Effect of two-week heat stress during grain filling on stem reserves, senescence, and grain yield of European winter wheat cultivars

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Journal of Agronomy and Crop Science published by Blackwell Verlag GmbH Institute for Crop and Soil Science, Julius Kühn-Institut, Braunschweig, Germany Institute of Agricultural Technology, Johann Heinrich von Thünen Institute, Braunschweig, Germany Institute for Application Techniques in Plant Protection, Julius Kühn-Institut, Braunschweig, Germany Strube Research, Söllingen, Germany


| INTRODUCTION
Ambient air temperature has significantly increased during the last decades and is expected to rise further (IPCC, 2018). The higher air temperature in conjunction with an increase of its variance will lead towards more frequent heat waves and record hot weather (IPCC, 2001;Porter & Semenov, 2005). Wheat is particularly sensitive to high temperatures during anthesis and grain filling (Farooq, Bramley, Palta, & Siddique, 2011;Satorre & Slafer, 1999). While heat stress during anthesis mainly affects the grain number, post-anthesis heat stress reduces the grain yield through a lower grain weight caused by a heat-induced premature leaf senescence and an associated shortened grain filling duration (Bergkamp, Impa, Asebedo, Fritz, & Jagadish, 2018). Heat stress induced yield losses in wheat, which is one of the world's most important food crops poses a serious threat to global food security. Already today, high temperatures are limiting wheat productivity in many countries worldwide (Farooq, Khaliq, & Mahmood, 2015;Narayanan, Prasad, Fritz, Boyle, & Gill, 2015).
It has been estimated that for each 1°C of further temperature increase, grain filling duration of wheat decreases by 2.8 days (Streck, 2005) and, as a consequence, wheat grain yield by about 6% (Asseng et al., 2015). An increase of the average ambient temperature does not only result from a rising maximum temperature, but also from a growing minimum temperature (Vose, Easterling, & Gleason, 2005). Lobell et al. (2005) observed an even stronger dependence of wheat grain yield on the minimum than on the maximum temperature and quantified the yield decrease as 10% for every 1°C rise of the night temperature.
The optimum temperature for grain filling in wheat ranges from 12 to 22°C (Farooq et al., 2011;Porter & Gawith, 1999). The maximum temperature for grain filling above which wheat growth stops is about 35°C and the upper lethal temperature is 47.5°C (Porter & Gawith, 1999). The extent of the heat stress reaction depends on the degree and speed of the temperature increase, as well as the duration of exposure to the high temperature (Wahid, Gelani, Ashraf, & Foolad, 2007). Temperatures of about 45°C represent a critical threshold, because temperatures above this threshold irreversibly damage the photosystem II (PSII), whereas at temperatures below 45°C the PSII functionality is able to recover more or less rapidly depending on the wheat genotype (Haque, Kjaer, Rosenqvist, Sharma, & Ottosen, 2014;Mathur, Jajoo, Mehta, & Bharti, 2011). One of the earliest symptoms of heat-induced premature leaf senescence is the decline in chlorophyll content which becomes apparent in leaf chlorosis caused by the concurrent physiological processes of heat-accelerated chlorophyll degradation and heat inhibition of chlorophyll biosynthesis (Jespersen & Huang, 2014). The carbohydrate depletion resulting from the decrease in photosynthesis and the increase in respiration exhausts the plant's energy reserves that are particularly needed for recovery after long periods of heat stress.
Heat stress in wheat is a particular issue in countries with tropical and sub-tropical climates. However, yield losses and quality deterioration such as small or deformed grains caused by above optimum temperatures during grain filling are a concern also in temperate climates. Based on long-term phenological and climate data of Germany, Rezaei, Siebert, and Ewert (2015) concluded that despite heat escape through an earlier onset of phenological phases (e. g. 14 days earlier heading), warming might still affect grain yield through a shortened grain filling period. Semenov and Shewry (2011) expect that the increase in frequency and degree of heat stress around anthesis will cause substantial yield losses for the heat-sensitive cultivars that are commonly grown in Northern Europe.
In the present field study, 32 mostly European winter wheat genotypes were exposed to high temperatures for 14 days during grain filling. The study aimed (a) to determine whether there exists genetic variability for terminal heat stress tolerance within the adapted primary gene pool of wheat, which could be used in breeding programmes, and (b) to identify and explain important physiological processes such as the mobilisation of stem carbohydrate reserves, chlorophyll degradation and premature senescence which are involved in the heat tolerance of wheat.

| Study site
The experiment was conducted on the experimental fields of the Strube Research wheat breeding station located at Söllingen

| Testing facility and experimental layout
The 32 genotypes were grown under two temperature regimes consisting of a 14-day post-anthesis heat stress treatment and a nearby non-stressed control. Each temperature treatment was laid out as a completely randomised block design with two replicates. The heat stress treatment was conducted in a mobile FILCLAIR 850 foil tunnel (Filclair Serren Industry). The tunnel frame was erected immediately after sowing, but the inflatable duplex polythene foil with about 70% light transmittance was mounted just before the heat stress treatment. The 14-day stress period started on 25 June in 2015 and on 9 June in 2016 and 2017. By that time, all genotypes had completed flowering, corresponding to growth stage 69 on the BBCH-scale (Meier, 2018). The tunnel was 50 m long, 8.5 m wide and had a ridge height of 3.2 m. Four ventilation flaps distributed evenly over the side length of the tunnel allowed for combined side and ridge ventilation. The flaps were opened manually as soon as the temperature in the tunnel approached 40°C. After the heat stress treatment had ended, the tunnel foil was detached so that the environmental conditions were again the same as those in the control.

| Crop management
The field trials were sown on 1 October 2014, 30 September 2015, and 5 October 2016 with a plot drill (Wintersteiger) at a density of 350 grains/m 2 . The 4.5 m 2 plots consisted of seven rows with 17.5 cm row spacing. Each year, mineral nitrogen fertiliser in the form of calcium ammonium nitrate was applied at a rate able to close the gap between the available soil mineral N and the target N level of 220 kg/ha. The N fertiliser was provided in split application at the BBCH stages 20, 30 and 47 to support tillering, booting and grain filling, respectively. Herbicides, insecticides, fungicides and growth regulators were applied according to local practice at the rates recommended by the manufacturer.

| Meteorological measurements
Ambient air temperature, air humidity, global radiation and CO 2 concentration in the foil tunnel and the non-stressed control were recorded every five minutes by an automatic weather station constructed by the Thünen Institute of Agricultural Technology. Air temperature and air humidity were determined by means of 16 EE071 sensors (E + E Elektronik) placed at ear height and equally distributed over the length of the experiment. The EE071 sensors were equipped with a radiation protection and a solar-powered ventilation. For monitoring the CO 2 concentration, one EE871 sensor each was installed in the centre of the tunnel and in the non-stressed control. The light intensity was determined with a CMP3 pyranometer (Kipp & Zonen). In each year, soil samples from the experimental field were taken in 10-cm intervals for determining the field capacity (FC) and the permanent wilting point (PWP). In the laboratory, the FC was determined in a vacuum chamber at a pF value of 1.8. The PWP was measured at a pF value of 4.2 in a pressure pot. The soil moisture was monitored by means of two Sentek EnviroSCAN sensors (Sentek Sensor Technologies). The sensors were installed between two wheat rows in a centrally situated plot, one sensor in the foil tunnel and one sensor in the control. The moisture readings were taken in 10-cm intervals to a depth of 60 cm. The PWP was used for calculating the plant-available water quantity in the soil (Blume et al., 2016) by subtracting the PWP from the measured soil moisture (mm/dm) and adding up the difference over the 60 cm soil horizon.

| Anthesis and grain yield
For each plot, the beginning of anthesis was recorded as the number of days from 1 January to the scoring day when 50% of the plants of a plot had reached BBCH growth stage 61. Each year, the entire experiment was harvested with a plot combine (Wintersteiger).
Although the heat-stressed plots always reached maturity prematurely, the plots in both environments were harvested on the same day, that is on 30 July 2015, 22 July 2016 and 17 August 2017. The grain yield is expressed on the basis of 14% water content.

| Green crop area
For the quantification of heat-induced chlorophyll degradation, digital colour photographs were taken from all experimental plots in the foil tunnel and control. The photographs were taken on three dates in 2016 and on five dates in 2017 during the heat stress treatment and, in both years, on the fourth day after completion of the heat stress treatment. In 2016, the photographs were taken with a Samsung smartphone (model SM-G388F) camera and in 2017 with a Canon EOS 600D camera, using the automatic white balance setting in either case. The photographs were mostly taken with nadir viewing direction and in few cases with oblique viewing angle (about 45° from nadir) and were analysed automatically by means of OpenCV 4 with Python. After conversion to the HIS colour space, very dark pixels (mainly shaded soil and dead lower leaves) as well as extremely light pixels (mostly ears) without reliable colour information were excluded from the analysis. Proper upper and lower limits for hue were chosen to classify and separate the green pixels, representing the photosynthetically active green crop area (Broge & Mortensen, 2002) which is equivalent to the green leaf area but considers all green plant organs including leaves, stems and ears. The green crop area percentage (GCAP) was calculated from the number of green pixels (n green ) and the number of non-green pixels (n yellow ) as follows:

| Water soluble carbohydrates
The concentration of water soluble carbohydrates (WSC) of the stems was determined at crop maturity in 2017. Twenty ear-bearing stems were clipped above the ground and stored in a portable cooler until further processing to minimise carbohydrate respiration losses. The ears were cut from the stem at the ear collar.
The samples were dried at 60°C for 72 hr. After removal of leaf blades and leaf sheaths, the stems were ground to 1 mm size using a Brabender mill (Brabender). A 1-g sample of the ground material was extracted using 70-ml demineralised water at 80°C for 30 min in a shaking water bath. The water soluble carbohydrate concentration was determined by means of high-performance liquid chromatography (HPLC) using a Kontron 400 HPLC system (Kontron Instruments) as described in detail by Menge-Hartmann, Soufan, and Greef (2009). In brief, the HPLC was equipped with a Rezex RPM monosaccharide separation column (Phenomenex) and a Shodex RI-71 refractive index detector (Showa Denko America) for identifying the different carbohydrates. The total WSC concentration was calculated as the sum of glucose, fructose, sucrose GCAP = n green * 100 n green + n yellow and fructans determined by means of peak area comparison with external standards of known concentrations.

| Statistical analysis
The analyses of variance for all measured traits were carried out for individual years and across years using R (R Core Team, 2018). In these analyses, year, temperature regime and genotype were considered fixed effects. The least significant differences (p < .05) were calculated using the function LSD.test (package "agricolae") with the Bonferroni correction. SigmaPlot version 13 (Systat Software Inc.) was used for creating the graphs and calculating the linear regression coefficients between different pairs of traits of interest. The relationship between green crop area and water soluble carbohydrate concentration for the heat stress treatment in Figure 1 was described by an exponential curve using the regression procedure of SigmaPlot.

| Climatic conditions
With the roof foil acting as a light barrier, the radiation intensity in the tunnel was reduced by about 30% compared to that in the ad- lethal maximum temperature for wheat (Porter & Gawith, 1999).

| Genotype response to heat stress treatment
Averaged across seasons, the 14-day post-anthesis heat stress reduced the grain yield by 57.3% from 10.4 to 4.3 t/ha (Table 1)  The French cultivars "Apache," "Solehio" and "Premio" also recorded above-average grain yields irrespective of the temperature regime.
The German pure-line cultivars revealed quite a mixed picture.
While "Lennox," "Tobak," "Julius," "Elixer" and "Rumor" in both environments were among the highest yielders, "Pegassos," "Memory," "Glaucus" and "Magister" performed well in the non-stressed control, but only ranked in the lower third when exposed to temporary heat stress. The experimental lines "Extreme dwarf" and "Straw type" as well as the eastern European cultivars "Moskovskaja56," "Viktoriya Odesskaya" and "Bohemia," produced below-average yields under both stress and non-stressed conditions. The eastern European cultivars "Psenica," "MV Lucilla" and "Viktorya Odesskaya" exhibited a small relative grain yield reduction, but they also only had a low grain yield potential.

| Days to anthesis and its relation to grain yield
The beginning of anthesis was used as a proxy indicator for earliness. This seems justified because the days to anthesis for 18 of the 32 wheat genotypes mentioned in the descriptive variety list of the German Federal Plant Variety Office (https://www.bunde ssort enamt.de/bsa/en/varie ty-testi ng/descr iptiv e-varie ty-lists /) did closely correlate (r = 0.753, p < .001) with the maturity score reported there. The beginning of anthesis had a range of 8 days in 2016 and of 11 days each in 2015 and 2017 (Figure 2). In the non-stressed control, the correlation coefficients between the beginning of anthesis and the grain yield were not statistically significant (p < .05).
Yet in the treatment with temporary heat stress, a significant negative correlation between these two traits was found in 2017 but no significant correlation existed in 2015 and 2016.

| Stay-green and its relation to maturity, grain yield and assimilate relocation
The

| Drawbacks of foil tunnels for field-based heat tolerance screening
For studying the response of wheat genotypes to high temperatures, different experimental set-ups can be used such as delayed sowing (Kumari, Pudake, Singh, & Joshi, 2013;Nahar, Ahamed, & Fujita, 2010;Trethowan & Mahmood, 2011), growth chambers (Rezaei et al., 2018;Yang, Sears, Gill, & Paulsen, 2002), foil tunnels or foliage greenhouses (Li et al., 2018;Talukder, McDonald, & Gill, 2014), artificially warmed field plots (Kimball et al., 2008), simulation modelling (Asseng, Foster, & Turner, 2011;Semenov & Shewry, 2011), satellite images (Lobell, Sibley, & Ortiz-Monasterio, 2012) and inherently warm environments (Reynolds, Nagarajan, Razzaque, & Ageeb, 2001). Most of the studies aimed at field-based measuring of heat tolerance conducted so far have used controlled environment facilities such as foil tunnels or similar devices (Bergkamp et al., 2018). Foil tunnels enable the simultaneous heat stress screening of numerous genotypes which is particularly important in practical breeding programmes (Wahid et al., 2007). However, though there is the desired effect of an increased air temperature, the microclimate in foil tunnels differs from field conditions particularly with regard to reduced radiation, altered carbon dioxide concentration, and higher air humidity. The possible effects of these environmental artefacts on grain yield will be briefly discussed below.
Xu, Tao, Wang, and Wang (2016) used different numbers of layers of polyethylene screens for achieving varying levels of shading of the wheat canopy from the beginning of anthesis to maturity. The use of two polyethylene layers with 67% light transmittance in their experiment caused an average grain yield reduction of 19.4%. Applied to the present study with a similar light transmittance of 70% and approximately half the shading duration, this corresponds to a roughly 10% grain yield reduction.
As is typical of closed greenhouses, the CO 2 concentration in the foil tunnel through plant respiration and microbial activities increased during the night far beyond the ambient level (Poudel & Dunn, 2017) of currently around 410 ppm. However, after sunrise, the excess CO 2 in the tunnel was completely used up by photosynthesis within a few hours, and often dropped below the ambient level during daytime. It is thus assumed that the CO 2 surplus in the morning and the temporary CO 2 deficit during the day have at least partly offset each other in their effect on grain yield.
The relative humidity of 86% in the foil tunnel during the day was markedly higher than the relative humidity of 66% in the control. The high relative humidity in the tunnel was caused by the good water supply of the deep loess soil which in turn was desirable to avoid mixing up effects of drought stress with those of heat stress. Rawson, Begg, and Woodward (1977) have shown that the relative humidity does not affect photosynthesis of individual leaves in winter wheat and various other crops. Overall, in

| heat stress treatment substantially decreased grain yield
The present study showed a decrease in grain yield of 57.3% when a two-week severe heat stress was applied during grain filling (Table 1). Nahar et al. (2010) reported a similarly large grain yield reduction of 53%-73% under late heat stress conditions generated by delayed wheat sowing in Bangladesh. Bergkamp et al. (2018) reported 2%-27% grain yield reduction under post-anthesis heat stress using field based "heat tents" in Kansas, USA. Li et al. (2018) measured  Asseng et al. (2011) which shows that a single heat event during grain filling has a maximum effect of about 5% on grain yield but can accumulate to a yield reduction of up to 60%.

| Relationship between earliness and grain yield
In the heat stress treatment of the present study, a significant negative correlation was observed between the beginning of anthesis and the grain yield in one of the three experimental years (Figure 2).
These results indicate a tendency towards higher yields of early genotypes if high temperatures are prevailing during grain filling. Tewolde, Fernandez, and Erickson (2006) also observed that, under the high temperature conditions of Southwest Texas, earlier heading cultivars performed better than later heading cultivars. Similarly, Midmore, Cartwright, and Fischer, (1984) reported negative correlations between grain yield and days to anthesis for spring wheat grown along an altitudinal transect in Central Mexico with earliness being most favourable at the warmest, least elevated site. Midmore et al. (1984) assumed that the lower yields of the later genotypes were attributable to an accelerated senescence and thus a shorter grain filling duration due to the late-season rise in temperature. In contrast to this, Reynolds et al. (2001) reported highly positive genetic correlations of grain yield with both days to anthesis and leaf chlorophyll retention during grain filling in CIMMYT's high temperature mega-environments. It is assumed that differences between the above cited studies with respect to the intensity and duration of heat stress might explain the inconsistent findings.

| Genotypes do not benefit from stay-green under severe heat stress
The physiological processes involved in yield reduction under heat stress are only poorly understood. Some crop physiologists emphasise the importance of stay-green traits for improving the heat tolerance (Cossani & Reynolds, 2012;Harris al., 2007;Lopes & Reynolds, 2012). This approach is based on the assumption that the wheat yield under heat stress is source-limited and that extending the green canopy duration by delaying leaf senescence and increasing photosynthesis is the key to improving the biomass and grain yield (Abdelrahman, Burritt, Gupta, Tsujimoto, & Tran, 2019). In the present study, however, the retention of green crop area was not significantly associated with grain yield. Li et al. (2018), too, found in only one of two crop seasons a positive, yet only weak association The transfer of photoassimilates from source to sink is a critical step during wheat grain filling (Abdelrahman et al., 2019). The WSC can become the predominant source of carbohydrates for the developing grain, if the ongoing photosynthesis is severely inhibited by heat stress (Blum, 1998;Talukder, McDonald, & Gill, 2013). While net photosynthesis is declining already at temperatures above 30°C (Wardlaw, 1974) and is irreversibly damaged at temperature > 45°C (Mathur et al., 2011), the relocation of assimilates from the stem reserves to the grains is not affected by high temperatures of up to 50°C (Wardlaw, 1974). It is therefore interesting to note that genotypes with a high concentration of WSC in their stems at maturity showed a higher green crop area percentage under post-anthesis heat stress than those with a low stem WSC concentration (Figure 1).
The capacity for photosynthesis lost due to the adverse effect of heat stress was probably compensated for by the transfer of WSCs from the stems into the grains. However, the delayed loss of green colour did not pay off in terms of higher grain yields. Genotypes like "Julius" thus resemble in a way the so-called non-functional (cosmetic) type C stay-green mutants (Thomas & Howarth, 2000;Thomas & Ougham, 2014) which retain chlorophyll but not their photosynthetic capacity. It should be noted, however, that the WSC concentration was only studied in 2017.
The results of the present study do not support the hypothesis that stay-green phenotypes with decreased rates of senescence are a worthwhile goal in breeding for high temperature environments.
This result applies at least to conditions of severe and long-lasting post-anthesis heat stress. For regions with regular harsh post-anthesis heat stress Blum (1998) proposed to select cultivars with a high capacity for stem reserve storage and utilisation for grain filling. In the present study, the WSC concentration correlated significantly positively with the beginning of anthesis (r = 0.704; p < .001) but significantly negatively with the grain yield (r = −0.431; p < .05) (data not shown). This indicates that early genotypes with an efficient remobilisation of the stem carbohydrate reserves are more valuable under late heat stress. For wheat grown under chronic post-anthesis heat stress and heat shock, Yang et al. (2002) obtained high yields regardless of the source of assimilates for grain filling, whether from photosynthesis or stem reserves. Even if both assimilate supply routes were equally effective in terms of grain yield, from the farmers' perspective the use of stem reserves appears preferable because a heat-induced rapid senescence allows an earlier harvest and thus somewhat greater flexibility in the subsequent land use.
Furthermore, the earlier harvest preserves the soil water due to lesser evaporation and transpiration losses.

| CONCLUSION
In this 3-year field study, 32 mostly European winter wheat genotypes were exposed to severe post-anthesis heat stress, both in terms of stress duration and intensity. The daytime air temperature during the 14-day heat stress treatment in the foil tunnel frequently exceeded 35°C and repeatedly was just below the lethality threshold of 47.5°C. The genotypes from eastern Europe exhibited a generally small yield reduction relative to the control, but were low yielding under heat stress and control conditions. On the other hand, a number of genotypes from western Europe not only were high yielding in the control but also performed above average under heat stress conditions. The genetic material studied differed markedly in its physiological response to heat stress. While some genotypes tolerated the high temperatures and retained their greenness after the heat treatment, high temperatures in other genotypes triggered a rapid transition to emergency ripening. Earliness was promoting this trigger but was not a precondition. In contrast to stress escape due to earliness, emergency ripening is a physiological response to a stress to which the plant is inescapably exposed. Emergency ripening is characterised by a premature loss of the green canopy, accompanied by the mobilisation of stem carbohydrate reserves and their relocation to the grains. Genotypes exhibiting this kind of heat response might represent a wheat ideotype for environments where extremely high temperatures occur towards the end of the cropping season.