Functional Characterization Of CsPSY And CsLCYb From Cara Cara Navel Orange And Effects Of The CrtB Transgene On Carotenoid Biosynthesis In Citrus

Carotenoids are the pigments responsible for the external and internal coloration of fruit of most citrus species. Recently, citrus red-fleshed cultivars have drawn the attention of consumers for their attractive pulp colour and their significant benefits to human health. Cara Cara, a bud mutation from the Washington Navel orange, has been characterized and identified as the unique navel orange dominantly accumulating lycopene andβ-carotene in the pink flesh. In the present research, three cDNAs encoding phytone synthase (CsPSY) and lycopeneβ-cyclase (CsLCYb1 and CsLCYb2) respectively, which were the key enzymes in the carotenoid biosynthesis pathway, were functionally characterized. The results could help us gain more knowledge on the mechanism involved in the formation of red-fleshed trait in citrus fruit. In addition, a bacterial phytoene synthase gene crtB was introduced into citrus embryogenic calluses and epicotyl segments by Agrobacterium-mediated transformation. Molecular analyses, phenotypic identifications, cytological observations and metabolite determination of transgenic lines were conducted. And the mechanism on enhancement of carotenoid biosynthesis in citrus calluses and embryoids transformed with crtB gene was discussed. The main results were as follows:1. Functional characterization of CsPSY gene from Cara Cara nave orange.1) The cDNA sequence of CsPSY contains an open-reading frame of 1308 bp in length, which is predicted to encode a putative protein of 436 amino acids with a calculated molecular weight of 49.5kD. The deduced amino acids sequence of CsPSY had a high homology with PSYs of plants (>70%), algae (54-58%), and cyanobacteria (45-47%), but not with bacteria (<30%), suggesting that PSY was a highly conserved protein during evolution. The CsPSY protein had putative transit sequence in the N-terminal residues 1-44 and a predicted transmembrane domain in 260-282 amino acids.2) Real time RT-PCR analysis revealed that the CsPSY expression was up-regulated during fruit development and maturation; the transcripts levels of CsPSY gene in falvedo, albedo and segment membrane of Cara Cara was higher than that of Washington, respectively. No significant difference in CsPSY transcripts in juice sacs between these two cultivars was observed.3) The full-length coding region of CsPSY cDNA was inserted into pET-28a (+) plasmid and then transferred into E. coli strain BL21. The results confirmed that CsPSY could encode a functional phytoene synthase which could catalyze a head-to-head condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene in a heterologous E. coli system engineered to accumulate GGPP.4) The plant expression vector pBI-CsPSY containing CsPSY was constructed and used to transform Hongkong kumquat (Fortunella hindsii Swingle), resulting in 5 independent transgenic plantlets. HPLC analysis showed that total fruit carotenoids of transgenic lines were 2.0-fold higher than the wide-type controls, whereas phytoene, lycopene,β-carotene andβ-crytoxanthin levels were increased 2.1-,2.5-,2.0- and 2.3-fold, respectively, suggesting that the over-expression of CsPSY enhanced the carotenoids content of transgenic Hongkong kumquat fruits. Real-time RT-PCR analysis revealed that transcript levels of carotenoids biosynthetic genes in the CsPSY overexpressed plants remained unaltered except that PDS and ZDS showed a minor increase, although there was an increase in PSY transcript by from 4 to 6 folds.2. Functional characterization of CSLCYb genes from Cara Cara nave orange.1) The cDNA sequences of both CsLCYb1 and CsLCYb2 contain an open-reading frame of 1512 bp in length, which is predicted to encode a putative protein of 504 amino acids with a calculated molecular weight of 56.5kD. Both CsLCYbl and CsLCYb2 shared 98% identity at the nucleotide and amino acid levels. Analysis of nucleotide sequence in the ORF of CsLCYb cDNAs revealed 23 nucleotide changes, causing 11 amino acid substitutions. The deduced amino acids of CSLCYb1 and CsLCYb2 had a high homology with LCYbs of plants (>70%), algae (50-54%), and cyanobacteria (35-46%), but not with bacteria (<30%), suggesting that LCYb is also a highly conserved protein during evolution. The CsLCYb proteins had four predicted transmembrane domains in the sites of 85-106, 206-227,373-391 and 460-480 amino acids in the CsLCYb1, and three in 85-106, 206-227 and 373-391 amino acids for the CsLCYb2.2) Real-time RT-PCR analysis revealed that CsLCYb1 and CsLCYb2 displayed similar expression trends during the fruit development, and the expression level of CsLCYb2 was higher than that of CsLCYbl in all tissues of both Cara Cara and Washington navel oranges. In the flavedo and albedo, the levels for the two genes transcripts were lower in Cara Cara than in Washington during early stages of fruit development (August and September). During later developmental stages, the transcript levels of two genes in the flavedo were still lower in Cara Cara than in Washington, while the transcripts in the albedo were higher in Cara Cara than in Washington. No significant difference in CsLCYbl and CsLCYb2 expression in segment membrane between these two cultivars was observed. In the juice sacs, although CsLCYb1 and CsLCYb2 displayed a similar expression pattern in both cultivars; the levels of the two genes expression were lower in Cara Cara than in Washington throughout the whole fruit ripening process.3) The full-length coding region of CsLCYbl and CsLCYb2 cDNA were cloned into pET-28a (+) plasmid and then transferred into E. coli strain BL21, respectively. The results indicated that CsLCYbl and CsLCYb2 could encode a functional lycopene P-cyclase which could catalyze the formation of twoβ-rings in lycopene to produce P-caotene in a heterologous E. coli system engineered to accumulate lycopene. Interestingly, in contrast to CsLCYb1, the catalytic activity of CsLCYb2 was lower, and a remarkable accumulation of y-carotene was observed in E. coli during the conversion of lycopene toβ-carotene.4) The plant expression vectors pBI-CsLCYbl and pBI-CsLCYb2 containing CsLCYbl and CsLCYb2 were constructed and used to transform tomato(Lycopersicon esculentum), respectively, resulting in 18 putatively independent transgenic plantlets of 12 with CsLCYbl and 6 with CsLCYb2. HPLC analysis revealed that CsLCYbl transgenic fruits displayed a bright orange coloration due to the accumulation of high amounts of P-carotene, while CsLCYb2 transgenic fruits did not show obvious phenotypic differences from those of the non-transformed controls during the fruit development and ripening. These results suggested that the over-expression of CsLCYbl facilitate a virtually complete conversion of lycopene intoβ-carotene of transgenic tomato fruis, while CsLCYb2 did not have significantly effects onβ-carotene production in transgenic tomato fruits.3. Construction the plant expression vector pBI-ts-rbcS-crtB and its functional characterization by tomato transformation. The plant expression vector pBI-ts-rbcS-crtB containing a bacterial phytoene synthase gene crtB was constructed and used to transform tomato (Lycopersicon esculentum), resulting in 15 independent transgenic plants. HPLC analysis showed that total fruit carotenoids of transgenic tomato were 1.3 to 2.5-fold higher than the wide-type controls, whereas phytoene, lycopene,β-carotene and a-carotene levels were increased 4.3-,1.8-,2.2-and 2.3-fold, respectively. These results suggested that the over-expression of crtB significantly enhanced the carotenoids biosynthesis and accumulation in transgenic tomato fruits. Real-time RT-PCR analysis revealed that the expression of GGPS was markedly decreased in the crtB transgenic lines. A simultaneous increase in the mRNA levels of PSY1,PSY2,PDS,ZDS,CRTISO,LCYb,BCH was observed in the transgenic lines, while no significant changes for LCYe and ZEP expression in most transgenic lines.4. Enrichment of carotenoids in citrus embryogenic calluses by overexpression of bacterial phytoene synthase gene crtB. A phytoene synthase (crtB) from the bacterium Erwinia herbicola has been overexpressed in citrus embryogenic calluses. In contrast to the normal white color of wide-type calluses, the crtB transgenic calluses and embryoids displayed a distinct yellow-orange and orange pigmentation due to the accumulation of high amounts ofβ-carotene. HPLC analysis showed thatβ-carotene of transgenic calluses and embryoids of GWZ were 6.1 and 20.9-fold higher than the wide-type controls, whereasβ-carotene levels in the transgenic calluses and embryoids of FXC were increased 4.1 and 9.8-fold. Light microscopic observations revealed that the crtB transgenic calluses and embryoids included orange bodies representing chromoplasts containing orange pigmentation. A large number of orange structures including helical sheets and fragments were also found in the suspension. Transmisson electron microscopic analysis indicated that the crtB transgenic lines have much plastoglobuli presumed to contain carotenoid within the chromoplasts, whereas the non-transformed controls contained much amyloplasts exclusively. These results suggested thatβ-carotene accumulation induced by the crtB transgene was related with an increased capacity of carotenoid sequestering. Real-time RT-PCR analysis revealed that the expression of endogenous carotenogenic genes PSY, PDS, ZDS, CRTISO,LCYb and LCYe were up-regulated in the transgenic calli and embryoids, whereas the transcript levels of BCH and ZEP genes were down-regulated. These results suggested thatβ-carotene accumulation induced by the crtB transgene was also related with an enhanced capacity of carotenoid biosynthesis.5. Production of transgenic plants with crtB gene in citrus. A bacterial phytoene synthase gene crtB was also introduced into Cocktail grapefruit and precocious trifoliate orange by Agrobacterium-mediated transformation. Following selection on the basis of kanamycin resistance, approximately 114 putatively independent transgenic plants (86 lines for Cocktail grapefruit and 36 lines for precocious trifoliate orange) were generated. PCR analysis showed that 4 and 12 out of these transgenic Cocktail grapefruit and precocious trifoliate orange lines were positive for NPTII and ts-rbcS-crtB genes, respectively. Southern analysis with the NPTII and crtB gene probe revealed the integration of 1-2 copies and 2-4 copies of the transgene in transgenic Cocktail grapefruit and precocious trifoliate orange, respectively. RT-PCR analysis indicated that the foreign crtB gene was expressed in these transgenic lines. HPLC analysis showed that total carotenoids in transgenic leaf of Cocktail grapefruit decreased by about 25-30% compared to the non-transformed controls, whereas a-carotene and P-carotene levels decreased by about 40-50% and 20-40%, respectively. However, the total amount of leaf carotenoids of transgenic precocious trifoliate orange was 1.3 to 1.7-fold higher than the non-transformed controls, whereas a-carotene andβ-carotene levels were increased 1.2-1.6 and 1.5-2.0 fold, respectively. These results suggested that the over-expression of crtB have differential effects on carotenoids biosynthesis and accumulation in transgenic Cocktail grapefruit and precocious trifoliate orange leaf, which may be relative to a strongly regulated and interdependent fashion of carotenoid and chlorophylls biosynthesis in citrus leaf.