Study On The Phloem Loading Mechanism Of Sucrose In Rice Leaves

Grain filling in rice(Orazy sativa L.)is a procedure of coordinated “Source-phloem transport-sink” system,including the loading of photoassimilates(mainly sucrose)form source leaves to source phloem,long-distance phloem transport of sucrose from source phloem to sink phloem and carbohydrates down-loading into the sink grains.As an important part in “Source-phloem transport-sink” system,the efficient of phloem loading affects the transport of sucrose and sink downloading directly.Nowadays,insufficient photoassimilates transport ability has been the main factor that leads to the low grain filling rate and limited grain yield in rice.However,there is little research about phloem loading in rice studied,and there is no clear conclusion about phloem loading strategy in rice.Recently,bio-information and sub-micro-structure analysis revealed that it’s highly possible that rice use apoplastic loading as major phloem loading strategy,while there is still no sufficient molecular and physiological evidence.Here,cell wall invertase(INV)was transformed into Nipponbare to study the characteristic of phloem under restrained apoplastic loading in rice.Structure and function analysis of Os SUTs,combined with physiological research were also conducted to reveal the major Os SUTs involved in apoplastic loading and further clarify the mechanism of phloem loading in rice.These researches will not only offer sufficient evidence to prove apoplastic loading strategy of photoassimilates in rice,but also provide theoretical basis for breeding and cultivation to gain higher yield in rice.The main findings are shown as follows:1.Physiological analysis shows that apoplastic loading is more suitable for rice.Traditional EDTA method was improved by covering the extracting leaves with sponge to decrease transpiration rate and take leaf sheath as control to decrease the side-effects caused by EDTA damage.With an increased efficiency of 33.66%,improved EDTA method was used to measure the phloem loading ability in rice.The result shows that with the treatment of PCMBS(a transmembrane transport inhibitor),the activity of Os SUTs were inhibited and phloem loading ability was restrained.For rice phloem cell structure,the abundance of plasmodesmata in rice is medium,while the abundance of plasmodesmata between companion cells and phloem parenchymal cells is significantly lower than the interface of other cells among the phloem loading pathway.The sucrose content in leaf mesophyll cells is also remarkably lower than that in leaf vein during night time.These results indicate that apoplasitc loading is more suitable for rice phloem loading and satisfies the characteristics of phloem physiology.2.Phloem loading in rice was restrained in 35S::PI-INV plants,indicating that rice use apoplastic loading as major phloem loading strategy.The most significant characteristic of apoplastic loading is the transmembrane transport process.Here,yeast invertase(INV)was expressed and located specifically on the cell wall of rice plants to interfere the transmembrane transport of sucrose in rice,the result shows that phloem loading rate was decreased sharply when the transmembrane transport of sucrose get suppressed,leading to the accumulation of carbohydrates in rice source leaves,and this would show a passive feedback to the photosynthesis ability in rice.With less photoassimilate exported,the vegetative growth and yield of rice plants are both restrained,showing that once apoplastic loading is restrained,the phloem loading is also inhibited in rice.This provides strong evidence to support that rice use apoplastic loading as major phloem loading strategy.3.Expressing 35S::PI-INV in rice enhenced carbohydrate metabolis and nitrogen metabolism,but less photoassimilates transported from source leaves to grains inhibited yield formation.According to the results of transcription analysis,accumulated photoassimilates in source leaves caused by restrained apoplastic loading might lead to upregulated sucrose-starch metabolism to maintain the content and composition of cellular soluble sugars.ETH pathway was also activated and regulates cellular respiration together with hexose signal pathway,which increases the consume of carbohydrates and provide energy for cell development and nitrogen metabolism.Meanwhile,ETH induces cellular senescence and programmed cell death,which in turn facilitates the remobilization and reuse of nitrogen in source leaves.In grains,cytoplasmic sucrose was digested into hexose with the function of cytoplastic INV.hexose signal stimulates the ETH metabolism and cellular respiration pathway,while hexose can also participate in respiration and nitrogen metabolism directly,improving the cellular development pathway significantly.However,ETH inhibits the development of grains and the expression level of sugar transporters are decreased,leading to less carbohydrates obtained by grains.These results indicate that the main factors that restrain the formation of yield in 35S::PI-INV lines are less photoassimilates transported from source leaves to grains.4.Os SUT1 might be major sucrose transporter involved in phloem loading in rice.In this study,we analyzed the primary structure,secondary structure,three dimension structure and the pairing models of sucrose and Os SUTs through the amino acid sequence of Os SUTs,the function and characteristic of Os SUTs were further predicted through their protein structure.According to our analysis,all the Os SUTs except Os SUT2 may play a role in phloem loading in rice,while Os SUT1 has the strongest binding capacity with sucrose and the sucrose transport efficiency in Os SUT4 is the highest.The expression analysis of Os SUTs showed that Os SUTs are all expressed along the phloem long distance transport pathway,and expression levels of Os SUT1 is much higher than that of other Os SUTs.GUS staining shows that Os SUT1 is expressed in the phloem of mature leaves.Meanwhile,the research of ossuts mutants shows that ossut1 and ossut4 mutants exhibit a much obvious retarded phenotype compared with ossut3 and ossut5 mutants.These results indicate that Os SUT1 might be the major sucrose transporter involved in apoplastic loading in rice.