New insights into carotenoid accumulation in tomato fruit = Nuevos aspectos sobre acumulación de carotenoides en el fruto del tomate

Abstract

Lycopene is an antioxidant carotenoid present in high amounts inside the ripe tomato fruit, in watermelon, red bell pepper and a few other red-pigmented fruits. Its health benefits, notably the prevention of prostate cancer and cardiovascular diseas, have cast attention on this molecule, justifying the study of its biosynthesis, regulation and accumulation. Biotechnological approaches aimed at increasing the lycopene content in tomato fruits or other plant leves have usually had limited results, which reflect to some extent the lack of knowledge regarding on how the accumulation occurs in vivo.

It has been long hypothesized that a number of “sink” structures must be formed during the ripening of the tomato fruit, as the green chloroplast differentiates into the red-coloured chromoplast.

In the present thesis, the elucidation of which are these sink structures is addressed. The subfractionation of the tomato fruit chromoplast has been addressed, in the hope of disambiguating the location of lycopene. Initially, a method for isolating tomato chromoplasts was pursued, but eventually a more general method has been set up with the aim of de-composing the whole chromoplast. So, a method was developed which allowed for the isolatiofng of the different membranous sub-fractions that are found inside the mature tomato fruit chromoplast.

The resulting fractions have been profiled at the level of carotenoids, tocopherols, galacto- and phopholipids, and protein. As well, a characterization of the fractions has been performed by electron microscopy. This has allowed for the identification of fractions containing pure plastoglobules, fractions containing a mixture of plastoglobules and lycopene crystals, and other membranous fractions with a more complex identity. It has determined that the majority of the lycopene was found in the crystal-containing fractions. It was as well found that plastoglobules contain a very high proportion of the carotenoid precursors phytoene and phytofluene. During the proteomics works, the concept “outlier proteome” is suggested, in complementareity to the concept of “core proteome”, and hypotheses are given regarding the finding of proteins in unexpected locations.

During the decourse of this work, a number of proteins involved in the accumulation of carotenoids inside the tomato chromoplast, as well as more generally related to plant stress resistance were identified. Those are the fibrillins, which had been reported to be a family of proteins which precisely bound to the possible sink structures that had been considered in this work. The lack of further structural, mechanistical and functional information on this family of proteins, has led to the bioinformatic, and biotechnological study of fibrillins. The quantitative and qualitative importance of some of them is strongly suggested during tomato fruit ripening. As well, the information vacuum regarding most of the family has been notably filled. Consensus sequences of al this family have been elaborated, and their characteristics elucidated. A general model describing the defining characteristic of the whole family has been elaborated, and a tentative structure is strongly suggested, even if only by bioinformatics means, supported by previous literature. This model casts new light into the study of these proteins, whose research evolved with slowness over recent years. The knowledge of their organization in regard to their function will lend a firm hand to boost further research in this field. In summary, it is believed that this work offers a number of novel apportations and hypothesis confirmations which give it value to empower further research.

El licopeno es un carotenoide antioxidante presente en altas cantidades en el interior del fruto de tomate maduro y algunos frutos rojos. Sus beneficios para la salud a nivel de prevención de cáncer de próstata y enfermedades cardiovasculares, han puesto de manifiesto el interés de la investigación sobre esta molécula.

Los enfoques biotecnológicos destinados a aumentar el contenido de licopeno en modelos vegetales por lo general han tenido pocos resultados. Aparentemente, el desconocimiento mecanístico de cómo y dónde se acumula el licopeno podría estar limitando el éxito de investigaciones centradas a nivel enzimático o biosintético.

En la presente tesis, se trata de confirmar y aclarar específicamente qué estructuras son las reponsables de la acumulación de licopeno en la maduración del fruto. Se ha desarrollado un método válido para aislar estructuras sumidero, y otras fracciones membranosas pertenecientes al cromoplasto. La caracterización de dichas fracciones ha permitido la asignación de una identidad conocida total o parcialmente, y ha permitido obtener conclusiones referidas a las estructuras sumidero estudiadas. Se ha determinado que la mayoría del licopeno encontrado estaba en fracciones que contienen cristales. Se ha determinado también que otra estructura, los plastoglobules está en fruto de tomate maduro muy enriquecida en precursores de la síntesis de carotenoides.

En el transcurso de este trabajo, se hallaron determinadas proteínas implicadas en la acumulación de carotenoides en los sumideros, así como relacionadas con la resistencia al estrés. Esta familia, las fibrillinas, ha sido caracterizada bioinformatica y biotecnológicamente, llenando un vacío existente en su campo. Se ha elucidad con éxito un modelo que describe de forma general esta familia, que hasta hace poco era conocida de forma muy limitada. Se considera que en este trabajo se han realizado aportaciones valiosas, respondiendo preguntas científicas que pueden potenciar nuevas labores de investigación en el campo de los carotenoides, y en la resistencia de la planta al estrés.