Coupling Virus-Induced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of ""sectoring"" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.

Contrasting phylogenetic signals and evolutionary rates in floral traits of Neotropical lianas

The diversity of floral forms has long been considered a prime example of radiation through natural selection. However, little is still known about the evolution of floral traits, a critical piece of evidence for the understanding of the processes that may have driven flower evolution. We studied the pattern of evolution of quantitative floral traits in a group of Neotropical lianas (Bignonieae, Bignoniaceae) and used a time-calibrated phylogeny as basis to: (1) test for phylogenetic signal in 16 continuous floral traits; (2) evaluate the rate of evolution in those traits; and (3) reconstruct the ancestral state of the individual traits. Variation in floral traits among extant species of Bignonieae was highly explained by their phylogenetic history. However, opposite signals were found in floral traits associated with the attraction of pollinators (calyx and corolla) and pollen transfer (androecium and gynoecium), suggesting a differential role of selection in different floral whorls. Phylogenetic independent contrasts indicate that traits evolved at different rates, whereas ancestral character state reconstructions indicate that the ancestral size of most flower traits was larger than the mean observed sizes of the same traits in extant species. The implications of these patterns for the reproductive biology of Bignonieae are discussed. (C) 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102, 378-390.

The Tnt1 family member Retrosol copy number and structure disclose retrotransposon diversification in different Solanum species

Eukaryotic genome expansion/retraction caused by LTR-retrotransposon activity is dependent on the expression of full length copies to trigger efficient transposition and recombination-driven events. The Tnt1 family of retrotransposons has served as a model to evaluate the diversity among closely related elements within Solanaceae species and found that members of the family vary mainly in their U3 region of the long terminal repeats (LTRs). Recovery of a full length genomic copy of Retrosol was performed through a PCR-based approach from wild potato, Solanum oplocense. Further characterization focusing on both LTR sequences of the amplified copy allowed estimating an approximate insertion time at 2 million years ago thus supporting the occurrence of transposition cycles after genus divergence. Copy number of Tnt1-like elements in Solanum species were determined through genomic quantitative PCR whereby results sustain that Retrosol in Solanum species is a low copy number retrotransposon (1-4 copies) while Retrolyc1 has an intermediate copy number (38 copies) in S. peruvianum. Comparative analysis of retrotransposon content revealed no correlation between genome size or ploidy level and Retrosol copy number. The tetraploid cultivated potato with a cellular genome size of 1,715 Mbp harbours similar copy number per monoploid genome than other diploid Solanum species (613-884 Mbp). Conversely, S. peruvianum genome (1,125 Mbp) has a higher copy number. These results point towards a lineage specific dynamic flux regarding the history of amplification/activity of Tnt1-like elements in the genome of Solanum species.

Genomic Analysis of Wild Tomato Introgressions Determining Metabolism- and Yield-Associated Traits

With the aim of determining the genetic basis of metabolic regulation in tomato fruit, we constructed a detailed physical map of genomic regions spanning previously described metabolic quantitative trait loci of a Solanum pennellii introgression line population. Two genomic libraries from S. pennellii were screened with 104 colocated markers from five selected genomic regions, and a total of 614 bacterial artificial chromosome (BAC)/cosmids were identified as seed clones. Integration of sequence data with the genetic and physical maps of Solanum lycopersicum facilitated the anchoring of 374 of these BAC/cosmid clones. The analysis of this information resulted in a genome-wide map of a nondomesticated plant species and covers 10% of the physical distance of the selected regions corresponding to approximately 1% of the wild tomato genome. Comparative analyses revealed that S. pennellii and domesticated tomato genomes can be considered as largely colinear. A total of 1,238,705 bp from both BAC/cosmid ends and nine large insert clones were sequenced, annotated, and functionally categorized. The sequence data allowed the evaluation of the level of polymorphism between the wild and cultivated tomato species. An exhaustive microsynteny analysis allowed us to estimate the divergence date of S. pennellii and S. lycopersicum at 2.7 million years ago. The combined results serve as a reference for comparative studies both at the macrosyntenic and microsyntenic levels. They also provide a valuable tool for fine-mapping of quantitative trait loci in tomato. Furthermore, they will contribute to a deeper understanding of the regulatory factors underpinning metabolism and hence defining crop chemical composition.

Withanolides from Aureliana fasciculata var. fasciculata

In the first phytochemical study of the Aureliana genus (Solanaceae), two new withanolides, 1 and 2, together with two known sterols, were isolated from the MeOH extract of the leaves of Aureliana fasciculata var. fasciculata. The structures were established as (4S,22R)-16 alpha-acetoxy-5 beta,6 beta-epoxy-4 beta,17 alpha-dihydroxy-1-oxowitha-2,24-dienolide (aurelianolide A) and (4S,22R)-16 alpha-acetoxy-4 beta,17 alpha-dihydroxy-1-oxowitha-2,5,24-trienolide (aurelianolide B). The new compounds possessed the unusual 16 alpha,17 alpha-dioxygenated group and were fully characterized by spectroscopic techniques, including (1)H- and (13)C-NMR (DEPT), as well as 2D-NMR (HMBC, HMQC, (1)H, (1)H-COSY, NOESY) experiments, and HR-MS.