Plasma membrane intrinsic proteins from maize cluster in two sequence subgroups with differential aquaporin activity

Abstract

The transport of water through membranes is regulated in part by aquaporins or water channel proteins. These proteins are members of the larger family of major intrinsic proteins (MIPs). Plant aquaporins are categorized as either tonoplast intrinsic proteins (TIPs) or plasma membrane intrinsic proteins (PIPs). Sequence analysis shows that PIPs form several subclasses. We report on the characterization of three maize (Zea mays) PIPs belonging to the PIP1 and PIP2 subfamilies (ZmPIP1a, ZmPIP1b, and ZmPIP2a). The ZmPIP2a clone has normal aquaporin activity in Xenopus laevis oocytes. ZmPIP1a and ZmPIP1b have no activity, and a review of the literature shows that most PIP1 proteins identified in other plants have no or very low activity in oocytes. Arabidopsis PIP1 proteins are the only exception. Control experiments show that this lack of activity of maize PIP1 proteins is not caused by their failure to arrive at the plasma membrane of the oocytes. ZmPIP1b also does not appear to facilitate the transport of any of the small solutes tried (glycerol, choline, ethanol, urea, and amino acids). These results are discussed in relationship to the function and regulation of the PIP family of aquaporins.

Figure 1

ZmPIP1a (accession no. X82633), ZmPIP1b, and ZmPIP2a sequence comparison. Amino acid sequences were compared with the MEGALIGN program (DNASTAR, Madison, WI). Identical amino acid residues to at least two sequences are in a box. Numbering refers to the respective amino acid sequence. Black dots indicate the putative phosphorylation sites.

Figure 2

Dendogram of the comparison between 43 plant PIPs, including ZmPIP1a, ZmPIP1b, and ZmPIP2. Amino acid sequences were compared using the MEGALIGN program (DNASTAR). The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events. A dotted line indicates a negative branch. The black dots indicate the maize ZmPIPs. Accession numbers are (in parentheses): BrMOD (X95640), BoMIPB (AF004293), AtPIP1b (X68293), BoMIPA (X95639), AtPIP1a (X75881), RsPAQ1 (AB012044), AtPIP1c (X75882), AtTMPC (D26609), McMIPb (L36097), SoPM28b (I. Johansson and P. Kjellbom, personal communication), NtNT2 (U62280), NePIPa (AB002149), NtAQUA1 (AJ001416), CpPIPb (AJ001293), HvMIP (S41194), OsPIP1c (AF022737), OsPIP1b (AB009665), ZmPIP1a (X82633), ZmPIP1b(AF131201), NePIPb (AB002147), McMIPa (L36095), BvMIP3 (U60149), Ps7a (X54357), McMIPD (U26537), HvBPW1 (AB009307), ZmPIP2a (AF130975), OsPIP 2a (AF062393), GmPIP1 (U27347), SsAQUA2 (AF067185), AtPIP2b (X75884), AtRD28 (D13254), AtPIP2a (X75883), RsPAQ2 (AB012045), BvMIP1 (U60147), McMIPC (U73466), SoPM28a (L77969), AcPIP (U18403), BvMIP2 (U60148), AtPIP3 (U78297), McMIPE (U73467), PaMIP2 (Z93764), and PmSB01 (AF051202).

Figure 3

Pf values of individual oocytes injected with water (H₂O) or cRNA encoding AtRD28, ZmPIP1a, ZmPIP1b, or ZmPIP2a, derived from volume change measurements. When indicated, the assay was performed in the presence of 1 mm HgCl₂ with a 10-min preincubation (HgCl₂) or mercaptoethanol after mercuric chloride treatment (HgCl₂ + β-ME: 10 min preincubation with 1 mm HgCl₂ followed by a 15-min preincubation and the assay, both in presence of 5 mm mercaptoethanol). Data are expressed as the means ± se of data from five to 12 cells.

Figure 4

Plasma membrane localization of AtRD28, ZmPIP1a, and ZmPIP1b in cRNA-injected oocytes. In vivo-labeled proteins contained in total membrane fraction (M) and plasma membrane fraction (PM) of water or cRNA-injected oocytes were prepared as described in “Materials and Methods.” Dots indicate the polypeptides resulting from cRNA injection. The positions of the molecular mass standards are indicated on the left.

Figure 5

Gel-blot analysis of ZmPIP1a, ZmPIP1b, and ZmPIP2a mRNA in different vegetative and reproductive organs. Total RNA (20 μg) was extracted from seeds (S), endosperm (E), embryos (Em), shoots (Sh), roots (R), 10-d-old maize plantlet leaves (L1, L2, and L3), developing tassels (T), and ears (Ea) and separated by gel electrophoresis. After transfer, the blots were hybridized with the indicated probes.

Figure 6

Localization of ZmPIP1b::sGFP(S65T) and sGFP(S65T) in root tip cells. Confocal microscopic images of transgenic tobacco root tips expressing ZmPIP1b::sGFP(S65T) (A–C) and sGFP(S65T) (D–F). A and D, Root tips; B and E, zone of cell elongation; C and F, elongated cells.

Figure 7

Immunodetection of ZmPIP1b::sGFP(S65T) and sGFP(S65T) in transgenic tobacco plants. Subcellular fractions of transgenic plants expressing ZmPIP1b::sGFP(S65T) and sGFP(S65T) were obtained as described in “Materials and Methods,” fractionated by SDS-PAGE, transferred to nitrocellulose, and immunostained using N. plumbaginifolia H⁺-ATPase (PMA) or GFP antisera. H, Homogenate; S, cytosolic supernatant; Mi, microsomal fraction; PM, plasma membrane-enriched fraction. The positions of the molecular mass standards are indicated on the right.