolites of the tricarboxylic acid (TCA) cycle, γ-aminobutyric acid (GABA-shunt pathway), glutathione ascorbate (GSH-ASC)-cycle, cell wall and sugar metabolism, abscisic acid (ABA), spermidine (Spd) content and expression of an aquaporin (AP) protein under deficit irrigation. Our results suggest that Pbz application could significantly improve tolerance in tomato plants under limited water availability through selective changes in morpho-physiology and induction of stress-related molecular processes.

Metabolic and Physiological Responses of Shiraz and Cabernet Sauvignon (Vitis vinifera L.) to Near Optimal Temperatures of 25 and 35 °C.

Shiraz and Cabernet Sauvignon (Cs) grapevines were grown at near optimal temperatures (25 or 35 °C). Gas exchange, fluorescence, metabolic profiling and correlation based network analysis were used to characterize leaf physiology. When grown at 25 °C, the growth rate and photosynthesis of both cultivars were similar. At 35 °C Shiraz showed increased respiration, non-photochemical quenching and reductions of photosynthesis and growth. In contrast, Cs maintained relatively stable photosynthetic activity and growth regardless of the condition. In both cultivars, growth at 35 °C resulted in accumulations of secondary sugars (raffinose, fucose and ribulose) and reduction of primary sugars concentration (glucose, fructose and sucrose), more noticeably in Shiraz than Cs. In spite of similar patterns of metabolic changes in response to growth at 35 °C, significant differences in important leaf antioxidants and antioxidant precursors (DHA/ascorbate, quinates, cathechins) characterized the cultivar response. Correlation analysis reinforced Shiraz sensitivity to the 35 °C, showing higher number of newly formed edges at 35 °C and higher modularity in Shiraz as compared to Cs. The results suggest that the optimal growth temperatures of grapevines are cultivar dependent, and allow a first insight into the variability of the metabolic responses of grapevines under varied temperatures.

Network-guided GWAS improves identification of genes affecting free amino acids

A metabolic network-guided genome-wide association study of seed free amino acids facilitates the identification of a histidine-specific transporter in Arabidopsis. Amino acids are essential for proper growth and development in plants. Amino acids serve as building blocks for proteins but also are important for responses to stress and the biosynthesis of numerous essential compounds. In seed, the pool of free amino acids (FAAs) also contributes to alternative energy, desiccation, and seed vigor; thus, manipulating FAA levels can significantly impact a seed’s nutritional qualities. While genome-wide association studies (GWAS) on branched-chain amino acids have identified some regulatory genes controlling seed FAAs, the genetic regulation of FAA levels, composition, and homeostasis in seeds remains mostly unresolved. Hence, we performed GWAS on 18 FAAs from a 313-ecotype Arabidopsis (Arabidopsis thaliana) association panel. Specifically, GWAS was performed on 98 traits derived from known amino acid metabolic pathways (approach 1) and then on 92 traits generated from an unbiased correlation-based metabolic network analysis (approach 2), and the results were compared. The latter approach facilitated the discovery of additional novel metabolic interactions and single-nucleotide polymorphism-trait associations not identified by the former approach. The most prominent network-guided GWAS signal was for a histidine (His)-related trait in a region containing two genes: a cationic amino acid transporter (CAT4) and a polynucleotide phosphorylase resistant to inhibition with fosmidomycin. A reverse genetics approach confirmed CAT4 to be responsible for the natural variation of His-related traits across the association panel. Given that His is a semiessential amino acid and a potent metal chelator, CAT4 orthologs could be considered as candidate genes for seed quality biofortification in crop plants.

The transporter GAT1 plays an important role in GABA-mediated carbon-nitrogen interactions in Arabidopsis.

Glutamate derived γ-aminobutyric acid (GABA) is synthetized in the cytosol prior to delivery to the mitochondria where it is catabolized via the TCA cycle. GABA accumulates under various environmental conditions, but an increasing number of studies show its involvement at the crossroad between C and N metabolism. To assess the role of GABA in modulating cellular metabolism, we exposed seedlings of A. thaliana GABA transporter gat1 mutant to full nutrition medium and media deficient in C and N combined with feeding of different concentrations (0.5 and 1 mM) of exogenous GABA. GC-MS based metabolite profiling showed an expected effect of medium composition on the seedlings metabolism of mutant and wild type alike. That being said, a significant interaction between GAT1 deficiency and medium composition was determined with respect to magnitude of change in relative amino acid levels. The effect of exogenous GABA treatment on metabolism was contingent on both the medium and the genotype, leading for instance to a drop in asparagine under full nutrition and low C conditions and glucose under all tested media, but not to changes in GABA content. We additionally assessed the effect of GAT1 deficiency on the expression of glutamate metabolism related genes and genes involved in abiotic stress responses. These results suggest a role for GAT1 in GABA-mediated metabolic alterations in the context of the C-N equilibrium of plant cells.

Correlation-Based Network Generation, Visualization, and Analysis as a Powerful Tool in Biological Studies: A Case Study in Cancer Cell Metabolism

In the last decade vast data sets are being generated in biological and medical studies. The challenge lies in their summary, complexity reduction, and interpretation. Correlation-based networks and graph-theory based properties of this type of networks can be successfully used during this process. However, the procedure has its pitfalls and requires specific knowledge that often lays beyond classical biology and includes many computational tools and software. Here we introduce one of a series of methods for correlation-based network generation and analysis using freely available software. The pipeline allows the user to control each step of the network generation and provides flexibility in selection of correlation methods and thresholds. The pipeline was implemented on published metabolomics data of a population of human breast carcinoma cell lines MDA-MB-231 under two conditions: normal and hypoxia. The analysis revealed significant differences between the metabolic networks in response to the tested conditions. The network under hypoxia had 1.7 times more significant correlations between metabolites, compared to normal conditions. Unique metabolic interactions were identified which could lead to the identification of improved markers or aid in elucidating the mechanism of regulation between distantly related metabolites induced by the cancer growth.

Sulfite Oxidase Activity Is Essential for Normal Sulfur, Nitrogen and Carbon Metabolism in Tomato Leaves

Plant sulfite oxidase [SO; E.C.1.8.3.1] has been shown to be a key player in protecting plants against exogenous toxic sulfite. Recently we showed that SO activity is essential to cope with rising dark-induced endogenous sulfite levels in tomato plants (Lycopersicon esculentum/Solanum lycopersicum Mill. cv. Rheinlands Ruhm). Here we uncover the ramifications of SO impairment on carbon, nitrogen and sulfur (S) metabolites. Current analysis of the wild-type and SO-impaired plants revealed that under controlled conditions, the imbalanced sulfite level resulting from SO impairment conferred a metabolic shift towards elevated reduced S-compounds, namely sulfide, S-amino acids (S-AA), Co-A and acetyl-CoA, followed by non-S-AA, nitrogen and carbon metabolite enhancement, including polar lipids. Exposing plants to dark-induced carbon starvation resulted in a higher degradation of S-compounds, total AA, carbohydrates, polar lipids and total RNA in the mutant plants. Significantly, a failure to balance the carbon backbones was evident in the mutants, indicated by an increase in tricarboxylic acid cycle (TCA) cycle intermediates, whereas a decrease was shown in stressed wild-type plants. These results indicate that the role of SO is not limited to a rescue reaction under elevated sulfite, but SO is a key player in maintaining optimal carbon, nitrogen and sulfur metabolism in tomato plants.