Rising night temperatures pose a significant threat to wheat productivity, yet the physiological basis of wheat adaptation to nocturnal warming remains poorly understood. We evaluated leaf photosynthetic and respiratory traits in ten Australian wheat cultivars released between 1901 and 2012 to warm nights under temperature-controlled environments. When exposed to warmer nights, rates of leaf net CO₂ assimilation measured at 25 °C (A_net²⁵) remained stable across cultivar release date despite declines in photosynthetic capacity (V_cmax and J₁₅₀₀) in newer cultivars. In most cultivars leaf respiratory CO₂ release in the dark (R_dark) exhibited divergent thermal responses: warm nights suppressed temperature-normalised night R_dark (R_{dark_night}) but stimulated or maintained R_dark in the daytime (R_{dark_day}). The results suggest that a century of yield-focused breeding may have inadvertently maintained A_net²⁵ under warmer nights in modern cultivars. This likely reflects the selection of genotypes with more efficient photosynthetic capacity (i.e. greater return per protein investment) under warm nights. It is also likely that modern cultivars exhibit reduced respiratory demand for maintenance of processes such as Rubisco protein turnover and synthesis. Our findings highlight trait-based targets for enhancing energy efficiency and climate resilience in wheat and opportunities to improve the parameterization of R_dark in Earth system models.

Materials and methods

The experiment was conducted in controlled-environment facilities (glasshouses and growth chambers) at the University of New England (UNE) in Armidale, Australia (30.48°S, longitude 151.63°E, elevation 1021.5 m a.s.l.) in 2022.

Plant materials and growth conditions

A set of 10 wheat genotypes (Supplementary Table 1) were used in this experiment. The genotypes included commercial Australian wheat varieties spanning over a century (1901-2012) of breeding. Seeds of ten Australian wheat cultivars were planted in 4.5 L plastic pots (18 cm diameter and 60 cm height) containing Professional premium potting mix with slow-release fertiliser (J.C. & A.T. Searle Pty. Ltd., Qld. Australia). Potted plants were raised in glasshouse bays set to day/night temperatures of 20/12 °C, 70% relative humidity and a natural photoperiod of 12 hours. The plants were grown for four weeks in the glasshouse until the tillering stage (Zadok 20-29) (Zadoks et al., 1974). During this period, watering was provided manually, and standard operating protocols used for plant husbandry in the glasshouse applied. At tillering, when all plants had a fully extended third leaf, wheat plants were moved into growth chambers (Conviron, Conviron Inc., Winnipeg, Canada), and raised for three weeks till their booting stage (Zadok 30-49).

The three growth chambers were equipped with incandescent and fluorescent lamps, and set to a 14-hour photoperiod with photosynthetic photon flux density (PPFD) of 800-850 µmol m^-2, and relative humidity of 60%. The experiment was a complete randomised design with two treatments and three replicates. However, each cultivar had six technical replicates (i.e. two pots per chamber per treatment). The treatments were the control temperature (25/12 °C, maximum day/minimum night) and high night temperature (25/22°C, maximum day/minimum night). The experiment was conducted in two sequential batches, with all control plants grown first, followed by all high night temperature plants. Each batch used the same three growth chambers, serving as biological replicates, to ensure consistent environmental conditions across treatments. Maximum and minimum temperatures were maintained for 8 hour each in all growth chambers with a transition period of 4 hour between maximum day/minimum night temperatures. Temperature and relative humidity (RH) at canopy level were recorded every five minutes using Tiny-tag Ultra 2 Hasting temperature/RH data loggers (Gemini Data Loggers UK LTD, UK) in all growth chambers during both treatments.

Photosynthesis and respiration

Gas exchange measurements were conducted on recently fully developed flag leaves on tagged main tillers in each treatment using portable infrared gas analyser (IRGA) units (Li-6400XT, LI-COR Inc., Lincoln, NE, USA). The net CO₂ assimilation (Anet²⁵) measurements were taken with Li-6400XT units with 6 cm leaf chamber and block temperatures set to 25 °C, CO₂ fixed at 400 ppm with a flow rate of 500 μmol s⁻¹ and light intensity of 1500 μmol m^{−2 s−1} of Photosynthetically Active Radiation (PAR). Dark-adapted temperature-normalised R_day and R_night were recorded with the same Li-6400XT units. Each chamber’s head block temperature was set to 25 °C for R_day, and 20 °C for R_night. The set chamber temperature resulted in average leaf temperature of 25.5 °C for A_net²⁵ and R_day and 21.3 °C for R_night. Measurements of A_net²⁵ and R_day (after at least 20 minutes of dark adaptation) were completed during the day (between 09:00 and 17:30 h) and R_night measured at night (after 20:00 h).  

Maximum Rubisco activity and electron transport rate

Two Li-6400XT units were used to assess photosynthetic capacity of the ten wheat genotypes. Photosynthetic CO₂ response curves (A:C_i curves) were generated, at constant irradiance of 1500 μmol photons m⁻² s⁻¹, by varying the CO₂ inside the Li-6400XT leaf chambers as follows: 400, 40, 60, 100, 150, 250, 400, 400, 600, 800, 1000, 1200, 1400 and 400 μmol mol⁻¹. Relative humidity within the chamber was maintained between 40 and 70% for all measurements. The maximum Rubisco activity at 25 °C, V_cmax²⁵, and electron transport rate at PAR of 1500 μmol m^{−2 s−1} and measured at 25 °C, J₁₅₀₀²⁵ were calculated using the leaf biochemical model of photosynthesis (Farquhar et al., 1980) with kinetic constants derived for wheat (Silva-Perez et al., 2018). Response curves of A_net²⁵ to the intercellular CO₂ concentration (C_i) were measured in the mid-section of the flag leaf when the plants reached Zadoks stage (40-49). The relationship between A_net²⁵ and CO₂ followed the FvCB model (Farquhar et al., 1980; von Caemmerer & Farquhar, 1981) with a simple function for limitation by triose phosphate utilization (TPU) (Sharkey et al., 2007). The approach of Gu and Jerome (2010) was used where all possible carboxylation limitation-state combinations were tested, given the required order of limitation states along the C_i axis (Rubisco limited <electron transport limited < TPU limited) and the minimum number of data necessary for each limitation state (n≥2 when Michaelis Menten constants for Rubisco catalysis of carboxylation, Kc, and oxygenation reactions, Ko; and the photosynthetic CO₂ compensation point in the absence of mitochondrial respiration in the light, Γ*, are fixed). The R Language and Environment function optim (R Core Team, 2023) was used to minimize the distribution-wise cost function, and the model with the lowest cost function value was accepted after checking for admissibility and, if necessary, testing for co-limited ‘swinging points’ (Gu & Jerome, 2010). These measurements were taken within a day between 09:00 and 17:30 hr (~40 min before sunset) for each plant.

Determination of leaf mass per area and nitrogen content

Leaf area (cm²) was calculated using the same leaf used for gas exchange measurements. Leaves were dried at 65 °C for 72 h or until constant dry weight was achieved then used to calculate leaf dry mass per unit area (LMA, g m^-2). Then, 1 mg of dry leaf section of each sampled tissue was weighed and analysed to calculate leaf N (%) using an automatic element analyser (Sercon Control “Callisto CF-IRMS” Version 30.0.11, Sercon Integra 2) coupled to a mass spectrometer (Elemental microanalysis LTD, United Kingdom). Using determined N content (%) and LMA, we estimated leaf nitrogen per area (N_area, g m^-2) and leaf nitrogen per dry mass (N_mass, mg g^-1).

Supplementary Table S1. Pedigrees, year of introduction, and susceptibility traits of the wheat cultivars used in this study. 

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