Chlorophyll a Fluorescence as an Indicator of Temperature Stress in Four Diverse Cotton Cultivars (Gossypium hirsutum L.)

Abstract

Heat stress has a detrimental effect on cotton (Gossypium hirsutum L.) production worldwide. The reproductive stage is especially vulnerable to heat stress, which will result in significant yield losses. Chlorophyll a fluorescence (ChlF) induction kinetics was used to investigate the heat tolerance of four cotton cultivars. Cultivars Arkot 9704, VH260, DP393, and DP 210 B2RF were subjected to 30°C and 40°C heat treatments. Plants were grown for 46 days up to the pinhead square stage whereafter plants were subjected to the two temperature regimes for a period of 6 hours. Decreases in the maximum quantum yield of PSII (Fv/Fm) and the performance indexes (PIABS and PITOTAL) reflected the negative impact of elevated temperature on photosynthesis in all four cultivars. In cultivar DP393 the lowest drop in values for Fv/Fm, PIABS, and PITOTAL, showed the genetic capacity of this cultivar to cope with heat stress. Cultivars VH260, DP210 and to a lesser extent Arkot 9704 were adversely affected by heat stress. Chlorophyll a fluorescence measurements and the interpretation of the functions within the chlorophyll transient proved to be a fast and accurate method of identifying heat-tolerant cotton cultivars.

1. Introduction

Cotton, the most important and widespread natural fiber globally, is produced in 75 countries, providing income for more than 250 million people [1]. Cotton is a significant agricultural commodity throughout the world used primarily for its fibers to manufacture textiles [2]. Approximately half of all textile products are made of cotton in the form of apparel, home textiles, and industrial products [1]. Global climate changes strongly influence plant production worldwide [3]. Crop growth and productivity are severely impacted when plants experience drought and heat stress [4]. Heat stress is defined as the rise in temperature beyond a threshold level for a sufficient period of time to cause irreversible damage to plant growth and development [5]. The effects of plant stress depend on the crops’ tolerance toward stress, timing (developmental stage), duration and severity of stress [6, 7].

High temperatures (>32°C) cause serious yield reduction in cotton by affecting its physiology, biochemistry, and quality [8]. Excessive temperatures (even 1°C) significantly limit the yield formation process, decrease boll retention, and reduce yield by 110 kg ha⁻¹. This decline in yield is attributed to smaller boll biomass and a low number of seeds produced in a boll which is caused by heat-induced pollen damage, low fertility and fertilization efficiencies [8].

Cotton is a warm-season crop, but a negative correlation was found between yield and high temperature during the reproductive stage [9, 10]. Research in Arizona and Mississippi in the United States indicates that the reproductive performance of Upland cotton declines once the mean crop temperature exceeds 28–30°C [11, 12, 13]. Excessive temperature (above 30°C) during the reproductive stage (flowering) detrimentally affects cotton yield potential [14]. Schlenker and Roberts [15] indicated that yield growth for corn, soybean, and cotton would gradually increase with temperatures up to 29–32°C and then sharply decrease with temperature increases beyond this threshold.

The thermal kinetic window (TKW) for enzyme activity in cotton (23.5–32°C) strongly correlates with optimal temperatures for general metabolism and growth for various species [16]. Photosynthesis in cotton is highly sensitive to temperatures above 35°C [17, 18, 19, 20]. Some physiological effects of high temperature include decreased efficiency of the photosystems [19, 21, 22]. In cotton, heat stress during flowering resulted in square and flower drops when day temperatures exceeded 30°C [13].

Photosynthesis is one of the processes that are sensitive to heat stress [23]. Because of its sensitivity, the plant’s photosynthetic efficiency is often measured to determine how sensitive the plant is to stress. Photosystem II (PSII) is the initial complex in the photosynthetic electron transport chain, responsible for water oxidation and the generation of molecular oxygen [24]. Heat stress causes changes in the reduction-oxidation properties of PSII acceptors. It reduces electron transport efficiency in the photosystems [25]. Heat stress causes several reactions, for example, increases in leaf senescence, reduction of photosynthesis, deactivation of photosynthetic enzymes, and generation of oxidative damages to the chloroplasts [26].

Chlorophyll fluorescence originates from chlorophyll a pigments [27]. The absorbed light energy can undergo one of three fates, namely, (a) drive photosynthesis, (b) dissipation of excess energy as heat, and (c) re-emitted as light at a longer wavelength (fluorescence). These three processes compete with each other, such that the increase in efficiency of one will lead to a decrease in the yield of the other two [27, 28]. Chlorophyll a fluorescence is defined as the loss of partial exit energy after the antennae have absorbed the light. This occurs in Photosystem II (PSII) through the radiation of red light with a wavelength of 680 nm. Therefore, chlorophyll a fluorescence is light re-emitted by chlorophyll molecules during the return from non-excited states and used as an indicator of photosynthetic conversion in higher plants.

The motivation for the study was to evaluate diverse cotton cultivars to identify a heat-tolerant cultivar. The research was done to assess whether or not chlorophyll a fluorescence is a useful method to indicate heat tolerance in a cultivar to ease breeders’ work to breed superior cultivars, capable of withstanding heat waves that are occurring more often due to climate change. The early identification of cotton cultivars tolerant toward heat stress is of great economic importance and an objective that many plant breeders prioritize. With this study on cotton, chlorophyll a fluorescence measurements are investigated as a useful tool to identify and quantify the impact of heat stress. With such data for different cultivars, negative impacts and crop losses can be avoided, ensuring more stable cotton production.

2. Material and methods

In this study, four diverse cotton cultivars, namely, Arkot 9704, VH260, DP393, and DP210 B2RF were planted in 10 pots in soil that consisted of a 50/50% mixture of coarse sand and black arcadia clay in a greenhouse study with 30/20°C day/night temperature. The pots were placed in a randomized block design. Plants were grown for five weeks up to the pinhead square stage and then subjected to heat stress. Plants were subjected to two treatments in two laboratory ovens (Scientific 2000) to create the 30°C control and 40°C heat stress. During the first study, five-week-old plants were subjected to heat stress for 2, 4, and 6 hours. During studies 2 to 4, only a 6-hour measurement was taken as this was the only treatment that showed significant differences in their chlorophyll fluorescence measurements.

Chlorophyll fluorescence measurements of intact dark-adapted cotton leaves were measured five weeks after emergence with a portable fluorometer (PEA-Plant Efficiency Analyzer, Hansatech Instruments, King’s Lynn, Norfolk, UK). One leaf per plant was measured at three different positions on the leave. The samples were dark-adapted for 6 hours before the measurements and then illuminated with continuous light (2400 μmol m⁻² s⁻¹, 650 nm peak wavelength, for 1 s provided by an array of six light-emitting diodes focused on a circle of 5 mm diameter of the sample surface. Biolyzer v.3.0.6 software (developed by R Rodriguez, University of Geneva) evaluated fluorescence induction transients. Table 1 is a summary of parameters measured via chlorophyll a fluorescence measurements, plus a description of the formulas with references to authors.

Parameter	Description	Formula
Fv/Fm; Maximum quantum yield	a quantitative measurement of maximum or potential photochemical efficiency [29]. Fv/Fm is the most widely used parameter in chlorophyll fluorescence research to document stress [30, 31] and [28] defined the boundary level for a fully functional PSII system to be 0.750	Fv/Fm = (Fm-F0) / Fm; F0 = minimal fluorescence,; Fm = maximal fluorescence; Fv = variable fluorescence.; Butler and Kitajima [32] were the first authors to calculate the maximum quantum yield of primary PSII photochemistry (Fv/Fm) based on the characteristics of the OJIP curve. Fv/Fm was however shown to be nonspecific [33] and insensitive [34].
PIABS	a multiparametric function representing three independent parameters contributing to photosynthesis, namely; (1) the density of fully active reaction centers (RC’s); $\frac{RC}{\left|\right|}$,; (2) efficiency of electron movement by trapped excitation into the electron transport chain beyond QA; and; (3) the probability that an absorbed photon will be trapped by RC’s. It therefore reflects the accumulation of all of PSII’s responses:	$P{I}_{\mathit{ABS}}=\frac{RC}{\mathit{ABS}}\frac{{\varphi }_{Po}}{1-{\varphi }_{Po}}\frac{{\psi }_o}{1-{\psi }_o}$; Strasser et al., [35] developed performance index of overall photochemistry (PIABS) by using three independent OJIP curve parameters (φPo—maximum quantum yield of primary PSII photochemistry, ψEo—efficiency with which a PSII trapped electron is transferred from Q − A to PQ: and RC/ABS—the density of PSII reaction centers) [35]. PIABS could reflect the state of plant photosynthetic apparatus more accurately than Fv/Fm [36] whereas PIABS was related only to the electron transport to the PQ pool [37].
PITOTAL	PITOTAL (relative photosynthetic performance) (one of the most sensitive OJIP parameters) [30]. include the four partial parameters that are related to the amount of active PSII reaction centers per absorbed energy (RC/ABS), the maximum energy flux reaching the PSII reaction centers per absorbed energy (jPo/(1 – jPo)), the probability that this energy will be conserved as redox energy and drive electron transport beyond QA (yEo/(1 – yEo)), and the probability that electrons from intermediate carriers finally reach the end acceptors of PSI (dRo/(1 – dRo)). PITOTAL is considered to be positively correlated with CO2 assimilation rates, hence to productivity based on photosynthesis [38].	Tsimilli-Michael and Strasser, [39], defines PITOTAL as: RC/ABS jPo/(1–jPo) yEo/(1–yEo) dRo/(1–dRo); [40] Hao et al., 2021 summarize, PITOTAL as calculated by PIABS and δRo (the efficiency of the electron from PQH2 is transferred to final PSI acceptors), which can fully describe the photochemical activity of the linear photosynthetic electron transfer chain

Table 1.

Parameters measured plus description.

3. Results

The F_v/F_m values were significantly lower in heat stress plants than in non-stressed plants in all four cultivars. The lowest drop for F_v/F_m in values between heat stress and non-stressed plants were in the leaves of cultivar DP393, meaning DP393 coped with the heat stress and exhibits heat tolerance. The highest drop in values was in cultivar VH260, then DP210 and Arkot 9704 (Figure 1). The decreased values of F_v/F_m in heat stress plants were likely due to damage to the PSII system and a consequent increase in non-photochemical quenching (NPQ) [41]. Increasing temperature damages PSII reaction centers and dissociates antennae pigment-protein complexes from the central core of the PSII light-harvesting apparatus, consequently impairing photosynthesis [42].

Figure 1.

The effect of 6 hours of continuous heat stress on the maximum quantum yield (Fv/Fm) of four cultivars (Arkot 9704, VH260, DP393 and DP 210 B2RF) treatment values not connected by the same letters are significantly different (P < 0.05).

3.1 The difference in relative variable fluorescence (ΔV_XX)

Double normalization of the OJIP curve between 0.03 ms and 2 ms reveals the presence of the ΔK-band (Figure 2). An increase in the amplitude of the ΔK-band is indicative of damage to the oxygen-evolving complex associated with photosystem II. Heat stress affected the oxygen-evolving complex much more of Arkot9704 than DP393. When ranking the genotypes according to heat tolerance using variable fluorescence, DP393 was the most heat tolerant, followed by VH260 and DP 210 B2RF, and Arkot 9704 was the most sensitive to heat.

Figure 2.

The difference in relative variable fluorescence (ΔVOJ) of cotton cultivars Arkot 9704, VH260, DP393, and DP210 B2RF after a 6-hour exposure to heat stress at 40°C (van der Westhuizen, 2017).

3.2 Performance index: PI_ABS

PI_ABS values were significantly (P ≤ 0.05) lower in the heat-stressed plants. The lowest decline in values between heat stress and non-stressed plants was in the leaves of cultivar Arkot 9704, followed by DP393, DP210. The highest difference in the values was in cultivar VH260 (Figure 3).

Figure 3.

The average of the PI_ABS taken over four studies for four cultivars (Arkot 9704, VH260, DP393, and DP 210 B2RF) at non stressed (30°C) and heat stress (40°C) treatments, at 6 hours after elevated temperatures. Treatment values not connected by the same letters are significantly different (P < 0.05).

3.3 Performance index: PI_TOTAL

The largest difference between the heat-stressed and the control plants was observed with VH260 plants. VH260 also had the largest PI_TOTAL value of all the cultivars at 30°C (Figure 4). Ranking the cultivars based on the difference in values, DP 393 showed the least difference in values, followed by Arkot 9704 and DP210.

Figure 4.

The average of the PI_TOTAL taken over four studies for four cultivars (Arkot 9704, VH260, DP393, and DP 210 B2RF) at non stressed (30°C) and heat stress (40°C) treatments, at 6 hours after elevated temperatures. Treatment values not connected by the same letters are significantly different (P < 0.05).

4. Discussion

High temperature is one of the most important environmental factors that affect plant growth and development [43]. Because of these rising temperatures, heat stress is becoming a more frequent occurrence posing serious risks to crops. Future cotton production is reported to be subjected to multiple abiotic stresses, including extreme and prolonged high temperatures [44]. In cotton, the sensitive stage for heat stress is the reproductive stage [9, 13, 22] resulting in fruit abscission, smaller bolls, and decreased yields [45].

Climate change has caused a shift in necessity toward the identification of plants tolerant to abiotic stresses. In this cotton study, heat stress decreases the F_v/F_m values in all four cultivars to below the 0.75 boundary level for fully functional PSII system. In similar studies, [46] found decreased F_v/F_m values in cotton under drought stress as did [47] in cotton under heat stress.

The decreases in performance indexes that were found in this cotton study was in accordance to results in a study by [48] where heat stress decreased the performance indexes of heat-treated alfalfa cultivars. The decrease in performance indexes could indicate the lower photochemistry of PSII [30]. Bange et al. [49] recommended that in regions where there is a significant risk of heat stress, cultivars that demonstrate resilience to these stresses should be considered and irrigation management adapted to help mitigate the negative effects of heat stress as crop’s capacity to moderate tissue temperature though transpirational cooling is dependent upon adequate moisture supply. Higher than optimum temperatures have long been known to adversely affect several physiological and metabolic processes with detrimental effects on plant growth and yield. To that end, extensive efforts have been undertaken and heat-tolerant cultivars have been introduced [50].

In a study done by [51] van der Westhuizen (2017), where the author evaluated different screening methods to detect heat stress in Growth Chamber Studies, it was found that decreases in chlorophyll a fluorescence were obtained when genotypes were subjected to heat stress. This is in agreement with research in cotton by [19, 52, 53] who recorded genotypic differences in F_v/F_m in response to heat stress. Bibi et al. [19] found that an increase in temperature from 30.0°C to 33.0°C did not affect F_v/F_m significantly, however, at 36°C and above, F_v/F_m decreased significantly. This is in agreement with research in cotton by [52, 53] who recorded genotypic differences in F_v/F_m in response to heat stress. Wu (2013) [50] found that based on selection by F_v/F_m measurements, it was clear that wild cotton accessions were more tolerant to heat stress than a set of random accessions and check genotypes in a growth chamber.

5. Conclusion

In this study, the impact of elevated temperature on photosynthesis was significant in all four cultivars as reflected in the decrease in the maximum quantum yield of PSII (Fv/Fm) as well as decreased performance indexes (PI_ABS and PI_TOTAL). In cultivar DP393 the lowest drop in values for F_v/F_m, PI_ABS and PI_TOTAL, showed the genetic capacity of this cultivar to cope with heat stress. Considering all results, the cultivars VH260, DP210, and to a lesser extent Arkot 9704 were adversely affected by heat stress and therefore heat sensitive. It is recommended that the cultivar DP393 can be used as the basis in further cotton breeding programs as a source of tolerance for high-temperature stress.

Acknowledgments

The authors want to thank the University of North West (Potchefstroom), the National Research Foundation, University of Arkansas, CottonSA and the Agricultural Research Council – Institute for Industrial Crops for funding.