JXB Advance Access originally published online on December 20, 2004
Journal of Experimental Botany 2005 56(412):725-736; doi:10.1093/jxb/eri044
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RESEARCH PAPER |
Interactive effects of carbon dioxide, temperature, and ultraviolet-B radiation on soybean (Glycine max L.) flower and pollen morphology, pollen production, germination, and tube lengths
1Department of Plant and Soil Sciences, 117 Dorman Hall, Box 9555, Mississippi State University, Mississippi State, MS 39762, USA
2USDA-ARS, Alternate Crops and Systems Laboratory, Bldg 001, Rm 342, BARC-W, 10300 Baltimore Ave., Beltsville, MD 20705, USA
* To whom correspondence should be addressed. Fax: +1 662 325 9461. E-mail: krreddy{at}ra.msstate.edu
Received 17 September 2004; Accepted 1 October 2004
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Abstract |
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Plant reproduction is highly vulnerable to global climate change components such as carbon dioxide concentration ([CO2]), temperature (T), and ultraviolet-B (UV-B) radiation. The objectives of this study were to determine the effects of season-long exposure to treatments of [CO2] at 360 (control) and 720 µmol mol–1 (+CO2), temperature at 30/22 °C (control) and 38/30 °C (+T) and UV-B radiation 0 (control) and 10 kJ m–2 d–1 (+UV-B) on flower and pollen morphology, pollen production, germination, and tube lengths of six soybean genotypes (D 88-5320, D 90-9216, Stalwart III, PI 471938, DG 5630RR, and DP 4933RR) in sunlit, controlled environment chambers. The control treatment had 360 µmol mol–1 [CO2] at 30/22 °C and 0 kJ UV-B. Plants grown either at +UV-B or +T, alone or in combination, produced smaller flowers with shorter standard petal and staminal column lengths. Flowers so produced had less pollen with poor pollen germination and shorter tube lengths. Pollen produced by the flowers of these plants appeared shrivelled without apertures and with disturbed exine ornamentation even at +CO2 conditions. The damaging effects of +T and +UV-B were not ameliorated by +CO2 conditions. Based on the total stress response index (TSRI), pooled individual component responses over all the treatments, the genotypes were classified as tolerant (DG 5630RR, D 88-5320: TSRI >–790), intermediate (D 90-9216, PI 471938: TSRI <–790 to >–1026), and sensitive (Stalwart III, DP 4933RR: TSRI <–1026). The differences in sensitivity identified among genotypes imply the options for selecting genotypes with tolerance to environmental stresses projected to occur in the future climates.
Key words: Carbon dioxide, pollen germination, pollen tube length, soybean, temperature, ultraviolet-B radiation
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Introduction |
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Reproduction plays an important role in the survival and succession of seeded crop plants. The onset of the reproductive phase, its duration, and the quality and quantity of reproductive products are regulated by abiotic factors. Of the various abiotic factors, atmospheric CO2 concentration ([CO2]), temperature, and ultraviolet-B (UV-B) radiation are projected to change in the near future. Current [CO2] of 360 µmol mol–1 could reach anywhere between 560 and 970 µmol mol–1 by the middle or later part of the 21st century (Houghton et al., 2001
). As a consequence of increased [CO2], the projected increase in global mean air temperature could range from 1.4 °C to 5.8 °C by 2100 (Houghton et al., 2001). Global terrestrial UV-B radiation levels range between 2 kJ and 12 kJ m–2 d–1 on a given day with near equator and mid-latitudes receiving higher doses (Total ozone mapping spectrometer 2002, http://toms.gsfc.nasa.gov/ery-uv/euv.html). This includes the 6–14% increase of UV-B radiation since the 1970s at the Earth's surface due to chlorofluorocarbons released into the atmosphere during the past few decades (UNEP, 2002). Relative to the 1979–1992 conditions, for the 2010–2020 time period, the GISS model results indicate a springtime enhancement of erythemal UV doses of up to 14% in the Northern hemisphere and 40% in the Southern hemisphere (Taalas et al., 2000).
Interactions between these climate stress factors may exacerbate the rate and direction of individual climate stress factors and their effects on terrestrial ecosystems. The yield increases experienced at elevated [CO2] (Reddy and Hodges, 2000, and references therein) were not observed when the plants were grown in combination with high temperature (Reddy et al., 1997; Wheeler et al., 2000; Prasad et al., 2003) or increased UV-B radiation (Teramura et al., 1990; Gwynn-Jones et al., 1997; Sullivan, 1997; Tosserams et al., 2001; Zhao et al., 2003). The resulting yield decrease due to high temperature and UV-B radiation could be due to the effect on reproduction at both organ and process levels. Recent in vitro studies showed that high temperature and elevated UV-B radiation inhibit pollen germination and pollen tube growth, and genotypes differ in their sensitivity (Huan et al., 2000; Kakani et al., 2002).
The climate change factors being tested in this study modify reproductive organs and processes. Elevated [CO2] and high temperature increased flower production in soybean (Nakamoto et al., 2001; Zheng et al., 2002) while elevated UV-B radiation altered cotton flower morphology, producing smaller flowers with fewer anthers (Kakani et al., 2003). Elevated [CO2] (720 µmol mol–1) increased stand-level ragweed-pollen production by 61% (Wayne et al., 2002), but did not affect peanut pollen viability (Prasad et al., 2003). By contrast, high temperature reduced pollen viability in peanut (Prasad et al., 2003). Similarly, both high temperatures (36 °C and 40 °C) and high doses of UV-B irradiation (900–1500 µW cm–2) reduced pollen viability in tall fescue (Wang et al., 2004). However, studies with Ipomea purpurea suggested that flavonoids (compounds that block UV-B radiation) alleviate the effect of heat stress on fertilization success (Coberly and Rausher, 2003). Therefore, studies are needed to provide insights into understanding and evaluating the reproductive performance of plants, so that suitable genotypes and management practices can be developed for future climates.
In reality, plants in nature are exposed to multiple environmental conditions concomitantly, and their performance can be assessed only when plants are grown in multiple stress conditions. The objectives of the research were to determine the interactive effects of atmospheric [CO2], temperature, and UV-B radiation on flower and pollen morphology, pollen production, pollen germination, and pollen tube lengths of soybean and to understand genotypic variation, if any, in the soybean response to multiple environmental stresses.
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Materials and methods |
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Experimental conditions, cultivars and plant husbandry
The study was conducted in eight sunlit Soil-Plant-Atmosphere-Research (SPAR) chambers located at the RR Foil Plant Science Research Facility of Mississippi State University (33°28' N, 88°47' W), Mississippi, USA in 2003. Each SPAR chamber consists of an upper Plexiglas unit that is 2.5 m tall by 2 m long by 1.5 m wide and a lower steel soil bin of 1 m deep by 2 m long by 0.5 m wide. The Plexiglas blocks solar UV radiation wavelengths below 385 nm, but transmits 96.6±0.5% of incoming PAR (wavelength 400–700 nm) (Zhao et al., 2003
). During the experiment, the ambient solar radiation (285–2800 nm) measured with a pyranometer (Model 4–48, The Eppley Laboratory Inc., Newport, Rhode Island, USA) was 21.2±0.5 MJ m–2 d–1. Each SPAR unit consists of a heating and a cooling system, and an environment monitoring and control system. The SPAR facility has the capability to control temperature and [CO2] precisely at predetermined set points for plant growth studies under near ambient levels of photosynthetically active radiation (PAR). Details of the operation and control of SPAR chambers have been described by Reddy et al. (1992, 2001).
Six soybean genotypes, Delsoy (D) 88-5320 (maturity group VI, non-glyphosate-tolerant), and D 90-9216 (maturity group VII, non-glyphosate-tolerant), Stalwart III (maturity group III, non-glyphosate-tolerant), Plant Introduction (PI) 471938 (maturity group V, non-glyphosate-tolerant), Deltagrow (DG) 5630RR (maturity group V, glyphosate-tolerant), and Deltapine (DP) 4933RR (maturity group IV, glyphosate-tolerant) were sown on 5 August 2003 in pots filled with fine sand. Thirty 2.5 l pots (five pots for each genotype) were arranged randomly in each SPAR chamber. Plants were watered three times a day with half-strength Hoagland's nutrient solution (Hewitt, 1952) delivered at 08.00, 12.00, and 16.00 h to ensure favourable nutrient and water conditions for plant growth through an automated and computer-controlled drip system. Variable-density black shade cloths around the edges of plants were adjusted regularly to match plant height in order to simulate natural shading in the presence of other plants.
Treatments
The chambers were maintained at temperatures of 30/22 °C (day/night) up to 10 d after sowing (DAS). Thereafter, each chamber was set at one of the eight treatments until 60 DAS. The treatments included combinations of two CO2 levels of 360 µmol mol–1 and 720 µmol mol–1 (+CO2), two levels of temperature 30/22 °C and 38/30 °C (+T) and two daily biologically effective UV-B radiation intensities of 0 and 10 (+UV-B) kJ m–2 d–1. Control treatment consists of 30/22 °C, 360 µmol mol–1 [CO2], and 0 kJ UV-B treatments. Air temperature in each SPAR chamber was monitored and adjusted every 10 s throughout the day and night and maintained within ±0.5 °C of the treatment set points measured with shielded, aspirated thermocouples. The daytime temperature was initiated at sunrise and returned to the night-time temperature 1 h after sunset. Constant humidity was maintained by operating solenoid valves that injected chilled water through the cooling coils located outside the air handler of each chamber. These cooling coils condensed excess water vapour from the chamber air in order to regulate the relative humidity at 60% (McKinion and Hodges, 1985).
The chamber [CO2] was measured with a dedicated infrared gas analyser (Li-Cor, model LI-6252, Lincoln, Nebraska, USA) from the gas sample that was drawn through the lines run underground from SPAR units to the field laboratory building. Moisture was removed from the gas sample by running the sample lines through a refrigerated water tap (4 °C) that was automatically drained and through a column of magnesium perchlorate. Chamber [CO2] was maintained by supplying pure CO2 from a compressed gas cylinder through a system that includes a pressure regulator, solenoid and needle valves, and a calibrated flow meter (Reddy et al., 2001).
Square-wave UV-B supplementation systems were used to provide the required UV-B radiation in this study under near ambient PAR. The UV-B radiation was delivered to plants for 8 h d–1, from 08.00 h to 16.00 h by eight fluorescent UV-313 lamps (Q-Panel Company, Cleveland, Ohio, USA) powered by 40 W variable dimming ballasts. The lamps were wrapped with presolarized 0.07 mm cellulose diacetate film to filter UV-C (<280 nm) radiation. The cellulose diacetate film was changed at 3–4 d intervals. The UV-B energy delivered at the top of the plant canopy was checked daily at 09.00 h with a UVX digital radiometer (UVP Inc., San Gabriel, California, USA) and calibrated against an Optronic Laboratory (Orlando, Florida, USA) Model 754 Spectroradiometer, which was used initially to quantify lamp output. The lamp output was adjusted, as needed, to maintain the respective UV-B radiation levels. A distance of 0.5 m from lamps to the top of plants was always maintained throughout the experiment. The actual biologically effective UV-B radiation was measured during the crop growth period at six different locations in each SPAR unit corresponding to the pots arranged in rows. The weighted total biologically effective UV-B radiation levels received at the top of the plants were 9.8±0.16 for +UV-B, 9.6±0.07 for +T+UV-B, 9.6±0.07 for +CO2+UV-B, and 9.5±0.10 kJ m–2 d–1 for +CO2+T+UV-B treatments using the generalized plant response action spectrum normalized at 300 nm (Caldwell, 1971).
Measurement of floral morphology
Soybean, belonging to the family Fabaceae and subfamily Papilionoideae, has a flower with a tubular calyx of five unequal sepal lobes and a five-parted corolla consisting of a posterior standard petal, two lateral wing petals, and two anterior keel petals in contact with each other but not fused (Carlson and Larsten, 1987). Lengths of flower, standard petal, and staminal column were measured on 20 fresh flowers randomly picked from five plants of each genotype, 60 DAS. Flower length was measured from the tip of the standard petal to the base of the calyx. The standard petal was stretched out before measuring the length, and the length was measured from the point of insertion to the distal end. The staminal column was separated from the flower and the length was measured.
Measurement of pollen number
Mature anthers were collected from five different flowers from five plants a day before anthesis to determine the number of pollen grains produced per anther, 60 DAS. Pollen number was counted by placing a single anther in a water drop on a glass slide and squashed with a needle, and the pollen grains dispersed in the drop of water were counted (Bennett, 1999). The number of pollen grains from five anthers per genotype under each treatment was counted.
Measurement of pollen germination and pollen tube lengths
Pollen germination was determined through in vitro pollen germination on a medium consisting of 15 g sucrose (C12H22O11), 0.03 g calcium nitrate [Ca(NO3)2· 4H2O], and 0.01 g boric acid (H3BO3) dissolved in 100 ml of deionized water (Gwata et al., 2003, with modifications). To this liquid medium, 0.6 g agar was added and slowly heated on a hot plate. After the agar was completely dissolved, 10 ml of the germinating medium was poured into five Petri dishes for each genotype in each treatment and allowed to cool for about 15 min so that the agar would solidify. Soybean flowers were randomly collected from five plants in each genotype in the morning between 09.00 h and 10.00 h 55–60 DAS, air-dried for 2 h and pollen from each flower was dusted onto the germination medium to allow a good uniform distribution of grains on the surface of the medium (Salem et al., 2004). The plates were then covered and incubated at 30 °C (Precision Instruments, New York, USA) for 3 h. Pollen grains were counted for pollen germination (five fields in each Petri dish), and a pollen grain was considered germinated when its tube length equalled the grain diameter (Luza et al., 1987) using a microscope (Nikon SMZ 800 microscope, Nikon Instruments, Kanagawa, Japan) with a magnification of 6.3x. Percentage pollen germination was calculated by counting the total number of pollen grains, an average of 140 pollen grains, and germinated pollen in each Petri dish. Pollen tube length was measured from 20 pollen tubes randomly selected from each Petri dish using a microscope. The lengths were measured with an ocular micrometer fitted to the eyepiece of the microscope. A total of 100 pollen tubes were measured for each treatment.
Cumulative stress response index and total stress response index
The cumulative stress response index (CSRI) is calculated as the sum of the relative individual component responses at each treatment and is similar to the combined response index quoted in other UV-B studies (Dai et al., 1994). CSRI was calculated to evaluate the reproductive response of soybean to the treatments under study (+CO2, +T, +UV-B, +CO2+UV-B, +CO2+T, and +CO2+T+UV-B). The CSRI was calculated as follows:
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Pollen morphology
Based on the TSRI, two genotypes (DP 5630RR and DP 4933RR) with low and high TSRI values were selected for pollen morphological studies. For this study, fresh flowers collected between 19.00 h and 21.00 h, 1 d before anthesis and stored in FAA (formaldehyde:glacial acetic acid:ethyl alcohol) solution were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2 overnight at 4 °C for SEM. After fixation, specimens were rinsed in buffer, post-fixed in 2% osmium tetroxide in 0.1 M phosphate buffer for 2 h, then rinsed in distilled water, dehydrated in an ethanol series, and critical point dried in a Polaron E 3000 Critical Point Dryer (Quorum Technologies, Newhaven, UK). Specimens were mounted on aluminium stubs, sputter-coated with gold in a Polaron E 5100 sputter coater (Quorum Technologies), and viewed in a LEO Stereoscan 360 SEM (LEO Electron Microscopy, Thornwood, NY, USA) at an accelerating voltage of 15 kV. Images were recorded on Polaroid Type 55 film (Polaroid, Cambridge, Massachusetts, USA).
Data analysis
To test the significance of atmospheric [CO2], temperature, UV-B radiation, and genotypes, and their interactive effects on flower morphology, pollen number per anther, pollen germination percentage, and pollen tube lengths, data were statistically analysed using analysis of variance (ANOVA) by Genstat 6 for Windows (Genstat 6 Committee, 1997). Pollen germination percentage data were transformed using the arcsin transformation before the analysis. The least significant difference (LSD) tests at P=0.05 were employed to distinguish treatment difference means of the parameters measured in this study. Genotypes were classified based on TSRI of all the treatments as tolerant (>minimum TSRI –1 standard deviation (sd)), intermediate (>minimum TSRI –2 sd and <minimum TSRI –1 sdv), and sensitive (<minimum TSRI – 2 sd).
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Results |
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Floral morphology
Plants grown under +UV-B conditions showed reduced standard petal lengths either alone or in combination with +T and +CO2 conditions (Fig. 1A). Similar responses were observed for staminal column lengths. The standard petals were significantly larger in the flowers produced in the plants grown at control and +CO2 treatments. Significant [CO2]xT, [CO2]xUV-B, and [CO2]xTxUV-B interactions were observed for standard petal length and the TxUV-B interaction was significant for staminal column lengths (Table 1). Significant differences between genotypes were observed in their response to these stressors. Flowers of DP 4933RR had significantly shorter staminal columns under +UV-B, +CO2+UV-B, +T+UV-B, and +CO2+T+UV-B conditions compared with the control. The reductions in staminal column with the +CO2+T+UV-B treatment ranged from 28% (D 90-9216) to 81% (DP 4933RR) (Fig. 1B) compared with the control.
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Similarly, plants grown under UV-B in combination with either +CO2 or +T or both produced smaller flowers with 31–38% less flower length than those produced under control conditions (Fig. 2A). Flowers produced on plants grown under elevated [CO2] conditions had significantly longer flower lengths in most of the genotypes, while temperature had no effect on flower length in all the genotypes, whereas UV-B radiation had significant effects on the flower length with significant genotypic differences (Table 1). Averaged across all the genotypes, UV-B decreased flower lengths by 28%. Significant [CO2]xUV-B and TxUV-B interactions were observed for flower length (Table 1).
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Pollen production
Significant TxUV-B and [CO2]xTxUV-B interactions were observed in the number of pollen produced per anther. Plants grown under +T+UV-B and +CO2+T+UV-B treatments produced significantly less pollen. In DG 5630RR, the 20% more pollen produced for plants grown at +CO2 was not observed when temperatures and UV-B were increased as well as [CO2]. Compared with control plants, pollen production in plants grown in the +CO2+T+UV-B treatment was significantly less, and reduced by 68% (DG 5630RR) and 18% (PI 471938) (Fig. 2B). When averaged over the genotypes, pollen production was reduced by about 30–50% with +T, +CO2+T, +T+UV-B, and +CO2+T+UV-B treatments. Genotypes varied significantly in their response to elevated [CO2] for pollen production. Elevated [CO2] either increased (7% and 20% in DG 5630RR and PI 471938, respectively), decreased (15% and 17% in D 88-5320 and DP 4933RR, respectively) or had no effect (D 90-9216 and Stalwart III) (Table 1). On the other hand, +T and +UV-B significantly decreased pollen production.
Pollen germination and tube lengths
Significant [CO2]xUV-B, TxUV-B, and [CO2]xTxUV-B interactions for pollen germination percentage and [CO2]xT, [CO2]xUV-B, and TxUV-B interactions for pollen tube length were observed. Maximum pollen germination percentage was observed at either control conditions (92, 86, and 92% for PI 471938, DG 5630RR, and DP 4933RR, respectively) and at +CO2 conditions (88, 93, and 82% for D 88-5320, D 90-9216, and Stalwart III, respectively). Both temperature and UV-B reduced pollen germination drastically when imposed alone or in combination with [CO2]. Temperature had a tremendous influence on pollen germination in D 90-9216, where more than 90% reduction was observed at +T, +CO2+T, +T+UV-B, and +CO2+T+UV-B treatments. Pollen produced under +CO2+T+UV-B conditions, had reductions of 73 (D 88-5320) and 96% (D 90-9216) in pollen germination percentage (Fig. 3A).
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Pollen produced with the +CO2+T+UV-B treatment had significantly shorter tube lengths, ranging from 126 µm (DG 5630 RR) to 235 µm (D 90-9216) (Fig. 3B). Pollen produced for plants grown under +CO2 treatment, when germinated, had longer pollen tubes than that of the control pollen and the tube lengths ranged from 223 µm (DP 4933RR) to 325 µm (D 90-9216). Genotypes varied significantly in their response to these treatments (Table 1).
Cumulative stress response index
CSRI, which is the sum of individual component relative responses, showed that soybean is sensitive to both temperature and UV-B radiation, and the impact is much more when they occur together. Most of the genotypes had positive CSRI under +CO2 condition, but when plants were grown under +CO2 in interaction with negative stressors such as +T and +UV-B radiation, the responses were negative in all the genotypes (Table 2). Out of all the treatments, averaged over all the genotypes, the highest negative CSRI was observed at both +T+UV-B (–184.2) and +CO2+T+UV-B (–206.3) compared with +T (–126.2) and +UV-B (–146.9) treatments. The reproductive ability of the plants was reduced in these treatments. Plants produced smaller flowers producing less pollen and with poor germination and tube growth. Based on TSRI, the total response over the treatments (Table 2), the genotypes were classified: DG 5630RR and D 88-5320 as tolerant (TSRI > –790); D 90-9216 and PI 471938 as intermediate (TSRI –790 to –1026); Stalwart III and DP 4933RR as sensitive (TSRI < –1026) (Table 2).
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Pollen morphology
Based on the CSRI, two genotypes, one tolerant (DG 5630RR) and one sensitive (DP 4933RR) were studied for pollen morphology. No morphological differences in the pollen grown under control conditions were visible between the sensitive and tolerant genotypes. Pollen abnormalities, however, were observed in both the genotypes with increasing temperatures and UV-B. These morphological differences were similar at both ambient and elevated [CO2] conditions, and therefore only differences under ambient [CO2] in combination with other environmental factors are presented. The abnormalities at +T, +UV-B, and +T+UV-B were more evident in DP 4933RR compared with DG 5630RR. A large proportion of the pollen was shrivelled under the +T+UV-B treatment compared with the pollen from plants grown under control conditions (Fig. 4D, H). There were no apertures on the pollen grains from +UV-B and +T plants (Fig. 4B, C, D, F, G, H), while the pollen produced in plants grown under control conditions were triporate, i.e. they had three protruding apertures (Fig. 4A, E). The abnormalities were more evident in the sensitive genotype rather than in the tolerant genotype. Differences in pollen exine structure were also observed (Fig. 5A–H). The columellae heads of the exine were less at +T and +UV-B treatments in both sensitive and tolerant genotypes, and the differences in the columellae heads were more pronounced with the +T+UV-B treatment. Both sensitive and tolerant genotypes had an altered appearance of the exine, while on sensitive genotype the abnormalities were more evident.
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Discussion |
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This is the first report to show that there were no beneficial interactions between the three important global change factors ([CO2], temperature, and UV-B radiation) on the reproductive processes of soybean. Flower morphology, pollen production, pollen germination, pollen tube lengths, and pollen morphology were all negatively affected by +T and +UV-B treatments alone or in combination at both control and +CO2. Similar to these results, previous investigations on the interactive effects of temperature and [CO2] on other legumes (Ahmed et al., 1992
; Prasad et al., 2002, 2003) showed that the positive interactions observed between [CO2] and temperature on vegetative growth cannot be translated to reproductive processes. Significant negative correlation between pollen production and temperature were found in groundnut (Prasad et al., 1999). Lower seed yield at high temperatures under both ambient and elevated [CO2] conditions was shown to be due to decreased pollen viability in groundnut and bean (Prasad et al., 2002, 2003).
Smaller flowers with smaller flower-component parts such as standard petal and staminal column lengths were observed in all the genotypes for plants grown in treatment conditions where +T or +UV-B were involved either alone or in combination. Similarly, flowers produced under these treatments also had reduced pollen production even under elevated [CO2] conditions. Kakani et al. (2003) showed similar flower length reductions in cotton under enhanced levels of UV-B radiation. Prasad et al. (1999) found strong negative linear relationships between day temperatures over the range of 28–48 °C and fruit number, fruit set, pollen production, and germination in peanut. When +T and +CO2 acted together, the combined effect on pollen number and pollen germination was additive, being higher than the single stress effects. This concurs with the studies of Prasad et al. (2002, 2003), where they showed that elevated [CO2] did not counteract the negative effects of high temperature on pollen production and viability.
High temperatures during micro and macrosporogenesis reduced pollen viability and pollen number (Prasad et al., 1999) at day temperatures >33 °C. Talwar and Yanagihara (1999) have also shown that day/night temperatures of 35/25 °C increased the length of the hypanthium and reduced the rate of pollen tube growth in heat-susceptible genotypes of groundnut when compared with 25/20 °C, both these factors reduce the chances of successful fertilization. In this study, significant reductions in pollen germination in all the genotypes were observed. It seems very likely, however, that pollen germination is affected by environmental stresses during the pre-anthesis period because this is the time during which development of the anther and pollen grains occur. Prasad et al. (2002) showed that groundnut flowers were most sensitive to high temperature during microsporogenesis and anthesis. Similar pollen germination inhibitions at high temperatures (Peet et al., 1998; Prasad et al., 2002, 2003; Cross et al., 2003; Young et al., 2004) and at higher UV-B levels (Demchik and Day, 1996; Torabinejad et al., 1998; Musil et al., 1999; Feng et al., 2000) were previously reported. Microspore meiosis occurs 9 d before anthesis and differentiation of the stomium, tapetum, middle layers, and endothecium occurs before meiosis (Goldberg et al., 1993). If the differentiation processes of those organs are sensitive to high temperature and UV-B radiation, then pollen viability could well be affected since the stomium is important for anther dehiscence (Koltunow et al., 1990). Pollen sterility and pollen production at high temperatures may also be associated with early degeneration of the tapetal layer of pollen (Porch and Jahn, 2001). The exact physiological reasons of pollen viability loss are not clearly known and need further investigation.
This study also demonstrated that the morphology of pollen was affected when plants were subjected to high temperatures and enhanced UV-B radiation alone or in combination with either control or +CO2 conditions, which resulted in flattened and collapsed microspores. The pollen produced under control conditions were equipped with three apertures that act as exits for the germinating pollen and also allows recognition of protein signals, which initiates development of a pollen tube (Heslop-Harrison, 1971). These apertures were missing in pollen produced under both high temperature and high UV-B radiation treatments. In addition, high temperature and enhanced UV-B had a marked effect on the exine ornamentation of both sensitive (DP 4933RR) and tolerant (DG 5630RR) genotypes. The pollen morphological aberrations observed at these climate change stress factors might have resulted in poor pollen germination and shorter tube lengths in the sensitive genotypes. High temperature stress (Cross et al., 2003) and water deficit stress (Shen and Webster, 1986) in Phaseolus vulgaris showed microsporogenesis to be the most detrimental process during reproductive development, resulting in abnormal exine with deeply pitted and smooth regions. The exine originates from the tapetum (Dickinson and Potter, 1976) and normal pollen development depends on a close interaction with the tapetal tissue that composes the innermost layer of the anther. Although premature degeneration of the tapetal layer (Ahmed et al., 1992) could explain the abnormalities observed. All these and other unknown factors might have contributed to the poor pollen germination observed in plants grown under high temperatures, and enhanced UV-B radiation. The stress-induced flower and pollen morphological changes may need further investigation.
These data also showed genotypic variation in the reproductive response to the environmental factors studied. Even though the direction of the stress effect was the same for all the six genotypes, the genotypes varied in the magnitude of their response. Based on TSRI, the six genotypes were classified into tolerant, intermediate, and sensitive. The differences can be due to different adaptive or defensive mechanisms to high temperatures and UV-B radiation, such as the inherent capacity of repair systems such as membrane stability of genotypes.
In summary, there were no beneficial interactions between [CO2], temperature, and UV-B radiation on reproductive developmental processes such as pollen production, germination, and tube lengths of soybean. Temperatures of 38/30 °C and UV-B radiation levels of about 10 kJ m–2 d–1 alone or in combination at both the control and +CO2 conditions produced smaller flowers and fewer pollen grains per flower together with lower pollen germination. In addition, pollen grains produced at these high temperature and UV-B treatments were shrivelled resulting in further deterioration in pollen germination. The clear effect of high temperature and UV-B radiation on pollen production and pollen grain germination will have major implications on the fertilization process and fruit set in sensitive crops under future climates. In conclusion, pollen germination can be used as a screening tool for selecting cultivars for high temperature and UV-B radiation environments as it is more responsive to those variables. Variability among soybean genotypes in their responses to [CO2], temperature, and UV-B radiation indicates the possibility of selecting soybean cultivars for higher yields and/or stability in a warmer temperature and higher UV-B, most likely in a future higher CO2 world. However, the genetic bases for these differences must be further examined.
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Acknowledgements |
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The project was funded in part by the USDA UV-B Monitoring Program and the RSTC-NASA. We thank D Brand, K Gourley, and W Ladner for their technical support and Bill Monroe, Electron Microscopy Center for his help during SEM studies. We thank Drs P Meints, B Sampson, and A Teramura for reviewing the manuscript and for their helpful comments. We acknowledge Drs R Smith and T Carter for providing the seed of the genotypes used in this study. Contribution from the Department of Plant and Soil Sciences, Mississippi State University, Mississippi Agricultural and Forestry Experiment Station paper no. J10509.
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References |
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