Juliana Almeida

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.

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.

Third-order nonlinear spectra and optical limiting of lead oxifluoroborate glasses

We have determined two-photon absorption and nonlinear refraction spectra of the 50BO(1.5) - (50-x)PbF(2) - xPbO glasses (with x = 25, 35, 50 cationic %) at the range of the 470 and 1550 nm. The replacement of fluor atoms by oxygen leads to an increase in the third-order susceptibility, due to the formation of non-bridging oxygens (NBO). The nonlinear index of refraction is one order of magnitude higher than the one for fused silica, and it increases almost twice for the sample with x = 50. This sample has also shown promising features for all-optical switching as well as for optical limiting.

Generation of copper nanoparticles induced by fs-laser irradiation in borosilicate glass

Glasses containing metallic nanoparticles are promising materials for technological applications in optics and photonics. Although several methods are available to generate nanoparticles in glass, only femtosecond lasers allow controlling it three-dimensionally. In this direction, the present work investigates the generation of copper nanoparticles on the surface and in the bulk of a borosilicate glass by fs-laser irradiation. We verified the formation of copper nanoparticles, after heat treatment, by UV-Vis absorption, transmission electron microscopy and electron diffraction. A preferential growth of copper nanoparticles was observed in the bottom of the irradiated region, which was attributed to self-focusing in the glass.

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.

Down-regulation of tomato PHYTOL KINASE strongly impairs tocopherol biosynthesis and affects prenyllipid metabolism in an organ-specific manner

Highlight Phytol kinase plays a key role in the regulation of isoprenoid metabolism in an organ-specific manner.

Metallic nanoparticles grown in the core of femtosecond laser micromachined waveguides

3D-waveguides containing silver nanoparticles have been fabricated in tungsten lead–pyrophosphate glass by femtosecond laser micromachining. Nucleation and growth of nanoparticles occur in a single step process when high repetition rate laser (MHz) is employed, while an additional annealing is required for the irradiation using kHz laser system. The presence of nanoparticles locally changes the refractive index, and, therefore, the elliptical structures produced by direct laser writing were able to guide light. By increasing the pulse energy applied during the micromachining, the waveguide size increased from 2 to 30 μm, while their propagation loss decrease from 1.4 to 0.5 dB/mm at 632.8 nm.

Fabrication of uniformly dispersed nanoparticle-doped chalcogenide glass

The dispersion of metallic nanoparticles within a chalcogenide glass matrix has the potential for many important applications in active and passive optical materials. However, the challenge of particle agglomeration, which can occur during traditional thin film processing, leads to materials with poor performance. Here, we report on the preparation of a uniformly dispersed Ag-nanoparticle (Ag NP)/chalcogenide glass heterogeneous material prepared through a combined laser- and solution-based process. Laser ablation of bulk silver is performed directly within an arsenic sulfide/propylamine solution resulting in the formation of Ag NPs in solution with an average particle size of less than 15 nm as determined by dynamic light scattering. The prepared solutions are fabricated into thin films using standard coating processes and are then analyzed using energy-dispersive X-ray spectroscopy and transmission electron microscopy to investigate the particle shape and size distribution. By calculating the nearest neighbor index and standard normal deviate of the nanoparticle locations inside the films, we verify that a uniformly dispersed distribution is achieved through this process.

Pheophytinase Knockdown Impacts Carbon Metabolism and Nutraceutical Content Under Normal Growth Conditions in Tomato.

Although chlorophyll (Chl) degradation is an essential biochemical pathway for plant physiology, our knowledge regarding this process still has unfilled gaps. Pheophytinase (PPH) was shown to be essential for Chl breakdown in dark-induced senescent leaves. However, the catalyzing enzymes involved in pigment turnover and fruit ripening-associated degreening are still controversial. Chl metabolism is closely linked to the biosynthesis of other isoprenoid-derived compounds, such as carotenoids and tocopherols, which are also components of the photosynthetic machinery. Chls, carotenoids and tocopherols share a common precursor, geranylgeranyl diphosphate, produced by the plastidial methylerythritol 4-phosphate (MEP) pathway. Additionally, the Chl degradation-derived phytol can be incorporated into tocopherol biosynthesis. In this context, tomato turns out to be an interesting model to address isoprenoid-metabolic cross-talk since fruit ripening combines degreening and an intensely active MEP leading to carotenoid accumulation. Here, we investigate the impact of PPH deficiency beyond senescence by the comprehensive phenotyping of SlPPH-knockdown tomato plants. In leaves, photosynthetic parameters indicate altered energy usage of excited Chl. As a mitigatory effect, photosynthesis-associated carotenoids increased while tocopherol content remained constant. Additionally, starch and soluble sugar profiles revealed a distinct pattern of carbon allocation in leaves that suggests enhanced sucrose exportation. The higher levels of carbohydrates in sink organs down-regulated carotenoid biosynthesis. Additionally, the reduction in Chl-derived phytol recycling resulted in decreased tocopherol content in transgenic ripe fruits. Summing up, tocopherol and carotenoid metabolism, together with the antioxidant capacity of the hydrophilic and hydrophobic fractions, were differentially affected in leaves and fruits of the transgenic plants. Thus, in tomato, PPH plays a role beyond senescence-associated Chl degradation that, when compromised, affects isoprenoid and carbon metabolism which ultimately alters the fruits nutraceutical content.