Functional Characterization And Regulation Of Key Genes Involved In Tomato Ascorbic Acid Biosynthesis

Ascorbate acid (AsA) is an important antioxidant in plants, playing important roles in various plant physiological processes. Humans have lost the ability to synthesis ascorbate and have to absorb ascorbate from diet including fresh fruits and vegetables, as they are the major sources of ascorbate. More attention has been paid to improving ascorbate content in plants especially in fruits and vegetables. Since ascorbic acid biosynthetic pathway in plant was proposed in 1998, researches on plant ascorbic acid biosynthsis and regulation are very limited, In tomato, only a few genes involved in ascorbate metabolism were cloned and identified, and little is known about ascorbate regulation especially in fruit.In this study, some genes related to ascorbate biosynthesis were cloned from tomato and strawberry. Then these genes were overxpressed or suppressed in tomato by Agrobacterium-mediated transformation. The ascorbate contents and stress tolerance of the obtained transgenic plants were assayed. We found that SlGME1, SIGME2, SIGMP and FaGalUR are good candidate genes in regulating tomato ascorbate biosynthesis. To further understand the gene function, gene expression profiles in fruits of SIGME1, SIGME2 and SIGMP over-expressing transgenic plants were analyzed by cDNA microarray. The main results are presented as following:1. Two GME genes (SIGME1 and SIGME2) involved in ascorbate Smirnoff biosynthesis pathway were cloned from tomato and the sequences data have been submitted to GenBank databases under the accession number GQ150164 and GQ150165, respectively. They both contain a complete open reading frame of of 1131bp, contain six exons and five introns and encode 376 amino acids. The nucleotide sequences of the two SIGME genes share 79% identity, whereas the two putative amino acid sequences have 92% similarity. FaGalUR, the key gene involved in ascorbate biosynthesis D-galacturonic acid pathway was isolated from strawberry fruit.2. The fusion expression vectors of SIGME1 and SIGME2 with GFP were constructed. Onion epidermal cells expressing the fusion proteins were established by particle bombardment. Results indicate that the fluorescence of both fusion proteins of SIGME 1::GFP and SlGME2::GFP are clearly visible in the cytoplasm.3. The expression levels of SlGME1, SlGME2 and SlGMP were analysed in various tissues of tomato. Results showed that SlGME1, SlGME2 and SlGMP were constitutively expressed in roots, stems, leaves, flowers and fruits, whereas different expression levels were found in different tissues.4. The sense vector of SlGME1, SlGME2 and FaGalUR were constructed and transformed to tomato variety AC using the Agrobacterium-mediated transformation. The sense and RNAi vector of SlGMP were also transformed to Ailsa Craig. The obtained putative transgenic plants were identified by PCR and Southern blot, results showed that the T-DNA was integrated into the tomato genome, with 1 to 3 copies.5. RT-PCR and Real-time RT-PCR were used to analyse the expression levels of the target genes in some transgenic tomato plants, results showed that the transcript levels of aimed genes were elevated significantly in over-expressing transgenic lines and suppressed significantly in SlGMP RNAi transgenic lines. In addition, co-suppression was found in SlGME2 sense transgenic lines, and the suppression of SlGME1 was more obvious.6. Total ascorbate content in leaves and fruits were measured in some transgenic tomato lines. Transgenic plants over-expressing SlGME1 and SlGME2 exhibited a significant increase in total ascorbic acid in leaves and red fruits compared with wild-type plants, whereas the total asorbate content in leaves and breaker fruits decreased remarkably in SlGME2 co-suppression transgenic lines, just 12% and 28% of that in wild-type plants, respectively. In addition, over-expression and suppression of SlGMEs both altered the transcript levels of some ascorbate biosynthesis related genes. These results indicated that the two SlGMEs genes played important roles in tomato ascorbate biosynthesis, and biosynthesis and accumulation of ascorbate could be regulated through transgenic approach.7. Similarity, SlGMP played an important role in regulating ascorbate biosynthesis in tomato. Alteration of the expression of SlGMP significantly changed the total ascorbate content in transgenic tomato plants. In SlGMP over-expressing transgenic lines, the ascorbate content increased 22% in leaves and 60% in fruits compare with the wild-type plants, and increase was more effective in fruits. In addition, the ascorbate contents in leaves and fruits were significantly decreased in SlGMP RNAi transgenic lines.8. Ectopic expression of strawberry GalUR in tomato significantly increased the total ascorbate content in fruits; the maximum was increased by 65% compared with that in wild-type plants. It demonstrated that it was feasible to increase tomato ascorbate content using FaGalUR. In addition, it revealed that tomato may biosynthesize ascorbate through D-galacturonic acid pathway. 9. Suppression of SIGMEs and SlGMP in tomato influenced plant growth and development. The inhibition of apical buds and early formation of axillary buds were found in SlGME2 co-suppression transgenic tomato seedlings. Cytological observation showed that cell apoptosis existed in apical meristem of main stem. For SlGMP RNAi transgenic tomato lines, some spontaneous lesions were developed on the surface of the leaves when the plants were at the seedling stage, and high concentration of H2O2 in transgenic tomato leaves lead to the formation of lesions.10. Tomato with increased ascorbate accumulation improved the tolerance to abiotic stresses. The over-exprssing SlGME1 and SlGME2 transgenic tomato plants enhanced oxidative stress, cold and salt tolerance. The transgenic plants over-exprssing SlGMP showed improved tolerance to oxidative stress. Ectopic expression of strawberry GalUR in tomato also exhibited enhanced salt tolerance.11. Gene expression profile in breaker fruits of SlGME1 over-expressing tomato plants were analyzed by cDNA microarray, and the results indicated that some genes involved in stress response, lipid metabolism and flavonoids biosynthesis were up-regulated in transgenic tomato breaker fruits. Many differently expressed genes both existed in SlGME1 and SlGME2 over-expressing tomato breaker fruits shared the same change trend. The expression profile in breaker and red ripe fruits of SlGMP over-exprssing tomato plants were analyzed, and the results showed that genes involved in cell component organization, lipid metabolism, ethylene biosynthesis and stress response were significantly up-regulated in transgenic tomato breaker and red ripe fruits.In summary, Smirnoff pathway was the major ascorbate biosynthesis pathway in tomato, and SlGMEs and SlGMP were the key genes in Smirnoff pathway in regulating tomato ascorbate content. In addition, tomato may biosynthesize ascorbate through D-galacturonic acid pathway.