The yellow `Pinalate´ sweet orange mutant shows altered carotenoid profile in fruit tissues during ripening and reduced carotenoid content in leaves
Changes in carotenoid content and composition were evaluated in flavedo and pulp of `Pinalate´ fruits at three ripening stages: immature green, breaker and fully ripe, and compared with those of its wild-type `Navelate´ (Fig. 2). In immature green flavedo the total carotenoid content in `Pinalate´ was about 25% lower (65 mg g-1) than in parental fruits (85 mg g-1) and differences in individual carotenoids were identified. As in parental fruit, the main carotenoid in `Pinalate´ green flavedo was the b,e-xanthophyll lutein (19.5 mg g-1 which represents about 30%) containing also 17 mg g-1 of b,b-xanthophylls (25%) (neoxanthin and violaxanthin) as well as a- and b-carotene (14.3 mg g-1). Interestingly, `Pinalate´ green flavedo contained more than 25% of the upstream carotenes (phytoene, phytofluene and z-carotene) which were barely detectable in the parental fruit. In agreement with previous data [46], different z-carotene isomers were detected in `Pinalate´ and in the context of this study it is important to mention that at this green stage the presence of 9,15, 9´tri-cis-z-carotene isomer was not detected and the predominant isomer was 9, 9´di-cis-z-carotene (2.7 mg g-1) (Fig. 2A). At the breaker stage, flavedo of both genotypes exhibited a light green coloration with yellow patches indicative of chlorophylls degradation and the initiation of chloroplasts to chromoplasts transition (Fig. 2A). Flavedo of `Pinalate´ fruit at this stage contained almost double carotenoid content (104 mg g-1) than those of `Navelate´ (53 mg g-1), and while in `Pinalate´ this amount involved an 80% increment over immature green fruits, contrastingly in `Navelate´ represented a 40% reduction. The carotenoid composition in the peel of both genotypes was different at breaker stage. In wild-type the profile was similar to green fruit but an increase in the proportion of b,b-xanthophylls was already evident, indicating the enhancement of carotenogenesis associated with fruit ripening. In `Pinalate´ the content of phytoene (46.8 mg g-1) and of z-carotene (9.4 mg g-1) was increased by 3-fold compared to green flavedo and, more importantly, the presence of 9,15,9´tri-cis-z-carotene was detected (1.2 mg g-1). At ripe stage, total carotenoid content in the flavedo of `Pinalate´ almost doubled that of the wild-type (235 and 126 mg g-1, respectively) and the composition was strikingly different. The flavedo of `Navelate´ contained more than 90% of b,b-xanthophylls, with 105.1 mg g-1 of violaxanthin and 14.7 mg g-1 of other b,b-xanthophylls (zeaxanthin, anteraxanthin and b-cryptoxanthin), while upstream carotenes, including phytoene and phytofluene, accounted for 5.2 mg g-1 and z-carotene was not detected. In `Pinalate´, phytoene and phytofluene were overrepresented with concentrations of 153 and 36 mg g-1, respectively, and several isomers of both carotenes were identified. Six different isomers were detected for z-carotene (Fig. S1), with concentrations of 5.1 and 20.3 mg g-1 for 9,15,9´tri-cis and 9,9´di-cis, respectively, and 8.9 mg g-1 for all other z-carotene isomers. Violaxanthin was the only xanthophyll identified in the flavedo of `Pinalate´ and its concentration was 11.8 mg g-1 which is approximately 10-fold lower than in `Navelate´.
In the pulp of both genotypes, significant differences in carotenoid content and composition were obvious since green immature stage (Fig. 2B). Carotenoids concentration in the pulp of green `Navelate´ fruit was negligible while in `Pinalate´ minor amounts of phytoene (1.8 mg g-1), phytofluene (0.4 mg g-1) and z-carotene (0.3 mg g-1) were detected. At breaker stage, `Navelate´ pulp displayed low concentration of carotenoids being phytoene the main carotene (2.6 mg g-1) but also contained a significant proportion of b,b-xanthophylls (0.8 mg g-1), particularly violaxanthin and anteraxanthin. In `Pinalate´ there was almost a 4-fold increase in the total carotenoid content compared to green stage with a carotenoid profile similar to green stage. Carotenoid content in the pulp of mature `Navelate´ fruit was 5-fold higher than at breaker, and was predominantly composed by b,b-xanthophylls (13.3 mg g-1) but also phytoene (3.5 mg g-1), phytofluene (0.5 mg g-1), and traces of 9,9´di-cis-z-carotene and b-carotene. In ripe `Pinalate´ pulp the concentration of total carotenes increased by 5-fold compared to breaker stage and it was almost 3-fold higher than in `Navelate´. Phytoene was the predominant carotenoid (27.7 mg g-1) representing more than 50% of the total content, followed by phytofluene (8.7 mg g-1 ) and z-carotene (3.7 mg g-1 of 9,9´di-cis isomer and 1.8 mg g-1 of 9,15,9´tri-cis). The concentration of b,b-xanthophylls was more than 2-fold lower (5.3 mg g-1) than in `Navelate´ with a major proportion of violaxanthin. Representative chromatograms illustrating the marked differences in carotenoid profile between the pulp of `Pinalate´ and Navalete ripe fruits are shown in Fig. S2.
The carotenoid composition in young leaves of both genotypes was very similar and typical of chloroplastic tissues, with a predominance of lutein and other b,b-xanthophylls (violaxanthin, neoxanthin, zeaxanthin) and a- and b-carotene (Fig. 2C). However, the carotenoid content was 40% lower in `Pinalate´ leaves compared to parental and, in contrast with fruit tissues, there was no accumulation of upstream carotenes. The chlorophyll content was also reduced by half in mutant leaves (545 mg g-1) compared to the parental ones (1146 mg g-1).
In the context of this study, it is relevant to mention that neither lycopene nor neurosporene were detected in either mutant and wild-type fruit or leaf extracts.
Light growing conditions affect carotenoid profile of `Pinalate´ fruit peel
The abnormal accumulation of colorless carotenes and z-carotene isomers in `Pinalate´ fruit tissues suggests a partial blockage either in ZDS or Z-ISO activity. It has been reported that Z-ISO activity, but not ZDS, can be partially compensated by light, and under dark growing conditions the biochemical blockage may be intensified [33]. In order to investigate the effect of light/dark conditions on `Pinalate´ carotenoids profiling, we covered `Pinalate´ fruits with black plastic at immature green stage and allowed them to develop and ripe under darkness until harvest [46]. As control, `Pinalate´ fruits directly exposed to sunlight from the external tree canopy were selected. Light and dark-grown fruits were harvested at the ripe stage and the carotenoid profile was determined in the flavedo. At harvest, the external color of the fruits grown under both conditions was different: light-grown fruit displayed a pale-orange coloration while dark-grown fruits where completely light-yellow with any signals of orange tint (Fig. 3). Total carotenoid content was higher in dark-grown (227 mg g-1) than in light-exposed fruit (195 mg g-1) and differences in carotenoid profile were also observed. Dark-grown `Pinalate´ flavedo contained a larger proportion (84% of the total carotenoids) and content of phytoene and phytofluene (191 mg g-1), and reduced xanthophylls (2.2 mg g-1 accounting for 0.97% of the total) compared to light-grown which contained 74% of colorless carotenes (143 mg g-1) and 16% of b,b-xanthophylls (33 mg g-1) (Fig. 3). Interestingly, the content and the ratio of 9,15,9´tri-cis:9,9´di-cis z-carotene isomers was also affected by light conditions. Thus, whereas in light-grown fruits the ratio tri-cis:di-cis was 0.17 in dark-grown increased up to 0.34. Moreover, the concentration of tri-cis was 3-fold higher in the flavedo of dark-grown compared to that of light-exposed fruits.
Transcriptional profiles of main carotenoid biosynthetic genes in `Pinalate´ do not correlate with its carotenoid composition
Since carotenoid composition in `Pinalate´ fruit suggests an impairment in the pathway flux toward the b,b-xanthophylls production, we performed a comparative transcriptional analysis of main genes involved in their synthesis: PSY, PDS, ZDS1, ZDS2 and ZDS3, bLCY1 and bLCY2, and bCHX, in flavedo, pulp and leaves of mutant and wild-type (Fig. 4). In both genotypes PSY, ZDS1, bLCY2 and bCHX genes were upregulated in flavedo and pulp during ripening while PDS gene was almost constitutively expressed in pulp. The expression of PDS in flavedo, and ZDS2, ZDS3 and bLCY1 in both fruit tissues, did not show clear trends or were constitutive (Fig. 4A, B). In general, the pattern and relative expression level for most of the genes was similar in `Pinalate´ and `Navelate´, and only some genes showed differences between genotypes: PSY, bLCY2 and bCHX transcript levels were significantly lower in flavedo of mature `Pinalate´ fruits, and expression of bCHX (breaker stage) and ZDS2 (ripe stage) were higher in `Pinalate´. The relative transcript level of additional genes such as PSY2 [9], CRTISO, CYP97A and ZEP were also determined in `Pinalate´ and `Navelate´ flavedo at breaker stage and no significant differences between both genotypes were detected (data not shown). In the pulp, the only difference between `Pinalate´ and `Navelate´ gene transcript levels was the reduced expression of ZDS3 in `Pinalate´ green fruits. Altogether, variations in the transcript levels between `Pinalate´ and `Navelate´ fruit tissues do not explain by themselves the differences in carotenoid composition between both genotypes.
Since young leaves of `Pinalate´ and `Navelate´ did not show differences in carotenoid profile but content was reduced by half in the mutant, we also analysed the relative expression levels of carotenoid biosynthetic genes (Fig. 4C). The expression of PSY and bLCY1 was approximately 50% lower in `Pinalate´ but other genes showed similar transcript levels in both genotypes (Fig. 4C). No expression of ZDS3 and bLCY2 genes was detected in leaves samples.
`Pinalate´ sweet orange mutant harbours a new Z-ISO allele with a single nucleotide insertion
The massive accumulation of early carotenes in `Pinalate´ fruit tissues together with the abnormal presence of 9,15,9´tri-cis-z-carotene without evident correlation with transcriptional profile of main carotenoid biosynthetic genes, and the partial rescue of flavedo wild-type phenotype when mutant fruits were exposed to light, strongly suggest a deficiency in the 15-cis-z-carotene isomerase, Z-ISO. In order to explore this hypothesis we identified the sweet orange Z-ISO gene by BLAST search in the Citrus sinensis v1.1 genome assembly in Plant Comparative Genomics portal Phytozome (https://phytozome.jgi.doe.gov/) using the Z-ISO from Arabidopsis (NP563879.1). A single gene was identified (orange1.1g017272m.g) with a length of 3189 nucleotides and similar genomic structure to Arabidopsis and maize homologues [26] (Fig. 5A). Sweet orange Z-ISO gene contains four exons and three introns encoding a predicted protein of 374 amino acids. The alignment of orange Z-ISO protein with homologues from Arabidopsis, maize and tomato showed a high degree of identity (76-71 % at protein level) and most of the variability was detected at the N-terminus which corresponds with the plastid transit peptide predicted by ChloroP1.1 (Fig. 5B). A model prediction of 3D structure of the sweet orange ZISO was generated in the PPMserver by using I-TASSER [47]. For modelling, two oxidoreductases: the integral membrane sterol reductase from Methylomicrobium alcaliphilum (Acc. PBD: 4QUV) and phenol hydroxylase-Regulatory Protein Complex from Pseudomonas (Acc. PBD: 2INP) were used [27]. The topology of sweet orange Z-ISO was also investigated by using MEMSAT3 [48]. The 3D model and topology analysis for orange Z-ISO showed a structure with seven transmembrane a-helices (S1 to S7) (Fig. 5C) suggesting that is an integral membrane protein as reported for maize Z-ISO [27]. The mechanistic study of maize Z-ISO showed that H150 (transmembrane domain 2) and, C263 and H266 (transmembrane domain 5) are critical residues for isomerization involved in the cofactor (heme) binding and reversible heme ligation. In sweet orange Z-ISO homologous residues were identified in transmembrane domains S2 (H158) and S5 (C271 and H274) (Fig. 5B).
Genomic sequencing of Z-ISO from wild-type `Navelate´ revealed a unique sequence for all clones isolated and identical to orange1.1g017272m.g available at the Citrus sinensis genome. By contrast, in `Pinalate´ mutant two different genomic sequences were isolated, one identical to wild-type and a second allele with a T insertion in the exon 2 at position 1588 (accession number MN417949) (Fig. 6A). Recently, it is has been reported that Z-ISO is a single copy gene in citrus [49] which is also compatible with information obtained from Citrus sinensis genome database; therefore, the presence of two different alleles indicates heterozygosity at Z-ISO locus in `Pinalate´.
The cDNA sequencing of Z-ISO from parental `Navelate´ sweet orange showed a single sequence with an ORF of 1125 nucleotides (Fig. 6B). PCR amplification of full-length Z-ISO cDNAs from `Pinalate´ samples (flavedo and pulp tissues at breaker and ripe stages) revealed the presence of three Z-ISO transcript variants in the mutant (Fig. 6B). Out of 71 independent cDNA clones from `Pinalate´, 36 corresponded to wild-type sequence (WT), 29 were identical to wild-type plus a nucleotide insertion (T) in exon 2 at position 1587_1588 (P6), and 6 sequences (P5) showed a deletion of the first 42 nucleotides of exon 2 (positions 1543-1585) in addition to the T insertion identified in P6 (Fig. 6A,B). Thus, approximately 50% of the `Pinalate´ Z-ISO transcripts showed an altered sequence (Fig. 6B): the P5 resulted in a shorter sequence (1082 nucleotides) and P6 contained one extra nucleotide compared to the wild-type. Both P5 and P6 give frameshift mutations with premature stop codons at 523 and 487 nucleotides and truncated proteins with 177 and 163 amino acids, respectively (Fig. 6B). The 42 nucleotides deletion in P5 transcripts can be explained by an aberrant splicing at the intron 1-exon 2 caused by the T nucleotide insertion. The sequence (CAGTTG) generated by T insertion in `Pinalate´ Z-ISO allele (Fig. 6A) highly resembles the intron 1-exon 2 junction (CAGGTTG) and might create a novel acceptor splice site skipping the first 42 nucleotides of exon 2.
The cDNA sequence data suggest that `Pinalate´ is heterozygous in the Z-ISO locus. This possibility was confirmed by direct sequencing genomic DNA of Z-ISO from `Pinalate´. The nucleotide sequencing trace up to the T insertion site was unblemished indicating that is identical in both alleles. From the T insertion and on, the sequence trace became blurred due to a frameshift in one allele (Supplemental Fig. S3).
The Z-ISO encodes a bona fide 15-cis-ζ-carotene isomerase but mutant variants (P5 and P6) are not functional
The 15-cis-ζ-carotene isomerization activity of Z-ISO variants was tested in E. coli cells carrying the pZETA vector that produced 9,15,9´tri-cis-ζ-carotene. To that end, the full-length cDNAs of Z-ISO isolated from wild-type `Navelate´ (WT) and `Pinalate´ mutant, (variants P5 and P6) were cloned in the plasmid vector pGEM-T. E. coli cells carrying pZETA alone (control), pZETA together with WT or mutant Z-ISO cDNAs, were selected following transfection to E. coli cells and selection with the appropriate antibiotics. Carotenoid composition in the bacteria grown in suspension cultures was analyzed following incubation in dark or light conditions. E. coli cells with pZETA produced 9,15,9´tri-cis-ζ-carotene and 9,9´di-cis-ζ-carotene at a ratio of 1:2, respectively (Fig. 7). In the presence of wild-type Z-ISO this ratio was changed to 1:1, but the mutant Z-ISO (P5 or P6) was inactive. Similar results were obtained when Z-ISO cDNAs from `Pinalate´ (P5 or P6) and `Navelate´ were expressed in E. coli with the plasmid pPROLYCOPENE (Fig. S4). These results demonstrate that citrus Z-ISO codes for a bona fide 15-cis-ζ-carotene isomerase and both cDNA variants from `Pinalate´ abolished Z-ISO activity.
The presence of di-cis-ζ-carotene in E. coli with pZETA alone may have resulted from a spontaneous chemical isomerization of the 15-cis double bond. This possibility is supported by the finding that when the E. coli cell cultures were exposed to dim white light (50 μmol photons m-2 s-1), the non-enzymatic conversion of tri-cis to di-cis-ζ-carotene increased even more than the enzymatic process (Fig. 7A, B).
However, conversion of tri-cis to di-cis with P5 and P6 mutant gene products in dark conditions was similar to empty vector pZETA, indicating no Z-ISO function for P5 and P6 while the functionality of WT Z-ISO sequence was demonstrated by a 25% increase in the proportion of di-cis-z-carotene (Fig 7A).
Expression level of Z-ISO is significantly down-regulated in `Pinalate´ mutant
The expression of Z-ISO gene was analyzed during fruit ripening and in young leaves from parental `Navelate´ and mutant `Pinalate´. In parental fruit tissues a significant up-regulation in Z-ISO expression occurred during ripening, more noticeable in flavedo than in pulp (Fig. 8). In `Navelate´ ripe fruit compared to immature stage, the expression of Z-ISO was almost 8 and 3-fold higher in flavedo and pulp, respectively (Fig. 8). By contrast, the Z-ISO expression only exhibited a moderate increase in `Pinalate´ fruit tissues during ripening. As a result, in mutant flavedo, pulp and leaves the Z-ISO expression was 3.8-, 2.5- and 5-fold lower than in wild-type, respectively, and only immature green peel of `Pinalate´ showed similar expression than parental (Fig. 8).