Intron and Polypeptide Evolution of Conserved NPA to NPA Motif Regions in Plant Aquaporins

Aquaporin proteins are part of an ancient family that functions as water transporting facilitators in all organisms. Phylogenetic and physiological analyses have revealed that plant aquaporins consist of two groups: the plasma membrane intrinsic proteins (PIPs) and the tonoplast intrinsic proteins (TIPs). Using the conserved asparagineproline-alanine (NPA) to NPA motif regions, we studied the evolution of 35 plant aquaporins that included nine of our newly cloned cucumber aquaporins and 26 from the GenBank database. Results indicated that NPA repeated regions were effective for phylogenetically characterizing the plant aquaporin family, and to accurately localize the introns. Phylogenetic analysis showed that 35 plant aquaporins fell into two distinct groups (except for the Arabidopsis gene AtMip)—PIPs and TIPs. The nine cucumber aquaporins belong to the PIP group that were localized further into two different sub-groups. The intron analysis showed that introns of plant aquaporins mainly consist of two types. Eighteen PIPs shared identical intron positions localized in connecting loop C between amino acids 95 and 96. Nine TIPs shared the other identical intron positions localized in connecting loop D between amino acids 44 and 45. Cucumber aquaporins CRB9 and CRB10 (with no intron in the repeated NPA regions) may be the result of intron loss events, while intronless rice (Orzya sativa) Os-TIP1 and Os-TIP2 may have resulted from other intron loss events. PIP11 and Os-PIP do not have the same amino acid number as major PIP members, but combined phylogenetic analysis results along with intron positions and phases showed that they belong to the PIP group. The phylogenetic tree and intron position information suggest that AtMip was mis-annotated as a member of aquaporin, and is a homologue of the glycerol facilitator-like protein. Introns share identical positions and phases within the PIP group (except PIP13) or the TIP group, but differ between the plasma and the tonoplast membrane aquaporins matching the phylogenetic analysis results. Intron positions of the repeated NPA regions of plant aquaporins that have stable inheritance can act as molecular markers for phylogenetic studies. (Reizer et al., 1993; Park and Saier, 1996). Site-directed mutagenesis on the loops containing the NPA motifs confi rmed that two NPA segments line the path of water permeation, and NPA and adjacent residues are essential for water transporting activity (Jung et al., 1994). In plants, aquaporins have been localized into vacuolar and plasma membranes and were phylogenetically divided into two distinct groups: the plasma membrane intrinsic proteins (PIPs) and the tonoplast intrinsic proteins (TIPs) (Chrispeels and Agre, 1994; Höfte et al., 1992; Kammerloher and Schäffner, 1993; Kjellbom et al., 1999; Weig et al., 1997; Yamada et al., 1995; Zardoya and Villalba, 2001). PIPs have extended N-termini as compared to TIPs (Johansson et al., 2000). Although much work has been done to characterize the functional localization and phylogenetic relationship of aquaporins in plants, we know of no reports of intron evolution, perhaps because of the small number of plant aquaporin genomic sequences that have been sequenced. Tobacco aquaporin TobRB7 was cloned by our group more than 10 years ago (Conkling et al., 1990; Yamamoto et al., 1991). Recently, we have cloned one full length aquaporin genomic sequence and eight genomic fragments which encoded the aquaporin conserved NPA to NPA regions in cucumber (Xie et al., 2002). The Arabidopsis and rice (Oryza sativa) genomic projects also provided sequences which were annotated as homologous to the aquaporins. In total, 35 plant aquaporin genes (fragments) are available for intron tracing within the repeated NPA regions. Characterization of intron information including number, position, and phase could provide additional information for investigation of molecular evolution of this gene family. The analysis of phylogenetic relationships and intron evolution in the repeated NPA regions has some advantages over other Received for publication 2 Jan. 2003. Accepted for publication 1 Apr. 2003. Research supported by a grant from Pickle Packers International. 1Department of Horticultural Science. 2Department of Genetics. 3Current addresses: Vector Research Ltd, 700 West Main Street, Durham, NC 27701; Kurt Wollenberg, Tufts Center for Vision Research, NEMC, 750 Washington Street, Boston, MA 02111. 4Correspondence author; e-mail [email protected]. Aquaporins belong to the ubiquitous Major Intrinsic Protein (MIP) family that functions as water-selective channels (Chrispeels and Agre, 1994; Chrispeels and Maurel, 1994; Engel et al., 1994; Park and Saier, 1996). Aquaporins together with transporters of glycerol and other small neutral molecules make up the MIP super-family (Johansson et al., 2000; Park and Saier, 1996). Owing to functional importance, a large number of aquaporin genes have been cloned and characterized in animals, plants, and microorganisms. Members of the aquaporin family are highly conserved from bacteria to mammals, all of which have similar sequences as well as similar molecular masses, ranging from 25 to 31 kDa (Pao et al., 1991). Structurally, the aquaporin polypeptides have similar topology, with all having six membrane-spanning α-helixes, fi ve loops, and a Cand Nterminus (Park and Saier, 1996; Reizer et al., 1993). The primary amino acid sequence of aquaporins shows that the Nand Cterminal halves of the protein have obverse symmetry and represent a tandem sequence repeat, each comprised of three bilayer-spanning domains with two connecting loops (Cheng et al., 1997; Jung et al., 1994; Walz et al., 1995). The most highly conserved sequences are two tripeptide (NPA) motifs in the two longest loops, B (cytoplasmic) and E (extracellular), which are conserved over diverse species, including bacteria and animals