A novel system for spatial and temporal imaging of intrinsic plant water use efficiency

Abstract

Instrumentation and methods for rapid screening and selection of plants with improved water use efficiency are essential to address current issues of global food and fuel security. A new imaging system that combines chlorophyll fluorescence and thermal imaging has been developed to generate images of assimilation rate (A), stomatal conductance (gs), and intrinsic water use efficiency (WUEi) from whole plants or leaves under controlled environmental conditions. This is the first demonstration of the production of images of WUEi and the first to determine images of g s from themography at the whole-plant scale. Data are presented illustrating the use of this system for rapidly and non-destructively screening plants for alterations in WUEi by comparing Arabidopsis thaliana mutants (OST1-1) that have altered WUEi driven by open stomata, with wild-type plants. This novel instrument not only provides the potential to monitor multiple plants simultaneously, but enables intra- and interspecies variation to be taken into account both spatially and temporally. The ability to measure A, gs, and WUEi progressively was developed to facilitate and encourage the development of new dynamic protocols. Images illustrating the instrument's dynamic capabilities are demonstrated by analysing plant responses to changing photosynthetic photon flux density (PPFD). Applications of this system will augment the research community's need for novel screening methods to identify rapidly novel lines, cultivars, or species with improved A and WUEi in order to meet the current demands on modern agriculture and food production.

Keywords: Chlorophyll fluorescence imaging; dynamic responses; leaf heterogeneity; screening; thermal imaging; water use efficiency..

Fig. 1.

Schematic diagram of the system used to image whole plants for chlorophyll a fluorescence and temperature under controlled conditions. The imaging system was modified to allow the attachment of two cameras, the thermal camera being directly positioned above the plant and the fluorescence camera situated at 90 ° to the thermal camera, utilizing a silver-backed mirror at 45 ° to capture images of F _q′/F _m′. The plant was positioned within an open-topped chamber (photograph insert) where concentrations of O₂, CO₂, and H₂O were maintained at a typical flow rate of ~0.87 l s^–1 N₂, with all gases passing through a humidifying system prior to entering the chamber. Concentrations of O₂, CO₂, and H₂O were measured every second by an IRGA and oxygen sensor.

Fig. 2.

Concentrations of (a) O₂ and (b) CO₂ (filled circles) and water vapour (triangles) in the measuring chamber during an experiment. Oxygen concentration was switched from atmospheric (210 mmol mol^–1) to 20 mmol mol^–1 five times while maintaining CO₂ and H₂O vapour concentrations.

Fig. 3.

Typical (a) F _q′/F _m′ and (b) temperature images of an A. thaliana plant illustrating the production of (c) CO₂ assimilation (A), (d) stomatal conductance (g _s), and (e) intrinsic WUE (IWUE_i) images. Calibrations were applied to convert pixel values within each image into A and g _s data, respectively. The wet and dry temperature standards used in the calculation of g _s are indicated by arrows on the temperature image. To produce an image of WUE_i (A/g _s=IWUE_i), the A image was rotated, scaled, and interpolated onto the image of g _s. The colour bar beneath each image shows the range of parameter values. For further details of this process, see Materials and methods.

Fig. 4.

The response of CO₂ assimilation (a) and PSII operating efficiency (b), estimated from F _q′/F _m′, to changes in internal CO₂ concentration (C _i) under 20 mmol mol^–1 O₂ and at 200 (open circles), 500 (grey circles), and 800 (filled circles) µmol m^–2 s^–1 PPFD. The correlation between CO₂ assimilation and PSII operating efficiency (c) was fitted using a linear regression for each PPFD intensity. All measurements were made on 5-week-old A. thaliana. Air temperature and VPD were 25 °C and 1.2 kPa, respectively. Data are the means with standard errors (n=3–5).

Fig. 5.

A comparison between stomatal conductance calculated from thermal images with that measured using an IRGA. To stimulate a range of conductances, leaves of Phaseolus vulgaris (filled circles) and A. thaliana thaliana Col-0 (grey circles) and WS-0 (triangles) were exposed to PPFDs between 200 µmol m^–2 s^–1 and 2000 µmol m^–2 s^–1. The solid line represents a 1:1 relationship (P < 0.0001, R _s=0.96).

Fig. 6.

Screening of mutant OST1-1 (left column of three plants) and wild-type Ler (right column of three plants) for differences in CO₂ assimilation (A), stomatal conductance (g _s), and IWUE_i. The plants were dark adapted for 20min before the PPFD was increased to 200 µmol m^–2 s^–1. Images were taken after 20min (a–c) and after 35min (d–f). Images of A (a and d), g _s (b and e), and IWUE_i (c and f) were calculated. All images were captured at 400 µmol mol^–1 [CO₂], 21 °C air temperature, and 1.1 kPa VPD. The colour bar between each image shows the range of parameter values.

Fig. 7.

Changes in measured (filled circles) and calculated (open circles) values of CO₂ assimilation, stomatal conductance, and WUE_i/IWUE_i were determined on leaves of 5-week-old A. thaliana plants during a stepwise increase in PPFD from 200 µmol m^–2 s^–1 to 800 µmol m^–2 s^–1 PPFD after 15min (↑). The asterisk (*) denotes data points presented as images in Fig. 9. Air temperature and VPD were 25 °C and 1.2 kPa, respectively. Data are means with standard errors (n=3).

Fig. 8.

Images of CO₂ assimilation, stomatal conductance, and IWUE_i taken at 9, 21, and 30min as shown in Fig. 7. Arrows indicate older leaves selected for analysis. Air temperature and VPD were 25 °C and 1.2 kPa, respectively. The colour bars on the right show the range of parameter values.

Fig. 9.

A comparison of IRGA measurements of WUE_i and calculated values of IWUE_i from images captured using the combined imaging system. WUE_i was measured from leaves (circles) of A. thaliana during a step-wise change in light at a CO₂ concentration of 400 µmol mol^–1 (see Fig. 8); data are means with standard errors (n=5–8). Measurements were taken from individual leaves (squares) at CO₂ concentrations between 100 µmol mol^–1 and 2000 µmol mol^–1. Air temperature and VPD were 25 °C and 1.2 kPa, respectively. The solid line represents a 1:1 relationship.