TY - JOUR
AU - Roe, Bruce A.
AB - Abstract Fescues (Festuca sp.) are major cool-season forage and turf grass species around the world. Heat stress is one of the limiting factors in the production of fescues as forage in the southern Great Plains of the US. Heat responsive gene transcripts were cloned by using suppression subtractive hybridization between a heat-tolerant and a heat-sensitive fescue genotype subjected to a slowly increased temperature mimicking the natural conditions. The temperature in the growth chamber containing the plants was gradually increased from 24 °C to 44 °C over a period of 2 weeks. Three subtractions were conducted between samples of the two genotypes collected after 12 h of exposure to 39, 42, and 44 °C. A total of 2495 ESTs were generated, of which 1800 clustered into 434 contigs and 656 were singlets. The putative functions of ESTs were predicted by BLASTX. Nearly 30% of the contigs and 39% of the singlets had no similarity to GenBank sequences. Differentially expressed genes selected by subtractions were classified into 10 broad categories according to their putative functions generated by BLAST analysis. Under heat-stress conditions, cell maintenance, chloroplast associated and photosynthesis-, protein synthesis-, signalling-, and transcription factor-related genes had higher expression levels in the heat-tolerant genotype. Genes related to metabolism and stress had higher expression in the heat-sensitive genotype. The expression of 17 selected gene transcripts were examined by RT-PCR using plant tissues of the two genotypes grown under heat stress and under optimal temperature conditions (24 °C) for fescue. Results from RT-PCR confirmed the differential expressions of these transcripts. The differential expressions of at least 11 of these genes were attributable to heat stress rather than to differences in the genetic backgrounds of the genotypes. Differential gene expression, fescue, heat stress, heat tolerance, SSH This paper is available online free of all access charges (see http://jxb.oupjournals.org/open_access.html for further details) The online version of this article has been published under an Open Access model. Users are entitled to use, reproduce, disseminate, or display the Open Access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and the Society for Experimental Biology are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirely but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org Introduction Fescues (Festuca sp.) are major cool-season forage and turf grasses grown in a wide range of soil and climatic conditions in the United States. Fescues are outcrossing species that belong to the grass family Poaceae, subfamily Pooideae, and tribe Poeae (Sleper, 1985). High-temperature stress is one of the limiting factors for forage production and turf management of fescue plants in the southern Great Plains of the US, and most of the genotypes lack persistence through the hot and dry summers in this area (Wallner et al., 1982). Higher plants exposed to excess heat exhibit a characteristic set of cellular and metabolic responses, including a decrease in the synthesis of normal proteins and an accelerated transcription and translation of heat shock proteins (HSPs). This response is observed when plants are exposed to temperatures at least 5 °C above their optimal growing conditions (Bray et al., 2000). High temperature may also cause large, reversible effects on the rate of photosynthesis (Weis and Berry, 1988). In addition to altering patterns of gene expression, heat also damages cellular structures, including organelles and the cytoskeleton, and impairs membrane function. Studies have demonstrated that cellular and metabolic changes are required for plants to survive high temperatures (Guy, 1999). Plants are often under high temperature stress even in their native habitats. Studies indicated that plants can acquire thermotolerance if subjected to a non-lethal high temperature for a few hours before encountering heat-shock conditions. The increase in thermotolerance above the basal level is caused by acclimation (Vierling, 1991). An acclimated plant can survive exposure to a temperature that would otherwise be lethal (Key et al., 1983; O'Mahony and Burke, 2000; Hong et al., 2003). It is clear that plants surviving heat-stress conditions have developed this thermotolerance mechanism, which is also a major factor governing the distribution of plants around the globe (Woodward, 1988). Acclimation to non-lethal heat treatment, therefore, may alter transcriptional regulation in a novel manner that may be different from the one caused by heat shock. Heat tolerance of plants is a complex trait, most probably controlled by multiple genes. Examination of the effects caused by temperature extremis can reveal useful information, since heat-stress responses of plants are common to several other forms of stress, such as cold or drought stress (Quinn, 1988; Bray et al., 2000; Rizhsky et al., 2002). Although the research on abiotic stress tolerance has advanced considerably in recent years, no study characterizing the genes responsive to acclimation to long-term (days and weeks) non-lethal high temperatures in forage and turf plants has been conducted. Understanding the molecular mechanisms of plant response to long-term heat stress will be helpful in the development of heat-tolerant Festuca cultivars. It has been shown that severe heat stress can cause significant and irreversible damage to photosystem II (PSII) and Rubisco activation (Markus et al., 1981; Crafts-Brandner and Salvucci, 2000; Bernacchi et al., 2002). As a C3 plant, fescue photosynthetic carbon fixation is catalysed by Rubisco (ribulose 1,5-bisphosphate carboxylase) through the Calvin cycle (Farquhar et al., 1980). The diffusion of CO2 from the atmosphere reaching the active site of Rubisco depends on several physical and environmental properties, including temperature (Farquhar and Sharkey, 1982; Harley and Tenhunen, 1991; Nobel, 1999). Therefore, high temperature stress may damage fescue plants by reducing or blocking photosynthesis. Subtractive hybridization is an attractive method for cloning enriched differentially expressed genes and was first used to purify phage T4 mRNA (Bautz et al., 1966). This method involves hybridization of cDNA from one sample population (tester) to an excess of mRNA (cDNA) from another sample population (driver) and then separation of the unhybridized fraction (target) from hybridized common sequences (Hara et al., 1991). Diatchenko et al. (1996) further introduced the technique of suppression subtractive hybridization (SSH), in which differentially expressed genes could be normalized and enriched over 1000-fold in a single round of hybridization. SSH has been applied in many studies to identify abiotic stress-regulated gene transcripts of plants (Bahn et al., 2001; Hinderhofer and Zentgraf, 2001; Mahalingam et al., 2003; Watt, 2003). Recently, the heat tolerance of 18 clonally propagated fescue genotypes (nine most persistent and nine least persistent genotypes in the southern Great Plains under field conditions) in controlled environments has been evaluated (Zwonitzer and Mian, 2002). The growth chamber temperature was gradually raised from 24 °C to 39 °C over a period of 2 weeks. The optimum temperature for fescue is considered to be 24 °C. The plants were then kept between 39 °C and 44 °C for a period of 20 d. The discolouration, desiccation, and death of leaves and tillers were recorded during and immediately after the heat stress period. The temperature in the growth chamber was then gradually lowered to 24 °C over a period of 7 d and the plants were allowed to recover for another 2 weeks under normal temperature conditions. The number of live tillers and shoot biomass of the plants were recorded (Zwonitzer and Mian, 2002). Based on the visual stress symptoms, survival, and regrowth after stress, PI297901 was found to be highly tolerant to heat stress (with very little visible symptoms and the best regrowth among the 18 genotypes) and PI283316 was identified to be highly sensitive to heat stress (the clones of this genotype were dead after the stress period). In the current study, gene transcripts differentially expressed under heat stress conditions were selected by SSH from these two fescue genotypes with different levels of heat tolerance. Heat tolerance-related gene transcripts were identified based on their putative functions. Materials and methods Plant materials and growth conditions Fescue seeds of PI297901 and PI283316 were placed onto moist blot paper in Petri dishes, sealed with parafilm, and then placed in a germinator at 24 °C for 12/12 h day/night. Three to five days after initial incubation, emerging seedlings were transplanted in 48-cell packs filled with Metro Mix 350 (Scotts, Marysville, OH). Newly planted seedlings were placed on a mist bench for 2–3 d to help with establishment. Plants were grown in 6-inch pots to the multiple tiller stage and were replicated by separating the tillers. Clonal replicates were grown in a greenhouse for 4 weeks before being placed in the growth chambers for the experiment. Heat treatments Fescue plants were placed in growth chambers in a controlled environment (light intensity at 375 μmol m−2 s−1, 16 h day length, and 60% relative humidity). Randomized complete block design was used for the two genotypes with 18 replications for both heat-treated and control plants. Plants were subjected to a slowly increased temperature to mimic the natural conditions: 24 °C (day) and 16 °C (night) for 10 d for adaptation, the temperature was then raised to 21 °C night and 29 °C day temperature for 3 d, then the night temperature was raised to 26 °C and the day temperature to 34 °C for 3 d, followed by a 31 °C night temperature and 39 °C day temperature for 3 d, which was considered the initial stage of the heat stress. The night temperature was then raised to 36 °C and the day temperature was 42 °C for 3 d. The final temperature regime used was 44 °C day and 36 °C night for another 2 d. Plants shoot samples were collected at 12 h after plants were exposed to 39 °C, 42 °C, and 44 °C. At each sampling time point, the entire shoot mass from three plants (replicates) of each genotype (under both heat-stressed and control conditions) were collected. Fescue plants were subjected to high heat with sufficient water to prevent drought-induced stress. To reduce the environmental variation, the same experiment was conducted twice with the two growth chambers switched for heat stress and control treatments and the plant tissues from two experiments were pooled together. The plants grown in the control (no heat stress) conditions were used for comparisons of gene expression by RT-PCR. The plants were arranged in a randomized complete block design within each growth chamber. RNA isolation After collection of samples from the three temperature treatments, the frozen shoots and leaves were ground to a fine powder with a mortar and pestle. Total RNA was extracted from ground leaf tissues from each sampling time by Tri-reagent (MRC, Cincinnati, OH) and the total RNA was quantified and was checked for quality. The mRNA was purified by Oligotex mRNA midi kit (Qiagen, Valencia, CA). Suppression subtractive hybridization (SSH) Heat-regulated differentially expressed genes between PI297901 (heat-tolerant) and PI283316 (heat-sensitive) exposed to 39 °C, 42 °C, and 44 °C for 12 h were identified by using PCR-Select cDNA Subtraction Kit (BD, Palo Alto, CA) starting with 4 μg of poly A+ RNA from the tissues being compared (PI297901 versus PI283316). Three sets of subtractions were performed, including both forward and reverse as well as control subtractions following the manufacturer's instructions. The forward subtraction identified clones in which mRNA from the heat-stressed plants of PI297901 (heat-tolerant) was used as the ‘tester’ and the mRNA from PI283316 (heat-sensitive) was used as the ‘driver’. This set of cDNA clones was enriched for genes over-expressed in the heat-tolerant genotype compared with the heat-sensitive genotype. To obtain clones that were over-expressed in the heat-sensitive genotype, a reverse subtraction was performed. In this case, mRNA from heat-stressed PI283316 was used as the ‘tester’ and mRNA from PI297901 were used as the ‘driver’. This set of cDNA clones was enriched for genes over-expressed in the heat-sensitive genotype. In the last step of secondary PCR, a 7 min 72 °C extension was applied to ensure that all PCR products were full length and 3′ adenylated. The subtracted cDNA populations were cloned into the TOPO TA cloning vector and transformed with One Shot TOP10F′ chemically competent cells (Invitrogen, Carlsbad, CA). Sequencing and EST assembling High-throughput sequencing and data analysis were conducted at the Advanced Center for Genome Technology at the University of Oklahoma to identify heat-responsive genes. Briefly, clones from all libraries were cultured in 384 well microtitre plates containing 70 μl TB plus salt supplemented with 100 μg ml−1 ampicillin for 22 h at 37 °C with shaking at 520 rpm. A double-stranded DNA template was isolated as described (http://www.genome.ou.edu/Zymark_384_well_isolation.html). All sequencing reactions were conducted with the M13 reverse primer. Sequencing electrophoresis was run in an ABI 3700 capillary sequencer. The trace files obtained from the ABI sequencers were then entered into a pipeline that entailed a semi-automatic process to perform the base calling using Phred (http://www.phrap.org). Each sequence was screened for overall base quality and contaminating vector, mitochondrial, ribosomal, and E.coli sequences were removed. High quality ESTs that passed these screenings were subjected to a BLASTX (Altschul et al., 1990) comparison to the GenBank nr database (Benson et al., 2000). All members of the assembled EST database, singlets and contigs, were examined for homology to the GenBank nr database by BLASTX batch analysis. The ESTs were also submitted to the GenBank dbEST (http://www.ncbi.nlm.nih.gov/dbEST). RT-PCR For expression study of fescue heat stress-related genes under high temperature, 4 μg of total RNA from each fescue sample were used to synthesize cDNA using the Ready-To-Go RT-PCR kit (Amersham, Piscataway, NJ). By following the two-step protocol suggested by the manufacturer, first-strand cDNA generated with lyophilized pd (T)12–18 was used as a template for polymerization. 2 μl of cDNA template were added into each PCR reaction with Arabidopsis Actin primers and fescue gene-specific primers. Fescue gene-specific primers were designed by using LUX™ Designer (Invitrogen, Carlsbad, CA). Primers were designed flanking the conserved region of fescue genes. All gene-specific primers amplified cDNA fragments with corresponding sizes predicted by the designed primer. The expression level of genes in each fescue sample was checked and calculated based on the intensity of the band (Volume CNTmm2) by Quantity One 4.4 1-D Analysis Software (BIO-RAD, Hercules, CA). Actin was used as an internal standard. The expression intensity was normalized by expression of the actin gene in an identical sample (expression level of fescue gene versus actin). The ratio of the expression intensity between pairs of samples was calculated and compared with copy numbers presented in subtraction libraries. General PCR was conducted with the following program: inactivated the reverse transcriptase at 95 °C for 5 min, then followed with 94 °C for 1 min, 52 °C for 1 min, 72 °C for 2 min with 20, 25 or 30 cycles. The PCR products were checked with 3% agarose gel in 1× TAE with EtBr. Results Identification of differentially expressed gene transcripts by SSH The clonally replicated plants of heat-sensitive fescue genotype PI283316 and heat-tolerant genotype PI297901 were used in this study. Three subtractions were conducted between shoot tissues of PI297901 (heat-tolerant) and PI283316 (heat-sensitive) plants treated under 39, 42, and 44 °C for 12 h to isolate differentially expressed genes from both heat-tolerant and sensitive genotypes. A set of 1311 clones that were over-expressed in the heat-tolerant genotype compared with the heat-sensitive genotype were identified by SSH, whereas 2000 clones that were over-expressed in the heat-sensitive genotype were selected by reverse subtractions (see Materials and methods). High throughput sequencing was conducted for all clones. With a 70% success rate, a total of 2495 ESTs were generated with an average length of 310 bp, and submitted to GenBank dbEST (http://www.ncbi.nlm.nih.gov/dbEST) with accession numbers from CK800817 to CK803266. After data assembly, 656 ESTs were found to be singlets, while the other 1839 ESTs clustered into 434 contigs, each contig had 2–28 ESTs with an average length of 591 bp (Table 1). The ESTs generated from each time-point library were also assembled and analysed separately. Including contigs and singlets, there were 299 transcripts identified at 39 °C, 477 transcripts at 42 °C, and 395 transcripts at 44 °C. Therefore, more differentially expressed genes that had higher level of expression in either PI297901 or PI283316 from both forward and reversed libraries were isolated with increased temperatures between these two genotypes. Table 1. Summary of ESTs from fescue heat-stressed SSH libraries . All SSH clones . 39 °C . 42 °C . 44 °C . No. of ESTs 2495 415 759 1312 No. of ESTs for multi-sequence clusters 1839 196 434 1118 No. of contigs 434 80 152 201 With hits 301 (69.4%) 56 (70%) 106 (69.7%) 131 (65.2%) Average contig size (bp) 591 498 609 520 No. of single- sequence clusters (singlet) 656 219 325 194 With hits 397 (60.5%) 144 (65.8%) 196 (60.3%) 85 (43.8%) No. of contigs and singlet 1090 299 477 395 . All SSH clones . 39 °C . 42 °C . 44 °C . No. of ESTs 2495 415 759 1312 No. of ESTs for multi-sequence clusters 1839 196 434 1118 No. of contigs 434 80 152 201 With hits 301 (69.4%) 56 (70%) 106 (69.7%) 131 (65.2%) Average contig size (bp) 591 498 609 520 No. of single- sequence clusters (singlet) 656 219 325 194 With hits 397 (60.5%) 144 (65.8%) 196 (60.3%) 85 (43.8%) No. of contigs and singlet 1090 299 477 395 EST sequences were assembled from clones for all three temperatures (All SSH clones) and for each temperature point (39 °C, 42 °C, and 44 °C) individually. Some transcripts were assembled from clones found at more than one temperature point. Numbers of ESTs listed at each temperature point includes genes selected from both forward and reverse libraries that were differentially expressed in either PI297901 or PI283316. Open in new tab Table 1. Summary of ESTs from fescue heat-stressed SSH libraries . All SSH clones . 39 °C . 42 °C . 44 °C . No. of ESTs 2495 415 759 1312 No. of ESTs for multi-sequence clusters 1839 196 434 1118 No. of contigs 434 80 152 201 With hits 301 (69.4%) 56 (70%) 106 (69.7%) 131 (65.2%) Average contig size (bp) 591 498 609 520 No. of single- sequence clusters (singlet) 656 219 325 194 With hits 397 (60.5%) 144 (65.8%) 196 (60.3%) 85 (43.8%) No. of contigs and singlet 1090 299 477 395 . All SSH clones . 39 °C . 42 °C . 44 °C . No. of ESTs 2495 415 759 1312 No. of ESTs for multi-sequence clusters 1839 196 434 1118 No. of contigs 434 80 152 201 With hits 301 (69.4%) 56 (70%) 106 (69.7%) 131 (65.2%) Average contig size (bp) 591 498 609 520 No. of single- sequence clusters (singlet) 656 219 325 194 With hits 397 (60.5%) 144 (65.8%) 196 (60.3%) 85 (43.8%) No. of contigs and singlet 1090 299 477 395 EST sequences were assembled from clones for all three temperatures (All SSH clones) and for each temperature point (39 °C, 42 °C, and 44 °C) individually. Some transcripts were assembled from clones found at more than one temperature point. Numbers of ESTs listed at each temperature point includes genes selected from both forward and reverse libraries that were differentially expressed in either PI297901 or PI283316. Open in new tab BLAST analysis using the GenBank non-redundant (nr) database revealed that 301 (69.4%) contigs had similarities to known genes and the rest (30.6%) had no hit. Out of 656 singlets, 60.5% had matches to known genes (Table 1). The results were similar to other reports indicating that about 30% of the clones in an SSH library had no similarity to sequences in the database (Desai et al., 2000). Functional classification of differentially expressed genes To understand the molecular mechanisms of heat tolerance of fescue plants, differentially expressed genes selected by subtractions were classified into different categories according to their putative functions generated by BLAST analysis. A total of 698 gene transcripts (301 contigs and 397 singlets) with hits to the GenBank nr database were grouped into 10 functional categories: (1) cell maintenance and development, (2) chloroplast associated and photosynthesis, (3) metabolism, (4) protein synthesis, (5) signalling, (6) stress related, (7) transcription factor, (8) transport, (9) unclassified proteins, and (10) others (Fig. 1). More than 33% of gene transcripts annotated with unknown function were included in the category of unclassified proteins. The largest group of genes with known function was metabolism-related genes and approximately 13% of the transcripts were included in this category. Stress-related as well as chloroplast-associated and photosynthesis genes seem to play important roles in plants under heat stress conditions. 84 (12%) and 72 (10%) gene transcripts that belong to these two categories, respectively, were identified from the three subtraction libraries. The entire list of genes with hits to the GenBank nr database is included in supplementary data file 1 at JXB online. Fig. 1. Open in new tabDownload slide Distribution of differentially expressed gene transcripts in fescue subtraction libraries including all heat treatments of both genotypes. Total 698 gene transcripts with hits to GenBank nr database were grouped into 11 functional categories. The percentage of gene transcripts in each group is listed with the function. Fig. 1. Open in new tabDownload slide Distribution of differentially expressed gene transcripts in fescue subtraction libraries including all heat treatments of both genotypes. Total 698 gene transcripts with hits to GenBank nr database were grouped into 11 functional categories. The percentage of gene transcripts in each group is listed with the function. Differentially expressed gene transcripts with known functions selected from either PI297901 or PI283316 were grouped to reveal the different response of the two fescue genotypes to high-temperature stress. Regardless of the copy number, there were 328 gene transcripts isolated from heat-tolerant genotype whereas 408 genes were from the heat-sensitive genotype considering all three temperatures tested. Percentages of gene transcripts that belonged to each functional category were calculated and compared for differentially expressed genes in either heat-tolerant or heat-sensitive fescue (Fig. 2). Under heat-stress conditions, the genes involved in cell maintenance and development, chloroplast associated and photosynthesis, protein synthesis, signalling, transcription factor, and transport had higher expression levels in the heat-tolerant fescue than the heat-sensitive fescue. Genes related to metabolism and stress responses had higher expression levels in the heat-sensitive genotype (Fig. 2). Fig. 2. Open in new tabDownload slide Differential gene expression of heat-tolerant genotype (PI297901) versus heat-sensitive genotype (PI283316) at all three temperatures with functional classification. The bars represent the percentages of gene transcripts for each genotype in each functional category: (1) cell maintenance and development, (2) chloroplast associated and photosynthesis, (3) metabolism, (4) others, (5) protein synthesis, (6) signalling, (7) stress related, (8) transcription factor, (9) transport, and (10) unclassified proteins. Fig. 2. Open in new tabDownload slide Differential gene expression of heat-tolerant genotype (PI297901) versus heat-sensitive genotype (PI283316) at all three temperatures with functional classification. The bars represent the percentages of gene transcripts for each genotype in each functional category: (1) cell maintenance and development, (2) chloroplast associated and photosynthesis, (3) metabolism, (4) others, (5) protein synthesis, (6) signalling, (7) stress related, (8) transcription factor, (9) transport, and (10) unclassified proteins. The percentages of differentially expressed genes were also compared between two genotypes at each temperature. Although some transcripts were found at more than one temperature point, similar patterns were obtained at all three temperatures (Table 2). Genes involved in cell maintenance and development, chloroplast associated and photosynthesis, and protein synthesis were consistently over-expressed in the heat-tolerant genotype, PI297901, in all three temperatures. Metabolism and stress-related gene transcripts had higher expression in the heat-sensitive genotype, PI283316, under heat stress. The percentages of differentially expressed genes in these two categories increased with increased temperature, although the same pattern was evident at each temperature. For example, there were 17% more metabolism-related genes in the heat-sensitive genotype at 39 °C (12.6% versus 10.8%), 45% at 42 °C (15.9% versus 11.0%), and 69% at 44 °C (14.5% versus 8.6%) compared with the heat-tolerant genotype. Table 2. Distribution of differentially expressed genes in two genotypes at three time points Functional classification . T39 . S39 . T42 . S42 . T44 . S44 . Cell maintenance and development 7 (6.9%) 6 (5.0%) 6 (4.1%) 8 (4.0%) 8 (7.6%) 5 (4.0%) Chloroplast associated and photosynthesis 13 (12.7%) 10 (8.4%) 16 (11.0%) 16 (8.0%) 18 (17.1%) 16 (12.9%) Metabolism 11 (10.8%) 15 (12.6%) 16 (11.0%) 32 (15.9%) 9 (8.6%) 18 (14.5%) Other 5 (4.9%) 12 (10.1%) 7 (4.8%) 22 (10.9%) 5 (4.8%) 10 (8.1%) Protein synthesis 14 (13.7%) 7 (5.9%) 10 (6.8%) 8 (4.0%) 11 (10.5%) 5 (4.0%) Signalling 6 (5.9%) 11 (9.2%) 9 (6.2%) 7 (3.5%) 7 (6.7%) 2 (1.6%) Stress related 10 (9.8%) 14 (11.8%) 16 (11.0%) 34 (16.9%) 16 (15.2%) 24 (19.4%) Transcription factor 4 (3.9%) 1 (0.8%) 5 (3.4%) 2 (1.0%) 1 (1.0%) 2 (1.6%) Transport 2 (2.0%) 4 (3.4%) 7 (4.8%) 10 (5.0%) 4 (3.8%) 5 (4.0%) Unclassified proteins 30 (29.4%) 40 (33.6%) 54 (37.0%) 62 (30.8%) 26 (24.8%) 37 (29.8%) Total 102 120 146 201 105 124 Functional classification . T39 . S39 . T42 . S42 . T44 . S44 . Cell maintenance and development 7 (6.9%) 6 (5.0%) 6 (4.1%) 8 (4.0%) 8 (7.6%) 5 (4.0%) Chloroplast associated and photosynthesis 13 (12.7%) 10 (8.4%) 16 (11.0%) 16 (8.0%) 18 (17.1%) 16 (12.9%) Metabolism 11 (10.8%) 15 (12.6%) 16 (11.0%) 32 (15.9%) 9 (8.6%) 18 (14.5%) Other 5 (4.9%) 12 (10.1%) 7 (4.8%) 22 (10.9%) 5 (4.8%) 10 (8.1%) Protein synthesis 14 (13.7%) 7 (5.9%) 10 (6.8%) 8 (4.0%) 11 (10.5%) 5 (4.0%) Signalling 6 (5.9%) 11 (9.2%) 9 (6.2%) 7 (3.5%) 7 (6.7%) 2 (1.6%) Stress related 10 (9.8%) 14 (11.8%) 16 (11.0%) 34 (16.9%) 16 (15.2%) 24 (19.4%) Transcription factor 4 (3.9%) 1 (0.8%) 5 (3.4%) 2 (1.0%) 1 (1.0%) 2 (1.6%) Transport 2 (2.0%) 4 (3.4%) 7 (4.8%) 10 (5.0%) 4 (3.8%) 5 (4.0%) Unclassified proteins 30 (29.4%) 40 (33.6%) 54 (37.0%) 62 (30.8%) 26 (24.8%) 37 (29.8%) Total 102 120 146 201 105 124 T39: PI297901 sample treated at 39 °C for 12 h. S39: PI283316 sample treated at 39 °C for 12 h. T42: PI297901 sample treated at 42 °C for 12 h. S42: PI283316 sample treated at 42 °C for 12 h. T44: PI297901 sample treated at 44 °C for 12 h. S44: PI283316 sample treated at 44 °C for 12 h. Numbers and percentage of gene transcripts are listed in each function category. EST sequences were assembled for each temperature point (39 °C, 42 °C, and 44 °C) individually. Some transcripts were found at more than one temperature points. Open in new tab Table 2. Distribution of differentially expressed genes in two genotypes at three time points Functional classification . T39 . S39 . T42 . S42 . T44 . S44 . Cell maintenance and development 7 (6.9%) 6 (5.0%) 6 (4.1%) 8 (4.0%) 8 (7.6%) 5 (4.0%) Chloroplast associated and photosynthesis 13 (12.7%) 10 (8.4%) 16 (11.0%) 16 (8.0%) 18 (17.1%) 16 (12.9%) Metabolism 11 (10.8%) 15 (12.6%) 16 (11.0%) 32 (15.9%) 9 (8.6%) 18 (14.5%) Other 5 (4.9%) 12 (10.1%) 7 (4.8%) 22 (10.9%) 5 (4.8%) 10 (8.1%) Protein synthesis 14 (13.7%) 7 (5.9%) 10 (6.8%) 8 (4.0%) 11 (10.5%) 5 (4.0%) Signalling 6 (5.9%) 11 (9.2%) 9 (6.2%) 7 (3.5%) 7 (6.7%) 2 (1.6%) Stress related 10 (9.8%) 14 (11.8%) 16 (11.0%) 34 (16.9%) 16 (15.2%) 24 (19.4%) Transcription factor 4 (3.9%) 1 (0.8%) 5 (3.4%) 2 (1.0%) 1 (1.0%) 2 (1.6%) Transport 2 (2.0%) 4 (3.4%) 7 (4.8%) 10 (5.0%) 4 (3.8%) 5 (4.0%) Unclassified proteins 30 (29.4%) 40 (33.6%) 54 (37.0%) 62 (30.8%) 26 (24.8%) 37 (29.8%) Total 102 120 146 201 105 124 Functional classification . T39 . S39 . T42 . S42 . T44 . S44 . Cell maintenance and development 7 (6.9%) 6 (5.0%) 6 (4.1%) 8 (4.0%) 8 (7.6%) 5 (4.0%) Chloroplast associated and photosynthesis 13 (12.7%) 10 (8.4%) 16 (11.0%) 16 (8.0%) 18 (17.1%) 16 (12.9%) Metabolism 11 (10.8%) 15 (12.6%) 16 (11.0%) 32 (15.9%) 9 (8.6%) 18 (14.5%) Other 5 (4.9%) 12 (10.1%) 7 (4.8%) 22 (10.9%) 5 (4.8%) 10 (8.1%) Protein synthesis 14 (13.7%) 7 (5.9%) 10 (6.8%) 8 (4.0%) 11 (10.5%) 5 (4.0%) Signalling 6 (5.9%) 11 (9.2%) 9 (6.2%) 7 (3.5%) 7 (6.7%) 2 (1.6%) Stress related 10 (9.8%) 14 (11.8%) 16 (11.0%) 34 (16.9%) 16 (15.2%) 24 (19.4%) Transcription factor 4 (3.9%) 1 (0.8%) 5 (3.4%) 2 (1.0%) 1 (1.0%) 2 (1.6%) Transport 2 (2.0%) 4 (3.4%) 7 (4.8%) 10 (5.0%) 4 (3.8%) 5 (4.0%) Unclassified proteins 30 (29.4%) 40 (33.6%) 54 (37.0%) 62 (30.8%) 26 (24.8%) 37 (29.8%) Total 102 120 146 201 105 124 T39: PI297901 sample treated at 39 °C for 12 h. S39: PI283316 sample treated at 39 °C for 12 h. T42: PI297901 sample treated at 42 °C for 12 h. S42: PI283316 sample treated at 42 °C for 12 h. T44: PI297901 sample treated at 44 °C for 12 h. S44: PI283316 sample treated at 44 °C for 12 h. Numbers and percentage of gene transcripts are listed in each function category. EST sequences were assembled for each temperature point (39 °C, 42 °C, and 44 °C) individually. Some transcripts were found at more than one temperature points. Open in new tab Differential gene expression revealed by contig analysis The sequence assembly process revealed that contigs contained multiple fragments isolated from the different conditions tested. This reflects the fact that different cDNA fragments have varied abilities to be enriched, and highly differentially expressed gene sequences are much more prevalent in subtracted libraries than others (Kuang et al., 1998). Therefore, ESTs assembled into the same contigs were analysed based on their copy numbers at each temperature in both genotypes (Fig. 3). There were genes that were expressed only at certain temperatures (65 genes at 39 °C, 121 genes at 42 °C, and 164 genes at 44 °C). As an example, one gene coding for senescence-associated protein was highly differentially expressed only in the heat-sensitive genotype at 44 °C. Fig. 3. Open in new tabDownload slide Venn diagrams showing contigs assembled among temperatures. ESTs assembled into same contigs were analysed based on their copy numbers at each temperature in both genotypes. Numbers of contigs that contain genes from only one temperature are listed under each temperature at 39, 42, and 44°C. Numbers of contigs belonging to genes expressed in different temperatures are located in the overlaying areas of the temperatures. Fig. 3. Open in new tabDownload slide Venn diagrams showing contigs assembled among temperatures. ESTs assembled into same contigs were analysed based on their copy numbers at each temperature in both genotypes. Numbers of contigs that contain genes from only one temperature are listed under each temperature at 39, 42, and 44°C. Numbers of contigs belonging to genes expressed in different temperatures are located in the overlaying areas of the temperatures. A large number of contigs were assembled from gene transcripts selected in more than one SSH library constructed at different temperatures (two or all three temperatures) (Fig. 3). The HSPs and JA-induced protein genes were expressed in all three temperatures for both genotypes, which may be induced by a general stress response. Both RuBisCo and ABC transporters had high levels of expression at all three temperatures, but were expressed only in the heat-tolerant and the heat-sensitive genotypes, respectively. Contig analysis demonstrated that there were more common genes (41 genes) between 42 °C and 44 °C compared with other pair-wise comparisons (16 common genes between 39 °C and 42 °C, and 9 common genes between 39 °C and 44 °C). Verification of heat-regulated differential gene expression To verify if the gene expression corresponding to ESTs generated by SSH were differentially expressed in two fescue genotypes under heat stress, expression of 17 gene contigs selected from subtraction libraries were examined by RT-PCR analysis in both heat-tolerant (PI297901) and heat-sensitive (PI283316) genotypes at all three temperatures. Expression of these genes in corresponding unstressed control plants were also tested by RT-PCR (Fig. 4). One gene each from signalling and transport, two genes each from photosynthesis and protein synthesis, three genes from metabolism, and eight genes from stress-related functional groups were randomly selected to design gene-specific primers based on their contig sequences (Table 3). Fig. 4. Open in new tabDownload slide Expression analysis of selected differentially expressed SSH genes by RT-PCR. Number of PCR cycles is listed on the right side. T39: PI297901 (heat-tolerant) sample treated at 39 °C for 12 h. S39: PI283316 (heat-sensitive) sample treated at 39 °C for 12 h. T42: PI297901 sample treated at 42 °C for 12 h. S42: PI283316 sample treated at 42 °C for 12 h. T44: PI297901 sample treated at 44 °C for 12 h. S44: PI283316 sample treated at 44 °C for 12 h. T39′, S39′, T42′, S42′, T44′, and S44′ were the corresponding untreated samples collected at the same time points with the collections of heat-treated samples. Gene function and corresponding expression patterns extracted by SSH are listed in Table 3. Fig. 4. Open in new tabDownload slide Expression analysis of selected differentially expressed SSH genes by RT-PCR. Number of PCR cycles is listed on the right side. T39: PI297901 (heat-tolerant) sample treated at 39 °C for 12 h. S39: PI283316 (heat-sensitive) sample treated at 39 °C for 12 h. T42: PI297901 sample treated at 42 °C for 12 h. S42: PI283316 sample treated at 42 °C for 12 h. T44: PI297901 sample treated at 44 °C for 12 h. S44: PI283316 sample treated at 44 °C for 12 h. T39′, S39′, T42′, S42′, T44′, and S44′ were the corresponding untreated samples collected at the same time points with the collections of heat-treated samples. Gene function and corresponding expression patterns extracted by SSH are listed in Table 3. Table 3. Differentially expressed transcript contigs of selected SSH genes validated by RT-PCR Primers . Transcript ID . Accession no. . Contig Copy Numbers in SSH . . . . . . Function classification . Genes function with annotation . . . . T39 . S39 . T42 . S42 . T44 . S44 . . . Rubisco Combine.Contig364 CK802928 0 0 6 0 0 0 Photosynthesis Ribulose bisphosphate carboxylase small subunit (Triticum aestivum) RubiSCP Combine.Contig361 CK802664 3 0 5 0 2 0 Photosynthesis Ribulose-bisphosphate carboxylase (EC 4.1.1.39) small chain precursor (Avena clauda) DHS Combine.Contig401 CK802607 7 1 1 0 1 1 Metabolism Deoxyhypusine synthase (Oryza sativa, japonica cultivar-group) FAH Combine.Contig265 CK800883 0 0 0 0 3 0 Metabolism Fumarylacetoacetate hydrolase-related protein (Arabidopsis thaliana) ICDH Combine.Contig337 CK801331 0 0 0 0 5 0 Metabolism NADP-specific isocitrate dehydrogenase (Oryza sativa) SF3B Combine.Contig273 CK802750 0 0 0 0 3 0 Protein synthesis Splicing factor 3B subunit 3 (Oryza sativa, japonica cultivar-group) TEFTu Combine.Contig281 CK803104 0 0 0 0 3 0 Protein synthesis Translational elongation factor Tu (Oryza sativa) IPS Combine.Contig300 CK802850 0 0 3 0 1 0 Signalling Myo-inositol phosphate synthase (Lolium perenne) CP Combine.Contig415 CK801752 0 0 0 3 0 13 Stress-related Cysteine proteinase [Hemerocallis hybrid cultivar] DegP Combine.Contig328 CK801142 0 2 0 2 0 0 Stress-related DegP protease (Arabidopsis thaliana) HSC70 Combine.Contig414 CK800824 6 5 5 0 0 0 Stress-related Heat shock protein cpHsc70-2 (hsc70-7) (Arabidopsis thaliana) LMW-HSP Combine.Contig426 CK801922 0 0 0 1 0 9 Stress-related Class I low-molecular-weight heat shock protein 17.9 (Oryza sativa) Rgene Combine.Contig340 CK802598 3 2 0 0 0 0 Stress-related Disease resistance protein (Oryza sativa) RNS2 Combine.Contig358 CK801125 0 0 0 6 0 0 Stress-related S-like ribonuclease RNS2 (Oryza sativa, japonica cultivar-group) Sense Combine.Contig420 CK801527 0 0 0 0 0 18 Stress-related Senescence-associated protein (Pisum sativum)] VBP Combine.Contig252 CK802953 0 1 0 0 2 0 Stress-related Victorin binding protein (Avena sativa) SAR Combine.Contig389 CK802034 0 2 0 0 0 7 Transcription factor SAR DNA binding protein (Oryza sativa) Primers . Transcript ID . Accession no. . Contig Copy Numbers in SSH . . . . . . Function classification . Genes function with annotation . . . . T39 . S39 . T42 . S42 . T44 . S44 . . . Rubisco Combine.Contig364 CK802928 0 0 6 0 0 0 Photosynthesis Ribulose bisphosphate carboxylase small subunit (Triticum aestivum) RubiSCP Combine.Contig361 CK802664 3 0 5 0 2 0 Photosynthesis Ribulose-bisphosphate carboxylase (EC 4.1.1.39) small chain precursor (Avena clauda) DHS Combine.Contig401 CK802607 7 1 1 0 1 1 Metabolism Deoxyhypusine synthase (Oryza sativa, japonica cultivar-group) FAH Combine.Contig265 CK800883 0 0 0 0 3 0 Metabolism Fumarylacetoacetate hydrolase-related protein (Arabidopsis thaliana) ICDH Combine.Contig337 CK801331 0 0 0 0 5 0 Metabolism NADP-specific isocitrate dehydrogenase (Oryza sativa) SF3B Combine.Contig273 CK802750 0 0 0 0 3 0 Protein synthesis Splicing factor 3B subunit 3 (Oryza sativa, japonica cultivar-group) TEFTu Combine.Contig281 CK803104 0 0 0 0 3 0 Protein synthesis Translational elongation factor Tu (Oryza sativa) IPS Combine.Contig300 CK802850 0 0 3 0 1 0 Signalling Myo-inositol phosphate synthase (Lolium perenne) CP Combine.Contig415 CK801752 0 0 0 3 0 13 Stress-related Cysteine proteinase [Hemerocallis hybrid cultivar] DegP Combine.Contig328 CK801142 0 2 0 2 0 0 Stress-related DegP protease (Arabidopsis thaliana) HSC70 Combine.Contig414 CK800824 6 5 5 0 0 0 Stress-related Heat shock protein cpHsc70-2 (hsc70-7) (Arabidopsis thaliana) LMW-HSP Combine.Contig426 CK801922 0 0 0 1 0 9 Stress-related Class I low-molecular-weight heat shock protein 17.9 (Oryza sativa) Rgene Combine.Contig340 CK802598 3 2 0 0 0 0 Stress-related Disease resistance protein (Oryza sativa) RNS2 Combine.Contig358 CK801125 0 0 0 6 0 0 Stress-related S-like ribonuclease RNS2 (Oryza sativa, japonica cultivar-group) Sense Combine.Contig420 CK801527 0 0 0 0 0 18 Stress-related Senescence-associated protein (Pisum sativum)] VBP Combine.Contig252 CK802953 0 1 0 0 2 0 Stress-related Victorin binding protein (Avena sativa) SAR Combine.Contig389 CK802034 0 2 0 0 0 7 Transcription factor SAR DNA binding protein (Oryza sativa) The copy numbers of each contig assembled from each subtraction library are listed under Contig copy numbers in SSH. ‘0’ represents no differential expression in one set of subtraction between PI297901 versus PI283316 at each temperature points. T39: PI297901 sample treated at 39 °C for 12 h. S39: PI283316 sample treated at 39 °C for 12 h. T42: PI297901 sample treated at 42 °C for 12 h. S42: PI283316 sample treated at 42 °C for 12 h. T44: PI297901 sample treated at 44 °C for 12 h. S44: PI283316 sample treated at 44 °C for 12 h. Open in new tab Table 3. Differentially expressed transcript contigs of selected SSH genes validated by RT-PCR Primers . Transcript ID . Accession no. . Contig Copy Numbers in SSH . . . . . . Function classification . Genes function with annotation . . . . T39 . S39 . T42 . S42 . T44 . S44 . . . Rubisco Combine.Contig364 CK802928 0 0 6 0 0 0 Photosynthesis Ribulose bisphosphate carboxylase small subunit (Triticum aestivum) RubiSCP Combine.Contig361 CK802664 3 0 5 0 2 0 Photosynthesis Ribulose-bisphosphate carboxylase (EC 4.1.1.39) small chain precursor (Avena clauda) DHS Combine.Contig401 CK802607 7 1 1 0 1 1 Metabolism Deoxyhypusine synthase (Oryza sativa, japonica cultivar-group) FAH Combine.Contig265 CK800883 0 0 0 0 3 0 Metabolism Fumarylacetoacetate hydrolase-related protein (Arabidopsis thaliana) ICDH Combine.Contig337 CK801331 0 0 0 0 5 0 Metabolism NADP-specific isocitrate dehydrogenase (Oryza sativa) SF3B Combine.Contig273 CK802750 0 0 0 0 3 0 Protein synthesis Splicing factor 3B subunit 3 (Oryza sativa, japonica cultivar-group) TEFTu Combine.Contig281 CK803104 0 0 0 0 3 0 Protein synthesis Translational elongation factor Tu (Oryza sativa) IPS Combine.Contig300 CK802850 0 0 3 0 1 0 Signalling Myo-inositol phosphate synthase (Lolium perenne) CP Combine.Contig415 CK801752 0 0 0 3 0 13 Stress-related Cysteine proteinase [Hemerocallis hybrid cultivar] DegP Combine.Contig328 CK801142 0 2 0 2 0 0 Stress-related DegP protease (Arabidopsis thaliana) HSC70 Combine.Contig414 CK800824 6 5 5 0 0 0 Stress-related Heat shock protein cpHsc70-2 (hsc70-7) (Arabidopsis thaliana) LMW-HSP Combine.Contig426 CK801922 0 0 0 1 0 9 Stress-related Class I low-molecular-weight heat shock protein 17.9 (Oryza sativa) Rgene Combine.Contig340 CK802598 3 2 0 0 0 0 Stress-related Disease resistance protein (Oryza sativa) RNS2 Combine.Contig358 CK801125 0 0 0 6 0 0 Stress-related S-like ribonuclease RNS2 (Oryza sativa, japonica cultivar-group) Sense Combine.Contig420 CK801527 0 0 0 0 0 18 Stress-related Senescence-associated protein (Pisum sativum)] VBP Combine.Contig252 CK802953 0 1 0 0 2 0 Stress-related Victorin binding protein (Avena sativa) SAR Combine.Contig389 CK802034 0 2 0 0 0 7 Transcription factor SAR DNA binding protein (Oryza sativa) Primers . Transcript ID . Accession no. . Contig Copy Numbers in SSH . . . . . . Function classification . Genes function with annotation . . . . T39 . S39 . T42 . S42 . T44 . S44 . . . Rubisco Combine.Contig364 CK802928 0 0 6 0 0 0 Photosynthesis Ribulose bisphosphate carboxylase small subunit (Triticum aestivum) RubiSCP Combine.Contig361 CK802664 3 0 5 0 2 0 Photosynthesis Ribulose-bisphosphate carboxylase (EC 4.1.1.39) small chain precursor (Avena clauda) DHS Combine.Contig401 CK802607 7 1 1 0 1 1 Metabolism Deoxyhypusine synthase (Oryza sativa, japonica cultivar-group) FAH Combine.Contig265 CK800883 0 0 0 0 3 0 Metabolism Fumarylacetoacetate hydrolase-related protein (Arabidopsis thaliana) ICDH Combine.Contig337 CK801331 0 0 0 0 5 0 Metabolism NADP-specific isocitrate dehydrogenase (Oryza sativa) SF3B Combine.Contig273 CK802750 0 0 0 0 3 0 Protein synthesis Splicing factor 3B subunit 3 (Oryza sativa, japonica cultivar-group) TEFTu Combine.Contig281 CK803104 0 0 0 0 3 0 Protein synthesis Translational elongation factor Tu (Oryza sativa) IPS Combine.Contig300 CK802850 0 0 3 0 1 0 Signalling Myo-inositol phosphate synthase (Lolium perenne) CP Combine.Contig415 CK801752 0 0 0 3 0 13 Stress-related Cysteine proteinase [Hemerocallis hybrid cultivar] DegP Combine.Contig328 CK801142 0 2 0 2 0 0 Stress-related DegP protease (Arabidopsis thaliana) HSC70 Combine.Contig414 CK800824 6 5 5 0 0 0 Stress-related Heat shock protein cpHsc70-2 (hsc70-7) (Arabidopsis thaliana) LMW-HSP Combine.Contig426 CK801922 0 0 0 1 0 9 Stress-related Class I low-molecular-weight heat shock protein 17.9 (Oryza sativa) Rgene Combine.Contig340 CK802598 3 2 0 0 0 0 Stress-related Disease resistance protein (Oryza sativa) RNS2 Combine.Contig358 CK801125 0 0 0 6 0 0 Stress-related S-like ribonuclease RNS2 (Oryza sativa, japonica cultivar-group) Sense Combine.Contig420 CK801527 0 0 0 0 0 18 Stress-related Senescence-associated protein (Pisum sativum)] VBP Combine.Contig252 CK802953 0 1 0 0 2 0 Stress-related Victorin binding protein (Avena sativa) SAR Combine.Contig389 CK802034 0 2 0 0 0 7 Transcription factor SAR DNA binding protein (Oryza sativa) The copy numbers of each contig assembled from each subtraction library are listed under Contig copy numbers in SSH. ‘0’ represents no differential expression in one set of subtraction between PI297901 versus PI283316 at each temperature points. T39: PI297901 sample treated at 39 °C for 12 h. S39: PI283316 sample treated at 39 °C for 12 h. T42: PI297901 sample treated at 42 °C for 12 h. S42: PI283316 sample treated at 42 °C for 12 h. T44: PI297901 sample treated at 44 °C for 12 h. S44: PI283316 sample treated at 44 °C for 12 h. Open in new tab The SSH (BD Clontech PCR-Select Kit) technique is expected to amplify differentially expressed genes between tester and driver RNA. The level of enrichment of a particular cDNA depends greatly on its original abundance, and therefore is biased towards the enrichment of genes with larger differences in expression (Desai et al., 2000). Some fragments of a given differentially expressed gene may be eliminated during the SSH, sub-cloning, or sequencing procedures, whereas other fragments of the same gene may be enriched and isolated. On the other hand, RT-PCR amplified the transcripts from both tester and driver genotypes. The only difference is in the amount of the PCR products, and thus the intensity of the PCR bands in the gel. It is often difficult to verify such differences in the band intensities, particularly with the naked eye. Therefore, there are some discrepancies between the data reported in Table 3 and Fig. 4. In most part, however, there were high levels of similarities detected between the expression patterns obtained from SSH and RT-PCR products of the 17 genes (Table 3; Fig. 4). SSH indicated higher expressions of the Rubisco homologue gene (RubiSCP) in heat-tolerant fescue, PI297901, under heat stress at all three temperatures (Table 3). However, the expression of this gene was also detected in the heat-sensitive genotype, PI283316, by RT-PCR (Fig. 4). The expressed transcript fragments of this gene in PI283316 were subtracted by identical fragments of the gene in PI297901, therefore, there was no copy of this gene transcript isolated from PI283316 by SSH. Genes coding for cysteine proteinase (CP) and SAR DNA binding protein (SAR) were isolated by SSH, and each showed higher expression in the heat-sensitive genotype under heat stress at two of the three temperature points. RT-PCR results indicated that these two genes were differentially expressed in heat-sensitive genotype at all three temperature points. SSH detected significantly higher expression of cysteine proteinase gene (13 copies) and SAR DNA binding protein gene (7 copies) at 44 °C in PI283316, however, the expression of CP at 39 °C and SAR at 42 °C were not detected. These differentially expressed gene transcripts were probably lost during subcloning or sequencing. SSH identified one stress-related gene, LMW-HSP (Combine.Contig426) encoding for class I low-molecular-weight heat shock protein only from the heat-sensitive genotype at 42 °C and 44 °C with 1 and 9 copies, respectively. RT-PCR indicated that this gene was expressed at all three temperatures points in both genotypes (Table 3; Fig. 4). Such discrepancies between SSH and RT-PCR results are again attributable to one or more of the factors mentioned above. The comparisons of expression of the 17 genes under heat-stress versus control environments revealed several interesting insights into the expression of these genes. Two genes (LMW-HSP and Sense) were expressed only in heat-treated plants, and were not detectable in non-stressed ones, therefore, were tightly associated with heat treatment (Fig. 4). Compared with the untreated control, three genes (ICDH, IPS, and RNS2) had higher expression in heat-treated plants. Genes coding for the ribulose bisphosphate carboxylase small subunit (Rubisco and RubiSCP), and victorin binding protein (VBP) were expressed in all conditions in both genotypes, but were significantly suppressed in the heat-sensitive genotype at 44 °C. Compared with non-stressed plants, expression of genes coding for disease resistance protein (Rgene) and myo-inositol phosphate synthase (IPS) were higher in the heat-tolerant genotype, PI297901, under high heat conditions (42 °C and 44 °C). The cysteine proteinase (CP) gene and the SAR DNA binding protein (SAR) gene had higher expression in heat-sensitive plants compared with heat-tolerant plants under heat stress and to a lesser extent under control environments. Both heat-shock protein homologue genes (HSC70 and LMW-HSP) were differentially expressed, however, with noticeably different patterns (Fig. 4). Discussion 1090 gene transcripts (434 contigs and 656 singlets) with differential expression in two fescue genotypes under high-temperature stress have been identified. These include genes encoding proteins that are involved in the general defence responses (CuZn-superoxide dismutase, ascorbate reductase, glutathione S-transferase, and thaumatin-like protein), heat shock, and other stress conditions (senescence-, wound- and pathogen-related stresses). This result suggests that plants may use some common molecular mechanisms (e.g. signal transductions and transcriptional regulations) in response to various stress conditions. Cell structure associated heat tolerance After exposure to 42 °C for 24 h, PI297901 and PI283316 showed clear visible difference in their stress levels (e.g. discolouration and drying of leaves). Therefore, it is not surprising to have more differentially expressed gene transcripts isolated from the subtraction libraries at higher temperatures than 39 °C. A previous study (Zhang et al., 2004) demonstrated that the morphology of PI297901 and PI283316 is different even under normal growing conditions. A histological study of leaf tissue illustrated distinctly different cell structures between these two genotypes. Heat-tolerant fescue PI297901 had larger cells and more sclerenchyma and collenchyma (supporting tissue) in between the vascular bundle and epidermal cells compared with PI283316 (Zhang et al., 2004). Genes coding for homologues of glycine-rich protein (CK803136) and extensin-like protein (CK803238) were selected from the subtraction library of PI297901 treated at 44 °C. These two proteins are well-characterized structural proteins known to determine the physical characteristics of the plant cell wall (Ringli et al., 2001; Bucher et al. 2002). Therefore, the strong cell structure of PI297901 might be associated with the heat tolerance of this genotype. However, the function of these two genes in heat tolerance needs to be studied in more detail to support the putative correlation between morphology and heat tolerance of PI297901. Acquired thermotolerance in fescue To mimic natural conditions, the growth chamber experiment was designed with slow step-wise temperature increases (see Materials and methods). Therefore, all plants were acclimatized to the heat conditions. Key et al. (1983) indicated that acclimated plants can acquire thermotolerance. The acclimation process is thought to involve new proteins synthesized in response to high temperature that confer thermotolerance to the organism. Results of this study indicated that heat-tolerant PI297901 had higher expression of photosynthesis as well as of protein synthesis genes compared with the heat-sensitive genotype under all three temperatures, which may help this genotype of fescue to maintain cellular functions under heat stress. Considering all three temperature points, there was significantly higher expression of transcription factor genes in heat-tolerant plants (Fig. 2), especially under the early heat condition at 39 °C (3.9% versus 0.8%) and 42 °C (3.4% versus 1.0%) (Table 2). Transcription factors usually play important roles in signal transduction pathways and are the earliest group of genes to respond to biotic and abiotic stresses (Shinozaki et al., 2003). Molecular and genomic studies have shown that various transcription factors are involved in the regulation of stress-inducible genes (Uno et al., 2000; Sakuma et al., 2002; Doubuzet et al., 2003). Heat-shock proteins (HSPs) are known to play important roles in protecting organisms under high heat-stress conditions and are grouped into five distinct classes named for their approximate molecular masses (Vierling, 1991). Except for HSP60, the other four classes of heat-shock proteins, including HSP100, HSP90, HSP70, and low molecular weight HSPs (HSP16.9 and HSP26.7) that differentially expressed between the two genotypes of fescues under heat stress were cloned in this study (see supplementary data file 1 for the original data at JXB online). HSP70 proteins are essential for normal cell function (Hartl, 1996). Some members of this family are expressed constitutively; others are induced by heat or cold (Karlin and Brocchieri, 1998). Several HSP70 protein gene transcripts (Combine.Contig414, NF17b04f42.r1, and NF25g03f44.r1) have been identified by SSH in this study, and all of them had higher expressions in PI297901. One of these HSP70 genes was probably induced by the high heat condition (44 °C) compared with unstressed plants as indicated by RT-PCR (Fig. 4, HSC70), although the expression levels were almost identical for heat-treated and untreated plants for each genotype at 39 °C and 42 °C. Study has shown that HSP70 is required for survival at moderately high temperatures, but not for surviving extreme temperatures in E. coli and yeast (Deloche et al., 1997). In addition, the expression of this gene was much higher in PI297901 under all conditions (heat-treated and untreated), therefore, the differential expression identified by SSH maybe due to the genetic background difference of PI297901 and PI283316. Low molecular weight heat shock proteins or small heat shock proteins (smHSPs) are produced ubiquitously in prokaryotic and eukaryotic cells upon heat (Waters et al., 1996). Six classes of smHSPs have been identified in plants, suggesting the special importance of these proteins (Scharf et al., 2001). Although the function of smHSPs have not been demonstrated definitively, there is strong evidence that they play a role as molecular chaperones that bind to partially folded or denatured substrate proteins and thereby prevent irreversible aggregation or promote correct substrate folding to protect cells from stress damage (Sun et al., 2002). However, there is no evidence that they are required for normal cellular function. Results of RT-PCR experiment in our study (Fig. 4) clearly confirmed there was no expression of this gene (LMW-HSP) in non-stressed condition and a strong activation in both genotypes under heat stress. In addition, there was a higher expression of this gene in the heat-sensitive genotype compared to the heat-tolerant genotype with heat treatment (Fig. 4). SSH cloned three other low molecular weight heat shock proteins (Combine.Contigs362, 417, 430) that differentially expressed in the sensitive genotype only, and an additional one (Combine.Contig432) were found in both genotypes, but only in the sensitive one at higher temperatures (see supplementary data file 1 for the original data at JXB online). This may imply that the repair and protection mechanisms were highly activated in heat-sensitive plants for surviving under stressful conditions. The effect of high temperature on higher plants is primarily on photosynthetic functions (Crafts-Brandner and Salvucci, 2000). The heat-tolerance limits of higher plants are determined by the thermal sensitivity of primary photochemical reactions occurring in the thylakoid membrane system (Vani et al., 2001). Studies have shown that long-term acclimations can be superimposed upon fast adaptive adjustment of the thermal stability (Weis and Berry, 1988). Results of this study met the expectation that more chloroplast-associated and photosynthesis-related genes would be expressed in the heat-tolerant genotype of fescue subjected to high temperature. Although the functions of these genes are not clear, they could play important roles in tolerant genotypes to sustain heat stress. Metabolism- and stress-related genes were found more frequently in the heat-sensitive genotype compared with the heat-tolerant genotype. Results of RT-PCR also identified stress-associated proteins, for example, cysteine proteinase as well as senescence-associated protein genes that were induced in PI283316. This result suggests that the heat-sensitive fescue plants were under great stress and they tried to survive by using more metabolites through glycolysis, and protein and lipid degradation. Interestingly, the percentages of stress-related genes were not increased gradually with higher temperatures. There were 20.4%, 53.6%, and 27.6% more stress-related genes expressed in heat-sensitive PI283316 compared with heat-tolerant PI297901 at 39 °C, 42 °C, and 44 °C, respectively. It is possible that heat-sensitive plants may encounter a critical stress condition at 42 °C that resulted in a large number of stress-related genes, and at 44 °C many of the defence mechanisms of this genotype may have collapsed leading to death of the plant. In summary, 2495 ESTs have been cloned and sequenced from three SSH libraries constructed by using the heat-stressed shoot tissues of a heat-tolerant and a heat-sensitive fescue genotype. A number of gene transcripts coding for known stress-related proteins, including cysteine proteinase, heat shock proteins, senescence associated proteins, and victorin binding proteins were cloned and sequenced in this study. In addition, a large number of novel genes with unknown functions that may have a potential role in heat tolerance in fescues were also cloned and sequenced. The expression patterns of 17 differentially expressed genes with known functions were confirmed by RT-PCR. By comparing the RT-PCR profiles of the heat-stressed and control plants, the differential expression of at least 11 of these genes could be attributed to heat stress rather than to the differences in the genetic backgrounds of the two genotypes. The expression patterns of the other differentially expressed genes reported in this study will also need to be confirmed by RT-PCR or microarray experiments. The primers designed from the heat responsive genes can be used for the identification of quantitative trait loci for heat tolerance in fescue through molecular mapping or association studies. We would like to thank Drs Mark E Sorrells and Kiran Mysore for reviewing the manuscript. 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TI - Differential gene expression in Festuca under heat stress conditions
JO - Journal of Experimental Botany
DO - 10.1093/jxb/eri082
DA - 2005-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/differential-gene-expression-in-festuca-under-heat-stress-conditions-HSN3UdyUzc
SP - 897
EP - 907
VL - 56
IS - 413
DP - DeepDyve
ER -