Expression of cassava Manihot Esculenta Crantz. sucrose transporter genes in yeast Saccharomyces Cerevisiae and characterization of the proteins

Abstract

Sucrose transporter proteins (SUTs) play a pivotal role in active sucrose transport processes in higher plants which affect the plant maturation and crop production. Four sucrose transporter genes in cassava (Manihot esculenta Crantz.) designated as MeSUT1, MeSUT2, MeSUT4 and MeSUT5, have been isolated from leaf and storage root cDNA libraries. In order to obtain the biochemical properties of each MeSUTs, plasmids containing each of MeSUT cDNAs were constructed and used for heterologous expression in an invertase deficient SUSY7/ura3 yeast (Saccharomyces cerevisiae) mutant. All four cassava sucrose transporter genes were successfully transformed and they were able to complement the yeast SUSY7/ura3 phenotypes, suggesting the proteins were functional in yeast. 14C-sucrose uptake by yeast expressing MeSUTs was pH dependent but their optimum activities were observed at different pH. MeSUT1 was highly active at pH 6, while the highest sucrose uptake activities of MeSUT2, MeSUT4 and MeSUT5 were observed at pH~7. MeSUT1 was a high affinity/low capacity sucrose transporter with the Km and Vmax of 1.50±0.11 mM and 0.06 nmol/mg FW/min, while those of MeSUT2 were 12.55±3.58 mM and 0.36±0.10 nmol/mg FW/min, indicating that MeSUT2 belonged to low affinity/high capacity type. MeSUT4 and MeSUT5, the isoforms of cassava SUT4, showed the Km and Vmax of 42.25±1.54 mM and 1.58±0.13 nmol/mg FW/min and 51.37±5.30 mM and 1.10±0.12 nmol/mg FW/min, respectively, suggesting that they were low affinity/high capacity sucrose transporters. The responses to various inhibitors and sugars of these four MeSUTs were different, however, all were strongly inhibited by CCCP and antimycin A, supporting the characteristics of active transport carriers. From the sensitivity towards pH and several metabolic inhibitors, it was proposed that MeSUT1 might be sucrose/H+-symporter, while MeSUT2, MeSUT4 and MeSUT5 were possibly sucrose/H+ -antiporters. The sucrose binding activity of plasma membrane of cassava tubers was pH dependent with the highest activity observed at pH 8 and its Km and Vmax were 19.16±6.44 mM and 1.47 ± 0.54 umol/mg protein/h, respectively. cRNA of all four MeSUTs were synthesized and expressed in Xenopus laevis oocytes. Expression of MeSUT2 was not successful whereas MeSUT1 and MeSUT5 expressing oocytes showed small induced currents but too small to be used for further analysis. MeSUT4 expressing oocytes showed clear inward currents and the K0.5 for sucrose transport was 12.35 mM, supporting its low affinity characteristics.