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

Abstract

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 beta-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 beta-carotene hydroxylase enzyme as well as flower- and fruit-specific geranylgeranyl diphosphate synthase, phytoene synthase, and lycopene beta-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 beta-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 beta-carotene, which accumulates as a result of the wf mutation but is not due to altered expression of genes in the biosynthetic pathway.

Figure 1.

Flowers of wf Mutants. (A) wf1-1 (F2 of a cross LA2370 × IL3-2) (left) and its nearly isogenic wild-type line (F2 of the same cross). (B) wf1-2 (e1827) (left) and its isogenic line M82. (C) Flower of wf1-2 (left) and the wild type (right) in developmental stages 1 to 3 analyzed in this work.

Figure 2.

Gene Structure of β-Carotene Hydroxylase Genes (CrtR-b). Tomato (Sl) CrtR-b1 and CrtR-b2, Arabidopsis (At) CrtR-b1 and CrtR-2, and rice (Os) CrtR-b2. Exons are depicted as boxes.

Figure 3.

Genetic Mapping of CrtR-b1, CrtR-b2, and wf on the Tomato Linkage Map. The linkage map was adapted from Eshed and Zamir (1995). The relevant chromosomal segments from Solanum pennellii that were introgressed into S. lycopersicum are represented by black bars with the names of the ILs that carry them. The high-resolution map of IL3-2, which includes the wf locus, is displayed next to chromosome 3. RFLP markers used in the fine mapping of wf and genetic distances between each pair are presented. r and B represents the loci of yellow-flesh (Psy1) and Beta (Cyc-B) mutations, respectively. cM, centimorgans.

Figure 4.

Deletions in CrtR-b2 mRNA of wf1-2 (e1827) and wf1-1 (LA2370). Total RNA was extracted from stage 3 flowers and used as template for RT-PCR amplification. The resulting DNA products were separated by electrophoresis on 1.0% agarose gel and stained with ethidium bromide. A full-length DNA fragment of 1058 bp was generated in the wild type. Small deletions of ∼60 bp were observed in the mutants. M, DNA size markers.

Figure 5.

Mutations in the CrtR-b2 Gene from wf1-1 and wf1-2. Genomic sequence of the first three exons of CrtR-b2 from tomato is presented. Exons are in boldface. The sequence deleted in allele wf1-1 is underlined. A transition mutation of G to A that causes skipping of the second exon in allele wf1-2 is shown at nucleotide 804.

Figure 6.

HPLC Analysis of Carotenoids in E. coli Cells Expressing CrtR-b1 and CrtR-b2 from Tomato. Carotenoids were extracted from E. coli cells carrying plasmid pBETA (top); pBETA + pCrtR-b1 expressing cDNA of CrtR-b1 from tomato (middle); or pBETA + pCrtR-b2 expressing cDNA of CrtR-b2 from tomato (bottom). Peak identification: 1, β-carotene; 2, β-cryptoxanthin; 3, zeaxanthin.

Figure 7.

Expression of CrtR-b1 and CrtR-b2 in Various Tomato Organs. Top panel: Steady state levels of mRNA of CrtR-b1, CrtR-b2, and Pds were measured concomitantly by RT-PCR amplification from the same samples of total RNA. PCR was performed with ³²P-labeled dCTP. Autoradiograms of PCR-amplified DNA fragments that were separated by polyacrylamide gel electrophoresis are exhibited. RNA from leaves (L) and petals from flower stages 1 (P1) and 3 (P3) are presented in the left panel. RNA from different tissues of stage 3 flowers is presented in the right panel: S, sepals; P, petals; A, anthers; and C, carpels. Samples Ax3 and Ax1/3 were amplified from anther RNA, 3-fold and one-third of the original concentration, respectively. Bottom panel: Relative levels of CrtR-b1 and CrtR-b2 mRNA from roots, leaves, flowers, and mature fruit were determined by real-time RT-PCR using gene-specific primers. Expression data were normalized to the expression of actin. Data shown are means + sd (n = 4).

Figure 8.

Expression of Isoprenoid and Carotenoid Biosynthesis Genes in Flowers of the Wild Type (M82) and Mutant wf1-2. Total RNA was extracted from flowers at developmental stages 1 to 3 (Figure 1). Levels of mRNA were determined by real-time RT-PCR using gene-specific primers for the following genes: Dxs, Ggps2, Psy1, Psy2, Pds, and Zds. Expression data were normalized to the expression of actin. Data shown are means + sd (n = 5). Black bars correspond to the wild type (M82) and white bars to wf1-2.

Figure 9.

Phylogenetic Tree Based on Amino Acid Sequences of β-Carotene Hydroxylases in Higher Plants. The following sequences have been analyzed: Arabidopsis 1, Arabidopsis 2, Crocus 1, Crocus 2, Pepper 1 (CRTR-B2), Pepper 2 (CRTR-B1), Rice 1, Rice 2, Tomato 1, and Tomato 2. Alignment of the amino acid sequences of these polypeptides is presented in Supplemental Figure 1 online.

Figure 10.

Carotenoid Biosynthesis Pathway in Tomato. Cyc-B, chromoplast-specific lycopene β-cyclase; Ggps, geranylgeranyl diphosphate synthase; Lcy-b, lycopene β-cyclase; Lcy-e, lycopene ɛ-cyclase; CrtR-b, β-ring hydroxylase; CrtR-e, ɛ-ring hydroxylase; Nxs, neoxanthin synthase; Pds, phytoene desaturase; Psy, phytoene synthase; Vde, violaxanthin de-epoxidase; NCED, 9-cis-epoxycarotenoid dioxygenase; Zds, ζ-carotene desaturase; Zep, zeaxanthin epoxidase.