Journal of Experimental Botany Advance Access published March 23, 2015 Journal of Experimental Botany doi:10.1093/jxb/erv102

Research Paper

A tomato chloroplast-targeted DnaJ protein protects Rubisco activity under heat stress Guodong Wang, Fanying Kong, Song Zhang, Xia Meng, Yong Wang and Qingwei Meng* College of Life Science, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an, Shandong 271018, PR China

Received 7 December 2014; Revised 5 February 2015; Accepted 10 February 2015

Abstract Photosynthesis is one of the biological processes most sensitive to heat stress in plants. Carbon assimilation, which depends on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is one of the major sites sensitive to heat stress in photosynthesis. In this study, the roles of a tomato (Solanum lycopersicum) chloroplast-targeted DnaJ protein (SlCDJ2) in resisting heat using sense and antisense transgenic tomatoes were examined. SlCDJ2 was found to be uniformly distributed in the thylakoids and stroma of the chloroplasts. Under heat stress, sense plants exhibited higher chlorophyll contents and fresh weights, and lower accumulation of reactive oxygen species (ROS) and membrane damage. Moreover, Rubisco activity, Rubisco large subunit (RbcL) content, and CO2 assimilation capacity were all higher in sense plants and lower in antisense plants compared with wild-type plants. Thus, SlCDJ2 contributes to maintenance of CO2 assimilation capacity mainly by protecting Rubisco activity under heat stress. SlCDJ2 probably achieves this by keeping the levels of proteolytic enzymes low, which prevents accelerated degradation of Rubisco under heat stress. Furthermore, a chloroplast heat-shock protein 70 was identified as a binding partner of SlCDJ2 in yeast two-hybrid assays. Taken together, these findings establish a role for SlCDJ2 in maintaining Rubisco activity in plants under heat stress. Key words: CO2 assimilation, heat stress, Hsp70, SlCDJ2, Rubisco, tomato.

Introduction Plant productivity is often challenged by environmental stresses, including high temperature and drought. Increased global temperature is predicted to have both ecological and agricultural consequences in the future. High temperature negatively affects plant growth, survival, and yield. Field studies and mathematical modelling have revealed that decadal variations in temperature have significant effects on crop productivity. Photosynthesis, the process through which plants accumulate biomass by converting inorganic carbon to carbohydrates using light energy, is a major target for improving crop productivity (Kurek et al., 2007). It is also one of the most heat-sensitive processes in plants (Salvucci, 2008).

There are at least three major heat stress-sensitive sites in the photosynthetic machinery: the photosystems, mainly photosystem II (PSII) with its oxygen-producing complex, the ATP-generating ATPase, and the carbon assimilation processes (Nishiyama et al., 2005, 2006; Murata et al., 2007; Mohanty et al., 2007). The primary site of heat stress inhibition in Calvin cycle activity is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), where heat stress reduces the enzyme’s activity and synthesis (Demirevska-Kepova et  al., 2005). The catalytic activity of Rubisco is regulated by the enzyme Rubisco activase (RCA), which is localized in the chloroplast and is a member of the AAA+ family of ATPases

Abbreviations: aldolase, fructose-bisphosphate aldolase; cpHsp70, chloroplast Hsp70; DAB, 3’,3-diaminobenzidine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SlCDJ2, Solanum lycopersicum chloroplast-targeted DnaJ protein 2; MDA, malondialdehyde; NBT, nitroblue tetrazolium; PFD, photon flux density; PSII, photosystem II; PVP, polyvinylpyrrolidone; RbcL, Rubisco large submit; RCA, Rubisco activase; REC, relative electric conductivity; ROS, reactive oxygen species; RT-qPCR, quantitative real-time PCR; Rubisco, ribulose-1, 5-bisphosphate carboxylase/oxygenase; sFBPase, stromal fructose-1, 6-bisphosphatase; WT, wild type. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected]

Downloaded from http://jxb.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on April 9, 2015

*  To whom correspondence should be addressed. E-mail: [email protected]

Page 2 of 14 | Wang et al. and facilitated its thermotolerance, whereas its suppression increased Rubisco damage and decreased its thermotolerance.

Materials and methods Plant materials and treatments Three sense (S4, S5, and S6) and three antisense (A4, A7, and A10) T2 transgenic lines of a tomato cultivar (Solanum lycopersicum cv. Zhongshu 6) were used as plant materials. Seeds were sterilized, sown on Murashige and Skoog medium, and incubated at 25 °C under a 16/8 h light/dark cycle for 10 d.  The young seedlings were planted in plastic pots filled with sterilized soil and grown in a greenhouse with a 25/20 °C day/night cycle and 16/8 h day/night photoperiod, 200 μmol m−2 s−1 photon flux density (PFD), and 50–60% relative humidity range. The plants were irrigated with Hoagland’s nutrient solution once per week. When the sixth leaf was fully expanded (in ~6 weeks), the plants were acclimated in an illuminated incubation chamber (GXZ-260C) for 2 d before treatment. To study the expression patterns of SlCDJ2 under heat stress, 6-week-old wild-type (WT) tomato plants were subjected to heat stress treatments at 30, 35, 38, 42, and 45  °C for 6 h. WT plants were also subjected to 42 °C for 0, 3, 6, 9, 12, and 24 h with a 16/8 h light/dark regime. For heat treatment, whole plants were treated at 42  °C for 12 h and 24 h in an illuminated incubation chamber with ~200  μmol m−2 s−1 PFD. All measurements of physiological and biochemical parameters were conducted on the youngest fully expanded leaves. Transformation and identification of transgenic tomato plants The open reading frame (ORF) of SlCDJ2 cDNA was subcloned into the pBI121 expression vector under the control of the 35S Cauliflower mosaic virus promoter (Supplementary Fig. S1 available at JXB online). The constructs were introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method and verified by PCR and sequencing analyses. T0 kanamycin-resistant transgenic tomato plants were generated by the A.  tumefaciensmediated leaf disk method. Sense and antisense transgenic plant DNAs were extracted and used to amplify the target gene by PCR. Plants that produced 100% kanamycin resistance in T2 generation progeny were considered homozygous and were used in subsequent studies. Real-time quantitative PCR (RT-qPCR) analysis Total RNAs were extracted from WT and transgenic tomato leaves following the manufacturer’s instructions using the RNeasy plant mini kit (Tiangen, Beijing, China). Real-time quantitative RT-PCR (RT-qPCR) was performed as described by Kong et  al. (2014b). EF-1α (GenBank accession no. LOC544055) was as the actin control. Template-free, negative, and single primer controls were established before RT-qPCR analysis. Three biological replicates (each with three technical replicates) were used for each sample, and the standard curve method was used for statistical analysis. The primers used for the RT-qPCRs are listed in Supplementary Table S1 at JXB online. Chloroplast, thylakoid membrane, and stromal protein isolation For the preparation of intact chloroplast, tomato functional leaves (~50 g) were homogenized in 20 ml of ice-cold isolation buffer (20 mM HEPES/KOH, 0.3 M sorbitol, 5 mM MgCl2, 5 mM EGTA, 5 mM EDTA, and 10 mM NaHCO3; pH 8.0). The homogenate was filtered through a double layer of Miracloth and centrifuged at 3000 g for 3 min; the precipitate was resuspended in 1 ml of isolation buffer. The resuspended chloroplasts were loaded onto a 20%/40%/80% (v/v/v) three-step Percoll gradient and centrifuged

Downloaded from http://jxb.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on April 9, 2015

associated with diverse cellular activities (Neuwald et  al., 1999; Portis, 2003). RCA can remove naturally occurring sugar-phosphate inhibitors from both active (carbamylated) and inactive (decarbamylated) Rubisco sites through protein– protein interactions and ATP hydrolysis, which is essential for maintaining Rubisco’s catalytic competency (Kurek et al., 2007). DnaJ proteins belong to a large family with several members: 22 in yeast (Walsh et  al., 2004), 41 in humans (Qiu et al., 2006), at least 89 in Arabidopsis (Miernyk et al., 2001), at least 19 in Arabidopsis chloroplasts (Chiu et  al., 2013), and 63 in tomato (Kong et al., 2014a). DnaJ proteins function as molecular chaperones, either alone or in association with their heat-shock protein 70 (Hsp70) partners, and are involved in various essential cellular processes, including protein folding/unfolding, assembly/disassembly, and degradation (Hennessy et al., 2005; Craig et al., 2006). They are the key components contributing to cellular protein homeostasis under normal and stress conditions (Wang et  al., 2004). Some DnaJ proteins that are not located in the chloroplast play important roles against heat stress. AtDjA2 and AtDjA3 improve Arabidoposis thaliana thermotolerance (Li et  al., 2007). Thermosensitive male-sterile J-protein (TMS1) plays an important role in thermotolerance of pollen tubes (Yang et  al., 2009). Moreover, AtDjB1 facilitates thermotolerance by protecting cells against heat-induced oxidative damage in A. thaliana (Zhou et al., 2012). Previous studies have revealed that chloroplast-targeted DnaJ proteins also play important roles in maintaining protein homeostasis. Brutnell et  al. (1999) reported that chloroplast-targeted bundle sheath defective 2, which includes a DnaJ-like domain bound to Rubisco large subunit (RbcL) polypeptides, regulates RbcL protein assembly. AtJ8, AtJ11, and AtJ20 mutants accumulated lower levels of Rubisco activase when grown under light, suggesting a possible role for these three chloroplast-targeted J-proteins in enzyme folding or assembly (Chen et  al., 2010). A  chloroplast-targeted LeCDJ1 protein contributes to the maintenance of PSII under chilling stress (Kong et al., 2014b). Pulido et al. (2013) reported that Arabidopsis J-protein J20 interacts with deoxyxylulose 5-phosphate synthase, the first enzyme in the plastidial isoprenoid pathway, and delivers it to protein quality control. Therefore, chloroplast-targeted DnaJ proteins may also play important roles in the regulation of key enzymes in other plastidial biosynthetic pathways under normal or stress conditions. A chloroplast-targeted DnaJ protein (SlCDJ2) was previously isolated from tomato by the authors’ group (Wang et al., 2014). SlCDJ2 does not have other structural features besides a typical HPD motif, which include a glycine/phenylalanine-rich region, zinc-finger structure, and four cysteine repeats in its C-terminus; therefore, it belongs to the simplest group of DnaJ proteins (type III) (Cheetham and Caplan, 1998). In this work, expression of SlCDJ2 was proven to be induced by heat stress. It contributed to stabilizing the levels of proteolytic enzymes during heat treatment and alleviated degradation of Rubisco. Overexpression of SlCDJ2 in tomato alleviated heat stress-induced damage of Rubisco

DnaJ protein protects Rubisco activity under heat stress  |  Page 3 of 14 on a swing-out rotor at 3500 g for 30 min. The intact chloroplasts appeared at the 40% and 80% Percoll interphase and were recovered, washed with isolation buffer, and centrifuged at 3000 g for 3 min. The precipitate was comprised of isolated chloroplasts. Thylakoid membranes and stromal proteins were prepared from isolated intact chloroplasts according to Stöckel and Oelmüller (2004). Measurements of physiological parameters Six-week-old WT and transgenic plants were treated at 42  °C for 24 h. Six-week-old WT and transgenic plants were simultaneously placed at 25 °C for 24 h as controls. Chlorophyll was extracted from the leaves (3.1 cm2) and the content measured as described by Kong et al. (2014b). Malondialdehyde (MDA) content and relative electric conductivity (REC) were also measured in the leaves as described by Kong et al. (2014b).

Measurement of key enzyme activity in the Calvin cycle To measure the activities of key enzymes in the Calvin cycle, 0.2 g of leaves was ground with liquid nitrogen and 1% polyvinylpyrrolidone (PVP) in a mortar and 1.9 ml ice-cold Rubisco extraction buffer (50  mM TRIS-HCl, 1  mM MgCl2, 1  mM EDTA, 12.5% glycerol, 10% PVP; pH 7.5) was added. The homogenate was centrifuged at 15 000 g for 10 min. The supernatant contained the extracted enzyme. Rubisco, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), stromal fructose-1, 6-bisphosphatase (sFBPase), and fructose-bisphosphate aldolase (aldolase) activities were measured according to the methods of Sawada et al. (2003), Innocenti et  al. (2002), Holaday et  al. (1992), and Haake et  al. (1998), respectively. Determination of the Rubisco activation state To measure the Rubisco activation state, 3.1 cm2 leaves were rapidly ground in 2 ml of ice-cold extraction medium (50 mM TRISHCl pH 7.5, 1 mM MgCl2, 1 mM EDTA, 12.5% glycerol, and 10% PVP). The resulting solution was immediately transferred to a 1.5 ml microfuge tube and centrifuged at 15 000 g for 10 min at 4 °C, and the supernatant was used as the initial extract for activity determinations. The initial activity was determined at 28  °C, and 100 μl of the supernatant was immediately added to 900 μl of reaction liquid [50 mM HEPES pH 8.0, 1 mM EDTA, 20 mM MgCl2, 2.5 mM dithiothreitol (DTT), 10 mM NaHCO3, 5 mM ATP, 5 mM phosphocreatine, 0.15 mM NADH2, 10 U ml–1 3-phosphoglycerate kinase, 10 U ml–1 NADP-GAPDH, 10 U ml–1 creatine kinase, 0.6 mM RuBP; Sigma, USA]. Then, absorbance at 340 nm was determined. After initial activity determinations were underway, 100 μl of supernatant was transferred to another tube and activated by addition of 200 μl of activated reaction liquid (final concentration: 33 mM TRIS-HCl pH 7.5, 0.67 mM EDTA, 33 mM MgCl2, 10 mM NaHCO3). After the mixture was maintained at 28 °C for 10 min and 900 μl of reaction liquid was added for determination of total activity, the absorbance at 340 nm was determined. The activation state was estimated as the ratio of the initial Rubisco activity to total Rubisco activity.

Histochemical staining and measurements of H2O2 and O2·− Six-week-old WT and transgenic plants were treated at 42  °C for 12 h. Six-week-old WT and transgenic plants were simultaneously placed at 25 °C for 12 h as controls. Hydrogen peroxide (H2O2) was stained with 3’,3-diaminobenzidine (DAB) according to the method of Giacomelli et  al. (2007). Superoxide radical (O2·−) was visually detected using nitroblue tetrazolium (NBT) as described by Rao and Davis (1999). Trypan blue staining was conducted as described by Choi et  al. (2007). The contents of H2O2 and O2·− in 6-weekold transgenic and WT lines were measured following the method described by Kong et al. (2014b). Protein extraction and western blot analysis Total proteins were extracted from the leaves following the method described by Kong et al. (2014b). The chloroplasts, thylakoid membranes, and stromal proteins were isolated as previously described. For western blot analysis, proteins were separated by SDS–PAGE using 15% (w/v) acrylamide gels with 6 M urea. After electrophoresis, proteins were electroblotted on a polyvinylidene fluoride membrane (Millipore, Watford, Herts, UK), and subsequently blocked with 5% milk or fatty acid-free bovine serum albumin (BSA). Rubisco, RCA, and Hsp70 antibodies were purchased from Abcam (UK). SlCDJ2 antibody was produced in the authors’ laboratory (Wang et al., 2014). The protein content was determined by conducting a dye binding assay. Quantitative image analysis of protein levels was performed with a Tanon Digital Gel Imaging Analysis System (Tanon-4100, Shanghai Tanon Science and Technology Co., Shanghai, China). Yeast two-hybrid assays Yeast two-hybrid assays were performed according to the Matchmaker™ Gold Yeast Two-Hybrid System User Manual. The cpHsp70 (GenBank accession no. EU195057.1) coding region was amplified with cDNA from tomato seedlings and ligated to BamHI/XhoII-digested pGBKT7 (Clontech). The SlCDJ2 coding region was amplified from a plasmid and then ligated to EcoRI/SacIdigested pGADT7 (Clontech). The expression vector pGADT7SlCDJ2 was co-transformed into the yeast strain Y187 (Clontech) with pGBKT7-cpHsp70 by the lithium acetate transformation method. Cells were plated onto a selective medium without leucine and tryptophan (SM-LW). Putative transformants were transferred to a selective medium without leucine, tryptophan, histidine, and adenine, but supplemented with X-α-Gal and aureobasidin (SM-LWHA/X/A). The interactions of pGADT7-T and pGBKT753 proteins were used as positive controls. The results were based on three independent biological repeats. Statistical analysis Data represents the mean ±standard deviation (SD) of three replicates. Statistical significance of the differences of WT and transgenic

Downloaded from http://jxb.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on April 9, 2015

Measurement of CO2 assimilation CO2 assimilation was determined using an open-gas portable photosynthesis system (CIRAS-2, PP Systems, Herts, UK) with artificial blue–red light-emitting diodes. The CO2 assimilation response to the PFD was obtained by varying the PFD from 2000 μmol m–2 s–1 to 0 μmol m–2 s–1 with 400 μmol mol–1 CO2. The CO2 assimilation response to CO2 concentration was determined under 1000  μmol m–2 s–1 PFD. The leaf temperature was kept at 25  °C. A  total of 10–15 plants were measured for both the light response curves and the CO2 response curves. All results were based on three independent biological repeats.

Measurement of proteolytic activity Proteolytic activity was measured according to Lin and Wittenbach (1981) with slight modifications. A 1.0 g aliquot of leaves was rapidly ground in 5 ml of ice-cold extraction medium (25 mM HEPES, 4 mM DTT, 1 mM EDTA-Na; pH 7.5). The resulting solution was immediately transferred to a 10 ml centrifuge tube and centrifuged at 10 000 g for 30 min at 4 °C, and the supernatant was run through small Sephadex G-25 columns pre-equilibrated with 25 mM HEPES (pH 7.5), 1 mM EDTA-Na, and 4 mM DTT. The protein eluting in the void volume was collected and used to assay for proteolytic activity. At the same time, leaf protein-citric acid buffer (2 mg ml–1) was added to each tube. The mixture was then incubated at 28 °C for 30 min and centrifuged at 4000 g for 30 min to remove the precipitate. Finally, ninhydrin was added to the supernatant. After placing it in a boiling water bath for 10 min, absorbance at 566 nm was measured.

Page 4 of 14 | Wang et al. plants with respect to the measured parameters was tested using SPSS 13.0 (Chicago, IL, USA). Significant differences with respect to the control are indicated by * (P

A tomato chloroplast-targeted DnaJ protein protects Rubisco activity under heat stress.

Photosynthesis is one of the biological processes most sensitive to heat stress in plants. Carbon assimilation, which depends on ribulose-1,5-bisphosp...
2MB Sizes 0 Downloads 6 Views