1021-4437/02/4906- $27.00 © 2002
Russian Journal of Plant Physiology, Vol. 49, No. 6, 2002, pp. 804–809. Translated from Fiziologiya Rastenii, Vol. 49, No. 6, 2002, pp. 904–910.
Original Russian Text Copyright © 2002 by Krasnyanskaya, Belova, Velikanov, Widholm, Lozovaya.
Chinese milk vetch (
) is an
important legume crop grown in Asia for green fodder.
Simple methods have been developed for
eration of Chinese milk vetch plants; for this reason, the
species is a feasible test-system for studying foreign
gene expression in regenerated plants and their prog-
eny. The protocol has been worked out for obtaining
hairy root cultures by infecting Chinese milk vetch with
. Insertion of the Ri-plas-
into the plant genome promotes
excessive root proliferation in infection sites (hairy root
disease) due to enhanced phytohormone production.
, hairy roots are cultured in a hormone-free
medium. In their structure, hairy roots resemble normal
roots, except there is higher epidermal root hair prolif-
eration in the former; hairy roots are completely differ-
entiated, consistently produce root-speciﬁc metabolites
through a long period of culturing, and when cultured
, grow at a rate similar or even exceeding that of
nontransformed roots. Moreover, the clonal constitu-
tion of hairy roots and their capacity for regeneration
demonstrate the feasibility of using this model system
for studying foreign gene expression and producing
secondary metabolites [2–4].
Among plant defense responses to biotic and abiotic
stress agents, we ﬁnd the activation of phenolic metab-
olism, particularly, the synthesis of PAL, the enzyme
catalyzing the ﬁrst and rate-limiting reaction of the phe-
nylpropanoid pathway: phenylalanine deamination.
PAL participates in the synthesis of the precursors of
salicylic acid, phytoalexins, and lignin monomers,
which strengthen the mechanical and chemical barriers
of plant cells . It follows that plant resistance
towards various stress factors can be enhanced by ele-
vating the enzyme activities of phenolic metabolism. In
addition, by manipulating the genes of phenolic metab-
olism in an experiment, one may elucidate the mecha-
nisms of regulating the alternative branches of the phe-
The objective of the present study was to obtain
roots by introducing the kidney
gene with the help of
and to fol-
low the metabolic (PAL activity and lignin accumula-
tion in cell walls) and electrophysiological characteris-
tics of the transformed root cells.
MATERIALS AND METHODS
Bacterial strain and plasmid vector.
L. plants, we used the
DC-AR2 (a kanamycin-sensitive mutant) derived from
the strain MAFF 301724 (renamed strain MAFF03-
01724 comprising a mikimopine-type pRi1724) iso-
lated from diseased melon plants in Japan .
Expression of the Kidney Bean Phenylalanine-Ammonia Lyase
Gene in the Hairy Roots of
V. Krasnyanskaya*, L. P. Belova**, G. A. Velikanov**, J. Widholm*, and V. V. Lozovaya*
*Department of Crop Science, University of Illinois, Urbana–Champaine,
288 ERLM, 1201 W. Gregory Drive, Urbana, IL 61801 United States;
fax: (217) 333-4777; e-mail: firstname.lastname@example.org
**Kazan Institute of Biochemistry and Biophysics, Kazan Research Center, Russian Academy of Sciences, ul. Lobachevskogo
2/31, Kazan, Tatarstan, 420503 Russia
Received June 26, 2001
plants were transformed with the kidney bean
gene coding for phenylalanine-ammonia lyase (PAL). The hairy root culture thus obtained manifested
enhanced PAL activity and lignin content in the cell walls; in addition, the transformed cells differed from the
wild-type ones in several electrophysiological indices. In particular, the diffusion component of the total mem-
brane potential of plasmalemma increased in the
-transformed roots. The authors presume that the volume
density of the protein-related negative charge of the cytoplasm increases in the transformed root cells along with
changes in the cytoplasmic pH and pCa
, the extent of coupling of these two indices, and the hydraulic con-
ductivity of plasmodesmata.
Key words: Astragalus sinicus - transformation -
- phenylalanine-ammonia lyase - electrogenesis
—the gene encoding
membrane potential; MS—Murashige and Skoog nutrient
—the gene encoding neomycin phosphotrans-
—the gene encoding phenylalanine-ammonia lyase;
PAL—phenylalanine-ammonia lyase; R
—input cell resistance.