Subcellular Localization and Multimerization Analyses of HbPIP2;7, a PIP Aquaporin from Hevea brasiliensis

Natural rubber is specifically synthesized in the laticifer of Hevea brasiliensis. Due to the absence of plasmodesmata between mature laticifer cells and surrounding parenchyma cells, water influx toward laticifers is mainly controlled by plasma membrane intrinsic proteins(PIPs) within the aquaporin(AQP) family. To investigate the molecular mechanism of water balance in laticifers, the coding sequence(CDS) of an abundant PIP gene denoted HbPIP2;7 was isolated using RT-PCR followed by subcellular localization and multimerization analyses of its coding protein. Sequence analysis revealed that the CDS length of HbPIP2;7 is 837 bp, putatively encoding 278 amino acids with the theoretical molecular weight of 29.56 kDa, the isoelectric point of 9.11, the instability index of 29.89, and the grand average of hydropathicity of 0.526. The protein was shown to contain 1 conserved MIP domain with 6 transmembrane helixes and may function in tetramers. Bioinformatics prediction and transient overexpression in Nicotiana benthamiana leaves revealed that HbPIP2;7 is localized in the plasma membrane. Both bimolecular fluorescence complementation and yeast two-hybrid analyses suggested that this protein could not form a homomultimer. Thereby, HbPIP2;7 is more likely to be involved in laticifer water balance in the form of hereotetramers.