Plant Molecular Biology 34: 111–118, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Identiﬁcation of a
-TIP cDNA clone and determination of related A and D
genome subfamilies in Gossypium species
David L. Ferguson, Rickie B. Turley
and Reiner H. Kloth
Cotton Physiology and Genetics, USDA-ARS, P.O. Box 345, Stoneville, MS 38776, USA (
Received 13 September 1996; accepted in revised form 4 February 1997
Key words: alloalleles, cotton, genome, Gossypium, TIP, tonoplast intrinsic protein
Tonoplast intrinsic proteins (TIPs) have been implicated in the process of cell elongation, such as occurs in the
developing cotton ﬁber. We have isolated a cDNA clone (997 bp in length) from a cotton (Gossypium hirsutum
L.) library which putatively encodes a protein of 248 residues (
25079) with 85% identity to Arabidopsis
-TIP. The derived amino acid sequence included two conserved sequences associated with major intrinsic proteins
(SGxHxNPA at residues 78 to 85, NPA residues at 197 to 199) and a cysteine residue at 116 which is reported to
bind mercury in Arabidopsis
-TIP. The polymerase chain reaction was used to generate partial genomic clones of
-TIP. In comparison to other genomic TIP sequences, the number (two) and position of the introns were
conserved in cotton. Comparing the TIP sequences from cotton revealed two subfamilies, which were consistently
distinguished by a Tsp45I restriction site polymorphism. This polymorphism was used to demonstrate that TIP
subfamilies were speciﬁc to either the A or D genomes of Gossypium.When
-TIP DNA fragments were ampliﬁed
from cDNA of ﬁber 14 days after anthesis, the A and D were found, indicating the presence of
-TIP transcripts in
these elongating cells.
Major intrinsic proteins (MIP) are a large family of
channel-forming proteins that are found in all living
organisms . Though the function of many of these
proteins is unknown, some have shown the capab-
ility of acting as membrane channels for water .
In plant vacuoles, these membrane channels are des-
ignated as tonoplast intrinsic proteins or TIPs .
There are four described TIPs [3, 10, 12], which are
related to the MIPs by their conserved amino acid
sequences SGxHxNPA and NPA . TIP isoforms
are expressed during speciﬁc stages of plant develop-
ment as well as in speciﬁc cell types [3, 10, 12, 14].
-TIP are found in cells that are elongating
and associated with the vascular system [3, 14–17].
Ludevid et al.  analyzed the expression pattern of
GenBank and DDBJ Nucleotide Sequence Databases under the
accession number U62778.
-TIP in Arabidopsis with in situ hybridization and
GUS expression controlledby a
-TIP promoter. They
root and shoot tissue and in the cells surrounding the
vascular bundles . Maeshima  isolated tono-
plast membranes from three regions of the mung bean
hypocotyl. A protein with high identity to
16] was found in greater amounts in the elongating
and mature regions of the hypocotyl compared to the
region composed of dividing cells . When Phillips
and Huttly  stimulated cell elongation in a dwarf
antlyincreasedoverunstimulatedcells. A recentreport
-TIP includedstudieson its expression
in different plant tissues . The expression pattern
-TIP differed to some degree from that of
however, the tissues expressing the two forms of TIP
overlapped [3, 14]. Using a
-TIP promoter to control
GUS expression, Daniels et al.  found the greatest
GUS activity in cells surrounding the vascular tissues
GR: 201001898, Pips nr. 135348 BIO2KAP
pla360us.tex; 14/04/1997; 7:09; v.7; p.1