Aquaporins constitute a large and highly divergent protein family in maize

Abstract

Aquaporins (AQPs) are an ancient family of channel proteins that transport water and neutral solutes through a pore and are found in all eukaryotes and most prokaryotes. A comparison of the amino acid sequences and phylogenetic analysis of 31 full-length cDNAs of maize (Zea mays) AQPs shows that they comprise four different groups of highly divergent proteins. We have classified them as plasma membrane intinsic proteins (PIPs), tonoplast intrinsic proteins, Nod26-like intrinsic proteins, and small and basic intrinsic proteins. Amino acid sequence identities vary from 16% to 100%, but all sequences share structural motifs and conserved amino acids necessary to stabilize the two loops that form the aqueous pore. Most divergent are the small and basic integral proteins in which the first of the two highly conserved Asn-Pro-Ala motifs of the pore is not conserved, but is represented by alanine-proline-threonine or alanine-proline-serine. We present a model of ZmPIP1-2 based on the three-dimensional structure of mammalian AQP1. Tabulation of the number of times that the AQP sequences are found in a collection of databases that comprises about 470,000 maize cDNAs indicates that a few of the maize AQPs are very highly expressed and many are not abundantly expressed. The phylogenetic analysis supports the interpretation that the divergence of PIPs through gene duplication occurred more recently than the divergence of the members of the other three subfamilies. This study opens the way to analyze the function of the proteins in Xenopus laevis oocytes, determine the tissue specific expression of the genes, recover insertion mutants, and determine the in planta function.

Figure 1

Model of the structure of an AQP showing the principal features of the protein. Alpha helices are represented as rectangles. There are six transmembrane domains (TM1–TM6) connected by five loops (A–E). Two helical domains (HB and HE) in different loops dip halfway into the membrane from opposite sides and form the aqueous pore. Loops B and E also contain the highly conserved NPA motifs that are part of the pore. In the three-dimensional structure (see Fig. 7), these two motifs are positioned one above the other.

Figure 2

Phylogenetic analysis of 31 maize AQP proteins. The distance scale represents the evolutionary distance, expressed in the number of substitutions per amino acid. National Center for Biotechnology Information accession numbers are shown in Table I.

Figure 3

Phylogenetic analysis of the maize ZmPIPs and other plant PIPs. Accession numbers of ZmPIPs are shown in Table I. Other plant PIP accession numbers are indicated in the tree or (in parentheses): NtAQP1 (AJ001416), AtPIP1c (AAF81320), AtPIP1b (AAC28529), AtPIP1a (CAB71073), AtRD28 (AAD18141), AtPIP2a (CAB67649), and AtPIP3 (CAA17774). The distance scale represents the evolutionary distance, expressed in the number of substitutions per amino acid.

Figure 4

Phylogenetic analysis of the maize ZmTIPs and other plant TIPs. Accession numbers of ZmTIPs are shown in Table I. Other plant TIP accession numbers are indicated in the tree or (in parentheses): Atα-TIP (AAF18716), AtβTIP (AAF97261), AtδTIP (BAB1264), AtγTIP (AAD31569), and NtTIPa (CAB40742).

Figure 5

Phylogenetic analysis of the maize ZmNIPs and other plant NIPs. Accession numbers of ZmNIPs are shown in Table I. Other plant NIP accession numbers are indicated in the tree or (in parentheses): LjLIMP2 (AAF82791), GmNOD26 (AAA02946), AtNLM1 (CAA16760), and AtNLM2 (CAB78893). The distance scale represents the evolutionary distance, expressed in the number of substitutions per amino acid.

Figure 6

Amino acid residues in the NPA motifs of ZmAQPs. The amino acid residues in the structural loops B and E of each maize subfamily are indicated. Residues in bold are found in 20 or more of 31 ZmAQPs.

Figure 7

Phylogenetic analysis of the maize and Arabidopsis SIPs. Accession numbers of ZmSIPs are shown in Table I. Arabidopsis accession numbers are indicated in the tree. The distance scale represents the evolutionary distance, expressed in the number of substitutions per amino acid.

Figure 8

A topological model of the maize ZmPIP1-2. The representation is based on the human AQP1 and bacterial GlpF structures (Fu et al., 2000; Murata et al., 2000), and shows the six transmembrane helices (TM1–TM6) and the two short helices in the structural loops B and E (HB and HE). Residues in yellow with thick red circles are highly conserved among ZmAQPs (found in 20 or more of the 31 ZmAQPs). Residues in pink indicate the position of a highly conserved residue present in 20 or more of the 31 ZmAQPs but absent from ZmPIP1-2 (⁹⁷Ser and ¹⁴⁰Met of ZmPIP1-2 are replaced by an Ala).