Functional Characterization Of Silicon Transporter Genes In Cucumis Sativus And Solanum Lycopersicum

Silicon is the second most abundant element on earth.It can not only exert benefical effects on plants when they are subjected to abiotic and biotic stresses,but also regulate plant growth and development and increase the yield.Silicon concentration in different plant varies considerably,which is mainly attributed to the differences in capacity of plant silicon uptake.Up to date,most studies on the mechanism of silicon uptake in plants have been focused on silicon accumulators especially monocots,little is understood on the molecular mechanism in low silicon accumulators like dicots.The reason of the distinction of silicon uptake ability among plants still remains unknown.Cucumber(Cucumis sativus)is a widely grown cucurbitaceous vegetable that exhibits a relatively high capacity for silicon(Si)accumulation,while tomato(Solanum lycopersicum)is a typical low silicon accumulator.The molecular mechanism for silicon uptake in cucumber and tomato remains to be clarified.In view of this,in this study,cucumber(‘Mch-4’)and tomato(‘Micro Tom’)were chosen to investigate the mechanism of silicon uptake in the root.Functional analysis of putative silicon transporters CsLsi1,CsLsi2,CsLsi6 in cucumber and SlLsi1 and SlLsi2 in tomato was conducted.The main results are as follows:1.CsLsi1 was isolated in cucumber ‘Mch-4’ by homology-based cloning.Bioinformatics analysis shows that CsLsi1 was located on chromosome 3 and the coding sequence were 867-bp long.CsLsi1 shares 55.70% and 90.63% homology with theLsi1 s of a monocot and dicot,rice(Oryza sativa)and pumpkin(Cucurbita moschata),respectively.CsLsi1 was predominantly expressed in the roots,and application of exogenous silicon suppressed its expression.Transient expression in cucumber protoplasts showed that CsLsi1 was localized in the plasma membrane.Heterologous expression in Xenopus laevis oocytes showed that CsLsi1 evidenced influx transport activity for silicon but not urea or glycerol.Expression of cucumber CsLsi1-mGFP under its own promoter showed that CsLsi1 was localized at the distal side of the endodermis and the cortical cells in the root tips as well as in the root hairs near the root tips.Heterologous expression of CsLsi1 in a rice mutant defective in silicon uptake and over-expression of this gene in cucumber further confirmed the role of CsLsi1 in silicon uptake.The results suggest that CsLsi1 is a silicon influx transporter in cucumber.2.CsLsi2 was a putative silicon efflux transporter isolated in cucumber ‘Mch-4’ by homology-based cloning.Bioinformatics analysis shows that CsLsi2 was also located on chromosome 3 and the coding sequence were 1644-bp long.CsLsi2 shares 53.27% and 89.95%identity with rice OsLsi2 and pumpkin CmLsi2-2,respectively.CsLsi2 was mainly expressed in the root and application of exogenous silicon increased its expression.Expression of CsLsi2-eGFP fusion sequence in onion epidermis cells showed that CsLsi2 was localized at the plasma membrane.Transient expression in Xenopus laevis oocytes showed that CsLsi2 demonstrated efflux but no influx transport activity for silicon,and the transport was energy-dependent.Heterologous expression of CsLsi2 in tomato increased silicon accumulation in the shoot,and white powders composed of silicon accumulated on tomato leaves.The expression of CsLsi2-mGFP under its own promoter evidenced that CsLsi2 was mainly expressed on endodermal cells,showing no polar distribution.The results suggest that CsLsi2 is a silicon efflux transporter in cucumber.3.CsLsi6 was isolated in cucumber ‘Mch-4‘ by homology-based cloning.Bioinformatics analysis shows that CsLsi6 was located on chromosome 3 and the coding sequence was 786-bp long.CsLsi6 shares 48.32% homology with OsLsi6 in rice.CsLsi6 was mainly expressed in the stem,and application of exogenous silicon suppressed its expression in stem.Transient expression of CsLsi6-eGFP fusion sequence in cucumber protoplasts showed that CsLsi6 was localized in the plasma membrane.Heterologous expression in Xenopus laevis oocytes showed that CsLsi6 evidenced influx transport activity for silicon and the transport activity was not affected by low temperature.Expression of cucumber CsLsi6-mGFP under its own promoter showed that CsLsi6 was localized at the distal side of the epidermis cells in the root.The results show that CsLsi6 was a silicon transporter in cucumber,which might be involved in silicon transport process in cucumber.4.SlLsi1 was isolated in tomato ‘Micro Tom‘ by in silico cloning.Bioinformatics analysis shows that SlLsi1 was located on chromosome 3 and the coding sequence was 852-bp long.SlLsi1 shares 53.33% and 64.85% identity with rice OsLsi1 and cucumber CsLsi1,respectively.SlLsi1 was mostly expressed in the root,and its expression was not affected by silicon supply.Transient expression of SlLsi1-eGFP in onion epidermal cells and tomato protoplasts indicated that SlLsi1 was localized at the plasma membrane.Heterologous expression in Xenopus laevis oocytes and a rice mutant defective in silicon uptake showed that SlLsi1 had a specific transport activity for silicon.Heterologous expression of SlLsi1 in a rice silicon uptake mutant lsi1 rescued the defect in silicon accumulation and growth.SlLsi1-eGFP fusion expression showed that SlLsi1 was mainly expressed on the exodermis cells.Overexpression of SlLsi1 in tomato increased silicon concentration in the root and root cell sap,but did not change the silicon concentrations in the shoot.These results indicate that SlLsi1 is a silicon influx transporter in the root and the cellular localization of SlLsi1 in the root may be associated with the low capacity of silicon uptake in tomato.5.Two putative Lsi2s – SlArsB-1 and Sl ArsB-2 in tomato were isolated by homology-based cloning,and the deduced amino acid sequence showed 55.09% and 57.14%homology with OsLsi2 in rice,respectively.Heterologous expression of Sl Ars Bs in Xenopus laevis oocytes showed no silicon transport activity.Considering that expressing CsLsi2 from cucumber in tomato significantly increased silicon accumulation in the shoot of transgenic tomato,the results suggest that,compared with that in other plants,the low capacity of silicon accumulation in tomato may be attributed to the lack of functional silicon efflux transporter Lsi2-an important member in the active uptake pathway of silicon in the root.In summary,CsLsi1,CsLsi2 and CsLsi6 in cucumber root cooperated to uptake silicon from the outer liquid and transport it further into the stele.The low capacity of silicon accumulation in tomato may be attributed to the lack of functional silicon efflux transporter Lsi2-an important member in the active uptake pathway of silicon in the root.The study shows that both functional silicon transporters and efficient cellular localization of silicon transporters are closely associated with the capacity of silicon uptake.