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.Fil: Quadrana, Leandro Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Almeida de Souza, Juliana Beatriz. Universidade de Sao Paulo; BrasilFil: Otaiza González, Santiago Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Duffy, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: da Silva, Junia V. Corrêa. Universidade de Sao Paulo; BrasilFil: de Godoy, Fabiana. Universidade de Sao Paulo; BrasilFil: Asis, Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Bermudez Salazar, Luisa Fernanda. Universidade de Sao Paulo; BrasilFil: Fernie, Alisdair R.. Institut Max Planck fur Molekulare Physiologie; AlemaniaFil: Carrari, Fernando Oscar. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Rossi, Maria Magdalena. Universidade de Sao Paulo; Brasi

The geometry of the forisome-sieve element-sieve plate complex in the phloem of Vicia faba L. leaflets

Forisomes are contractile protein bodies that appear to control flux rates in the phloem of faboid legumes by reversibly plugging the sieve tubes. Plugging is triggered by Ca2+ which induces an anisotropic deformation of forisomes, consisting of a longitudinal contraction and a radial expansion. By conventional light microscopy and confocal laser-scanning microscopy, the three-dimensional geometry of the forisome–sieve element–sieve plate complex in intact sieve tubes of leaflets of Vicia faba L. was reconstructed. Forisomes were mostly located close to sieve plates, and occasionally were observed drifting unrestrainedly along the sieve element, suggesting that they might be utilized as internal markers of flow direction. The diameter of forisomes in the resting state correlated with the diameter of their sieve elements, supporting the idea that radial expansion of forisomes is the geometric basis of reversible sieve tube plugging. Comparison of the present results regarding forisome geometry in situ with previously published data on forisome reactivity in vitro makes it questionable, however, whether forisomes are capable of completely sealing sieve tubes in V. faba leaves