Evaluation of heat tolerance of hybrid wheat by chlorophyll fluorescence

Wheat ( Triticum aestivum L.) is a C 3 plant that thrives well in cool environments. But it is a widely grown crop from temperate, irrigated to dry and high rainfall areas and from warm, humid to dry cold environments (Reynolds et al ., 2001). It is critical to human nutrition, accounting for about 18% of all calories and up to 19% of protein consumed (Erenstein et al., 2022). However, the production of this important cereal is limited by a number of abiotic stress factors such as drought, heat and salinity. But heat stress is the most important stress factor that affects between 25 and 30 million hectares of wheat annually in the world and thereby causing significant grain yield reduction (Battisti and Naylor, 2009). It has thus posed a severe threat to wheat production in many countries, particularly when it occurs during reproductive and grain filling phases. For healthy wheat growth and a good yield, the range of the optimum temperatures was 18 to 24°C. Temperatures above 28 to 32°C for short periods (e.g.,5 to 6 days) found to cause about 20% or more wheat yield losses. This is because heat stress causes an array of physiological, biochemical and morphological changes in wheat which reduce tillering capacity, shortens grain filling period and accelerates crop senescence (Elbashier et al ., 2012). . In relation to climate change, this effect would be more worsen and heat stress will be a major global challenge for a sustainable production of wheat in near future.

Photosynthesis is very sensitive to heat stress and it is one of the key physiological processes affected by heat stress in plants. Photosynthesis is often the first process that is affected by high temperature stress. Various techniques have been used to measure the photosynthetic response of plants to stress. One important technique is chlorophyll fluorescence, which have been used successfully to measure responses to high temperature in higher plant and the advantages of the technique are that it is quick and non-destructive (Xu et al., 1995). In recent years, the technique of chlorophyll fluorescence has become ubiquitous in plant ecophysiology studies. No investigation into the photosynthetic performance of plants under field conditions seems complete without some fluorescence data. Chlorophyll fluorescence (ChlF) is a powerful, non-destructive tool for screening wheat genotypes for heat and drought tolerance, acting as an indicator of photosystem II (PSII) functionality. Tolerant genotypes exhibit higher Fv/Fm (maximum quantum yield), (PI (performance index), and regulated NPQ (nonphotochemical quenching) compared to sensitive ones, which show significant stress-induced reductions(Maxwell1 and Johnson 2000). It is generally considered that heat-induced downregulation of pho tosynthesis is due to impairment of photochemistry in the light reaction and the reduction in activation of Rubisco in the dark reaction (Haldimann and Feller 2004). In the photosynthetic apparatus, photosystem II (PSII) is regarded the most sensitive and heat labile component, which primarily limits photochemistry in response to environmental per turbations and stresses including heat, light, etc. (Baker and Rosenqvist, 2004; Baker 2008). With advancement in crop stress physiology, chlorophyll fluorescence has emerged as a common tool to study in vivo stress responses in plants, particularly in PSII (Maxwell and Johnson, 2000; Baker and Rosenqvist, 2004; Baker 2008). This technique has been used to detect and quan tify damage in PSII in response to temperature stress in several crops including barley (Rizza et al., 2011), legumes (Herzog and Chai-Arree, 2012) and maize (Sinsawat et al., 2004).

Chlorophyll fluorescence has been used to detect genotypic differences in response to heat stress (Sharma et al., 2012). The ratio F v / F m provides an estimate of the maximum photochemical efficiency of PSII and has been widely used to detect stress induced perturbations in the photosynthetic apparatus (Baker and Rosenqvist, 2004). Other parameters commonly used are quantum yield of PSII, non-photochemical quenching, NPQ and the redox state of PSII, q L (Kramer et al., 2004). The quantum yield of PSII (also known as PSII operating efficiency), F ′ q / F ′ m , measures the proportion of light absorbed by chlorophyll associated with PSII that is used in photochemistry (Baker and Rosenqvist, 2004). The NPQ (heat dissipation) compares the light-induced F ′ m level to the dark-adapted F m and monitors the apparent rate constant for non-radiative decay (heat loss) from PSII and its antennae (Baker and Rosenqvist, 2004). The q L is a parameter estimating the fraction of PSII centres in open states with a high connectivity of PSII units (Kramer et al., 2004).

Heat tolerance is an important trait crop improvement. Information concerning the ability of wheat to acclimate to elevated temperature and the mechanism of heat stability as well as recovery after heat stress, however, is scanty. Therefore, cultivar with superior photosynthetic traits including chlorophyll fluorescence and high resistance to heat stress is important to wheat production. For this purpose we have selected 12 wheat genotypes including 9 hybrids (a wheat hybrid variety with potentially higher yield than its parents may be a wheat hybrid variety with potentially photosynthetic capacity than its parents) and 3 high yielding commercial cultivars. Higher photosynthetic capacity in hybrid wheat is associated with higher CO 2 assimilation rate, PS II efficiency and Rubisco activity (Chen et al., 2011). The proposed research aims was to identify differences in heat tolerance of tested hybrid and commercial cultivars of wheat by examining their chlorophyll fluorescence under heat stress and control conditions.

MATERIALS AND METHODS

The experiment was conducted in a controlled environment greenhouse at Plant Breeding Institute, University of Sydney, Camden, Australia. Twelve wheat genotypes including 9 hybrids (F1(2)- AR5/PBICR-08-TC005-#16, F1(3)- R26 3-1 / PBI09C009-BC-DH16, F1(4)-AR22-4 / FERROU -2/POTAM*2KS811261-8//ZEMAMRA-8, F1(5)-AR31-2 / PBI09C009-BC-DH16, F1(6)- AR4-5 / Suntop, F1(7)- AR5-9 / PBICR-08-TC005-#16, F1(8)- AR46-2 / PBI09C009-BC-DH16, F1(9)- AR20-2/PBI07C101-BC-DH13, F1(10)- AR22-2/FERROU -2/POTAM*2KS811261-8//ZEMAMRA-8) and 3 commercial cultivars (Suntop, Sunmate and Spitfire) were used as study materials. Suntop is the male parent of F1(6) hybrid. All seeds were received from I.A. Watson Plant Breeding Institute, University of Sydney, Narrabri, NSW, Australia. Seeds were sown in 5 L plastic pots (18 cm height) filled with Osmocote Professional Premium Potting Mix, fertilised regularly Osmocote (NPK of 19.4 : 1.6 : 5 and contains trace elements). Temperature in the glasshouse during plant growth was set 22/15 ºC (day/night) with 50% humidity. Plants was heated at 35/22 ºC (day/night) for three days at anthesis. Heat treated plants were transferred to growth cabinets, when the majority of culms in each pot was reach anthesis, for a 3 day exposure to high temperature stress. After the three day heat stress in the cabinets, pots were returned to the glass house to mature. Plants grown continuously at 22/15 ºC were serve as the controls.

Plants of each genotype were chosen for analysis of physical parameters. Best agronomic practice and pest control was carried out as required. Since tolerance to high temperature was the only variable to evaluate, plants in the experiment was irrigated daily to ensure that water was not a limiting factor. The experiment was laid out in a factorial completely randomized design with two factors (temperature and wheat genotypes) and three replications.

Measurements

Chlorophyll fluorescence measurements was taken from the main stem flag leaf of plants using the Li-6400 fluorescence detector (LI-COR inc., lincoln, NE, USA). The sample leaves were dark adapted with a leaf clip for 30 min before measurement and then pulsed with a weak modulated measuring light for determining initial chlorophyll fluorescence (Fo ). A saturating light pulse was then applied for 1s to determine the maximum chlorophyll fluorescence (Fm ) . Variable chlorophyll fluorescence ( F v ) is the difference between F 0 and F m. The potential quantum yield of photosystem II (PS-II) was determined by means of the ratios of variable to maximum fluorescence (Fv/ Fm= (Fm – Fo)/ Fm ). In addition the PhiPS2 (PSII efficiency, ETR (Electron transport rate), qP(Photochemical quenching) were recoded. The measurements were taken 3 times: heat-stress (just after the heat treatment), control (at anthesis of untreated plant) and post-heat (at the 3^rd day after heat treatment).

Statistical analysis

Data were statistically evaluated by analysis of variance (ANOVA) and mean values were compared using least significance differences (LSD), for a probability of P = 0.05.

RESULTS

Dark chlorophyll fluorescence (Fv/Fm): Dark chlorophyll fluorescence (Fv/Fm) was significantly affected (p<0.01) by the interaction effect of heat treatments and wheat genotypes (Table 1). The main effect of heat treatments and wheat genotypes were also significant. At heat stress condition all the genotypes showed reduced Fv/Fm compared to control and post heat conditions. Under control condition the range of Fv/Fm was higher in hybrid genotypes than the commercial cultivars. The Fv/Fm for hybrid varieties was 0.803- 0.820, where as it was 0.806-0.818 for commercial cultivars. In this control condition hybrid variety F1(7) attained the highest Fv/Fm (0.820) followed by F1(8) and the lowest Fv/Fm (0.803) was obtained by F1(4). At heat stress condition hybrid variety F1(5) had the higher Fv/Fm (0.796) and F1(10) had the lowest Fv/Fm (0.768) which was at par with Spitfire (0.770). At post-heat condition, the range of Fv/Fm for hybrid varieties was 0.800-0.816 and for commercial cultivars it was 0.798-0.805. In this post heat condition the hybrid variety F1(8) had the highest Fv/Fm (0.816) and the commercial cultivar Spitfire showed the lowest Fv/Fm (0.798).

Light chlorophyll fluorescence (Fv'/Fm'): Heat stress significantly affected the light chlorophyll fluorescence in all the twelve wheat genotypes (Table 1). The main effect and also the interaction effect on Fv'/Fm' of wheat genotypes and heat treatments were significant (p<0.01). At heat stress condition all the genotypes irrespective of hybrid and commercial cultivars showed reduced Fv'/Fm' compared to control and post-heat conditions. Hybrid genotypes maintained always higher range of Fv'/Fm' compared to commercial cultivars. Under control condition the range of Fv'/Fm' for hybrid varieties was 0.550-0.620, where as it was 0.513- 0.506 for commercial cultivars. In this control condition hybrid variety F1(8) had the highest Fv'/Fm' (0.620), whereas commercial cultivar Spitfire showed the lowest Fv'/Fm' (0.513). At heat stress condition the range of Fv'/Fm' for hybrid varieties was 0.485-0.539, whereas for commercial cultivars it was 0.461-0.510. In this heat stress condition F1(3) had the highest Fv'/Fm' (0.539) and the commercial cultivar Spitfire showed the lowest Fv'/Fm' (0.505). Under post-heat condition the hybrid varieties attained the higher range of Fv'/Fm' (0.522-0.592) compared to commercial cultivars. In this condition hybrid variety F1(3) showed the highest Fv'/Fm'(0.592), whereas commercial cultivar Spitfire had the lowest Fv'/Fm' (0.505).

PSII efficiency (PhiPS2): Results showed that the interaction effect of heat treatments and wheat genotypes on PSII efficiency (PhiPS2) was significant (p<0.01) (Table 2). The main effects of heat treatments and wheat genotypes on PhiPS2 were also statistically significant. At heat stress condition all the wheat genotypes showed higher PhiPS2 compared to control and post-heat conditions except F1(10). Hybrid variety F1(10) had slightly lower PhiPS2 at heat stress condition compared to control and post-heat conditions. Hybrid variety F1(3) had the highest PhiPS2 (0.341), whereas F1(10) showed the lowest PhiPS2 (0.211). In this heat stress condition F1(6) maintained higher PhiPS2 (0.272) than its male parent Suntop. At control condition hybrid variety maintained higher PhiPS2 (0.242) and the commercial cultivar Sunmate had the lower PhiPS2 (0.184). In this control condition hybrid variety F1(6) again showed the higher performance (0.242) than its male parent (0.195). At the post-heat condition hybrid variety F1(5) showed the highest PhiPS2 (0.263), whereas F1(3) showed the lowest PhiPS2 (0.189). Hybrid variety F1(6) also had the higher PhiPS2 (0.256) than its male parent commercial cultivar Suntop (0.193).

Electron transport rate (ETR): The electron transport rate was significantly affected (p<0.01) by the combined effect of heat treatments and wheat genotypes (Table 2). The main effect of heat treatments and wheat genotypes were also significant. At heat stress condition all the wheat genotypes showed higher ETR compared to control and post-heat conditions. But the differences between control and post-heat conditions were much lower than the differences between heat stress and control/post-heat. At heat stress condition hybrid genotype F1(7) had the highest ETR (167.5) and the F1(10) had lowest ETR (110.0). Hybrid variety F1(6) maintained higher ETR (142.8) than its male parent Suntop (126.8) at this heat stress condition. Under control condition hybrid variety F1(10) showed the highest ETR (131.0), whereas commercial cultivar Sunmate had the lowest ETR (97.0). In this control condition F1(6) also had much higher ETR (127.7) that its male parent Suntop (103.3). At post-heat condition hybrid variety F1(8) maintained higher ETR (136.7), whereas F1(2) showed the lowest ETR (99.7). In this condition F1(6) also showed the higher ETR (135.3) than its male parent Suntop (101.6).

Photochemical quencing (qP): Results showed that heat treatments significantly affected photochemical quencing (qP) of different wheat genotypes (Table 3). The interaction and the main effects of wheat genotypes and heat treatments were significant (p<0.01). All the wheat genotypes showed higher qP at heat stress condition compared to control and post-heat condition. At heat stress condition hybrid variety F1(3) had the highest qP, whereas F1(10) showed the lowest qP. At this heat stress condition difference between in qP of F1(6) and its male parent Suntop is not significant. At control condition, F1(6) was the highest qP producer, where as its male parent commercial cultivar Suntop was the lowest producer of qP. Under post-heat condition hybrid variety F1(8) attained highest qP value and the F1(3) had the lowest qP. Here the F1(6) had significantly higher qP value than its male parent Suntop.

Non-photochemical quencing (qN): The effect of heat treatments on non-photochemical quencing (qN) of twelve wheat genotypes is shown in Table 3. Results showed that the interaction effect of heat treatments and wheat genotypes on qN was significant (p<0.01). The main effect of heat treatments and wheat genotypes were also significant. At heat stress condition all wheat genotypes showed lowest qN compared to control and post-heat conditions. The difference between in qN of control and post-heat condition were much lower than the difference between heat stress and control/post-heat condition. At heat stress condition hybrid variety F1(6) had highest qN (0.747) and F1(3) showed the lowest qN (0.431). Under control condition hybrid variety F1(3) maintained highest qN (0.707), whereas F1(4) had the lowest qN (0.526). At post-heat condition F1(9) had the highest qN (0.719) and F1(5) showed lowest qN (0.614).

Table 1: Dark adapted chlorophyll fluorescence ( Fv/Fm) and light adapted chlorophyll fluorescence (Fv’/Fm’) of twelve wheat genotypes at different heat-stress

Wheat enotypes		Fv/Fm			(Fv’/Fm’)
		Control	Heat- stress	Post-heat	Control	Heat Stress	Post-heat
F 1 (2)		0.817	0.795	0.816	0.559	0.511	0.538
F 1 (3)		0.812	0.790	0.811	0.589	0.539	0.592
F 1 (4)		0.803	0.777	0.804	0.607	0.512	0.528
F 1 (5)		0.810	0.796	0.813	0.593	0.518	0.583
F 1 (6)		0.810	0.777	0.811	0.550	0.497	0.559
F 1 (7)		0.820	0.791	0.811	0.567	0.515	0.565
F 1 (8)		0.819	0.795	0.816	0.551	0.539	0.542
F 1 (9)		0.805	0.793	0.808	0.620	0.509	0.552
F 1 (10)		0.807	0.768	0.800	0.565	0.485	0.522
Suntop		0.818	0.791	0.804	0.566	0.468	0.542
Sunmate		0.810	0.782	0.805	0.536	0.510	0.544
Spitfire		0.806	0.770	0.798	0.513	0.461	0.505
Mean		0.811	0.785	0.808	0.567	0.505	0.547
LSD (p≤ 0.05)	enotype ( )	0.004			0.026
	Treatment (T)	0.002			0.013
	× T	0.008			0.047

Table 2: PSII efficiency (PhiPS2) and electron transport rate (ETR) of twelve wheat genotypes at different heat-stress

Wheat enotypes		PSII efficiency (PhiPS2)			Electron transport rate (ETR)
		Control	Heat- stress	Post-heat	Control	Heat- Stress	Post-heat
F 1 (2)		0.236	0.319	0.216	124.3	167.0	113.4
F 1 (3)		0.221	0.341	0.189	116.0	178.8	99.7
F 1 (4)		0.210	0.283	0.226	109.9	145.4	119.0
F 1 (5)		0.218	0.302	0.263	114.4	154.5	138.3
F 1 (6)		0.242	0.272	0.256	127.7	142.8	135.3
F 1 (7)		0.223	0.320	0.252	117.8	167.5	132.4
F 1 (8)		0.221	0.264	0.259	116.6	138.4	136.7
F 1 (9)		0.211	0.283	0.230	111.3	148.4	121.3
F 1 (10)		0.248	0.211	0.237	131.0	110.7	125.2
Suntop		0.195	0.242	0.193	103.0	126.8	101.6
Sunmate		0.184	0.299	0.217	97.6	156.7	114.2
Spitfire		0.203	0.240	0.204	106.3	123.0	107.1
Mean		0.217	0.281	0.228	114.7	146.7	120.4
LSD (p≤ 0.05)	enotype ( )	0.031			16.18
	Treatment (T)	0.015			8.09
	× T	0.054			28.02

Table 3: Photochemical quenching (qP) and Non-photochemical quenching (qN) of twelve wheat genotypes at different heat stress

Wheat enotypes		Photochemical quenching (qP)			Non-photochemical quenching (qN)
		Control	Heat- stress	Post-heat	Control	Heat- Stress	Post-heat
F 1 (2)		0.4286	0 .6238	0.4003	0.6675	0.5959	0.6841
F 1 (3)		0.3767	0 .6335	.3284	0.7056	0.4313	0.6800
F 1 (4)		0.3486	0.5680	0.4276	0.5265	0.5673	0.6744
F 1 (5)		0.3687	0.5824	.4539	0.6050	0.5557	0 .6142
F 1 (6)		0.4411	0.5455	0.4595	0.6955	0.7477	0.6569
F 1 (7)		0.3935	0.6211	0.4468	0.6675	0.5959	0.6841
F 1 (8)		0.4016	0.5123	.4772	0.6865	0.6579	0.6771
F 1 (9)		0.3447	0.5559	0.4178	0.6392	0.4695	0.7192
F 1 (10)		0.4402	0.4528	0.4541	0.6655	0.7424	0.6874
Suntop		0.3447	0 .5165	0.3570	0.7485	0.5647	0.7420
Sunmate		0.3449	0.5866	0.3984	0.6675	0.5959	0.6841
Spitfire		0.3949	0.5173	0.4003	0.7360	0.6255	0.7064
Mean		0.385	0.559	0.418	0.667	0.595	0.684
LSD (p≤ 0.05)	enotype( )	0.055			0.053
	Treatment(T)	0.027			0.026
	× T	0.095			0.093

DISCUSSION

Chlorophyll a fluorescence is a tool to investigate the physiological state of PSII. It quantifies the efficiency of PSII in utilisation of the light energy absorbed by chlorophyll and also the extent to which the efficiency is reduced by stress (Maxwell and Johnson, 2000; Baker and Rosenqvist, 2004). The Fv/Fm ratio is one of the important chlorophyll a fluorescence parameters equivalent to the intrinsic maximum photochemical efficiency of PSII (Adams et al. , 1990).The reaction centre of PSII is one of the primary targets of various environmental constraints such as heat, cold, light, nutrients. Therefore, PSII plays a crucial role in the response of leaf photosynthesis to environmental perturbations and stresses (Baker, 1991).

The main objective in the present study was to assess genetic variation for heat tolerance among selected wheat hybrids and commercial cultivars by evaluating differences in their different chlorophyll florescence parameters. In our investigation, heat-stress significantly affected both the dark and light chlorophyll fluorescence. At the heat stress condition all the wheat genotypes irrespective of hybrid and commercial cultivars showed reduced chlorophyll florescence compared to control and post-heat condition. At all the three heat treatments hybrid varieties showed higher chlorophyll fluorescence than commercial cultivars. Similar results on wheat under heat stress were found by Sharma et al., (2015). They found reduced chlorophyll fluorescence at heat stress compared to control. Stress-induced decrease in Fv/Fm could have various causes including structural alternations in the PSII super-complex that hinders energy transfer from the light harvesting antenna complex to PSII reaction centre (Misra and Singhal, 1992), the physical separation between the PSII reaction centres and the peripheral antennae (Bukhov et al., 1990), inactivation of oxygen evolving complex (Nash et al., 1985), non-photochemical quenching or photo protective regulating mechanisms (Murchie and Niyogi, 2011) and inhibition of photosynthetic electron transfer chain (Krause and Weis, 1991). Furthermore, changes in the fluorescence induction and its derived parameters (Fo, Fm, Fv) can also be used to evaluate the thermo-tolerance of wheat genotypes (Babani and Mathis, 1995). They exposed five wheat cultivars to 40 ºC for 4 h and reported a large decrease in variable fluorescence parameters and PSII efficiency. It was also concluded that changes in fluorescence induction to estimate damage caused by high temperature stress in wheat. However, Balota and Lichenthaler (1999) reported the effect of moderate heat stress (35 ºC) in wheat seedlings under field conditions and have shown that such temperatures affected the chlorophyll fluorescence measurements and net photosynthesis in wheat.

Mufti (2005) reported heat-tolerant cultivars were considered those which showed a minimum decrease in Fv/Fm ratios after 6 h of heat-stress of 40 ºC, while heat susceptible genotypes showed maximum decrease in Fv/Fm ratios after heat-stress. He found this ratios was approximately 0.820 to 0.830 in leaves of wheat seedlings before heat stress. After the heat-stress it usually decreased in the range of 0.795 to 0.780 in heat-tolerant genotypes, while it reduce more than 0.760 to 0.750 in heat-sensitive genotypes, commonly. However, detailed experiments have revealed that recovery of Fv/Fm ratios was also important factor in determining the heattolerance or heat-sensitivity of the genotype. The Fv/Fm ratios recovered relatively more quickly in heat-tolerant genotypes than heat-sensitive genotypes. Therefore, Fv/Fm ratios were found to be a useful criterion for selecting heat-tolerant wheat cultivars under control conditions. In general, he observed heat-stress affected all chlorophyll fluorescence parameters including initial fluorescence (Fo), variable fluorescence (Fv), maximum fluorescence (Fm) and ratio of variable to maximum fluorescence (Fv/Fm) and time to reach maximum fluorescence (Tm). Initial fluorescence (Fo) increased drastically even after one hour of heat-stress in all genotypes and particular in heat-sensitive genotypes. The drastic changes in all in vivo chlorophyll fluorescence measurements most probably indicates the physical dissociation of PSII reaction centres from light harvesting complexes, a substantial accumulation of inactivated PSII centres as well as photo inhibition. However, changes in chlorophyll fluorescence during and after removing heatstress indicated reversibility of damage to photosystems. These changes were more likely involved in the relatively fast recovery of PSII efficiency in heat-tolerant genotypes and slow recovery in heat-sensitive genotypes. All these findings are an agreement of our results of present study.

CONCLUSION

From the overall results it might be concluded that genetic variations in chlorophyll fluorescence and its derived parameters were very prominent among studied wheat genotypes under heat treatments. Most of the wheat genotypes showed increased values of chlorophyll fluorescence control condition compared to heat-stress and post-heat conditions, whereas at heat-stress conditions all the studied wheat genotypes attained decreased chlorophyll fluorescence’s parameters compared to control condition. At post-heat condition all the genotypes recovered the stress induced damage compared to heat stress condition. But the capacities of damage and recovery among wheat genotypes were not similar. Hybrid varieties exhibited lesser damage and more recovery compared to commercial cultivars. Hybrid variety F1(6) was found to be superior in maximum studied characteristics than its male parent Suntop.

ACKNOWLEDGEMENTS

The author expresses his profound gratitude to Australian overnment for granting Endeavour Research Fellowship (Endeavour ID Number: ERF_PDR_5288_2016) to conduct this post-doctoral research at the University of Sydney, Australia. The present paper is an important part of whole research work He is grateful to Dr Daniel, Associate Professor, Faculty of Agriculture and Environment, University of Sydney to being his host supervisor and cordial help, fruitful advice and guidance during his whole fellowship period. The author also expresses his gratitude and indebtedness to Professor Richard Trethowan, Director, IA Watson Research Centre, Narrabri, University of Sydney to provide valuable seeds of this study and cordial cooperation during his fellowship period.

CONFLICTS OF INTEREST

All authors declare that they have no conflicts of interest.