Phloem Loading In Cucumber:Combined Symplastic And Apoplastic Strategies

Phloem loading,as the first step of transporting photoassimilate from mesophyll cell to the sieve element-companion cell complex,creates the driving force for long-distance photoassimilates transport.Cucumber as a typical stachyose transport species is thought that employ the polymer-trapping loading pathway.However,since sucrose transporters expressed highly in mature leaves,we proposed that apoplastic phloem loading pathway may be contained in cucumber leaves.For proving this hypothesise,we observed the types and propotion of companion cells by electron microscopy,determined the categary and percentage of sugars in phloem stream by 14C labeling experiment and reverse genetics methods to demonstrate systematically that polymer-trapping and apoplastic loading pathway co-exist in noninfected cucumber leaves.This finding providing the most comprehensive analysis of mixed loading in any other species.The results as below:1.The results from anatomical observation indicate that there are three kinds of companion cells,they are ordinary cells(OCCs),intermediary cells(ICs)and transfer cells(TCs).In the fifth order veins,almost all companion cells(CCs)are ICs accompanied by a small number of OCCs,and there are no TCs;in the third order veins,the percentage of OCCs and ICs are almost equal,and ICs are seen at the periphery of the phloem,but there are no TCs;In the main veins,most CCs are OCCs with a small number of ICs and TCs.The results from qPCR and immunolocalization showed that galactinol synthase gene(CsGolS1)is expressed mainly in the fifth and third order veins and localized in CCs;while sucrose transporter gene(CsSUT2)is expressed mainly in the third order veis and localized in the CCs and sieve elements(SEs).These are the basic of molecular and anatomic in cucumber loading strategy.2.Suppressing CsGolSl,the CsGolS1i plant height and leaf area are obviously smaller than in wild type plants;the synthesis of stachyose,raffinose and galactinol significantly decreases,sucrose(the substrate of stachyose synthase)content are elevated and accumulate high level starch;After 14CO2 labeling,the percentage of radiolabeled stachyose is obviously lower in CsGolSli plants than WT,however,the percentage of radiolabeled sucrose and monosaccharides increased obviously.These results showed that poymer-trapping loading strategy is the mainly phloem loading way in cucumber.3.In CsGolSli plant,the expression of CsSUTs and CsSWEETs are highly upregulated,thus,we proposed that sucrose transporters maybe involved in the phloem loading in cucumber leaves.For verifying this hypothesis,we constructed the CsSUT2i plants.Distinct from weak growth in the CsGolSli plants,the phenotypes of CsSUT2i plants are similar with WT.Suppressing CsSUT2,sucrose and monosaccharide levels,especially sucrose,are lower in the leaves of CsSUT2i plants than in WT,whereas RFOs,galactinol and starch levels,especially stachyose and starch,are significantly higher in CsSUT2i plant;After 14CO2 labeling,[14C]sucrose export decreases and[14C]stachyose export increases into petioles in CsSUT2i plants.These results showed that CsSUT2 play a role in cucumber phloem loading.4.Similarly,after treating the leaves of CsGo1S1i-1 and CsGo1S1i-2 plants with 0.5 mM PCMBS,an inhibitor of the sucrose transporter,and kept in the dark for 20 h after normal light for 16 h,more starch retention is observed in leaves of CsGolSl1i plants treated by PCMBS compared with in control(treated by water),but no such retention is detected in WT.This suggests that downregulating CsGolS1 and inhibiting the activity of CsSUTs reduces export of carbohydrate and makes it more difficult for leaves to export carbohydrates accumulated in transient starch reserves.