Carotenoid biosynthesis in plants has been described at the molecular level for most of the biochemical steps in the pathway. However, the cis-trans isomerization of carotenoids, which is known to occur in vivo, has remained a mystery since its discovery five decades ago. To elucidate the molecular mechanism of carotenoid isomerization, we have taken a genetic map-based approach to clone the tangerine locus from tomato. Fruit of tangerine are orange and accumulate prolycopene (7Z,9Z,7 Z,9 Z-tetra-cis -lycopene) instead of the all-trans -lycopene, which normally is synthesized in the wild type. Our data indicate that the tangerine gene, designated CRTISO , encodes an authentic carotenoid isomerase that is required during carotenoid desaturation. CRTISO is a redox-type enzyme structurally related to the bacterial-type phytoene desaturase CRTI. Two alleles of tangerine have been investigated. In tangerine mic , loss of function is attributable to a deletion mutation in CRTISO , and in tangerine 3183 , expression of this gene is impaired. CRTISO from tomato is expressed in all green tissues but is upregulated during fruit ripening and in flowers. The function of carotene isomerase in plants presumably is to enable carotenoid biosynthesis to occur in the dark and in nonphotosynthetic tissues.

SUMMARYPlant cuticles are broadly composed of two major components: polymeric cutin and a mixture of waxes, which infiltrate the cutin matrix and also accumulate on the surface, forming an epicuticular layer. Although cuticles are thought to play a number of important physiological roles, with the most important being to restrict water loss from aerial plant organs, the relative contributions of cutin and waxes to cuticle function are still not well understood. Tomato (Solanum lycopersicum) fruits provide an attractive experimental system to address this question as, unlike other model plants such as Arabidopsis, they have a relatively thick astomatous cuticle, providing a poreless uniform material that is easy to isolate and handle. We identified three tomato mutants, cutin deficient 1 (cd1), cd2 and cd3, the fruit cuticles of which have a dramatic (95-98%) reduction in cutin content and substantially altered, but distinctly different, architectures. This cutin deficiency resulted in an increase in cuticle surface stiffness, and in the proportions of both hydrophilic and multiply bonded polymeric constituents. Furthermore, our data suggested that there is no correlation between the amount of cutin and the permeability of the cuticle to water, but that cutin plays an important role in protecting tissues from microbial infection. The three cd mutations were mapped to different loci, and the cloning of CD2 revealed it to encode a homeodomain protein, which we propose acts as a key regulator of cutin biosynthesis in tomato fruit.

Major improvements in proteomic techniques in recent years have led to an increase in their application in all biological fields, including plant sciences. For all proteomic approaches, protein extraction and sample preparation are of utmost importance for optimal results; however, extraction of proteins from plant tissues represents a great challenge. Plant tissues usually contain relatively low amounts of proteins and high concentrations of proteases and compounds that potentially can limit tissue disintegration and interfere with subsequent protein separation and identification. An effective protein extraction protocol must also be adaptable to the great variation in the sets of secondary metabolites and potentially contaminating compounds that occurs between tissues (e.g., leaves, roots, fruit, seeds and stems) and between species. Here we present two basic protein extraction protocols that have successfully been used with diverse plant tissues, including recalcitrant tissues. The first method is based on phenol extraction coupled with ammonium acetate precipitation, and the second is based on trichloroacetic acid (TCA) precipitation. Both extraction protocols can be completed within 2 d.

A hydrophobic cuticle consisting of waxes and the polyester cutin covers the aerial epidermis of all land plants, providing essential protection from desiccation and other stresses. We have determined the enzymatic basis of cutin polymerization through characterization of a tomato extracellular acyltransferase, CD1, and its substrate, 2-mono(10,16-dihydroxyhexadecanoyl)glycerol (2-MHG). CD1 has in vitro polyester synthesis activity and is required for cutin accumulation in vivo, indicating that it is a cutin synthase.