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.