Ethylene inhibiting compound 1-MCP delays leaf senescence in cotton plants. under abiotic stress conditions 1

Transcription

1 Ethylene inhibiting compound 1-MCP delays leaf senescence in cotton plants under abiotic stress conditions 1 CHEN Yuan 1, J. T. Cothren 1, CHEN De-hua 2, Amir M.H. Ibrahim 1, Leonardo Lombardini 3, 1 Department of Soil and Crop Sciences, Texas A&M University, College Station, TX, USA 2 Jiangsu Provincial Key Laboratory of Crops Genetics and Physiology, Yangzhou University, Yangzhou, P.R. China 3 Department of Horticultural Sciences, Texas A&M University, College Station, TX, USA 1 Correspondence Yuan Chen, Tel: ; fax: ; address: chenyua3@tamu.edu 1

2 Abstract Cotton (Gossypium hirsutum L.) plants produce more ethylene when subjected to abiotic stresses, such as high temperatures and drought, which result in premature leaf senescence, reduced photosynthetic efficiency, and thus decreased yield. This study was conducted to test the hypothesis that the ethylene-inhibiting compound 1-methylcyclopropene (1-MCP) treatment of cotton plants can delay leaf senescence under high temperature, drought, and the aging process in controlled environmental conditions. Potted cotton plants were exposed to 1-MCP treatment at the early square stage of development. The protective effect of 1-MCP against membrane damage was found on older compared to younger leaves, indicating 1-MCP could lower the stress level caused by aging. Application of 1-MCP resulted in reduction of lipid peroxidation, membrane leakage, soluble sugar content, and increased chlorophyll content, in contrast to the untreated plants under heat stress, suggesting that 1-MCP treatment of cotton plants may also have the potential to reduce the effect of heat stress in terms of delayed senescence. Application of 1-MCP caused reductions of lipid peroxidation, membrane leakage, and soluble sugar content, together with increases in WUE, water potential, chlorophyll content, and fluorescence quantum efficiency, compared to the untreated plants under drought, suggesting that 1-MCP treatment of cotton plants may also have the ability to reduce the level of stress under drought conditions. In conclusion, 1-MCP treatment of cotton should have the potential to delay senescence under heat and drought stress, and the ageing process. Additionally, 1-MCP is more effective under stress than non-stress conditions. Key words: 1-MCP; ethylene; membrane leakage; lipid peroxidation; chlorophyll fluorescence; total soluble sugar Introduction Leaf senescence is mainly regulated by the developmental age of the plant. However, various internal and environmental signals are integrated with the age information to impact the senescence process. The environmental factors include extreme temperature, drought, shading, pathogen infection, and nutrient deficiency (Bleecker and Kende, 2000; Dong et al., 2006; Dong et al., 2014; Dong et al., 2009; Gan and Amasino, 1997; Lim et al., 2007; Zhao et al., 2005). Leaf senescence is characterized by loss of chlorophyll content and increased membrane leakage (Jiang et al., 2002) and often occurs during the boll-filling stage in cotton. Premature senescence caused a lower photosynthetic rate and less carbon accumulation and thereby decreased yield in cotton production (Wright, 1999). Thus, it is desirable to protect yield by delaying senescence. 2

3 Ethylene is a mediator in the senescence process in which cells undergo programmed cell death induced by developmental and environmental signals (Gan and Amasino, 1997; Pierik et al., 2007; Young et al., 2004). When ethylene function was blocked, lower electrolyte leakage was detected, indicating delayed senescence in flower petals (Faragher et al., 1987; Sisler et al., 1996; Sisler and Serek, 1997; Suttle and Kende, 1980; Thompson et al., 1982) and leaves (Wills et al., 2002). Enhanced chlorophyll degradation associated with ethylene production has also been observed in many other studies (Bleecker and Patterson, 1997; Gepstein and Thimann, 1981; Jiang et al., 2002; Kao and Yang, 1983). Thus, it is desirable to protect yield by antagonizing the ethylene effect to delay senescence. In addition, ethylene synthesis can be elicited by factors such as high temperature, freezing and chilling, water stress including drought and waterlogging, exposure to chemicals including herbicides, physical wounding including bruising, cutting and insect biting; and pathogens (Lieberman, 1979; Morgan and Durham, 1973). Also, the relation between leaf age and ethylene evolution has been reported in cotton plants (Morgan and Durham, 1973; Suttle and Hultstrand, 1991), together with the facts that environmental stress and developmental age all accelerate leaf senescence. Therefore, reducing ethylene s effect on senescence is very important, especially under stress conditions and ageing processes. The plant growth regulator 1-methylcyclopropene (1-MCP) inhibits ethylene action and has been proven to be a valuable product in industry to improve quality and shelf life of horticultural products (Blankenship and Dole, 2003). 1-MCP, a gas at room temperature, has a formula of C 4 H 6. It has been shown that 1-MCP occupies the ethylene receptor site and has an affinity 10 times greater for the site than that of ethylene (Blankenship and Dole, 2003; Sisler et al., 1996). Thus, 1- MCP may inhibit ethylene action by competing with ethylene for the ethylene receptor to inhibit binding. High temperature, especially during reproductive development, has an adverse effect on cotton yields (Pettigrew, 2008). High temperature (above 35 ºC) often occurs during cotton reproductive development in the U.S. Cotton Belt, whereas the optimal day temperature for cotton is approximately 30 ºC (Burke et al., 1988; Reddy et al., 1992). To further complicate this issue, 45% of the world s agricultural land is prone to drought, and over 50% of arable land is expected to be impacted by drought in the next 50 years (Boote et al., 2011). Cotton plants respond negatively to drought (McWilliams, 2003), therefore, due to the widespread occurrence of high temperature and drought conditions in the U.S. Cotton Belt, it is desirable to provide optimal growing conditions for minimizing the deleterious effects of environmental stresses on cotton. The corresponding subtending leaves of bolls provide more than 60% of the total carbohydrates of developing reproductive organs (Wullschleger and Oosterhuis, 1990). The photosynthetic rate of leaves subtending bolls declines during active fruit growth, with the leaves 3

4 subtending bolls reaching peak photosynthesis around the time of anthesis. Because the photosynthetic capacity of the subtending leaf is out of phase with bolls for assimilate demands (Constable and Rawson, 1980; Wullschleger and Oosterhuis, 1990), delaying senescence caused by developmental age to extend the photosynthetically active period for assimilates accumulation to meet these demands is also desirable. Thus in this paper the effect of 1-MCP on leaf senescence was studied under aging, drought and heat stress on cotton. We hypothesized that prevention of ethylene perception and elicitation signal transduction through use of the ethylene receptor inhibitor 1-MCP would alleviate the adverse impacts caused by ethylene on leaf senescence under ageing, drought and heat stress. The objectives of the study were to determine the effect of 1-MCP on premature senescence of cotton plants by using 1-MCP as an agent to reduce the adverse effects of aging, drought and heat stress on senescence. Results 1-MCP Effect on Leaves of Different Ages in Cotton Plants Electrolyte leakage is impacted by both plant and leaf age as well as leaf position (Bandurska and Gniazdowska-Skoczek, 1995; Martineau et al., 1979; Premachandra and Shimada, 1987). The effect of 1-MCP on leaves of different main stem positions was examined in pot study in a growth chamber and the effect of 1-MCP on leaves of different fruiting branch positions was examined in field study. Leaves at the 2nd (MP2), 3rd (MP3), 4th (MP4), and 5th (MP5) nodes on the main stem were collected for electrolyte leakage measurement. Leaf position significantly impacted electrolyte leakage in our study. Membrane damage of the top fully expanded leaves of the canopy (MP5 and MP4) was lower than that of the bottom leaves (MP2) (Fig. 2a). Membrane damage of the leaves close to the main-stem was numerically greater than those of distal leaves (Fig. 2b). Thus, the effect of 1-MCP on membrane damage was examined at different leaf positions. Leaves at the 2nd (MP2), 3rd (MP3), 4th (MP4), and 5th (MP5) nodes on the main stem that were treated with 1-MCP exhibited a numerically lower electrolyte leakage compared to the untreated control. However, significant differences were only observed at the 2nd (MP2) and 3rd (MP3) nodes, which were older leaves than those leaves at the 4th (MP4) and 5th (MP5) nodes (Fig. 3a). 1-MCP treatment decreased the membrane leakage at leaf positions 1 (FP1) and 2 (FP2) on the 6th nodes of fruit branches which were also older leaves compared to leaves at position 3 (FP3) (Fig. 3b). In addition, older leaves showed greater membrane leakage in contrast to younger leaves. Thus, this finding suggests that 1-MCP worked more effectively on older leaves which were potentially more stressed, indicating 1-MCP could delay senescence caused by developmental age. 1-MCP Effect on Leaf Senescence of Heat Stressed Cotton Plants 4

5 Membrane damage and lipid peroxidation are among typical biochemical traits of premature leaf senescence (Liu and Huang, 2000). A significant effect of heat stress on membrane damage of cotton leaves was noted at 3 and 4 DAT, with heat-stressed (HT) plants exhibiting higher membrane damage compared with those under optimum temperature (OT) (Fig. 4a). In contrast, 1- MCP decreased membrane damage at 3 DAT under high temperature conditions (Fig. 4a). MDA, a decomposition product of polyunsaturated fatty acid hydroperoxides that is widely used to quantify lipid peroxidation, was measured in our study. Greater MDA content in response to a higher temperature regime was noted under both 1-MCP untreated and treated conditions at 3 DAT and 4 DAT, indicating a detrimental effect of high temperature on membrane integrity (Fig. 4b). Application of 1-MCP lowered MDA content compared to the untreated control at 3 and 4 DAT under high temperature, whereas no significant effect was detected under the optimum temperature (Fig. 4b). Greater thylakoid membrane damage reflected by lower chlorophyll fluorescence yield and chlorophyll loss, are the typical physiological traits of premature leaf senescence. Cotton plants exposed to high temperature exhibited a lower fluorescence yield compared to those grown under optimum temperature on all recorded days, which indicates a lower PSII photochemistry efficiency in stressed plants (Fig. 4c). However, 1-MCP treatment failed to impact fluorescence yield when compared to the untreated control either under optimum or high temperature (Fig. 4c). Although a numerical difference was detected with 1-MCP treatment, it was not enough to make a statistically significant difference in photosystem efficiency. A significant temperature regime effect was observed at 4 DAT; HT plants exhibited lower chlorophyll content than the OT-treated plants (Fig. 4d). Significant effects of 1-MCP were noted on chlorophyll content in the present study under high temperature. 1-MCP treatment enhanced chlorophyll content compared to the untreated control under high temperature at 4 DAT (Fig. 4d). Sugar accumulation usually triggers senescence (Sekhon et al., 2012; Wingler et al., 2009; Wingler et al., 2006). Soluble sugar content was very sensitive to high temperature in the present study, showing that high temperature increased soluble sugar under both 1-MCP untreated and treated conditions at 2, 3, and 4 DAT (Fig. 5). 1-MCP decreased soluble sugar at 3 and 4 DAT under high temperature stress, suggesting that plants treated with 1-MCP were at a lower stress level than the untreated plants (Fig. 5). 1-MCP Effect on Leaf Senescence of Water Stressed Cotton Plants Water potential was detected to assess the drought stress level. Significant difference was observed at all three days after 1-MCP treatment (DAT) between water-stressed and well-watered treatments, with the water-stressed treatments exhibiting decreased water potential under both 1-MCP untreated and treated conditions. A statistical difference in water potential under the water-stressed conditions 5

6 was detected at 3 DAT; that is, 1-MCP increased water potential compared to the untreated control under drought (Fig. 6). Less negative water potential caused by 1-MCP treatment indicated a lower stress level, which means plants are maintained at a comparatively healthier state. No significant differences were noted in water potential between 1-MCP and the untreated control under wellwatered conditions (Fig. 6), which suggests 1-MCP may be more efficient in stressed conditions. A significant water regime effect on membrane integrity was noted at 3 DAT under both 1- MCP untreated and treated conditions. Water-stressed plants showed higher membrane damage when compared to the untreated plants. The 1-MCP treatment lowered membrane damage, indicating better membrane integrity, at 1, 2, and 3 DAT under water deficit. Additionally at 3 DAT, lower membrane damage caused by 1-MCP was also detected under well-watered conditions (Fig. 7a). The MDA content was increased under water deficit at 3 DAT, indicating more peroxidation and damage of membrane lipids. 1-MCP treatment decreased the MDA content at 2 DAT under wellwatered conditions and at 3 DAT under water stress (Fig. 7b). Lower chlorophyll fluorescence yield was observed under water stress compared to wellwatered conditions at 2 and 3 DAT under both 1-MCP untreated and treated conditions, indicating decreased photosystem II quantum efficiency. 1-MCP increased fluorescence yield at 3 DAT under water deficit (Fig. 7c). Additionally, chlorophyll content detected at 4 DAT showed that water stress decreased plant chlorophyll content and 1-MCP increased it (Fig. 7d). The water-stressed plants had greater soluble sugar at 2 and 3 DAT compared to wellwatered plants under 1-MCP untreated conditions (Fig. 8). This increase in soluble sugar level under water stress would cause a decrease of osmotic potential, and consequently an increase of pressure potential, to maintain stable water potential. This stable pressure potential contributed to normal plant growth and function. At 2 and 3 DAT, application of 1-MCP caused a statistical difference compared to the untreated control under water deficit. The 1-MCP treatment decreased soluble sugar content compared to the untreated control (Fig. 8). This measurement indicated that 1-MCP treated plants were in a less stressed conditions than untreated plants as confirmed by less negative water potential. Discussion These experiments strengthened the concept that 1-MCP could delay aging induced leaf senescence as reflected by less membrane damage in 1-MCP-treated leaves. Also, 1-MCP worked more efficiently on older leaves. It has been reported that ethylene induce senescence only when age-controlled developmental changes are present. In other words, ethylene does not control the onset of leaf senescence, but regulates the timing of senescence (Grbić and Bleecker, 1995). This 6

7 may contribute to the reason why 1-MCP works more efficiently in maintaining membrane integrity in older leaves. Only in older leaves, when the developmental changes are present, 1-MCP inhibits the ethylene-induced senescence. The effect of ethylene on senescence is especially meaningful for cotton yield improvement. The square retention rate for cotton is 60% at the first fruiting position, 30% at the second position, and 15% at the third position, with fruiting branches 3 to 8 contributing 75% of the yield (Hake et al., 1992). Thus, the fruit located on the lower branches and inner positions (first and second fruiting position) of the cotton plants are more important for yield. However, the subtending leaves in these positions that provide most carbohydrates for the developing bolls (Constable and Rawson, 1980; Wullschleger and Oosterhuis, 1990) are comparatively older and more stressed by developmental age. Therefore, protection of older leaves from aging-induced senescence is important for extending the active photosynthetic period and providing more carbon assimilation for developing bolls. Indeed, 1-MCP appears to function in this capacity of delaying aging-induced senescence and could benefit the cotton yield by extending the active photosynthetic period in older leaves. Our present study showed that 1-MCP treatment could decrease membrane damage caused by aging, heat stress, and water stress. This result agrees with recent studies which suggested that 1-MCP treatment decreased leaf membrane leakage in cotton plants (Kawakami et al., 2010) and soybean (Djanaguiraman et al., 2011). Thus, 1-MCP treatment had a beneficial effect on membrane integrity, as reflected in less electrolyte leakage. 1-MCP enhanced membrane integrity, especially under stress conditions. Heat stress was reported to cause the degradation of chlorophyll pigment in a previous study in creeping bentgrass (Agrostis palustris Huds.) (Liu and Huang, 2000). In contrast, dryland cotton leaves had 19% greater chlorophyll content than the irrigated leaves. This increased chlorophyll content was due to reduced leaf size in dryland plants and thus greater specific leaf weight (Pettigrew, 2004). Similar results have been reported for higher chlorophyll content being observed in water stressed plants with smaller leaves (da Costa and Cothren, 2011). In both studies, plants were exposed to water stress gradually for enough time to affect leaf size, whereas in our present study with a lower chlorophyll content detected in water stressed plants, water stress was imposed rapidly over a short time. 1-MCP treated plants exhibited higher chlorophyll contents under heat stress and drought stress. This result is in accordance with findings of Djanaguiraman, who found greater chlorophyll content in 1-MCP-treated soybeans (Djanaguiraman et al., 2011). Enhanced chlorophyll degradation associated with ethylene production has been observed in many studies (Bleecker and Patterson, 1997; Gepstein and Thimann, 1981; Jiang et al., 2002; Kao and Yang, 1983). Thus, 1-MCP might delay chlorophyll degradation by inhibiting ethylene activity. It was reported that cytokinin could decrease the chlorophyll loss by increasing chlorophyll synthesis, and 7

8 reducing chlorophyll degradation of carnations (Dianthus caryophyllus L.) (Genkov et al., 1997); thus, it is of interest to further investigate the effect of ethylene on chlorophyll metabolism. 1-MCP treatment maintained a more efficient photosynthetic apparatus under drought stress. Numerically higher photosynthetic quantum efficiency was detected in 1-MCP treated plants under heat stress. PSII has been identified as the major site in the photosynthesis process that responds to environmental stress (Baker, 1991; Havaux, 1992). Environmental stress such as heat, drought, and salt stress are believed to result in damage in the reaction center of PSII (Giardi et al., 1996; Liu et al., 2006; Yan et al., 2012; Yang et al., 1996). Reactive oxygen species are mainly generated in chloroplasts (Asada, 2006; Edreva, 2005). Additionally thermotolerance of different cultivars of cotton was found to be dependent on antioxidant enzyme content prior to heat stress for antioxidant protection of the photosynthetic apparatus (Snider et al., 2010). Thus, the reduced photosynthetic efficacy might be partially due to ROS production under stress that may be ameliorated by 1-MCP and its ability to alter antioxidant enzyme levels. Sugar accumulation can accelerate leaf senescence (Sekhon et al., 2012; Wingler et al., 2009; Wingler et al., 2006). Our present studies showed that both water stressed and heat stressed plants exhibited higher soluble sugar content and 1-MCP decreased sugar content under stress. This result is in accordance with Djanaguiraman, who showed that 1-MCP treatment reduced total soluble sugars (Djanaguiraman et al., 2011). Thus, 1-MCP has a potential to delay senescence in terms of decreased sugar contents. These conclusions were drawn from studies conducted in environmental controlled conditions. It is also of interest to validate our environmentally controlled study results under field conditions. 1-MCP treatment responses were not always consistent in cotton plants. More thorough studies on uptake of 1-MCP are needed. Radioactive-labeled 1-MCP could be used to investigate the uptake and translocation of the chemical in leaf tissues. By this method, we could obtain data about the time needed for 1-MCP uptake, the 1-MCP uptake pathway (cuticle or stomatal pores), and the effect of temperature, solution ph, relative humidity, spray volume, and the chemical spraying position (abaxial or adaxial epidermis) on chemical efficiency. A 1-MCP and ethylene receptor binding assay as well as a better comprehension of ethylene receptor regeneration after 1- MCP spray are also important aspects to help us understand the mechanisms. Another important question pertaining to spray application is the chemical spray position and coverage of cotton plants. The present spray method usually covers the upper canopy; however, according to our research, 1- MCP works more efficiently on older leaves located in the lower canopy. Thus, a better spray method that incorporates finding of 1-MCP uptake research could lead to increased 1-MCP efficiency. It is also of interest to test response times to 1-MCP treatments at the molecular level by studying genes involved in ethylene production or the ethylene downstream signal. Then we can 8

9 predict the time needed for the molecular level response to be reflected at the physiological trait level. Conclusion Our findings indicate that the greater beneficial effects of 1-MCP were observed under stress in terms of decreased soluble sugar and MDA content, declined membrane leakage, and higher chlorophyll levels. Moreover, 1-MCP impacted plants most efficiently under moderate stress level at 3 DAT when water potential was about -1.8 MPa. Kawakami et al., (2010a) (Kawakami et al., 2010) agreed with our present study that 1-MCP was more efficient at an increased water stress level. Similar results were observed in soybean with greater beneficial effect of 1-MCP detected under high temperature stress compared to optimum temperature (Djanaguiraman et al., 2011). In addition, it is the first time reported that 1-MCP works more effectively in older leaves, which is under greater developmental stress. Thus, the protective effect afforded by 1-MCP toward decreased senescence was greater under stress conditions. Materials and Methods Plant Materials and Growth Conditions The studies were conducted in 2011 in growth chambers under the following growth conditions: 30/20 ºC day/night temperature; 14 h/day photoperiod at a photon flux density of 200 μmol m -2 s -1 ; and 45% relative humidity. Temperature and humidity measurements were made with a Center 315 Meter (Center Technology Corp., Taiwan). Photosynthetic photon flux density (PPFD) was determined by the quantum sensor of the infrared gas analyzer (LI-6400 XT, LI-COR Inc., Lincoln, NE) during gas exchange measurements. Plants were grown in 189 ml cone-tainers with 45 g of Sunshine potting mix (Sun Gro Horticultural Distribution Inc., Bellevue, WA) for aging and drought studies. Additional cotton plants were grown in 500-mL pots with 500 g Sunshine potting mix (Sun Gro Horticultural Distribution Inc., Bellevue, WA) for high temperature studies. Plants were watered thoroughly on a daily basis with deionized water and fertilized twice a week with a quarter strength complete balanced Peter s (%N, P, and K as N, P 2 O 5, and K 2 O equivalents) nutrient solution (Miller Greenhouse Special, Miller Chemical and Fertilizer Co., Hanover, PA). Experimental Design In the aging study, water was withheld from plants with eight true leaves for one day before the 1- MCP application. The study was conducted using a completely randomized design. Six individual plants per treatment were measured and each pot was considered as an experimental unit. To determine the effect of 1-MCP on leaf membrane integrity of different leaf positions, plants were sprayed with a solution of 12.5 g a.i. ha -1 (gram active ingredient per hectare )1-MCP and a % 9

10 rate of Silwet L77 (organosilicone surfactant )(Rohm & Haas, Philadelphia, PA). At 4 h after the 1- MCP treatment, the 2nd to 5th nodal position leaves on the main stem were sampled for membrane leakage (Fig. 1a). For the field study, the study was arranged as a randomized complete block design with four replications. Subtending leaves from fruiting positions 1, 2, and 3 located at the 6th node were sampled in the field grown cotton (100 days after planting) at one day after 1-MCP treatment (10 g a.i. ha -1 with % Silwet L77) (Fig. 1b). In 2011 the application daily temperature was 37/23 ºC maximum/minimum, and in the year 2012 the application daily temperature was 39/24 ºC maximum/minimum. There was no precipitation in the following one week in 2011 and a precipitation of 0.25 and 6.35 mm, respectively, at 5 and 6 days after application in In the high temperature study, the experiments were performed utilizing a completely randomized design that consisted of two 1-MCP rates (0 and 10 g a.i. ha -1 ) and two temperature regimes (optimum temperature: 30/20 ºC day/night and high temperature: 40/25 ºC day/night). The treatments were initiated when plants reached 7-true leaves, which approximated the early square stage of development. The 1-MCP was applied with a spray chamber at a 10 g a.i. ha -1 rate of 1- MCP in combination with a % rate of Silwet L77. Four hours after treatment, plants were transported to two growth chambers with different temperature settings (30/20 ºC day/night and 40/25 ºC day/night). The measurements commenced at one day after treatment (1 DAT) and ended at 4 DAT, which was the second day plants were subjected to 1-MCP treatments. Measurements were taken daily on 20 plants with 5 plants per each treatment, and a new set of 20 plants was used for the measurement on the next day. In the drought study, the experiment was performed using a completely randomized design that consisted of two 1-MCP rates (0 and 10 g a.i. ha -1 ) and two water regimes (well-watered and water-stressed). Each pot was treated as an experimental unit. At early square stage (7-true leaf), 1-MCP was applied according to treatments mentioned above before the start of drought. The 1- MCP was applied with a spray chamber at a 10 g a.i. ha -1 rate of 1-MCP and a % (v/v) rate of Silwet L77. Immediately after 1-MCP treatment, water was withheld from plants subjected to drought stress. Plants under well-watered conditions were watered thoroughly in the morning at least 1 h before the measurements. After drought initiation, fertilization of the plants was suspended. Measurements commenced at one day after treatment (1 DAT), which was the second day plants were subjected to 1-MCP and water regime treatments, and ended at 4 DAT. Measurements were taken daily on 20 plants with 5 plants per each treatment, and a new set of 20 plants was used for the measurements on the next day. Membrane Leakage Membrane leakage was measured to show the level of plasma membrane integrity using the method of Djanaguiraman et al. (2011) (Djanaguiraman et al., 2011) with some modification. Five 1-cm- 10

11 diameter leaf discs from the 5th fully extended uppermost leaf were sampled and incubated in 12-mL of double distilled water (ddh 2 O) in glass tubes at room temperature. After 1 h of incubation, initial electrical conductivity (IEC) was taken using a calibrated conductivity meter (Oaklon CON11, EUTECH Instrument, IL). Leaf discs were then autoclaved at 120 ºC for 15 min. After the solution cooled to room temperature, final electrical conductivity (FEC) was measured and the membrane damage was calculated from the equation: Membrane damage = (IEC/FEC)*100. Lipid Peroxidation Malondialdehyde (MDA, the final product of lipid peroxidation) was measured to test the level of lipid peroxidation. A 0.8 g leaf sample from the 6th uppermost fully-extended leaves was homogenized in 10 ml of 10% trichloroacetic acid (TCA) and centrifuged for 10 min at 4,000 g. A 3-mL portion of the supernatant was mixed with 3 ml of 10% TCA containing 0.6% 2,4,6-tribromoanisole (TBA). The mixture was incubated in a 100 ºC water bath for 15 min. Samples were centrifuged for 10 min at 4,000 g after a 5-min ice bath. The absorbance for the supernatant was determined spectrophotometrically (DU Series 500, Beckman Instruments Inc., CA) at 532 nm, 600 nm, and 450 nm to calculate the MDA content. Lipid peroxidation was expressed as the MDA concentration in µm g -1 FW. Chlorophyll Fluorescence Chlorophyll fluorescence was obtained in leaves at the 4th position from the uppermost fullyexpanded leaves with a fluorometer (PAM-2100, Walz, Germany). The measuring light intensity and saturation light intensity were both changed to level 9, and the modulation frequency was changed to 20 khz, and other settings were standard in PAM The value of quantum yield (Φ PSII ), used to reflect photosystem II effective quantum efficiency. Chlorophyll Content Chlorophyll content was measured by the ethanol extraction method according to Djanaguiraman et al. (2011)(Djanaguiraman et al., 2011). Six 1 cm-diameter leaf discs from the 5th uppermost fullyextended leaves were incubated in 10 ml of 100% ethanol for 24 h in the dark. Absorbance of the extract was recorded at 645 nm and 663 nm with a spectrophotometer (DU series 500, Beckman Instruments Inc., CA). Total Soluble Sugar The procedure of Hodge and Hofreiter (1962) (Hodge and Hofereiter, 1962) was followed to determine total soluble sugar with some modifications. The 6th uppermost fully-expanded leaves were used for this measurement. Water Potential Water potential was determined daily by psychrometry (PSYPRO Water Potential System, Wescor Inc., Utah). One 0.6 cm-diameter leaf disc from newly collected 4th uppermost fully-expanded leaves 11

12 were incubated in the chamber for 40 min and then water potential was recorded at room temperature. Data Analysis The statistical significance between means were determined by analysis of variance (ANOVA) and multiple mean comparisons were analyzed by LSD (α=0.05) in SAS 9.3 (SAS Institute, NC). At least two independent series of each experiment were performed and similar tendencies were detected. Data presented hereafter are pooled from the repeated series. Acknowledgement We are grateful for the financial support from AgroFresh. We also would like to thank Clayton Lewis and the entire Cotton Physiology Workgroup for their assistance for the field study, and the technical assistance provided by Dr. Hongwen Su and Dr. Yujin Wen. Reference Asada K Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiology, 141, Baker N R A possible role for photosystem-ii in environmental perturbations of photosynthesis. Physiologia Plantarum, 81, Bandurska H, Gniazdowska-Skoczek H Cell membrane stability in two barley genotypes under water stress condisitons. Acta Societatis Botanicorum Poloniae, 64, Blankenship S M, Dole J M methylcyclopropene: A review. Postharvest Biology and Technology, 28, Bleecker A B, Kende H Ethylene: A gaseous signal molecule in plants. Annual review of cell and developmental biology, 16, Bleecker A B, Patterson S E Last exit: Senescence, abscission, and meristem arrest in arabidopsis. The Plant Cell, 9, Boote K J, Ibrahim A M H, Lafitte R, McCulley R, Messina C, Murray S C,... Giese J H Position statement on crop adaptation to climate change. Crop Science, 51, Burke J J, Mahan J R, Hatfield J L Crop-specific thermal kinetic windows in relation to wheat and cotton biomass production. Agronomy Journal, 80, Constable G A, Rawson H M Carbon production and utilization in cotton - inferences from a carbon budget. Australian Journal of Plant Physiology, 7, da Costa V A, Cothren J T Drought effects on gas exchange, chlorophyll, and plant growth of 1-methylcyclopropene treated cotton. Agronomy Journal, 103,

13 Djanaguiraman M, Prasad P V V, Al-Khatib K Ethylene perception inhibitor 1-mcp decreases oxidative damage of leaves through enhanced antioxidant defense mechanisms in soybean plants grown under high temperature stress. Environmental and Experimental Botany, 71, Dong H, Li W, Tang W, Li Z, Zhang D, Niu Y Yield, quality and leaf senescence of cotton grown at varying planting dates and plant densities in the yellow river valley of china. Field Crops Research, 98, Dong H, Mao S, Zhang W, Chen D On boll-setting optimization theory for cotton cultivation and its new development. China Agriculture Science, 47, Dong H, Niu Y, Kong X, Luo Z Effects of early-fruit removal on endogenous cytokinins and abscisic acid in relation to leaf senescence in cotton. Plant Growth Regulation, 59, Edreva A Generation and scavenging of reactive oxygen species in chloroplasts: A submolecular approach. Agriculture, Ecosystems & Environment, 106, Faragher J D, Wachtel E, Mayak S Changes in the physical state of membrane lipids during senescence of rose petals. Plant Physiology, 83, Gan S, Amasino R M Making sense of senescence (molecular genetic regulation and manipulation of leaf senescence). Plant Physiology, 113, Genkov T, Tsoneva P, Ivanova I Effect of cytokinins on photosynthetic pigments and chlorophyllase activity in in vitro cultures of axillary buds of dianthus caryophyllus l. Journal of Plant Growth Regulation, 16, Gepstein S, Thimann K V The role of ethylene in the senescence of oat leaves. Plant Physiology, 68, Giardi M T, Cona A, Geiken B, Kucera T, Masojidek J, Mattoo A K Long-term drought stress induces structural and functional reorganization of photosystem ii. Planta, 199, Grbić V, Bleecker A B Ethylene regulates the timing of leaf senescence in arabidopsis. Hake K, Carter F, Mauney J, Namken N, Heitholt J, Kerby T, Pettigrew B Sqaure retention. Cotton Phsiology Today Newletter. National Cotton Council, Memphis, TN, Technical Service. Havaux M Stress tolerance of photosystem-ii invivo - antagonistic effects of water, heat, and photoinhibition stresses. Plant Physiology, 100, Hodge J E, Hofereiter B T Determination of reducing sugars and carbohydrates. In RL Whistler, JL Wolfrom, eds, methods in carbohydrate chemistry. Acedemic Press, New York. p Jiang W, Sheng Q, Zhou X-J, Zhang M-J, Liu X-J Regulation of detached coriander leaf senescence by 1-methylcyclopropene and ethylene. Postharvest Biology and Technology, 26,

14 Kao C H, Yang S F Role of ethylene in the senescence of detached rice leaves. Plant Physiology, 73, Kawakami E M, Oosterhuis D, Snider J Physiological effects of 1-methylcyclopropene on well-watered and water-stressed cotton plants. Journal of Plant Growth Regulation, 29, Lieberman M Biosynthesis and action of ethylene. Annual Review of Plant Physiology and Plant Molecular Biology, 30, Lim P O, Kim H J, Nam H G Leaf senescence. Annual review of plant biology. Annual Reviews, Palo Alto. p Liu W J, Yuan S, Zhang N H, Lei T, Duan H G, Liang H G, Lin H H Effect of water stress on photosystem 2 in two wheat cultivars. Biologia Plantarum, 50, Liu X Z, Huang B R Heat stress injury in relation to membrane lipid peroxidation in creeping bentgrass. Crop Science, 40, Martineau J R, Specht J E, Williams J H, Sullivan C Y Temperature tolerance in soybeans.1. Evaluation of a technique for assessing cellular membrane thermostability. Crop Science, 19, McWilliams D Drought strategies for cotton. CES Circular 583. Morgan P W, Durham J I Leaf age and ethylene-induced abscission. Plant Physiology, 52, Pettigrew W T Physiological consequences of moisture deficit stress in cotton. Crop Science, 44, Pettigrew W T The effect of higher temperatures on cotton lint yield production and fiber quality. Crop Science, 48, Pierik R, Sasidharan R, Voesenek L Growth control by ethylene: Adjusting phenotypes to the environment. Journal of Plant Growth Regulation, 26, Premachandra G S, Shimada T The measurement of cell-membrane stability using polyethylene-glycol as a drought tolerance-test in wheat. Japanese Journal of Crop Sciense, 56, Reddy K R, Hodges H F, Reddy V R Temperature effects on cotton fruit retention. Agronomy Journal, 84, Sekhon R S, Childs K L, Santoro N, Foster C E, Buell C R, de Leon N, Kaeppler S M Transcriptional and metabolic analysis of senescence induced by preventing pollination in maize. Plant Physiology, 159,

15 Sisler E C, Dupille E, Serek M Effect of 1-methylcyclopropene and methylenecyclopropane on ethylene binding and ethylene action on cut carnations. Plant Growth Regulation, 18, Sisler E C, Serek M Inhibitors of ethylene responses in plants at the receptor level: Recent developments. Physiologia Plantarum, 100, Snider J L, Oosterhuis D M, Kawakami E M Genotypic differences in thermotolerance are dependent upon prestress capacity for antioxidant protection of the photosynthetic apparatus in gossypium hirsutum. Physiologia Plantarum, 138, Suttle J C, Hultstrand J F Ethylene-induced leaf abscission in cotton seedlings - the physiological bases for age-dependent differences in sensitivity. Plant Physiology, 95, Suttle J C, Kende H Ethylene action and loss of membrane integrity during petal senescence in tradescantia. Plant Physiology, 65, Thompson J E, Mayak S, Shinitzky M, Halevy A H Acceleration of membrane senescence in cut carnation flowers by treatment with ethylene. Plant Physiology, 69, Wills R B H, Ku V V V, Warton M A Use of 1-methylcyclopropene to extend the postharvest life of lettuce. Journal of the Science of Food and Agriculture, 82, Wingler A, Masclaux-Daubresse C, Fischer A M Sugars, senescence, and ageing in plants and heterotrophic organisms. Journal of Experimental Botany, 60, Wingler A, Purdy S, MacLean J A, Pourtau N The role of sugars in integrating environmental signals during the regulation of leaf senescence. Journal of Experimental Botany, 57, Wright P R Premature senescence of cotton (gossypium hirsutum l.) - predominantly a potassium disorder caused by an imbalance of source and sink. Plant Soil, 211, Wullschleger S D, Oosterhuis D M Photosynthetic carbon production and use by developing cotton leaves and bolls. Crop Science, 30, Yan K, Chen P, Shao H, Zhao S, Zhang L, Xu G, Sun J Responses of photosynthesis and photosystem ii to higher temperature and salt stress in sorghum. Journal of Agronomy and Crop Science, 198, Yang G, Rhodes D, Joly R Effects of high temperature on membrane stability and chlorophyll fluorescence in glycinebetaine-deficient and glycinebetaine-containing maize lines. Australian Journal of Plant Physiology, 23, Young T E, Meeley R B, Gallie D R Acc synthase expression regulates leaf performance and drought tolerance in maize. Plant Journal, 40,

16 Zhao D, Reddy K R, Kakani V G, Koti S, Gao W Physiological causes of cotton fruit abscission under conditions of high temperature and enhanced ultraviolet-b radiation. Physiologia Plantarum, 124, Fig. 1 Graph shows leaf position on main stem (a) and on the fruiting branch located on 6th nodes (b) 296x162mm (150 x 150 DPI). 16

17 Fig. 2 The effect of leaf position on main stem (main stem position: MP) (a) and leaf position on sympodial branch (fruiting position: FP) (b) on membrane damage of the untreated cotton plants. Same letters above histograms within each leaf position represent non-significant differences (P=0.05). Vertical bars indicate SE. 128x190mm (150 x 150 DPI). 17

18 Fig. 3 The effect of leaf position on main stem (main stem position: MP) (a) and leaf position on sympodial branch (b) on membrane damage of the untreated control and 1-MCP-treated cotton 18

19 plants. Same letters above histograms within each leaf position represent non-significant differences (P=0.05). Vertical bars indicate SE. Control = control cotton plants without 1-MCP treatment; 1-MCP = cotton plants treated with 1-MCP 163x190mm (150 x 150 DPI). Fig. 4 The effect of 1-MCP on membrane damage (a), MDA content (b), leaf chlorophyll fluorescence (c), and leaf chlorophyll content (d) of cotton plants under optimum temperature (OT: 30/20 ºC day/night temperature) and high temperature (HT: 40/25 ºC day/night temperature). Same letters above histograms within each time interval represent non-significant differences (P=0.05). Vertical bars indicate SE. OT = cotton plants without 1-MCP treatment under optimum temperature; OT+1-MCP = cotton plants treated with 1-MCP under optimum temperature; HT =cotton plants without 1-MCP treatment under high temperature; HT+1-MCP = cotton plants treated with 1-MCP under high temperature 223x170mm (96 x 96 DPI). 19

20 Fig. 5 The effect of 1-MCP on soluble sugar content of cotton plants under optimum temperature (30/20 ºC day/night temperature) and high temperature (40/25 ºC day/night temperature). Same letters above histograms within each time interval represent non-significant differences (P=0.05). Vertical bars indicate SE. OT = cotton plants without 1-MCP treatment under optimum temperature; OT+1-MCP = cotton plants treated with 1-MCP under optimum temperature; HT = cotton plants without 1-MCP treatment under high temperature; HT+1-MCP = cotton plants treated with 1-MCP under high temperature 152x118mm (150 x 150 DPI). 20

21 Fig. 6 The effect of 1-MCP on leaf water potential of well-watered and water-stressed cotton plants. Same letters below histograms within each time interval represent non-significant differences (P=0.05). Vertical bars indicate SE. WW = well-watered cotton plants without 1-MCP treatment; WW+1-MCP = well-watered cotton plants treated with 1-MCP; WS = water-stressed cotton plants without 1-MCP treatment; WS+1-MCP = water-stressed cotton plants treated with 1-MCP 152x118mm (150 x 150 DPI) 21

22 Fig. 7 The effect of 1-MCP on membrane damage (a), MDA content (b), leaf chlorophyll fluorescence (c), and leaf chlorophyll content (d) of well-watered (WW) and water-stressed (WS) cotton plants. Same letters above histograms within each time interval represent non-significant differences (P=0.05). Vertical bars indicate SE. WW = well-watered cotton plants without 1- MCP treatment; WW+1-MCP = well-watered cotton plants treated with 1-MCP; WS = waterstressed cotton plants without 1-MCP treatment; WS+1-MCP = water-stressed cotton plants treated with 1-MCP 227x169mm (96 x 96 DPI). 22

23 Fig. 8 The effect of 1-MCP on soluble sugar content of well-watered (WW) and water-stressed (WS) cotton plants. Same letters above histograms within each time interval represent nonsignificant differences (P=0.05). Vertical bars indicate SE. WW = well-watered cotton plants without 1-MCP treatment; WW+1-MCP = well-watered cotton plants treated with 1-MCP; WS = waterstressed cotton plants without 1-MCP treatment; WS+1-MCP = water-stressed cotton plants treated with 1-MCP 152x118mm (150 x 150 DPI). 23