PlantCell Reports
Plant Cell Reports (1992) 11: 5 7 6 - 580
9 Springer-Verlag 1992
Genotype-specific heat shock proteins in two maize inbreds Julie A. Jorgensen t, Jian Weng 1, Tuan-hua David Ho 2, and Henry T. Nguyen 1 1 Plant Genetics Laboratory, Mail Stop 2122, Texas Tech University, Lubbock, TX 79409, USA 2 Department of Biology, Washington University, St. Louis, MO 63130, USA Received January 20, 1992/Revised version received August 6, t992 - Communicated by J. M. Widhohn
Summary. Leaf blade tissue of maize inbred lines B73 and Mo17 was analyzed for intraspecific genetic variability in the heat shock response. The maize inbreds were characterized for acquired thermal tolerance and patterns of heat shock protein synthesis. The leakage conductivity assay of membrane stability during stress indicated that Mo17 possesses greater potential than B73 to acquire thermal tolerance, Poly(A)+ RNA, extracted from leaf blades, was translated in vitro in the presence of 35Smethionine and the translation products separated by twodimensional gel electrophoresis. Major genotypic differences were observed in the translation products. Mo17 synthesized twelve unique heat shock proteins in the 15-18 kD range, but B73 synthesized only three unique heat shock proteins in the same range. DNA polymorphisms were observed between the maize lines using 32_p labeled heat shock protein gene probes.
Abbreviations: HKT, Heat-killing time - HS, Heat shock - HSP, Heat shock protein - HMW, High molecular weight - LMW, Low molecular weight
Introduction Plants exposed to high temperature stress display a dramatic alteration in gene expression. The HS response is characterized by a reduction in normal gene expression and a rapid induction of HSP gene transcription. The synthesis and accumulation of HSPs have been correlated with acquired thermal tolerance in many species such as yeast (Sanchez and Lindquist 1990); fibroblast cells (Riabowol et al. 1988) soybean (Lin et al. 1985); and wheat (Krishnan et al. 1989). In general, HMW HSPs are more abundantly synthesized in yeast, insect and animal cells than LMW HSPs. In contrast, plants synthesize a higher level of HSPs in the range of 15-27kD. The abundance and complexity of LMW HSPs in plants have stimulated great interest in determining their role in thermal tolerance (Vierling 1991).
Previous studies of the HS response in maize have addressed the aspects of induction temperature, tissue specificity, HSP profiles, and intracellular localization (Baszczynski and Walden 1982; Cooper and Ho 1983; Cooper et at. 1984; Cooper and Ho 1987; Atkinson et al. 1989). Very few investigations have dealt with HSP genetic diversity in maize (Yacoob and Filion 1986; Frova et al. 1988; Nguyen et at. 1989). Although these reports indicated the existence of HSP genetic variability in maize, the major limitation of the data was the use of onedimensional gel electrophoresis for protein separation. Further analysis using two-dimensional gel electrophoresis is required to reveal more detailed qualitative and quantitative differences in HSP profiles among maize genotypes. The identification of intraspecific variation in HSP synthesis will facilitate genetic analysis of the role of lISP genes in plant thermal tolerance. Genetic variability in HSP synthesis has been previously reported in cotton (Fender and O'Connell 1989) and wheat (Krishnan et al. 1989; Vierling and Nguyen 1990). Our goal was to examine the relationship between several molecular characteristics of HSPs and acquired thermal tolerance in maize. In this paper, we report qualitative and quantitative genetic variation in HSP profiles between two maize inbred lines differing in acquired thermal tolerance. Furthermore, DNA polymorphisms were detected between these maize lines using HSP gene probes.
Materials and methods Plant Material. lnbreds B73 and Mo17 were chosen because they are
of genetically different backgrounds and are commonly used in genetic studies. Green leaf blades from ten-day-old seedlings were used in all experiments. Seeds obtained from Pioneer Hi-Bred International, Inc., were germinated and grown at 26~ with high light in a Conviron E-15 growth chamber (Conviron, AsheviUe, NC) until HS, as described by Vierling and Nguyen (1990). Thermal tolerance assay. In order to determine inherent thermal tolerance, seedling leaf blades were exposed to 50~ (normally lethal) without a HS pre-treatment. To analyze acquired thermal tolerance, seedlings were acclimated for 24 h at the optimum HSP gene induction
Contribution of the College o f Agricultural Sciences, Texas Tech University, Journal No. T-4-333 Correspondence to." H. T. Nguyen
577 temperature (40~ (Cooper and Ho 1983) in an incubator with high humidity. A heat acclimation of 24 h was chosen to maximize the potential for acquisition of thermal tolerance (Chen et at. 1982). Heat-acclimated leaf blades were then treated at 50~ The 50~ treatment in both the inherent and acquired thermal tolerance assays consisted of 0, 30, 60, 120 or 240 min in a water bath. At each timepoint, three independent observations of each genotype were taken. An observation consisted of two individual leaf blades. Ion conductivity of heat-treated leaf blades was measured using a conductivity bridge (YSI Model 31, Yellow Springs Instrument Company. Yellow Springs, OH). Ion leakage greater than 50% of the total was measured as it is a standard estimation of irreversible membrane damage. The time required to impose this level of damage was defined as the HKT (Chen et al. 1982; Chaisompongpan et a1.1990). Mean values of three independent HKT observations for each treatment and each genotype were analyzed by best-fit simple linear regression analysis, according to the equation y = mx + b, with x = time of heat treatment as the independent variable and y = injury level as the dependent variable. Comparisons between genotypes were made by testing the differences between independentregressions for the same treatment (Steel and Torrie 1980).
lISP Synthesis Study. For induction of HSP synthesis, seedlings were heat shocked at 40~ for 0, 30, 60, 120 or 240 min in an incubator with high humidity. Leaf blades were immediatelyfrozen in liquid N2 and stored at -70~ until RNA extraction. RNA was isolated by LiC1 precipitation and the samples were enriched for poly(A)+ RNA by oligo(dT) chromatography according to Sambrook et al. (1989). Poly(A)+ RNA was translated in the presence of 35S-methionine using rabbit reticulocyte lysate (Promega Corporation, Madison, WI), according to the manufacturer's instructions. TCA-insoluble counts were determined according to Mans and Novelli (1961). IEFSDS/PAGE and fluorography were performed according to Vierling and Nguyen (1990). Equal counts were loaded per gel and X-ray films were exposed for equal times.
Southern Blot Hybridization.
DNA was extracted according to Sambrook et al. (1989). DNA (10mg/sample) was digested with BamH I, EcoR I and Hind FII, respectively, separated on 0.7% agarose gels by electrophoresis and transferred by vacuum blotting to ZetaProbe (Bio-Rad, Richmond, CA) membranes. 32p-dCTP labeled probes for HSP18, HSP26 and HSP70 were prepared using a random primer labeling kit (United States Biochemical Corporation, Cleveland, OH) and hybridized with blots for 16 h in 6 X SSC, 5 X Denhardt's Solution, 0.5% SDS, 100mg/ml salmon speonn DNA and 50% formamide at 42~ C. The blots were washed at 65 C under the following conditions: 2 X SSC 0.1% SDS 2 X 20 min, 1 X SSC 0.1% SDS 2 X 20 rain, 0.1 X SSC 0.1% SDS 2 X 20 min, and 0.05 X SSC 0.1% SDS 2 X 20 rain. Preparation of probes, blotting and hybridizationparameters were as described in Sambrook et al. (1989).
Results
and
discussion
Membrane Leakage Assay Regression coefficients (slopes) indicated that Mo17 and B73 m a i z e lines differ s i g n i f i c a n t l y in their heat acclimation potentials (Table 1). H K T following heat treatments were determined by substituting for "y" the 50% irreversable injury value into the equation y = mx + b. The H K T for B73 and Mo17 following a 50~ heat treatment with no pretreatment were 26.7 and 28.0 min, respectively. This variation is insignificant and indicates that these two inbreds do not differ in levels of inherent thermal tolerance at 50~ Both genotypes had an increased HKT if first acclimated at 40~ prior to the 50 ~ C heat treatment. HKT for B73 increased to 37.5 min from 26.7 min while the H K T of Mo17 increased to 55.6 min from 28.0 min. Although
both genotypes had the capacity to acquire thermal tolerance, Mo17 had significantly greater potential and was therefore classified as being more heat tolerant than B73. These results indicate genetic variability in acquired thermal tolerance and i n v i t e i n v e s t i g a t i o n into the molecular basis of the variation. Table 1. Electrolyte leakage conductivity in heat-shocked Mo17 and B73 leaf tissue. Slope coefficients (m), intercepts (b) and coefficients of determination (r2) obtained by regression of membrane stability values over time, as measured by leakage conductivity. Each timepoint utilized in the simple linear regression analyses (y = mx + b) was the mean of three observations (Steel and Torrie, 1980).
SLOPE1
r2
Direet HS Mo17 B73
.0092a .010a
.149 .154
.90 .92
Acclim~edHS Mo17 B73
.0042a .0075b
.268 .217
.90 .94
l Slope coefficients within each treatment followed by different letters were significantly different (P< 0.10) as deten'nined by slope coefficient T-test.
HSP Synthesis Study B73 and Mo17 HSP in vitro synthesis patterns were compared over the HS timecourse and are presented in Fig. 1. A HSP was defined as a protein whose synthesis can first be detected or which is synthesized at dramatically increased levels during HS. Patterns of control tissue in vitro t r a n s l a t i o n p r o d u c t s r e v e a l e d some protein differences between genotypes, and several common heatinducible proteins at 14.5kD (Fig. l a and lb). After 30 rain of 40~ HS, in vitro translatable m R N A patterns were d r a m a t i c a l l y d i f f e r e n t from control patterns. Reductions and disappearance of m a n y control protein products were detected after 30 rain of HS. In vitro translation products c o m m o n to both genotypes were detected at 92kD, 70kD, 42kD, 29kD, 15-18 and 14.5kD. Furthermore, each genotype synthesized a number of unique HSPs. In B73 this included several HSPs at 70kD, two in the 15-18kD range and one at 14.5kD (Fig. lc). The Mo17 profile was strikingly different with many unique HSPs; one at 70kD, one at 42kD, and nine in the 15-18kD range (Fig. ld). At 60 m i n of HS, levels of in vitro translatable HSP m R N A species increased in both genotypes. The most obvious changes from the 30 min sampling were observed in the 15-18kD range, with induction of unique HSPs and increases in HSPs c o m m o n to both genotypes. Most normal protein product levels were further reduced (data not shown). After 120 min of HS, relative levels of several in vitro translatable L M W HSP m R N A declined in B73 including several in c o m m o n with Mo17, and in the unique HSPs around 15-18kD (Fig. le). Mo17, however, appeared to maintain levels of several c o m m o n HSPs, as well as to
578 undergo induction of three new unique HSPs around 1518kD (Fig. If). HSP in vitro translation products specific to Mo17 reached maximum numbers and totaled twelve at this timepoint, compared to only two in B73. Both genotypes displayed additional HSPs around 14.5kD. At this HS timepoint most normal protein product levels continued to decrease in both genotypes. Although both genotypes had reduced levels of in vitro translatable mRNA after 240 min of HS, B73 exhibited
the greatest decline, with reductions across all major HSP classes, but especially in the 15-18kD range. Mo17 levels of in vitro translatable mRNA were also reduced across the 15-18kD group. An exception to reductions in mRNA products noted in both genotypes was the increasing numbers and amounts of HSPs at 14.5kD. Normal protein products in both genotypes were drastically reduced at this HS timepoint. These data reflected the same trends of previous timepoints in both samples (data not shown).
579
Fig.1 a-f. In vitro translation products of (a) B73 and (b) Mo17 control; (c) B73 and (d) Mo17, heat shocked for 30 minutes at 40~ (e) B73 and (f) Mo17, heat shocked for 120 minutes at 40~ Similar results were obtained in at least one repetition of each data point. KEY to symbols used in Figure 1: circles represent HSPs common to both genotypes, squares represent HSPs unique to Mo17 and triangles represent HSPs unique to B73.
The HS response in maize inbreds B73 and Mo17 was characterized by the induction of numerous HSPs, and reduced but continued normal protein synthesis. Our data is consistent with data from earlier studies in maize (Cooper and Ho 1983; Atkinson et al. 1989). Nearly all HSPs in B73 and Mo17 were newly synthesized. However, the group of 14.5kD HSPs were detectable in control protein patterns, while their levels were dramatically increased during HS. Unlike many other HSPs observed in this study, the levels of these HSPs did not decrease even after 240 min of HS. Genotypic variation in maize HSPs was established by the differential expression of in vitro translation products over the HS timecourse. Several gene products common to both genotypes were induced at different times of HS, some of which subsequently disappeared from the profile at various times, possibly indicating genotype and genespecific differences in regulation and requirements for a HSP. However, the most interesting observation was the variation in the synthesis of numerous HSPs in the 1518kD range common to one genotype. The synthesis of unique HSPs should stimulate investigation into their role in thermal tolerance. Southern Blot Analysis Southern blots were analyzed to investigate genomic organization within B73 and Mo17 HSP genes (Fig. 2). Probes were made from cDNAs for HSP18, HSP26 and HSP70 to focus on organizational patterns of genes corresponding to protein products in the range of 18kD
26kD and 70kD, respectively. Comparison of hybridization patterns with all three HSP probes revealed distinct variation between the genotypes. A blot using HSP18 as a probe confirmed a multigene family in both genotypes (several signals were observed in each) and the patterns revealed high levels of DNA polymorphism (Fig. 2a). Patterns of hybridization to HSP26 also were different between the genotypes; only one HSP26 signal was detected in B73 and two signals were detected in Mo17 (Fig. 2b). There were two hybridization signals for HSP70 in B73 while in Mo17 there were three signals (Fig. 2c). These data support findings of qualitative variation of in vitro translatable mRNA for HSPs. Further examination of the novel genes corresponding to HSPs synthesized will necessitate their isolation, sequencing and comparative genetic analysis. Several maize HSP genes have been sequenced: HSP70 (Rochester et al. 1986), HSP26 (Nieto-Sotelo et al. 1990) and three members of the HSP18 family (Dietrich et al. 1991; Goping et al. 1991). Future analysis should focus on indepth characterization of individual members of a multigene HSP family. Whether the intraspecific molecular variation in HSP genes is causally related to thermal tolerance remains to be determined. Substantiation of a causal relationship between specific HSPs and thermal tolerance will require formal genetic analyses of B73 X Mo17 F 2 plants. Strategies for exploration of HSP roles in thermal tolerance could include analysis with gene-specific probes, analysis of HSP mutants, or overexpression of HSP genes in transgenic plants. Investigations utilizing the latter option will be simplified when maize transformation becomes routine. Once the role of individual HSPs in thermal tolerance is established, there may be significant natural diversity in HSP genes among maize germplasm available for exploitation in crop improvement.
580 HSPI8 B
~ 2' q T P
kb
23.1
E
HSP26 B
H
q---2,
H
i ! 1
HSP70
E 2
B
! i 1
2
1
2
1 1
E 2 I
H
I 1
2 I
I I
2 I
--
W 9.4--
qm
W
I,,
wqD
6.6-4.4--
~
q~
11,,
2.3--
U
W
O
w
O!B ii~
~'W
Fig. 2 a-c. Southern blots of genomic DNA hybridizing with HSP18, HSP26, and HSP70, respectively. Similar results were obtained in at least one repetition of each experiment. Restriction enzyme digestion of DNA prior to hybridization was as follows: digestions with BamH I (B), E c o R I (E) and Hin d Ill (H); column 1 of each blot represents B73 digestion and column 2 of each blot represents Mo17.
~
2.0~
A c k o w l e d g e m e n t s . We thank R. Sinibaldi (Zoecon Research Institute, Palo Alto, CA) for the pHSP18 clone, and Monsanto Co. (St. Louis, MO) for the HSP70 clone. Thanks to Dr. Kerry L. Hendershot for assistance with the thermal tolerance evaluations, and to Dr. Richard Vierling and Dr. Chandrashekhar P. Joshi for critical review of this manuscript. This work was supported in part by a grant from Pioneer Hi-Bred International, Inc.
References Atkinson BG, Liu L, Goping IN, Walden DB (1989) Expression of the genes encoding hsp73, hspl8, and ubiquitin in radicles of heat- shocked maize seedlings. Genome 31:698-704 Baszczynski CL, Walden DB (1982) Regulation of gene expression in corn (Zea mays L.) by heat shock. Can J Biochem 60:569-579 Chaisompongpan N, Li PH, Davis DW, Markhart AH III (1990) Photosynthetic responses to heat stress in common bean genotypes differing in heat acclimation potential. Crop Sci 30:100-104 Chen H-H, Shen Z-Y, Li PH (1982) Adaptibility of crop plants to high temperature stress. Crop Sci 22:719-725 Cooper P, Ho T-HD (1983) Heat shock proteins in maize. Plant Physiol 71:215-222 Cooper P, Ho T-HD, Hauptmann RM (1984) Tissue specificity of the heat-shock response in maize. Plant Physiol 75:431-441 Cooper P, Ho T-HD (1987) Intracellular localization of heat shock proteins in maize. Plant Physiol 84:1197-1203 Dietrich, PS, Bouchard RA, Casey ES, Sinibaldi RM (1991) Isolation and characterization of a small heat shock protein gene from maize. Plant Physiol 96:1268-1276 Fender, SE, O'Connell MA (1989) Heat shock protein expression in therrnotolerant and thermosensitive lines of cotton. Plant Cell Reports 8:37-40 Frova C, Taramino G, Binelli G, Ottaviano E (1988) Heat-shock protein variability in maize. Maydica 33:65-76 Goping IS, Frappier JR, Walden DB, Atkinson BG (1991) Sequence, identification and characterization of cDNA encoding two different members of the 18kDa heat shock family of Zea mays L. Plant Mol Biol 16:699-711 Krishnan M, Nguyen HT, Burke JJ (1989) Heat shock protein synthesis and thermal tolerance in wheat. Plant Physiol 90:140145
Lin CY, Chen YM, Key JL (1985) Solute leakage in soybean seedlings under various heat shock regimes. Plant Cell Physiol 26(8):1493-149 M a n s RJ, N o v e l l i G D ( 1 9 6 1 ) M e a s u r e m e n t o f the incorporation of radioactive amino acids into protein by a filter paper disk method. Arch Biochem Biophys 94:48-53 Nguyen HT, Krishnan M, Burke JJ, Porter DR, Vieding RA (1989) Genetic diversity of heat shock protein synthesis in cereal plants. In: Cherry JH (ed) Environmental stress in plants. Springer-Verlag Berlin Heidelberg, pp319-330 Nieto-Sotelo J, Vierling E, Ho T-HD (1990) Cloning, sequence analysis, and expression of a cDNA encoding a plastidqocalized heat shock protein in maize. Plant Physiol 93:1321-1328 Riabowol KT, Mizzen LA, Welch WJ (1988) Heat shock is lethal to fibroblasts microinjected with antibodies against HSP70. Science 242:433-436 Rochester DF, Winer JA, Shah DM (1986) The structure and expression of maize genes encoding the major heat shock protein, hsp70. EMBO 5 (3):451-458 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning A Laboratory Manual 2nd edn. Cold Spring Harbor Laboratory Press Sanchez Y, Lindquist SL (1990) HSP104 Required for induced thermotolerance. Science 248:1112-1115 Steel RGD, Torrie JH (1980) Principles and procedures of statistics. A biometrical approach. 2nd ed. Mc-Graw-Hill New York pp239271 Vierling E (1991) The roles of heat shock proteins in plants. Annu Rev Plant Phys Plant Mol Biol 42:579-620 Vierling RA, Nguyen HT (1990) Heat-shock protein synthesis and accumulation in diploid wheat. Crop Sci 30:1337-1342 Yacoob RK, Filion WG (1986) Temperature-stress response in maize: a comparison of several cuhivars. Can J Genet Cytol 28:11251131
Plant Cell Reports (1992) 11: 5 7 6 - 580
9 Springer-Verlag 1992
Genotype-specific heat shock proteins in two maize inbreds Julie A. Jorgensen t, Jian Weng 1, Tuan-hua David Ho 2, and Henry T. Nguyen 1 1 Plant Genetics Laboratory, Mail Stop 2122, Texas Tech University, Lubbock, TX 79409, USA 2 Department of Biology, Washington University, St. Louis, MO 63130, USA Received January 20, 1992/Revised version received August 6, t992 - Communicated by J. M. Widhohn
Summary. Leaf blade tissue of maize inbred lines B73 and Mo17 was analyzed for intraspecific genetic variability in the heat shock response. The maize inbreds were characterized for acquired thermal tolerance and patterns of heat shock protein synthesis. The leakage conductivity assay of membrane stability during stress indicated that Mo17 possesses greater potential than B73 to acquire thermal tolerance, Poly(A)+ RNA, extracted from leaf blades, was translated in vitro in the presence of 35Smethionine and the translation products separated by twodimensional gel electrophoresis. Major genotypic differences were observed in the translation products. Mo17 synthesized twelve unique heat shock proteins in the 15-18 kD range, but B73 synthesized only three unique heat shock proteins in the same range. DNA polymorphisms were observed between the maize lines using 32_p labeled heat shock protein gene probes.
Abbreviations: HKT, Heat-killing time - HS, Heat shock - HSP, Heat shock protein - HMW, High molecular weight - LMW, Low molecular weight
Introduction Plants exposed to high temperature stress display a dramatic alteration in gene expression. The HS response is characterized by a reduction in normal gene expression and a rapid induction of HSP gene transcription. The synthesis and accumulation of HSPs have been correlated with acquired thermal tolerance in many species such as yeast (Sanchez and Lindquist 1990); fibroblast cells (Riabowol et al. 1988) soybean (Lin et al. 1985); and wheat (Krishnan et al. 1989). In general, HMW HSPs are more abundantly synthesized in yeast, insect and animal cells than LMW HSPs. In contrast, plants synthesize a higher level of HSPs in the range of 15-27kD. The abundance and complexity of LMW HSPs in plants have stimulated great interest in determining their role in thermal tolerance (Vierling 1991).
Previous studies of the HS response in maize have addressed the aspects of induction temperature, tissue specificity, HSP profiles, and intracellular localization (Baszczynski and Walden 1982; Cooper and Ho 1983; Cooper et at. 1984; Cooper and Ho 1987; Atkinson et al. 1989). Very few investigations have dealt with HSP genetic diversity in maize (Yacoob and Filion 1986; Frova et al. 1988; Nguyen et at. 1989). Although these reports indicated the existence of HSP genetic variability in maize, the major limitation of the data was the use of onedimensional gel electrophoresis for protein separation. Further analysis using two-dimensional gel electrophoresis is required to reveal more detailed qualitative and quantitative differences in HSP profiles among maize genotypes. The identification of intraspecific variation in HSP synthesis will facilitate genetic analysis of the role of lISP genes in plant thermal tolerance. Genetic variability in HSP synthesis has been previously reported in cotton (Fender and O'Connell 1989) and wheat (Krishnan et al. 1989; Vierling and Nguyen 1990). Our goal was to examine the relationship between several molecular characteristics of HSPs and acquired thermal tolerance in maize. In this paper, we report qualitative and quantitative genetic variation in HSP profiles between two maize inbred lines differing in acquired thermal tolerance. Furthermore, DNA polymorphisms were detected between these maize lines using HSP gene probes.
Materials and methods Plant Material. lnbreds B73 and Mo17 were chosen because they are
of genetically different backgrounds and are commonly used in genetic studies. Green leaf blades from ten-day-old seedlings were used in all experiments. Seeds obtained from Pioneer Hi-Bred International, Inc., were germinated and grown at 26~ with high light in a Conviron E-15 growth chamber (Conviron, AsheviUe, NC) until HS, as described by Vierling and Nguyen (1990). Thermal tolerance assay. In order to determine inherent thermal tolerance, seedling leaf blades were exposed to 50~ (normally lethal) without a HS pre-treatment. To analyze acquired thermal tolerance, seedlings were acclimated for 24 h at the optimum HSP gene induction
Contribution of the College o f Agricultural Sciences, Texas Tech University, Journal No. T-4-333 Correspondence to." H. T. Nguyen
577 temperature (40~ (Cooper and Ho 1983) in an incubator with high humidity. A heat acclimation of 24 h was chosen to maximize the potential for acquisition of thermal tolerance (Chen et at. 1982). Heat-acclimated leaf blades were then treated at 50~ The 50~ treatment in both the inherent and acquired thermal tolerance assays consisted of 0, 30, 60, 120 or 240 min in a water bath. At each timepoint, three independent observations of each genotype were taken. An observation consisted of two individual leaf blades. Ion conductivity of heat-treated leaf blades was measured using a conductivity bridge (YSI Model 31, Yellow Springs Instrument Company. Yellow Springs, OH). Ion leakage greater than 50% of the total was measured as it is a standard estimation of irreversible membrane damage. The time required to impose this level of damage was defined as the HKT (Chen et al. 1982; Chaisompongpan et a1.1990). Mean values of three independent HKT observations for each treatment and each genotype were analyzed by best-fit simple linear regression analysis, according to the equation y = mx + b, with x = time of heat treatment as the independent variable and y = injury level as the dependent variable. Comparisons between genotypes were made by testing the differences between independentregressions for the same treatment (Steel and Torrie 1980).
lISP Synthesis Study. For induction of HSP synthesis, seedlings were heat shocked at 40~ for 0, 30, 60, 120 or 240 min in an incubator with high humidity. Leaf blades were immediatelyfrozen in liquid N2 and stored at -70~ until RNA extraction. RNA was isolated by LiC1 precipitation and the samples were enriched for poly(A)+ RNA by oligo(dT) chromatography according to Sambrook et al. (1989). Poly(A)+ RNA was translated in the presence of 35S-methionine using rabbit reticulocyte lysate (Promega Corporation, Madison, WI), according to the manufacturer's instructions. TCA-insoluble counts were determined according to Mans and Novelli (1961). IEFSDS/PAGE and fluorography were performed according to Vierling and Nguyen (1990). Equal counts were loaded per gel and X-ray films were exposed for equal times.
Southern Blot Hybridization.
DNA was extracted according to Sambrook et al. (1989). DNA (10mg/sample) was digested with BamH I, EcoR I and Hind FII, respectively, separated on 0.7% agarose gels by electrophoresis and transferred by vacuum blotting to ZetaProbe (Bio-Rad, Richmond, CA) membranes. 32p-dCTP labeled probes for HSP18, HSP26 and HSP70 were prepared using a random primer labeling kit (United States Biochemical Corporation, Cleveland, OH) and hybridized with blots for 16 h in 6 X SSC, 5 X Denhardt's Solution, 0.5% SDS, 100mg/ml salmon speonn DNA and 50% formamide at 42~ C. The blots were washed at 65 C under the following conditions: 2 X SSC 0.1% SDS 2 X 20 min, 1 X SSC 0.1% SDS 2 X 20 rain, 0.1 X SSC 0.1% SDS 2 X 20 min, and 0.05 X SSC 0.1% SDS 2 X 20 rain. Preparation of probes, blotting and hybridizationparameters were as described in Sambrook et al. (1989).
Results
and
discussion
Membrane Leakage Assay Regression coefficients (slopes) indicated that Mo17 and B73 m a i z e lines differ s i g n i f i c a n t l y in their heat acclimation potentials (Table 1). H K T following heat treatments were determined by substituting for "y" the 50% irreversable injury value into the equation y = mx + b. The H K T for B73 and Mo17 following a 50~ heat treatment with no pretreatment were 26.7 and 28.0 min, respectively. This variation is insignificant and indicates that these two inbreds do not differ in levels of inherent thermal tolerance at 50~ Both genotypes had an increased HKT if first acclimated at 40~ prior to the 50 ~ C heat treatment. HKT for B73 increased to 37.5 min from 26.7 min while the H K T of Mo17 increased to 55.6 min from 28.0 min. Although
both genotypes had the capacity to acquire thermal tolerance, Mo17 had significantly greater potential and was therefore classified as being more heat tolerant than B73. These results indicate genetic variability in acquired thermal tolerance and i n v i t e i n v e s t i g a t i o n into the molecular basis of the variation. Table 1. Electrolyte leakage conductivity in heat-shocked Mo17 and B73 leaf tissue. Slope coefficients (m), intercepts (b) and coefficients of determination (r2) obtained by regression of membrane stability values over time, as measured by leakage conductivity. Each timepoint utilized in the simple linear regression analyses (y = mx + b) was the mean of three observations (Steel and Torrie, 1980).
SLOPE1
r2
Direet HS Mo17 B73
.0092a .010a
.149 .154
.90 .92
Acclim~edHS Mo17 B73
.0042a .0075b
.268 .217
.90 .94
l Slope coefficients within each treatment followed by different letters were significantly different (P< 0.10) as deten'nined by slope coefficient T-test.
HSP Synthesis Study B73 and Mo17 HSP in vitro synthesis patterns were compared over the HS timecourse and are presented in Fig. 1. A HSP was defined as a protein whose synthesis can first be detected or which is synthesized at dramatically increased levels during HS. Patterns of control tissue in vitro t r a n s l a t i o n p r o d u c t s r e v e a l e d some protein differences between genotypes, and several common heatinducible proteins at 14.5kD (Fig. l a and lb). After 30 rain of 40~ HS, in vitro translatable m R N A patterns were d r a m a t i c a l l y d i f f e r e n t from control patterns. Reductions and disappearance of m a n y control protein products were detected after 30 rain of HS. In vitro translation products c o m m o n to both genotypes were detected at 92kD, 70kD, 42kD, 29kD, 15-18 and 14.5kD. Furthermore, each genotype synthesized a number of unique HSPs. In B73 this included several HSPs at 70kD, two in the 15-18kD range and one at 14.5kD (Fig. lc). The Mo17 profile was strikingly different with many unique HSPs; one at 70kD, one at 42kD, and nine in the 15-18kD range (Fig. ld). At 60 m i n of HS, levels of in vitro translatable HSP m R N A species increased in both genotypes. The most obvious changes from the 30 min sampling were observed in the 15-18kD range, with induction of unique HSPs and increases in HSPs c o m m o n to both genotypes. Most normal protein product levels were further reduced (data not shown). After 120 min of HS, relative levels of several in vitro translatable L M W HSP m R N A declined in B73 including several in c o m m o n with Mo17, and in the unique HSPs around 15-18kD (Fig. le). Mo17, however, appeared to maintain levels of several c o m m o n HSPs, as well as to
578 undergo induction of three new unique HSPs around 1518kD (Fig. If). HSP in vitro translation products specific to Mo17 reached maximum numbers and totaled twelve at this timepoint, compared to only two in B73. Both genotypes displayed additional HSPs around 14.5kD. At this HS timepoint most normal protein product levels continued to decrease in both genotypes. Although both genotypes had reduced levels of in vitro translatable mRNA after 240 min of HS, B73 exhibited
the greatest decline, with reductions across all major HSP classes, but especially in the 15-18kD range. Mo17 levels of in vitro translatable mRNA were also reduced across the 15-18kD group. An exception to reductions in mRNA products noted in both genotypes was the increasing numbers and amounts of HSPs at 14.5kD. Normal protein products in both genotypes were drastically reduced at this HS timepoint. These data reflected the same trends of previous timepoints in both samples (data not shown).
579
Fig.1 a-f. In vitro translation products of (a) B73 and (b) Mo17 control; (c) B73 and (d) Mo17, heat shocked for 30 minutes at 40~ (e) B73 and (f) Mo17, heat shocked for 120 minutes at 40~ Similar results were obtained in at least one repetition of each data point. KEY to symbols used in Figure 1: circles represent HSPs common to both genotypes, squares represent HSPs unique to Mo17 and triangles represent HSPs unique to B73.
The HS response in maize inbreds B73 and Mo17 was characterized by the induction of numerous HSPs, and reduced but continued normal protein synthesis. Our data is consistent with data from earlier studies in maize (Cooper and Ho 1983; Atkinson et al. 1989). Nearly all HSPs in B73 and Mo17 were newly synthesized. However, the group of 14.5kD HSPs were detectable in control protein patterns, while their levels were dramatically increased during HS. Unlike many other HSPs observed in this study, the levels of these HSPs did not decrease even after 240 min of HS. Genotypic variation in maize HSPs was established by the differential expression of in vitro translation products over the HS timecourse. Several gene products common to both genotypes were induced at different times of HS, some of which subsequently disappeared from the profile at various times, possibly indicating genotype and genespecific differences in regulation and requirements for a HSP. However, the most interesting observation was the variation in the synthesis of numerous HSPs in the 1518kD range common to one genotype. The synthesis of unique HSPs should stimulate investigation into their role in thermal tolerance. Southern Blot Analysis Southern blots were analyzed to investigate genomic organization within B73 and Mo17 HSP genes (Fig. 2). Probes were made from cDNAs for HSP18, HSP26 and HSP70 to focus on organizational patterns of genes corresponding to protein products in the range of 18kD
26kD and 70kD, respectively. Comparison of hybridization patterns with all three HSP probes revealed distinct variation between the genotypes. A blot using HSP18 as a probe confirmed a multigene family in both genotypes (several signals were observed in each) and the patterns revealed high levels of DNA polymorphism (Fig. 2a). Patterns of hybridization to HSP26 also were different between the genotypes; only one HSP26 signal was detected in B73 and two signals were detected in Mo17 (Fig. 2b). There were two hybridization signals for HSP70 in B73 while in Mo17 there were three signals (Fig. 2c). These data support findings of qualitative variation of in vitro translatable mRNA for HSPs. Further examination of the novel genes corresponding to HSPs synthesized will necessitate their isolation, sequencing and comparative genetic analysis. Several maize HSP genes have been sequenced: HSP70 (Rochester et al. 1986), HSP26 (Nieto-Sotelo et al. 1990) and three members of the HSP18 family (Dietrich et al. 1991; Goping et al. 1991). Future analysis should focus on indepth characterization of individual members of a multigene HSP family. Whether the intraspecific molecular variation in HSP genes is causally related to thermal tolerance remains to be determined. Substantiation of a causal relationship between specific HSPs and thermal tolerance will require formal genetic analyses of B73 X Mo17 F 2 plants. Strategies for exploration of HSP roles in thermal tolerance could include analysis with gene-specific probes, analysis of HSP mutants, or overexpression of HSP genes in transgenic plants. Investigations utilizing the latter option will be simplified when maize transformation becomes routine. Once the role of individual HSPs in thermal tolerance is established, there may be significant natural diversity in HSP genes among maize germplasm available for exploitation in crop improvement.
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Fig. 2 a-c. Southern blots of genomic DNA hybridizing with HSP18, HSP26, and HSP70, respectively. Similar results were obtained in at least one repetition of each experiment. Restriction enzyme digestion of DNA prior to hybridization was as follows: digestions with BamH I (B), E c o R I (E) and Hin d Ill (H); column 1 of each blot represents B73 digestion and column 2 of each blot represents Mo17.
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A c k o w l e d g e m e n t s . We thank R. Sinibaldi (Zoecon Research Institute, Palo Alto, CA) for the pHSP18 clone, and Monsanto Co. (St. Louis, MO) for the HSP70 clone. Thanks to Dr. Kerry L. Hendershot for assistance with the thermal tolerance evaluations, and to Dr. Richard Vierling and Dr. Chandrashekhar P. Joshi for critical review of this manuscript. This work was supported in part by a grant from Pioneer Hi-Bred International, Inc.
References Atkinson BG, Liu L, Goping IN, Walden DB (1989) Expression of the genes encoding hsp73, hspl8, and ubiquitin in radicles of heat- shocked maize seedlings. Genome 31:698-704 Baszczynski CL, Walden DB (1982) Regulation of gene expression in corn (Zea mays L.) by heat shock. Can J Biochem 60:569-579 Chaisompongpan N, Li PH, Davis DW, Markhart AH III (1990) Photosynthetic responses to heat stress in common bean genotypes differing in heat acclimation potential. Crop Sci 30:100-104 Chen H-H, Shen Z-Y, Li PH (1982) Adaptibility of crop plants to high temperature stress. Crop Sci 22:719-725 Cooper P, Ho T-HD (1983) Heat shock proteins in maize. Plant Physiol 71:215-222 Cooper P, Ho T-HD, Hauptmann RM (1984) Tissue specificity of the heat-shock response in maize. Plant Physiol 75:431-441 Cooper P, Ho T-HD (1987) Intracellular localization of heat shock proteins in maize. Plant Physiol 84:1197-1203 Dietrich, PS, Bouchard RA, Casey ES, Sinibaldi RM (1991) Isolation and characterization of a small heat shock protein gene from maize. Plant Physiol 96:1268-1276 Fender, SE, O'Connell MA (1989) Heat shock protein expression in therrnotolerant and thermosensitive lines of cotton. Plant Cell Reports 8:37-40 Frova C, Taramino G, Binelli G, Ottaviano E (1988) Heat-shock protein variability in maize. Maydica 33:65-76 Goping IS, Frappier JR, Walden DB, Atkinson BG (1991) Sequence, identification and characterization of cDNA encoding two different members of the 18kDa heat shock family of Zea mays L. Plant Mol Biol 16:699-711 Krishnan M, Nguyen HT, Burke JJ (1989) Heat shock protein synthesis and thermal tolerance in wheat. Plant Physiol 90:140145
Lin CY, Chen YM, Key JL (1985) Solute leakage in soybean seedlings under various heat shock regimes. Plant Cell Physiol 26(8):1493-149 M a n s RJ, N o v e l l i G D ( 1 9 6 1 ) M e a s u r e m e n t o f the incorporation of radioactive amino acids into protein by a filter paper disk method. Arch Biochem Biophys 94:48-53 Nguyen HT, Krishnan M, Burke JJ, Porter DR, Vieding RA (1989) Genetic diversity of heat shock protein synthesis in cereal plants. In: Cherry JH (ed) Environmental stress in plants. Springer-Verlag Berlin Heidelberg, pp319-330 Nieto-Sotelo J, Vierling E, Ho T-HD (1990) Cloning, sequence analysis, and expression of a cDNA encoding a plastidqocalized heat shock protein in maize. Plant Physiol 93:1321-1328 Riabowol KT, Mizzen LA, Welch WJ (1988) Heat shock is lethal to fibroblasts microinjected with antibodies against HSP70. Science 242:433-436 Rochester DF, Winer JA, Shah DM (1986) The structure and expression of maize genes encoding the major heat shock protein, hsp70. EMBO 5 (3):451-458 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning A Laboratory Manual 2nd edn. Cold Spring Harbor Laboratory Press Sanchez Y, Lindquist SL (1990) HSP104 Required for induced thermotolerance. Science 248:1112-1115 Steel RGD, Torrie JH (1980) Principles and procedures of statistics. A biometrical approach. 2nd ed. Mc-Graw-Hill New York pp239271 Vierling E (1991) The roles of heat shock proteins in plants. Annu Rev Plant Phys Plant Mol Biol 42:579-620 Vierling RA, Nguyen HT (1990) Heat-shock protein synthesis and accumulation in diploid wheat. Crop Sci 30:1337-1342 Yacoob RK, Filion WG (1986) Temperature-stress response in maize: a comparison of several cuhivars. Can J Genet Cytol 28:11251131
