AB - Marigold (Tagetes erecta L.) flower petals synthesize and accumulate carotenoids at levels greater than 20 times that in leaves and provide an excellent model system to investigate the molecular biology and biochemistry of carotenoid biosynthesis in plants. In addition, marigold cultivars exist with flower colors ranging from white to dark orange due to >100-fold differences in carotenoid levels, and presumably similar changes in carbon flux through the pathway. To examine the expression of carotenoid genes in marigold petals, we have cloned the majority of the genes in this pathway and used these to assess their steady-state mRNA levels in four marigold cultivars with extreme differences in carotenoid content. We have also cloned genes encoding early steps in the biosynthesis of isopentenyl pyrophosphate (IPP), the precursor of all isoprenoids, including carotenoids, as well as two genes required for plastid division. Differences among the marigold varieties in the expression of these genes suggest that differences in mRNA transcription or stability underlie the vast differences in carotenoid synthesis and accumulation in the different marigold varieties.
We identified 67 carotenoid biosynthetic genes in B. rapa, which were orthologs of the 47 carotenoid genes in A. thaliana. A high level of synteny was observed for carotenoid biosynthetic genes between A. thaliana and B. rapa. Out of 47 carotenoid biosynthetic genes in A. thaliana, 46 were successfully mapped to the 10 B. rapa chromosomes, and most of the genes retained more than one copy in B. rapa. The gene expansion was caused by the whole-genome triplication (WGT) event experienced by Brassica species. An expression analysis of the carotenoid biosynthetic genes suggested that their expression levels differed in root, stem, leaf, flower, callus, and silique tissues. Additionally, the paralogs of each carotenoid biosynthetic gene, which were generated from the WGT in B. rapa, showed significantly different expression levels among tissues, suggesting differentiated functions for these multi-copy genes in the carotenoid pathway.
Regulation of carotenoid biosynthetic genes expression …
The WGT event in the B. rapa genome provides a model for the study of the evolutionary fate of multi-copy genes and the effects of polyploidy in economically important crops. To investigate the evolutionary relationship of carotenoid biosynthetic genes in B. rapa, separate neighbor-joining trees were generated for the enzyme PSY by aligning the protein sequence with the corresponding orthologs in Arabidopsis and other plant species (Fig. ). The phylogenetic analysis of PSY in B. rapa, Arabidopsis, and other monocot and dicot plant species revealed that PSYs cluster into two separate monocot- and dicot-specific clades, where most of the members show a monophyletic pattern of origin. In the Brassicaceae family, the species were separated into two specific clades. As shown in Fig. , PSY1, PSY2, and PSY3 sequences from B. rapa each clustered into groups with their respective B. oleracea orthologs on a separate branch. Interestingly, B. rapa, B. oleracea, and Schrenkiella parvula are clustered on one branch, which indicates that the Brassica are closer to S. parvula than to Arabidopsis and that the Brassiceae triplication event occurred near the time of the divergence between Brassiceae and Schrenkiella . However, three B. napus PSYs were clustered on one specific branch with the Arabidopsis and Thellungiella PSYs.
to study carotenoid biosynthesis ..
According to the expression analysis results, most B. rapa carotenoid biosynthetic genes appeared to have similar roles to their orthologs in other species. For example, it has been reported that the PSY, ZDS, PDS, and ZEP genes play crucial roles in carotenoid biosynthesis in A. thaliana . In B. rapa, these genes exhibited either predominant or specific expression patterns in leaves, such as BrZEP1, which was highly expressed in flowers and leaves. A. thaliana contains a family of 12 genes that are similar to GGPS, but only five GGPS genes have been shown to be expressed in different tissues during plant development . Although there are 13 duplicated BrGGPS genes in B. rapa, only BrGGPS1.1 was highly expressed in the six organs we examined. Moreover, most of the BrGGPS genes did not have detectable expression levels, which may be due to functional divergence after the triplication event or because they are specifically expressed at other developmental stages.
Carotenoid Metabolism: Biosynthesis, Regulation, and …
The B. rapa carotenoid biosynthetic genes could be divided into two clusters based on their expression patterns (Fig. ; Additional file : Table S2). Cluster 1 was composed of genes that were expressed in all six organs, while Cluster 2 contained genes that had low expression levels and some organ-specific expression patterns. Cluster 1 included two expression groups. The first group showed a high expression level in all six organs. The second group exhibited a higher expression level in flowers, leaves, and siliques, indicating their roles in the carotenoid biosynthetic pathway in these tissues, than in roots, calli, and stems. The genes belonging to Cluster 2 had lower expression levels and could be subdivided into three groups. The first group was composed of 10 genes that had low expression levels in siliques, roots, calli, and stems. The genes in the second group exhibited low but stable expression levels in all six organs, except BrZEP2, which was highly expressed in flowers. This result was consistent with a previous study and indicates that BrZEP2 plays an important role in the synthesis and accumulation of carotenoids in B. rapa flowers . Most genes belonging to the third group exhibited low or undetectable expression levels in all six organs. Interestingly, the gene BrCCD8 was expressed in stems and roots but could not be detected in flowers, leaves, siliques, or calli, and BrNCED9.1 was highly expressed in siliques.