Cell and Tissue Journal

Abstract Aim: Movement of water across cellular membranes is facilitated by the presence of water channels named aquaporins. Plant plasma membrane intrinsic proteins (PIPs) fall into two groups, PIP1s and PIP2s that exhibit different water channel activities. So PIP1s are inactive, whereas PIP2s induce a marked increase in the membrane osmotic water permeability coefficient, p < /em>_f. This difference may be due to their subcellular localization. The aim of this research was to determine the subcellular localization of NtPIP1;1 and NtPIP2;1 in living tobacco cells.
Material and methods: In this experimental study, NtPIPs cDNA sequences were fused to green fluorescent protein gene and expressed transiently in tobacco mesophyll protoplasts. Then, cellular localization of aquaporins was investigated using fluorescent microscope under blue excitation wavelength.
Results: When expressed alone, NtPIP1;1 fusion protein was retained in the internal membrane structures, whereas NtPIP2;1 was found in membrane structure within the cell. To identify the internal structures containing NtPIP1;1 fusion proteins, NtPIP1;1-GFP was co-expressed in tobacco protoplasts with the YFP::HDEL protein, an ER marker. As shown, NtPIP1;1-GFP and YFP::HDEL colocalized.
Conclusion: NtPIP2;1 was transported to plasma membrane, but NtPIP1 was retained in the ER of tobacco protoplasts so showed no water transport activity.
 

Abstract Aim: Movement of water across cellular membranes is facilitated by the presence of water channels named aquaporins (AQPs). Mercurial compounds are potent blockers of plant and animal aquaporins. However some aquaporin isoforms such as NtAQP1 in tobacco are mercury-insensitive. The aim of this research was to determine the sensitivity of tobacco NtPIP2;1 to mercury.
Materials and methods: In this experimental study, after invitro transcription of NtPIP2;1 and cRNA synthesis, cRNA was injected to Xenopus oocytes by microinjection. Two days after incubation, oocytes were subjected to 1 mM mercury chloride for 10 min. Then, oocyte volume swelling assay in hypotonic medium was conducted and the membrane osmotic water permeability coefficient (Pf ) was determined.
Results: Pf for control and mercury chloride-treated oocytes expressing NtPIP2;1 was respectively calculated 0.99×10^-2 and 0.98×10^-2 cm s^-1 , which was not significant (P>0.05). Comparing the amino acid sequence of NtPIP2;1 with NtAQP1, a mercury-insensitive aquaporin, revealed the similar to  NtAQP1 replacement of cys with Thr in 233 position near the aquaporin pore in NtPIP2;1.
Conclusion: NtPIP2;1 encodes a mercury-insensitive aquaporin in tobacco which could be due to cysteine replacement with threonin near the aquaporin pore.