Sucrose Apoplastic Loading Pattern In Cassava Leaf

The majority of higher plants transport sucrose as mobile carbohydrate for phloem loading.There are three strategies of phloem loading:passive diffusion that relies on plasmodesmatal densities;polymer trapping by loading via symplstic plasmodesmatal and being converted into raffinose and stachyose;apoplastic step driven by plasma-membrane transporter and energized by proton motive force.The energized apoplastic loading step promotes energy utilization,furthermore may help plants response to carbon loss by herbivors.To understand the relationship between the features of high biomass and high starch production of cassava（Manihot esculent）and sucrose phloem loading strategy,the physiologic structure and biochemical action of cassava leaf were studied and then the apoplastic phloem loading step in cassava leaf was demonstrated.Besides,relative gene family,sucrose transporter（SUT）,and other members of sugar transporters（SWEET,Sugars Will Eventually be Exported Transporters）,which are playing multiple roles in cassava were characterized.1 Plasmodesmata fruquence at interface between vein cells were calculated.Few plasmodesmata present at all interfaces in the minor vein phloem of cassava leaves suggesting that the movement of sucrose from mesophyll into phloem in cassava（Manihot esculent）doesn’t rely on symplastic passive way.Cassava leaf tissue exposed to exogenous 14C-Sucrose or 14CO2 accumulated radiolabel in the minor veins and showed a web vein map,as determined by macroautoradiography.This study revealed that sucrose phloem loading is apoplastic in cassava.This energized apoplastic loading step in cassava promotes utilization efficiency to maintain high photosynthetic efficiency and high biomass,furthermore may help cassava response to carbon loss by herbivors.2 Depended on information from Genbank,literature and cassava genome data from phytozome,6 cassava sucrose transporters（MeSUTs）were cloned.MeSUTs is a complex gene family full of homologs with SNP site difference and alternative splicing caused by premature termination.MeSUTla presented high transcript level in cassava leaves when tested by real-time quantitative PCR.Defect yeast transformed with 6 MeSUT grown using sucrose as single carbon,which suggested MeSUT1a could strongly uptake sucrose.Furthermore GUS stain analysis pointed out MeSUT1a and MeSUT2 located in cassava leaf vein.All of characters predict MeSUTla certainly involves in sucrose apoplastic loading.3 To fully understand the response of MtN3/saliva/SWEET family to cassava physiological activity,29 MeSWEETs were obtained from cassava genome.Most of them do not transcript in normal grown samples.7 of them were cloned and identified in yeast strain EBY4000,which is deficient to uptake hexose.MeSWEETlb could strongly uptake fructose,glucose and mannose while MeSWEETla intensely uptakes glucose but weakly uptake mannose.4 To solve the trouble of unstable reference genes in cassava samples when performing real-time quantitative PCR,a series of candidate reference genes were evaluated via 384 amplification platform and SYBR green quantitative PCR technology.After analyzed by two algorithms,geNorm suggested the combination of cassava4.1₀17977 and cassava4.1₀06391 for many different samples.NormFinder predicted cassava4.1₀06884（26S proteasome regulatory complex,subunit N10）as a super reference gene.This job mainly demonstrated sucrose apoplastic phloem loading strategy in cassava,raised hypothesis and explanation,finally filled this blank.