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Cytokinins (CKs) are known to regulate leaf senescence and affect heat tolerance, but mechanisms underlying CK regulation of heat tolerance are not well understood. A comprehensive proteomic study was conducted to identify proteins altered by the expression of the adenine isopentenyl transferase (ipt) gene controlling CK synthesis and associated with heat tolerance in transgenic plants for a C3 perennial grass species, Agrostis stolonifera. Transgenic plants with two different inducible promoters (SAG12 and HSP18) and a null transformant (NT) containing the vector without ipt were exposed to 20°C (control) or 35°C (heat stress) in growth chambers. Two-dimensional electrophoresis and mass spectrometry analysis were performed to identify protein changes in leaves and roots in response to ipt expression under heat stress. Transformation with ipt resulted in protein changes in leaves and roots involved in multiple functions, particularly in energy metabolism, protein destination and storage, and stress defence. The abundance levels of six leaf proteins (enolase, oxygen-evolving enhancer protein 2, putative oxygen-evolving complex, Rubisco small subunit, Hsp90, and glycolate oxidase) and nine root proteins (Fd-GOGAT, nucleotide-sugar dehydratase, NAD-dependent isocitrate dehydrogenase, ferredoxin-NADP reductase precursor, putative heterogeneous nuclear ribonucleoprotein A2, ascorbate peroxidase, dDTP-glucose 4–6-dehydratases-like protein, and two unknown proteins) were maintained or increased in at least one ipt transgenic line under heat stress. The diversity of proteins altered in transgenic plants in response to heat stress suggests a regulatory role for CKs in various metabolic pathways associated with heat tolerance in C3 perennial grass species.
Heat stress is a major factor influencing the growth of cool-season plant species during summer. Plant adaptation to heat stress involves changes in various processes, including hormone metabolism (Wahid et al., 2007). Heat stress inhibits synthesis and causes degradation of cytokinins (CKs), hormones known to regulate various growth and development processes, including cell division, leaf senescence, and root growth (Nooden and Leopold, 1988; Hoth et al., 2003; Heintz et al., 2006). CKs may play important roles in regulating plant responses to heat stress.
Exogenous application of CKs has been found to be effective to suppress leaf senescence (Singh et al., 1992) and mitigate root mortality and root electrolyte leakage, leading to improved heat tolerance (Liu et al., 2002; Xu and Huang, 2009). Overexpression of a gene isolated from Agrobacterium tumefaciens that encodes the enzyme adenine isopentenyl transferase (ipt), which catalyses the rate-limiting step in CK biosynthesis, has also demonstrated positive effects of elevated levels of CK in delaying leaf senescence and improving stress tolerances in various plant species (Gan and Amasino, 1995; Veselov et al., 1995; Clark et al., 2004; Huynh et al., 2005; Rivero et al., 2007), including heat tolerance in perennial grass species (Xu et al., 2009). The expression of the ipt gene in creeping bentgrass (Agrostis stolonifera), a heat-sensitive C3 perennial grass species, improved heat tolerance, as manifested by increases in tiller formation and root production and delay in leaf senescence under heat stress (Xu et al., 2009; Xing et al., 2010). Despite physiological studies demonstrating the positive effects of CKs on stress tolerance, the biochemical and molecular mechanisms of CK regulation of plant stress tolerance, particularly heat tolerance, are largely unknown.
Recent advances in proteomics have made it possible to perform large-scale, quantitative measurements of protein composition in plants, providing a powerful approach to discovering the genes and pathways that are crucial for stress responsiveness and tolerance (Chen and Harmon, 2006; Yoshimura et al., 2008). Proteomic analysis using two-dimensional gel electrophoresis (2D-GE) and mass spectrometry has identified many stress-responsive proteins in various plant species in response to a wide range of abiotic stresses, including heat stress (Swidzinski et al., 2004; Ferreira et al., 2006; Lee et al., 2007; Valcu et al. 2008; Xu et al., 2008). Previous studies have also detected some proteins responsive to changes in CK production through exogenous application of CKs or overexpression of the ipt gene to increase CK synthesis. Wingler et al. (1998) reported that ipt transgenic tobacco plants that produced more CK delayed the decline in Rubisco content in senescent tissues. Zavaleta-Mancera et al. (2007) reported that incubation of wheat leaves (Triticum aestivum) with 6-benzylaminopurine (BAP) reduced the degradation of Rubisco large and small subunits during dark-induced senescence. However, a large-scale analysis of proteomic changes associated with changing endogenous production of CKs, particularly in relation to heat stress tolerance, is lacking. Knowledge of proteins conferring stress tolerance that may be regulated by CKs may provide further insights into the molecular mechanisms of CK-regulated stress tolerance.
The objectives of this study were to identify protein changes associated with increases in endogenous CK production through ipt transformation in creeping bentgrass and determine the proteomic mechanisms underlying CK regulation of C3 perennial grass responses to heat stress. Specifically, the study was conducted to compare differentially expressed proteins in leaves and roots between ipt transgenic creeping bentgrass lines and a non-ipt transgenic control line [a null transformant (NT) containing the vector without ipt] exposed to heat stress. Transgenic lines were generated using two inducible promoters from Arabidopsis thaliana (SAG12 and HSP18) to avoid overproduction of CKs with constitutive promoters. The SAG12-ipt construct has an autoregulatory feature in that the transcription of SAG12-ipt is activated in response to leaf senescence, leading to CK production, which in turn suppresses senescence; the SAG12 promoter then attenuates ipt transcription and subsequent enzyme production, which prevent overproduction of CKs (Gan and Amasino, 1995, 1996). Rivero et al. (2007) used the promoter from a senescence-associated receptor protein kinase gene (SARK) as a promoter for ipt and found that expression of SARK-ipt delayed drought-induced leaf senescence in tobacco. The small heat shock protein (Hsp) gene promoter in the HSP18-ipt line is heat inducible, with an optimum induction temperature at 35–37°C (Takahashi et al., 1992; Yoshida et al., 1995). Hsp promoters have been used to control gene transcription to increase CK synthesis in tobacco (Schmülling et al., 1989; Smart et al., 1991; Smigocki, 1991; Van Loven et al., 1993). In this study, 2D-GE followed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was used to identify proteins that are differentially expressed between the ipt lines and the NT after plants were exposed to 10d of heat stress at 35°C. The putative roles of the identified proteins in senescence and heat tolerance of the plants are discussed.
Transgenic creeping bentgrass cv. Penncross expressing SAG12-ipt or HSP18-ipt was generated from stolons of a single plant in order to produce transgenic plants with an identical genetic background. The ipt gene is from the Ti plasmid of A. tumefaciens. The construct pCAMBIA1300-SAG12-ipt was created from pSG516 (Gan and Amasino, 1995) and pCAMBIA 1300, a binary plasmid containing the gene for hygromycin resistance. The construct pCAMBIA1301-HSP18-ipt-GUS was created from pUC-HSP18, pSG516, and pCAMBIA 1301, a binary plasmid containing the genes for hygromycin and β-glucuronidase (GUS). Both constructs were introduced into A. tumefaciens LBA4404 by electroporation. The pCAMBIA1300 and pCAMBIA1301 without ipt were used to generate NT control lines; see Xu et al. (2009) and Xing et al. (2010) for details. Two ipt-transgenic lines, SAG12-ipt (S41) and HSP18-ipt (H31), were selected after northern and CK analyses confirmed ipt expression and elevated CK production under stress, compared with an NT line containing the pCAMBIA1301 vector without ipt. These lines were chosen because they were representative of typical heat stress responses among nine transgenic lines evaluated from each category (Xing et al., 2010).
Plants of S41, H31, and NT were vegetatively propagated in plastic pots (15cm in diameter and 20cm deep) filled with sterilized sand and established in a greenhouse. The greenhouse had natural light averaging 600μmol m−2 s−1 photosynthetic photon flux density at canopy height for a 12h photoperiod and an average air temperature of 21°C/14°C (day/night). Plants were watered daily and fertilized twice a week with half-strength Hoagland's solution (Hoagland and Arnon, 1950). After 42d of establishment in the greenhouse, clonal plants were transferred to controlled-environment growth chambers (Conviron, Winnipeg, Canada) with a temperature of 20°C/15°C (day/night), 12h photoperiod, 60% relative humidity, and 500μmol m−2 s−1 photosynthetic photon flux density at canopy height. Plants were allowed to acclimate to growth chamber conditions for 7d before temperature treatments were imposed.
Plants of S41, H31, and NT were exposed to 35°C/30°C (day/night) (heat stress) or 20°C/15°C (day/night) (control) in growth chambers for 10d. Each temperature treatment was repeated in three growth chambers. Plants were arranged randomly inside each chamber and relocated within and among chambers every 3d to minimize environment differences among and within the chambers. Plants were watered twice daily until free drainage occurred from the bottom of the container to prevent water deficit during the heat treatment period, and fertilized weekly using half-strength Hoagland's nutrient solution (Hoagland and Arnon, 1950).
Leaf chlorophyll content was measured to evaluate leaf senescence in transgenic and NT plants. Leaf chlorophyll was extracted from ~0.2g of fresh leaves using dimethylsulphoxide. The absorbance of leaf extracts was determined using a spectrophotometer (Spectronic Genesys2, Spectronic Instruments, Rochester, NY, USA.). Chlorophyll content was calculated based on the absorbance at 663nm and 645nm using the formulae described by Arnon (1949).
Leaf isopentenyladenosine (IPA), the form of CK whose production was directly controlled by the ipt gene, was quantified by an indirect competitive enzyme-linked immunosorbent assay (ELISA). Extraction and quantification of hormones followed the method described by Setter et al. (2001) with some modifications (Wang et al., 2003). Briefly, samples were extracted in 80% (v/v) methanol and isolated with reverse phase C18 columns. Hydrophilic contaminants were washed out with 200ml of 20% solvent [20% methanol, 80% aqueous triethylamine (TEA, 10mM, pH 3.5)]. The CK-containing fraction was eluted using 200μl of 30% solvent (30% methanol, 70% aqueous TEA). A mouse monoclonal antibody against IPA (Agdia, Inc., Elkhart, IN, USA) and a goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma, St Louis, MO, USA) were used as the primary and secondary antibodies, respectively.
At the end of the treatment, root growth response to heat stress was evaluated. The root system was washed free of sand and organic matter. Root fresh weight was measured after blotting dry.
Total RNA was extracted from leaves or roots using TRIZOL Reagent (Invitrogen, USA). A 10μg aliquot of RNA from each leaf sample or 25μg of RNA from each root sample were size fractionated in a 1.2% (w/v) agarose gel for 3h at 65V and transferred to a nylon membrane using a capillary blot method. The membranes were UV cross-linked. Different gene fragments were separately labelled using a random primed labelling kit (Ambion, USA). The labelled probes were purified using NICK™ columns (GE Healthcare, Sweden). Hybridization was carried out at 42°C overnight in a northernMax pre-hybridization/hybridization buffer (Ambion, USA). The membranes were washed with 2× SSC, 0.2% (w/v) SDS at 42°C for 10min, and then with 0.1× SSC, 0.1% SDS at 42°C for 10min. Membranes were exposed to X-ray film (Fuji photo film, Japan) at –80°C for signal detection. The northern blots were repeated three times for each sample. The best representative image from the three replicates was presented.
Bulk shoot and root samples (comprising both mature and young tissues) were harvested from each pot at the end of the treatment (10d) and immediately frozen in liquid nitrogen. The samples were then ground into fine powder and stored at –80°C before analysis. Proteins were extracted using the trichloroacetic acid/acetone method described by Xu et al. (2008). About 0.5g of leaf or 1g of root samples were homogenized on ice in 10ml of precipitation solution (10% trichloroacetic acid and 0.07% 2-mercaptoethanol in acetone) for 10min and incubated at –20°C for 2h. The protein pellet was collected and washed with cold acetone containing 0.07% 2-mercaptoethanol until the supernatant was colourless. The pellet was then vacuum-dried and suspended in resolubilization solution (8M urea, 2M thiourea, 2% CHAPS, 1% dithiothreitol, and 1% pharmalyte). The suspension was centrifuged at 21000g for 20min and the supernatant was collected for protein quantification.
Protein content was determined using the method of Bradford (1976). A 10μl aliquopt of protein extract was mixed with 0.5ml of a commercial dye reagent (diluted five times) (Bio-Rad Laboratories, Hercules, CA, USA). The absorbance was measured spectrophotometrically at 595nm between 5min and 30min after reaction. A standard curve was made from bovine serum albumin.
An IPGPhor apparatus (GE Healthcare, Piscataway, NJ, USA) was used for isoelectric focusing (IEF). Portions of the extracts containing 300μg of protein were subjected to IEF in immobilized pH gradient strips (pH 3.0–10.0, linear gradient, 13cm), formed by rehydrating the strips for 12h at room temperature in 250μl of rehydration buffer (8M urea, 2M thiourea, 2% CHAPS, 1% dithiothreitol, 1% pharmalyte, and 0.002% bromophenol blue). The voltages for IEF were 500V for 1h, 1000V for 1h, 5000V for 1h, and 8000V for 80kVh. Following IEF, the strips were equilibrated for 15min twice at room temperature in equilibration buffer I (50mM TRIS-HCl pH 8.8, 6M urea, 30% glycerol, 2% SDS, and 1% dithiothreitol), then transferred to equilibration buffer II (50mM TRIS-HCl pH 8.8, 6M urea, 30% glycerol, 2% SDS, and 2.5% iodoacetamide). The second dimension electrophoresis was performed on a 12.5% SDS–polyacrylamide gel using a Hoefer SE 600 Ruby electrophoresis unit (GE Healthcare, USA). The running conditions were 5mA per strip for 30min followed by 20mA per strip for 5h. The gels were stained with Coomassie brilliant blue (CBB) G-250 and scanned using a Personal Densitometer (GE Healthcare, USA).
Gel images were analyzed using Progenesis software (Nonlinear Dynamics, Durham, NC, USA). Manual correction and editing of spot features created by automatic default spot analysis settings were included. The spot volumes were normalized as a percentage of the total volume of all spots on the gel in order to correct the variability due to staining. Data were subjected to analysis of variance to test the treatment effects on each transgenic line. Means were separated by Fisher's protected least significance difference (LSD) test (P <0.05).
Selected protein spots were manually excised from gels and subjected to digestion with trypsin. The peptides were analyzed by MALDI-TOF-MS as described by Xu and Huang (2008). Data were searched against the NCBI database using a local MASCOT search engine (V1.9) on a GPS (V. 3.5, ABI) server. Proteins containing at least two peptides with a confidence interval value >95% were considered as being identified. The obtained sequence was also manually assigned to perform another search in the Swiss-Prot and TrEMBL databases using FASTA.
The experiment was considered to be a completely randomized split-plot design, with temperature as the main plots and plant lines as the subplots. Two-way analysis of variance was performed based on the general linear model procedure of SAS (SAS Institute Inc., Cary, NC, USA). Temperature effects on each line and genotypic differences under each temperature treatment were analysed separately because of significant interactions of temperature treatments and genotypes. Treatment means and differences between transgenic and NT control plants for each physiological parameter were separated by LSD test at the 0.05 probability level to compare differential temperature responses for each line and compare differences among the NT and two transgenic lines under control temperature and heat stress.
Both ipt transgenic lines produced more tillers and had more vertical shoot growth than the NT plants, as shown in Fig. 1. No significant differences in leaf chlorophyll content were observed among the three lines grown at 20°C. After 10d at 35°C, the chlorophyll content in S41 and H31 was maintained at the respective control levels (20°C); however, the chlorophyll content in NT declined by 30%, and was 29% and 32% lower than that in S41 and H31, respectively (Fig. 2A).
Leaf IPA content was comparable among the three lines when they were grown at 20°C (Fig. 2B). Heat treatment (35°C for 10d) led to a 53% decrease in leaf IPA content in NT. In contrast, there was no significant decline in leaf IPA content in H31, and the content was even increased by 31% in S41 after the same treatment. Both transgenic lines maintained significantly higher leaf CK (IPA) content than the NT plants under heat tress.
Total root weight did not differ among the three lines when grown at 20°C, but significantly decreased in all three lines when the temperature was increased to 35°C for 10d (Fig. 2C). Heat-induced reduction in root growth was the most severe in NT (73%), followed by S41 (54%) and H31 (40%). Both transgenic lines had significantly greater root weight than the NT plants under heat tress.
Low levels of ipt gene expression were detected in the leaves of S41 and in both leaves and roots of H31 when they were grown at optimum temperature (20°C), and the expression levels were enhanced by heat stress (35°C for 10d) in both transgenic lines (Fig. 3). No ipt gene expression was detected in the NT plants grown at optimum temperature or under heat stress.
A total of ~300 protein spots were detected on each gel for each leaf sample. They were reproducibly observed in three independent replications. No significant differences were observed in the expression levels of all proteins among the three lines at 20°C. However, 15 protein spots exhibited differential response patterns to heat stress at 35°C between transgenic lines and the NT control line. These 15 spots were excised from the gels for protein identification in order to examine differential protein changes caused by the ipt transformation in response to heat stress.
Fourteen of the differentially expressed proteins were identified by MALDI-TOF-MS and their respective positions were marked in the protein map shown in Fig. 4. The identities of these proteins and the respondse patterns of their expression levels to heat stress in each line are summarized in Table 1. The amino acid sequences of the trypsin-digested peptides (CI >95%) for shoot protein identification are listed in Supplementary Table S1 available at JXB online. The normalized volume (abundance) of each spot was used to analyse the differential level of protein expression, presented as the percentage volume of each protein spot in heat-stressed plants compared with unstressed plants. The expression levels of four protein spots (#22, 40, 110, and 192) at 20°C and 35°C in each line are shown in Fig. 5 as examples. Spot #22 was identified as a Hsp90, whose expression level was increased only in S41, but not changed in H31 or NT under heat stress (Fig. 5A). Spot #40 was identified as an Hsp70; its expression level was increased in both S41 and H31, but not changed in NT under heat stress (Fig. 5A). Spot #110 (Fig. 5B) and 192 (Fig. 5C) were identified as glycolate oxidase and a putative oxygen-evolving complex (OEC) protein, respectively; the expression levels of both proteins were decreased in NT, not changed in S41, and increased in H31 under heat stress.
The functions of the 14 identified proteins were categorized using the criteria described in Bevan et al. (1998) and summarized in Fig. 8A. Among 15 proteins, 60% of the proteins belonged to the energy category, mainly for glycolysis [enolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] and photosynthesis [Rubisco large subunit, Rubisco small subunit, Rubisco large subunit-binding protein subunit a, chloroplast chlorophyll a/b-binding protein precursor, oxygen-evolving enhancer protein 2 (OEE 2), a putative OEC, and photosystem II (PSII) polypeptide]; 20% of the proteins belonged to the category of protein destination and storage, including two Hsp90s and one Hsp70; 13% of the proteins belonged to the category of disease/defence; these were two antioxidant enzymes [catalase-1 (CAT-1) and glycolate oxidase (GOX)]. One protein (7%) belonged to the category of unclear classification.
Over 600 protein spots were reproducibly detected and clearly separated by 2D-GE in roots of creeping bentgrass. A total of 43 protein spots were found to be differentially expressed among the three lines in response to 10d of heat treatment, and their positions were marked in the protein map shown in Fig. 6. Among the 43 proteins, 38 were successfully identified by MALDI-TOF-MS. Their identities as well as the patterns of their expression levels in response to heat stress in each line are summarized in Table 2. The amino acid sequences of the trypsin-digested peptides (CI >95%) for root protein identification are listed in Supplementary Table S2 at JXB online. The expression levels of six spots (#118, 264, 267, 316, 322, and 324) at 20°C and 35°C in each line are shown in Fig. 7 as examples. Spot #316 and 322 were identified as two glutathione S-transferases (GSTs), whose expression levels were only increased in S41, but not changed in NT or H31 under heat stress (Fig. 7A). Spot #324 was identified as a small GTP-binding protein (RAN); its expression level was only increased in NT, but not changed in the transgenic lines under heat stress (Fig. 7A). Spot #118 was identified as an Hsp90, whose expression level was increased only in S41, but not changed in NT or H31 (Fig. 7B). Spots #264 and 267 were identified as two GAPDHs; their expression levels were increased only in H31, but not changed in NT or S41 under heat stress (Fig. 7C).
The functions of the 43 proteins are summarized in Fig. 8B. Sixteen percent of the proteins fell in the category of metabolism, such as ferredoxin-dependent glutamate synthase (Fd-GOGAT) and methionine synthase; 26% of the proteins belonged to the category of energy and most of them play roles in glycolysis [GAPDH, triosephosphate isomerase (TPI), fructose-bisphosphate aldolase, and glucose-6-phosphate isomerase (GPI)], respiration (NADH dehydrogenase), and the tricarboxylic acid (TCA) pathway [NAD-dependent isocitrate dehydrogenase (IDH) and aconitate hydratase]; 16% of the proteins were related to protein destination and storage, including two Hsps and one calreticulin; 9% of the proteins belonged to the category of disease/defence, including three antioxidants [ascorbate peroxidase (APX) and two GSTs]. Proteins in the other categories included transcription (5%), protein synthesis (5%), intracellular traffic (2%), cell structure (2%), signal transduction (2%), secondary metabolism (5%), and unclear classification (2%).
The subcellular location of each identified protein was predicted based on computer analysis using the Swiss-Prot protein knowledge base (http://ca.expasy.org/sprot/). Among the 15 differentially expressed proteins in leaves between NT and the ipt transgenic lines, the largest portion was localized in the chloroplast (53%), followed by the cytoplasm (20%), peroxisome (13%), plastid (7%), and unknown (7%) (Fig. 9A). Among the 43 differentially expressed proteins in roots between NT and the ipt transgenic lines, the largest portion was localized in the cytoplasm (33%), followed by plastid (16%), nucleus (14%), unknown (14%), endoplasm (9%), mitochondrion (9%), and peroxisomse (5%) (Fig. 9B).
The two ipt transgenic lines were able to maintain shoot growth and exhibited no leaf senescence following 10d of heat stress at 35°C (Fig. 1). Both S41 and H31 had maintained higher leaf chlorophyll content and root mass than NT after 10d of heat stress. The results suggested that the transformation of creeping bentgrass with the ipt gene using the SAG12 or HSP18 promoter improved heat tolerance associated with suppression of leaf senescence and promotion of root growth. These results could be related to enhanced expression of ipt transcripts and increased production of CKs (Xu et al., 2009; Xing et al., 2010). Consistently, enhanced expression of ipt transcripts in both leaves and roots of the two transgenic lines under heat stress was confirmed by northern blots in the current study. Leaf IPA, the form of CK whose production is directly controlled by the ipt gene, was maintained in H31 and increased in S41 under heat stress, in contrast to a decrease in the NT plants, indicating the active functioning of the ipt gene ligated to either promoter in stimulating CK production under heat stress. It is worth noting that increased root production may be an indirect effect of ipt expression in the shoots of the plants, as no differences in the number of roots and total root length on a per tiller basis were found between the SAG12-ipt line and the NT control line (Xu et al., 2009). No difference in the levels of leaf IPA was detected among the three lines when they were grown at optimal temperature, though low expression of ipt transcripts was detected in both ipt transgenic lines. It suggests that both inducible promoters functioned effectively to prevent overexpression of ipt transcripts when there was no senescence or heat shock signal and CK contents were adequate. The up-regulation of ipt transcripts under the heat shock promoter did not cause significant increases in leaf IPA concentration, but helped to maintain the IPA concentration of heat-stressed plants at the pre-stress level.
Chloroplast proteins represent a major fraction (53%) of the differentially expressed proteins in leaves detected in the present study, and most of them had relatively higher expression levels after heat stress in at least one ipt transgenic line. Caers et al. (1985) studied the effects of heat stress on photosynthetic activity and chloroplast ultrastructure in correlation with the endogenous CK concentration in maize (Zea mays cv. Fronica) seedlings, and found the inhibition of photosynthetic activity and chlorophyll accumulation by heat stress could be reversed when levels of CKs were elevated by adding benzyladenine.
Root tissue was more severely affected by heat stress. Xu and Huang (2000) reported that high soil temperature was more detrimental than high air temperature, and roots may mediate shoot responses to high temperature stress in creeping bentgrass. A total of 28 more differentially expressed proteins between NT and the ipt transgenic lines were observed in the roots of the three lines than in the leaves under heat stress. The differentially expressed proteins detected in roots included a high percentage (33%) of proteins located in the cytoplasm, suggesting the importance of cytosolic proteins in root tolerance to heat stress. Differentially expressed proteins in leaves or roots in responses to heat stress between the transgenic lines and the NT control are discussed below according to functional categories.
Energy category: Many identified proteins in this category are related to photosynthesis. Photosynthesis is among the processes most sensitive to elevated temperature. Changes in various transcripts and proteins involved in photosynthetic reactions have been reported in response to heat stress (Kim and Portis, 2005; Allakhverdiev et al., 2008; Pushpalatha et al., 2008).
The OEC along with the associated cofactors in PSII and carbon fixation by Rubisco are two primary targets of heat damage in plants (Allakhverdiev et al., 2008). Two OEC-related protein spots (#178 and 192) were identified. Spot #178 was OEE 2 that contained a conserved domain of the extrinsic protein PsbP. Its expression level was reduced 30% only in NT, but not in S41 or H31 under heat stress. Spot #192 was a putative OEC precursor that contained a conserved domain of the extrinsic protein PsbQ. Its expression level was decreased 70% in NT, not changed in S41, and increased in H31 (1.9-fold) under heat stress. Protection of OEC by the extrinsic proteins of PSII was shown to be essential for development of cellular thermotolerance (Kimura et al., 2002), thus maintenance or up-regulation of the extrinsic proteins in PSII may be attributed to the superiority of the two transgenic lines over the NT control line growing under heat stress.
Rubisco large and small subunits often degrade with leaf senescence or maturation (Wilson et al., 2002). Wingler et al. (1998) reported that the senescence-related decline in Rubisco was delayed in transgenic tobacco plants that produced more CKs. Zavaleta-Mancera et al. (2007) also found that incubation of wheat leaves (Triticum aestivum) with BAP reduced the degradation of Rubisco large and small subunits during dark-induced senescence. Decreased expression levels of both Rubisco large and small subunits were observed in NT and S41 under heat stress. There was no reduction in the expression level of Rubisco small subunit in H31, indicating improved thermal stability of Rubisco protein in this line. The difference could be due to the direct effect of the response of the HSP18 promoter in H31 to heat stress on chloroplast protein levels by effectively controlling the ipt gene to regulate CK production under heat stress, compared with NT without the ipt gene or S41 with the SAG12 promoter that responded to senescence signals caused by heat stress.
The expression level of a chloroplast chlorophyll a/b-binding protein (LHCP) belonging to the light-harvesting complex was increased the most in NT (2.2-fold), followed by H31 (1.2-fold), and not changed in S41 under heat stress. This protein functions as a light receptor that captures and delivers excitation energy to PSII and PSI. Under heat stress, carbon fixation is inhibited, which does not require much light energy and may result in oxidative damage to the photosynthetic apparatus (Dinar et al., 1983). Therefore, the lower or no induction of LHCP in H31 and S41 compared with NT indicated reduced light absorption capacity to avoid excessive excitation energy under heat stress that could damage the photosynthetic apparatus. An alternative explanation can be that in the ipt transgenic plants there is still a balance between NADPH production in the light reactions and its use in the Calvin cycle, thus there is no need for more LHCP, while the increase in LHCP by NT may be an attempt to dissipate excess light energy. The regulatory role of CKs in LHCP was reported by Flores and Tobin (1988), who detected a decline in the mRNA level of LHCP during dark-induced senescence whereas CK pre-treatment slightly increased the mRNA abundance. There may be a discrepancy between the abundance of LHCP mRNA and protein as reported by Barak et al. (2000) in the chlorophyll-deficient tobacco mutants, which accumulated normal levels of LHCP transcript but failed to accumulate the protein.
Several proteins functioning in the glycolytic pathway were differentially expressed between NT and the ipt transgenic lines, including enolase and GAPDH. Enolase and GAPDH are enzymes catalysing the ninth and sixth steps of glycolysis, respectively. A proteomic study conducted by Ferreira et al. (2006) in Populus euphratica leaves found that the abundance of enolase and GAPDH was transiently increased after 30h of heat stress, followed by a reduction after 54h of heat stress, suggesting an early acceleration of the glycolytic pathway upon exposure to high temperature. In the present study, the expression level of a leaf cytosolic enolase was increased in S41 (2.0-fold), maintained in H31, but decreased in NT (0.7-fold) after 10d of heat stress; the expression level of a cytosolic GAPDH was increased to similar extents in NT (1.5-fold), S41 (1.4-fold), and H31 (1.5-fold), respectively. The higher enolase expression in the leaves of both transgenic lines carrying the ipt gene than in the NT control line after 10d of heat stress may have resulted from CK activation of the glycolytic pathway. The response of enolase to CK was also found in the leaves of common ice plants (Mesembryanthemum crystallinum) by Forsthoefel et al. (1995), who detected increased enolase transcripts upon treatment of unstressed plants with BAP.
Protein destination and storage category: A plastid Hsp90, a cytoplasmic Hsp90, and a chloroplast Hsp70 were identified in this category. Proteins in the Hsp90 family act as chaperonins with ATPase activity and interact with proteins involved in transcription regulation and signal transduction pathways (Majoul et al., 2003). Hsp70 has a known function in preventing protein aggregation and assisting refolding of non-native proteins under both normal and stress conditions. The expression of Hsps has been correlated with the acquisition of thermotolerance in many plant species (Vierling, 1991), including creeping bentgrass (Park et al., 1996; He et al., 2005). In the current study, an increase in the abundance of a plastid Hsp90 in S41 (2.8-fold) and a decrease in the abundance of a cytoplasmic Hsp90 in NT (70%) were observed under heat stress. A chloroplast Hsp70 was up-regulated 1.5-fold and 2.0-fold in S41 and H31, respectively, but not in NT. These results suggest a regulatory role for CKs in Hsp metabolism in the leaves of the ipt transgenic lines under heat stress, which may be related to the inhibition of Hsp degradation or activation of Hsp production. In agreement with this, Veerasamy et al. (2007) previously reported that foliar spray of zeatin riboside increased the content of several Hsps and alleviated heat-induced leaf senescence in creeping bentgrass.
Disease/defence category: Two proteins belonging to this category were identified, GOX and CAT-1. GOX is a key enzyme involved in the photorespiratory pathway, which helps dissipate excessive energy and protect photosynthetic membranes. CAT, whose primary function is to catalyse the decomposition of H2O2, is essential for the protection of GOX against photoinactivation (Schafer and Feierabend, 2000). Both GOX and CAT represent the major constituents of the peroxisomal matrix in photosynthetic tissues. Ludt and Kindl (1990) found that both GOX and CAT mRNAs decreased during leaf senescence in lentil (Lens culinaris). Transgenic Pssu-ipt tobacco with an elevated content of endogenous CKs showed increased GOX activity compared with non-transformed control plants (Synkova et al., 2004). The expression level of GOX was significantly decreased by 70% in NT, whereas it was increased in H31 (1.7-fold) but did not change in S41 under heat stress. These results indicate that improved heat tolerance regulated by CKs in the transgenic lines could involve maintenance or enhancement of expression of such glycolate pathway enzymes as GOX. The expression level of CAT-1 was increased to a greater extent in NT (2.3-fold) than in S41 (1.4-fold) and H31 (1.6-fold) under heat stress. Stress-induced CAT-1 expression was related to the triggering of H2O2 production, as proved by the observation that CAT-1 expression was induced by applied H2O2 (Xing et al., 2007). Thus the lower CAT-1 expression in the ipt transgenic lines could reflect less severe oxidative damage caused by heat stress than that in the NT control line. This down-regulatory role of CKs in CAT was also reported by Toyama et al. (1995) in a large-scale differential hybridization study, which detected a decreased level of CAT cDNA in etiolated cotyledons of cucumber (Cucumis sativus) treated with N6-benzyladenine.
Metabolism category: Fd-GOGAT is an enzyme functioning in the glutamine synthetase (GS)/GOGAT ammonium assimilation pathway, which regulates nitrogen assimilation (Tempest et al., 1970). A significant decline in the expression level of Fd-GOGAT protein was observed in roots of NT and H31, but not in S41 under heat stress. The maintenance of Fd-GOGAT in roots of S41 could sustain active assimilation of nitrogen, suggesting that the SAG12 promoter was effective in responding to the signals triggered by impaired nutrient uptake under heat stress, which correlates with the recent findings that SAG12-ipt expression suppressed leaf senescence induced by N deficiency in creeping bentgrass (authors’ unpublished results).
Serine hydroxymethyltransferase (SHMT) and phosphoserine aminotransferase (PSAT) are two enzymes functioning in one-carbon compound metabolic pathway. Both were detected in soybean (Glycine max) root nodules and involved in the purine biosynthetic pathway required for transport of the assimilated nitrogen from the nodules (Mitchell et al., 1986; Reynolds and Blevins, 1986). Xu and Huang (2008) recently reported that the expression level of SHMT decreased whereas that of PSAT increased under heat stress in two Agrostis grass species. In the current study, the decline of SHMT was greatest in NT (80%), intermediate in H31 (60%), and least in S41 (30%) after 10d of heat stress. Increased PSAT expression was only observed in NT (1.5-fold), but not in S41 or H31. The less severe decline of SHMT and more stable production of PSAT in both transgenic lines may improve nitrogen transport and utilization to enable better growth under heat stress compared with the NT control line.
The expression level of a nucleotide-sugar dehydratase was decreased 80% only in NT under heat stress, but not changed in either ipt transgenic line. Production of this enzyme may assist in maintaining adequate sugar metabolism for growth of the transgenic lines under heat stress. This may relate to decreased sugar sensitivity resulting from activation of CK signalling as reported by Franco-Zorrilla et al. (2005).
Energy category: Increased expression levels of two phosphorylated cytoplasmic GAPDHs were detected only in roots of H31 (1.2- and 1.7-fold, respectively) under heat stress. GAPDH has been regarded as a target of CK action in regulating glycolytic activity by modifying the phosphorylation state of GAPDH (Heintz et al., 2006). GPI and TPI are enzymes catalysing the second and fifth steps of glycolysis, respectively. An increased level of a cytoplasmic GPI expression was observed only in the roots of H31 (1.7-fold) under heat stress. The expression level of a cytoplasmic TPI was increased in all three lines after heat treatment, but the increase was greater in both transgenic lines (2.1-fold) than in NT (1.5-fold). Dorion et al. (2005) has reported that cytoplasmic TPI activity and protein levels appeared to be regulated during development and are important in the supply of carbon to respiratory and biosynthetic pathways during active growth of potato. Hence, CK regulation of GPI and TPI could play a role in the balance of metabolic fluxes in plant primary metabolism that is required for sustained plant growth under stress conditions.
NADH dehydrogenase is an enzyme located in the mitochondrion that catalyses the transfer of electrons from NADH to coenzyme Q in the respiratory pathway. Two NADH dehydrogenase proteins whose expression levels were both decreased only in H31 under heat stress were identified. Rachmilevitch et al. (2006) reported that total root respiration rate and specific respiratory costs for maintenance and ion uptake increased with increasing soil temperatures in two Agrostis grass species and the increases were less pronounced in the tolerant species than in the sensitive species. The lower NADH in H31 than in NT may reflect the inhibitory effects of CKs on respiratory carbon metabolism, which could lower respiratory carbon consumption in H31, an important factor controlling root survival under high temperature. The repression of the respiratory rise by CKs such as kinetin and BAP was also reported by Tetley and Thimann (1974). Moreover, Miller (1980) suggested that there may be a point located between NADH dehydrogenase and cytochrome b of the electron transport system that is inhibited by CKs.
IDH is an enzyme functioning in the TCA cycle and catalyses the oxidative decarboxylation of isocitrate while converting NAD+ to NADH. It has been proposed that mitochondrial IDH can serve as the enzymatic origin of 2-oxoglutarate, the carbon skeleton required for plant ammonium assimilation through the GS/GOGAT pathway (Hodges et al., 2003). Lancien et al. (1999) found that IDH mRNA levels were increased by the addition of nitrate or NH4+ to N-starved tobacco roots. Decreased levels of IDH expression were detected in roots of NT and H31 under heat stress, while the level in S41 was maintained. The relatively higher level of IDH expression in roots of S41 could reflect better assimilation of nitrogen for sustained growth under heat stress compared with the NT control line, which is consistent with the maintenance of Fd-GOGAT expression in the same line under heat stress as discussed above.
The expression level of a ferredoxin-NADP reductase precursor (FNR) was reduced 60% in the roots of NT, but maintained in both transgenic lines under heat stress. FNR in the non-photosynthetic tissues probably supports ferredoxin-dependent biosynthetic processes such as nitrogen assimilation (Morigasaki et al., 1990; Green et al., 1991). In that case, maintenance of FNR expression under heat stress could also reflect a superior nitrogen assimilation capacity of the transgenic lines over the NT control line that facilitates their growth under heat stress, in accordance with the present findings on Fd-GOGAT and IDH expression.
Protein destination and storage category: The expression levels of a protein disulphide-isomerase (PDI), two Hsp90s, and a putative t-complex protein 1 theta chain were increased in roots of S41 under heat stress. PDI catalyses the formation, cleavage, and isomerization of disulphide bonds, and is involved in regulating the folding and deposition of storage proteins (Johnson and Bhave, 2004). Up-regulation of PDI as well as expression of Hsp90s in S41 may be related to protein folding and deposition of damaged proteins. The function of the putative t-complex protein 1 theta chain in stress tolerance has not been well documented.
Cyclophilins catalyse the isomerization of peptide bonds from the trans form to the cis form at proline residues and facilitate protein folding. Pan et al. (2008) reported that cyclophilin A is required for activation, export, and translocation of some important nuclear proteins. In this study, the expression level of a cyclophilin A-2 protein was increased 3.7-fold in both transgenic lines and 2.7-fold in NT under heat stress. Greater accumulation of this protein under heat stress could contribute to improved heat tolerance of the transgenic lines by reinforcing proper protein export and translocation.
Calreticulin is a multifunctional protein that binds Ca2+ ions. It binds to misfolded proteins and prevents them from being exported from the endoplasmic reticulum to the Golgi apparatus. An increased level of calreticulin expression was observed only in roots of H31 (1.4-fold) under heat stress, which may also support the survival of this transgenic line under heat stress by facilitating proper protein export and translocation.
Disease/defence category: Heat stress induces the production of reactive oxygen species (ROS). The induction of ROS-related enzymes is involved in the protection of root tissues from oxidative damage under stress conditions (Yoshimura et al., 2008). Among them are GST and APX. Increased expression of two GSTs was detected only in S41 (1.4-fold), and decreased expression of an APX only in NT (80%). The accumulation of GST and maintenance of APX may be partially responsible for the capability of transgenic lines to sustain root growth under heat stress through activating the ROS-scavenging system. Similarly, Roxas et al. (2000) reported that overexpression of GST in tobacco seedlings increased glutathione-dependent peroxide scavenging and alterations in glutathione and ascorbate metabolism that led to reduced oxidative damage and enhanced growth under heat stress.
The expression level of a molybdenum cofactor (Moco)-containing protein was increased in S41 (1.6-fold) under heat stress. Moco-containing enzymes play roles in basic metabolic reactions in the nitrogen, sulphur, and carbon cycles, catalysing oxygen atom transfer in a two-electron transfer redox reaction mediated by Moco (Kisker et al., 1997). Four major groups of them have been found in plants: nitrate reductase, catalysing the key step in inorganic nitrogen assimilation; aldehyde oxidase, catalysing the last step in the biosynthesis of abscisic acid; xanthine dehydrogenase, involved in purine catabolism and stress reactions; and sulphite oxidase, involved in detoxifying excess sulphite (Mendel and Hansch, 2002). Although the function of the Moco protein identified in S41 has not been specified, up-regulation of this Moco protein by CKs may facilitate plant survival by assisting in the oxidation–reduction processes involved in heat stress responses.
Transcription category: The expression level of a putative heterogeneous nuclear ribonucleoprotein (hnRNP) A2 was decreased only in roots of NT (50%) but maintained in both ipt transgenic lines under heat stress. hnRNPs are complexes formed by the nuclear precursors of mRNAs and specific proteins, which are major constituents of the nucleus and are important elements in the post-transcriptional pathway of the expression of genetic information (Swanson and Dreyfuss, 1988). The maintenance of this hnRNP in transgenic plants could enable proper gene expression for plants growing under stress conditions.
A decreased expression level of a nuclear RNA binding protein A-like protein was observed only in H31 (30%) under heat stress. RNA-binding proteins participate in synthesizing, processing, editing, modifying, and exporting RNA molecules from the nucleus (Fedoroff, 2002). Some of the RNA-binding proteins have been reported to be hormone receptors and mediate a subset of hormone actions (Razem et al., 2006). How nuclear RNA-binding proteins are involved in stress tolerance is not well understood.
Transformation with ipt induced protein changes involved in multiple functional groups. The diversity of the differentially expressed proteins in response to heat stress suggests a regulatory role for CKs in various metabolic pathways for heat tolerance. Among the differentially expressed proteins, a remarkably high percentage of them function in energy, protein destination and storage, and disease/defence categories in both leaves and roots. The expression levels of many of these proteins were maintained or increased in at least one ipt transgenic line under heat stress while they were decreased in the NT control line. CK regulation of heat tolerance of creeping bentgrass could involve complex mechanisms operating at the transcriptional, post-transcriptional, and post-translational levels. Further research may be conducted to confirm the expression of differentially expressed proteins using western blot analysis and identify genes encoding those proteins altered by ipt expression in order to reveal specific metabolic pathways and molecular mechanisms of CK regulation of heat tolerance in perennial grass species.
Supplementary data are available at JXB online.
Table S1. The amino acid sequences of the trypsin-digested peptides (CI >95%) for shoot protein identification.
Table S1. The amino acid sequences of the trypsin-digested peptides (CI >95%) for root protein identification.
The authors would like to thank Rutgers Center for Turfgrass Science for funding support, and Dr Chenping Xu for technical assistance in protein analysis. Thanks are also due to Dr Chenping Xu and Emily Meriwitz for critical review of the manuscript.