Doctoral Degrees (Viticulture and Oenology)

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Browsing Doctoral Degrees (Viticulture and Oenology) by browse.metadata.advisor "Asaph, Aharoni"

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    Cross species identification of genetic factors that determine fruit surface characteristics
    (Stellenbosch : Stellenbosch University, 2015-12) Lashbrooke, Justin Graham; Asaph, Aharoni; Costa, Fabrizio; Vivier, Melane A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: The plant cuticle is a typically waxy layer that covers the entire above ground part of higher plants and performs a number of important roles in vegetative organs and during fruit development and ripening, including protection from a range of abiotic and biotic stresses. This complex hydrophobic layer consists of a cutin matrix of predominantly fatty acids embedded with long chain waxes, synthesized in (and secreted from) the epidermal cells. Suberin, an aliphatic polymer which is higher in phenolic content than cutin and less elastic, may be formed due to wounding and/or cuticle damage. While the synthesis and transport of cutin and wax have been investigated for a number of decades, there are still steps in these pathways that are yet to be elucidated. In the case of suberin biosynthesis, much less has been described. The understanding of the regulatory mechanisms for these processes has only recently received attention, and has already proven to be an area of significant interest for plant scientists, particularly with regard to the interaction between the regulation of epidermal cell identity and cuticle development. In terms of fruit production the cuticle can be linked to many quality parameters, especially postharvest storage. This is particularly true for apple, a crop dependent on a long shelf life. Apple is also susceptible to the formation of a cuticle failure disorder known as russet. Apple russet results from micro-cracking of the cuticle and the formation of a corky suberized layer. During normal growth suberin can be found in potato tubers, roots, bark, and seed coat. However, on most fruit, suberin is typically an undesirable consumer trait, and in the case of apples can have a negative impact on the postharvest storage. In this work, the current understanding of plant cuticles is built upon with particular emphasis on fleshy fruit cuticle assembly and the regulation of the biosynthetic pathways generating its constituents. Research goals focused on identifying and characterizing genetic factors involved in fruit surface formation, particularly with regard to cuticle biosynthesis. Further attention is paid to understanding the regulatory mechanisms of cuticular pathways, including the emerging concept of co-regulation of epidermal cell differentiation and cuticle biosynthesis. Tomato and apple were the species in which these investigations were focused. This was due to the fact that tomato is seen as a model species for fruit surface biology research, while the applied aspects of apple surface research have far reaching impact. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface the tomato SlMIXTA-like gene was investigated. MIXTA/MIXTA-like proteins, initially described in snapdragon petals, are regulators of epidermal cell differentiation. In an effort to understand these processes in fruit, tomato was transgenically silenced for SlMIXTA-like expression. Plants displayed defects in the patterning of conical epidermal cells of fruit, and also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling, coupled with comprehensive microscopy, revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes acting downstream of SlMIXTA-like included oxidases, transferases and transporters involved in cutin synthesis and assembly. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, it was found that SlMIXTA-like acts downstream of the cutin biosynthesis regulator SlSHN3 and possibly co-operates with homeodomain-leucine zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly and epidermal cell patterning. While russeting may occur in apples after cuticle damage, it is also a heritable trait, and therefore is to some extent under genetic control. In order to identify genetic factors controlling cuticle biosynthesis in apple (and thus preventing russet), a QTLmapping survey was performed on a full-sib population. Two genomic regions located on chromosome 2 and 15 that could be associated with russeting were identified. Apples with compromised cuticles were identified through a novel and high throughput tensile analysis of the skin, while histological analysis confirmed cuticle failure in a subset of the progeny. Additional genomic investigation of the determined QTL regions identified a set of underlying genes involved in cuticle biosynthesis. Candidate gene expression profiling by qRT-PCR on a subset of the progeny highlighted the specific expression pattern of a SHN1/WIN1 transcription factor (termed MdSHN3) on chromosome 15. The MdSHN3 transcription factor displayed extremely low expression in lines with improper cuticle formation suggesting it to be a fundamental regulator of cuticle biosynthesis in apple fruit, and thus necessary for the prevention of suberized fruit surfaces (russet). In an effort to gain a greater understanding of the mechanisms underlying suberin biosynthesis in fruit, and in plants in general, transgenic tomato were generated with compromised cutin formation. This was achieved via the transcriptional silencing of SlDCR, an orthologue to AtDCR which was previously identified as a key step in cutin biosynthesis in Arabidopsis. Silencing of this BAHD acyltransferase resulted in an almost total elimination of the major monomer from the fruit cutin (C16-9,10-dihydroxy fatty acid), and the plants developed fruit with a suberized surface. This provided an excellent opportunity for transcriptome and chemical characterization of the suberization process in fleshy fruit. In parallel an apple clone that developed a russeted fruit surface was identified, and characterized. A large scale comparative transcriptomic analysis of these tomato and apple mutants was performed, generating a list of candidate genes for suberin deposition. Increasing the comparison to include data mined from literature resulted in the elucidation of a multi-species gene expression signature for suberin biosynthesis, and allowed for the identification and characterization of novel genetic elements, including those involved in the regulation of suberin formation and its deposition. Of these genetic elements, MYB107, was demonstrated to be a positive regulator of suberin accumulation in Arabidopsis seed coat. In totality this study has produced a greater understanding of genetic mechanisms governing cuticle biosynthesis, particularly in developing fruit. This was achieved primarily through the functional characterization of regulatory elements (MIXTA-like, SHN3 and MYB107 transcription factors) controlling the synthesis of cutin and suberin matrices. Additionally the DCR enzyme was demonstrated to be a crucial step in fruit cutin biosynthesis. Finally the intricate relationship between epidermal cell development and cuticle biosynthesis has been further highlighted.