Experimental design
Four adjacent glasshouse bays, each containing 20 randomly selected plants, were used to study the effects of growth temperature, water deficit and heatwave on plant physiology and biomass productivity. Growth temperatures in these compartments were 28/18oC, 28/22oC, 32/22oC and 32/26oC (midday/night), representing two basal temperature regimes (i.e. 28/18oC and 32/22 oC) and their corresponding nocturnal warming (+4oC) scenarios (i.e. 28/22 oC and 32/26 oC). Temperature was changed five times over 24 h to simulate the daily temperature cycle in the field. Humidity in the compartments was not controlled. Throughout the experimental period, daily mean relative humidity (RH, %) across chambers varied between 64.5±0.1% and 69.5±0.1%, with the daily mean atmospheric vapor pressure deficit (VPD, kPa) range being 0.98~1.07 kPa. CO2 was maintained at ambient level (420 ppm). Pots were moved and rotated routinely to minimize the environmental heterogeneity within each bay. Soil water content was maintained at field capacity until the water deficit stress was applied.
Within each temperature treatment, ten plants were randomly selected for water deficit treatment. Instead of using a priori defined values of soil water content for all plants in the water deficit treatment, water deficit stress was applied as 50% of the water loss (relative to field capacity) required to induce leaf wilting, to each individual plant. This approach ensured that plants were equally water limited regardless of the temperature treatment given that the water demand of individual plants can vary both within and among temperature treatments (Broughton et al., 2017). For plants assigned to the water deficit treatment, pots were first weighed in the morning on the next day following irrigation. Water was then withheld until leaves were visually wilting. Pots were weighed again, and 50% of the water lost during the dry-down was added to the pots, and that soil water content was maintained until the end of the experiment. For the well-watered treatment, the soil water content was maintained at field capacity by adding the water loss from the previous day back into the pots. Individual-specific soil water content was maintained by weighing all pots daily in the early morning to determine the total water loss in the previous day, and water loss was supplemented to maintain the soil water content at the desired level during the experiment. Given that elevated growth temperature can accelerate the development of cotton plants (Reddy, Baker, et al., 1991), the water treatment was initiated at 46 days after planting (DAP) for cool temperature regimes (i.e. 28/18oC and 28/22oC) and 38 DAP forwarm temperature regimes (i.e. 32/22oC and 32/26oC) to minimize the confounding effects associated with growth stage.
The heatwave treatment commenced at the beginning of the flowering stage (59 and 79 DAP for warm and cool grown plants, respectively). Five plants within each water × temperature treatment combination were randomly selected and were transferred into an adjacent bay, within which air temperature was maintained at 40/26oC (midday/night). Plants were exposed to the heatwave for 5 days, and then pots were moved back to their original growth temperature bays until the end of the experiment. For plants exposed to the heatwave, soil water content was controlled as usual despite potentially higher water consumption.