Chlorophyll fluorescence rise kinetics OJIP curves
For the treatments with ATZ only, the level of J-step of fluorescence rise kinetics increased for all ATZ concentrations (Fig. 1A); the I- and P-step levels also increased at ATZ concentrations of 0.1, 0.5, and 1.0 mg·L-1, but declined at ATZ concentrations of 2.0 mg·L-1. ATZ at concentrations of 1.0 and 2.0 mg·L-1 led to the disappearance of J-I and I-P phases. In addition, the uptrend in Fo and downtrend in Fv/Fo with the increase of ATZ concentration were observed, and the change trend showed a significant dose effect relationship (Fig. 1B). Fm decreased significantly in treatment with 2.0 mg·L-1 ATZ. It can be seen that the decrease in Fv/Fo was mainly attributable to the rise of Fo when ATZ concentrations were 0.5 and 1.0 mg·L-1; while it was associated not only with the increase of Fo but also with the decrease of Fm when ATZ concentration was 2.0 mg·L-1. ATZ alone at 0.1 mg·L-1 did not differ significantly from the control in Fo, Fm and Fv/Fo.
For treatments with Cd alone, Cd at concentrations of 5 mg·L-1 caused slight increases in J-, I- and P-step with obvious J-I and I-P phases (Fig. 1C), Fo, Fm and Fv/Fo did not change significantly compared with the control (Fig. 1D); When Cd concentration increased to 10 mg·L-1, however, the J-step level increased while the P-step level decreased (Fig. 1E), Fm and Fv/Fo were significantly lower than those of the control (Fig. 1F).
For treatments with ATZ plus Cd, OJIP curves showed visible J-, I- and P-step with lower Fo and higher Fv/Fo in comparison with ATZ alone (Fig. 1C, E); the levels of J-, I- and P-step were higher and the magnitude of J-I phase was still lower than those of the control except for 0.1 mg·L-1 ATZ combined with 5 mg·L-1 Cd; when ATZ concentrations were 1.0 and 2.0 mg·L-1, Fo and Fm increased significantly, and Fv/Fo did not showed significant compared with those of the control.
The double normalized fluorescence curves (presented as relative variable fluorescence Vt on a logarithmic time scale) show invisible features of OJIP curve (Guo et al. 2020). Compared with the control, J-step increased drastically under ATZ stress whether in presence or absence of Cd, and ATZ alone induced a greater increase than ATZ combined Cd did (Fig. 2A, C, E). Especially in the case of ATZ alone at concentrations above 0.1 mg·L-1, J-step was closed to P-step with a disappearance of the IP phase of the OJIP curve; at the same time, a remarkable lift in I-step and a flattened I-step was observed (Fig. 2A). The initial slope of the relative variable fluorescence kinetics, denoted as M0, expresses the net rate of the RCs' closure (Strasser et al. 2004). The M0 values of single and combined treatments of ATZ and Cd were significantly larger than that of the control (Fig. 2A, C, E; Table 2). ATZ alone at concentrations above 0.1 mg·L-1 had stronger effects on the M0 value than ATZ combined with Cd did; and the difference was significant when Cd concentration was 10 mg·L-1. The ANOVA result showed that ATZ and Cd were significant factors for the M0 value and had statistically significant interaction effects (Table 2). VJ, VI and FJ/FI further reflected the initial action site of ATZ. VJ and VI reflect the accumulation of QA- (Sun et al. 2020). Both single ATZ and ATZ combined Cd increased significantly the values of VJ, VI and FJ/FI compared with the control. ATZ only at levels above 0.1 mg·L-1 showed significant differences in values of VI, VI and FJ/FI from ATZ combined with 10 mg·L-1 Cd, whereas this difference was not observed when Cd concentration was 5 mg·L-1 (Fig. 2B, D, F). Under single ATZ and combined stresses of ATZ and Cd, VJ showed higher increase magnitude than VI.
The initial slope of the standardized fluorescence transient Ft/Fo
To ascertain the differences in the rate of the electron transfer from P680 to QA between ATZ-Cd treated and control plants, the initial slopes of the standardized fluorescence transient (the fluorescence values were expressed as Ft/Fo (Strasser et al. 2004)) at a linear time scale from 0.02 to 0.15 ms were presented in Fig. 3. The initial slope of single ATZ at the level of 0.1 and 0.5 mg·L-1 was significantly greater than that of the control, while it markedly decreased when ATZ concentration was up to 2.0 mg·L-1 (Fig. 3A, E). In contrast to the control, the plant treated with single Cd showed a significant increase in the initial slope, and there was significant difference among two Cd concentrations (Fig. 3B, C, E). The initial slope of ATZ combined with Cd was significantly larger than that of the control, and was significantly larger than that of ATZ alone when ATZ concentrations were 1.0 and 2.0 mg·L-1.
L-band
L-band is known as an indicator of the grouping of the PSII units or energetic connectivity between antenna and PSII RCs (Guo et al. 2020). For single ATZ treatment, the L-band increased with increasing ATZ concentrations. ATZ at the concentration of 1.0 and 2.0 mg·L-1increased L-band most obviously (Fig. 4A) with significantly higher WL and FL/FJ (Fig. 4B); ATZ at other concentrations yielded negative L-band values, and the levels of WL and FL/FJ for 0.1 mg·L-1 ATZ were significantly different from those of the control. For the treatments with Cd alone, L-band slightly increased (Fig. 4C, E), and there was no significant difference in WL and FL/FJ compared with the control (Fig. 4D, F). ATZ combined with Cd, except for 0.1 mg·L-1 ATZ combined with 5 mg·L-1 Cd, decreased L-band (Fig. 4C, E) with significantly smaller WL and FL/FJ compared with the control (Fig. 4D, F). In addition, when ATZ concentrations were 1.0 and 2.0 mg·L-1, the WL and FL/FJ values for ATZ alone were significantly larger than those of ATZ combined with Cd.
K-band
The inhibition of the donor side of PSII results in the appearance of K-step in the chlorophyll a fluorescence rise (Lazár 2006). The double normalized chlorophyll a fluorescence curve in the 0.02–2 ms time range revealed the changes of K-band (Fig. 5). I. pseudacorus exhibited a prominent rise of fluorescence intensity at K points of the transient curve in response to ATZ alone at concentrations of 1.0 and 2.0 mg·L-1 (Fig. 5A), and the values of WK and FK/FJ were significantly higher than those of the control (Fig. 5B). The exception was the treatments with ATZ alone at 0.1 mg·L-1 which showed a negative K-band with a significantly decreased FK/FJ (Fig. 5A, B), but this significant difference was not observed in WK (Fig. 5B). Compared with the control, Cd alone induced the occurrence of K-band, and only Cd at 10 mg·L-1 increased significantly the values of WK and FK/FJ. It was observed that the K-band was negative for 2.0 mg·L-1 ATZ combined with Cd (Fig. 5C, E), and FK/FJ decreased significantly compared to that of the control, while WK did not increase pronouncedly (Fig. 5D, F). On the other hand, when ATZ concentrations were 1.0 and 2.0 mg·L-1, the FK/FJ values for ATZ alone were significantly larger than those of ATZ combined with Cd, but WK did not show significant changes.
Energy conservation and photosynthetic performance
In the JIP-test parameters, the absorbed light energy (ABS/RC), captured light energy (TR0/RC), thermally dissipated light energy (DI0/RC), and the energy used for electron transfer (ET0/RC) on the unit of active center, were used to indicate the energy transformation in the RSII (Liu et al. 2020). For the treatment with single ATZ, there was no significant difference in ABS/RC (Fig. 6A) and DI0/RC (Fig. 6D) between ATZ at levels below 0.5 mg·L-1 and the control, while ATZ at levels of 1.0 and 2.0 mg·L-1 increased significantly ABS/RC and DI0/RC; ATZ at the level of 1.0 mg·L-1 also caused a significant increase in TR0/RC (Fig. 6B); ET0/RC decreased significantly at all ATZ concentrations (Fig. 6C). For the treatment with single Cd, ABS/RC, TR0/RC and DI0/RC increased significantly, while ET0/RC did not differ from the control. For the combined treatment with ATZ and Cd, ABS/RC and DI0/RC at high level ATZ (1.0 and 2.0 mg·L-1) were significantly lower than those of corresponding single ATZ treatments; ET0/RC of 1.0 and 2.0 mg·L-1 ATZ was significantly higher than that of ATZ alone when Cd concentration was 10 mg·L-1, but this significant difference was not observed when Cd concentration was 5 mg·L-1.
PIabs is the performance index for energy conservation from exciton to the reduction of intersystem electron acceptors, and PItotal is performance index for energy conservation from exciton to the reduction of PSI end acceptors (de Souza et al. 2020). For the treatment of single ATZ or single Cd, PIabs (Fig. 7A) and PItotal (Fig. 7B) declined significantly compare with the control. For the combined treatment with ATZ and Cd, the values of PIabs and PItotal were greater than those of the corresponding single ATZ treatment when ATZ concentrations were at levels 1.0 and 2.0 mg·L-1.