Degradation of Rubulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves During Dark-induced Senescence

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1 Acta Botanica Sinica 2004, 46 (2): Rapid Communication Degradation of Rubulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves During Dark-induced Senescence RUI Qi, XU Lang-Lai * (College of Life Sciences, Nanjing Agricultural University, Nanjing , China) Abstract: The degradation of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC ) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves during dark-induced senescence was studied. An in vivo degradation product of Rubisco large subunit (LSU) with molecular weight of 50 kd was detected by SDS-PAGE and immunobloted with antibody against tobacco Rubisco. This fragment could also be detected in natural senescence. The result also suggested that the Rubisco holoenzyme had not dissociated when LSU hydrolyzed from 53 kd to 50 kd. And LSU could be fragmented to 50 kd at and at ph 7.5 in crude enzyme extracts of wheat leaves dark-induced for 48 h, which suggested that maybe LSU was degraded to 50 kd by an unknown protease in chloroplast. Key words: Rubisco (EC ); protease; proteolysis; leaf senescence; wheat Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC ), accounting for more than 50% of the soluble protein in the mature leaves of C 3 plants, is the most abundant protein in plant cells (Makino et al., 1983). It is a bifunctional enzyme that catalyzes two competing reactions, photosynthetic CO 2 assimilation and photorespiratory carbon oxidation in the stroma of the chloroplasts. Thus the degradation of Rubisco is closely related to both the rate of photosynthesis and nitrogen economy in leaves. Although the synthesis, assembly, structure and regulation of Rubisco have been studied extensively, the mechanism of Rubisco degradation in leaves are not clearly known yet (IIoutz and Portis, 2003). Rubisco is a stromal protein that exists in chloroplasts. The decrease of the amount of Rubisco was much faster than the decrease of chloroplast number in many leaves such as wheat and barley during senescence (Martinoia et al., 1983; Mae et al., 1984). It has been suggested that the initial step of Rubisco degradation in leaves must occur within the chloroplasts and that the degradation may be triggered by a Rubisco-specific protease or by a specific modification of Rubisco protein, followed by general proteolysis. Some researchers think that reactive oxygen species (ROS) plays an important role in the Rubisco degradation. Mehta (1992) observed that oxidative aggregation between large subunits (LSU) and then translocation to the chloroplast membrane were crucial for its degradation. But Peng and Peng (2000) pointed out that it was not a necessary step for Rubisco breakdown. Now many researchers used isolated chloroplasts or chloroplasts lysates as model system, and treated them with some conditions to study the degradation of Rubisco. Different degradation products could be obtained under different conditions (Mae et al., 1989; Desimone et al., 1996; Ishida et al., 1997; Ishida et al., 1998; Kokubun et al., 2002). But so far, there have been no reports in which in vivo degradation products of Rubisco could be detected in leaves. Proteolysis during leaf senescence is a genetically controlled process. The results obtained using isolated chloroplast as material cannot completely represent the conditions in vivo. Using senescencing leaves as materials is more important for studying the Rubisco degradation in vivo. In this study, we investigated the degradation of Rubisco in wheat leaves during dark-induced senescence. An LSU fragment having an apparent molecular mass of 50 kd were detected in vivo. 1 Materials and Methods Wheat (Triticum aestivum L. cv. Yangmai 158) seeds were planted in pots with sandy clay and watered with Hoagland s complete nutrient solution. They grew in the natural environment. Samples were harvested at approximately four weeks after planting and the fully expanded leaves were used. Dark treatment was followed according to Rui and Xu (2003). The leaves in vitro were stored in dark at 25 to accelerate the senescence. To study the Rubisco degradation in natural senescence, we selected the leaves on different leaf positions as material (Zhang et al., 2001). The wheat seedlings with five leaves were used. The leaves from down to up were named as 1st, 2nd, 3rd, Received 16 Sept Accepted 10 Nov Supported by the National Natural Science Foundation of China ( ). * Author for correspondence.

2 4th and 5th leaf and the 4th leaf was fully expanded. The 1st leaf to the 4th leaf were selected. The crude enzyme extract was prepared by grinding at a ratio of 1 g leaf : 5 ml Tris-HCl buffer (50 mmol L, ph 7.5) containing 1 mmol L EDTA, 3 mmol/l β-mercaptoethanol, 1% PVP and some sand quarts in ice bath. Homogenates were centrifuged at 4 for 30 min at g. The supernatant was used as the crude enzyme extract or as sample for SDS-PAGE after boiling for 5 min in equal volume of 2 Laemmli sample buffer (Laemmli, 1970). The running gel and stacking gel contained 12.5% and 3.5% acrylamide, respectively. After electrophoresis, the gels were subjected to staining with Coomassie Brilliant R-250 (CBB), or to immunoblotting. If necessary, the gels stained with CBB were scanned at 595 nm with a dual-wavelength TLC scanner CS-930 (Shimadzu). For immunoblotting, the separated polypeptides on the gel were electrophoretically transferred to a nitrocellulose membrane with an electroblotting apparatus (Biometra B33). The membrane was reacted with antiserum raised against the tobacco Rubisco (gifted from Dr. CHEN G-Y), and further reacted with the second antibody with horseradish peroxidase-conjugated goat anti-rabbit IgG. Rubisco was purified from wheat leaves dark-induced for 0 h and 48 h as follows. The crude extracts were subjected to natural PAGE (5%) (Davies, 1964). The region that included the Rubisco was cut out from the gel plate and the protein was electrophoretically recovered from the gel cut. After elution, the protein was concentrated with concentrator (Amicon Microcon Devices) and then resolved by SDS-PAGE. The temperature effect on the formation of the degradation product was examined by incubation the crude extract at 4, 25, 30, 35, 40, 45, 50 and 55 at ph 7.5 for 20 min before SDS -PAGE. Soluble protein content was determined as described by Bradford (1976) using bovine serum albumin as a standard. Every experiment was carried out at least three times. 2 Results and Discussion The contents of both total soluble protein and Rubisco in wheat leaves decreased greatly during dark-induced senescence (Figs.1, 2). The degradation of intrinsic Rubisco large subunit (LSU) was analyzed by SDS-PAGE and immunoblotting. An in vivo degradation product of LSU, with an apparent molecular mass of 50 kd, appeared when dark incubating for 24 h and its amount clearly increased with dark treatment time from 24 h to 48 h (Fig.3A C). Since Fig.1. The changes of protein content during wheat leaf senescence. Fig.2. The changes of Rubisco in wheat leaves during darkinduced senescence. Natural PAGE (5%) was used, in which about 30 µl sample was applied. The gel was stained with Coomassie Brillient R-250 (CBB). Lanes 1 5 represent wheat leaves darkinduced for 0, 24, 48, 72 and 96 h, respectively. LSU and its degradation product have very near molecular weight, they cannot be resolved clearly when the content of LSU is high, especially when dark-induced for 24 h (Fig. 3A). While the applied sample was reduced to 5 µl, the 50 kd product in 24 h-dark-induced leaves was faintly visible (Fig.3B). The antibody used in this experiment is against tobacco Rubisco holoenzyme, which has high affinity to LSU, but has no affinity to small subunit (SSU) (personal communication with Dr. CHEN G-Y). So the Rubisco SSU could not be detected by immunoblotting. The degradation of Rubisco during natural senescence was also performed. The 50 kd fragment could be detected in the 1st, 2nd and 3rd leaves of wheat with five leaves (Fig.3D, E), but not in the 4th leaves. That is to say, LSU was hydrolyzed to 50 kd in both natural and dark-induced senescence. Many researchers had reported Rubisco degradation, but none of them detected the in vivo degradation product. For example, oxidative treatment could stimulate partial degradation of LSU to 36 kd in isolated barley chloroplasts (Desimone et al., 1996) and reactive oxygen species could

3 RUI Qi et al.: Degradation of Rubulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves During Dark-induced Senescence directly fragment LSU to 37 kd and 16 kd (Ishida et al., 1997). Additionally, EP1, a metalloprotease purified from the pea chloroplast, could hydrolyze the LSU to 36 kd (Bushnell, et al., 1993). Recently Kokubun et al. (2002) detected a 44 kd fragment in wheat chloroplast when isolated chloroplast was incubated in darkness for some time. Obviously, our result is different from all the above and the fragment which we detected is an in vivo product although Kokubun et al. (2002) reported no in vivo degradation products of Rubisco could be detected in the extracts from darkinduced or naturally senescing wheat leaves. Rubisco is composed of eight LSUs and eight SSUs. During leaf senescence, whether Rubisco was dissociated or degraded firstly was not reported. In this study, Rubisco holoenzyme was purified from natural gel and then subjected to SDS-PAGE. From Fig.3 F and G, it could be seen that only 53 kd was detected from Rubisco purified from leaves dark-induced for 0 h; but for Rubisco purified from leaves dark-induced for 48 h, both 53 kd and 50 kd fragments could be detected. It implied that the fragment was formed while the subunit was still integrated in the holoenzyme complex. Since these kinds of complexes have very near molecular mass, they could not be obviously separated by PAGE with low concentration (Fig.2). Rubisco was rapidly degraded during leaf senescence. But the degradation process is not very clear. Since the 50 kd fragment was not detected in young leaves, it might be that LSU degradation from 53 kd to 50 kd should be a key process for rapid Rubisco breakdown during leaf senescence and the protease catalyzing this reaction should be senescence-associated. To study the protease that functioned during LSU degraded from 53 kd to 50 kd, effects of temperature on the appearance of the 50 kd fragment were tested. The optimal temperature for 50 kd fragment formation was Fig.4. When the crude extracts were pretreated at 100 for 5 min and then incubated at 35 for 12 h, the amount of LSU fragment did not increase, which suggesting the involvement of a protease in the appearance of the 50 kd fragment. The cleavage was triggered by this unknown protease in the cell. And whether Fig.3. Degradation of Rubisco during dark-induced senescence. Degradation product of Rubisco was detected by staining the gels (12. 5%) with Coomassie Brilliant R-250 (CBB) (A, B, D, F) or immunoblotting (C, E, G) after SDS-PAGE. For A, D and F, 20 µl sample was applied to the lanes, but 5 µl of sample was applied to the lanes of B. For corresponding immunoblotting (C, E, G), 5 µl of sample was applied. Lanes 1 5 in A, B and C represent wheat leaves dark-induced for 0, 24, 48, 72 and 96 h, respectively. Lanes 1 4 in D represent the 4th leaf to 1st leaf, respectively. Lanes 1 and 2 in E represent the 4th leaf and the 3rd leaf. Lanes 1 and 2 in F and G represent purified Rubisco from wheat leaves dark-induced for 0 and 48 h. LSU, larger subunit; M, mark; SSU, small subunit.

4 the protease was expressed during senescence or existed in the cell as a proenzyme was unknown. While the temperature was above 45 the amount of both 53 kd and 50 kd decreased, and an emerging fragment with the molecular mass about 44 kd appeared (Fig.4). Whether it was the fragment reported by Korimoto (2002) was not studied in this study. Since the decrease of the amount of Rubisco was much faster than the decrease of chloroplast number during leaf senescence, it was suggested that the initial step of Rubisco degradation must occur within the chloroplasts (Martinoia et al., 1983; Mae et al., 1984). To preliminarily study the location of this unknown protease, which hydrolyzed LSU Fig.4. Effect of temperature on the appearance of 50 kd fragment. The crude enzyme extract was incubated at different temperatures for 20 min before SDS-PAGE. Abbreviations are the same as in Fig.3. Fig.5. Effect of ph on the appearance of 50 kd fragment. Lanes 1 4 represent the crude enzyme extract incubated at ph 5 (Naacetate buffer) for 0, 30, 60 and 300 min before SDS-PAGE, respectively. Lanes 5 8 represent the crude enzyme extract incubated at ph 7.5 (Hepes) for 0, 30, 60 and 300 min before SDS- PAGE, respectively. Abbreviations are the same as in Fig.3. to 50 kd fragment, we incubated the crude enzyme extract at ph 5 and ph 7.5 for some time to observe the degradation of Rubisco. When 48 h dark-induced crude extracts was incubated at ph 5 for 30 min, the 53 kd and 50 kd fragments were degraded and could not be detected by SDS-PAGE clearly (Fig.5). It had been tested that the endopeptidase activity increased greatly and a number of endopeptidase isoenzymes expressed during leaf senescence, most of which have acidic ph values, about ph 5 (Rui and Xu, 2002). So proteases activity at acidic ph was responsible for the quick degradation of Rubisco when 48 h-darkinduced crude enzyme extract was incubated at ph 5. While at ph 7.5, near the physiological ph of the chloroplast stroma, the LSU was hydrolyzed to 50 kd fragment in 30 min. And the amount of 50 kd fragment did not decrease with further incubation, even for 5 h (Fig.5). It maybe that the acidic proteases in the extract could not show their activities greatly at ph 7.5, and an unknown protease with optimal ph at 7.5 could specially hydrolyze LSU to 50 kd fragment. This unknown protease with neutral ph value that functioned during leaf senescence was probably located in the chloroplast since chloroplast could supply such environment. That is to say maybe LSU was initially degraded from 53 kd to 50 kd in the chloroplast and then Rubisco dissociated and were hydrolyzed by other proteases in the cell. Further research to test this point is under progress in our laboratory. Rubisco is a major source of leaf nitrogen. During senescence, it is degraded and the nitrogen is exported to younger parts of the plant (Feller, 1986). But the mechanism of Rubisco degradation is still in question. Studying the enzymes that function in this important recycling event is of great interest. In our study, future work will emphasize on determination the site of the split in the Rubisco LSU and identification of this unknown protease. Acknowledgements: We are grateful to Dr. CHEN Gen- Yun (Institute of Plant Physiology and Ecology, The Chinese Academy of Sciences) for the generous gift of anti- Rubisco antibody. We wish to thank Prof. PENG Xin-Xiang from South China Agricultural University Prof. WEI Jia- Mian from Institute of Plant Physiology and Ecology for constructive advice and help, Prof. YAN Qing and Ms. CHEN Min from Nanjing Agricultural University for technical assistance during our experiments. References: Bradford M M A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem, 72:

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