Localization and abundance of chloroplast protein synthesis elongation factor (EF-Tu) and heat stability of chloroplast stromal proteins in maize

Plant Science 166 (2004) 81–88

Localization and abundance of chloroplast protein synthesis elongation factor (EF-Tu) and heat stability of chloroplast stromal proteins in maize I. Momcilovic, Z. Ristic∗ Department of Biology, The University of South Dakota, 414 East Clark Street, Vermillion, SD 57069, USA Received 23 June 2003; received in revised form 15 August 2003; accepted 27 August 2003

Abstract Chloroplasts from a line of maize with high levels of chloroplast protein synthesis elongation factor (EF-Tu), ZPBL 1304, display greater heat stability than chloroplasts from a line with lower levels of EF-Tu, ZPL 389. We hypothesize that the greater heat stability of chloroplasts from ZPBL 1304 line may partly be attributed to EF-Tu, which may be protecting chloroplasts from heat injury. In this study, we investigated the subcellular distribution of EF-Tu and the heat stability of chloroplast stromal proteins in ZPBL 1304 and ZPL 389. Immunogold labeling and transmission electron microscopy (TEM) revealed that the chloroplast EF-Tu is localized mostly in the stroma and that under normal conditions agranal chloroplasts have a higher relative level of this protein than granal chloroplasts. Light scattering experiments with chloroplast stromal extracts showed that stromal proteins from the maize line with higher levels of EF-Tu, ZPBL 1304, display greater heat stability than stromal proteins from the line with lower levels of EF-Tu, ZPL 389. The results support the hypothesis that maize EF-Tu plays a role in the development of heat tolerance, possibly by acting as a molecular chaperone and protecting chloroplast stromal proteins from thermal aggregation. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Chloroplast EF-Tu; Chloroplast stromal proteins; Heat stress; Protein heat stability; Zea mays

1. Introduction Chloroplast protein synthesis elongation factor (EF-Tu) is a protein (45–46 kDa [1]) that plays a central role in the elongation phase of protein synthesis by promoting the GTP-dependent binding of aminoacyl-tRNA to the A site of the ribosome [2–4]. In most higher plants, this protein is encoded by the nuclear genome and synthesized in the cytosol [5]. Chloroplast EF-Tu is highly conserved as it shows a high sequence similarity to prokaryotic EF-Tu [1,4]. Recently our laboratory found that a heat-tolerant maize line, ZPBL 1304, synthesizes and accumulates increased amounts of EF-Tu under heat stress conditions [1]. Isolated chloroplasts from heat-stressed ZPBL 1304 plants also have been shown to have higher levels of EF-Tu as compared to non-stressed chloroplasts [1]. ∗ Corresponding author. Tel.: +1-605-677-6170; fax: +1-605-677-6557. E-mail address: [email protected] (Z. Ristic).

It has been hypothesized that maize EF-Tu may play a role in the development of heat tolerance [1,6]. Three lines of evidence support this hypothesis. First, the heat-induced synthesis and accumulation of EF-Tu is not seen in a heat-sensitive maize line, ZPL 389 [7,8] (it should be noted that in previous studies [7,8], maize EF-Tu was referred to as a 45–46 kDa heat shock protein (HSP) since the identity of this protein was not known until the report of Bhadula et al. [1]). Second, the heat-induced accumulation of EF-Tu associates with the heat tolerance phenotype [8,9]. And third, the over-expression of maize EF-Tu increases Escherichia coli viability under heat stress [6]. Previous studies have shown that chloroplasts from a line of maize with higher levels of EF-Tu, ZPBL 1304, display greater heat stability than chloroplasts from a line with lower levels of EF-Tu, ZPL 389 [10,11]. We speculate that the greater heat stability of chloroplasts from the ZPBL 1304 line may partly be attributed to EF-Tu, which may be protecting thylakoids from heat-stress. Indeed, the thylakoid membranes of ZPBL 1304 display greater heat stability than

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thylakoid membranes of ZPL 389 [10,11]. If the thylakoids of the ZPBL 1304 line are protected from heat stress by EF-Tu, it is reasonable to expect this protein may be associated with thylakoid membranes during exposure to stress. One of the objectives of this study was to investigate this possibility. We examined the subcellular distribution and abundance of EF-Tu in ZPBL 1304 and ZPL 389 maize under normal (control) and heat stress conditions using immunogold labeling and transmission electron microscopy (TEM). It is also possible that EF-Tu confers heat tolerance to chloroplasts by protecting chloroplast proteins from heat-induced denaturation and aggregation. Prokaryotic EF-Tu has been shown to protect heat-labile proteins from thermal damage by acting as a molecular chaperone [12,13]. Prokaryotic and maize EF-Tu are much alike (>80% amino acid identity) [1], and it is entirely possible that maize EF-Tu may show activity similar to prokaryotic EF-Tu. If maize EF-Tu protects heat-labile proteins from heat-induced damage, it is then reasonable to expect that chloroplast proteins from maize with higher levels of EF-Tu will show greater heat stability than chloroplast proteins from maize with lower levels of EF-Tu. In this study, we investigated this possibility. We assessed the heat stability of chloroplast stromal proteins in two lines of maize (ZPBL 1304 and ZPL 389) differing in heat tolerance and endogenous levels of EF-Tu.

2. Materials and methods 2.1. Plant material and heat-stress treatment Kernels of heat-tolerant, ZPBL 1304, and heat-sensitive, ZPL 389, maize (Zea mays L.) lines [11], were sown in pots containing Miracle-Gro Potting Mix (Miracle-Gro Lawn Products, Inc., NY, USA). Plants were grown under laboratory conditions (25 ◦ C/20 ◦ C: day/night temperatures, 12 h photoperiod (PPF: 280 ␮mol m−2 s−1 ), 70% RH) and were watered daily. Three-week-old whole plants were divided into control and experimental groups. The control group was maintained at 25 ◦ C and the experimental group was exposed to 45 ◦ C heat stress in a growth chamber (100% RH) for 3 h [14]. The temperature was gradually increased from 25 to 45 ◦ C over 1 h. The exposure time for the heat stress treatment was measured from the moment when the temperature reached 45 ◦ C. Following heat stress, fully expanded leaves were collected and used to investigate the subcellular distribution of EF-Tu (immunogold labeling and transmission electron microscopy) and heat stability of chloroplast stromal proteins. 2.2. Immunogold labeling and transmission electron microscopy Leaf samples (2–3 mm2 ) were obtained from the fourth leaf blades of five randomly selected seedlings from both control and heat-stressed plants. Leaf tissue was prepared

for immunogold labeling and TEM according to Hayat [15]. Samples were fixed in 3% (v/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde in 25 mM phosphate buffer (pH 7.0) for 1 h. Following fixation, samples were washed in 25 mM phosphate buffer, dehydrated in a graded series of ethanol (50–100% (v/v)), and embedded in LR White (London Resin Company, Berkshire, UK). The embedding was completed in 24 h at 50 ◦ C. Ultrathin sections (100 nm) were labeled with a rabbit anti-EF-Tu polyclonal antibody [1] and postlabeled with a goat anti-rabbit secondary antibody conjugated with 10-nm gold particles (British Biocell International Ltd., UK). Labeled sections were postfixed with 1% (v/v) glutaraldehyde, stained with 2% (w/v) aqueous uranyl acetate and 0.2% (w/v) aqueous lead citrate, and viewed with a JEM-1210 TEM (JOEL, Tokyo, Japan). Between 20 and 30 granal and 20 and 30 agranal chloroplasts [16] were examined in each leaf/plant. The cross-sectional area of examined chloroplasts was then determined [17], and the relative amount of EF-Tu was estimated by counting the number of gold particles in the chloroplasts and expressing the counts (gold/EF-Tu abundance) per chloroplast unit (1 ␮m2 ) area. Data obtained from each leaf sample were averaged, and the averages from five plants were used for statistical analysis. 2.3. Isolation and fractionation of chloroplasts Chloroplasts were isolated and purified as described by Bhadula et al. [1]. Phase contrast microscopy showed that the chloroplasts were intact (phase bright). Isolated chloroplasts were ruptured by osmotic shock using chloroplast lysis buffer [18] in the presence of 1% (v/v) protease inhibitor cocktail (Sigma–Aldrich, USA) followed by ultrasonication [1]. Chloroplasts lysates were then centrifuged for 5 min at 14,000×g. The supernatant containing the stromal (soluble) fraction and the pellet containing the membrane fraction were collected, and protein concentrations were determined using the Bradford Assay (Bio-Rad, CA). The stromal fraction was then used to assess the heat stability of stromal proteins. In addition, stromal and membrane fractions from ZPBL 1304 were analyzed for EF-Tu using western blotting; stromal and membrane fractions were analyzed in ZPBL 1304 only because this line had a much higher level of this protein than ZPL 389 ([1], this study). 2.4. Western blot analysis Aliquots of chloroplast stromal or membrane fractions from the ZPBL 1304 line were mixed with SDS-buffer and separated on 10% polyacrylamide gels [19]. Equal amounts of protein were loaded in each lane. Following electrophoresis, proteins were transferred to a nitrocellulose membrane (Bio-Rad, CA), and blots were probed for EF-Tu using an ECL western blot kit (Amersham-Pharmacia Biotech) and maize anti-EF-Tu polyclonal antibody [1]. The relative level (amount) of EF-Tu was estimated by determining band density, using Quantity One software (Bio-Rad).

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2.5. Heat stability of chloroplast stromal proteins The heat stability of chloroplast stromal proteins was assessed as their ability to remain soluble at high temperature, according to Lee et al. [20]. Aliquots (1 ml; protein concentration: 200 ␮g ml−1 ) of stromal fractions were incubated at 55 ◦ C for 30 min, and the heat stability of stromal proteins was estimated by monitoring light scattering at 320 nm [20,21].

3. Results

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and agranal chloroplasts were preserved and their membranes were distinct (Fig. 1). Heat stress, however, affected some chloroplasts in ZPL 389 line (Fig. 2). In affected chloroplasts, thylakoids and grana (Fig. 2C) and stroma lamellae (stroma thylakoid membranes) (Fig. 2D) were not well defined, and some agranal chloroplasts appeared swollen and irregular in shape and had broken envelopes (Fig. 2E). This appearance of affected chloroplasts was repeatedly apparent in two independent experiments. 3.2. Immunogold localization and abundance of chloroplast EF-Tu

3.1. Chloroplast structure after exposure to heat stress Exposure to heat stress did not cause significant changes in chloroplast structure in ZPBL 1304 (Fig. 1). Granal

The immunogold labeling and TEM revealed the subcellular distribution of EF-Tu in ZPBL 1304 and ZPL 389. As expected, the 10-nm gold particles, which indicate EF-Tu,

Fig. 1. Immunogold localization of chloroplast protein synthesis elongation factor (EF-Tu) in granal (A and C) and agranal (B and D) chloroplasts of ZPBL 1304 maize line. Three-week-old plants were exposed to 45 ◦ C heat stress for 3 h. A and B, control plants (plants not exposed to 45 ◦ C); C and D, heat-stressed plants. Arrows indicate 10-nm gold particles/EF-Tu; cw: cell wall; c: cytosol; e: chloroplast envelope; g: granum; f: stroma lamellae (stroma thylakoid membranes); m: mitochondrion.

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Fig. 2. Immunogold localization of chloroplast protein synthesis elongation factor (EF-Tu) in granal (A and C) and agranal (B, D, and E) chloroplasts of ZPL 389 maize line. Three-week-old plants were exposed to 45 ◦ C heat stress for 3 h. A and B, control plants (plants not exposed to 45 ◦ C); C–E, heat-stressed plants. Arrows indicate 10-nm gold particles/EF-Tu; arrowhead indicates broken chloroplast envelope (E); cw: cell wall; c: cytosol; e: chloroplast envelope; g: granum; f: stroma lamellae (stroma thylakoid membranes).

were predominantly observed in chloroplasts (Figs. 1 and 2). A few authentic gold particles/EF-Tu were seen in the cytosol (Figs. 1 and 2) but no gold was detected in mitochondria (Fig. 1A), nucleus or any other cell organelle. The presence of EF-Tu in the cytosol was not surprising since EF-Tu is known to be encoded by nuclear gene(s) and synthesized in the cytosol [5]. The distribution of gold particles signifying the presence of EF-Tu within chloroplasts was uniform in both lines. Most particles were noticed in the stroma and few appeared associated with the thylakoids/grana. A significant difference in the abundance of gold particles/EF-Tu was observed between the chloroplasts of two lines. ZPBL 1304 chloroplasts had a higher abundance of gold/EF-Tu (P < 0.05) than ZPL 389 chloroplasts (Fig. 3); on average, ZPBL 1304 chloroplasts (combined granal and agranal chloroplasts) had 2.7- and 4.8-fold

higher abundance of gold/EF-Tu than ZPL 389 chloroplasts under control and heat stress conditions, respectively (Fig. 3). Exposure to heat stress affected the abundance of gold/EF-Tu in the two lines differently. No significant change in the abundance of gold/EF-Tu was noticed in ZPL 389. In contrast, an increase in the abundance of gold was seen in ZPBL 1304 (Fig. 3). Granal chloroplasts from heat-stressed ZPBL 1304 plants had 38% higher abundance of gold/EF-Tu (P < 0.05) than granal chloroplasts from control plants (Fig. 3). A comparison of gold/EF-Tu abundance between granal and agranal chloroplasts within each maize line also revealed differences (Fig. 3). Under control conditions, agranal chloroplasts of both lines had a higher abundance of gold (P < 0.05) than granal chloroplasts. Under heat stress conditions, on the other hand, no significant difference in

2

Number of gold particles per µm of chloroplast cross-sectional area

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35 30

Granal chloroplasts Agranal chloroplasts

25 20 15 10 5 0 25˚C

45˚C

ZPL 389

25˚C

45˚C

ZPBL 1304

Fig. 3. Abundance of gold particles, indicating chloroplast EF-Tu, in granal and agranal chloroplasts of control (25 ◦ C) and heat-stressed (45 ◦ C) ZPBL 1304 and ZPL 389 maize plants. Three-week-old plants were exposed to 45 ◦ C for 3 h. Data were obtained from five plants; for each plant, 20–30 granal and 20–30 agranal chloroplasts were examined and gold particles were counted. Data obtained from each plant were averaged and used for statistical analysis (Tukey’s test). Bars indicate standard errors (n = 5). For details, see Section 2.

the abundance of gold was noticed between granal and agranal chloroplasts in both lines. 3.3. Western blot analysis Since the relative level of EF-Tu seemed to be much higher in the heat-tolerant line (ZPBL 1304) than in the heat-sensitive (ZPL 389) maize line, we further investigated the localization of this protein in the tolerant line. We analyzed proteins from chloroplast stromal and membrane fractions from ZPBL 1304 using western blotting and the anti-EF-Tu antibody. Western blotting revealed that the EF-Tu was predominantly located in the stromal fraction and a relatively small amount of this protein was apparent in the membrane fraction (Fig. 4). This pattern of EF-Tu distribution was seen under both control and heat stress conditions. In addition, the intensity of the EF-Tu band indicated that chloroplast stromal fraction from heat-stressed plants had higher relative level of EF-Tu than chloroplast stromal fraction from control plants (Fig. 4).

under both control and heat-stressed conditions. When the treatments within each line were compared (control versus heat stress), a significant difference in protein aggregation was also observed. In both lines, protein aggregation was lower in chloroplasts lysates from heat-stressed plants than in chloroplast lysates from control plants (Fig. 5). This decrease in protein aggregation, however, was much more prominent in ZPBL 1304 than in ZPL 389. Compared to protein aggregation in respective controls, 61% of stromal proteins from heat-stressed plants were aggregated in ZPL 389 and only 14% in ZPBL 1304 after 30 min at 55 ◦ C.

3.4. Heat stability of chloroplast stromal proteins 55 ◦ C,

When heated at chloroplast stromal proteins from both lines began to form insoluble aggregates, indicated by an increase in relative light scattering (Fig. 5). Aggregation of stromal proteins, however, was dramatically different between the two lines. Proteins from the line with the higher level of EF-Tu, ZPBL 1304, showed much less aggregation than proteins from the line with the lower level of EF-Tu, ZPL 389. This pattern of protein aggregation was evident

Fig. 4. Western blot analysis of proteins from chloroplast stromal and membrane fractions from the control (25 ◦ C) and heat-stressed (45 ◦ C) plants of the ZPBL 1304 maize line. Three-week-old plants were exposed to heat stress for 3 h. Equal amount of protein was loaded in each lane. The numbers at the bottom of the blot indicate the relative band density of the EF-Tu protein (considering control as 100%). Similar pattern in band density was observed in a duplicate blot. Note that most of the EF-Tu protein is located in the stromal fraction. Note: the relative band density of EF-Tu was determined using Quantity One software (Version 4.0.1) (Bio-Rad); because of the relatively low band density, Quantity One did not detect EF-Tu band in a membrane fraction.

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Relative light scattering at 320 nm

ZPL 389 C

0.3

ZPL 389 HS ZPBL 1304 C

0.25 ZPBL 1304 HS

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0.15

0.1

0.05

0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

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Time at 55ºC (min) Fig. 5. Relative light scattering of chloroplast stromal protein fractions from ZPBL 1304 and ZPL 389 maize lines. Three-week-old plants were exposed to 45 ◦ C heat stress for 3 h. Following heat stress, chloroplasts were isolated and purified and fractioned into stromal (soluble) and membrane fractions. Stromal (soluble) fractions (1 ml; protein concentration: 200 ␮g ml−1 ) were incubated at 55 ◦ C for 30 min, and light scattering was monitored at 320 nm during incubation [20]. Increase in relative light scattering indicates protein aggregation, which, in turn, indicates protein heat stability [20,21]. Note that stromal proteins from ZPBL 1304 had greater heat stability (lower protein aggregation) than stromal proteins from ZPL 389. Data represent averages of two independent experiments. Bars indicate standard errors. C: stromal fraction from control plants; HS: stromal fraction from heat-stressed plants.

4. Discussion Chloroplasts are key sites of damage caused by various environmental stresses including high temperature or heat [11,22,23]. The main damage to the chloroplast caused by heat mostly comes from detrimental effects on the chloroplast envelope [10,24] and thylakoid membranes [10,23]. High temperatures, however, can affect chloroplasts differently in genotypes with contrasting tolerance to heat stress [10,25]. In this study, the high temperature had different effects on chloroplast structure in our maize lines. Chloroplasts of a heat-tolerant line, ZPBL 1304, were not significantly affected by heat stress; in contrast, some chloroplasts of a heat-sensitive line, ZPL 389, suffered structural damage. This observation was similar to that reported by Ristic and Cass [10] who noted that exposure to 6-h high temperature stress (45 ◦ C) did not affect chloroplasts in ZPBL 1304 but damaged chloroplasts in ZPL 389. Immunogold labeling and TEM revealed the localization of EF-Tu in ZPBL 1304 and ZPL 389. As expected, the EF-Tu was mostly found in chloroplasts. Interestingly, under normal conditions agranal chloroplasts had higher relative levels of this protein than granal chloroplasts. We do not know the significance, if any, of the unequal distribution of EF-Tu between these two types of chloroplasts. Agranal and granal maize chloroplasts are structurally and metabolically

different [16]. Agranal chloroplasts lack grana (stacked thylakoids) and have low Photosystem II (PSII) activity, and granal chloroplasts, in contrast, have high PSII activity but no or very low Rubisco activity [16]. We speculate that unequal distribution of EF-Tu may be related to functional properties of these chloroplasts. Further studies are needed to address this possibility. Immunogold labeling and TEM also confirmed that the heat-tolerant line had a substantially higher level of EF-Tu than the heat-sensitive line (Fig. 3). The heat-tolerant line also accumulated EF-Tu under heat stress and the heat-sensitive line did not (Fig. 3). This pattern of EF-Tu accumulation corroborates previous studies, which showed that ZPBL 1304 synthesizes and accumulates EF-Tu under heat stress, whereas ZPL 389 does not [7,8]. Combined, this and previous studies [7,8] suggest that EF-Tu may play a role in the development of heat tolerance, possibly by protecting chloroplasts from the effects of heat stress. We tested the hypothesis that EF-Tu may be associated with thylakoid membranes, thereby protecting them from heat injury. The results of our study, however, did not support this hypothesis. The EF-Tu was mostly localized in chloroplast stroma (Figs. 1, 2 and 4) suggesting that this protein may be protecting stromal proteins rather than thylakoid membranes from heat stress. We investigated the possibility that EF-Tu may be related to heat stability of chloroplast stromal proteins. The results

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of our experiments supported this possibility. As indicated by relative light scattering, stromal proteins from ZPBL 1304 were more heat stable (displayed lower aggregation at 55 ◦ C) than stromal proteins from ZPL 389 (showed greater aggregation at 55 ◦ C) (Fig. 5). ZPBL 1304 has higher levels of EF-Tu than ZPL 389 under both control and heat stress conditions (Fig. 3). It is, therefore, highly possible that greater heat stability of stromal proteins from ZPBL 1304 may partly be attributed to EF-Tu. The EF-Tu may be protecting stromal proteins from thermal aggregation by acting as a molecular chaperone. This hypothesis is supported by Kudlicki et al. [26], Caldas et al. [12], and Malki et al. [13] who demonstrated chaperone-like activity for bacterial EF-Tu. Also, our preliminary in vitro experiments have shown that the purified recombinant maize EF-Tu displays chaperone properties as it protected heat-labile proteins, citrate synthase and malate dehydrogenase, from thermal aggregation and inactivation (Rao et al. unpublished). It could be argued that HSPs may possibly stabilize chloroplast stromal proteins in ZPBL 1304 and ZPL 389 during heat stress. Chloroplasts accumulate some low-molecular mass (LMM) HSPs (15–30 kDa) during heat stress [27–29], and it has been hypothesized that some chloroplast HSPs are involved in heat tolerance [30,31]. Previous studies have shown that ZPBL 1304 and ZPL 389 synthesize LMM HSPs [7,8], and it is, therefore, possible that LMM HSPs had an effect on the heat stability of chloroplast stromal proteins in our maize, as both lines displayed some increased thermal stability of stromal proteins after heat stress (Fig. 5). However, previous studies have also shown that ZPBL 1304 and ZPL 389 do not differ in the pattern of synthesis of LMM HSPs [7,8]. In fact, the heat-sensitive line, ZPL 389, synthesizes greater amounts of some LMM HSPs than the heat-tolerant line, ZPBL 1304 [7]. This suggests that the observed differences in the heat stability of chloroplast stromal proteins between ZPBL 1304 and ZPL 389 (Fig. 5) cannot be attributed solely to LMM HSPs. Rather, we suggest that the increased level of EF-Tu may partly be responsible for the almost complete prevention of thermal aggregation in the stromal proteins from ZPBL 1304. In summary, the results of this study showed that maize EF-Tu is localized mostly in the chloroplast stroma and that under normal conditions agranal chloroplasts have a higher relative level of this protein than granal chloroplasts. The results confirmed previous observations [7,8] that EF-Tu accumulates in maize genotypes that have greater tolerance to heat stress. Importantly, our study revealed that chloroplast stromal proteins from maize with higher levels of EF-Tu display greater heat stability than chloroplast stromal proteins from maize with lower levels of EF-Tu. The results support the hypothesis that maize EF-Tu plays a role in the development of heat tolerance, possibly by acting as a molecular chaperone and protecting chloroplast stromal proteins from thermal aggregation.

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Acknowledgements We acknowledge financial support for this research from the United States Department of Agriculture grant (Agreement no. 99-35100-8550) to Z. Ristic. The authors are thankful to Dr. Gary D. Small, Division of Basic Biomedical Sciences, The University of South Dakota School of Medicine, Dr. Karen L. Koster, Department of Biology, The University of South Dakota, and Dr. Thomas E. Elthon, Department of Biological Sciences, The University of Nebraska, Lincoln, NE for critical reading of the manuscript.

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