Figures legends
FIGURE 1 Schematic representation showing the experimental
setting of Li-cor 6400 together with Dual-PAM-100, through a chamber
holding on the emitter-detector system of the PAM, for controlling
CO2 level (390 or 2000 μL L-1) and
using an oxygen source equipped with an oxymeter to adjust oxygen flux
from the appropriate O2 source (2 or 21%) to the
chamber and accordingly be able to monitor the CO2 and
O2 concentrations in the chamber during measurements.
See also video for setting. IRGA and PAM mean infrared gas analyzer and
pulse amplitude modulation, respectively.
FIGURE 2 A ‘spider plot’ of selected parameters derived from
the chlorophyll fluorescence OJIP curves for Setaria (left column, A-C)
and Spartina (right column, D-F) treated with 0, 50 and 100 mM (A-C) and
0, 100, 250, 400 and 550 mM (D-F) NaCl for 12 days. All data of JIP test
parameters were normalized to the reference 0 mM NaCl and each variable
at the reference was standardized by giving a numerical value of the
unit (1).
FIGURE 3 Changes in leaf Na+ (A-B),
K+ (C-D) contents and
K+/Na+ ratio (E-F) over time in
Setaria (A, C and E) and Spartina (B, D and F). One-month old Setaria
and 2 month-old Spartina were exposed to salt for up to 2 weeks. Plants
were subjected to: 0, 50, and 100 mM NaCl for Setaria and 0, 100, 250,
400, and 550 mM NaCl for Spartina. Data represent the means of 4 to 5
replicates ±SE.
FIGURE 4 The effect of salt on the leaf total chlorophyll
content in Setaria (A) and Spartina (B). One month-old Setaria and two
month-old Spartina were exposed to different NaCl levels as described in
Fig. 2. Leaves were collected 12 days after initiating salt treatment to
determine chlorophyll concentration. For chlorophyll each data bar
represents the mean of at least 10 replicates ± SE. Fast- and
slow-relaxing components of NPQ (NPQf and NPQs) in
leaves of Setaria (C) and Spartina (D) exposed to to 0 and 100 (C) or 0
and 400 mM NaCl (D). Measurements were carried out 16 days after
initiating salt treatment at 25°C in the presence of 390 μL
L-1 CO2. Leaves were illuminated with
800 μmol m-2 s-1 actinic light. Each
data bar represents the means of at least 6 replicates ±SE.
FIGURE 5 Oxygen dependence of electron transport:
ETRII (A-B), P700 oxidation ratio (C-D),g ETC (E-F), and ETRI (G-H),
measured in leaves of Setaria (left column, A, C, E and G) and Spartina
(right column, B, D, F and H) endured NaCl (triangles): 100 mM for
Setaria and 250 mM for Spartina. Control plants (circles) were
maintained in a NaCl-free medium. The measurements were performed, 12
days after initiating salt treatment, under saturating
CO2 (2000 μL L-1), at 25°C and in the
presence of 21% (open symbols) or 2% (closed symbols) oxygen. Each
data point represents the means of at least 6 replicates ±SE.
FIGURE 6 NDH-dependent CEF pathway assessed as the
post-illumination Fo rise in plants grown on either
salt-free medium (ctrl) or subjected to 50 or 250 mM NaCl for Setaria
and Spartina, respectively, during 12 days. The post-illumination
Fo rsie was recorded in the dark after switching off 5
min illumination with actinic light (325 μmol m-2s-1). Each data bar represents the means of at least
10 replicates taken on different leaves ±SE.
FIGURE 7 Effects of n -PG and DBMIB on PSII photochemical
efficiency ΦPSII measured in leaves of either Setaria (A
and C) or Spartina (B and D) detached from plants subjected to: 0 and 50
(for SV ) ; or 0 and 250 mM NaCl (for SA ). Measurements
were carried out 12 days after initiating salt treatment at 25°C in the
presence of 390 μL L-1 CO2. Leaves
were illuminated with 800 μmol m-2s-1 red actinic light. Leaves were vacuum infiltrated
with water (white bars) or with 5 mM n -PG (A-B) or 50 μM DBMIB
(C-D) in the presence of 21% (gray bars) and 2% (black bars) oxygen.
Each data point exhibits the means of at least 6 replicates ±SE.
FIGURE 8 Effect of salt treatment on PTOX protein expression
(A) and the PTOX gene expression level assessed by q-PCR analysis (B) in
leaves of Setaria and Spartina subjected to 0 and 50 (for SV ) or
to 0 and 250 mM NaCl (for SA ). For protein expression (A), leaves
from control and salt-treated plants were collected 12 days after
initiating salt treatment for immune-detection after SDS-PAGE,
separation of 29 μg protein from the thylakoid membrane samples, and
electrophoretic transfer to nitrocellulose membrane. Western-blot band
size was quantified by TanonImage technology software. For gene
expression level (B), leaves from control and salt-treated plants for 12
days were collected and immediately stored in liquid nitrogen for RNA
isolation and purification using PureLink RNA Mini Kit Invitrogen (see
materials and methods). The synthesized cDNA was used for the q-PCR
analysis of PTOX. Data points represent the mean of around five
replicates for western SDS-PAGE ±SE and 6 replicates for qRT-PCR. Insert
of panel A shows typical bands from an original blot, loaded on an equal
protein basis.
FIGURE 9 Histochemical staining of Setaria (C-D) and Spartina
(A-B) leaves obtained from control untreated (A and C) or salt treated
(B and D) for Setaria at 50 mM and Spartina at 250 mM during 12 days
with 6 mM NBT (nitroblue tetrazolium). Dark-blue staining reveals the
interaction of superoxide radical
(.O-2) with NBT (500
μmol m-2 s-1) in leaves following
salt stress treatment.
FIGURE 10 Histochemical staining of Setaria (C-D) and Spartina
(A-B) leaves obtained from control untreated (A and C) or salt treated
(B and D) for Setaria at 50 mM and Spartina at 250 mM during 12 days
with 5 mM DAB (diaminobenzidine). Brown spots reflect the interaction of
hydrogen peroxide (H2O2) with DAB under
light (500 μmol m-2 s-1) in leaves
following salt stress treatment.