Investigating the function of carotenoid isomerase-like genes in Arabidopsis

Carotenoids are essential pigments of the photosynthetic apparatus in plants, algae, and cyanobacteria and are also produced by many bacteria and fungi. They serve indispensable functions in plants providing health benefits for humans and animals. In plants, it was considered that the conversion of C40 carotenoid (isoprenoid molecule/compound) to all-trans lycopene required two desaturases (PDS; phytoene desturase & ZDS; Zeta-carotene desaturase) plus a third distinct enzyme activity, the carotenoid isomerase (CRTISO). However, the specificity and role of cis-trans-isomerization of carotenoids and the requirement for other isomerase proteins remained unresolved. Currently, after the genome completion of Arabidopsis, the collected genome data is being converted into gene-function information, by adding more value to the assortment of nucleotide sequences. Thus, by knowing the exact sequence and location of all genes, it has been made possible to predict the function of unknown genes based on sequence comparison of already known genes. In the present study two putative genes, named here AtCRTISO-like2 (AtCRTIL2) and AtCRTISO-like3 (AtCRTIL3) sharing significant protein homology to CRTISO gene were identified in Arabidopsis. Therefore, to further decipher the carotenoid biosynthesis pathway these genes were characterized. AtCRTIL2 and 3 are localized in chloroplast and mitochondria, respectively. In addition, AtCRTIL2 is more highly expressed in photosynthetic tissues. AtCRTIL2 orthologues were found in higher plants, algae and cyanobacteria while the phylogenetic analysis of AtCRTIL3 revealed the existence of plant, bacterial and animal homologues. Based on subcellular localization of AtCRTIL2 in chloroplast the function of AtCRTIL2 was investigated further hypothesising that it could possibly be involved in carotenoid biosynthesis or catabolism. While considering the amino acid sequence similarities observed in AtCRTISO, AtCRTIL3 and its homologues, AtCRTIL3 was thought to be engaged in the processes that could potentially be related to carotenoid metabolism or possibly retinoid biosynthesis. In this study, transgenic plants including over-expression lines, hpRNAi knock-down lines and TILLING (Targeted Induced Local Lesions in Genomes) knock-out mutations were generated and analysed. Different molecular, physiological and biochemical approaches were used to characterise these genes. The detailed phenotypic analysis and plant architecture studies of AtCRTIL2 and 3 mutants and transgenic plants did not demonstrate significant variations and all the lines were found to be virtually indistinguishable from the wild-type plants throughout their entire life cycles. The carotenoid pigment analysis of mutants and transgenic lines indicated no obvious differences compare to the wild-type. Additionally, both AtCRTIL2 and 3 plants appeared to have normal photosynthetic activity as well as the photomorphogenic responses. Moreover, both of the genes showed drought and highlight stress responses comparable to wild-type. Interestingly, though, over-expression lines of AtCRTIL2 and 3 genes exhibited a cold tolerant phenotype as compared to the mutants and wild-type control, when grown under cold conditions. Taken together, the findings presented here did not provide enough evidence on the predicted role of AtCRTIL2 and 3 in carotenoid biosynthesis. However, a role in cold stress tolerance of AtCRTIL2 and 3 genes is indicated. - provided by Candidate