Menadione treatments

1
Salinity induces expression of Vvgdha in Vitis
vinifera L. cell suspension cultures Eleni D.
Pliakoni, Damianos Skopelitis, and Kalliopi A.
Roubelakis Angelakis Department of Biology,
University of Crete, P.O. Box 2208, 714 09
Heraklion, Crete, Greece
INTRODUCTION Glutamate dehydrogenase (GDH, EC
1.4.1.2) catalyzes the reductive amination of
a-ketoglutarate to glutamate (synthetic
reaction), as well as the oxidative deamination
of L-glutamate to a-ketoglutarate and ammonia
(catabolic reaction). Although, GDH has been
biochemically and molecularly characterized in
several plant species, the physiological function
still remains largely speculative. Salinity, a
major type of abiotic stress in plants, inhibits
plant growth by lowering soil water potential,
causing ion toxicity and ionic imbalance within
the tissue. ?n this work we have attempted to
determine the potential contribution of GDH in
ammonia detoxification during salinity stress.
Vitis vinifera L. suspended cells were cultured
in the presence of 0, 50 and 100 mM NaCl for 0,
24, 48, 72 and 96h. In addition, cells were
cultured in the presence of 25mM NH4Cl as a sole
nitrogen source. During a period of 96 to 144 h,
the generation of the Reactive Oxygen Species
(ROS,) superoxide anions and hydrogen peroxide,
intracellularly and extracellularly, were
monitored, as well as GDH activity,
immunoreactive protein and transcript size of
Vvgdha, which encodes for the a-subunit of GDH.
Moreover, GDH specific activity was determined in
cell suspension cultures treated with menadione,
as a ROS generating source. This enzyme could be
related to ammonia detoxification under stress
conditions since the amino acid sequence
similarity of Vvgdha to the extremophilic archeal
enzyme conforms to each stress related function
(15). Also, its increased activity during aging
supports that increased intracellular ammonia
induces its expression in a accordance with the
present results (3-9, 12, 14).
Menadione treatments Cells incubated with 40
µ? menadione for 24h, produced significant levels
of ROS. This did not lead to an equivalent
increase in GDH specific activity (Fig
5). Figure 5. AOS produced
(A) and GDH specific activity (B) in menadione
treated grapevine cell suspension cultures
CONCLUSIONS Our data suggest that both
salinity and ammonia ions induce GDH specific
activity, a-subunit synthesis and assembly of the
more anodic isoenzymes. GDH may act towards
ammonia detoxification produced by proteolysis
events, which are often triggered under severe
stress conditions However, ROS do not seem to
contribute directly to the signal transduction
pathway for the expression of GDH (Fig.
6). REFERENCES 1. Linsmaier and
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Figure 1b. GDH specific activity in
NH3Cl treated grapevine cell suspension
cultures Effect of salinity and ammonia ions on
protein levels and isoenzymic pattern of GDH
Western blot analysis revealed an increase in GDH
total protein by NaCl, parallel to the specific
activity (Fig. 2B), and was accompanied by a
change in the isoenzymic pattern with the more
anodic isoenzymes of GDH prevailing (Fig. 2A).
Similar results were found when the cells were
supplemented with 25mM NH3Cl for 144h (Fig. 3A,
3B). Figure 2. GDH isoenzymic
analysis (A) and immunolabelled protein (B) in
salt treated grapevine cell suspension
cultures Figure 3. GDH isoenzymic
analysis (A) and immunolabelled protein (B) in
NH3Cl treated grapevine cell suspension
cultures Reactive oxygen species Salinity is
often associated with the production of ROS. ROS
were detected extracellularly in the cell culture
medium at 72h after in the presence of 100mM
NaCl. In contrast, no significant levels were
detected intracellularly (Fig. 4). In ammonia
treatments no significant levels of ROS were
detected.
MATERIALS AND METHODS Cell suspension culture
treatments Cell suspension cultures were grown
on LS (1) medium at 25 oC in the dark for 2 days.
Thereafter, cells were incubated with 0, 50 and
100 mM NaCl for 0, 24, 48, 72 and 96h. In
addition, cell cultures were supplied with 25mM
NH4Cl as a sole nitrogen source for 0, 24, 48,
72, 96, 120 and 144h. Furthermore, cell
suspension culture was treated with 40 µ?
Menadione for 0, 6, 9 and 24h. ROS and GDH
specific activity was measured. Enzyme extraction
and enzyme assays Total proteins were extracted
from cells suspension culture according to
Loulakakis et al. (3). GDH and CAT activities
were determined according to Loulakakis et al.
(2) and Siminis et al. (16), respectively.
Protein concentration was determined by the
method of Lowry (9). Native gels and western
blot analysis Native-PAGE and immunodetection
were performed according to Loulakakis et al.
(2). O2- and H2O2 detection assays The
generation of O2- and H2O2 from grapevine cell
cultures was determined by the chemiluminescence
assay of lucigenin and luminol, respectively
(12,13). RESULTS AND DISCUSSION GDH enzymic
activity Grapevine cell suspension cultures
treated with 0, 50 and 100 mM NaCl for 0-96h
showed an increase in GDH specific activity in a
dose response manner. The highest activity was
observed after 96h in 100mM NaCl and increased by
2-fold compared to control (Fig. 1a).
Figure 1a. GDH specific activity
in salt treated grapevine cell suspension
cultures Cell suspension cultures grown in
the presence of 25mM ??3Cl, as sole nitrogen
source, exhibited a 2- to 3- fold increase in GDH
specific activity at 120h (Fig. 1b).