Physiological Mechanism Of Accumulating High Level L-Ascorbic Acid And Molecular Cloning And Expression Of Its Key Biosynthetic Enzyme In Rosa Roxburghii Tratt

Cili (Rosa roxburghii Tratt), a lately-domesticated fruit tree, was native to China and widely distributed in the south, south-west, central-south and north-west province of the country. This special fruit tree has brought about great interest worldwide due to its extremely high content of L-ascorbic acid (vitamin C, AsA) in fruit. Furthermore, it is also grown as an ornamental plant in China for its colorful, and multi-corolla flowers. Intensive efforts were focused on its exploitation, especially on the fruit exploitation and utilization because of its high AsA content, high superoxide dismutase (SOD) activity, and attractive senescence-retarding and cancer-preventing effects on human health, in the past decades. However, very little is known about why and how the highest level of AsA was accumulated in this plant, especially in its fruit.In present study, in order to clarify the physiological and molecular mechanism of AsA accumulation in R. roxburghii Tratt, AsA accumulating process and characteristics during development, the main AsA biosynthetic pathways in different organs and the AsA biosynthetic, turnover, and recycling enzyme activities were physiologically studied. Also, the molecular cloning and characterization of L-galactono-l,4-lactone dehydrogenase (EC 1.3.2.3, GalLDH), a key enzyme catalyzing the terminal step of AsA biosynthesis in higher plants, was reported and its expression in fruits during development was described for the first time. Furthermore, based on this, plant expression vector of GalLDH was constructed and its genetic transformation was preliminarily carried out, as well. The main results were summarized as follows.1. Different AsA accumulating characteristics in fruits and leaves during development, a) Different amounts of AsA accumulated in fruits and leaves: in mature fruits, AsA of approximately 1200 mg 100g-1 FW was accumulated, which was higher dozens of times than that in other familiar fruits or vegetables. In leaves, as compared, it had 174 mg 100g-1 FW of AsA which almost equal to just one seventh of that in the fruits, b) Different accumulating patterns: AsA accumulated continually with a slow, fast and slow pattern in the fruit during the whole development, in which the period from the end of June to the early of August was the most important stage in the AsA accumulation, due to the AsA content accumulated in this stage accounting for nearly 90% of the final level. However, the highest accumulation of reduced AsA occurred in young leaves and decreased slightly in mature ones. It had the biggest size of AsA pool (AsA+DHA) in fully-expanded leaves, most of it was, however, DHA. c) Different redox status of AsA: reduced AsA accounted for over 99% in fruits as compared to 40-70% in leaves.2. Biochemical properties of GalLDH and relationship between GalLDH activity and AsA accumulation during fruit development. Protein structure analysis and enzyme assay showed that Rosa roxburghii GalLDH, in accordance with all GalLDHs characterized to date, also sublocated in mitochondria. Of the inhibitors tested, the divalent metal salts CuSO4 and ZnSO4 had inhibiting effects on the GalLDH activity by about 30% at a concentration of 1 mmol/L. The organic inhibitors, acriflavine and lycorine, were able to partially inhibit the GalLDH activity but at different degrees.Acriflavine was able to inhibit extremely the GalLDH activity as compared to lycorine had a very slight influence on it even at concentration of up to 340 nmol/L. GalLDH presented approximately 40% activity when D-glucuronic acid was used as reaction substrate. Furthermore, the GalLDH activities in various organs showed a positive correlation with the total AsA levels, suggesting that the AsA pool size was, at least partly, determined by the GalLDH activity.3. AsA biosynthetic pathways in fruits and in leaves. AsA was able to be synthesized effectively in fruits using L-gaIactono-l,4-lactone or D-glucuronic acid as substrate in vitro, but the capacity of AsA using L-galactono-l,4-lactone substrate was much higher than that using D-glucuronic acid. This result, together with the most significant correlation between the GalLDH average activity and AsA accumulation per day (R2=0.783** ) in vivo, suggesting that AsA biosynthesis in fruits mainly proceeded via the L-galactose pathway. By contrast, incubations of leaves with D-glucuronic acid stimulated AsA biosynthesis more efficiently than that with L-galactono-l,4-lactone, indicating that leaf tissue synthesized AsA primarily by means of uronic acids conversion pathway. And, as a further proof, no correlation between the GalLDH activity and AsA accumulation in leaves was found. To the authors' knowledge, this is the first report that the two biosynthetic pathways existed in fruits and the uronic acids conversion pathway worked predominantly in leaves.4. Physiological mechanism of accumulating high content of AsA in the fruits. The highest level of AsA was accumulated in fruits caused by several mechanisms working in the organ: Firstly, high-level expression and long-time activity of the key biosynthetic enzyme, namely, GalLDH, tend to be an important reason of persistent biosynthesis of AsA during development since fruit synthesizing AsA mainly by the L-galactose pathway. Secondly, both L-galactose pathway and uronic acids conversion pathway working in the fruits indeed contribute to high amounts of AsA biosynthesis, although the latter appears not to a main biosynthetic means. Finally, only transitory and low activities of AAO and APX in the fruit were detected in the initial developing stage, suggesting that a little AsA was degraded by the two oxidative enzymes during the whole development.5. Isolation, sequence analysis and expression in E. coli. of full-length GalLDH cDNA. Full-length cDNA was isolated from Rosa roxburghii fruit using RT-PCR and rapid amplification of cDNA ends (RACE) techniques, designated as RrGalLDH (GeneBank accession number: AY643403). Sequence analysis indicated that it represents an open reading frame of 1 767 bp encoding 589 amino acids. The amino acid sequence showed high homology (75%~91.4%) with the GalLDH amino sequence known from other plants. Furthermore, the translated product of RrGalLDH has a putative mitochondrial targeting signal, a FAD-binding region and two predictable transmembrane regions. Southern blot analysis revealed that 2~3 copies of RrGalLDH gene existing in the genomic. The complete ORF was subcloned into pET-30a and transformed into E. coli cell, then induced by addition of IPTG. The expressing product showed an additional 56 kDa protein band by SDS-PAGE and specific enzyme activity of GalLDH as compared to the control. This is the firstreport on the cloning of full-length GalLDH gene from the perennial woody plants.6. Spatio-temporal expression of RrGalLDH and the relationship with AsA accumulation.Northern blot showed that the transcript of RrGalLDH in the fruits was significantly abundant than that of in the flowers, leaves, stems and roots. Accordingly, AsA content in fruits was as much as 60 times or more than that in the other organs. Furthermore, the RrGalLDH expression trait in various organs was correlated with RrGalLDH activity and AsA levels, apart from in the roots. The RrGalLDH massage accumulation was detected in fruits of various developing stage, although a weak, strong and weak pattern, which correlated with AsA accumulating rate, was observed. Namely, lower RrGalLDH transcript level in fruit was detected during initial (5-27th of May) and last (5-25th of August) developing stage, and much higher RrGalLDH transcript occurred during the long period from the early of June to the end of July. It should be noted, however, that the RrGalLDH expression patterns in fruits were not associated with onset of ripening, indicated by ACO mRNA levels. This is the first report on the GalLDH expression model in developing fruits.7. AsA contents and activities of some antioxidant enzymes during senescence, and their potential effects on anti-senescence in Rosa roxburghii leaves. No obvious change of AsA level observed in fully-expanded and mature leaves since there was no aggravating oxidative stress occurred, indicated by O2- generation rate, MDA content and plasma membrane permeability. As the leaves senescence progressively and anti-oxidation aggravating, AsA declined rapidly. The changes of O2- generation rate correlated significantly and negatively with AsA contents (r= -0.9723*), suggesting the important role of AsA in scavenging ROS generated by leaf senescence. Moreover, The significantly negative correlation between the POD and CAT activities and the plasma membrane permeability suggests that the two enzymes played important roles in alleviating membrane damage caused by natural senescence of leaves.8. Optimization of genetic transformation system and RrGalLDH transformation for strawberry leaf. Based on the stable and efficient regeneration system established before, genetic transformation system for strawberry leaf discs was optimized in order that the potential transgenetic plants could be screened with Hygromycin B. Result showed that 5 mg/L of Hygromycin B was the proper concentration for screening transgenetic shoots. The expression vector, designated as pCAMBIA 1301 /GalLDH, was constructed by introducing full-length RrGalLDH cDNA, which was located under control of the cauliflower mosaic virus 35S in sense orientation aforehand, into pCAMBIA1301. This expression vector was then used to genetic transformation for the above transformation system by Agrobacterium-mediation.