Abstract

The roles of cysteine proteinases (CP) in leaf protein accumulation and composition were investigated in transgenic tobacco (Nicotiana tabacum L.) plants expressing the rice cystatin, OC-1. The OC-1 protein was present in the cytosol, chloroplasts, and vacuole of the leaves of OC-1 expressing (OCE) plants. Changes in leaf protein composition and turnover caused by OC-1-dependent inhibition of CP activity were assessed in 8-week-old plants using proteomic analysis. Seven hundred and sixty-five soluble proteins were detected in the controls compared to 860 proteins in the OCE leaves. A cyclophilin, a histone, a peptidyl-prolyl cis-trans isomerase, and two ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase isoforms were markedly altered in abundance in the OCE leaves. The senescence-related decline in photosynthesis and Rubisco activity was delayed in the OCE leaves. Similarly, OCE leaves maintained higher leaf Rubisco activities and protein than controls following dark chilling. Immunogold labelling studies with specific antibodies showed that Rubisco was present in Rubisco vesicular bodies (RVB) as well as in the chloroplasts of leaves from 8-week-old control and OCE plants. Western blot analysis of plants at 14 weeks after both genotypes had flowered revealed large increases in the amount of Rubisco protein in the OCE leaves compared to controls. These results demonstrate that CPs are involved in Rubisco turnover in leaves under optimal and stress conditions and that extra-plastidic RVB bodies are present even in young source leaves. Furthermore, these data form the basis for a new model of Rubisco protein turnover involving CPs and RVBs.

Introduction

Climate change and ongoing ecosystem degradation necessitate the development of food and bio-energy crops that can support future increasing environmental fluctuations. Endogenous plant cysteine proteinase inhibitors or phytocystatins can be used to minimize insect attack (Christou et al., 2006) and improve the yields of useful bio-engineered proteins such as vaccines (Rivard et al., 2006). However, while second generation multiple proteinase inhibitor-containing insect-resistant plants are already in production (Christou et al., 2006) little is known about the effects of such manipulations on plant productivity. Cysteine proteinases (CP) are involved with a variety of proteolytic functions in higher plants (Granell et al., 1998), particularly those associated with the processing and degradation of seed storage proteins (Shimada et al., 1994; Toyooka et al., 2000), and fruit ripening (Alonso and Granell, 1995). They are also induced in response to stresses such as wounding, cold, and drought (Schaffer and Fischer, 1988; Koizumi et al., 1993; Linthorst et al., 1993; Harrak et al., 2001) and in programmed cell death (Solomon et al., 1999; Xu and Chye, 1999). Like their CP targets, phytocystatins are regulated by developmental (Lohman et al., 1994) and environmental cues (Botella et al., 1996; Pernas et al., 2000; Belenghi et al., 2003; Diop et al., 2004).

Two novel tobacco CP-coding sequences have previously been identified in tobacco including a KDEL-type CP NtCP2 (Beyene et al., 2006). The C-terminal KDEL motif, present in some cysteine proteinases, is an endoplasmic reticulum retention signal for soluble proteins that allows CP propeptides to be stored either in a special organelle, called the ricinosome (Schmid et al., 1999), or in KDEL vesicles (KV) before transport to vacuoles through a Golgi complex-independent route (Okamoto et al., 2003). The relatively acidic pH optima of many of the endogenous plant CPs indicate that they are localized in the vacuole (Callis, 1995). A papain-like sequence, termed NtCP1, was isolated from senescent tobacco leaves (Beyene et al., 2006). Papain-like cysteine proteinases are often found in senescing organs particularly leaves (Lohman et al., 1994; Ueda et al., 2000; Gepstein et al., 2003), flowers (Eason et al., 2002), legume nodules (Kardailsky and Brewin, 1996) as well as in germinating seeds (Ling et al., 2003). Senescence-associated genes (SAGs) are up-regulated during leaf senescence (Lohman et al., 1994; Quirino et al., 1999; Swidzinski et al., 2002; Gepstein et al., 2003; Bhalerao et al., 2003; Lin and Wu, 2004). Of these the SAG12 cysteine proteinase is one of the very few SAGs that are highly senescence-specific (Lohman et al., 1994).

The expression of many photosynthesis genes such as those encoding the chlorophyll a/b binding protein and the subunits of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) decreases during senescence and are hence they are classed as senescence down-regulated genes (Humbeck et al., 1996). Rubisco degradation can occur both inside and outside the chloroplast (Irving and Robinson, 2006). Inside the chloroplast, oxidation of critical cysteine residues on the Rubisco protein modifies the proteolytic susceptibility of these or associated amino acids, causing the protein to adhere to the chloroplast envelope and ‘marking’ the protein for degradation (Garcia-Ferris and Moreno, 1994). Recent evidence suggests that the 26S proteasome is activated by carbonylation and hence this protein degradation pathway is enhanced when the cellular environment becomes even mildly oxidizing (Basset et al., 2002). Vacuolar endopeptidases and globules or vesicles released from the chloroplasts into the cytosol have been implicated in Rubisco catabolism, but key questions have remained regarding the extent to which Rubisco is degraded outside the chloroplast and how Rubisco degradation is controlled (Feller et al., 2007). In the chloroplast Rubisco is protected against degradation by 2-carboxyarabinitol 1-phosphate (CA-I-P) but how this modulates degradation outside the chloroplast is unknown (Khan et al., 1999).

Little information is available on the effects of ectopic phytocystatin expression on plant growth and development (Masoud et al., 1993; Guttiérrez-Campos et al., 2001; Van der Vyver et al., 2003) as most studies have concentrated on effects on insect resistance or protein production (Christou et al., 2006; Rivard et al., 2006). The phenotype resulting from expression of the rice cystatin, OC-1, in transformed tobacco plants has been described previously (Masoud et al., 1993; Guttiérrez-Campos et al., 2001; Van der Vyver et al., 2003). However, increased biomass production resulting from cystatin expression under field conditions is often attributed to enhanced insect resistance rather than to direct effects of the cystatin on endogenous protein turnover in the plant tissues. Transgenic OC-1 expressing tobacco lines (OCE) grow more slowly with an extended vegetative phase compared to the wild type or empty vector controls (Van der Vyver et al., 2003). They are also more resistant to chilling-induced inhibition of photosynthesis (Van der Vyver et al., 2003). The following study was undertaken in order to determine how the constitutive expression of the rice cystatin, OC-1, in the cytosol of tobacco leaves alters leaf protein content and composition and exerts effects on photosynthesis in leaves at different stages of development.

Materials and methods

Plant material and growth conditions

OCE line T4/5 and wild-type control tobacco (Nicotiana tabacum L.) plants were grown in compost in pots in controlled environment chambers (Controlled Environments Ltd., Winnipeg, MB, Canada, R3H 0R9) and rooms under a 15/9 h light/dark regime (with a light intensity of 800–1000 μmol m−2 s−1) and a 26/20 °C day/night temperature cycle (Van der Vyver et al., 2003). The leaf ranking at 14 weeks is denoted from the base to the tip of the stem. Hence, leaf one is the oldest leaf on the stem.

Chilling stress treatments

The attached shoots of 6-week-old OCE and control tobacco plants were chilled in darkness at 5 °C for seven consecutive nights. At the end of each dark period the chilled plants were returned to optimal temperatures for the subsequent light period. Leaf discs were harvested from fully expanded leaves of OC-1 transformed (line T4/5) and wild-type plants at the start of the experiment (day 0) and again following 7 d of growth at 26/20 °C or 26/5 °C. Metabolism was arrested in each leaf disc by freeze-clamping at liquid nitrogen temperatures. At the end of the experiment, leaf discs were also collected from young expanding leaves that developed during the 7 d treatment period. Sampling occurred 4 h after the start of the light period under full illumination.

Gas exchange measurements

CO2 assimilation was measured in fully expanded leaves of OCE and control tobacco plants. CO2 assimilation rates were measured with a portable photosynthesis system (CIRAS-2, PP-systems, Hertz, UK) at a light intensity of 1200 μmol m−2 s−1 and a leaf temperature of 26 °C. Carbon dioxide response curves were generated and used for the calculation of ACE (apparent carboxylation efficiency) and Jmax (maximal rates of photosynthesis at high CO2 concentrations). Leaf discs were collected from fully expanded leaves for the measurement of initial and maximum Rubisco activity according to the radiometric method previously described (Keys and Parry, 1990).

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities

Initial and total Rubisco activities were measured in soluble protein extracts from the leaf samples according to Keys and Parry (1990). Initial activity is defined here as the activity of the enzyme under the growth conditions at the time of sampling. Total activity is defined here as the activity measured following activation of the extracted enzyme with bicarbonate. The total soluble protein content of extracts was determined according to the method of Bradford (1976).

Measurements of Rubisco degradation using in vitro assays

The effect of OC-1 expression on protein degradation in vitro was determined according to the method of Yoshida and Minamikawa (1996). Soluble protein extracts (30 μg) from leaves of either wild type or OC-1 transformed plants were incubated at 37 °C for 0–4 h in the presence or absence of 50 μM E64 (an inhibitor of cysteine proteinases) in 50 mM sodium acetate (pH 5.4) containing 10 mM β-mercaptoethanol. After incubation, samples were immediately loaded onto a native polyacrylamide gel (6%). Proteins were separated and the gel stained according to the method of Rintamäki et al. (1988).

CP activity measurements

CP activity was measured in leaf discs extracted in citrate phosphate buffer (0.1 M, pH 6.5) as previously described (Barrett, 1980).

In-gel protease activity assays

Proteolytic activity was detected in plant extracts after mildly denaturing gelatine-PAGE as previously described (Michaud et al., 1993).

Western blot analysis

Leaf discs were extracted in buffer containing 50 mM TRIS–HCl (pH 7.8), 1 mM EDTA, 3 mM DTT, 6 mM PMSF, and 30 mg insoluble PVPP. Proteins were separated by standard SDS-PAGE procedures. After transfer to nitrocellulose membranes (Hybond C-extra, Amersham Pharmacia Biotech, UK) protein detection was conducted using antibodies directed against Rubisco, Rubisco activase and glutamine synthetase (Foyer et al., 1993).

Electron microscopy and immunogold labelling

Leaf samples were fixed at 4 °C in 3% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2.5 h. The samples were dehydrated with a graded ethanol series and embedded in London Resin White (LR White) acrylic resin. Ultrathin sections (60–70 nm) were made on a Leica EM UC6 Ultramicrotome (Leica Microsystems GMBH, Wetzlar). Ultrathin sections on coated nickel grids were incubated for 30 min in PBS plus 5% (w/v) BSA to block non-specific protein binding on the sections. They were then incubated for 3 h with either anti-RbcL (Rubisco Form I and Form II) antibody raised in rabbit (Agrisera, Vännäs, Sweden) diluted 1:250 for the RbcL antibody and 1:100 for the OC-1 antibody with phosphate buffered saline (PBS) plus 5% (w/v) bovine serum albumin (BSA) or with OC-1 antibody raised in rabbit (Van der Vyver et al., 2003). After washing with PBS plus 1% (w/v) BSA, the sections were incubated for 1.5 h with the secondary antibody goat anti-rabbit IgG gold labelled (10 nm, British BioCell International) diluted 1:50 with PBS plus 1% (w/v) BSA and 1% (w/v) Goat Serum (Sigma). The sections were washed sequentially with PBS (two washes) and distilled water (five washes). Ultrathin sections were then stained with uranyl acetate followed by lead citrate and observed in Philips Tecnai 12 transmission electron microscope.

Two-dimensional (2-D) gel electrophoresis

In the following analysis the proteome of leaf 16 only from C and OCE tobacco plants was investigated. Leaf 16 extracts were compared by 2D electrophoresis according to instructions in the handbook, 2-D electrophoresis: principles and methods (GE Healthcare). Three technical replicates were prepared from each extraction. Proteins were precipitated after grinding leaf material in liquid nitrogen. Ground leaf material (200–250 mg) was incubated over-night at –20 °C in precipitation buffer (1 ml) containing TCA (10%, w/v) and β-mercaptoethanol (0.07% v/v) in acetone (100%, v/v). Precipitated protein was pelleted by centrifuging for 25 min at 4 °C at 20 000 g and washed six times with ice-cold washing buffer containing acetone (90%, v/v) and β-mercaptoethanol (0.07% v/v) in Milli-Q water. Proteins were solubilized in sample buffer (1 ml) containing 8 M urea, 2% (w/v) CHAPS, 61 mM DTT, and 0.5% (v/v) IPG buffer (pH 3–10) (GE Healthcare), by sonication in an ultrasonic water bath for 1 h, with vortexing at 15 min intervals. Samples were then incubated in a heating block for 1.5 h at 30 °C with vortexing at 15 min intervals before overnight incubation at room temperature for optimal protein solubilization. Cell debris was removed by centrifugation for 25 min at 20 000 g. Solubilized proteins were quantified using the Bradford assay and ovalbumin (Sigma) as standard (Ramagli, 1999).

Samples were diluted in sample buffer containing a few grains of bromophenol blue to a concentration of 0.6 μg μl−1. Isoelectric focusing was performed after active rehydration on 150 μg protein using Immobiline DryStrip immobilized pH gradient (IPG) strips (13 cm) (GE Healthcare) and the Ettan IPGphor apparatus (GE Healthcare), with voltage being increased stepwise as follows: 30 V (12 h; for rehydration of strip), 100 V (1 h), 500 V (1 h), 1000 V (1 h), 5000 V (1 h), and 8000 V (19 000 Vh) to obtain a total of 26 000 Vh. IPG strips were then equilibrated for 15 min each in equilibration buffer (6 M urea, 50 mM TRIS–HCl pH 8.8, 30% v/v glycerol, 2% w/v SDS, a few grains of bromophenol blue) containing 65 mM DTT followed by equilibration in equilibration buffer containing iodoacetamide (25 mg ml−1).

Second dimension focusing of proteins was performed by SDS-PAGE on a 1 mm, 12% resolving gel with migration at 25 mA gel−1 for 20 min followed by 30 mA gel−1 for approximately 4 h or until the blue dye front had reached the bottom of the gel. Proteins were fixed in the gel overnight by incubation in fixing solution (50% methanol, v/v, 10% acetic acid, v/v) on a rocking platform at low speed.

After fixing of protein, gels were rinsed three times in Milli-Q water before being stained for 24 h in GelCode Blue (Pierce) on a rocking platform at low speed. Gels were rinsed three times in Milli-Q water before being scanned on a flatbed scanner for image analysis. Images were captured using the ImageMaster Labscan software, and analysed using Phoretix 2D Expression v2005 software.

SELDI-TOF MS and LC-MS/MS

Spots of interest were excised from polyacrylamide gels after 2-D electrophoresis for peptide fingerprint analysis by surface-enhanced laser desorption ionization–time of flight mass spectrometry (SELDI-TOF MS) or serial mass spectrometry (LC-MS/MS). LC-MS/MS was performed on excised spots at the McGill Proteomics Platform (McGill University, Montreal, Quebec) using an ESI-Quad-TOF mass spectrometer. For SELDI-TOF MS the procedure according to Jensen et al. (1999) was followed. Peptide extracts (1–2 μl) from each tryptic digest were spotted onto an H4 ProteinChip array (Ciphergen) and mixed with α-cyano-4-hydroxycinnamic acid (CHCA) [20%; in acetonitrile (5%)/TFA (0.1%)]. Samples were analysed by SELDI-TOF MS in the Ciphergen SELDI-TOF mass spectrometer (GE Healthcare). Spectra were calibrated against CHCA peaks (643.360 D, 1059.5 D, and 1475.48 D). Peptide peaks with a signal-to-noise ratio >5 were identified using the Ciphergen ProteinChip Software v3.2.0, and used to identify proteins with the Mascot search engine (www.matrixscience.com; Perkins et al., 1999). The type of search performed was a peptide mass fingerprint search at the NCBInr database as on 15 June 2007 (Viridiplantae only), with trypsin as enzyme, carbamidomethyl (C) as fixed modification, oxidation (M) as variable modification, using average mass values, a peptide mass tolerance of ±1 Da, and a maximum of one missed cleavage. LC-MS/MS results were obtained from the McGill proteomics portal online (http://portal.proteomics.mcgill.ca/portal). An LC-MS/MS ion search was performed using the Mascot search engine and a database containing all available nucleotide sequences as on 31 January 2007 in order to find protein homologues, with search specifications of trypsin as enzyme, carbamidomethyl (C) as fixed modification, oxidation (M) as variable modification, using monoisotopic mass values, a peptide mass tolerance and fragment mass tolerance of ±0.5 Da, and maximum of one missed cleavage.

Statistical analysis

The data was statistically analysed using parametric tests at a stringency of P < 0.05. The significance of variation in mean values for growth parameters and pigment and protein determinations was determined using a t test. The significance of the data for immunogold labelling measurements was analysed using ANOVA and Tukey HSD tests.

Results

The expression of OC-1 in transgenic tobacco plants decreased plant growth, development rate, and protected photosynthesis from chilling-induced inhibition (Van der Vyver et al., 2003). These effects were previously documented in three independent transgenic lines compared to the wild type and empty vector controls confirming that the slower development, growth, and delayed senescence traits were linked to the expression of the transgene as was the protection of photosynthesis from chilling-induced inhibition (Van der Vyver et al., 2003). Since Van der Vyver et al. (2003) demonstrated unequivocally that the altered traits under investigation are related to the expression of the transgene, the present study focused on the mechanisms by which altered leaf CP activity influences leaf protein composition, photosynthesis, Rubisco protein content and activity, and leaf and plant senescence in one transgenic line (line T4/5) compared to wild-type controls.

Leaf protein composition and turnover

To determine whether leaf protein composition was modified in the OCE plants, leaf proteins were extracted from the youngest mature leaves (number 16) of 8-week-old control and OCE plants and separated using 2-D gel electrophoresis (Fig. 1). Leaf proteins were extracted and precipitated by standard proteomic procedures, in which the Rubisco large subunit (LSU) has only limited solubility (Ramagli, 1999). Since Rubisco generally accounts for 30–60% of total soluble proteins in the leaves of C3 species, it is important to use this selective procedure to limit the amount of the Rubisco LSU on the gels, so that other proteins of lower abundance are not obscured.

Fig. 1.

The effect of inhibition of CP activity leaf protein on tobacco and composition. Proteins were extracted from leaf 16 of control and OCE plants at 8 weeks and were separated on bi-dimensional gels. Proteins with major differences in abundance are indicated (1–51) in upper panels. The position of the proteins with the greatest differences: two Rubisco activase forms (NTRA and NTRA2), histone 4 (H4) and putative pepyidylprolyl isomerise (PPI) are indicated in the lower panels.

Fig. 1.

The effect of inhibition of CP activity leaf protein on tobacco and composition. Proteins were extracted from leaf 16 of control and OCE plants at 8 weeks and were separated on bi-dimensional gels. Proteins with major differences in abundance are indicated (1–51) in upper panels. The position of the proteins with the greatest differences: two Rubisco activase forms (NTRA and NTRA2), histone 4 (H4) and putative pepyidylprolyl isomerise (PPI) are indicated in the lower panels.

The Phoretix 2-D gel analysis software identified 765 protein spots in the extracts from control leaves and 860 protein spots in extracts from OCE leaves. Key parameters (spot volume, pI, and MW) were calculated for all spots. Fifty-one spots were chosen for more intensive characterization based on visible differences in spot volume. Of the 51 spots, 13 were not statistically different in volume between C and OCE plants, seven spots had significantly greater volume in C plants, 26 spots had significantly greater volume in OCE plants, two spots were below the level of detection in OCE plants, and three spots were only detected in OCE plants (see Supplementary Table 1 at JXB online). Two spots showing a difference in volume (Fig. 1, upper panels, spots 4 and 5) were identified using SELDI-TOF MS. These proteins were highly homologous to Rubisco activase 2 (accession number Q40565) (spot 4) and Rubisco activase (accession number 1909374A) (spot 5) (Table 1). Spot 4 also showed significant homology to Rubisco activase (accession number number 1909374A) and Rubisco activase 1 (accession number Q40460), while spot 5 showed significant homology to Rubisco activase 1 (accession number Q40460) and Rubisco activase 2 (accession number Q40565). The normalized volumes for spots 4 and 5 in OCE extracts were, respectively, 2.42 and 2.99 times greater than those found in C extracts. In OCE protein extracts, spot 4 had a larger volume than spot 5 (1.3 times).

Table 1.

Identification of protein spots showing different abundance in control and OCE lines after 2D electrophoresis

Spot Identification method Accession Protein name Score e-value Queries matched Peptide sequence (MS/MS) 
NTRA2 (4) SELDI-TOF MS Q40565 Rubisco activase 2 (RA 2) 98 6.80E-05  
  1909374A Rubisco activase 75 1.30E-02  
  Q40460 Rubisco activase 1 (RA 1) 71 3.30E-02  
NTRA (5) SELDI-TOF MS 1909374A Rubisco activase 117 7.70E-07 15  
  Q40460 Rubisco activase 1 (RA 1) 108 6.10E-06 15  
  Q40565 Rubisco activase 2 (RA 2) 94 1.40E-04 13  
H4 (23) LC-MS/MS P08436 Histone H4 195  ISGLIYEETR 
       DNIQGITKPAIR 
       TVTAMDVVYALK 
  HSWT93 histone H2A.3 48  AGLQFPVGR 
  AAF07182 H2A protein 48  AGIQFPVGR 
PPI (24) LC-MS/MS NP_001054392 Os05g0103200 357  16 TFKDENFK 
       DFMIQGGDFDK 
       VYFDISIGNPVGK 
       HVVFGQVIEGMDIVK 
       DFMIQGGDFDKGNGTGGK 
  CAC05440 peptidylprolyl 105  TFKDENFK 
   isomerase-like protein     
Spot Identification method Accession Protein name Score e-value Queries matched Peptide sequence (MS/MS) 
NTRA2 (4) SELDI-TOF MS Q40565 Rubisco activase 2 (RA 2) 98 6.80E-05  
  1909374A Rubisco activase 75 1.30E-02  
  Q40460 Rubisco activase 1 (RA 1) 71 3.30E-02  
NTRA (5) SELDI-TOF MS 1909374A Rubisco activase 117 7.70E-07 15  
  Q40460 Rubisco activase 1 (RA 1) 108 6.10E-06 15  
  Q40565 Rubisco activase 2 (RA 2) 94 1.40E-04 13  
H4 (23) LC-MS/MS P08436 Histone H4 195  ISGLIYEETR 
       DNIQGITKPAIR 
       TVTAMDVVYALK 
  HSWT93 histone H2A.3 48  AGLQFPVGR 
  AAF07182 H2A protein 48  AGIQFPVGR 
PPI (24) LC-MS/MS NP_001054392 Os05g0103200 357  16 TFKDENFK 
       DFMIQGGDFDK 
       VYFDISIGNPVGK 
       HVVFGQVIEGMDIVK 
       DFMIQGGDFDKGNGTGGK 
  CAC05440 peptidylprolyl 105  TFKDENFK 
   isomerase-like protein     

Peptide fingerprint analysis (NTRA2 and NTRA) and/or ion analysis (H4 and PPI) using the Mascot search engine was used to establish protein identities.

To characterize the Rubisco activase isoforms present in these studies further, alignments were performed with three GenBank tobacco Rubisco activase sequences and two Arabidopsis Rubisco activase isoforms (Table 1; see Supplementary Fig. 1 at JXB online). While the highest scores for spots 4 and 5 were Rubisco activase 2 and Rubisco activase from tobacco, the comparison with Arabidopsis revealed the absence of the C-terminal amino acids characteristic of the long isoform of this gene in Arabidopsis. Instead of the final 36 C-terminal amino acids present in the long Arabidopsis isoform, the Arabidopsis short isoform has only eight amino acids (TEEKEPSK: Werneke et al., 1989), a difference that is considered to result from alternative splicing. The Arabidopsis large isoform has a MW of 46 kDa while the small isoform is approximately 43 kDa. Spot 4 has the highest homology to NTRA2 (RA2; see Supplementary Fig. 1 at JXB online) which lacks the C-terminal amino acids FAS. Spot 5 had the highest homology to another identified tobacco Rubisco activase (1909374A, RuAct; see Supplementary Fig. 1 at JXB online). This form lacks the first 59 amino acids in the N-terminus and also contains three additional amino acids at the C-terminal (FAS) when compared to RA2. Spot 5 also shows high homology with NTRA1 (RA1; see Supplementary Fig. 1 at JXB online) which has the full-length N-terminal sequence but also has the extra amino acids at the C-terminal.

Spot number 23 on Fig. 1 upper panels has very low abundance in the OCE proteome (see Supplementary Table 1 at JXB online) and was identified by LC-MS/MS analysis to be highly homologous to volvox histone H4 (P08436), histone H2A.3 from wheat (HSWT93), and rice H2A protein (AAF07182) (Table 1). Spot number 24 in the OCE proteome, which is below detection in C extracts, was identified by LC-MS/MS and was significantly homologous to rice Os05g0103200 (NP_001054392), which is described as a chloroplast precursor (EC 5.2.1.8) of peptidyl-prolyl cis-trans isomerase TLP20. This protein contains a cyclophilin ABH-like region. Spot 24 is also significantly homologous to an Arabidopsis peptidylprolyl isomerase-like protein (CAC05440), which also has a strong similarity to the chloroplast stromal cyclophilin, ROC4.

The relative abundance of the Rubisco LSU and Rubisco activase proteins was determined in leaves at different positions on the stem of 14-week-old plants (Fig. 2) using western blot analysis. In the control plants, the amount of Rubisco LSU protein was highest in the mature source leaves and least abundant in the youngest (18) and oldest (1) leaves (Fig. 2). However, the relative abundance of the Rubisco LSU protein was much higher in the leaves of the OCE plants at all ranks on the stem, even in the oldest leaves (Fig. 2). Inhibition of leaf CP activity resulted in a development-dependent difference in the Rubisco activase protein bands (Fig. 2). Two bands of Rubisco activase protein were observed on western blots using specific antibodies in all but the oldest senescent leaves of the control plants where only the lower band was detected (Fig. 2). In marked contrast, only the higher molecular weight band of Rubisco activase protein was detected in the young leaves of OCE plants, with two bands becoming evident only in the oldest leaves from leaf rank 10 and below (Fig. 2). It is important to note that we cannot directly relate the two bands of Rubisco activase protein observed after SDS-PAGE in Fig. 2 to those observed on 2-D gels in Fig. 1. For example, the spots characterized after 2-D gel electrophoresis in Fig. 1 are of similar molecular weights. A more exhaustive analysis of the different Rubisco activase proteins separated by 2-D gel electrophoresis is required as other Rubisco activase proteins are probably present.

Fig. 2.

Western blot analysis of the abundance of the Rubisco large subunit, and Rubisco activase in leaves at different positions on the stem of 14-week-old plants. Soluble proteins were extracted from leaves at the positions on the stems as indicted, with leaf 1 being at the bottom of each plant and leaf 18 or 28 being the youngest mature leaf on the control and OCE plants, respectively. 10 μg and 30 μg aliquots of leaf protein were loaded per well for the detection of Rubisco and Rubisco activase proteins, respectively.

Fig. 2.

Western blot analysis of the abundance of the Rubisco large subunit, and Rubisco activase in leaves at different positions on the stem of 14-week-old plants. Soluble proteins were extracted from leaves at the positions on the stems as indicted, with leaf 1 being at the bottom of each plant and leaf 18 or 28 being the youngest mature leaf on the control and OCE plants, respectively. 10 μg and 30 μg aliquots of leaf protein were loaded per well for the detection of Rubisco and Rubisco activase proteins, respectively.

Rubisco degradation and leaf CP activity

To determine whether tobacco Rubisco is susceptible to degradation by endogenous tobacco CPs in vitro assays were conducted comparing Rubisco degradation in OCE extracts with that in C extracts in the absence or presence of the CP inhibitor, E64 (Fig. 3A). Rubisco was protected from degradation by endogenous CPs in OCE extracts compared to control extracts; an effect that could be mimicked by inclusion of the CP inhibitor, E64, in the assays of the control extracts (Fig. 3A). The CP activities of leaf extracts, scrutinized by activity staining after SDS-PAGE, revealed that OCE leaves had much higher CP activities than controls (Fig. 3B), indicating the presence of feedback modulation of CP expression that enhances CP production when activity is impaired by constitutive cystatin expression.

Fig. 3.

Protection of Rubisco from degradation by OC-1 in OCE plants and by E64 in control (C) plants in vitro assays. (A) The abundance of the Rubisco holoenzyme protein was detected in soluble protein extracts from 4-week-old control (C) and OCE plants on non-denaturing PAGE gels stained with Coomassie Brilliant Blue. (B) In-gel activity assay showing degradation of the gelatine substrate by endogenous proteinases from extracts of leaves of 14-week-old OCE and control (C) plants. Extracts were prepared from leaves at the bottom (3), middle (8, 14), and top (18, 27) leaf ranks. Equal amounts of soluble protein (40 μg per well) extracted from C and OCE plants were compared in all instances.

Fig. 3.

Protection of Rubisco from degradation by OC-1 in OCE plants and by E64 in control (C) plants in vitro assays. (A) The abundance of the Rubisco holoenzyme protein was detected in soluble protein extracts from 4-week-old control (C) and OCE plants on non-denaturing PAGE gels stained with Coomassie Brilliant Blue. (B) In-gel activity assay showing degradation of the gelatine substrate by endogenous proteinases from extracts of leaves of 14-week-old OCE and control (C) plants. Extracts were prepared from leaves at the bottom (3), middle (8, 14), and top (18, 27) leaf ranks. Equal amounts of soluble protein (40 μg per well) extracted from C and OCE plants were compared in all instances.

Natural senescence and chilling-dependent inhibition of photosynthesis, decreased Rubisco content and activity

Dark chilling inhibited photosynthesis in a comparable manner in three independent transgenic lines relative to the wild type and empty vector controls and that effects of constitutive OC-1 expression on parameters such as the CO2 saturated rates of photosynthesis (Jmax) and apparent carboxylation efficiency (ACE) were linked to expression of the transgene (Van der Vyver et al., 2003). Leaf ACE and Jmax values attained maximal values 2 weeks after emergence in both OCE and control plants (Table 2). To investigate the effect of decreased CP activity on leaf senescence as determined by the age-dependent decrease in photosynthesis, leaf ACE and Jmax values were measured on the same leaves from 2–6 weeks (Table 2). The OCE leaves had greater ACE and Jmax values than controls at equivalent stages of development. Moreover, the senescence-related decline in photosynthesis was delayed in the OCE leaves (Table 2).

Table 2.

Senescence-related decreases in apparent carboxylation efficiency (ACE) and CO2 saturated rates of photosynthesis (Jmax) in wild-type controls (C) and OCE tobacco leaves

Time after leaf emergence C plants OCE plants C plants OCE plants 
(weeks) ACE ACE Jmax Jmax 
 (mol m−2 s−1(mol m−2 s−1(μmol m−2 s−1(μmol m−2 s−1
0.078±0.004 0.107±0.007 20.3±1.0 23.2±0.8 
0.069±0.004 0.110±0.013 15.1±1.0 21.7±1.1 
0.040±0.004 0.064±0.002 13.6±1.3 18.5±1.7 
0.034±0.004 0.063±0.005 9.6±0.6 16.6±0.6 
0.014±0.001 0.045±0.003 3.1±0.6 11.19±0.1 
Time after leaf emergence C plants OCE plants C plants OCE plants 
(weeks) ACE ACE Jmax Jmax 
 (mol m−2 s−1(mol m−2 s−1(μmol m−2 s−1(μmol m−2 s−1
0.078±0.004 0.107±0.007 20.3±1.0 23.2±0.8 
0.069±0.004 0.110±0.013 15.1±1.0 21.7±1.1 
0.040±0.004 0.064±0.002 13.6±1.3 18.5±1.7 
0.034±0.004 0.063±0.005 9.6±0.6 16.6±0.6 
0.014±0.001 0.045±0.003 3.1±0.6 11.19±0.1 

Measured ACE and Jmax values, which were highest in both lines 2 weeks after leaf emergence, were measured in the same leaves for up to 6 weeks. The values represent the means ±SE of four replicates per experiment.

Photosynthesis (Fig. 4A), extractable Rubisco activities, and activation states (Fig. 4B) were compared in the leaves of 6-week-old plants maintained at either optimal growth temperatures or exposed to seven consecutive nights of chilling. At the beginning of the experiment (day 0), fully expanded leaves of control (Fig. 4A, upper panel) and OCE plants (Fig. 4A, lower panel) grown at optimal temperatures had very similar rates of photosynthesis. At this stage the OCE plants had higher total Rubisco activities but lower activation states than controls (Fig. 4B). Seven days later, the control leaves maintained at optimal temperatures had about 20% lower photosynthetic rates than the OCE plants (Fig. 4A). Over the same period, total Rubisco activity in the control leaves maintained at optimal temperatures had also decreased (Fig. 4B) with significant effect on Rubisco activation state (Fig. 4B). While Rubisco activities were similar in OCE plants at both time points the Rubisco activation state was slightly increased at day 7 (Fig. 4B). The chilling-dependent increase in Rubisco activation state in the OCE lines is surprising given that the overall rate of photosynthesis declined and that the initial slope of the photosynthesis: intercellular CO2 response is also slightly decreased. However, these effects are very small compared to the large effect of chilling on photosynthesis rates in the control line, where Rubisco activation state was unchanged.

Fig. 4.

Effects of dark chilling on photosynthesis, Rubisco activity and activation state and relative abundance of Rubisco, Rubisco activase, and glutamine synthetase in the leaves of 6-week-old OCE and control tobacco plants. CO2 response curves for photosynthesis (A) in control and OCE leaves were measured at day 1 (closed circle), after 7 d of growth under optimal conditions (Opt: closed square) and after 7 nights of dark chilling (Chilled; open square). Initial and total Rubisco activities and the Rubisco activation state in control and OCE leaves measured at day 1, after 7 d of growth under optimal conditions and after 7 nights of dark chilling (B). Immunodetection of Rubisco, Rubisco activase, and glutamine synthetase (C) in soluble protein extracts from control and OCE leaves at the beginning of the experiment (lanes 1 and 3) and after 7 nights of dark chilling (lanes 2 and 4).

Fig. 4.

Effects of dark chilling on photosynthesis, Rubisco activity and activation state and relative abundance of Rubisco, Rubisco activase, and glutamine synthetase in the leaves of 6-week-old OCE and control tobacco plants. CO2 response curves for photosynthesis (A) in control and OCE leaves were measured at day 1 (closed circle), after 7 d of growth under optimal conditions (Opt: closed square) and after 7 nights of dark chilling (Chilled; open square). Initial and total Rubisco activities and the Rubisco activation state in control and OCE leaves measured at day 1, after 7 d of growth under optimal conditions and after 7 nights of dark chilling (B). Immunodetection of Rubisco, Rubisco activase, and glutamine synthetase (C) in soluble protein extracts from control and OCE leaves at the beginning of the experiment (lanes 1 and 3) and after 7 nights of dark chilling (lanes 2 and 4).

The leaves of different independent OCE lines contain about 20% more total soluble protein than those of controls at 6 weeks old (Van der Vyver et al., 2003). Consistent with this observation, the amounts of Rubisco LSU and SSU proteins (Fig. 4C) were similar in the leaves of 6-week-old control and OCE plants at this stage. However, dark-chilling stress led to a pronounced decrease in the abundance of Rubisco LSU and SSU proteins (Fig. 4C) in the leaves of control plants. In contrast, dark-chilling had no effect on the amount of detectable Rubisco LSU and SSU proteins in the OCE plants. Similar trends were observed in the contents of Rubisco activase protein, but not in the glutamine synthetase protein (Fig. 4C).

Intracellular localization of Rubisco protein in chloroplasts and vesicular bodies in the palisade cells of young leaves

Electron microscopy and immunogold labelling with specific polyclonal antibodies to the Rubisco LSU were used to determine the intracellular distribution of the Rubisco protein in the youngest mature leaves of control and OCE tobacco at 6 weeks old (Fig. 5). Label was detected in the chloroplasts of the palisade cells of control (Fig. 5B) and OCE leaves (Fig. 5C). In addition, Rubisco protein was also observed in vesicular bodies outside the chloroplast (Fig. 5B, C). The relative amounts of label were quantified in the chloroplasts and in the Rubisco vesicular bodies (RVB) of both control and OCE leave (Table 3). No differences were observed in the relative localization of Rubisco protein in the chloroplasts relative to the RVBs of both control and OCE leaves (Table 3).

Table 3.

Quantitation of gold particles (GP) after immunogold labelling of Rubisco large subunit in ultrathin leaf sections of C and OCE tobacco

 GP in chloroplast (μm−2GP in RVB (μm−2GP in cytosol (μm−2
Control 0.4±0.2 0.2±0.1 0.4±0.2 
OCE 27.9±3.3* 31.4±6.5* 1.4±0.8 
26.7±2.8* 29.1±5.3* 1.4±0.7 
 GP in chloroplast (μm−2GP in RVB (μm−2GP in cytosol (μm−2
Control 0.4±0.2 0.2±0.1 0.4±0.2 
OCE 27.9±3.3* 31.4±6.5* 1.4±0.8 
26.7±2.8* 29.1±5.3* 1.4±0.7 

Gold particles were counted in chloroplasts, Rubisco vesicular bodies (RVB) and cytosol. Values obtained were compared to samples (Control) of both lines in the absence of antibodies. Mean values ±SE (n=30). The means were compared by analysis of variance and by using the Tukey multiple range test at P <0.05. Significant differences between treatments are indicated by an asterisk.

Fig. 5.

Immunogold labelling detection of Rubisco protein in palisade cells of 6-week-old control and OCE tobacco leaves. High magnification of a cross-section of a control leaf showing the structure of the palisade cells with immunogold labelling (A), and higher magnification images of the intracellular structure showing the compartmention of the label in wild-type and OCE leaves (B, C). The presence of Rubisco protein in chloroplasts and vesicular bodies outside the chloroplast is indicated in (B) and (C). Areas of immunogold are indicated by black arrows. CY, cytosol; IS, intercellular space; P, plastoglobulus; RVB, Rubisco vesicular body; S, stroma; SG, starch grain; T, thylakoid membranes; V, vacuole.

Fig. 5.

Immunogold labelling detection of Rubisco protein in palisade cells of 6-week-old control and OCE tobacco leaves. High magnification of a cross-section of a control leaf showing the structure of the palisade cells with immunogold labelling (A), and higher magnification images of the intracellular structure showing the compartmention of the label in wild-type and OCE leaves (B, C). The presence of Rubisco protein in chloroplasts and vesicular bodies outside the chloroplast is indicated in (B) and (C). Areas of immunogold are indicated by black arrows. CY, cytosol; IS, intercellular space; P, plastoglobulus; RVB, Rubisco vesicular body; S, stroma; SG, starch grain; T, thylakoid membranes; V, vacuole.

Intracellular localization of OC-1 protein in the cytosol, chloroplasts, and vacuoles in the palisade cells of young leaves

Electron microscopy and immunogold labelling with specific polyclonal antibodies to the OC-1 protein were used to determine the intracellular distribution of the OC-1 protein in the youngest mature leaves of control and OCE tobacco at 6 weeks old (Fig. 6). The OC-1 protein was mainly located in the cytosol which had the highest relative gold particle concentrations (71±8 μm2; n=9). However, label was also detected in the vacuole at a gold particle concentration of 5.5±2 μm2 (n=9) and in the chloroplasts which had a gold particle concentration of 20.6±4.2 μm2 (n=9). Interestingly, the chloroplasts that showed imunogold labelling for the presence of the OC-1 protein also had an alteration to the structure at the periphery of the chloroplast either beneath or adjacent to the chloroplast envelope (Fig. 6A, C). A higher magnification of the chloroplast periphery shows that this structure possibly has a fibrillar or membranous nature (Fig. 6B, D). These samples had not been fixed with osmium and therefore lipids were not stained in these images. This structure is highly stained, suggesting perhaps that it might have a low lipid content. The chloroplasts containing these structures clearly show label (Fig. 6B, D, inserts), but further studies are required to explore the nature of this new structure and how it is formed by inhibition of CP activity.

Fig. 6.

Transmission electron micrographs of immunogold labelling detection of OCI protein in the palisade cells of 6-week-old control and OCE tobacco leaves. Chloroplasts in OCE leaves with an unusual fibril structure below the chloroplast envelope (A, C); enhanced images of the unusual structure below the chloroplast envelope (B, D). In both cases the chloroplasts (A, C) show labelling for OC-1 protein (B, D). A section of the OCE leaf shows the presence of a cystatin inclusion body (E) together with a higher magnification image showing that the cystatin inclusion body is present in the cytosol. (F) and has a crystalline structure with positive labelling for the OC-1 protein (G). The presence of immunogold label in the cytosol of the OCE leaves (H, I). C, cystatin inclusion body; Chl, chloroplast; Cyt, cytosol; L, unusual chloroplast structure; S, starch; T, thylakoid; V, vacuole.

Fig. 6.

Transmission electron micrographs of immunogold labelling detection of OCI protein in the palisade cells of 6-week-old control and OCE tobacco leaves. Chloroplasts in OCE leaves with an unusual fibril structure below the chloroplast envelope (A, C); enhanced images of the unusual structure below the chloroplast envelope (B, D). In both cases the chloroplasts (A, C) show labelling for OC-1 protein (B, D). A section of the OCE leaf shows the presence of a cystatin inclusion body (E) together with a higher magnification image showing that the cystatin inclusion body is present in the cytosol. (F) and has a crystalline structure with positive labelling for the OC-1 protein (G). The presence of immunogold label in the cytosol of the OCE leaves (H, I). C, cystatin inclusion body; Chl, chloroplast; Cyt, cytosol; L, unusual chloroplast structure; S, starch; T, thylakoid; V, vacuole.

Some of the OCE cells also show the presence of cytosolic inclusion bodies (Fig. 6E, F), which has a crystalline structure (Fig. 6G). This structure contains label (Fig. 6G, insert). It is tempting to suggest that it is formed by the strong interaction of the OC-1 protein with the endogenous cytosolic CPs, because similar inclusion bodies in the cytosol have been observed previously in transgenic plants expressing a wound- and methyl jasmonate-inducible 87 kDa tomato cystatin (Madureira et al., 2006). The high level of label in the cytosol (Fig. 6H, I, inserts) is consistent with the mode of expression of the OC-1 protein in these studies, where the protein lacked sequences for specific organellar targeting.

Inhibition of CP activity effects on lifespan and leaf protein and chlorophyll contents after flowering

The OCE plants have a slow growth phenotype compared to wild type or empty vector controls (Van der Vyver et al., 2003). In the present experiments, the control plants flowered at 58.33±1.20 d, at which point vegetative growth ceased. The OCE plants also sustained vegetative growth until flowering, but in this case, vegetative development ceased at 80.67±1.45 d (Fig. 7). Hence, at the point where the OCE lines reached sexual maturity (14 weeks in the OCE lines) the OCE lines were much taller (Fig. 7A), with greater numbers of larger and heavier leaves than the controls (Fig. 7B–H). The effects of inhibition of CP activity on leaf protein accumulation were much more pronounced in 14-week-old (Fig. 8A) than they were in 6–8-week-old tobacco plants (Van der Vyver et al., 2003). The increase in leaf protein in OCE plants that had flowered depended on the position on the stem (Fig. 8A). Chlorophyll was also increased but only in the youngest tobacco leaves (Fig. 8B). Maximal extractable leaf CP activities were greatly decreased in OCE leaves compared to controls at all positions on the stem (Fig. 8C), suggesting that the OC-1 remains bound to the CP during the extraction and spectrophotometric assay procedures, whereas it is removed by the in-gel assay methods used in Fig. 3B.

Fig. 7.

Comparisons of growth and development in OCE and control plants. Phenotypes of control and OCE plants are shown at (A) 8 weeks and (B) 14 weeks. The white vertical bar indicates a height of 0.5 m. After both genotypes had flowered at 14 weeks the following parameters were measured: plant height (C); leaf number (D); total leaf area (E); leaf weight (F); average area per leaf (G); average leaf weight per leaf area (H). Mean values ±SE (n=3). Significant differences between control and OCE plants are indicated by asterisk (P <0.05); ns, not significant.

Fig. 7.

Comparisons of growth and development in OCE and control plants. Phenotypes of control and OCE plants are shown at (A) 8 weeks and (B) 14 weeks. The white vertical bar indicates a height of 0.5 m. After both genotypes had flowered at 14 weeks the following parameters were measured: plant height (C); leaf number (D); total leaf area (E); leaf weight (F); average area per leaf (G); average leaf weight per leaf area (H). Mean values ±SE (n=3). Significant differences between control and OCE plants are indicated by asterisk (P <0.05); ns, not significant.

Fig. 8.

Comparisons of leaf soluble protein and chlorophyll contents and cysteine proteinase activities in 14-week-old OCE and control tobacco plants. Soluble protein content (A); chlorophyll content (B); cysteine proteinase (CP) activity (C). Open and closed symbols represent control and OCE plants, respectively. Mean values ±SE (n=3 for A and B; n=1 for C).

Fig. 8.

Comparisons of leaf soluble protein and chlorophyll contents and cysteine proteinase activities in 14-week-old OCE and control tobacco plants. Soluble protein content (A); chlorophyll content (B); cysteine proteinase (CP) activity (C). Open and closed symbols represent control and OCE plants, respectively. Mean values ±SE (n=3 for A and B; n=1 for C).

Discussion

The results presented here demonstrate that chloroplast proteins, particularly Rubisco and Rubisco activase, are major targets of leaf CPs. Moreover, the degradation of Rubisco involves interactions with the cytosol, as it can be blocked by endogenous cytosolic CP inhibitors such as OC-1. The demonstration of Rubisco-containing vesicles outside the chloroplast suggests a link between the protein turnover machinery in the chloroplasts, cytosol, and vacuoles. A detailed discussion of the results and logic corroborating the above conclusions is provided below.

While there are relatively few reports on tobacco leaf proteomics in the literature (Cooper et al., 2003; Franceschetti et al., 2004; Giri et al., 2006), the technique has been used successfully to study the proteome of leaf plasma membranes (Rouquié et al., 1997), trichomes (Amme et al., 2005), and apoplast (Dani et al., 2005). Proteome information is available for tobacco BY2 cell suspension cultures (Laukens et al., 2004) and for plastids isolated from these cultures (Baginsky et al., 2004). Rubisco activase sequences (Q40565, CAA78703, or 1909374A) have been identified previously in Nicotiana attenuata leaves (Giri et al., 2006). In this study concerning proteins elicited by oral secretions from Manduca sexta, seven spots with homology to Rubisco activase were identified (Giri et al., 2006). Of these, four spots had kDa/pI values similar to reported regulators of complementary activation proteins, with comparable molecular weights but different pI values. In the present study, spots 4 and 5 in Fig. 1 occupy similar positions to the Rubisco activase spots reported by Giri et al. (2006). Differences in the positions of the Rubisco activase spots on the gels might arise from proteolytic cleavage (Giri et al., 2006). The data presented in Figs 1–3 not only implicate CPs in Rubisco activase degradation but also in the relative abundance of different forms of the protein present in leaves. The relative abundance of the two Rubisco activase protein isoforms detected on western blots was changed by inhibition of CP activity, with greater abundance of the higher molecular weight form. Rubisco activase is a crucial regulator of Rubisco activation state (Zhang et al., 2002). This nuclear-encoded chloroplast protein consists of two isoforms in Arabidopsis, produced by alternative splicing (Zhang et al., 2002). In Arabidopsis, the longer isoform houses critical cysteine residues that are modulated by redox changes in the stroma, particularly under limiting light intensities (Zhang et al., 2002). However, the tobacco Rubisco activase isoform sequences examined to date in the present study lack the critical cysteine residues that characterize the longer isoform sequence in Arabidopsis.

While the mechanisms of turnover and degradation of Rubisco activase remain poorly characterized, there have been a large number of studies on the turnover of Rubisco and glutamine synthetase. Oxidation is known to enhance cleavage of these proteins (Garcia-Ferris and Moreno, 1994). For example, chloroplastic glutamine synthetase has been shown to be rapidly degraded under conditions that cause photo-oxidative stress in leaves and this has been linked to oxidative carbonylation of histidine residues on the glutamine synthetase protein (Palatnik et al., 1999; Ishida et al., 2002). Oxidatively-modified proteins are generally not repaired and are removed by proteolytic degradation. Within the chloroplast, oxidation of critical cysteine residues enhances the binding of the Rubisco protein to the chloroplast envelope membranes, marking the protein for degradation (Marín-Navarro and Moreno, 2006). While chaperone-mediated autophagy pathways for oxidatively-modified proteins remain to be demonstrated in plants, stress-induced non-specific autophagasitic pathways of protein degradation have been described (Xiong et al., 2007). Once outside the chloroplast, Rubisco occurs in the vesicular transport system and involves cytoplasmic vacuole-type compartments (Chiba et al., 2003). Thereafter, Rubisco degradation products appear in the vacuoles (Huffaker, 1990). While this process has only previously been considered to be important in senescing leaves where chloroplast lysis occurs (Mae et al., 1984; Ono et al., 1995; Hörtensteiner and Feller, 2002), the data presented here in Table 3 and Fig. 5 show that they are present even in young leaves. These results demonstrate the existence of cytoplasmic vacuole-type compartments that we have called Rubisco vesicular bodies (RVBs) even in young leaves. While the properties of the RVBs and their interactions with other compartments are not yet well known, circumstantial evidence as outlined below suggests their involvement in Rubisco protein turnover.

The data presented here allow the formulation of a new model for Rubisco degradation of the type illustrated in Fig. 9, where vesicles containing CPs continuously interact with those from functional chloroplasts to remove proteins marked for degradation. Rubisco protein is present in vesicles as well as in the chloroplasts in both young wild-type and OCE leaves (Fig. 5; Table 3). Interestingly, RVBs have recently been shown to arise from the stromules that are continuously produced by chloroplasts even in young leaves (Ishida et al., 2007). While the function of stromules remains a matter of debate (Foyer and Noctor, 2007), available evidence suggests that stromules forge associations between chloroplasts and other organelles including the vacuole. The similar pattern of Rubisco labelling in the RVBs and chloroplasts observed in the present study is entirely consistent with the chloroplast–stromule origin of RVBs. The tobacco chloroplasts observed in the present study show protrusions that are like stromules (data not shown). The demonstration of chloroplast–stromule–RVB associations together with the observations of OC-1 protein in the chloroplasts and vacuoles provide new insights into how chloroplast Rubisco becomes available for degradation by CPs that are absent from the chloroplast. The binding of oxidized Rubisco protein to the chloroplast envelope (Desimone et al., 1996) might facilitate the accumulation of the protein at the sites where stromules and hence vesicles form. The maximal extractable CP activity of leaves is greatly decreased by the presence of OC-1 (Fig. 8C) unless the cystatin is removed by procedures such as gel electrophoresis. These observations are consistent with a role for extra-chloroplastic CPs in Rubisco protein turnover. While, the major targets for leaf CPs are considered to be vacuolar or cytosolic storage proteins, plant cells contain several different types of vacuole-like compartments with distinct functions (Chrispeels and Herman, 2000). Vesicle trafficking has been traditionally viewed as a housekeeping process, but recent findings in plant, yeast and animal cells show that it can also play an important role in stress responses (Chrispeels and Herman, 2000; Herman and Schmidt, 2004). The physiological and biochemical identity of vacuoles is largely determined by correct targeting of vesicles and their cargo (Vitale and Pedrazzini, 2005). Since the data presented here indicate that the vesicles operate in the routine turnover of chloroplast proteins such as Rubisco, the name RVB seems appropriate. RVBs might be formed as part of the autophagasitic process and engulf cytosolic as well as chloroplastic proteins, explaining how cystatins in the cytosol can effectively inhibit vacuole CPs. The OC-1 protein is found largely in the cytosol, but also in the vacuole and in the chloroplasts, consistent with this view.

Fig. 9.

A hypothetical model for Rubisco degradation via autophagy and the plant vesicle trafficking system. AA, amino acids; CP, cysteine proteinase; ER, endoplasmic reticulum; OC-1, oryzacystatin-1; RVB, Rubisco vesicular body.

Fig. 9.

A hypothetical model for Rubisco degradation via autophagy and the plant vesicle trafficking system. AA, amino acids; CP, cysteine proteinase; ER, endoplasmic reticulum; OC-1, oryzacystatin-1; RVB, Rubisco vesicular body.

While the data presented here concerning RVBs are limited to Rubisco, it may be that other chloroplast proteins such as Rubisco activase are also present in the RVBs. This aspect is currently under investigation, together with the role of CPs and cystatins in the formation of peripheral structure adjacent to the chloroplast envelope that has been observed when CP activity is inhibited. To date, only the number of RVBs have been counted in the leaves of young plants prior to flowering, and values appear to be similar regardless of leaf CP activity. However, the number may increase as the leaf develops and senesces. Despite the absence of this information at present, our data suggest that the Rubisco turnover cycle involving RVBs must have a feedback interaction with the chloroplast that requires CP activity, as inhibition of CPs allows the chloroplast to maintain a high level of Rubisco activity and photosynthesis upon exposure to stress. These interactions may be controlled by redox regulation. In the control plants CP activity is regulated by cellular reductants such as glutathione. The much decreased CP activity in the OCE lines will allow time for the re-reduction of reversibly oxidized amino acids on the Rubisco protein such as cysteine and methionine residues by thiol-disulphide exchange modulators, liberating them from the chloroplast envelope and allowing return of the repaired protein to the stroma for photosynthetic purposes. While many gaps remain between the hard facts and the hypothetical model described in Fig. 9, this scheme provides a simple dynamic mechanism for the turnover of chloroplast proteins outside the plastid that can be tested readily with appropriate molecular and cell biology tools.

The processes that regulate plant development and senescence have been widely studied, the concept of regulation of plant lifespan is very poorly developed in comparison to animals, where a reduction in nutrient intake or dietary restriction extends lifespan in species as diverse as worm (C. elegans), fly (D. melanogaster), yeast (S. cerevisiae), and mouse (Mus musculus). In these organisms, an alteration of nutrient sensing via modulation by insulin-signalling genes has been shown to extend lifespan. Evolutionary theory suggests that nutrient sensing regulates key genetic pathways that shift the metabolic investment that any organism makes from reproduction and growth toward somatic maintenance, allowing the organism to survive under harsh environments until there are suitable reproductive conditions. While these theories remain to be tested in plants, the data presented here show that the lifespan of tobacco plants can be extended by inhibition of CP activity and that this manipulation also enables the plants to increase biomass production prior to sexual maturity and to avoid stress-induced premature senescence.

Supplementary data

Supplementary data can be found at JXB online.

Supplementary Table 1. 2-D PAGE analysis of the leaf proteome of control and OCE tobacco leaves (leaf 16). Normalized spot volume, isoelectric point (pI), molecular weight (MW), and significant differences in spot volume was calculated by Phoretix 2-D Expression v2005 software from reference gels generated for both control and OCE tobacco plants (n=3 for both lines). All numbers represent: mean value (SD). Spots are numbered according to Fig. 3 in the text.

Supplementary Fig. 1. Pair-wise alignment of RuBiSCO activase isoforms from Arabidopsis and three RuBiSCO activase proteins identified in tobacco. RA1, RuBiSCO activase 1 (tobacco); RA2, RuBiSCO activase 2 (tobacco); RuAct, RuBiSCO activase (tobacco); AthL, RuBiSCO activase long isoform (Arabidopsis); AthS, RuBiSCO activase short isoform (Arabidopsis).

Abbreviations

    Abbreviations
  • 2-D

    2-dimensional

  • BSA

    bovine serum albumin

  • CP

    cysteine proteinases

  • GS

    glutamine synthetase

  • LSU

    large subunit

  • OC-1

    oryzacystatin-1

  • OCE

    oryzacystatin-1 expressing plants

  • PBS

    phosphate buffered saline

  • Rubisco

    ribulose-1,5-bisphosphate carboxylase/oxygenase

  • RVBs

    Rubisco vesicular bodies

  • SDS-PAGE

    sodium dodecyl sulphate polyacrylamide gel electrophoresis

  • SSU

    small subunit

  • TCA

    trichloroactetic acid

This work was funded by a Royal Society (UK)–National Research Foundation (South Africa) joint project (GUN 2068793). AP thanks the Association of Commonwealth Universities for a Split-site PhD fellowship. We are grateful to Dominique Michaud for critical reading of the manuscript.

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