Role of the tomato TAGL1 gene in regulating fruit metabolites elucidated using RNA sequence and metabolomics analyses.

Fruit ripening is a complex biological process affecting fruit quality. In tomato the fruit ripening process is delicately regulated by transcription factors (TFs). Among these, the TOMATO AGAMOUS-LIKE 1 (TAGL1) gene plays an important role in both the development and ripening of fruit. In this study, the TAGL1 gene was successfully silenced by virus-induced gene silencing technology (VIGS), and the global gene expression and metabolites profiles of TAGL1-silenced fruits were analyzed by RNA-sequence analysis (RNA-seq) and liquid chromatography-mass spectrometry (LC-MS/MS). The TAGL1-silenced fruits phenotypically displayed an orange pericarp, which was in accordance with the results expected from the down-regulation of genes associated with carotenoid synthesis. Levels of several amino acids and organic acids were lower in the TAGL1-silenced fruits than in the wild-type fruits, whereas, α-tomatine content was greatly increased (more than 10-fold) in the TAGL1-silenced fruits compared to wild-type fruits. The findings of this study showed that TAGL1 not only regulates the ripening of tomato fruits, but also affects the synthesis and levels of nutrients in the fruit.

Transgenic Suppression of AGAMOUS Genes in Apple Reduces Fertility and Increases Floral Attractiveness.

We investigated the ability of RNA interference (RNAi) directed against two co-orthologs of AGAMOUS (AG) from Malus domestica (domestic apple, MdAG) to reduce the risks of invasiveness and provide genetic containment of transgenes, while also promoting the attractiveness of flowers for ornamental usage. Suppression of two MdAG-like genes, MdMADS15 and MdMADS22, led to the production of trees with highly showy, polypetalous flowers. These "double-flowers" had strongly reduced expression of both MdAG-like genes. Members of the two other clades within in the MdAG subfamily showed mild to moderate differences in gene expression, or were unchanged, with the level of suppression approximately proportional to the level of sequence identity between the gene analyzed and the RNAi fragment. The double-flowers also exhibited reduced male and female fertility, had few viable pollen grains, a decreased number of stigmas, and produced few viable seeds after cross-pollination. Despite these floral alterations, RNAi-AG trees with double-flowers set full-sized fruit. Suppression or mutation of apple AG-like genes appears to be a promising method for combining genetic containment with improved floral attractiveness.

α-synuclein aggregation reduces nigral myocyte enhancer factor-2D in idiopathic and experimental Parkinson's disease.

α-Synuclein is an abundant neuronal protein that has been linked to both normal synaptic function and neurodegenerative disease, in particular, Parkinson's disease (PD). Evidence from both in vitro and in vivo studies indicate that increased wild type or mutant α-synuclein can cause PD, but the molecular mechanisms that underlie α-synuclein-mediated neurotoxicity remain poorly understood. We reported here that myocyte enhancer factor 2D (MEF2D), a nuclear transcription factor known to promote neuronal survival, is down regulated in response α-synuclein accumulation and aggregation. Our data demonstrated that levels of cytoplasmic and nuclear MEF2D were significantly decreased in PD nigral neurons when compared to the nigra of age-matched controls and Alzheimer's disease (AD) cases. This decrease was significantly greater in the nigral neurons with α-synuclein inclusions. Viral vector-mediated overexpression of human α-synuclein in rats resulted in significantly decreased MEF2D in nigral neurons similar to what was seen in PD. The decline of MEF2D-immunoreactivity was associated with a reduction in TH-immunoreactivity. These results indicate that the neuronal survival factor MEF2D is decreased in human and experimental PD, and this decrease is specifically associated with α-synuclein accumulation and aggregation.

Regulatory polymorphism in transcription factor KLF5 at the MEF2 element alters the response to angiotensin II and is associated with human hypertension.

Krüppel-like factor 5 (KLF5) is a zinc-finger-type transcription factor that mediates the tissue remodeling in cardiovascular diseases, such as atherosclerosis, restenosis, and cardiac hypertrophy. Our previous studies have shown that KLF5 is induced by angiotensin II (AII), although the precise molecular mechanism is not yet known. Here we analyzed regulatory single nucleotide polymorphisms (SNPs) within the KLF5 locus to identify clinically relevant signaling pathways linking AII and KLF5. One SNP was located at -1282 bp and was associated with an increased risk of hypertension: subjects with the A/A and A/G genotypes at -1282 were at significantly higher risk for hypertension than those with the G/G genotype. Interestingly, a reporter construct corresponding to the -1282G genotype showed much weaker responses to AII than a construct corresponding to -1282A. Electrophoretic mobility shift, chromatin immunoprecipitation, and reporter assays collectively showed that the -1282 SNP is located within a functional myocyte enhancer factor 2 (MEF2) binding site, and that the -1282G genotype disrupts the site and reduces the AII responsiveness of the promoter. Moreover, MEF2 activation via reactive oxygen species and p38 mitogen-activated protein kinase induced KLF5 expression in response to AII, and KLF5 and MEF2 were coexpressed in coronary atherosclerotic plaques. These results suggest that a novel signaling and transcription network involving MEF2A and KLF5 plays an important role in the pathogenesis of cardiovascular diseases such as hypertension.

Identification of pharmacological inhibitors of the MEK5/ERK5 pathway.

We have identified two novel MEK5 inhibitors, BIX02188 and BIX02189, which inhibited catalytic function of purified, MEK5 enzyme. The MEK5 inhibitors blocked phosphorylation of ERK5, without affecting phosphorylation of ERK1/2 in sorbitol-stimulated HeLa cells. The compounds also inhibited transcriptional activation of MEF2C, a downstream substrate of the MEK5/ERK5 signaling cascade, in a cellular trans-reporter assay system. These inhibitors offer novel pharmacological tools to better characterize the role of the MEK5/ERK5 pathway in various biological systems.

Involvement of the histone acetyltransferase AtHAC1 in the regulation of flowering time via repression of FLOWERING LOCUS C in Arabidopsis.

Histone acetylation is an important posttranslational modification correlated with gene activation. In Arabidopsis (Arabidopsis thaliana), the histone acetyltransferase AtHAC1 is homologous to animal p300/CREB (cAMP-responsive element-binding protein)-binding proteins, which are the main histone acetyltransferases participating in many physiological processes, including proliferation, differentiation, and apoptosis. The functions of p300/CREB-binding proteins in animals are well characterized, whereas little is known about the roles of AtHAC1 in developmental control in Arabidopsis. Lesions in AtHAC1 caused pleiotropic developmental defects, including delayed flowering, a shortened primary root, and partially reduced fertility. Analysis of the molecular basis of late flowering in hac1 mutants showed that the hac1 plants respond normally to day length, gibberellic acid treatment, and vernalization. Furthermore, the expression level of the flowering repressor FLOWERING LOCUS C (FLC) is increased in hac1 mutants, indicating that the late-flowering phenotype of hac1 mutants is mediated by FLC. Since histone acetylation is usually associated with the activation of gene expression, histone modifications of FLC chromatin are not affected by mutations in HAC1 and expression levels of all known autonomous pathway genes are unchanged in hac1 plants, we propose that HAC1 affects flowering time by epigenetic modification of factors upstream of FLC.

The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis.

The acceleration of flowering by a long period of low temperature, vernalization, is an adaptation that ensures plants overwinter before flowering. Vernalization induces a developmental state that is mitotically stable, suggesting that it may have an epigenetic basis. The VERNALIZATION2 (VRN2) gene mediates vernalization and encodes a nuclear-localized zinc finger protein with similarity to Polycomb group (PcG) proteins of plants and animals. In wild-type Arabidopsis, vernalization results in the stable reduction of the levels of the floral repressor FLC. In vrn2 mutants, FLC expression is downregulated normally in response to vernalization, but instead of remaining low, FLC mRNA levels increase when plants are returned to normal temperatures. VRN2 function therefore stably maintains FLC repression after a cold treatment, serving as a mechanism for the cellular memory of vernalization.