TONOPLAST INTRINSIC PROTEINS FROM CAULIFLOWER (BRASSICA-OLERACEA L. VAR. BOTRYTIS) - IMMUNOLOGICAL ANALYSIS, CDNA CLONING AND EVIDENCE FOR EXPRESSION IN MERISTEMATIC TISSUES

The vacuolar membrane (tonoplast) of plant cells contains aquaporins, protein channels that facilitate the selective transport of water. The se tonoplast intrinsic proteins (TIPs) of 23-29 kDa belong to the anci ent major intrinsic protein (MIP) family. A monospecific polyclonal an tiserum directed against a 26 kDa intrinsic protein from the tonoplast of meristematic cells from cauliflower (Brassica oleracea L. var. bot rytis) was used to screen a cDNA library. Two distinct cDNAs have been isolated. Both clones, c26-1 and c26-2, encode closely related TIPs. The c26-1 insert, consisting of 933 bp upstream of the poly(A) tail, i s a full-length cDNA with an open reading frame encoding a protein of 251 amino acids with a calculated M-r of 25 500. The c26-2 insert is a 5' truncated cDNA. The two cDNAs share 90.5% sequence identity within their overlapping coding regions but only 35% sequence identity in th e 3' untranslated regions, indicating that highly related TIP-encoding genes are expressed in meristematic cells. Although TIPs have previou sly been found in a variety of cell types, they have not been found in meristems. The derived amino acid sequences (BobTIP26-1 and BobTIP26- 2, respectively) closely resemble the aquaporin gamma-TIP from Arabido psis thaliana. Northern blot analysis and in situ hybridization show t hat BobTIP26 mRNAs preferentially accumulate in highly meristematic ce lls, mostly before and during cell enlargement, and in the living cell s of the xylem. This differential pattern of expression is also found by immunodetection of BobTIP26 polypeptides. The gene expression patte rns are discussed with respect to the probable function of the gene pr oducts.