Effect Of Myo-inositol Metabolism On Abiotic Stress Resistance And Ascorbic Acid Formation In Kiwifruit

Myo-inositol(MI) is a low molecular weight polyol ubiquitously existed in organisms, involving in multiple aspects of life process. MI derivatives such as phosphatidylinositol and inositol phosphates, can act as sphingolipid signal molecules, functioning in several metabolic processes, e.g. regulation of gene expression, storage of phosphate, contact of auxin and receptor, membrane tethering, stress resistance, biosynthesis of polysaccharide(galactinol) and regulation of cell programmed death(sphingolipid). The oxygenased product of MI(D-glucuronate) is used as a non-cellulose precursor for synthesizing cell wall pectin, or in some organisms, formation of ascorbic acid(AsA) – a major antioxidant in plant cells. MI together with its methylated derivatives react as osmotic protectors involved in osmotic regulation in organisms. Thus the biosynthesis and metabolism of MI have essential effects on various and important pathways in plants. In kiwifruit, MI is one of its most important carbohydates, occupying 20%-60% of the total fruit sugars, being an important quality trait of kiwifruit.All organisms share the same biosynthesis pathway of MI. D-glucose-6-phosphate is catalyzed by myo-inositol-1-phosphate synthase(MIPS) to form myo-inositol-1-phosphate, which then produces free MI under the effect of myo-inositol-1-phosphate phosphatase. MIPS is the key enzyme in this process. In catalysis of MI, the key enzyme is myo-inositol oxygenase(MIOX) which catalyzes MI to form D-glucuronate, involving in subsequent reactions. In this study, we cloned MIPS sequences from Actinidia deliciosa cv. ‘QinMei’, Actinidia eriantha, Actinidia rufa and Actinidia arguta and changes of AdMIPS genes expression and enzyme activity under drought and salt stresses were researched. We analyzed changes of MIPS gene expression and enzyme activity from different Actinidia species in their fruit development stages, levels of MIPS gene expression in various Actinidia tissues of these species, detected and compared MI and other soluble carbohydrates concentrations in these Actinidia species during fruit development. AdMIPS was used for RNA interference in Actinidia deliciosa. Using RACE, we cloned 3’ sequence and partial 5’ sequence of MIOX gene in Actinidia deliciosa, and analyzed its expression in different tissues of Actinidia. The main results are as follows:1. MIPS cDNA sequences were cloned from Actinidia deliciosa, Actinidia rufa, Actinidia eriantha and Actinidia arguta, each of which comprises the largest open reading frame(ORF) of 1533 bp, encodes 510 amino acid with a protein molecular weight of 56.3 kD. The acquired sequences were named as AdMIPS(Actinidia deliciosa), ArMIPS(Actinidia rufa), AeMIPS(Actinidia eriantha) and AaMIPS(Actinidia arguta), and deposited to Genbank with accession numbers being: JX122766, KF114870, KF114869 and KF114872. The isoelectric point, hydrophilicity, secondary structure and 3-D structure were analyzed and predicted using bioinformatics tools. The Actinidia MIPS gene sequences share high similarity of 98.94%, besides their amino acid sequences are highly conserved alighed with MIPS amino acid sequences from other organisms. MIPS transcripts were detected from various Actinidia tissues. In the fruit developmental stages, the highest MIPS transcription level and enzyme activity presented at young fruit stage; a similar pattern was observed in different Actinidia species.2. Concentrations of MI, sucrose, glucose and fructose were detected in fruit developmental stages of A. deliciosa, A. eriantha, A. rufa and A. arguta. In general MI concentrations were lower than that of other measured sugars in A. deliciosa, A. eriantha, and A. rufa, but in young fruit of A. arguta, its concentration reached up to 6 fold that of other sugars. Through the calculation of MI / sucrose, MI / sum of glucose and fructose, we found that these two ratios were both higher in A. arguta than that in the other three species. The results revealed that MI is a main carbohydrate in young fruit of A. arguta, and the conversion efficiency of sucrose, glucose and fructose to MI may be higher in A.arguta than in the other three Actinidia species evaluated here.3. Drought and salt stresses were conducted using A. deliciosa seedlings. The result displayed that under drought treatment AdMIPS gene expression increased followed by a decrease, but the enzyme activity kept declining; under salt treatment with different duration, the transcription and enzyme activity of AdMIPS were both first induced and then inhibited, but when imposed with different concentrations of NaCl, AdMIPS gene expression and enzyme activities in leaf, phloem and root of kiwifruit were induced to different degrees. These results indicate that AdMIPS could response to drought and salt stresses, suggesting significant functions of A. deliciosa MI in stresses.4. The recombinant vectors of interference of AdMIPS were constructed and introduced into A. deliciosa. After transformation and using kanamycin resistance positive lines were selected. DNA and cDNA of positive lines were checked trough PCR, and corresponding bands could be found. The result of Southern blot indicated the interference fragments were successfully integrated into the genome of A. deliciosa. The results revealed that MI concentration in transgenic lines was lower than in the untransformed line; however AsA content in transgenic lines was higher than in the untransformed line, indicating a complex change of MI metabolism and AsA metabolism pathways. Drought and NaCl treatment were applied to the transgenic kiwifruit seedlings and the result indicated that under these two stress treatments, the MI content and stress resistance in transgenic lines were lower than that in untransformed seedlings.5. The 5’ and 3’ end of Actinidia MIOX4 EST sequence predicted from Genbank were amplified using RACE. Relatively clear bands were obtained through nest PCR. After sequencing these contigs were spliced and showed a sequence of 1255 bp. Real-time quantitative PCR was used for analyzing MIOX4 gene expression in different Actinidia species, the result revealed the highest transcript level in flower of A. deliciosa and A. eriantha.