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Published online 7 November 2007
Published in Crop Sci 47:2513-2520 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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TURFGRASS SCIENCE

Protein Changes during Heat Stress in Three Kentucky Bluegrass Cultivars Differing in Heat Tolerance

Yali Hea and Bingru Huangb,*

a Dep. of Plant Sciences, School of Agriculture and Biology, Shanghai Jiao Tong Univ., Shanghai 201101, P.R. China
b Dep. of Plant Biology and Pathology, Rutgers Univ., New Brunswick, NJ 08901

* Corresponding author (huang{at}aesop.rutgers.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Supraoptimal temperature limits growth and metabolic activities of cool-season turfgrasses. Understanding genetic variations and mechanisms in turfgrass heat tolerance would facilitate breeding and management programs to improve turf quality under summer stress. The objective of this study was to investigate protein changes associated with heat tolerance in three Kentucky bluegrass (Poa pratensis L.) cultivars. Plants of ‘Brilliant’, ‘Midnight’, and ‘Eagleton’ were subjected to 20°C (day/night, control) or 40°C (day/night, heat stress) in growth chambers. Eagleton maintained higher chlorophyll content and fewer yellow leaves than Brilliant, while Midnight was intermediate for both parameters at 28 d of heat stress. The content of cytoplasmic and membrane proteins declined during heat stress, to a greater extent in Brilliant than in Midnight and Eagleton. The content of both types of proteins in Brilliant were significantly lower than in Eagleton, but not different from that in Midnight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that Brilliant exhibited more severe protein degradation than other two cultivars. Heat stress induced expression of several heat shock proteins (HSPs) in the cytoplasm (64, 78, and 85 kDa) and membranes (39, 45, and 66 kDa) in all three cultivars, but the induction occurred 7 to 14 d earlier in Eagleton or Midnight than in Brilliant. An additional membrane protein of 68 kDa was induced in Midnight under heat stress. The results suggested that better heat tolerance in the Kentucky bluegrass was associated with induction of HSPs during the early phase of heat stress and maintenance of higher protein content and less severe protein degradation during prolonged periods of heat stress.

Abbreviations: DW, dry weight • FW, fresh weight • HS, heat stress • HSP, heat shock protein • KBG, Kentucky bluegrass • kDa, kiloDalton • MW, molecular weight • PAGE, polyacrylamide gel electrophoresis • SDS, sodium dodecyl sulfate • Tris, Tris (hydroxymethyl) aminomethane • YLI, yellow leaf index

Protein Changes during Heat Stress in Three Kentucky Bluegrass Cultivars Differing in Heat Tolerance

Yali Hea and Bingru Huangb,*

a Dep. of Plant Sciences, School of Agriculture and Biology, Shanghai Jiao Tong Univ., Shanghai 201101, P.R. China
b Dep. of Plant Biology and Pathology, Rutgers Univ., New Brunswick, NJ 08901

* Corresponding author (huang{at}aesop.rutgers.edu).

Supraoptimal temperature limits growth and metabolic activities of cool-season turfgrasses. Understanding genetic variations and mechanisms in turfgrass heat tolerance would facilitate breeding and management programs to improve turf quality under summer stress. The objective of this study was to investigate protein changes associated with heat tolerance in three Kentucky bluegrass (Poa pratensis L.) cultivars. Plants of ‘Brilliant’, ‘Midnight’, and ‘Eagleton’ were subjected to 20°C (day/night, control) or 40°C (day/night, heat stress) in growth chambers. Eagleton maintained higher chlorophyll content and fewer yellow leaves than Brilliant, while Midnight was intermediate for both parameters at 28 d of heat stress. The content of cytoplasmic and membrane proteins declined during heat stress, to a greater extent in Brilliant than in Midnight and Eagleton. The content of both types of proteins in Brilliant were significantly lower than in Eagleton, but not different from that in Midnight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that Brilliant exhibited more severe protein degradation than other two cultivars. Heat stress induced expression of several heat shock proteins (HSPs) in the cytoplasm (64, 78, and 85 kDa) and membranes (39, 45, and 66 kDa) in all three cultivars, but the induction occurred 7 to 14 d earlier in Eagleton or Midnight than in Brilliant. An additional membrane protein of 68 kDa was induced in Midnight under heat stress. The results suggested that better heat tolerance in the Kentucky bluegrass was associated with induction of HSPs during the early phase of heat stress and maintenance of higher protein content and less severe protein degradation during prolonged periods of heat stress.

Abbreviations: DW, dry weight • FW, fresh weight • HS, heat stress • HSP, heat shock protein • KBG, Kentucky bluegrass • kDa, kiloDalton • MW, molecular weight • PAGE, polyacrylamide gel electrophoresis • SDS, sodium dodecyl sulfate • Tris, Tris (hydroxymethyl) aminomethane • YLI, yellow leaf index


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HIGH TEMPERATURE is detrimental to the growth of cool-season plant species in many temperate areas during the summer or in subtropical regions. Heat stress can cause changes in various metabolic processes, such as protein denaturation, inhibition of synthesis of normal cellular proteins, and induction of some heat shock proteins (HSPs) (DiMascio and Danneberger, 1990). Protein degradation is accelerated under heat stress and this, in turn, may lead to accelerated leaf senescence (Al-Khatib and Paulsen, 1984; Ueda et al., 2000; Zavaleta-Mancera et al., 1999). In contrast, the synthesis of HSPs in response to heat shock contributes to the improved thermotolerance in various plant species (Burke, 2001; Hong and Vierling, 2000; Malik et al., 1999; Queitsch et al., 2000). The HSPs are proposed to act as molecular chaperones (Gething, 1997). They function in the stabilization of proteins and membranes, and can assist in protein refolding under stress conditions (Wang et al., 2004).

Proteins vary in size, solubility properties, localization, and distribution within cells (Loponen et al., 2004). Salt- or water-soluble proteins are localized in the cytoplasm (as cytoplasmic proteins), while most salt-insoluble proteins are found in cellular membranes (as membrane proteins) (Loponen et al., 2004). Changes in induction and degradation of different proteins are associated with variation in plant heat tolerance (Bhadula et al., 1998; Gulen and Eris, 2004; Kochhar and Kochhar, 2005; Ristic et al., 1991; Rousch et al., 2004). There exists large genetic variability in heat tolerance for cool-season, perennial turfgrass species. However, only limited work has been done to examine protein changes in relation to heat tolerance in turfgrass species (He et al., 2005a, b; Park et al., 1996, 1997). Turfgrass cultivars differing in heat tolerance may vary in the extent of degradation and induction of different proteins. Identification of proteins regulated by heat stress would provide insights into proteins or genes controlling heat tolerance in turfgrass. The objective of this study was to examine and compare changes in protein induction and degradation under heat stress among three cultivars of Kentucky bluegrass cultivars differing in the degree of heat tolerance. Three cultivars, Eagleton, Midnight, and Brilliant were chosen for this study based on our preliminary test in a growth chamber, showing that Eagleton was the most heat tolerant; Midnight, the medium heat tolerant and Brilliant, the most susceptible to heat stress among six cultivars examined. Evaluation of cultivars in fields in New Jersey showed the similar ranks of summer tolerance among the three cultivars (Bonos, personal communication, 2005).


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Materials and Growth Conditions
Sod plugs of Brilliant, Eagleton, and Midnight were collected from 3-yr-old field plots at the Rutgers University Horticulture Farm II, North Brunswick, NJ in May 2004. They were transferred into polyvinyl chloride (PVC) pots (20 cm in diameter and 25 cm in height, with holes at the bottom for drainage) filled with a mixture (3:1, v/v) of soil and sand. Plants were maintained in a greenhouse for 60 d and then moved into growth chambers. The chambers were set at 20/20°C (day/night temperature), 75% of relative humidity, 14-h photoperiod, and 400 µM photons m–2 s–1 of photosynthetically active radiation. Plants were fertilized once a week with 40 mL Hoagland's solution (Hoagland and Arnon, 1950) and cut twice a week to keep the height at approximately 10 cm. Plants were watered three times a week until drainage occurred at the bottom of the pot at each irrigation.

Heat Stress Treatments
Plants were allowed to acclimate to growth chamber conditions (as described above) for 14 d (30 July–12 Aug. 2004) before temperature treatments were imposed. All plants were cut to the canopy height of 10 cm 2 d before heat stress treatment was imposed to start with a uniform turf canopy. Twelve pots of plants (four pots for each cultivar) were maintained at 20°C as control plants (0 d of heat stress) while another set of 12 pots of plants (four pots for each cultivar) were exposed to 40°C (day/night temperature). Plants exposed to 40°C were watered twice a day to avoid water deficit. Other environmental conditions and fertility are the same as during the growing period described above.

Plants were treated under heat stress for 28 d between 12 August and 14 September in 2004. Nondestructive observation was made, and leaf samples were collected every 7 d, which is on 0 d (control at 20°C), 7, 14, 21, and 28 d of heat stress treatment. Fresh leaf samples were collected to measure chlorophyll content, protein content, and tissue dry weight. Leaf samples for protein analysis were frozen in liquid N and kept at –70°C before final analysis.

Heat Injury Determination
The severity of heat injury in whole plants was measured as yellow leaf index. Yellow leaf index was defined as the ratio of total yellow leaf length (the length of leaves that exhibited leaf senescence) to total leaf length per plant. The length of yellow leaves and total leaf length per plant was measured in 10 individual plants that were randomly sampled in each pot. Leaf injury was evaluated by measuring chlorophyll content per unit dry weight of leaves. Chlorophyll was extracted by soaking 0.1 g of leaves in 10 mL of dimethyl sulfoxide for 72 h. Absorbance of chlorophyll extracts were measured at 663 and 645 nm with a spectrophotometer (Spectronic Genesys 2, Spectronic Instruments, Inc. NY). Chlorophyll content was calculated using the formula of Arnon (1949).

Protein Extraction and Quantification
Extraction of proteins from shoots was performed following the method described by Shimoni et al. (1997) with slight modifications. Frozen leaves (0.5 g fresh weight) were ground in liquid N2 to fine powder. The powder was extracted in 2 mL of buffer containing 0.10 mM Tris-HCl (pH 7.6) and 0.15 M NaCl on ice bath (around 4°C) and then centrifuged twice at 16,000 gn at 4°C for 30 min. The resulting supernatant, containing the salt-soluble proteins, was kept for further analysis. The pellet was washed several times with the extraction buffer and subsequently resuspended in 2 mL of the buffer containing 2% (w/v) Triton X-100, 0.10 mM Tris-HCl (pH 7.6), and 0.15 M NaCl. Following extraction for 1 h at 4°C with agitation, the suspension was centrifuged as described above and the supernatant, termed "Triton-soluble protein", was kept. Protein content of the supernatant was determined by the method of Bradford (1976). Briefly, 100 µL of protein extraction (salt-soluble protein extraction, diluted 30 times and triton-soluble protein extraction diluted 15 times) was mixed with 3 mL of Coomassie G-250 reagent (0.01% Coomassie brilliant blue G, 4.75% ethanol, and 8.5% phosphoric acid), and the absorbance was measured at 595 nm between 5 and 30 min after reaction using a spectrophotometer (Spectronic Genesys 2, Spectronic Instruments, Inc. Rochester, NY). Bovine serum albumin was used as a standard.

Protein Gel Electrophoresis
Proteins were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970) with modifications. Protein extraction was dissolved in SDS-PAGE sample buffer containing 75 mM Tris-HCl (pH 6.8), 50% (w/v) sucrose, 10% (w/v) SDS, 20% (v/v) µ-mercaptoethanol, and 1% bromophenol blue at the sample to buffer volume ratio of 4:1. An equal amount of protein (45 µg for salt-soluble protein and 30 µg for triton-soluble protein) was loaded in each lane and was separated by discontinuous SDS-PAGE with a PROTEIN III electrophoresis unit (Bio-Rad, La Jolla, CA) using a 6% stacking gel and 8% resolving gel for salt-soluble protein separation and 12% resolving gel for triton-soluble protein separation. Electrophoresis was performed at 50 V in stacking gel and 80 V in resolving gel at room temperature (20–22°C). Gels were stained overnight with Coomassie brilliant blue R. Proteins with known molecular weights (MW) were used as markers to identify the MW of proteins in the samples in each gel. As the Log (MW) and the migration rate (distance the band migrated from the top of the separating gel divided by the distance migrated by the tracking dye of bromophenol) is linearly related when protein MW is between 12 and 200 kDa (Viney and Fenton, 1998), the distance and migration rate were used to calculate the MW of protein bands in the samples based on the linear regression of the markers in the same gel. By comparing protein profiles between control and high-temperature treated samples, the proteins that were present only in treated samples, but were not present in the control were defined as heat shock proteins. The proteins existing under normal temperature are defined as constitutive proteins. The SDS-PAGE was repeated six times for each treatment and the results shown in this paper are one of the representions of the gels that were run in the study.

Experimental Design and Statistical Analysis
Three cultivars were arranged in a randomized complete block design with four replicates (four pots for each cultivar) with repeated measurements over the duration of heat treatment. Plants of each cultivar were exposed to heat stress in four growth chambers (four replicates). Plants of three cultivars were randomly rearranged inside each growth chamber, and swamped between chambers once a week to minimize effects of other environmental factors in the chambers. Protein and chlorophyll content were measured for three subsamples of leaves taken from each pot (replicate). The mean of the three subsamples was used to represent a single replicate in the analysis of variance. Data were analyzed with analysis of variance using Microsoft Excel 2000 (Microsoft, Redmond, WA) (Levine et al., 2001) and mean separations were performed with the Fisher's protected least significance difference test at P = 0.05 (Steel and Torrie, 1980).


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cultivar Variation in Heat Injury
One of the most obvious symptoms of heat injury is leaf yellowing, which was determined by yellow leaf index and chlorophyll content. The proportion of yellow leaves to the total leaves increased with stress duration for all three cultivars, with a significant increase being observed at 7 d of heat stress for Midnight and Brilliant and at 28 d for Eagleton (Fig. 1 ). Yellow-leaf proportion was highest for Brilliant, intermediate for Midnight, and lowest for Eagleton by the end of the treatment period (at 28 d).


Figure 1
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  		Figure 1. Changes in the proportion of yellow leaves in three Kentucky bluegrass cultivars during heat stress. Bars on the top indicate LSD values (p = 0.05) for comparison among cultivars and on the right represent LSD values for comparison between duration of heat stress for a given cultivar.

 
Chlorophyll content was significantly higher in Brilliant than in Eagleton from 0 to 7 d of heat stress (Fig. 2 ). However, chlorophyll content in Brilliant decreased rapidly with a prolonged duration of heat stress and dropped to only 60% of the initial level (0 d) by 28 d of heat stress. Midnight maintained relatively constant chlorophyll content from 0 to 21 d and then significantly decreased by 28 d. Chlorophyll content in Eagleton did not change significantly during the 28-d heat stress. Chlorophyll content at 28 d in Eagleton was significantly higher than in Brilliant and in Midnight by 28 and 22%, respectively.


Figure 2
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  		Figure 2. Changes in total chlorophyll content in leaves of three Kentucky bluegrass cultivars during heat stress. Bars on the top indicate LSD values (p = 0.05) for comparison among cultivars and on the right represent LSD values for comparison between duration of heat stress for a given cultivar.

 
Changes in Cytoplasmic Protein Content during Heat Stress
Cytoplasmic protein content increased during heat stress in all three cultivars, which was 12, 35, and 68% higher at 28 d than their respective initial levels (at 0 d) for Brilliant, Midnight, and Eagleton, respectively (Fig. 3 ). At 0 d of heat stress, the content of cytoplasmic proteins in Brilliant was 22% higher than in Eagleton and 32% higher than in Midnight; however, it was significantly higher in Eagleton than in Midnight and Brilliant at 21 and 28 d of heat stress. No significant difference in cytoplasmic protein content was detected between Midnight and Brilliant during most of the treatment period.


Figure 3
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  		Figure 3. Changes in cytoplsmic protein content in leaves of three Kentucky bluegrass cultivars during heat stress. Bars on the top indicate LSD values (p = 0.05) for comparison among cultivars and on the right represent LSD values for comparison between duration of heat stress for a given cultivar.

 
Changes in Membrane Protein Content during Heat Stress
Membrane protein content decreased following prolonged period of stress in all three cultivars (Fig. 4 ). Following 28 d of heat stress, membrane protein content decreased in Brilliant and Midnight, which were 23 and 9% lower than their initial levels at 0 d of stress. For Eagleton, no significant difference in membrane content was detected between 0 and 28 d of heat stress.


Figure 4
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  		Figure 4. Changes in membrane protein content in leaves of three Kentucky bluegrass cultivars during heat stress. Bars on the top indicate LSD values (p = 0.05) for comparison among cultivars and on the right represent LSD values for comparison between duration of heat stress for a given cultivar.

 
At the initiation of heat stress (0 d), the content of membrane proteins was significantly higher in Brilliant than in Eagleton. However, by the end of heat stress treatment (28 d), the content of membrane proteins was significantly higher in Eagleton than in Midnight and Brilliant.

Changes in Protein Expression during Heat Stress
Under normal temperature (20°C), cytoplasmic proteins in Brilliant exhibited one unique (150 kDa) and one stronger band (66 kDa) compared to the other two cultivars (Fig. 5A ). No cultivar differences in the expression of membrane proteins were detected at 20°C (Fig. 5B).


Figure 5
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  		Figure 5. Protein profiles of cytoplasmic (A) and membrane (B) proteins in leaves of three Kentucky bluegrass cultivars under normal temperature (20°C). Lanes: (1) ‘Midnight’, (2) ‘Eagleton’, (3) ‘Brilliant’; MW, molecular weight; kDa, kiloDalton; Marker-standard protein with known molecular weight.

 
The 150 kDa protein expressed at 20°C in Brilliant (Fig. 6A , Lane 1) was no longer detectable when plants exposed to high temperature for 7 to 28 d (Fig. 6A, Lane 2–5). For all three cultivars, cytoplasmic proteins of 66 kDa expressed in control plants at 20°C were not detected under heat stress (Fig. 6, Lane 1–5). Two proteins of 85 and 78 kDa were induced at 7 d and present through 28 d of heat stress in all three cultivars (Lanes 2–5 in Fig. 6A, B, C). A 64 kDa protein band was detected at 21 and 28 d of heat stress in Brilliant, at 14, 21, and 28 d in Midnight, and at 7 to 28 d in Eagleton.


Figure 6
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  		Figure 6. Protein profiles of cytoplasmic protein in leaves of three Kentucky bluegrass cultivars (A, ‘Brilliant’, B, ‘Eagleton’, and C, ‘Midnight’) during heat stress. Lanes: (1) 20°C for 7 d; (2) 40°C for 7 d; (3) 40°C for 14 d; (4) 40°C for 21 d; (5) 40°C for 28 d; and (6) marker protein. Equal amounts of protein (45 µg) were loaded in each lane. Solid arrows indicate newly induced proteins during heat stress and hollow arrows indicate degraded constitutive proteins during heat stress. MW, molecular weight; kDa, kiloDalton; Marker, standard protein with known molecular weight.

 
Three membrane proteins of 66, 45, 39 kDa were detected in leaves of all three cultivars during heat stress (Fig. 7 ). All three HSPs (66, 45, 39 kDa) were detected first at 7 d of heat stress in Eagleton, but were not induced until 21 d in Brilliant. In Midnight, 39 kDa proteins were induced at 21 d, while 66 and 45 kDa proteins were detected as early as 7 d of heat stress. A unique protein of 68 kDa was induced in Midnight at 21 and 28 d of heat stress.


Figure 7
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  		Figure 7. Protein profiles of membrane protein in leaves of three Kentucky bluegrass cultivars (A, ‘Brilliant’; B, ‘Eagleton’; and C, ‘Midnight’) during heat stress. Lanes: (1) 20°C for 7 d; (2) 40 °C for 7 d; (3) 40 °C for 14 d; (4) 40 °C for 21 d; (5) 40 °C for 28 d; and (6) marker protein. Equal amounts of protein (30 µg) were loaded in each lane. Solid Arrows indicate newly induced proteins during heat stress. MW, molecular weight; kDa, kiloDalton; Marker, standard protein with known molecular weight.

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In response to heat stress, Brilliant exhibited loss of leaf chlorophyll while Eagleton maintained relative constant chlorophyll content. In addition, following prolonged periods (28 d) of heat stress, Eagleton maintained highest chlorophyll content and lowest proportion of yellow leaves, Brilliant had the lowest chlorophyll content and highest amount of yellow leaves, and Midnight exhibited intermediate levels for both parameters. Whole-plant or leaf senescence is a typical symptom of heat stress injury in turfgrass, and is a good indicator of the level of heat tolerance. Our results suggest that compared among the three cultivars, Eagleton was most heat tolerant, Brilliant was most susceptible, and Midnight has moderate tolerance. A previous study showed that chlorophyll content in Kentucky bluegrass cultivars decreased during the combined stress of heat and drought and the decline was more severe in Brilliant than that in Midnight (Wang and Huang, 2004). The cultivar variation in heat tolerance could be explained at least partially by the differences in protein content and expression in response to heat stress.

Under normal temperature (20°C or 0 d of heat stress), heat-tolerant Eagleton possessed lower amount of both cystoplasmic and membrane proteins than the other two cultivars, suggesting that heat tolerance was not associated with the basal level of proteins. Following 21 d of heat stress, Eagleton maintained similar levels of cell membrane proteins and higher levels of cytoplasmic proteins compared to the initial levels (0 d of heat stress). Conversely, membrane protein content in Brilliant exhibited decline (by 30%). In addition, the content of both cytoplasmic and membrane proteins in Eagleton was significantly higher than in Brilliant and Midnight at 21 and 28 d of stress. Our data suggest that the maintenance of high cytoplasmic and membrane protein content could contribute to heat tolerance in Kentucky bluegrass cultivars. The level of a given protein in a plant cell is the result of a balance between protein synthesis and degradation (Callis, 1995). A study with three Kentucky bluegrass, annual bluegrass (Poa annua L.), and perennial ryegrass (Lolium perenne L.) showed that incorporation of radio-labeled leucine into proteins declined by an average of 69% in plants previously heated at 43°C in compared to plants held at 27°C, suggesting that protein synthesis is heat sensitive (Wehner and Watschke, 1984). Whether the cultivar variations in protein content observed in this study were due to difference in protein synthesis or degradation was unknown and deserves further investigation.

Proteins of different sources seemed to vary in their sensitivity to heat stress in all three cultivars. Following 28 d of heat stress, cytoplasmic protein content in Brilliant maintained at the initial level or slightly increased, while membrane protein content decreased significantly, suggesting that membrane proteins were more sensitive to heat stress than cytoplasmic proteins in the heat-susceptible Kentucky bluegrass cultivar. He et al. (2005b) also reported that membrane protein degradation was more sensitive to heat stress than cytoplasmic protein in creeping bentgrass leaves (Agrostis stolonifera). Furthermore, heat-tolerant Eagleton had more stable membrane proteins than heat-susceptible Brilliant under heat stress. Together, these results suggest that protecting membrane proteins is an important factor for plant tolerance to heat stress.

During heat stress, the synthesis of normal cellular proteins may be reduced or stopped completely and proteins are degraded, while an increase in the number of heat shock proteins may occur (DiMascio and Danneberger, 1990). The three cultivars differing in heat tolerance exhibited different patterns of protein expression and the expression of different types of proteins also varied in response to heat stress. Severe degradation in 150 and 66 kDa proteins were detected in Brilliant during 7 to 28 d of heat stress. Proteins of 150 kDa have been found to be a subunit of various different enzymes, including dark-induced nitrate reductase (NR), a protein kinase phosphorylating NR in response to dark treatments (Nakamura et al., 2002), aldehyde oxidase in leaf and seed extracts of barley (Hordeum vulgare L.) (Omarov et al., 1999), xanthine dehydrogenases in Glycine max and Phaseolus vulgaris (Montalbini, 2000), and kinases regulating the activities of sucrose-phosphate synthase and nitrate reductase of spinach (Spinacia oleracea L.) leaf (McMichael et al., 1995). A 66 kDa polypeptide has been found to be involved in the ozone-elicited self-defense response pathway(s) in rice (Oryza sativa L.) (Agrawal et al., 2002), a subunit of galactan galactosyltransferase involved in the biosynthesis of the long-chain raffinose family of oligosaccharides in Ajuga reptans leaves (Haab and Keller, 2002), and induced by osmotic stress treatment with abscisic acid and jasmonate (Lehmann et al., 1995). Heat-induced degradation of these constitutive proteins can adversely affect activities of the enzymes involved, and thus may account for the heat susceptibility of Brilliant in this study.

Decrease in constitutive protein production is often coupled with an increase in stress- inducible HSPs (Schlesinger et al., 1982). Heat shock proteins play a crucial role in protecting plants against stress and in the re-establishment of cellular homeostasis by stabilizing proteins and membranes and assisting in protein refolding under stress conditions (Burke, 2001; Hong and Vierling, 2000; Wang et al., 2004). Some HSPs were induced in cytoplasm (64, 78, and 85 kDa) and membranes (39, 45, and 66 kDa) in all three cultivars during 7 to 28 d heat stress, but induction in these proteins occurred 7 to 14 d earlier during heat stress in Eagleton or Midnight than in Brilliant. In addition, membrane proteins of 68 kDa were detected only in Midnight exposed to heat stress. It appeared that earlier induction of HSPs was associated with better heat tolerance in Kentucky bluegrass. Heat shock protein induction has been correlated with acquired thermotolerance in other plant species (Kimpel and Key, 1985; Krishnan et al., 1989; Lin et al., 1984; Lindquist, 1986). Park et al. (1996) reported that heat-tolerant line ‘SB’ of creeping bent grass synthesized two additional HSPs of 25 kDa that did not accumulate in nonheat tolerant line ‘NSB’. Creeping bentgrass plants with the additional HSP25 polypeptides recovered from HS faster and were able to resume typical levels of protein synthesis approximately 2 h earlier than those without the new protein expression (Park et al., 1997). Another study with ‘Penncross’ creeping bentgrass showed that heat-induced expression of 57 and 54 kDa proteins was associated with acquired thermotolerance (He et al., 2005b). Proteins of different sizes may be induced in different plant species due to the genetic variation and differences in temperature level or duration. However, it can be inferred that those early-induced HSPs or unique membrane HSPs expressed under heat stress may be used as protein molecular markers in the selection for heat-tolerant plants.

How these heat-induced HSPs in Kentucky bluegrass are involved in heat stress tolerance is unclear, and warrants investigation. These proteins have been found to be involved in stress adaptation in other plant species. Western blotting showed that the 64 kDa polypeptide is a member of the HSP60 family (Singh and Lakhotia, 2000). Glycoproteins 64 kDa with affinity to the lectin ConA may be involved in pollen heat tolerance in tobacco (Nicotiana tabacum L.) (Hrubá et al., 2005). It has been reported that 42 kDa proteins induced in the cytoplasm in Saccharomyces cerevisiae form ordered oligomers with a barrel-like structure and act as a chaperone either in vivo or in vitro, which suppressed the aggregation of one-third of the cytosolic proteins ((Haslbeck et al., 2004; Haslbeck, 2006). The 68 kDa HSPs have been reported to be constitutively expressed, but its synthesis was increased during heat stress in tomato (Lycopersicon peruvianum L.) (Neumann et al., 1993). These proteins are hydrophilic, ATP-binding and localized in the mitochondrial matrix. The deduced amino-acid sequences show strong relationships to the DnaK (HSP70)-like proteins from bacteria and organelles of eukaryotic cells. The HSPs of 45 kDa have been detected in the heat-tolerant maize (Zea mays L.) line, ZPBL 1304, but were not found in the drought and heat-sensitive line, ZPL 389, when both were exposed to heat stress (Bhadula et al., 1998; Ristic et al., 1991). Investigation of the functions of heat-inducible or unique HSPs in Kentucky bluegrass would provide insight into the mechanisms of acquired or putative thermotolerance in cool-season grasses.

In summary, heat stress caused degradation of some constitutive proteins and induced some HSPs, but to different extents for cultivars differing in heat tolerance. Maintaining higher and/or relatively constant level of protein content, less degradation of constitutive proteins, and more and earlier induction of HSPs may contribute to better heat tolerance in relatively heat-tolerant Kentucky bluegrass cultivars (Eagleton and Midnight). The structure and function of heat-induced HSPs in Kentucky bluegrass requires further study to elucidate their involvement in heat tolerance.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Received for publication December 20, 2006.


   REFERENCES
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 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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