Isolation and characterization of the Z-ISO gene encoding a missing component of carotenoid biosynthesis in plants

Abstract

Metabolic engineering of plant carotenoids in food crops has been a recent focus for improving human health. Pathway manipulation is predicated on comprehensive knowledge of this biosynthetic pathway, which has been extensively studied. However, there existed the possibility of an additional biosynthetic step thought to be dispensable because it could be compensated for by light. This step, mediated by a putative Z-ISO, was predicted to occur in the sequence of redox reactions that are coupled to an electron transport chain and convert the colorless 15-cis-phytoene to the red-colored all-trans-lycopene. The enigma of carotenogenesis in the absence of light (e.g. in endosperm, a target for improving nutritional content) argued for Z-ISO as a pathway requirement. Therefore, understanding of plant carotenoid biosynthesis was obviously incomplete. To prove the existence of Z-ISO, maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) mutants were isolated and the gene identified. Functional testing of the gene product in Escherichia coli showed isomerization of the 15-cis double bond in 9,15,9'-tri-cis-zeta-carotene, proving that Z-ISO encoded the missing step. Z-ISO was found to be important for both light-exposed and "dark" tissues. Comparative genomics illuminated the origin of Z-ISO found throughout higher and lower plants, algae, diatoms, and cyanobacteria. Z-ISO evolved from an ancestor related to the NnrU (for nitrite and nitric oxide reductase U) gene required for bacterial denitrification, a pathway that produces nitrogen oxides as alternate electron acceptors for anaerobic growth. Therefore, plant carotenogenesis evolved by recruitment of genes from noncarotenogenic bacteria.

Figure 1.

Carotenoid biosynthesis in plants showing 15-cis-double bond isomerization catalyzed by putative Z-ISO enzyme or alternatively photoisomerized by light. GGPP, Geranylgeranyl pyrophosphate. [See online article for color version of this figure.]

Figure 2.

Greening phenotype of Z-ISO mutants in maize (y9) and Arabidopsis (zic1-1) compared with normal (wild type [wt]) counterparts. Maize greenhouse-grown plants are shown at top left, and Arabidopsis plants are shown in the remaining panels. Arabidopsis seeds were germinated in the dark and/or exposed to long-day growth (16 h of light/8 h of dark) for 1 or 7 d as noted.

Figure 3.

HPLC scans of carotenoid extracts showing light-mediated isomerization of 9,15,9′-tri-cis-ζ-carotene (Tri) to 9,9′-di-cis-ζ-carotene (Di). Top, extracts prepared from etiolated leaves of y9 (left) and dark-cultured E. coli producing 9,15,9′-tri-cis-ζ-carotene (right). Bottom, carotenoid extracts as in the top panels were exposed to 12 h of light. Insets show spectra of major peaks in the panels. Additional spectra can be found in Figure 4. Peak ζ2, unknown, similar spectrum to 9,15,9′-tri-cis-ζ-carotene; peak ζ3, unknown, similar spectrum to 9,15,9′-tri-cis-ζ-carotene; peak ζ5, unknown, similar spectrum to 9,9′-di-cis-ζ-carotene; peak ζ6, unknown, similar spectrum to 9,9′-di-cis-ζ-carotene. AU, Absorbance units. [See online article for color version of this figure.]

Figure 4.

HPLC analysis of ζ-carotene extracted from etiolated leaves of maize y9 and Arabidopsis zic1-1. Left, HPLC scans. Right, Peak spectra. Peaks are labeled as in Figure 3. AU, Absorbance units; Di, 9,9′-di-cis-ζ-carotene; Tri, 9,15,9′-tri-cis-ζ-carotene. [See online article for color version of this figure.]

Figure 5.

Fine-mapping of the Z-ISO locus, map-based cloning, and transcript expression pattern of the Arabidopsis Z-ISO gene (AtZ-ISO). The Z-ISO locus was mapped to chromosome 1 (Chr1) between markers T16B5 and T19P16M2. The Z-ISO locus cosegregated with marker T19D16M1. The numbers at the bottom indicate the number of recombinants identified from F2 plants. BACs refer to bacterial artificial chromosome clones in the region with associated markers identified. The round spot denotes the centromere. cM, Centimorgan.

Figure 6.

Gene structure of Arabidopsis At1g10830 (AtZ-ISO1.1) and the location of Z-ISO mutations. Exons are shown as gray boxes and introns are shown as white boxes. 5′ and 3′ untranslated regions are indicated by black lines. The start codon (ATG) and the stop codon (TAA) are indicated. The mutated sites of the six Z-ISO alleles are shown. zic1-1 has a 10-bp deletion in the fourth exon, creating a stop codon; zic1-2 has a 20-bp deletion at the end of intron 1, resulting in abnormal splicing. zic1-3 to zic1-6 are T-DNA lines obtained from TAIR, and the insertion sites are as shown.

Figure 7.

The structures of the AtZ-ISO1.1 gene transcript from the wild type (W.T.) and two Z-ISO alleles, zic1-1 and zic1-2. A, Structure of wild-type AtZ-ISO1.1 gene transcript. Numbers below indicate sites of primers 2590/2591 and 2567/2589 used for amplification of mutated AtZ-ISO1.1 transcripts in zic1-1 and zic1-2, respectively. B, Structure of AtZ-ISO1.1 transcript in zic1-1; the 10-bp deletion in the fourth exon creates a new stop codon. C, Structures of AtZ-ISO1.1 transcripts in zic1-2; the 20-bp deletion in the first intron causes alternative splicing resulting in the multiple transcripts shown in Figure 8D, including a trace amount of wild-type transcript (1,104 nucleotides).

Figure 8.

Structures and expression levels of AtZ-ISO transcripts. A, The structures of two types of AtZ-ISO gene transcripts, AtZ-ISO1.1 and AtZ-ISO1.2. B, Sequence alignment of the C termini of AtZ-ISO1.1 and AtZ-ISO1.2. AtZ-ISO1.2 has an additional 89-bp fragment from the third intron and the fourth exon is not included, resulting in a premature stop codon and encoding a protein with the variant C terminus shown. C, mRNA levels of AtZ-ISO1.1 and AtZ-ISO1.2 in green (“light”) and etiolated (“dark”) seedlings of the wild type were measured by quantitative RT-PCR. Primers 2686/2687, used for AtZ-ISO1.1, are located in the third and fourth exons, respectively; primers 2684/2685, used for AtZ-ISO1.2, are located in the end of the third exon. For each set of amplifications, the levels were normalized to actin and presented relative to the level of AtZ-ISO1.1 in light-grown plants. The fold differences between AtZ-ISO1.1 and AtZ-ISO1.2 transcripts are indicated above the bars. Error bars represent the sdof three biological replicates. D, AtZ-ISO transcripts produced in the two zic1 alleles compared with the wild type (wt). For amplification of transcripts in each mutant, primers on both sides of the mutation site were used for cDNA synthesis from leaves of 3-week-old plants of Col-0 (wt), zic1-1, and zic1-2.The zic1-1 gene has a 10-bp deletion and produces a transcript close in size to the wild type; the mutant zic1-2 produces multiple transcripts as a result of abnormal splicing, as verified by sequence analysis.

Figure 9.

Chromosome mapping of maize Z-ISO (ZmZ-ISO), gene structure of wild-type and mutant alleles, and mutant transcript analysis. Z-ISO was mapped to chromosome (Chr.) 10, contig ctg394, the locus of y9 (from the Maize Genetics and Genomics Database). The ZmZ-ISO gene has four exons and three introns, as indicated by gray and white boxes, respectively. Z-ISO genotyping of y9 alleles showed that all were caused by insertion of a Mu transposon in exon 1. Alleles carrying a 2,199-bp Mu7 insertion were as follows: X34D, X34E, X34F, X34G, X34H, X34I, X34K, X07CC, 5705B, and 5705E. Alleles carrying a Mu8 insertion were as follows: X34L and X07CB. Both Mu insertions also caused insertion site duplication. BAC, Bacterial artificial chromosome.

Figure 10.

Maize Z-ISO transcript levels in the wild type and mutants. A, Expression level of ZmZ-ISO in etiolated leaves from the wild type (B73) and the y9 allele (5705B) as shown by quantitative RT-PCR. A 117-bp fragment of ZmZ-ISO cDNA located in the second and third exons was amplified. Transcript levels were normalized for actin levels and presented relative to the corresponding wild-type level. Data represent averages and SD of three biological replicates. B, Expression levels of ZmZ-ISO in different maize tissues as measured by quantitative RT-PCR. Transcript levels were normalized for actin levels and presented relative to levels in leaves. Etiolated leaves (L), roots (R), embryo at 20 d after pollination (Em), and endosperm at 20 d after pollination (En) of wild-type B73 were used. Data represent averages and SD of three biological replicates.

Figure 11.

Phylogenetic tree showing that Z-ISO in plants is evolved from a common ancestor with bacterial NnrU sequences. The neighbor-joining tree was made using amino acid sequences analyzed by MEGA3.1. The confidence in the tree was determined by analyzing 1,000 bootstrap replicates, and bootstrap values are shown for each group. Branch lengths are drawn to the scale shown, indicating 0.2 amino acid substitutions per site. GenBank accessions are listed in Supplemental Table S2. [See online article for color version of this figure.]

Figure 12.

Gene clustering in bacteria implies function. Genome organization was taken from the SEED database (http://theseed.uchicago.edu/FIG/index.cgi). [See online article for color version of this figure.]

Figure 13.

Functional analysis of Z-ISO in E. coli. E. coli encoding bacterial GGPPS, PSY, and maize PDS accumulate 9,15,9′-tri-cis-ζ-carotene and were additionally transformed with the genes denoted or empty vector (pCOLADuet-1) as a control, and carotenoids were extracted and analyzed by HPLC. Under dark culturing conditions, Z-ISO from Arabidopsis (AtZ-ISO1.1) and maize (ZmZ-ISO1.1) convert most of the 9,15,9′-tri-cis-ζ-carotene (Tri) to 9,9′-di-cis-ζ-carotene (Di). Panels on the right show nonfunctioning proteins: truncated versions of Z-ISO missing the C terminus, such as the product of the short Arabidopsis transcript (AtZ-ISO1.2), transcript from the zic 1-1 mutant (AtZ-ISO1.1M), and NnrU from a denitrifying bacterium, S. meliloti 1021. Peaks are labeled as in Figure 3. [See online article for color version of this figure.]