Rapid, noninvasive screening for perturbations of metabolism and plant growth using chlorophyll fluorescence imaging

Abstract

A rapid, noninvasive technique involving imaging of chlorophyll fluorescence parameters for detecting perturbations of leaf metabolism and growth in seedlings is described. Arabidopsis seedlings were grown in 96-well microtitre plates for 4 d and then treated with eight herbicides with differing modes of action to induce perturbations in a range of different metabolic processes. Imaging of chlorophyll fluorescence emissions from 96 seedlings growing on a microtitre plate enabled images of a number of fluorescence parameters to be rapidly and simultaneously produced for the plants in each well. Herbicideinduced perturbations in metabolism, even in metabolic reactions not directly associated with photosynthetic metabolism, were detected from the changes in the images of fluorescence parameters considerably before any visual effects on seedling growth were observed. Evaluations of seedling growth were made from measurements of the area of chlorophyll fluorescence emission in images of plants growing in the 96-well plates. Decreased seedling growth related directly to herbicideinduced changes in the imaged chlorophyll fluorescence parameters. The applicability of this rapid-screening technique for metabolic perturbations in monocotyledonous species was demonstrated by treating Agrostis tenuis seedlings with Imazapyr, an inhibitor of branched-chain amino acid synthesis.

Figure 1.

Chlorophyll fluorescence trace illustrating the terminology and sequence of events leading to the acquisition of the raw fluorescence images from the Arabidopsis plants that are required for the construction of images of the fluorescence parameters F_v/F_m, 1–(F_o/F_p), 1–(F_o/F_i), and 1–(F_i/F_p). Plants were dark-adapted for 15 min before being exposed to weak measuring pulses for the measurement of F_o. After 20 s, a saturating pulse of approximately 4,000 μmol m^–2 s^–1 was applied for 0.8 s to allow imaging of F_m. The plants were then left in the weak measuring beam for approximately 10 min before being exposed to an actinic PPFD of 30 μmol m^–2 s^–1, and images of F_i and F_p were taken. Traces are shown for control, untreated leaves (•) and for leaves treated with 4 (□) and 8 (▴) mm Imazapyr for 24 h.

Figure 2.

Detection of the effects of the herbicide Imazapyr on plant metabolism using chlorophyll fluorescence imaging before the appearance of visual effects on plant growth. A, Four-day-old Arabidopsis plants growing in a 96-well plate immediately before treatment with 0.4 (rows 5 and 11), 0.8 (rows 4 and 10), 4 (rows 3 and 9), and 8 (rows 2 and 8) mm Imazapyr in 50% (v/v) acetone containing 0.1% (v/v) Tween; plants in rows 6 and 12 were untreated controls, and plants in rows 1 and 7 were treated with 50% (v/v) acetone containing 0.1% (v/v) Tween. Plants treated as described for A are shown after 24 and 48 h in B and C, respectively. Images of the chlorophyll fluorescence parameter, F_v/F_m, for the plants shown in A, B, and C are shown in D, E, and F, respectively. G and H, Enlargements of the plants and images of F_v/F_m outlined by the yellow boxes in B and E, respectively. The data in the images of F_v/F_m shown in D, E, F, and H respectively have been mapped to the color palette shown below H.

Figure 3.

The effects of Imazapyr on chlorophyll fluorescence induction parameters. Changes in the fluorescence parameters F_v/F_m (A), 1–(F_o/F_p) (B), 1–(F_i/F_p) (C), and 1–(F_o/F_i) (D) in Arabidopsis plants with time are shown after treatment with 0.8 mm Imazapyr (▴), 8 mm Imazapyr (○), and 50% (v/v) acetone containing 0.1% (v/v) Tween (□). Untreated controls are indicated by (▪). Data are the means of eight replicates; ses are shown when larger than the symbol.

Figure 4.

Effect of increasing the number of replicate data samples on the variability of the decrease in F_v/F_m for Arabidopsis plants after 6 h of treatment with 0.8 mm Imazapyr. Data from eight replicates of untreated plants (control) are shown, together with data from eight, 16, 32, and 64 replicates of treated plants. ses of the means are given. After 6 h, data from eight replicate plants were not significantly different to the controls; data from 16, 32, and 64 replicate plants were similar with significant differences at P < 0.01.

Figure 5.

Effects of a range of herbicides with differing modes of action on F_v/F_m, 1–(F_i/F_p), 1–(F_o/F_p), and 1–(F_o/F_i) of Arabidopsis plants. Plants were treated with Asulam (As), Atrazine (At), Bifenox (B), 2,4-D (D), Diclofop-methyl (Di), Glyphosate (G), Imazapyr (I), and Paraquat (P) at the recommended concentrations for average field applications (see Table I) and measurements made after 6 and 48 h. Data for control, untreated plants (C) are given. * and ** indicate that the herbicide treatment produced a significant difference from the control at P < 0.01 and P < 0.05, respectively.

Figure 6.

Detection of the effects of the herbicide Imazapyr on plant metabolism in A. tenuis using chlorophyll fluorescence imaging. A, Four-day-old plants growing in a 96-well plate after treatment for 48 h with 0.4 (rows 5 and 11), 0.8 (rows 4 and 10), 4 (rows 3 and 9), and 8 (rows 2 and 8) mm Imazapyr in 50% (v/v) acetone containing 0.1% (v/v) Tween; plants in rows 6 and 12 were untreated controls, and plants in rows 1 and 7 had been treated with 50% (v/v) acetone containing 0.1% (v/v) Tween. Images of the chlorophyll fluorescence parameter, F_v/F_m, for the plants shown in A are shown in B. C and D, Enlargements of the plants and images of F_v/F_m outlined by the yellow boxes in A and B, respectively. The data in the images of F_v/F_m shown in B and D, respectively, have been mapped to the color palette shown below D. E, Changes in F_v/F_m with time after treatment of plants with 0.8 mm Imazapyr (▴), 8 mm Imazapyr (○), and 50% (v/v) acetone containing 0.1% (v/v) Tween (□). Untreated controls are indicated by (▪). Data are the means of eight replicates; ses are shown when larger than the symbol.

Figure 7.

The relationship between F_v/F_m and leaf area for Arabidopsis plants treated with 0.4, 0.8, and 4 mm Imazapyr for 6 and 48 h. Each point represents the mean values of eight replicates of F_v/F_m and fluorescent leaf area calculated from images of F_v/F_m (as shown in Fig. 3) and F_m. The relationship between F_v/F_m and fluorescent leaf area is given by y = 0.816x + 0.09 with R² = 0.851.