1022-7954/04/4002- © 2004
Russian Journal of Genetics, Vol. 40, No. 2, 2004, pp. 216–218. Translated from Genetika, Vol. 40, No. 2, 2004, pp. 282–285.
Original Russian Text Copyright © 2004 by Kochetov, Titov, Kolodyazhnaya, Komarova, Koval, Makarova, Il’yinskyi, Trifonova, Shumny.
The proline content is a factor determining plant
resistance to stress. It increases in response to drought,
high salinity, and low temperature . Studying regula-
tion of proline metabolism is essential for understand-
ing fundamental mechanisms of stress response and
plant adaptation to abiotic factors. The results of these
studies can be practically used in breeding and biotech-
nology. Nowadays, the methods of gene engineering
have been extensively applied to regulate the proline
content and examine the relationships between this
parameter and plant resistance to various stressful fac-
tors. Although genetically modiﬁed plants with altered
proline content have been developed, the role of proline
in osmotolerance formation is evaluated within a range
from extremely important [2–8] to insigniﬁcant [9, 10].
In general, proline functioning in the complex multi-
component process of plant adaptation to abiotic stress
still remains unclear [10, 11].
Here we describe a construction, an antisense sup-
pressor of proline dehydrogenase (PDH), prepared on
the basis of an
gene. As shown previously,
the constructions similar in structure enhance the pro-
line content in transformants of
osmotolerance of these transformants was evaluated as
increased  or unchanged . We have obtained
transformants of another plant species, tobacco (
SR1). The antisense suppressor prepared
on the basis of the
gene proved to increase
the proline content in tobacco transformants. The cyto-
plasm osmotic pressure in the transformants was also
signiﬁcantly higher than in control plants growing
under the same conditions.
Antisense PDH suppressor.
A fragment of the ﬁrst
exon of proline dehydrogenase gene (GeneBank:
AB028614, U59508) was isolated from
genomic DNA using polymerase chain reaction (PCR;
primers: (1) 5'-aacaaactggatccggcgatcttac-3', positions
21–45 relative to start ATG codon; (2) 5'-gagatgttg-
gtctagatttggcagc-3', positions 541–565; contain restric-
tion sites for cloning). The PCR fragment containing
545 nucleotides was digested with
endonuclease (a site within primer 1) and with
(internal site) and cloned along with 260-nucleotide
fragment in plasmid pBlueScript KSII (Stratagene) for
the same restriction sites. After intermediate cloning in
plasmid pRT104  and joining with 35S promoter of
CMV, the fragment was transferred into a binary vector
pBi101 ; a scheme of the ﬁnal construction is shown
in the ﬁgure. A fragment of
PDH is linked to
beta-glucuronidase gene from
, which leads to pro-
duction of extended hybrid mRNA (according to ,
these hybrid mRNAs are more efﬁcient as suppressors).
To generate transformants, leaves of three-week
plants of tobacco
SR1 were used.
Agrobacterial transformation (strain LBA4404) was
conducted as previously described ; transformants
were obtained on selective media containing kanamy-
cin (100 mg/ml). In total, 12 independent transformants
have been obtained.
The proline content in tobacco plant leaves was mea-
sured by the Bates method . Cytoplasm osmotic pres-
sure was determined in cells of lower epidermis of leaves.
The samples were placed in sucrose solutions of various
concentrations (from 0 to 5 MPa). The degree of plasmol-
ysis was assessed after 40–50 min. The osmotic pressure
was determined from isotonic coefﬁcient .
Analysis of tobacco transformants.
The proline con-
tent was determined in 6–10-week plants growing in
the greenhouse. Leaf cuttings similar in size were
Tobacco Transformants Bearing Antisense Suppressor of Proline
Dehydrogenase Gene, Are Characterized by Higher Proline
Content and Cytoplasm Osmotic Pressure
A. V. Kochetov
, S. E. Titov
, Ya. S. Kolodyazhnaya
, M. L. Komarova
, V. S. Koval
N. N. Makarova
, Yu. Yu. Il’yinskyi
, E. A. Trifonova
, and V. K. Shumny
Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, 630090 Russia;
fax: (3832) 33-12-78; e-mail: email@example.com
Novosibirsk State University, Novosibirsk, 630090 Russia
Received April 11, 2003
—The antisense suppressor was constructed for proline dehydrogenase gene (PDH; a fragment of
in antisense orientation and under the control of 35S promoter of cauliﬂower mosaic
virus, CMV). In
SR1 tobacco transformants bearing antisense suppressor for PDH, the pro-
line content and the cytoplasm osmotic pressure were increased. The proline content in these transformants var-
ied, whereas cytoplasm osmotic pressure was stable, which seems to reﬂect complicated relationships between
these characteristics of the plant cell.