Crystal Sweetman

Regulation of malate metabolism in grape berry and other developing fruits

Organic acids are present in all plants, supporting numerous and varied facets of cellular metabolism. The type of organic acid found, and the levels to which they accumulate are extremely variable between species, developmental stages and tissue types. Acidity plays important roles in the organoleptic properties of plant tissues, where examples of both enhanced and reduced palatability can be ascribed to the presence of specific organic acids. In fruits, sourness is generally attributed to proton release from acids such as citric, malic, oxalic, quinic, succinic and tartaric, while the anion forms each contribute a distinct taste. Acidity imposes a strong influence on crop quality, and is an important factor in deciding the harvest date, particularly for fruits where acidity is important for further processing, as in wine grapes. In the grape, as for many other fruits, malate is one of the most prevalent acids, and is an important participant in numerous cellular functions. The accumulation of malate is thought to be due in large part to de novo synthesis in fruits such as the grape, through metabolism of assimilates translocated from leaf tissues, as well as photosynthetic activity within the fruit itself. During ripening, the processes through which malate is catabolised are of interest for advancing metabolic understanding, as well as for potential crop enhancement through agricultural or molecular practices. A body of literature describes research that has begun to unravel the regulatory mechanisms of enzymes involved in malate metabolism during fruit development, through exploration of protein and gene transcript levels. Datasets derived from a series of recent microarray experiments comparing transcript levels at several stages of grape berry development have been revisited, and are presented here with a focus on transcripts associated with malate metabolism. Developmental transcript patterns for enzymes potentially involved in grape malate metabolism have shown that some flux may occur through pathways that are less commonly regarded in ripening fruit, such as aerobic ethanol production. The data also suggest pyruvate as an important intermediate during malate catabolism in fruit. This review will combine an analysis of microarray data with information available on protein and enzyme activity patterns in grapes and other fruits, to explore pathways through which malate is conditionally metabolised, and how these may be controlled in response to developmental and climatic changes. Currently, an insufficient understanding of the complex pathways through which malate is degraded, and how these are regulated, prevents targeted genetic manipulation aimed at modifying fruit malate metabolism in response to environmental conditions.

Transcriptome analysis at four developmental stages of grape berry (Vitis vinifera cv. Shiraz) provides insights into regulated and coordinated gene expression

BackgroundVitis vinifera berry development is characterised by an initial phase where the fruit is small, hard and acidic, followed by a lag phase known as veraison. In the final phase, berries become larger, softer and sweeter and accumulate an array of organoleptic compounds. Since the physiological and biochemical makeup of grape berries at harvest has a profound impact on the characteristics of wine, there is great interest in characterising the molecular and biophysical changes that occur from flowering through veraison and ripening, including the coordination and temporal regulation of metabolic gene pathways. Advances in deep-sequencing technologies, combined with the availability of increasingly accurate V. vinifera genomic and transcriptomic data, have enabled us to carry out RNA-transcript expression analysis on a global scale at key points during berry development.ResultsA total of 162 million 100-base pair reads were generated from pooled Vitis vinifera (cv. Shiraz) berries sampled at 3-weeks post-anthesis, 10- and 11-weeks post-anthesis (corresponding to early and late veraison) and at 17-weeks post-anthesis (harvest). Mapping reads from each developmental stage (36-45 million) onto the NCBI RefSeq transcriptome of 23,720 V. vinifera mRNAs revealed that at least 75% of these transcripts were detected in each sample. RNA-Seq analysis uncovered 4,185 transcripts that were significantly upregulated at a single developmental stage, including 161 transcription factors. Clustering transcripts according to distinct patterns of transcription revealed coordination in metabolic pathways such as organic acid, stilbene and terpenoid metabolism. From the phenylpropanoid/stilbene biosynthetic pathway at least 46 transcripts were upregulated in ripe berries when compared to veraison and immature berries, and 12 terpene synthases were predominantly detected only in a single sample. Quantitative real-time PCR was used to validate the expression pattern of 12 differentially expressed genes from primary and secondary metabolic pathways.ConclusionsIn this study we report the global transcriptional profile of Shiraz grapes at key stages of development. We have undertaken a comprehensive analysis of gene families contributing to commercially important berry characteristics and present examples of co-regulation and differential gene expression. The data reported here will provide an invaluable resource for the on-going molecular investigation of wine grapes.

Manipulation of alternative oxidase can influence salt tolerance in Arabidopsis thaliana.

The growth and development of plants can be limited by environmental stresses such as salinity. It has been suggested that the non-phosphorylating alternative respiratory pathway in plants, mediated by the NAD(P)H dehydrogenase [NAD(P)H DH] and alternative oxidase (AOX), is important during environmental stresses. The involvement of this alternative pathway in a stress response may be linked to its capacity to uncouple carbon metabolism from adenylate control and/or the minimization of the formation of destructive reactive oxygen species (ROS). Salinity stress is a widespread, adverse environmental stress, which leads to an ionic imbalance, hyperosmotic stress and oxidative stress, the latter being the result of ROS formation. In this study, we show that salinity stress of Arabidopsis thaliana plants resulted in the formation of ROS, increased levels of Na+ in both the shoot and the root and an increase in transcription of Ataox1a, Atndb2 and Atndb4 genes, indicating the formation of an abridged non-phosphorylating electron transport chain in response to salinity stress. Furthermore, plants constitutively over-expressing Ataox1a, with increased AOX capacity, showed lower ROS formation, 30-40% improved growth rates and lower shoot Na+ content compared with controls, when grown under salinity stress conditions. Thus, more active AOX in roots and shoots can improve the salt tolerance of Arabidopsis as defined by its ability to grow more effectively in the presence of NaCl, and maintain lower shoot Na+ content. AOX does have an important role in stress adaptation in plants, and these results provide some validation of the hypothesis that AOX can play a critical role in cell re-programming under salinity stress.

Metabolic effects of elevated temperature on organic acid degradation in ripening Vitis vinifera fruit

Summary Experiments conducted under controlled conditions in vineyards and growth chambers demonstrated day- and night-specific responses of grape berry organic acid levels through altered TCA cycle and amino acid metabolism.

Annotation of gene function in citrus using gene expression information and co-expression networks

BackgroundThe genus Citrus encompasses major cultivated plants such as sweet orange, mandarin, lemon and grapefruit, among the world’s most economically important fruit crops. With increasing volumes of transcriptomics data available for these species, Gene Co-expression Network (GCN) analysis is a viable option for predicting gene function at a genome-wide scale. GCN analysis is based on a “guilt-by-association” principle whereby genes encoding proteins involved in similar and/or related biological processes may exhibit similar expression patterns across diverse sets of experimental conditions. While bioinformatics resources such as GCN analysis are widely available for efficient gene function prediction in model plant species including Arabidopsis, soybean and rice, in citrus these tools are not yet developed.ResultsWe have constructed a comprehensive GCN for citrus inferred from 297 publicly available Affymetrix Genechip Citrus Genome microarray datasets, providing gene co-expression relationships at a genome-wide scale (33,000 transcripts). The comprehensive citrus GCN consists of a global GCN (condition-independent) and four condition-dependent GCNs that survey the sweet orange species only, all citrus fruit tissues, all citrus leaf tissues, or stress-exposed plants. All of these GCNs are clustered using genome-wide, gene-centric (guide) and graph clustering algorithms for flexibility of gene function prediction. For each putative cluster, gene ontology (GO) enrichment and gene expression specificity analyses were performed to enhance gene function, expression and regulation pattern prediction. The guide-gene approach was used to infer novel roles of genes involved in disease susceptibility and vitamin C metabolism, and graph-clustering approaches were used to investigate isoprenoid/phenylpropanoid metabolism in citrus peel, and citric acid catabolism via the GABA shunt in citrus fruit.ConclusionsIntegration of citrus gene co-expression networks, functional enrichment analysis and gene expression information provide opportunities to infer gene function in citrus. We present a publicly accessible tool, Network Inference for Citrus Co-Expression (NICCE, http://citrus.adelaide.edu.au/nicce/home.aspx), for the gene co-expression analysis in citrus.

Two key polymorphisms in a newly discovered allele of the Vitis vinifera TPS24 gene are responsible for the production of the rotundone precursor α-guaiene

Highlight VvGuaS, a novel allele of the VvTPS24 gene, is responsible for the production of the rotundone precursor α-guaiene. Two specific polymorphisms distinguish VvGuaS from its non-guaiene-producing homologue VvPNSeInt.

New insights into the evolutionary history of plant sorbitol dehydrogenase

BackgroundSorbitol dehydrogenase (SDH, EC 1.1.1.14) is the key enzyme involved in sorbitol metabolism in higher plants. SDH genes in some Rosaceae species could be divided into two groups. L-idonate-5-dehydrogenase (LIDH, EC 1.1.1.264) is involved in tartaric acid (TA) synthesis in Vitis vinifera and is highly homologous to plant SDHs. Despite efforts to understand the biological functions of plant SDH, the evolutionary history of plant SDH genes and their phylogenetic relationship with the V. vinifera LIDH gene have not been characterized.ResultsA total of 92 SDH genes were identified from 42 angiosperm species. SDH genes have been highly duplicated within the Rosaceae family while monocot, Brassicaceae and most Asterid species exhibit singleton SDH genes. Core Eudicot SDHs have diverged into two phylogenetic lineages, now classified as SDH Class I and SDH Class II. V. vinifera LIDH was identified as a Class II SDH. Tandem duplication played a dominant role in the expansion of plant SDH family and Class II SDH genes were positioned in tandem with Class I SDH genes in several plant genomes. Protein modelling analyses of V. vinifera SDHs revealed 19 putative active site residues, three of which exhibited amino acid substitutions between Class I and Class II SDHs and were influenced by positive natural selection in the SDH Class II lineage. Gene expression analyses also demonstrated a clear transcriptional divergence between Class I and Class II SDH genes in V. vinifera and Citrus sinensis (orange).ConclusionsPhylogenetic, natural selection and synteny analyses provided strong support for the emergence of SDH Class II by positive natural selection after tandem duplication in the common ancestor of core Eudicot plants. The substitutions of three putative active site residues might be responsible for the unique enzyme activity of V. vinifera LIDH, which belongs to SDH Class II and represents a novel function of SDH in V. vinifera that may be true also of other Class II SDHs. Gene expression analyses also supported the divergence of SDH Class II at the expression level. This study will facilitate future research into understanding the biological functions of plant SDHs.

Alternative Respiratory Pathway Component Genes (AOX and ND) in Rice and Barley and Their Response to Stress

Plants have a non-energy conserving bypass of the classical mitochondrial cytochrome c pathway, known as the alternative respiratory pathway (AP). This involves type II NAD(P)H dehydrogenases (NDs) on both sides of the mitochondrial inner membrane, ubiquinone, and the alternative oxidase (AOX). The AP components have been widely characterised from Arabidopsis, but little is known for monocot species. We have identified all the genes encoding components of the AP in rice and barley and found the key genes which respond to oxidative stress conditions. In both species, AOX is encoded by four genes; in rice OsAOX1a, 1c, 1d and 1e representing four clades, and in barley, HvAOX1a, 1c, 1d1 and 1d2, but no 1e. All three subfamilies of plant ND genes, NDA, NDB and NDC are present in both rice and barley, but there are fewer NDB genes compared to Arabidopsis. Cyanide treatment of both species, along with salt treatment of rice and drought treatment of barley led to enhanced expression of various AP components; there was a high level of co-expression of AOX1a and AOX1d, along with NDB3 during the stress treatments, reminiscent of the co-expression that has been well characterised in Arabidopsis for AtAOX1a and AtNDB2.

Genomic structure and expression of alternative oxidase genes in legumes: Legume Alternative Oxidase

Mitochondria isolated from chickpea (Cicer arietinum) possess substantial alternative oxidase (AOX) activity, even in non-stressed plants, and one or two AOX protein bands were detected immunologically, depending on the organ. Four different AOX isoforms were identified in the chickpea genome: CaAOX1 and CaAOX2A, B and D. CaAOX2A was the most highly expressed form and was strongly expressed in photosynthetic tissues, whereas CaAOX2D was found in all organs examined. These results are very similar to those of previous studies with soybean and siratro. Searches of available databases showed that this pattern of AOX genes and their expression was common to at least 16 different legume species. The evolution of the legume AOX gene family is discussed, as is the in vivo impact of an inherently high AOX capacity in legumes on growth and responses to environmental stresses.