RESEARCH
High Day- or Nighttime Temperature Alters Leaf Assimilation, Reproductive Success, and Phosphatidic Acid of Pollen Grain in Soybean [Glycine max (L.) Merr.] M. Djanaguiraman, P. V. V. Prasad,* and W. T. Schapaugh Abstract Soybean [Glycine max (L.) Merr.] is often exposed to high daytime and nighttime temperatures during critical growth stages. Threshold mean daily temperature for photosynthesis, respiration, and reproductive process in soybean is ³26°C. In future, the magnitude of increase in nighttime temperatures will be greater than in daytime temperatures. The objectives were to determine effects of high daytime or nighttime temperatures on (i) leaf photosynthetic and respiration rates; (ii) pollen germination, pod-set, and seed weight; and (iii) pollen phospholipids profile. Soybean plants were exposed to high daytime temperature (39/20°C), high nighttime temperatures (30/23°C, 30/26°C, and 30/29°C), or optimum temperature (30/20°C) for 10 d at flowering stage. High daytime temperature (39/20°C) or nighttime temperatures (30/29°C) increased leaf respiration rates and decreased leaf chlorophyll content, photosynthetic rate, photochemical quenching, and electron transport rate compared to optimum temperature. Likewise, high temperature decreased pollen viability and germination. Lower pollen germination at high temperature may be due to decreased levels of saturated phospholipids and phosphatidic acid in pollen grains compared with optimum temperature. Pod-set and seed weight were decreased by high daytime or nighttime temperature. In conclusion, high daytime (39/20°C) or nighttime (30/29°C) temperature decreased leaf photosynthetic rate and pollen germination, leading to lower pod-set and seed weight.
Dep. of Agronomy, 2004 Throckmorton Plant Science Center, Kansas State Univ., Manhattan, KS 66506. Received 22 July 2012. *Corresponding author (
[email protected]). Abbreviations: DGDG, digalactosyldiacylglycerol; ETR, electron transport rate; MGDG, monogalactosyldiacylglycerol; NPQ, non–photochemical quenching; PA, phosphatidic acid; PC, phosphatidylcholine; PI, phosphatidylinositol; PSII, Photosystem II; qP, photochemical quenching; FPSII, quantum yield of Photosystem II.
S
oybean [Glycine max (L.) Merr.] is an important legume crop often exposed to high temperatures during reproductive stages of crop development in semiarid regions. It is predicted that global mean surface air temperatures will increase in the range of 1.5 to 4.5°C by the end of this century (IPCC, 2007). The magnitude of increase in temperatures will be greater during nighttime than in daytime (Peng et al., 2004). Several studies have investigated the combined effects of high daytime and nighttime temperatures (Prasad et al., 1999, 2006a, 2006b, 2008a) or nighttime temperatures (Manunta and Kirkham, 1996; Prasad et al., 2008b; Mohammed and Tarpley, 2009a, 2009b) on various crop species; however, studies comparing the effects of high daytime or nighttime temperatures on physiological processes and reproductive function are limited. Combination of high daytime and nighttime temperature significantly decreased the chlorophyll content, Photosystem II (PSII) quantum efficiency (FPSII), photosynthetic rate, and yield in soybean (Djanaguiraman et al., 2011a) and cotton (Gossypium hirsutum L.; Snider et al., 2009). Similarly, combination of high daytime and nighttime temperatures decreased pollen production and pollen viability, leading to decreased seed set and seed numbers in rice (Oryza sativa L.; Prasad et al., 2006b), groundnut (Arachis hypogaea
Published in Crop Sci. 53:1594–1604 (2013). doi: 10.2135/cropsci2012.07.0441 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
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L.; Prasad et al., 1999), and grain sorghum [Sorghum bicolor (L.) Moench] (Prasad et al., 2006a, 2008a). Likewise, soybean oil content increased with temperature, reaching a plateau at 22°C or 25 to 28°C, and decreased when temperatures exceed these levels. Seed protein concentration of soybean was unaltered at temperatures between 16 and 25°C but increased at temperatures above 25°C (Gibson and Mullen, 1996). Peters et al. (1971) found that a rise in night temperature from 9 to 26°C, reduced grain yield of wheat (Triticum aestivum L.) almost by half by reducing the period of grain filling at maturity phase. The response of photosynthesis to high nighttime temperatures was found to be highly variable, showing a positive (in Populus spp.; Turnbull et al., 2002), negative (in spring wheat; Prasad et al., 2008b), and no effect (in rice; Mohammed and Tarpley, 2009a). Manunta and Kirkham (1996) reported decreased plant height and increased respiration rate at high nighttime temperature in grain sorghum. High nighttime temperature during flowering stage decreased pollen germination in rice (Mohammed and Tarpley, 2009a) and pollen viability and seed set in grain sorghum (Prasad and Djanaguiraman, 2011) compared to optimum nighttime temperatures. However, there is limited information available on soybean. Lower pollen viability in grain sorghum at higher temperatures was mainly due to sucrose utilization and subsequent lack of sucrose biosynthesis by pollen grains (Jain et al., 2010). Similarly, decreases in starch and sugar concentration were observed in pollen grains of tomato (Lycopersicon esculentum L.; Pressman et al., 2002). Apart from sugars, lipids are other important reserves of pollen grain. Membrane fluidity is determined by lipid molecular species, degree of saturation, and temperature of the growing condition. Temperature-induced change in membrane fluidity is one of the immediate consequences of high temperature stress (Horwath et al., 1998). The importance of membrane fluidity under high temperature stress in a soybean mutant deficient in fatty acid unsaturation was described by Alfonso et al. (2001). Our earlier study on grain sorghum showed that high nighttime temperature decreased phospholipid saturation level and increased reactive oxygen species in pollen grains, resulting in membrane damage (Prasad and Djanaguiraman, 2011). Elevated production of reactive oxygen species coupled with membrane damage in pollen grains may be responsible for decreased pollen germination and seed set in grain sorghum (Prasad and Djanaguiraman, 2011). Although studies in soybean have shown decreases in pollen viability at high temperatures (Salem et al., 2007), phospholipid species variation in pollen grains and their relationship to pollen viability is not understood. Overall, there is limited information available on relative effects of high daytime or nighttime temperature on leaf photosynthesis and reproductive processes of soybean. Therefore, our objectives were to determine relative effects crop science, vol. 53, july– august 2013
of high daytime or nighttime temperatures on (i) leaf photosynthetic and respiration rates; (ii) pollen germination, pod-set, and seed yield; and (iii) pollen phospholipids profile. We hypothesize that high daytime or nighttime temperature during flowering stage will increase the rate of respiration while decreasing photosynthetic rate and pollen germination in soybean.
MATERIALS AND METHODS Plant Material and Growing Conditions Two separate experiments were conducted in controlled environment facilities at the Department of Agronomy, Kansas State University, Manhattan, KS. Soybean plants (short-day genotype S76-L9) were grown in 3.8-L pots (top and bottom pot diameters were 15 and 13 cm, respectively) containing 1.8 kg of Metromix 200 (Hummert International, Topeka, KS). Five seeds per pot were sown at a 4-cm depth. After emergence, plants were thinned to two plants per pot, which were maintained until maturity. The growing medium was fertilized with Osmocote at 3 g pot-1 (controlled-release plant food, 14:14:14% N:P2O5:K 2O, respectively; Hummert International, Topeka, KS) at sowing. After seedling emergence, a systemic insecticide (Marathon1% G, Imidacloprid, Hummert International, Topeka, KS) was applied to each pot at 2 g pot-1 to control sucking pests during the seedling stage. Plants were irrigated daily in the morning up to field capacity from sowing to harvest to avoid water stress. Soybean plants were grown from sowing to full bloom (growth stage, R2 stage; open flower at one of the two uppermost nodes; Fehr et al., 1971) at 30/20°C (daytime maximum/ nighttime minimum temperature) in growth chamber (Conviron Model CMP 3244, Winnipeg, MB). The daytime and nighttime temperature regimes in the growth chamber were held for 10 and 14 h, respectively, with a 30-min transition period between the daytime maximum and nighttime minimum temperatures and vice versa. The photoperiod was 10 h, and photon flux density (400 to 700 nm) provided by cool-fluorescent lamps was about 720 µmol m-2 s-1 at the top of the plant canopy. Relative humidity in the chambers was set at 85%. Air temperature and relative humidity were continuously monitored at 20-min intervals in all growth chambers throughout the duration of the experiment using HOBO data logger (Onset Computer Corporation, Bourne, MA). At full bloom (R2 stage; open flower at one of the two uppermost nodes), five temperature regimes—optimum temperature (30/20°C), daytime maximum/nighttime minimum temperature, high nighttime temperatures (30/23°C, 30/26°C, and 30/29°C), and high daytime temperature (39/20°C)— were imposed to five randomly selected growth chambers. The plants were maintained in the above temperatures for 10 d. Each temperature treatment had five replicated pots. The daytime and nighttime temperature regimes, photoperiod, and relative humidity were similar as described before. The quality of temperature controls in various growth chambers are given in Supplementary Fig. 1. After the completion of Experiment I, the same growth chambers were used for Experiment II with the same temperature, relative humidity, and light settings. The crop husbandry, temperature regimes, and traits recorded were the same as described below.
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Chlorophyll Content, Chlorophyll a Fluorescence, and Gas Exchange Measurement Chlorophyll fluorescence is often used to evaluate the health of the photosynthetic system in chloroplast membranes under various stresses (Chen et al., 2010). In both experiments, the various physiological traits (chlorophyll content, chlorophyll a fluorescence, and gas exchange measurements) were measured on the attached leaf of third trifoliate from the main-stem apex of three tagged plants from each treatment at Day 2 and Day 9 after the start of temperature treatments at midday (between 10:00 and 14:00 h). Chlorophyll content was measured using a self-calibrating chlorophyll meter (Soil Plant Analytical Device [SPAD], Model 502, Spectrum Technologies, Plainfield, IL). Chlorophyll a fluorescence parameters were measured using a modulated fluorometer (OS5p, OptiSciences, Hudson, NH). The minimal fluorescence (Fo), maximum fluorescence (Fm), and photochemical efficiency of PSII (Fv/Fm; ratio of variable to maximum fluorescence after dark adaptation, which represents maximum quantum yield of PSII) was measured in 30-min dark-adapted leaves. Decrease in Fv/Fm ratio indicates stress and increase in Fo/Fm ratio indicates damage to thylakoid membranes (Prasad et al., 2008a). For other fluorescence measurements, the leaves were dark adapted for 2 h, then the leaves were continuously irradiated with white actinic light to measure the initial fluorescence in leaves acclimated to irradiation (Fo¢), steady-state fluorescence yield (Fs), and maximum fluorescence yield (Fms) of irradiated leaves. By using the above parameters the following chlorophyll a fluorescence parameters were calculated: effective quantum yield of PSII (FPSII = [Fms − Fs]/Fms); apparent rate of photochemical transport of electrons through PSII (ETR = FPSII × PAR × 0.5 × 0.84), where PAR was incident photosynthetically active radiation in leaf; 0.5 corresponds to the proportion of absorbed quanta used by PSII reaction centers; and 0.84 represents the proportion of incident irradiance absorbed by leaf, (i.e., 84% of incident photosynthetically active photon flux density is assumed to be absorbed by leaves). This assumption may be reasonable for many mature green leaves (Baker, 2008). The coefficient of photochemical quenching (qP = [Fms − Fs]/[Fms − Fo]), and the coefficient of non–photochemical quenching of excitation energy (NPQ = [Fm − Fms]/Fms) was calculated by the instrument software (Van Kooten and Snel, 1999; Maxwell and Johnson, 2000). In addition, leaf-level gas exchange measurements (photosynthesis and stomatal conductance) were measured in three leaves using a LICOR 6400 portable photosynthesis system (LICOR, Lincoln, NE). Gas exchange measurements were taken at daytime growth temperature and ambient CO2 conditions (360 μmol mol−1). Constant temperature within the chamber was maintained, using the built-in software of the instrument, at the daytime growth temperature. The internal light-emitting diode (LED) light source in the LICOR 6400 was set at 1600 µmol m-2 s-1 to ensure a constant, uniform light across all measurements.
Respiration Rate Leaf-level respiration rate was measured using a LICOR 6400 portable photosynthesis system (LICOR, Lincoln, NE). Respiration rate was measured on the three attached leaf of third trifoliate from the main-stem apex between 23:00 and 01:00 h. The 1596
photosynthetic photon flux density, provided by a 6400-02 LED light source, was set at 0 µmol m-2 s-1 (dark environment). The CO2 concentration in the leaf cuvette was set at 360 µmol, and temperature was set at the nighttime minimum temperature of each treatment. Constant temperature within the chamber was the nighttime growth temperature.
Pollen Viability and Germination Pollen viability and germination was estimated in both the experiments. Pollen viability was tested using 2% (w/v) triphenyl tetrazolium chloride stain. Flowers that anthesed on Day 7 after high temperature stress (corolla opened slightly; and bright pink color) were collected from three tagged plants in each temperature regime between 09:00 and 10:00 h. Flowers collected were air-dried for 1 h in laboratory (~25°C) and pollen from a single flower was dusted on 2% triphenyl tetrazolium chloride solution by brushing anthers with a nylon hairbrush. Tetrazolium chloride stains the live pollen with reddish-purple color due to the formation of insoluble red formazan. The numbers of pollen grains stained was recorded 30 min after staining. A total of about 100 to 200 grains were counted on each slide. Pollen viability was estimated by counting the total number of pollen grains and number of stained pollen grains in a random microscopic field on each microscopic slide using a compound light microscope (Nikon Instruments Inc., Melville, NY). The percentage of viable pollen was estimated as the percentage of total pollen that was stained. Similarly, to determine pollen germination, flowers were collected as above and air-dried for 1 h in laboratory (~25°C) and pollen from each flower was dusted on germination medium by brushing anthers with a nylon hairbrush. The germination medium was prepared by dissolving 15 g of sucrose, 0.03 g of calcium nitrate, and 0.01 g of boric acid in 100 mL of deionized water (Salem et al., 2007). To this liquid, 0.5 g of agar was added and slowly heated on a hot plate. After the agar was completely dissolved, 3 mL of the germinating medium was poured on the required number of clean glass slides and allowed to cool for about 15 min to let the agar solidify. Each glass slide layered with germinating medium was kept in an empty petri dish lined with moistened filter paper to provide a humid atmosphere and incubated at 28°C for 30 min in incubator (Echotherm). A total of about 100 to 200 grains were counted on each slide. The percentage of pollen germination was estimated by counting the total number of pollen grains and number of germinated pollen grains in a random microscopic field on each microscope slide using a compound light microscope (Nikon Instruments Inc., Melville, NY). Pollen was considered germinated if the length of the pollen tube was greater than the diameter of the pollen grain (Prasad et al., 2006a).
Phospholipid Profiling in Pollen Grains At each temperature regime, flowers that that anthesed on Day 7 after high temperature stress were collected from three tagged plants between 09:00 and 10:00 h. Pollen grains were extracted and transferred to 3 mL of isopropanol with 0.01% (w/v) butylated hydroxytoluene maintained at 75°C within 15 to 20 s. The contents were kept in heating block for 15 min to inactivate the phospholipase D activity within the above said time. Then, 1.5 mL of chloroform and 0.6 mL of water were added. The tubes were shaken for 1 h, followed by removal of the extract. The pollen grains
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Table 1. Result from ANOVA (P values) on effects of high daytime and nighttime temperatures on soybean leaf and pollen response variables†. Source Experiment (E)
Temperature (T)
Time
E×T
Chlorophyll content (Soil Plant Analytical Device [SPAD] units) Thylakoid membrane damage (Fo/Fm ratio; dimensionless)
Variable
0.982 0.388
