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Page 1: Ubiquitin Conjugating Activity in Leaves and Isolated Chloroplasts from Avena sativa L. during Senescence

J. PlantPhysiol. Vol. 138. pp. 608-613 (1991)

Introduction

Ubiquitin Conjugating Activity in Leaves and Isolated Chloroplasts from A vena sativa L. during Senescence

BJARKE VEIERSKOVI and I. B. FERGUSON

DSIR Fruit & Trees, Mt Albert Research Centre, Private Bag, Auckland, New Zealand

1 Present address: Department of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark

ReceivedJanuary 17, 1991 . Accepted May 5,1991

Summary

Proteolysis was studied in oat (Avena sativa L.) leaves undergoing senescence in the dark, and in chloro­plasts isolated from oat leaves and allowed to senesce in vitro. Isolated chloroplasts were found to senesce in vitro at the same rate as attached leaves. In vivo labelling of leaf proteins with 35S-methionine increased over the first two days of senescence, and then decreased. During this time a small decrease was observed in the level of total leaf protein. The regulatory polypeptide ubiquitin stimulated in vitro breakdown of newly synthesized proteins. In senescing, isolated chloroplasts, protein and chlorophyll breakdown oc­curred in a pattern similar to that in senescing attached leaves. In vitro conjugation of ubiquitin to chloro­plast proteins was investigated. Time- and ATP-dependent ubiquitin conjugation with endogenous chlo­roplast proteins was found, and was maintained over the senescing period. Labelling of Western blots with ubiquitin antibodies showed increases in in vivo conjugation of ubiquitin to specific polypeptides during the 2 day period of senescence of isolated chloroplasts. These results suggest that ubiquitin may have be involved in protein breakdown in senescing green leaves, and particularly in chloroplasts.

Key words: Ubiquitin, senescence, chlorophyll breakdown, oat, proteolysis, protease, chloroplast.

Abbreviations: BCIP = 5-bromo-4-chloro-3-indolylphosphate p-toluidine; DTT = dithiothreitol; EDTA = ethylenediaminetetraacetic acid; HEPES = N-2-hydroxyethylpiperazine; MES = 2-{N-mor­pholino)ethanesulphonic acid; NBT = nitroblue tetrazolium chloride; RuBPCase = ribulose-1,5-bis­phosphate carboxylase; SDS-PAGE = sodium dodecyl sulphate polyacrylamide gel electrophoresis; Tris = 2-amino-2-{hydroxymethyl)-1,3-propanediol.

One of the unresolved problems in plant senescence is the need to explain the pattern of protein degradation in green leaves in terms of both the levels of cellular protease activity and the compartmentation of proteases and major cell pro­teins such as RuBPCase. Since proteolytic activity has been found in chloroplasts (Waters et al., 1982; Dalling et al., 1983; Casano et al., 1989; Musgrove et al., 1989), it is becom­ing accepted that chloroplast proteins can be degraded by proteases in situ (Dalling, 1987), although the identified pro­teases seem not to be involved in increased proteolysis dur-

ing senescence. The presence of proteolytic activity within the chloroplast is supported by the findings that gene expres­sion may occur without protein accumulation (Rochaix and Erickson, 1988), that etioplasts contain many short lived proteins (Mullet et al., 1990), and that although nuclear coded proteins have transit peptides for the transport into the chloroplast, these polypeptides as well as excess tran­scripts never accumulate (Liu and Jagendorf, 1984, 1985; Malek et al., 1984).

It has recently been accepted that protein turnover is con­trolled by ATP-dependent proteases in mammalian cells (Goldberg et al., 1985). Results showing the presence of

© 1991 by Gustav Fischer Verlag, Stuttgart

Page 2: Ubiquitin Conjugating Activity in Leaves and Isolated Chloroplasts from Avena sativa L. during Senescence

ATP-dependent proteolysis in pea chloroplasts are therefore of particular interest (Liu and Jagendorf, 1984, 1985; Malek et aI., 1984).

One pathway for ATP-dependent breakdown of proteins involves the small regulatory polypeptide ubiquitin. In this pathway, proteins are committed to degradation by ATP-de­pendent ligation to ubiquitin, and degraded to amino acids by ATP-dependent proteases. This pathway has been iden­tified in plants (Vierstra et aI., 1985; Vierstra, 1987; Veierskov and Ferguson, 1991). Oat leaf chloroplasts contain the enzyme system which allows conjugation of ubiquitin to chloroplast proteins, and in vitro breakdown of RuBPCase stimulated by ubiquitin has also been demonstrated (Veierskov and Ferguson, 1991).

The accumulating evidence for proteolytic activity within the chloroplast raises the question of what is controlling chloroplast protein degradation during greening and se­nescence. This has led us to investigate the possibility of fol­lowing senescence in isolated chloroplasts, and to look into the possible involvement of ubiquitin in leaf protein break­down, particularly during senescence.

Materials and methods

Plant material

Oats (Avena sativa L. cv. Makuru) were germinated in a general seedling mixture (Western Nurseries Ltd., Auckland N.Z.), and grown in a 16 h photoperiod for 8 days. The temperature was 23 ± 2°C, the irradiance at plant level 300 Itmol m - 2 S - I, and the light source a 400W M400/C/BU-HOR lamp (Sylvania-Canada).

Chloroplasts

Chloroplasts were isolated from oat leaves and purified as de­scribed previously (Veierskov and Ferguson, 1991). Intactness of the chloroplasts was determined by the protein/chlorophyll ratio (Lil­ley et aI., 1975). Only chloroplasts with a ratio of 20 were used, in­dicating an intactness close to 100 %. To follow senescence in isolat­ed chloroplasts, they were resuspended after preparation in 3 mL of sterile ginding media (0.35 M sucrose, 0.05 M HEPES, 0.01 M KHC03, 0.002M NaEDTA, O.OlM KCl, 0.005M N<4P2 0 7,

O.OOlM MgCh, O.OOlM MnCh, 0.004M DTT, 0.0002M ADP, pH 7.5 with KOH). The chloroplasts were held in the dark at room temperature, and there were no indications of bacterial contamina­tion. At daily intervals, 0.6 mL of the chloroplast preparation was sampled and divided into 200 ItL fractions. From each fraction, 30ltL was used for protein determination (Bradford, 1976). The re­maining volume was centrifuged at 6000 g for 1 min, and the pellet was made up to 300 ItL with water, and 1.2 mL acetone added. Chlo­rophyll levels were determined by measuring absorbance at 645.7 and 663.2nm (Lichtenthaler, 1987).

Plant senescence

Eight day old oat plants were placed in darkness, and samples were taken at appropriate intervals. The leaves were excised at soil level, and five 4 cm subapical segments were homogenized in a glass grinder in 1 mL Tris/MES/KOH buffer (0.1 M, pH 7.2). Samples of 50 ItL and 300 ItL was used for protein and chlorophyll measure­ments respectively, as described above.

Ubiquitin and senescence in oat leaves and chloroplasts 609

Protein labelling

Oat seeds were germinated and grown in vermiculite for 4 or 6 days, whereafter 50 plants were selected and placed in a beaker for either 3 days or 15 h containing 10 mL tap water and 20 ItL e5Sl­methionine (30TBq mmol- I

). After this time, the leaves were ex­cised above the seed and placed upright in a beaker in darkness with the basal 1 cm in tap water. At daily intervals 10 leaves were se­lected, homogenized in a glass homogenizer in 0.5 mL buffer (Tris/ MES 0.1 M pH 7.2), filtered through one layer of miracloth, cen­trifuged at 12,000 g for 2 min, and the supernatant desalted on a Sephadex G-25 column (0.5 x 5 cm) and eluted with HEPES buffer (0.01 M, pH 8.2). Protein contents and 35S activity were determined in the eluate.

Ubiquitin radiolabelling and binding assay

Radiolabelling of bovine ubiquitin (Sigma) with 1251, and the ubi­quitin binding assay was performed as described by Veierskov and Ferguson (1991). The conjugation reaction was carried out at 30°C for various times. After the reaction, most of the unbound ubiquitin was separated from the reaction mixture by passing the solution through a small Sephadex G-100 column. After sampling to de­termine cpm of ubiquitin-protein conjugates, the rest of the sample was freeze-dried for later identification of conjugates by SDS-PAGE (Veierskov and Ferguson, 1991).

lmmunoblotting 0/ ubiquitin

Extracts from senescing chloroplasts were prepared as described above. Samples containing 30 Itg protein were separated by SDS­PAGE (13% gels). Western blots were performed by transferring proteins from gels to nitrocellulose paper with a Proto blot appa­ratus (Biorad). The nitrocellulose papers were then held in boiling water for 30 min, and analysed using a polyclonal, monospecific an­tibody for ubiquitin (rabbit antiubiquitin, Dakopatts, Denmark). Antibodies bound to the blot were detected by secondary binding with alkaline phosphatase-conjugated swine anti-rabbit immunoglo­bulin (Dakopatts, Denmark), using BCIP/NBT (BRL) as substrate.

Results

When 10 day old oat plants were placed in darkness, the leaf protein level began to decrease after one day, whereas

40

o 2 3 T lme. days

Fig. 1: Dark-induced senescence of attached 10-day-old oat leaves. 5 leaves were sampled daily, and protein (0) and chlorophyll (e) con­tents determined as given in Material and Methods. Values for 100 % were 23.7 mg g - 1 FW and 307 Itg g - 1 FW for protein and chloro­phyll respectively. Data are means from 4 replications.

Page 3: Ubiquitin Conjugating Activity in Leaves and Isolated Chloroplasts from Avena sativa L. during Senescence

610 BJARKE VEIERSKOV and I. B. FERGUSON

Table 1: Breakdown of 35S-labeled proteins, and extractable protein content in leaves during senescence in the dark. Oat plants were labeled 3 days prior to excision, and the proteins extracted as given in Materials and Methods. Data are then means of 3 determinations ±SD.

Time Cpmmg Protein (days) protein- I (mgg-I)FW

° 13,959± 131 1183±14 1 21,193±564 1025±25 2 20,226± 15 978± 10 3 10,461±648 485±27

kD

200-

97.4-

66.2-

42.7-

31.0-

21.5-

1 2 3 4 5

Fig.2: Ubiquitin-dependent breakdown of 35S-labeled proteins ex­tracted from 8-day-old oat plants. The plants were labeled for 15 h prior to excision. 74 Jtg protein was used in each assay in the pres­ence of 0, 0.5, 1, 5 and 10 Jtg ubiquitin (lanes 1-5 respectively). Assay time was 60 min.

the chlorophyll level was stable for the first two days before decreasing (Fig. 1). In order to determine changes in protein turnover during senescence, young plants were grown hy­droponically with [35S}methionine for 3 days. Senescence was induced by excision and holding the leaves in the dark. During the first day after excision the level of 35S-labeled pro­teins increased by almost 50 %, and stayed at this level until day three, when both specific activity of labeled protein, and total protein content dropped by about 50 % (Table 1). When an extract of newly synthesized proteins (lsS-labeled for 15 h prior to excision) was incubated for 60 min with ubiquitin, disappearance of the labelled proteins was found

: 100.~ ;:: :~.\ ~ o~.~. :. 40 o~

o

o 2 3 Time. days

Fig. 3: Senescence of chloroplasts isolated from 8-day-old oat seed­lings and maintained in the dark. Samples were taken daily for pro­tein (0) and chlorophyll (e) measurements as given in Materials and Methods. Values for 100% averaged 501 JtgmL -I and 23 Jtg mL -I for protein and chlorophyll respectively. Results are the means of 2 experiments, 3 determinations being made in each.

to be dependent on ubiquitin concentration (Fig. 2). Pro­teolysis occurred equally for all 35S-labeled proteins.

When intact, isolated chloroplasts were incubated in the dark, protein and chlorophyll levels decreased (Fig. 3). Over four days in darkness, the chlorophyll content decreased to 50 % of the initial level, whereas the protein content de­creased to 28 %. Although initial reduction of protein and chlorophyll was faster in the isolated chloroplasts, the extent of the reduction at 4 days was not too dissimilar from that in whole leaves (d. Figs. 1 and 3).

We have shown previously (Veierskov and Ferguson, 1991) that ubiquitin is able to bind proteins in lyzed chloro­plasts. A comparison of ubiquitin conjugation in chloro­plasts immediately after preparation and after 24 h se­nescence of isolated chloroplasts in the dark shows that the capacity for conjugation is retained, with ATP-dependence increasing with time (Fig. 4). Over 2 days of senescence, when the protein content of isolated chloroplasts had ap­proximately halved (Fig. 3, Table 2), the ubiquitin binding enzymes were found to be still active (Table 2).

Immunoblots of proteins extracted from senescing chloro­plasts, using ubiquitin antibodies, revealed in vivo conjuga­tion of ubiquitin to specific polypeptides which increased over the 2 day senescence period (Fig. 5). Particularly strong binding increases were found for bands with relative molecu­lar weights of 69, 46, 39 and 33 kDa. There was a loss in conjugation in a polypeptide of M, 61 kDa.

Discussion

The patterns of protein and chlorophyll breakdown that we found in attached oat leaves senescing in the dark (Fig. 1) are very similar to those found in detached leaves (Veierskov and Thimann, 1988). Although it is possible that detach­ment induces senescence, for instance by stopping cytokinin supply (van Loon et al., 1987; Bollmark et aI., 1988), dark-in­duced senescence of oat leaves does appear to have a degree of autonomy. Rates of protein degradation in detached leaves are sometimes faster (van Loon et aI., 1987). However,

Page 4: Ubiquitin Conjugating Activity in Leaves and Isolated Chloroplasts from Avena sativa L. during Senescence

kD

97.4

66.2

31.0 -

21.5-

1 2 3 4 5 6

Fig. 4: Conjugation of [125I]-ubiquitin to proteins from lyzed chloro­plasts which had been held in the dark for 0 (lanes 1 - 3) or 24 h (lanes 4-6). The conjugation assay was performed in the presence (lanes 1, 2, 4, 5) or absence (lanes 3, 6) of ATP. Assay time was 0 (lanes 1,4) or 60 (lanes 2, 3, 5, 6) min. 75 p,g protein was used in each assay.

Table 2: ATP-dependent conjugation of ubiquitin to proteins ex­tracted from senescing chloroplasts. Isolated chloroplasts were held in darkness, and at the indicated times, samples were lyzed and e25I]-ubiquitin conjugation and protein content determined as given in Materials and Methods. Data are the mean of duplicate deter­minations.

Time Cpm Protein (days) +ATP -ATP (p,g ml- 1

)

0 2357 1115 750 1 1861 725 600 2 2143 1020 360

measurements of protease aCtIVIty in detached/attached leaves in the light and dark have given little guide as to the validity either of attached or detached systems (van Loon et ai., 1987; Veierskov and Thimann, 1988). Although we mainly used attached leaves in our study, the above consid­erations are important with regard to isolated senescing chlo­roplasts.

Though protein levels begin to decrease 24 h after oat plants are placed in darkness (Fig. 1), synthesis continues as shown by 35S-methionine labeling (Table 1). It has been shown (Veierskov and Thimann, 1988) that senescing excised oat leaves incorporate more amino acids into proteins in darkness than in the light. Since there seems to be a large va-

Ubiquitin and senescence in oat leaves and chloroplasts 611

kD

69-61-

46-39-

33-

a b

Fig. 5: Immunoblots of ubiquitin conjugates from isolated oat leaf chloroplasts senescing in vitro for 2 days. Equal protein (30 p,g) was applied to each lane. Lane a = day 0, lane b = day 2.

riety of proteins, including RuBPCase, that are synthesized (data not shown), it is unlikely to be senescing proteins only that become labeled. This is likely to be stronger in the in­duced senescence of young tissues (as is the present case) than in old. Our results showing ubiquitin-dependent breakdown of newly synthesized proteins in vitro suggest that the ubi­quitin pathway may be responsible for turnover of newly synthesized proteins during dark-induced senescence of leaves. We have shown previously (Veierskov and Ferguson, 1991) that the ubiquitin-conjugating system is present in green leaves, and is optimal at pHs likely to be found in the chloroplast rather than the cytosol or vacuole.

It has been generally believed that chloroplasts do not con­tain proteolytic enzymes, and they therefore are unable to senesce if isolated (Choe and Thimann, 1975). However, it is now accepted that proteins can not cross the chloroplastic membrane without a transit peptide attached (Della-Cioppa et aI., 1987), and it is therefore becoming increasingly un­likely that the movement of proteins across the envelope is associated with proteolysis.

Our results showed that isolated chloroplasts held in the dark were able to senesce at rates similar to those of attached leaves in terms of protein and chlorophyll breakdown (Fig. 3). It is unlikely that there has been a de novo synthesis of proteases in isolated chloroplasts due to a lack of amino acids indicating that the proteolytic system was present in suffi­cient quantities at the time of isolation. This is in agreement with the findings of van Loon et al. (1987) who showed se­nescence to occur without increased proteolytic activity. We did not have any indications that the proteolytic activity was

Page 5: Ubiquitin Conjugating Activity in Leaves and Isolated Chloroplasts from Avena sativa L. during Senescence

612 BJARKE VEIERSKOV and I. B. FERGUSON

caused by contamination or bacterial infection. The rates of protein and chlorophyll loss we observed were greater than those found by Choe and Thimann (1975), and closer to those in our attached leaves than Choe and Thimann (1975) found in their comparison with detached leaves. The closer agreement in our results may be due to preparative tech­niques which highlight rapidity and high protein/ chloro­phyll ratios whereas the protein/chlorophyll ratio of the chloroplasts of Choe and Thimann (1975) was only about 8.

Since the chloroplast contains the major single protein in green leaves, the control of chloroplast protein breakdown, is of special interest. Although levels of neutral and acidic protease activity in leaves may change during senescence, no evidence has been obtained showing that these non-specific proteases, particularly vacuolar ones, control protein turn­over during senescence (van Loon et a1., 1987; Veierskov and Thimann, 1988). This is particularly the case with endoge­nous proteins of the chloroplast. It is now assumed that chlo­roplasts are able to degrade their constituent proteins (Dalling, 1987; Thayer et a1., 1987), and our results with iso­lated chloroplasts also suggest that the necessary proteases are present in the organelle. Chloroplasts have been shown to contain proteases that are able to degrade the unassembled small subunit of RuBPCase to amino acids with a half-life of less than 7 min (Schmidt and Mishkind, 1983), and which can degrade newly synthesized proteins in an ATP-dependent system (Liu and Jagendorf, 1984; Malek et a1., 1984). When isolated chloroplasts were lyzed it was found that they con­tained the necessary enzymes for binding [l25I}ubiquitin to proteins, not only at the time of isolation, but also after 24 h in darkness (Fig. 4). The capacity to carry out ATP-depend­ent binding of [I25I}ubiquitin to chloroplast proteins lasted for at least the first two days in darkness (Table 2). Although the ubiquitin conjugation was not fully ATP-dependent, marked ATP-induced differences were found, especially in proteins with a molecular weight of 39 - 46 kDa (Fig. 4). In vitro binding of [l25I]-ubiquitin demonstrates the capacity for conjugation, but not the extent of in vivo binding. This can be assessed by immunoblotting with ubiquitin antibodies. When this was done (Fig. 5), we found strong binding in a number of bands which increased during senescence of the isolated chloroplast. Thus, not only is the capacity for con­jugation present, but it is also taking place, indicating that ubiquitin is without doubt present in chloroplasts, and may be involved in protein turnover within the chloroplast dur­mg senescence.

ATP concentrations increase during dark-induced se­nescence of excised oat leaves (Trippi et al., 1989), which sug­gests that ATP availability might not be a problem if protein breakdown by ATP-dependent proteases were involved in senescence. Apart from the ATP-dependent chloroplast pro­teases mentioned above, the only other report of such en­zymes in green plant tissues is that of Hammond and Preiss (1984). Our results indicate that with the potential role of ubiquitin in chloroplast protein breakdown, this type of protease may playa key role in controlling proteolysis dur­mg senescence.

The ubiquitin-dependent proteolytic pathway may not only be reponsible for the observed degradation of chloro­plast proteins, but could also be the cause for the observed

chlorophyll degradation. Work on the catabolism of chloro­phyll has shown that the detachment of chlorophyll from the chlorophyll-binding protein is an ATP-dependent pro­cess, and that the initial steps of chlorophyll degradation take place within the chloroplast (Matile et a1., 1988; Schel­lenberg et a1., 1990). Although these authors were unable to identify any ATP-dependent proteases, the ubiquitin-de­pendent proteolytic pathway, that we have shown to be pre­sent in isolated chloroplasts, may be a candidate for the ATP­dependent proteolytic pathway that degrades the chloro­phyll-binding protein.

Acknowledgements

We wish to thank Trish Grant and Mike Lay Yee for assistance and advice, and the Danish Agricultural and Veterinary Research Council for support to BV (grant no. 13-4090-M).

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