Molecular Cloning, Water Channel Activity and Tissue Specific Expression of Two Isoforms of Radish Vacuolar Aquaporin1

Abstract

A major membrane intrinsic protein (VM23) in vacuoles of radish (Raphanus) tap root was investigated. The cDNAs for two isoforms of VM23, gamma- and delta-VM23, encode polypeptides of 253 and 248 amino acids, respectively. gamma- and delta-VM23 correspond to the gamma- and delta-TIP (tonoplast intrinsic protein) of Arabidopsis. The deduced amino acid sequences of the two VM23 isoforms were 60% identical. The amino-terminal sequence of gamma-VM23 showed agreement with the direct sequence of the purified VM23, suggesting that gamma-VM23 is the most abundant molecule among the VM23 isoforms. When mRNAs of gamma- and delta-VM23 were injected into Xenopus oocytes, the osmotic water permeability of oocytes increased 6-fold (60 to 200 microns s-1) of the control oocytes. The transcripts of both isoforms were detected in a high level in growing hypocotyls and young leaves, but delta-VM23 was not detected in seedling roots. Light illumination enhanced the transcription of two genes of VM23 in cotyledons and roots but suppressed their expression in hypocotyls the growth of which was inhibited by light. These findings suggest that the expression of VM23 is tightly related to cell elongation.

Sorting of proteins to vacuoles in plant cells

“…Phillips, and J.C. Rogers, unpublished data). In immunofluorescence experiments with pea root tip cells, the anti-α-TIP peptide antibodies co-localize with antibodies raised to purified α-TIP [53] and the anti-γ -TIP peptide antibodies co-localize with antibodies to purified γ -TIP from radish [36]. The anti-δ-TIP peptide antibodies, however, identify a separate population of vacuoles present in cells that also contain vacuoles labeling with anti-α-TIP or anti-γ -TIP.…”

“…However, TIPs are very abundant proteins. In radish tap root, γ -TIP makes up 30-50% of total tonoplast protein, while vacuolar pyrophosphatase and H + -ATPase enzymes account for only about 10% of tonoplast protein [36]. This abundance of γ -TIP would seem to be in excess of the amount needed for water transport and suggests a structural function for the protein (M. Maeshima, personal communication).…”

Keep an Eye on PPi: The Vacuolar-Type H+-Pyrophosphatase Regulates Postgerminative Development inArabidopsis

“…After electrophoresis, the proteins were transferred to an Immobilon-P membrane (Millipore). The primary antibodies used were prepared previously: antibodies to V-PPase (antigen peptide sequence, DLVGKIERNIPEDDRN; Kobae et al, 2006), V-ATPase subunit A (TKAREVLQREDDLNEI; Kobae et al, 2006), V-ATPase subunit a (ELVEINANNDKLQRSYNEL; Kobae et al, 2004), BIP (SKDNKALGKLRREC; Kobae et al, 2004), and TIP1s (antibody to the purified protein; Higuchi et al, 1998). Antigen on the membrane was visualized with horseradish peroxidase-coupled protein A and chemiluminescent reagents ECL (GE Healthcare).…”

Early Effects of Salinity on Water Transport in Arabidopsis Roots. Molecular and Cellular Features of Aquaporin Expression

“…For this, we used antibodies raised against peptides derived from AtPIP1;1 and AtPIP2;1 , and g-VM23, a radish TIP homolog (Higuchi et al, 1998). Because of sequence conservation between aquaporin homologs, these antibodies are predicted to cross-react with several Arabidopsis isoforms of the PIP1 subfamily (PIP1;1-PIP1;4), the PIP2 subfamily (PIP2;1-PIP2;3), and the TIP1 subfamily (TIP1;1 and TIP1;2), respectively.…”