Biology Reference
In-Depth Information
to be enhanced to about 2.5-3.0 and 3-4 fold higher, respectively in the cells overexpressing the
ApPGDH, ApGSMT and ApDMT than the control and the cells expressing ApGSMT and ApDMT.
A measurement of intracellular levels of serine and glycine during salt stress of
A
.
halophytica
cells
revealed that the levels of both these amino acids decreased. However an exogenous supply of these
amino acids during salt stress contributed to the enhanced levels of glycine betaine synthesis and to
the salt tolerance as well. The activity of ApPGDH was found to be enhanced by 1.5-2.3 fold upon
increase of salinity from 0.5 to 2.5 M NaCl. A molecular characterization of the protein of ApPGDH
suggested it be closer to PGDH from
Synechocystis
sp. strain PCC 6803 (~79% identity) with a sequence
of 526 amino acid residues and a molecular weight of 56,410 Da. Besides possessing three domains
such as nucleotide-binding, substrate-binding and regulatory domains, the ApPGDH protein consists
of a C-terminal domain resembling the PGDHs of
Arabidopsis
and mammals.
A. halophytica
contains a betaine transporter (BetT) that is active at alkaline pH and high salinity.
BetT of
A
.
halophytica
(BetT
A
.
halophytica
)
belongs to the BCCT (betaine-choline-carnitive transporter)
family and is distinctive in having an isoelectric pH in the acidic range (4.58) in contrast to the
isolelectric pH in the basic range (9.54) of a typical BCCT transporter (opnD) like that of
Bacillus
subtilis
. BetT
A
.
halophytica
is specifi c for the transport of betaine and its activity was enhanced in presence
of sodium and thus it is suggested to be a Na
+
-betaine symporter. Betaine uptake by the cells of
A
.
halophytica
was enhanced two and half times in presence of 2.0 M NaCl than in presence of 0.5
M NaCl. Maximum uptake of betaine occurred at a pH of 9.0. Betaine transporter gene from
A
.
halophytica
has been cloned and used in the transformation of
S. elongatus
PCC 7942 (that lacks a
BetT). Transformants of
S. elongatus
PCC 7942 overexpressing this gene exhibited kinetic properties
of BetT similar to that of
A
.
halophytica
cells. Studies on the overexpression of the BetT gene of
A
.
halophytica
in
E
.
coli
mutant MKH13 (defi cient in
betT
,
putPA
,
proP
and
proU
genes) and its kinetic
properties also confi rmed that the BetT is a Na
+
-betaine symporter (Laloknam
et al
., 2006).
Another path way for the synthesis of glycine betaine is through oxidation of choline mediated
by choline oxidase. This pathway is characteristic of the soil bacterium
Arthrobacter globiformis
that
does not require any co-factors. The gene for this enzyme,
codA
has been cloned and used in the
transformation of
S. elongatus
PCC 7942. The transformants exhibited higher levels of glycine betaine
accumulation (60-80 mM) and improved tolerance to salt stress expressed in terms of growth,
chlorophyll content and photosynthetic activity (Deshnium
et al
., 1995).
The protective effects of glycine betaine reside in its dipole characteristics at physiological pH
values and the presence of three hydrophobic methyl groups at the positively charged end of the
molecule. The dipole seems to mask the Na
+
and Cl
-
that enter the cells under salt stress and the
hydrophobic methyl groups help in the stabilization of hydrophobic domains of proteins and thus
prevent their distortion (Nomura
et al
., 1998). The interaction of glycine betaine with hydrophilic
and hydrophobic domains of macromolecules has been tested by examining the effects of salt stress
and betaine synthesis on photoinhibition of PSII in transformants of
S. elongatus
PCC 7942 with
codA
gene (Deshnium
et al
., 1995). The photoinhibition of PSII induced by strong light was enhanced due
to salt stress (220 mM NaCl) and glycine betaine synthesized in the transformant cells protected PSII
against photoinhibition. However, neither salt stress nor betaine synthesis affected photodamage to
PSII. On the contrary, salt stress inhibited repair of photodamaged PSII and glycine betaine reversed
this inhibitory effect of salt stress. Pulse-chase labelling experiments with [S
35
]-Met revealed that
under control conditions (20 mM NaCl)
de novo
synthesis of thylakoid membrane proteins was
unaffected in presence of choline. In contrast, under salt stress (220 mM NaCl) the synthesis of
these proteins decreased but their levels returned to control levels in presence of choline. Likewise,
betaine did not affect the levels of radioactively labelled D1 protein in light under the non-stress
