Magdalena Rossi

Genomic distribution and characterization of EST-derived resistance gene analogs (RGAs) in sugarcane.

A large sugarcane EST (expressed sequence tag) project recently gave us access to 261,609 EST sequences from sugarcane, assembled into 81,223 clusters. Among these, we identified 88 resistance gene analogs (RGAs) based on their homology to typical pathogen resistance genes, using a stringent BLAST search with a threshold e-value of e−50. They included representatives of the three major groups of resistance genes with NBS/LRR, LRR or S/T KINASE domains. Fifty RGAs showed a total of 148 single-dose polymorphic RFLP markers, which could be located on the sugarcane reference genetic map (constructed in cultivar R570, 2n=~115). Fifty-five SSR loci corresponding to 134 markers in R570 were also mapped to enable the classification of the various haplotypes into homology groups. Several RGA clusters were found. One cluster of two LRR-like loci mapped close to the only disease resistance gene known so far in sugarcane, which confers resistance to common rust. Detailed sequence comparison between two NBS/LRR RGA clusters in relation to their orthologs in rice and maize suggests their polyphyletic origins, and indicates that the degree of divergence between paralogous RGAs in sugarcane can be larger than that from an ortholog in a distant species.

Natural occurring epialleles determine vitamin E accumulation in tomato fruits

Vitamin E (VTE) content is a low heritability nutritional trait for which the genetic determinants are poorly understood. Here, we focus on a previously detected major tomato VTE quantitative trait loci (QTL; mQTL(9-2-6)) and identify the causal gene as one encoding a 2-methyl-6-phytylquinol methyltransferase (namely VTE3(1)) that catalyses one of the final steps in the biosynthesis of γ- and α-tocopherols, which are the main forms of VTE. By reverse genetic approaches, expression analyses, siRNA profiling and DNA methylation assays, we demonstrate that mQTL(9-2-6) is an expression QTL associated with differential methylation of a SINE retrotransposon located in the promoter region of VTE3(1). Promoter DNA methylation can be spontaneously reverted leading to different epialleles affecting VTE3(1) expression and VTE content in fruits. These findings indicate therefore that naturally occurring epialleles are responsible for regulation of a nutritionally important metabolic QTL and provide direct evidence of a role for epigenetics in the determination of agronomic traits.

A candidate gene survey of quantitative trait loci affecting chemical composition in tomato fruit

In tomato, numerous wild-related species have been demonstrated to be untapped sources of valuable genetic variability, including pathogen-resistance genes, nutritional, and industrial quality traits. From a collection of S. pennellii introgressed lines, 889 fruit metabolic loci (QML) and 326 yield-associated loci (YAL), distributed across the tomato genome, had been identified previously. By using a combination of molecular marker sequence analysis, PCR amplification and sequencing, analysis of allelic variation, and evaluation of co-response between gene expression and metabolite composition traits, the present report, provides a comprehensive list of candidate genes co-localizing with a subset of 106 QML and 20 YAL associated either with important agronomic or nutritional characteristics. This combined strategy allowed the identification and analysis of 127 candidate genes located in 16 regions of the tomato genome. Eighty-five genes were cloned and partially sequenced, totalling 45 816 and 45 787 bases from S. lycopersicum and S. pennellii, respectively. Allelic variation at the amino acid level was confirmed for 37 of these candidates. Furthermore, out of the 127 gene-metabolite co-locations, some 56 were recovered following correlation of parallel transcript and metabolite profiling. Results obtained here represent the initial steps in the integration of genetic, genomic, and expressional patterns of genes co-localizing with chemical compositional traits of the tomato fruit.

Survey of transposable elements in sugarcane expressed sequence tags (ESTs)

The sugarcane expressed sequence tag (SUCEST) project has produced a large number of cDNA sequences from several plant tissues submitted or not to different conditions of stress. In this paper we report the result of a search for transposable elements (TEs) revealing a surprising amount of expressed TEs homologues. Of the 260,781 sequences grouped in 81,223 fragment assembly program (Phrap) clusters, a total of 276 clones showed homology to previously reported TEs using a stringent cut-off value of e-50 or better. Homologous clones to Copia/Ty1 and Gypsy/Ty3 groups of long terminal repeat (LTR) retrotransposons were found but no non-LTR retroelements were identified. All major transposon families were represented in sugarcane including Activator (Ac), Mutator (MuDR), Suppressor-mutator (En/Spm) and Mariner. In order to compare the TE diversity in grasses genomes, we carried out a search for TEs described in sugarcane related species O.sativa, Z. mays and S. bicolor. We also present preliminary results showing the potential use of TEs insertion pattern polymorphism as molecular markers for cultivar identification.

Genetic dissection of vitamin E biosynthesis in tomato

Vegetables are critical for human health as they are a source of multiple vitamins including vitamin E (VTE). In plants, the synthesis of VTE compounds, tocopherol and tocotrienol, derives from precursors of the shikimate and methylerythritol phosphate pathways. Quantitative trait loci (QTL) for α-tocopherol content in ripe fruit have previously been determined in an Solanum pennellii tomato introgression line population. In this work, variations of tocopherol isoforms (α, β, γ, and δ) in ripe fruits of these lines were studied. In parallel all tomato genes structurally associated with VTE biosynthesis were identified and mapped. Previously identified VTE QTL on chromosomes 6 and 9 were confirmed whilst novel ones were identified on chromosomes 7 and 8. Integrated analysis at the metabolic, genetic and genomic levels allowed us to propose 16 candidate loci putatively affecting tocopherol content in tomato. A comparative analysis revealed polymorphisms at nucleotide and amino acid levels between Solanum lycopersicum and S. pennellii candidate alleles. Moreover, evolutionary analyses showed the presence of codons evolving under both neutral and positive selection, which may explain the phenotypic differences between species. These data represent an important step in understanding the genetic determinants of VTE natural variation in tomato fruit and as such in the ability to improve the content of this important nutriceutical.

Transcriptional regulation of tocopherol biosynthesis in tomato.

Tocopherols, compounds with vitamin E (VTE) activity, are potent lipid-soluble antioxidants synthesized only by photosynthetic organisms. Their biosynthesis requires the condensation of phytyl-diphosphate and homogentisate, derived from the methylerythritol phosphate (MEP) and shikimate pathways (SK), respectively. These metabolic pathways are central in plant chloroplast metabolism and are involved in the biosynthesis of important molecules such as chlorophyll, carotenoids, aromatic amino-acids and prenylquinones. In the last decade, few studies have provided insights into the regulation of VTE biosynthesis and its accumulation. However, the pathway regulatory mechanism/s at mRNA level remains unclear. We have recently identified a collection of tomato genes involved in tocopherol biosynthesis. In this work, by a dedicated qPCR array platform, the transcript levels of 47 genes, including paralogs, were determined in leaves and across fruit development. Expression data were analyzed for correlation with tocopherol profiles by coregulation network and neural clustering approaches. The results showed that tocopherol biosynthesis is controlled both temporally and spatially however total tocopherol content remains constant. These analyses exposed 18 key genes from MEP, SK, phytol recycling and VTE-core pathways highly associated with VTE content in leaves and fruits. Moreover, genomic analyses of promoter regions suggested that the expression of the tocopherol-core pathway genes is trancriptionally coregulated with specific genes of the upstream pathways. Whilst the transcriptional profiles of the precursor pathway genes would suggest an increase in VTE content across fruit development, the data indicate that in the M82 cultivar phytyl diphosphate supply limits tocopherol biosynthesis in later fruit stages. This is in part due to the decreasing transcript levels of geranylgeranyl reductase (GGDR) which restricts the isoprenoid precursor availability. As a proof of concept, by analyzing a collection of Andean landrace tomato genotypes, the role of the pinpointed genes in determining fruit tocopherol content was confirmed. The results uncovered a finely tuned regulation able to shift the precursor pathways controlling substrate influx for VTE biosynthesis and overcoming endogenous competition for intermediates. The whole set of data allowed to propose that 1-deoxy-D-xylulose-5-phosphate synthase and GGDR encoding genes, which determine phytyl-diphosphate availability, together with enzyme encoding genes involved in chlorophyll-derived phytol metabolism appear as the most plausible targets to be engineered aiming to improve tomato fruit nutritional value.

Different mechanisms are responsible for chlorophyll dephytylation during fruit ripening and leaf senescence in tomato

Phytol hydrolysis during chlorophyll breakdown in tomato leaves is specifically catalyzed by pheophytinase, while during fruit ripening other, so far unknown, hydrolases are active. Chlorophyll breakdown occurs in different green plant tissues (e.g. during leaf senescence and in ripening fruits). For different plant species, the PHEOPHORBIDE A OXYGENASE (PAO)/phyllobilin pathway has been described to be the major chlorophyll catabolic pathway. In this pathway, pheophorbide (i.e. magnesium- and phytol-free chlorophyll) occurs as a core intermediate. Most of the enzymes involved in the PAO/phyllobilin pathway are known; however, the mechanism of dephytylation remains uncertain. During Arabidopsis (Arabidopsis thaliana) leaf senescence, phytol hydrolysis is catalyzed by PHEOPHYTINASE (PPH), which is specific for pheophytin (i.e. magnesium-free chlorophyll). By contrast, in fruits of different Citrus spp., chlorophyllase, hydrolyzing phytol from chlorophyll, was shown to be active. Here, we enlighten the process of chlorophyll breakdown in tomato (Solanum lycopersicum), both in leaves and fruits. We demonstrate the activity of the PAO/phyllobilin pathway and identify tomato PPH (SlPPH), which, like its Arabidopsis ortholog, was specifically active on pheophytin. SlPPH localized to chloroplasts and was transcriptionally up-regulated during leaf senescence and fruit ripening. SlPPH-silencing tomato lines were impaired in chlorophyll breakdown and accumulated pheophytin during leaf senescence. However, although pheophytin transiently accumulated in ripening fruits of SlPPH-silencing lines, ultimately these fruits were able to degrade chlorophyll like the wild type. We conclude that PPH is the core phytol-hydrolytic enzyme during leaf senescence in different plant species; however, fruit ripening involves other hydrolases, which are active in parallel to PPH or are the core hydrolases in fruits. These hydrolases remain unidentified, and we discuss the question of whether chlorophyllases might be involved.

Nitric Oxide, Ethylene, and Auxin Cross Talk Mediates Greening and Plastid Development in Deetiolating Tomato Seedlings.

Light-evoked cotyledon greening and chloroplast differentiation in deetiolating tomato seedlings are orchestrated by regulatory feedback loops involving phytochromes, nitric oxide, auxins, and ethylene. The transition from etiolated to green seedlings involves the conversion of etioplasts into mature chloroplasts via a multifaceted, light-driven process comprising multiple, tightly coordinated signaling networks. Here, we demonstrate that light-induced greening and chloroplast differentiation in tomato (Solanum lycopersicum) seedlings are mediated by an intricate cross talk among phytochromes, nitric oxide (NO), ethylene, and auxins. Genetic and pharmacological evidence indicated that either endogenously produced or exogenously applied NO promotes seedling greening by repressing ethylene biosynthesis and inducing auxin accumulation in tomato cotyledons. Analysis performed in hormonal tomato mutants also demonstrated that NO production itself is negatively and positively regulated by ethylene and auxins, respectively. Representing a major biosynthetic source of NO in tomato cotyledons, nitrate reductase was shown to be under strict control of both phytochrome and hormonal signals. A close NO-phytochrome interaction was revealed by the almost complete recovery of the etiolated phenotype of red light-grown seedlings of the tomato phytochrome-deficient aurea mutant upon NO fumigation. In this mutant, NO supplementation induced cotyledon greening, chloroplast differentiation, and hormonal and gene expression alterations similar to those detected in light-exposed wild-type seedlings. NO negatively impacted the transcript accumulation of genes encoding phytochromes, photomorphogenesis-repressor factors, and plastid division proteins, revealing that this free radical can mimic transcriptional changes typically triggered by phytochrome-dependent light perception. Therefore, our data indicate that negative and positive regulatory feedback loops orchestrate ethylene-NO and auxin-NO interactions, respectively, during the conversion of colorless etiolated seedlings into green, photosynthetically competent young plants.

Crop yield: challenges from a metabolic perspective

Considering the dual use of plants, as bio-factories for foods and feedstock for bio-refining, along with a rising world population, the plant biotechnology field is currently facing a dramatic challenge to develop crops with higher yield. Furthermore, convergent studies predict that global changes in climate will influence crop productivity by modifying most yield-associated traits. Here, we review recent advances in the understanding of plant metabolism directly or indirectly impacting on yield and provide an update of the different pathways proposed as targets for metabolic engineering aiming to optimize source-sink relationships.

Galacturonosyltransferase 4 silencing alters pectin composition and carbon partitioning in tomato

Pectin is a main component of the plant cell wall and is the most complex family of polysaccharides in nature. Its composition is essential for the normal growth and morphology pattern, as demonstrated by pectin-defective mutant phenotypes. Besides this basic role in plant physiology, in tomato, pectin structure contributes to very important quality traits such as fruit firmness. Sixty-seven different enzymatic activities have been suggested to be required for pectin biosynthesis, but only a few genes have been identified and studied so far. This study characterized the tomato galacturonosyltransferase (GAUT) family and performed a detailed functional study of the GAUT4 gene. The tomato genome harbours all genes orthologous to those described previously in Arabidopsis thaliana, and a transcriptional profile revealed that the GAUT4 gene was expressed at higher levels in developing organs. GAUT4-silenced tomato plants exhibited an increment in vegetative biomass associated with palisade parenchyma enlargement. Silenced fruits showed an altered pectin composition and accumulated less starch along with a reduced amount of pectin, which coincided with an increase in firmness. Moreover, the harvest index was dramatically reduced as a consequence of the reduction in the fruit weight and number. Altogether, these results suggest that, beyond its role in pectin biosynthesis, GAUT4 interferes with carbon metabolism, partitioning, and allocation. Hence, this cell-wall-related gene seems to be key in determining plant growth and fruit production in tomato.