Preliminary Studies Of The Relationships Between The14-3-3Proteins And Sucrose Metabolism During Tomato Development

The synthesis and cleavage of sucrose in plant not only influence the balance betweensucrose and starch content in source but also the carbohydrates partioning in sink. Currently,the way of SPS-SPase is regarded as the main pathway of sucrose synthesis in sucrosemetabolism. SPS（E.C.2.4.1.14）is the rate-limiting enzyme for this pathway and catalyzes thereaction, to wit, UDPG+fructose-6-phosphateâ†'UDP+sucrose-6-phosphate.With the complex consisted of SPS and SPP (sucrose-phosphate phosphatase) in vivo,the reaction is irreversible in fact. Therefore, the activity of SPS is directly related to sucrosesynthesis and accumulation and thus affects the quality of tomato fruit. Most serine residuesof SPS can be phosphorylated and thus its activity is regulated by covalentmodification.Fourteen-three-three protein is a kind of modulator with small molecule andinvolves a variety of physiological processes. The present evidence showed that14-3-3protein can bind to the phosphorylated serine site (s) on SPS in many crops, in turn altering itsactivity. However, we know nothing about the modulation of the14-3-3protein to SPS andeven more about the relationship between the14-3-3protein and sucrose metabolism. Herewe use the tomato cultivar-Liaoyuanduoli as research materials, with the regulation of14-3-3protein to SPS as a breakthrough, to explore the relationships between14-3-3proteins andsucrose metabolism in tomato.Firstly the coding sequences of tft11and tft12which are not published in public database(GenBank) were determined through the bioinformatics methods. Then the isoforms whichcan interact with SPS in1214-3-3protein isoforms were determined by bioinformaticsanalysis. Meanwhile, the content of soluble sugar (sucrose, glucose,fructose) and starch aswell as the activities of sucrose metabolizing enzymes(acid invertase, neutral invertase,sucrose synthase, sucrose phosphate synthase) in different development stages of tomato leafand fruit were tested with traditional plant physiological methods to clear the relationshipsbetween sugar content and enzyme activity. Finally the expression of14-3-3genes and sps indifferent development stages of tomato leaf and fruit was examined with real-time PCR.Results are listed as following:1. The coding sequence of tft11is cloned with in silico cloning;2. Of1214-3-3protein isoforms (TFT1-TFT12), TFT1and TFT10most likely interact with SPS which contains phosphoserine sites to alter its activity;3. The transient accumulation of soluble sugar and starch is present in the early developmentstage (after anthesis,0-10d) of tomato leaf. Besides the content of hexose is higher than thatof sucrose, in which the content of fructose occupies the first place;4. During the tomato leaf development, the activity of sucrose synthase is significant negativecorrelated with the content of glucose (P<0.05),significant negative withfructose(P<0.05),highly significant positive with sucrose(P<0.01),respectively.The activity ofacid invertase is found to be highly significant positive correlated with the content ofglucose(P<0.01), highly significant positive with fructose(P<0.01), highly significant negativewith starch(P<0.01), respectively.The activity of neutral invertase is significant positivecorrelated with the content of glucose(P<0.01), highly significant positive withfructose(P<0.01),respectively.5. During the tomato leaf development, the expression of tft1is positive correlated with theexpression of sps, while tft10is negative.6. During the tomato fruit development, the transient accumulation of soluble sugar and starchis present in the early development stage (after anthesis0-10d) of tomato fruit. Besides thecontent of hexose is higher than that of sucrose, in which the content of fructose occupies thefirst place.7. During the tomato fruit development, the activity of sucrose synthase is found to be highlysignificant negative correlated with the content of glucose(P<0.01), significant negativecorrelated with fructose (P<0.05), highly significant positive correlated with sucrose (P<0.01),significant positive correlated with starch(P<0.05), respectively. The activity of neutralinvertase is highly significant negative correlated with the content of sucrose (P<0.01).8. During the tomato fruit development,the expression of tft1is highly significant positivecorrelated with the activity of sucrose synthase(P<0.01), highly significant positive withneutral invertase(P<0.01), highly significant positive with sucrose phosphate synthase(P<0.01), respectively. The expression of tft10is significant positive correlated with theactivity of sucrose synthase (P<0.05), highly significant positive with neutral invertase(P<0.01), highly significant positive with sucrose phosphate synthase (P<0.01), respectively.9. During the tomato fruit development, neutral invertase and sucrose synthase performcritical roles in the early development stage (after anthesis0-10d) with the appearance of highactivities.