JXB Advance Access originally published online on March 21, 2006
Journal of Experimental Botany 2006 57(6):1431-1443; doi:10.1093/jxb/erj123
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RESEARCH PAPER |
Two new cysteine proteinases with specific expression patterns in mature and senescent tobacco (Nicotiana tabacum L.) leaves*
1Forestry and Agricultural Biotechnology Institute, Botany Department, University of Pretoria, Hillcrest, Pretoria 0002, South Africa
2Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK
To whom correspondence should be addressed. E-mail: karl.kunert{at}fabi.up.ac.za
Received 21 September 2005; Accepted 13 January 2006
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Abstract |
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Cysteine proteinases are involved in various physiological and developmental processes in plants. Two cDNAs from senescent and non-senescent tobacco leaves were isolated with degenerate primers designed from conserved regions of plant senescence-associated cysteine proteinases using rapid amplification of cDNA ends (RACE). Both sequences encode papain-like cysteine proteinases: the 833 bp fragment (NtCP1) encoding a C-terminus partial sequence of a putative tobacco cysteine proteinase gene whereas the 1300 bp fragment (NtCP2) is a full-length cysteine proteinase. On the amino acid sequence level, NtCP1 has a high similarity with other senescence-associated cysteine proteinases. It is expressed only in senescent leaves. It is not induced in mature green leaves upon exposure to drought or heat. These results suggest that it might be a good developmental senescence marker in tobacco. By contrast, NtCP2 has a high similarity to KDEL-tailed cysteine proteinases and is expressed in mature green leaves. Both drought and heat decreased NtCP2 transcript abundance in mature green leaves. It is concluded that NtCP1 is a senescence-specific cysteine proteinase whereas NtCP2 fulfils roles in green leaves that might be similar to those of KDEL-tailed cysteine proteinases involved, for example, in programmed cell death.
Key words: Cysteine proteinase, KDEL motif, senescence markers, tobacco
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Introduction |
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Cysteine proteinases are involved in a variety of proteolytic functions in higher plants (Granell et al., 1998
). Many of the endogenous plant cysteine proteinases identified to date have acidic pH optima in vitro, suggesting that they are localized to the vacuole in vivo (Callis, 1995). Cysteine proteinase expression has been intensively studied with various expression patterns reported for different stages of plant development (Buchanan-Wollaston and Ainsworth, 1997; Guerrero et al., 1998; Xu and Chye, 1999). Such proteinases are involved in processing and degradation of seed storage proteins (Shimada et al., 1994; Toyooka et al., 2000), fruit ripening (Alonso and Granell, 1995) as well as in legume nodule development (Naito et al., 2000). They are also implicated in responses to stresses such as wounding, cold, and drought (Schaffer and Fischer, 1988; Koizumi et al., 1993; Linthorst et al., 1993; Harrak et al., 2001) as well as in programmed cell death (Solomon et al., 1999; Xu and Chye, 1999). Some cysteine proteinases have specific characteristics such as a C-terminal KDEL motif. This motif, which is an endoplasmic reticulum retention signal for soluble proteins, allows cysteine proteinase 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).
A number of genes encoding papain-like cysteine proteinases have also been isolated from senescing organs including leaves (Lohman et al., 1994; Ueda et al., 2000; Gepstein et al., 2003), flowers (Eason et al., 2002), legume nodules (Kardailsky and Brewin, 1996), and germinating seeds (Ling et al., 2003). In general, senescence is characterized by the breakdown of proteins (Callis, 1995) in senescing organs and nutrient remobilization to other developing parts of the plant (Noodén, 1988). There is considerable evidence from screening of cDNA libraries derived from senescent leaf tissues that the expression of the vast majority of genes is down-regulated during senescence (Bhalerao et al., 2003). Senescence down-regulated genes (SDGs) include photosynthesis genes such as those encoding the chlorophyll a/b binding protein and the ribulose-1, 5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit (Humbeck et al., 1996). However, a number of senescence-associated genes (SAGs) are up-regulated during leaf senescence (Lohman et al., 1994; Quirino et al., 1999; Swidzinski et al., 2002; Bhalerao et al., 2003; Gepstein et al., 2003; Lin and Wu, 2004). These SAGs are either expressed exclusively during senescence (Class I SAGs) or their expression increases during senescence from a continuous basal level during leaf development (Class II SAGs) (Gan and Amasino, 1997). Of the few SAGs that are highly senescence-specific, SAG12 encodes a cysteine proteinase. It is highly abundant in senescing leaves but is undetectable in non-senescent leaves (Lohman et al., 1994).
Although cysteine proteinases have been extensively characterized in Arabidopsis, only some cysteine proteinases have been investigated in other plant species. In tobacco, several cysteine proteinases have been identified (Linhorst et al., 1993; Ueda et al., 2000; V Senyuk et al., unpublished data, GenBank accession number: CAB44983). However, neither KDEL nor exclusively senescence-related cysteine proteinases have so far been isolated and characterized from tobacco.
The objective of this study was therefore to isolate senescence-related cysteine proteinase genes from tobacco. The isolation and characterization of two novel tobacco cysteine proteinase coding sequences is described here. These sequences, termed NtCP1 and NtCP2, were isolated from senescent and mature green tobacco leaves, respectively. NtCP1 and NtCP2 are differentially expressed in response to abiotic stress. They belong to two distinct subgroups within the papain-like family of cysteine proteinases. In addition, they are phylogenetically distant from other tobacco cysteine proteinase coding sequences described to date.
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Plant material
Nicotiana tabacum L. cv. Samsun plants were grown in a greenhouse and maintained at a 26/20 °C day/night temperature cycle and a 12/12 h light/dark cycle. Photosynthetic photon flux density during the light phase was 600±50 µmol m–2 s–1 and plants were grown at a relative humidity of 60%. For measurements, senescent and non-senescent, fully expanded mature green leaves from 3-month-old plants were used. Non-senescent, green leaves used in these experiments had the highest chlorophyll content of all leaves on the plant and they had no visible symptoms of yellowing. Leaves were considered senescent when they showed yellowing and their chlorophyll content was lower than 40% of the chlorophyll content of a green mature leaf without signs of yellowing.
Plant treatment
For drought stress, watering of tobacco plants was withheld for 10 d. For heat stress, plants were moved to a growth chamber maintained at temperature of 38/28±2 °C day/night temperature cycle for 10 d. A combination of heat and drought stress was carried out by withholding water for 3 d from plants grown at 38/28±2 °C. For all stress experiments, photosynthesis photon flux density in the growth chambers was 300±20 µmol m–2 s–1. For analysis, leaf samples were collected from stressed and non-stressed plant of the same age and samples were either immediately used after freezing in liquid nitrogen or kept after freezing by storing at –80 °C until needed.
Chlorophyll determination
Leaf chlorophyll content of leaves was measured from three leaf discs each with 8 mm diameter. Spectrophotometric determination of total chlorophyll content in 80% acetone was done according to the calculation described by MacKinney (1941).
Proteinase determination
For the determination of proteinase activity, leaf samples were homogenized in extraction buffer (50 mM TRIS–HCl, pH 7.4) in the presence of liquid nitrogen without the addition of a proteinase inhibitor during homogenization. Samples stored on ice were processed immediately and all extraction steps were performed on ice to minimize any proteinase action. Homogenates were centrifuged at 13 000 rev min–1 at 4 °C for 15 min and the supernatant was used for the different assays. Protein content of supernatant was quantified according to the method described by Bradford (1976) using BSA as a standard.
Proteinase determination with gelatine SDS-PAGE
Proteinase-containing supernatants from leaf homogenates with 10 µg of total protein were added to an equal volume of a loading buffer (90 mM TRIS–HCl, pH 6.8, 20% glycerol, 2% SDS, and 0.2% bromophenol blue). To determine the proteinase composition in the samples, different proteinase inhibitors were added to the protein samples before the addition of the loading buffer. Samples were incubated with the different inhibitors for 15 min at 37 °C with final concentrations of 100 µM of E-64 (inhibitor of cysteine proteinases), 100 µM of BBTI (soybean Bowman–Birk inhibitor of serine proteinases), or 5 mM of PMSF (inhibitor of serine and cysteine proteinases). After incubation, samples were separated on a 10% resolving sodium dodecylsulphate polyacrylamide (SDS-PAGE) gel (Laemmli, 1970) containing 0.1% gelatine (type I from porcine skin) (Sigma, USA) with a 5% stacking gel according to the method outlined by Michaud et al. (1993). Gels were run at 150 V for 1 h at 4 °C. Proteinases present on the gels were re-natured in a 2.5% Triton X-100 solution at room temperature with gentle shaking for 30 min. Respective proteinase inhibitors at similar concentrations as those used for sample incubations were added to the proteinase re-naturation and development buffers. However, all gels containing inhibitor-free samples were treated with proteinase inhibitor-free buffers for loading, re-naturation, and development. After proteinase re-naturation, gels were rinsed in an excess of distilled water and developed overnight at 37 °C in proteolysis buffer (100 mM citrate phosphate buffer, pH 6.0; 10 mM L-cysteine). Gels were then stained in 0.05% Coomassie Brilliant Blue R-250 in 10% acetic acid and 25% iso-propanol and de-stained with the same solution without addition of the dye. Areas of proteinase activity were identified as clear bands against a blue background.
Azocasein assay
Total proteolytic activity of leaf extracts from senescent leaves was determined using azocasein (Sigma, USA) as a proteinase substrate according to the method of Hines et al. (1992). Different extract samples (50 µl sample–1) containing 50 µg protein were incubated in 200 µl proteolysis buffer (100 mM citrate phosphate buffer, pH 6.0 and 10 mM L-cysteine) for 30 min at 37 °C with and without the addition of proteinase inhibitors. Inhibitors with final concentrations in the incubation mixture were E-64, BBTI, and pepstatin A each at 100 µM, PMSF at 5 mM, and EDTA at 20 mM. Azocasein (2%) was dissolved in proteolysis buffer and 200 µl of azocasein solution was then added to the incubation mixture and incubated for 24 h at 37 °C. After incubation, the reaction was stopped by adding an equal volume of 10% trichloroacetic acid (TCA) to the reaction mixture, which was followed by incubation on ice for 30 min and centrifugation of the mixture at 12 000 rev min–1 for 5 min. After centrifugation, the supernatant (500 µl) was added to an equal volume of 1 M NaOH for colour development and the absorbance of the mixture was measured at 440 nm in a spectrophotometer. As blanks, identical reactions were set up, but reactions were immediately stopped by the addition of TCA.
Fluorimetric measurement of cysteine proteinase activity
Cysteine proteinase activity was measured with the fluorescence substrate Z-phe-arg-AMC with or without the addition of a cysteine proteinase inhibitor using a modified method described by Abrahamson (1994). For determination, a plant protein extract (50 µl) with 30 µg of protein/sample was mixed with 325 µl proteolysis buffer (100 mM citrate phosphate pH 6.0 and 10 mM L-cysteine). The mixture was preincubated for 10 min at 37 °C with or without the addition of a cysteine proteinase inhibitor (E-64 at 100 µM and PMSF at 5 mM) before adding 125 µl of proteinase substrate (20 µM Z-phe-arg-AMC) diluted in proteolysis buffer. Similar reactions were set without the plant extract as a blank. The reaction mixture was then incubated for 10 min at 37 °C and was stopped by the addition of 1.0 ml stopping buffer (10 mM sodium monochloroacetate, 30 mM sodium acetate, and 70 mM acetic acid, pH 4.3). Release of fluorescent AMC was determined using a fluorescence spectrophotometer (Model F-2000; Hitachi, Japan) using an excitation and an emission wavelength of 370 nm and 460 nm, respectively.
Isolation of cysteine proteinase coding sequences
Total RNA was extracted from leaf material using the TriPure total RNA isolation kit according to the manufacturer's recommendation (Roche, Germany) and contaminant genomic DNA was digested by RNase-free DNase. Total RNA (10 µg) was used for cDNA synthesis using oligo-(dT)15 for priming poly(A) RNA and AMV reverse transcriptase for reverse transcription followed by second strand synthesis according to the outline given by the manufacturer (Roche, Germany). Synthesized double-stranded cDNA was used as a template for gene isolation by the polymerase chain reaction (PCR).
For isolation of cysteine proteinase coding sequences from synthesized cDNAs, a forward degenerate primer with the sequence 5'-AGAATCAAGGACAATGTGGATGY(C/T)TGY(C/T)TGGGC-3' and a reverse degenerate primer with the sequence 5'-TCCCCAAGAATTCTTAATAATCCAR(A/G)TAY(C/T)TT-3' were used. The design of both primers was based on sequence information for the conserved regions of senescence-associated cysteine proteinases from Brassica napus (GenBank accession number AAD53011), Arabidopsis thaliana (GenBank accession number AAK64131), and Gossypium hirsutum (GenBank accession number AAT34987) using the CODEHOP (Consensus Degenerate Hybrid Oligonucleotide Primers) program (Rose et al., 2003). For amplification of coding sequences by PCR, a primer annealing temperature of 50 °C was used in a standard PCR reaction containing 20 mM TRIS-HCl, pH 8.4, 50 mM KCl, 0.25 mM each dNTPs, 5 units of Taq polymerase (Roche, Germany), and 0.2 µM of the degenerate primer mixture. The PCR cycles were 2 min at 94 °C followed by 35 cycles at 30 s at 94 °C, 30 s at 50 °C, and 60 s at 72 °C followed by an extension for 10 min at 72 °C.
Either a full-length or a partial cDNA clone for cysteine proteinases were obtained by performing 5' and 3' RACE using the GeneRacerTM kit according to the manufacturer's instructions (Invitrogen, USA) along with gene-specific primers. Gene-specific forward primer 5'-CATGGCTGAAGGTGGCGAGTGTGA-3' and two nested reverse primers with the sequences 5'-CCTTAGGTGCTGTTGCAGGAGACCCTGT-3' (external primer) and 5'-CATTCAGGTCCCCACGAGTTCCTCAC-3' (internal primer) were used for isolation of a full-length cysteine proteinase coding sequence from synthesized cDNA derived from non-senescent leaf material. Two forward primers with the sequences 5'-TTCATGGGGCAGTAAATGGGGTGACA-3' (external primer) and 5'-TGGGGCAGTAAATGGGGGACAGTGG-3' (internal primer) were used for isolation of a 3'-end of a cysteine proteinase coding sequence from synthesized cDNA derived from senescent leaf material. The 5' RACE, 5' nested, 3' RACE, and 3' nested primers were provided with the GeneRacerTM kit (Invitrogen, USA) that were used together with the gene-specific primers. All amplified PCR products were finally cloned into the vector pGEM-T Easy vector system II (Promega, USA).
Sequence analysis
Sequencing of the inserts were performed by using the BigDye® Terminator Cycle Sequencing FS Ready Reaction Kit, v 3.1 on ABI PRISM® 3100 automatic DNA-Sequencer (Applied Biosystems, USA). The BLASTN and BLASTP programs (Altschul et al., 1997) were used for the gene sequence homology search. Amino acid sequences of selected plant papain-like cysteine proteinases including known N. tabacum proteinases were aligned using Clustal W (Thompson et al., 1994). A phylogenetic tree was constructed from aligned sequences using maximum likelihood parsimony with 100 bootstrap re-sampling methods of the Phylip 3.6 package (Felsenstein, 1989). ExPASy (Gasteiger et al., 2003) web site and programs therein were used for the prediction of amino acid features of NtCP1 and NtCP2.
Southern blot analysis
Genomic DNA (20 µg) was digested for 12 h with 100 units of EcoRI and XbaI and digested DNA was separated on a 1% (w/v) agarose gel at 50 V for 5 h. Separated DNA was transferred to a Hybond N+ membrane using a standard protocol as outlined by Sambrook and Russell (2001). The gene-specific DNA probe for NtCP1 was prepared by amplifying a 659 bp fragment and probe for NtCP2 was prepared by amplifying a 604 bp DNA fragment. Probes were labelled using a random-prime labelling kit according to the manufacturer's instructions (Amersham, UK). Prehybridization for 2 h and hybridization of probes with membrane-bound DNA were performed overnight at 60 °C in a hybridization buffer containing 0.5 M Na2HPO4, pH 7.2, 7% (w/v) SDS, and 1 mM EDTA. Three subsequent stringency washes were performed at 60 °C for 15 min each. The first washing solution contained 0.1% SDS (w/v), 2x SSC, the second 0.1% SDS (w/v), 1x SSC, and the third washing 0.5x SSC and 0.1% SDS. Detection of hybridization products was carried out with the Gene ImagesTM CDP–StarTM system (Amersham, UK) followed by exposure to a HyperTM film (Amersham, UK).
Northern blot analysis
Total RNA was extracted from leaf material using the TriPure total RNA isolation kit according to the manufacturer's recommendations (Roche, Germany) and contaminant genomic DNA was digested by RNase-free DNase. Northern blotting was carried out as described by Sambrook and Russell (2001). For blotting, total RNA (20 µg) was first size-separated on a 1.2% agarose gel containing 2.2 M formaldehyde, transferred to a Hybond-N+ membrane (Amersham, UK), and then UV cross-linked. Prehybridization and hybridization of RNA-containing membranes was carried out at 65 °C. Specific probes for NtCP1 and NtCP2 were produced by PCR amplification of a NtCP1 fragment (positions 500–833) and a 596 bp NtCP2 fragment (positions 705–1300) from cloned products and labelling of probes using a random-prime labelling kit according to the manufacturer's instructions (Amersham, UK). Hybridization and stringency washes were carried out as outlined under Southern blot analysis. As an internal control for equal loading, a 598 bp N. tabacum 18S ribosomal RNA probe (GenBank accession number AJ236016) was amplified from genomic DNA of tobacco using the forward primer 5'-CCTGAGAAACGGCTACCACATCCA-3' and reverse primer 5'-CGAGCCCCCAACTTTCGTTCT-3'.
Statistical analysis
All estimates of sample variability are given in terms of the SD of the mean. The significance of differences in chlorophyll and soluble protein content and proteinase activity of tobacco leaves of different age was determined by the Student's two-tailed t test. P values 
0.05 were considered significant.
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Results |
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Leaf chlorophyll, soluble protein content and proteinase activity
Tobacco leaves were harvested from plants at the development stage shown in Fig. 1A. The leaves were ranked from the top of the plant to the bottom. The third and the seventh leaf were denoted as markers for green and senescent leaves, respectively (Fig. 1B). Leaf number 7 had significantly less (P
0.05) chlorophyll (60%) and protein (49%) than leaf number 3 (Fig. 1C).
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Senescent leaves (S) had higher maximal extractable proteinase activities than mature green (G) leaves (Fig. 2A) when assayed by the gelatine SDS-PAGE method. Addition of either the cysteine proteinase inhibitor, E-64 or a serine proteinase inhibitor (BBTI) inhibited proteinase activity in extracts from both types of leaves. Treatment of plant extracts with PMSF, which inhibits cysteine and serine proteinases, completely inhibited proteinase activity in both types of leaf extract (data not shown).
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Similarly, the addition of E-64, BBTI, the aspartic-proteinase inhibitor, pepstatin A, or PMSF to leaf extracts, analysed in the azocasein assay system significantly inhibited activity (P
0.05) by 35%, 51%, 13%, and 72%, respectively (Fig. 2B). No inhibition of activity was observed when EDTA was added to extracts to prevent metallo-proteinase activity. This indicates that senescent tobacco leaves have at least three different classes of proteinase activity (cysteine, serine, and aspartic proteinases), but metallo- proteinases were not detected under this experimental system.
Cysteine proteinase activity was also determined using Z-phe-arg-AMC (Fig. 2C). These analyses showed that cysteine proteinase activity was significantly higher (P 0.05) in senescent leaves (630±47 FU mg–1 protein) than mature green leaves (430±7 FU mg–1 protein). Proteinase activity in extracts of both types of leaves was significantly (P 0.05) inhibited by E-64 (64% for green leaves and 99% for senescent leaves) or by PMSF (34% for green leaves and 74% for senescent leaves).
Isolation and analysis of NtCP1 and NtCP2
Two cDNA fragments with sizes of 514 bp and 507 bp were isolated from tobacco cDNAs that were synthesized from total RNA from senescent and mature green leaves, respectively. Degenerate primers were designed from sequence information for the conserved regions of plant senescence-associated cysteine proteinase sequences in the available databases. The isolated 514 bp cDNA fragment from senescent leaves had 86% similarity at the nucleotide level with the Petuniaxhybrida cysteine proteinase, CP10 (GS Chaffin et al., unpublished results; GenBank accession number AY662996). By comparison, the 507 bp cDNA fragment from non-senescent leaves had 83% similarity with Petuniaxhybrida cysteine proteinase CP6 (GS Chaffin et al., unpublished results; GenBank accession number AY662992). Applying the RACE technique and gene-specific primers designed according to the sequence information obtained from the 514 bp and 507 bp fragments, only a 3' partial 833 bp sequence, denoted as NtCP1, was obtained from the original 514 bp fragment derived from senescent leaves. This 833 bp sequence of NtCP1 had a 224 bp 3' untranslated region (UTR) and a partial open reading frame (ORF) of 609 bp (Fig. 3A). By contrast, a full-length 1300 bp sequence, named NtCP2, was obtained from the original 507 bp fragment derived from green, non-senescent leaves and NtCP2 had a 23 bp 5' and a 194 bp 3' UTR with an ORF of 1083 bp (Fig. 3B).
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A predicted co-translational N-terminal signal peptide with a hydrophobic core was identified in the deduced amino acid sequence of NtCP2 (Fig. 3B; positions M1-S20) derived from non-senescent leaves. Such signal peptide carrying hydrophobic residues are typical of sequences allowing endoplasmic reticulum (ER) targeting. Further, the pro-domain of NtCP2 (Fig. 3B; positions E53-N72) contains a conserved non-contiguous ERFNIN motif (EX3RX3FX2NX3I/VX3N) typical for cysteine proteinases in the Cathepsin L and H like proteinases (Karrer et al., 1993
). A GCNGG motif was identified in both NtCP2 (G188–G192; Fig. 3B) and NtCP1 (Fig. 3A; G48–G52). With the exception of the central Asn (N) residue, this GCNGG motif is invariant in all ERFNIN proteinases and also in the cathepsin B-like proteinases (Karrer et al., 1993). In papain-like proteinases, the Cys residue in the GCNGG motif is involved in the formation of a disulphide bridge. In addition, NtCP2 has a C-terminus KDEL motif (Fig. 3B), which is absent from NtCP1. Both NtCP1 and NtCP2 contain the conserved cysteine proteinase catalytic triade Cys, His and Asn as well as the conserved Glu residue (Figs 3, 4).
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The NtCP2 and the partial NtCP1 sequences have an identity of 46% and 59% on the nucleotide and the amino acid sequence level respectively, with each other. In addition, using a BLASTN and a BLASTP search, both NtCP1 and NtCP2 were identified to have higher homology to the group of papain-like cysteine proteinases. Alignment of NtCP1 derived from senescent leaves against already reported cysteine proteinase amino acid sequences in the NCBI database revealed a similarity to several papain-like cysteine proteinases (Fig. 4A). This includes 67–70% identity with Daucus carota DcCysP2 (Mitsuhashi et al., 2004
), Ipomoea batatas SPG31 (Chen et al., 2002), Arabidopsis thaliana SAG12 (Lohman et al., 1994), and Brassica napus SAG12-1 (Noh and Amasino, 1999). NtCP1 is less related to other tobacco cysteine proteinases reported to date. These include CPR1 from germinating tobacco seeds (55% similarity; GenBank accession number Z99173), drought-inducible CPR2 (39%; GenBank accession number AJ242994), NTCP-23 (40%; Ueda et al., 2000) and wound-inducible CYP-7 (39%; Linthorst et al., 1993), and CYP-8 (39%; Linthorst et al., 1993).
By comparison, the similarity search using NtCP2 derived from mature green leaves revealed a rather high amino acid similarity (68–72%) with KDEL-tailed plant cysteine proteinases (Fig. 4B). These include similarity to Ricinus communis Cys-EP (Schmid et al., 1998), Phaseolus vulgaris EP-C1 (Tanaka et al., 1991), Glycine max CysP1 (Ling et al., 2003), and Vigna mungo sulphydryl-endopeptidase (SH-EP; Akasofu et al., 1989). NtCP2 has, however, a rather low similarity to other already identified Nicotiana cysteine proteinases. On the amino acid level this includes a 31% similarity with tobacco NTCP-23, a 30% and 31% similarity with CYP-7 and CYP-8, a 30% similarity with CPR2 and 49% similarity with CPR1. Database searches also revealed that NtCP2 has a very high (94%) similarity with a partial N-terminal sequence for a cysteine proteinase derived from tobacco anthers (TP Beals, RB Goldberg, unpublished results; GenBank accession number U57824). However, none of the database tobacco sequences have such a well-defined KDEL tail as NtCP2.
A phylogenetic tree, constructed to identify the relatedness of amino acid sequences of NtCP1 and NtCP2 to other members of the papain-like cysteine proteinase sub-family (Fig. 5) revealed that the two sequences are localized in two separate groups. NtCP1 is grouped in group 4 or C1A-4 according to Beers et al. (2004). This group includes leaf senescence-specific proteinases, such as Arabidopsis SAG12, Brassica napus SAG12-1, and Ipomoea batatas SPG31. By contrast, NtCP2 is located in group 2. This group contains KDEL-tailed family members, such as Ricinus communis Cys-EP and Vigna mungo SH-EP.
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Copy number and expression of NtCP1 and NtCP2
Southern blot analysis of isolated tobacco genomic DNA showed that multiple copies of NtCP1 and NtCP2 exist in the tobacco genome (Fig. 6). Two distinct hybridization products and two overlapping products were found with labelled NtCP1 and genomic DNA digested with XbaI and EcoRI, respectively. By contrast, four hybridization products were found with labelled NtCP2 and XbaI. More than six products were found with EcoRI digested genomic DNA.
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Northern blot analysis revealed that NtCP1 was present in senescent leaves. By contrast, no NtCP1 transcripts were detected in mature green leaves. NtCP2 transcripts were detected in mature green leaves (Fig. 7A), but not in senescent leaves. NtCP1 expression was not induced in mature green leaves by exposure to abiotic stress. In the present study, including drought, heat, and a combination of both stresses, NtCP1 transcripts had a similar low abundance in all conditions (Fig. 7B). NtCP2 transcripts were much decreased in mature green leaves following drought treatment, they fell below the level of detection following heat treatment or a combination of both stresses (Fig. 7B).
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Discussion |
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NtCP1 and NtCP2 sequence analysis
In the present study, two novel tobacco coding sequences encoding cysteine proteinases, denoted as NtCP1 and NtCP2 have been identified. According to their deduced amino acid sequences both proteinases can be classified as belonging to the sub-family C1A of papain-like cysteine proteinases (MEROPS peptidase database, http://merops.sanger.ac.uk; Rawlings et al., 2004
). Based on nucleotide as well as amino acid analysis, NtCP2 is phylogenetically distant from NtCP1. Except for a partial N-terminal sequence from tobacco anthers, which is related to NtCP2, the data presented here provide evidence that these are novel coding sequences that are only distantly related to each and other tobacco cysteine proteinases reported to date.
NtCP2 is (to our knowledge) the first KDEL-motif-containing cysteine proteinase to be isolated from tobacco. No KDEL motif was found in NtCP1. Papain-like cysteine proteinases with a KDEL motif are involved in programmed cell death (Guerrero et al., 1998; Schmid et al., 1998; Gietl and Schmid, 2001; Ling et al., 2003). Since NtCP2 belongs to the KDEL cysteine proteinase group, it might also play a role in programmed cell death. There is also considerable evidence that KDEL proteinases accumulate in cell vesicles, such as ER-derived ricinosomes (Schmid et al., 2001) or in KDEL vesicles (Toyooka et al., 2000), before being transported to the vacuoles. Okamoto et al. (2003) suggested that the KDEL motif of KDEL proteinases could act as enhancers for vacuolar transport, because the KDEL motif appears to be directly involved in the formation of KDEL vesicles and vacuole transportation. Transgenic Arabidopsis plants expressing a mutant SH-EP proteinase lacking the KDEL motif were unable to develop KDEL vesicles, the mutant SH-EP being mainly secreted into the intercellular spaces of the transgenic plants, which showed abnormal development and accelerated death (Okamoto et al., 2003).
NtCP1 characterization
Senescent tobacco leaves expressing NtCP1 had increased proteinase activity. The inhibitor studies showed that at least three classes of proteinase (cysteine, serine, and aspartic proteinases) are present in senescent tobacco leaves. Moreover, increased proteinase activity was accompanied by decreases in chlorophyll and protein content. NtCP1 transcripts were only detectable in senescent tobacco leaves. Therefore, it would appear that NtCP1 is not expressed in mature green leaves. Similarly, NtCP1 transcripts were not detected in mature green leaves following exposure to drought or heat or a combination of both stresses. NtCP1 was only expressed in senescent leaves, a result comparable with that obtained for another cysteine proteinase, SAG12. This proteinase, previously identified by Lohman et al. (1994) in Arabidopsis, exhibits one of the highest levels of induction during Arabidopsis leaf senescence. It is also often used as a senescence marker in studies in plant development as it is not induced by stress-induced programmed cell death, unlike other types of SAG genes (Brodersen et al., 2002). Phylogenetic analysis revealed that NtCP1 clusters with SAG12 and also with BnSAG12-1 (Noh and Amasino, 1999). Since both Arabidopsis SAG12 and Brassica BnSAG12-1 are considered to be senescence-specific genes these data support the notion that NtCP1 is a developmental marker for tobacco leaf senescence (Lohman et al., 1994; Noh and Amasino, 1999; Wan et al., 2002). Hence, NtCP1 could be a good marker for developmental senescence in tobacco as it is not induced in mature green leaves in optimal or stress conditions.
Like SAG12, NtCP1 belongs to class I type SAGs. In general, SAGs can be separated into two classes based on their temporal gene expression pattern during leaf senescence (Gan and Amasino, 1997). Class I type SAGs are expressed only during leaf senescence. Therefore, they are denoted as being senescence-specific. By contrast, class II type SAGs have a low basal expression throughout leaf development, but at the onset of senescence their expression is significantly enhanced. Unlike NtCP1, the tobacco NTCP-23 cysteine proteinase sequence shows a typical class II type SAG expression pattern (Ueda et al., 2000).
A recent study also showed that SAG12 and possibly other senescence-associated cysteine proteinases are restricted to senescence-associated vacuoles (SAVs; Otegui et al., 2005). These SAVs are more acidic than the central vacuoles. The specific development of SAV in cells containing chloroplasts might indicate the possible involvement of SAVs in the degradation of chloroplast proteins (Otegui et al., 2005). While the exact cellular localization of NtCP1 is unknown, it has been observed that Rubisco degradation is prevented in tobacco leaves expressing a rice cysteine proteinase inhibitor (OCI), suggesting that cysteine proteinases are important for the degradation of chloroplast proteins such as Rubisco (authors own unpublished data).
NtCP2 characterization
NtCP2 transcripts were only detected in mature green leaves. Moreover, NtCP2 transcripts were significantly decreased in leaves following exposure to drought or heat stress or a combination of both stresses. Such drought-induced down-regulation of cysteine proteinase expression has also been reported for other cysteine proteinases (Weaver et al., 1998). The NtCP2 clusters with a group of cysteine proteinases whose expression profiles are rather variable between species. For example, SEN102 and SEN11 transcripts from Hemerocallis spp flowers (Valpuesta et al., 1995; Guerrero et al., 1998) that are closely related to NtCP2 accumulate at high levels in senescing flowers, but in leaves a higher level of accumulation was found in green leaves and their expression was lower in senescing leaves. Moreover, two KDEL-tailed cysteine proteinases also belonging to this group CysP1 and CysP2 have been found in senescent soybean cotyledons, flowers, roots, and pods, as well as in young leaves (Ling et al., 2003). In tobacco, it was found that NtCP2 expression was higher in mature green leaves and significantly decreased in senescent leaves. This perhaps suggests that different species use either similar or identical gene-products to modulate the development of different organs including leaves.
In conclusion, two novel tobacco leaf cysteine proteinases have been identified, which are differentially expressed during development and in response to stress. While to date there is no precise information concerning their cellular function or localization, the data presented here indicate different roles in leaf development; since NtCP1 is also only expressed in senescent tobacco leaves, NtCP1, like SAG12, can be used as a specific molecular marker for age-mediated leaf senescence in tobacco.
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Acknowledgements |
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This work is supported by a grant to G Beyene from the Ethiopian Government and by a SA Innovation Fund grant (project number 41421) to KJ Kunert. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the UK.
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* Nucleotide sequence data reported in this paper are available in the DDBJ/EMBL/GenBank under accession numbers AY881010 and AY881011.
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