Gil Ronen

Cloning of tangerine from Tomato Reveals a Carotenoid Isomerase Essential for the Production of β-Carotene and Xanthophylls in Plants

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 tangerinemic, loss of function is attributable to a deletion mutation in CRTISO, and in tangerine3183, 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.

Suppression of Arabidopsis vesicle-SNARE expression inhibited fusion of H2O2-containing vesicles with tonoplast and increased salt tolerance.

Intracellular vesicle trafficking performs essential functions in eukaryotic cells, such as membrane trafficking and delivery of molecules to their destinations. A major endocytotic route in plants is vesicle trafficking to the vacuole that plays an important role in plant salt tolerance. The final step in this pathway is mediated by the AtVAMP7C family of vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs) that carry out the vesicle fusion with the tonoplast. Exposure to high-salt conditions causes immediate ionic and osmotic stresses, followed by production of reactive oxygen species. Here, we show that the reactive oxygen species are produced intracellularly, in endosomes that were targeted to the central vacuole. Suppression of the AtVAMP7C genes expression by antisense AtVAMP711 gene or in mutants of this family inhibited fusion of H2O2-containing vesicles with the tonoplast, which resulted in formation of H2O2-containing megavesicles that remained in the cytoplasm. The antisense and mutant plants exhibited improved vacuolar functions, such as maintenance of ΔpH, reduced release of calcium from the vacuole, and greatly improved plant salt tolerance. The antisense plants exhibited increased calcium-dependent protein kinase activity upon salt stress. Improved vacuolar ATPase activity during oxidative stress also was observed in a yeast system, in a ΔVamp7 knockout strain. Interestingly, a microarray-based analysis of the AtVAMP7C genes showed a strong down-regulation of most genes in wild-type roots during salt stress, suggesting an evolutionary molecular adaptation of the vacuolar trafficking.

A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato white-flower locus

Carotenoids and their oxygenated derivatives xanthophylls play essential roles in the pigmentation of flowers and fruits. Wild-type tomato (Solanum lycopersicum) flowers are intensely yellow due to accumulation of the xanthophylls neoxanthin and violaxanthin. To study the regulation of xanthophyll biosynthesis, we analyzed the mutant white-flower (wf). It was found that the recessive wf phenotype is caused by mutations in a flower-specific β-ring carotene hyroxylase gene (CrtR-b2). Two deletions and one exon-skipping mutation in different CrtR-b2 wf alleles abolish carotenoid biosynthesis in flowers but not leaves, where the homologous CrtR-b1 is constitutively expressed. A second β-carotene hydroxylase enzyme as well as flower- and fruit-specific geranylgeranyl diphosphate synthase, phytoene synthase, and lycopene β-cyclase together define a carotenoid biosynthesis pathway active in chromoplasts only, underscoring the crucial role of gene duplication in specialized plant metabolic pathways. We hypothesize that this pathway in tomato was initially selected during evolution to enhance flower coloration and only later recruited to enhance fruit pigmentation. The elimination of β-carotene hydroxylation in wf petals results in an 80% reduction in total carotenoid concentration, possibly caused by the inability of petals to store high concentrations of carotenoids other than xanthophylls and by degradation of β-carotene, which accumulates as a result of the wf mutation but is not due to altered expression of genes in the biosynthetic pathway.