Targeted gene disruption coupled with metabolic screen approach to uncover the LEAFY COTYLEDON1-LIKE4 (L1L4) function in tomato fruit metabolism

Key message Functional analysis of tomato L1L4 master transcription factor resulted in important metabolic changes affecting tomato fruit quality. Abstract Tomato fruits from mutant lines bearing targeted disruption of the heterotrimeric nuclear transcription factor Y (NF-Y) transcription factor (TF) gene LEAFY-COTYLEDON1-LIKE4 (L1L4, NF-YB6), a master regulator of biosynthesis for seed storage proteins and fatty acids, were evaluated for metabolites content and morphology. Metabolic screens using LC-MS/MS-based analysis and physico-chemical methods in different L1L4 mutants of the fourth generation allowed a comparative assessment of the effects of the TF disruption. Mutagenesis resulted in fruits phenotypically similar to wild-type with subtle shape differences in the distal end protrusion and symmetry. Conversely, mutant fruits from independent lines had significant variation in moisture content, titratable acidity and overall metabolite profiles including oxalic and citric acid, fructose, beta-carotene, total polyphenols and antioxidants. Lines 6, 7 and 9 were the richest in beta-carotene and antioxidant activity, line 4 in ascorbic acid and lines 4 and 8 in succinic acid. The reduced content of the anti-nutrient oxalic acid in several mutant fruits suggests that L1L4 gene may regulate the accumulation of this compound during fruit development. Detailed LC-MS/MS analysis of mutant seeds showed substantial differences in bioactive compounds compared to wild-type seeds. Taken together, the results suggest that the L1L4 TF is a significant regulator of metabolites both in tomato fruit and seeds providing a molecular target for crop improvement. Elucidation of the candidate genes encoding key enzymes in the affected metabolic pathways aimed to facilitate the L1L4 gene network exploration and eventually lead to systems biology approaches in tomato fruit quality.European funded COST ACTION [FA1106

GSK-3 kinases enhance calcineurin signaling by phosphorylation of RCNs

The conserved RCN family of proteins can bind and directly regulate calcineurin, a Ca(2+)-activated protein phosphatase involved in immunity, heart growth, muscle development, learning, and other processes. Whereas high levels of RCNs can inhibit calcineurin signaling in fungal and animal cells, RCNs can also stimulate calcineurin signaling when expressed at endogenous levels. Here we show that the stimulatory effect of yeast Rcn1 involves phosphorylation of a conservedserine residue by Mck1, a member of the GSK-3 family of protein kinases. Mutations at the GSK-3 consensus site of Rcn1 and human DSCR1/MCIP1 abolish the stimulatory effects on calcineurin signaling. RCNs may therefore oscillate between stimulatory and inhibitory forms in vivo in a manner similar to the Inhibitor-2 regulators of type 1 protein phosphatase. Computational modeling indicates a biphasic response of calcineurin to increasing RCN concentration such that protein phosphatase activity is stimulated by low concentrations of phospho-RCN and inhibited by high concentrations of phospho- or dephospho-RCN. This prediction was verified experimentally in yeast cells expressing Rcn1 or DSCR1/MCIP1 at different concentrations. Through the phosphorylation of RCNs, GSK-3 kinases can potentially contribute to a positive feedback loop involving calcineurin-dependent up-regulation of RCN expression. Such feedback may help explain the large induction of DSCR1/MCIP1 observed in brain of Down syndrome individuals

Comprehensive approaches reveal key transcripts and metabolites highlighting metabolic diversity among three oriental tobacco varieties

Tobacco is an economically important crop with leaves that are a significant source of aromatic, bioactive compounds such as phenolics and alkaloids. In the present study un-biased transcriptomics and metabolomics approaches were employed to identify and quantify individual changes in transcript and metabolite profiles in leaves of three oriental tobacco varieties. Based on next generation sequencing (NGS) and gas chromatography mass spectrometry (GC–MS) technologies, a wide variety of transcripts and metabolites was detected and the metabolic diversity among varieties was determined. Genes with largest expression differences were identified in the leaves of the three varieties; among them three were commonly over-expressed in two varieties in comparison with the third variety. Notably, significant expression differences were recorded in phenylalanine ammonia lyase (PAL) genes that are key genes of phenylpropanoid biosynthesis. Following transcriptomics, metabolomics analysis has shown that polyphenolic compounds varied widely among the three varieties. Furthermore, statistically significant differences in soluble sugars, alcohols, organic acids, amino acids and other metabolites were also revealed. The integration of the two -omics datasets in determining diversity of tobacco varieties offers important readouts for the genetic control of metabolite production and constitutes a resource for future studies in the area of plant biotechnology for improving tobacco specific traits