Expressed Sequence Tags Analysis And The ABA Metabolism In Fruits Of Sweet Orange Red-Flesh Mutants

The red pigment lycopene is absent in common citrus varieties. The lycopene-accumulating mutants possess greater market competence for the special red pulp color and the increased nutritional value. As for research, these mutants are excellent materials for the investigation of carotenogenesis and regulation of carotenoid end-products (such as ABA, vitamin A, etc.) biosynthesis.’Cara Cara’ navel orange is the scarce red-fleshed navel orange reported so far;’Hong Anliu’ sweet orange is a newly identified bud sport with lycopene accumulating. This study was presented as four parts: Based on the EST sequences from the pulp of red-fleshed mutant’Cara Cara’ navel orange, the compostion and expression profiles of transcripts related to citrus fruit ripening was well elucidated, and then the differentially expressed genes between the mutant and the ordinary navel’Washington’ fruits were identified by digital analysis; The expression profiles and potent hydroxyl radical-scavenging activity of three abundant metallothionein proteins from citrus fruit were analyzed; Genes involved in ABA biosynthetic and catabolic pathway in’Cara Cara’ navel orange were isolated; ABA concentration and expression levels of ABA pathway genes were examined in juice sacs of the mutant’Hong Anliu’’Cara Cara’ and their respective wild-type’Anliu’ ’Newhall’. The main results were as follows:1 Comprehensive analysis of expressed sequence tags (ESTs) from the pulp of the red-fleshed mutant’Cara Cara’ navel orange (Citrus sinensis Osbeck)(1) More than 1540 clones were randomly selected and sequenced from the cDNA library of’Cara Cara’ navel orange. Interpretation of the EST datasets revealed that the mutant pulp transcriptome held a high section of stress responses related genes such as the type III metallothionein gene (6.0%), heat shock protein (1.7%), Cu/Zn superoxide dismutase (0.6%), late embryogenesis abundant protein 5 (0.5%), etc.(2) Potential SSR and SNP loci were surveyed in the transcripts of this library for cultivar-specific markers. Trinucleotide SSRs were the most abundance at 40.9% of the total repeats. One hundred and eleven SSR primers were successfully designed and several sets were selected for polymporphism detection among 16 citrus varieties. As a result,21 out of 46 sets which were polymorphic can be used for genetic mapping. SNPs analysis found that transitions (73%) outnumbered transversions (27%). A total of 17 potential cultivar-specific and 387 heterozygous SNP loci were detected from’Cara Cara’ and’Washington’ EST pool.(3) To identify genes differentially expressed during fruit development between the mutant and the wild-type, we collected 12658 ESTs from 10’Washington’ navel orange fruit cDNA libraries representing different tissues and developmental stages. By digital expression analysis,133 transcripts were detected to be differentially expressed between the red mutant and its orange-color wild genotype’Washington’. Among them, genes involved in metabolism, defence/stress, signal transduction, photosynthetic and transport were statistical overrepresented. Fifteen transcription factors, composing of NAC, MYB, MYC, bHLH, bZIP domain members, were also included. Eight differentially expressed genes analyzed in digital were validated by quantitative real-time PCR. CAT and aldehyde dehydrogenase shared relatively higher expression in’Cara Cara’fruits, while lower expression of late embryogenesis abundant protein 5 (LEA5) was detected.2 Identification of three abundant metallothionein proteins from citrus fruitThree metallothionein genes (MT) with abundant expression were identified from fruits of’Cara Cara’and’Washington’navel orange. CsMT-1 was a newly isolated MT gene in citrus. One or two bands were detected in southern blot analysis of CsMT-1, suggesting its single copy property in the citrus genome. CsMT-1 was comprised of two exons and three introns, encoding only 63 amino acids. There were many light-regulated and tissue-specific elements located in the 5’flanking region of CsMT-1 promoter, however, the core metal-responsive element (MRE) and ethylene-responsive element (ERE) were absent although they were often oberserved in the 5’upstream regions of plant MT genes. Each CsMT showed a tissue-preferential expression pattern; the mRNA levels of CsMTs were higher in fruit sacs of’Newhall’than in that of the mutant’Cara Cara’navel orange; and their expression were also induced in calli by exogenous ABA. Besides, hydroxyl radical-catalyzed degradation of citrus genomic DNA could be efficiently suppressed by CsMTs in vitro.3 Isolation and sequence anlysis of ABA pathway genes from fruits of’Cara Cara’ navel orange(1) Isolation and sequence analysis of ABA biosynthetic genes. Alternative intron retention of CsZEP generated four different transcripts:a wild-type transcript, other three transcripts with early terminations resulting in lack of partial function domains. Southern blot analysis suggested that CsZEP gene was present at one locus or two different loci in the citrus genome. The full length coding region of four transcripts were inserted into pET-28a (+) plasmid and then transferred into E. coli strain BL21, respectively. The functional complementation of each recombinant protein was performed in a heterologous E. coli system. Meanwhile, complete or partial mRNA sequences of ABA pathway genes were isolated from ’Cara Cara’ navel orange and designated as CsVDE (GenBank accession no. HM036682), CsABA4 (GenBank accession no. HM036683), CsABA2 (GenBank accession no. HM036684), CsAAO3 (GenBank accession no. HM036685) and CsABA3 (GenBank accession no. HM036685).(2) Isolation and sequence analysis of ABA catabolic genes. The full-length cDNAs for CsCYP707A1 (1825 bp), CsCYP707A2 (1834 bp), CsCYP707A3 (1490 bp) and CsCYP707A4 (1729 bp) encoding ABA 8’-hydroxylase were isolated from Cara Cara navel orange. The deduced polypeptide is predicted to be 477,477,471, and 470 amino acids in length respectively. CsCYP707As showed significantly differential expression among different organ and tissue types. The mRNA levels of CsCYP707As in juice sacs of ’Cara Cara’ and its wild-type ’Newhall’ were regulated by endogenous ABA during fruit development. Exogenous ABA treatment could induce higher accumulation of CsCYP707As mRNA in calli. Besides, the full-length sequence of CsAOG encoding ABA glucosyltransferase isolated from fruits of ’Cara Cara’ navel orange was 1464 bp, containing an open reading frame of 1446 bp which is predicted to encode a putative protein of 481 amino acids.4 Accumulation of ABA in fruits of citrus red-fleshed mutants(1) The ABA content was analyzed in the juice sacs of ’Hong Anliu’,’Cara Cara’ and their respective wild-type ’Anliu’, ’Newhall’ at different developmental stages. Results showed that changes in ABA content during fruit maturation were simiar for all genotypes, containing two ABA accumulation peaks in the ripening process of orange fruits while the first one was detected at 150 DAFB and another one was in late stages of maturation (210 DAFB or 240 DAFB). The level of ABA in red-fleshed sweet oranges was much lower than that in ordinary ones at all sampling time points.(2) Carotenoid profiles in mature juice sacs of lycopene-accumulating mutants and their control varieties were investigated. Juice sacs of the mutants contained 4-50 times higher concentration of carotenoids than those of ordinary citrus. Aside from the dramatic accumulation of lycopene, the red-fleshed fruits also exhibited relatively higher leve1 of a-carotene, lutein,β-carotene as well as ABA precursor 9-z-violaxanthin than ordinary ones. The data indicated that the reduction of ABA level in the red-fleshed mutants was not due to a decrease of its substrates.(3) Expression profiles of ABA biosynthetic genes in juice sacs of ’Hong Anliu’ and its wild-type ’Anliu’ sweet orange were determined. In both genotypes, expression of these pathway genes was well correlated to the enhancement of ABA content during the maturation. The transcriptional level of CsNCED2 and CsNCED3 were relatively lower in ’Hong Anliu’ than in ’Anliu’, while other biosynthetic genes (CsZEP, CsVDE, CsABA4, CsABA2, CsAAO3 and CsABA3) were expressed at higher levels. The reciprocal association of decreased CsNCEDs transcription and impaired ABA synthesis suggested a potential factor that was responsible for the ABA phenotype in the mutant. Besides, sequence comparison of CsNCEDs showed no differences in either coding or promoter regions between red-fleshed and ordinary sweet oranges, which helped us to attribute the differential expression of CsNCEDs to the alteration of their regulatory genes.(4) Expression profiles of ABA catabolic genes in juice sacs of ’Hong Anliu’ and ’Anliu’ sweet orange were investigated. Four CsCYP707As and CsAOG remained at a relatively higher level in ’Hong Anliu’ than in ’Anliu’ at all developmental stages except at 150 DAFB, implying the important contribution of its increased catabolic rate to the reduced accumulation of ABA.Taken together, compared to ordinary sweet oranges, the red-fleshed mutants exhibited a mild ABA deficiency phenotype, which was a consequence of the reduced transcription of CsNCEDs and the enhanced expression of ABA catabolic genes.Finally, the possible crosstalk between ABA and carotenoid was further discussed.