Crop Science 41:1351-1355 (2001)
© 2001 Crop Science Society of America
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Evaluation of a ninhydrin procedure for measuring membrane thermostability of wheat
Cemal Cekica and
Gary M. Paulsen*,b
a Transitional Zone Agric. Res. Inst., Adana, Turkey
b Dep. of Agronomy, Kansas State Univ., Manhattan, KS 66506-5501
* Corresponding author (gmpaul{at}ksu.edu)
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ABSTRACT
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Membrane thermostability (MT) is used widely to measure high-temperature tolerance of wheat (Triticum aestivum L.). However, the method, which measures conductivity of electrolytes released from injured cells, is affected by environmental factors for growing plants and is not effective on maturing plants. Amino acids exosmose like inorganic electrolytes from cells that are damaged by high temperature but, unlike electrolytes, typically occur only in living cells. These studies determined whether measuring leakage of amino acids effectively assessed high-temperature injury to wheat in comparison with the conductivity method, and if it was suitable for evaluating maturing plants. Membrane thermostability was measured by conductivity and ninhydrin methods on seedlings and maturing plants grown under controlled conditions and on 12 genotypes at two field locations. Relative injury (RI) values from hardened seedlings differed significantly among 12 genotypes. Rankings of the genotypes were similar for both methods in seedlings (r = 0.94), and values of both methods were significantly correlated in maturing plants (r = 0.90). The RI values by the ninhydrin method but not the conductivity method differed significantly among the 12 genotypes and identified the top-yielding cultivars at both field locations, but results of neither method correlated with grain yields. The results showed that the ninhydrin method measures high-temperature injury of young plants as successfully as the conductivity method, but neither method is suitable for maturing plants.
Abbreviations: MT, membrane thermostability • RI, relative injury
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INTRODUCTION
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MEMBRANE THERMOSTABILITY is used widely as a measure of high-temperature tolerance of crops because adaptation to stress is associated with viability of cells (Gusta and Chen, 1987). Membrane thermostability usually is determined by measuring conductivity of electrolytes that leak from leaves subjected to high temperature (Saadalla et al., 1990). Blum and Ebercon (1981) proposed that hardened winter wheat seedlings could be utilized to determine genetic differences in MT, which were associated with field performance of other crops under high temperature conditions (Martineau et al. 1979; Sullivan and Ross, 1979). Shanahan et al. (1990) showed that tolerant lines selected for MT had a yield advantage in high temperature regions. Although the procedure held promise for selecting high temperature-tolerant germplasm, some problems were associated with the method (Saadalla et al., 1990). Genetic differences in MT depended on environmental factors and the type of hardening treatment or temperature to which plants were exposed prior to sampling (Blum and Ebercon, 1981; Chen et al., 1982). The method also was ineffective on maturing plants (Saadalla et al., 1990), possibly because nonviable cells in senescing tissues contain electrolytes that interfere with measurements of living cells (Siminovitch et al., 1964).
Maintenance of cell membrane integrity at high temperature varied among wheat genotypes (Blum and Ebercon, 1981; Saadalla et al., 1990; Porter et al., 1995). Blum and Ebercon (1981) found no significant differences in MT among field-grown bread wheat, durum wheat (Triticum durum Desf.), triticale (x Triticosecale Wittmack), and barley (Hordeum vulgare L.) as a group. Galiba et al. (1997), however, indicated that MT of durum wheat was significantly greater than MT of bread wheat. The association between rankings of MT of spring wheat genotypes grown under two different field conditions was low, and rankings of MT of field-grown wheat and seedlings also differed (Saadalla et al., 1990). Saadalla et al. (1990) also found that yields of high temperature-tolerant, intermediate, and sensitive wheat cultivars grown at two different environments were similar.
Free amino acids exosmose like inorganic electrolytes from cells that are damaged by high temperature or other stresses, but unlike inorganic electrolytes, typically occur only in living cells (Thimann, 1987). Siminovitch et al. (1964) found that leakage of free amino acids from black locust (Robinia pseudoacacia L.), alfalfa (Medicago sativa L.), and wheat correlated significantly with freezing injury to all three species. They suggested that measuring exosmosis of amino acids from living cells instead of conductivity of electrolytes might give a more accurate indication of injury. The method has never been applied to measurement of high temperature injury or to maturing plants, however, where it might be most effective. The objectives of this study were to determine the feasibility of measuring amino acid exosmosis by ninhydrin as a test for high temperature injury to wheat and evaluate the suitability of the method for use on maturing plants.
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Materials and Methods
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Evaluation of Seedling Plants
Two sets of seedlings of 12 wheat cultivars were prepared, one for a hardening treatment and the other as a control. Twenty-five foundation class seeds of each cultivar (Big Dawg, TAM 107, Karl 92, KS95HW62-6, Scout 66, Coronado, KS85W663, KS84063-2W, 2163, Dominator, 2137, and Jagger) were wrapped in paper towels and germinated in darkness at 15°C for each treatment. When the first leaves were approximately 4 cm long after 10 to 12 d, one set of 25 seedlings of each cultivar was hardened under room lighting (about 80 µmol m-2s-1) for 48 h by immersing the roots in a water bath set at 34°C and covered with transparent plastic to maintain a uniform temperature and high humidity (Saadalla et al., 1990), and the other set of 25 seedlings was held at 15°C. Membrane thermostability was measured by the conductivity and ninhydrin methods on duplicate 0.5-g subsamples of leaf blades from each set of seedlings. A randomized complete block design with four replications of 25 seedlings in all treatments was used.
Evaluation of Maturing Plants
Foundation class seed of three winter wheat cultivars from different regions [TAM 107 (Texas), Jagger (Kansas), and Scout 66 (Nebraska)] was germinated in vermiculite moistened with 0.1-strength Hoagland solution (Hoagland and Arnon, 1950). One-week-old seedlings were vernalized at 5°C and 12-h photoperiod for 5 wk. Two vernalized seedlings of each cultivar were transplanted into individual 16-cm-diam pots containing 2 kg of equal volumes of peat, sand, and soil [Kahola silt loam (fine-sity, mixed mesic Cumulic Hapudolls)] and fertilized with 120 mg kg-1 N and 40 mg kg-1 P. Plants were watered daily and grown to anthesis in a greenhouse at 20/15°C. Sunlight was supplemented by high pressure sodium lamps to maintain a 14-h day/10-h night photoperiod and a minimum irradiance of about 600 µmol m-2 s-1. An additional 80 mg kg-1 N was added to each pot at the blooming stage. Humidity was not controlled. One week after 50% of the plants flowered, the pots were placed randomly into growth chambers (Conviron PGW 36, Asheville, NC) at 20/15 and 32/27°C. Relative humidity was set at 50% during daytime and 70% during nighttime. A 16-h photoperiod was maintained, and irradiance was 980 µmol m-2 s-1 at the top of the plants.
Membrane relative injury and grain growth were measured on separate two-plant samples from each treatment when the differential temperatures were imposed; 1, 2, 3, 4, and 5 wk after anthesis at 20/15°C; and 1, 2, and 3 wk after anthesis at 32/27°C, which caused the plants to mature early. Plant flag leaves were used for measuring relative injury by the conductivity and ninhydrin methods. Grain was thrashed manually from the spikes, dried at 60°C for 48 h, and weighed. Treatments were arranged in a split split-plot design with three replications. Temperature treatments were main plots, cultivars were subplots, and sampling times were sub-subplots. A total of 36 and 24 plants of each cultivar were sampled at the low and high temperatures, respectively, during the experiment (2 plants sample-1 x 6 and 4 sampling dates x 3 replications).
Evaluation of Field-Grown Plants
The 12 winter wheat cultivars used for the seedling experiment were grown at Manhattan and Hutchinson, KS, during the 1997-1998 season. At Manhattan, the soil type was a Reading silt loam (fine-silty, mixed, mesic Cumulic Hapudells), the planting date was 7 Sept. 1997, the seeding rate was 280 seeds m-2, and fertilizer rates were 84 kg N ha-1 and 30 kg P ha-1 at planting and 60 kg N ha-1 on 10 March 1998. At Hutchinson, the soil type was an Ost silt loam (mixed, thermic typic Arguistolls), the planting date was 18 Sept. 1997, the seeding rate was 220 seeds m-2, and fertilizer rates were 90 kg N ha-1 and 48 kg P ha-1 at planting and 60 kg N ha-1 on 16 March 1998. Plot dimensions were 1.4 by 4 m at Manhattan and 1.5 by 8.8 m at Hutchinson. Randomized complete block designs with four replications were used at both locations.
One week after 50% of the plants flowered on 18 May and 20 May 1998 at Manhattan and Hutchinson, respectively, 15 flag leaf blades were sampled from each plot. The leaves were placed in plastic bags containing moist paper towels to prevent desiccation, immediately transported to the laboratory, and cut into 1-cm segments. The relative injury of all samples was measured by the conductivity and amino acid methods as described below.
Wheat cultivars matured early. Areas of 1.4 by 3 m and 1.5 by 7.8 m were harvested with a plot combine at Manhattan and Hutchinson on 3 July and 17 June 1998, respectively. Yields were calculated at 125 g moisture kg-1 grain.
Favorable conditions contributed to excellent stands in the fall at both locations. Spring conditions were near ideal except for a slight deficiency of moisture in April and May. Extremely hot winds on May 29 and 30 rapidly senesced flag leaves, and only one sampling was made from each location. Soil water contents between 0- and 60-cm depths were low, 163 and 149 g kg-1 at Manhattan and Hutchinson, respectively, on the dates that plants were sampled. Temperature was near normal during spring, except for extremely high temperatures during late May and early June, which shortened the grain-filling period.
Membrane Thermostability Assays
Leaf blades were cut into 1-cm-long sections immediately after sampling for all studies. Two 0.5-g leaf samples were placed into vials, one for a control and the other for a heat treatment. The samples were washed three times with distilled water to remove electrolytes adhering to leaves or released from the cut ends of the tissues. Distilled water (10 mL) was added to each vial. Control vials were covered and held in a water bath at 25°C, and treatment vials were heated at 50°C for 1 h. Both control and treatment vials were held at 10°C for 24 h to allow exosmosis and then warmed to room temperature and gently mixed. Electrolytes were measured directly by an electrical conductivity meter (Model 32, YSI, Yellow Springs, OH), and free amino acids were measured with ninhydrin. A 0.1-mL aliquot from each vial was transferred into a 10-mL Pyrex tube, and 0.9 mL of distilled water and 1 mL of ninhydrin reagent (0.70 M H2PO4, 0.30 M Na2HPO4, 0.03 M ninhydrin, and 0.02 M fructose) (Rosen, 1957) were added. The tubes were heated in boiling water for 10 min and cooled to room temperature, and the contents were diluted with 5 mL of a solution containing 0.01 M KIO3 and 400 mL 950 mL ethanol L-1. Absorbance of the samples was measured at 570 nm on a spectrophotometer (Hitachi U-1100, Tokyo, Japan). After the 0.1-mL aliquots were removed, samples were boiled for 15 min to kill plant tissues and then held at 10°C for 24 h. The conductivity and ninhydrin assays were repeated as described above to determine total electrical conductivity and total free amino acid content. Membrane thermostability was expressed as relative injury (RI) (Martineau et al., 1979):
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where T1 is the conductivity or absorbance value for treated samples heated at 50°C, C1 is the value for control samples held at 25°C, and T2 and C2 are values for treated and control samples, respectively, after boiling.
Statistical Analysis
Least significant differences (P = 0.05) in RI among cultivars were calculated by analysis of variance (SAS Institute, Cary, NC). Phenotypic relationships among RI values by the conductivity and amino acid methods and grain yields were determined by Spearman correlation analyses.
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Results
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Seedling Plants
The RI values of nonhardened seedlings averaged 91.0% for the conductivity method and 88.0% for the ninhydrin method and were similar for all 12 cultivars (Table 1). The RI values of hardened seedlings ranged from 35.4 to 58.8% with a mean of 46.5% for the conductivity method and from 34.7 to 55.7% with a mean of 43.5% for the ninhydrin method. Values differed significantly among the 12 cultivars (P < 0.05) after hardening and were always significantly lower than values for nonhardened seedlings. Rankings of the hardened cultivars were similar for the two methods (r = 0.94, P < 0.01). Hardened seedling of KS85W663 Exp. and Big Dawg had high RI values by the conductivity and ninhydrin methods, respectively, whereas Dominator had low RI values by both methods.
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Table 1. Relative high-temperature injury (RI) of nonhardened and hardened seedlings of 12 wheat cultivars measured by conductivity and ninhydrin methods.
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Maturing Plants
Relative injury and grain growth were similar (P > 0.05) at each temperature during maturation of the three cultivars, and only mean values are shown (Table 2). Membrane injury averaged 95.8% for the conductivity method and 93.8% for the ninhydrin method over all dates when the plants were grown at 20/15°C. Mean RI values of 69.9% for the conductivity method and 59.0% for the ninhydrin method for plants grown at 32/27°C were significantly lower (P < 0.01) than for plants at the lower temperature. The RI values measured by the conductivity and ninhydrin methods were correlated significantly in maturing plants (r = 0.90, P < 0.05) as they were in the seedlings.
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Table 2. Mean relative high-temperature injury (RI) measured by conductivity and ninhydrin methods and grain yield of Jagger, TAM 107, and Scout 66 wheat cultivars grown at 20/15 and 32/27°C during maturation.
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High temperature greatly accelerated senescence and advanced physiological maturity of the three cultivars (Table 2). Grain filling ceased after 3 wk at 32/27°C and 5 wk at 20/15°C, and yield per spike differed more than 3-fold between the two treatments at maturity. None of the RI measurements correlated significantly with grain growth or final yields.
Field-Grown Plants
The RI values determined by the conductivity method were similar for all cultivars and averaged 87.1% at Manhattan. At Hutchinson, values ranged from 79.6% for Big Dawg to 100% for Karl 92, with a mean of 89.3% (P < 0.05) (Table 3). The RI determined by the ninhydrin method differed significantly among cultivars at both locations (P < 0.05); values ranged from 79.1 (2137) to 94.4% (Dominator) with a mean of 86.7 at Manhattan, and from 71.0 (Jagger) to 88.1% (Karl 92) with a mean of 81.4% at Hutchinson.
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Table 3. Relative injury (RI) measured by conductivity and ninhydrin methods and grain yield of 12 wheat cultivars grown at Manhattan and Hutchinson, KS, during 1998.
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Grain yield differed significantly (P < 0.01) among the 12 wheat cultivars and ranged from 1908 to 4096 kg ha-1 at Manhattan and from 2509 to 3900 kg ha-1 at Hutchinson (Table 3). Yields and rankings of the 12 cultivars were similar and highly correlated (r = 0.63) (P < 0.01) between the two locations. Cultivar 2137 yielded high at Manhattan, Jagger yielded high at Hutchinson, and Scout 66 yielded low at both locations. Test weights also differed significantly among the 12 cultivars (P < 0.05) (data not shown) and correlated highly (r = 0.44) (P < 0.01) between the two locations. Relative injury values of the 12 cultivars by the conductivity and ninhydrin methods were not correlated with each other or with grain yields at either Manhattan or Hutchinson.
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Discussion
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The ninhydrin method measured injury to young plants as successfully as the conductivity method and, as shown here, would be a valuable alternative that is indicative of damage to living cells. Similar rankings among hardened seedlings of the 12 wheat cultivars and the high correlation between the two methods indicated that the ninhydrin method accurately measured high-temperature injury. The results demonstrated that amino acids exosmose like inorganic electrolytes when cells are damaged by high temperature as they do when cells are damaged by low temperature (Siminovitch et al.,1964). Mean RI values of the two methods of approximately 50% for hardened seedlings were suitable for determining genetic differences among cultivars (Chen et al., 1982). During maturation, when plants were senescing, measuring amino acids would be more valid, at least in theory, than measuring inorganic electrolytes (Thimann, 1987). Although it detected differences among cultivars, the ninhydrin method appeared to suffer some of the same deficiencies as the conductivity method on maturing plants.
The conductivity and ninhydrin methods both successfully measured injury on young plants, because membrane-associated processes were most vulnerable to high temperature at that stage. Photosystem II, which is associated with thylakoid membranes, for instance, was highly susceptible to high temperature (Al-Khatib and Paulsen, 1984). The marked difference between RI values of nonhardened and hardened seedlings demonstrated that elevated growing temperatures increased resistance of membranes to thermal injury as measured by the ninhydrin method in the present studies and also shown by the conductivity method in previous studies (Saadalla et al., 1990). The results also reinforced the conclusion that exposure to elevated temperatures was essential for expression of differences among the cultivars (Shanahan et al., 1990). Tolerance to high temperatures was similar for resistant and susceptible cultivars under nonstress environments in the present studies as in previous studies (Chen et al., 1982). Acclimation to high temperatures, which might be effective only over a narrow temperature range (Blum and Ebercon, 1981), however, separated the cultivars in the present and previous studies (Saadalla et al., 1990). These results suggest that innate differences are as important as the ability to acclimate to stress in high-temperature tolerance of wheat.
The ninhydrin method was expected to be more applicable than the conductivity method for measuring high-temperature injury during maturation. Although significant differences among field-grown cultivars were detected at both locations by the ninhydrin method, neither method was satisfactory for maturing plants. The ninhydrin method identified the high-yielding cultivars at both locations, but the lack of any overall relationship between results of either method and grain yields of the 12 cultivars indicated that other components were more susceptible than leaf-cell membranes to high temperature. Integrity of leaf-cell membranes probably was not the most sensitive factor to high temperature during maturation, and other components or processes that were more labile might have been damaged earlier. Enzymes that function in biosynthesis of starch in the developing grain, for instance, are extremely sensitive to high temperature (Paulsen, 1994; Wardlaw and Wrigley, 1994). The extreme sensitivity caused grain processes to be the most susceptible of all plant parts to injury during maturation (Stone and Nicolas, 1995). Thus, it is unlikely that measuring injury to leaf-cell membranes would distinguish susceptible and tolerant cultivars.
We concluded that the ninhydrin method is a valuable alternative to the conductivity method. It was as successful as the conductivity method for young, hardened plants but had the same disadvantages for maturing, field-grown plants. Measurement of other traits that correlate with grain yield under high-temperature conditions should be employed for maturing plants (Reynolds et al., 1994).
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NOTES
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Contribution no. 00-41-J from the Kansas Agric. Exp. Stn.
Received for publication September 2, 1999.
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REFERENCES
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ABSTRACT
INTRODUCTION
