JXB Advance Access originally published online on October 24, 2005
Journal of Experimental Botany 2005 56(422):3111-3120; doi:10.1093/jxb/eri308
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RESEARCH PAPER |
Cucurbit phloem serpins are graft-transmissible and appear to be resistant to turnover in the sieve element–companion cell complex
1Department of Plant Biology, Royal Veterinary and Agricultural University (KVL), Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
2Biochemistry and Nutrition Group, BioCentrum, Building 224, Technical University of Denmark, DK-2800 Lyngby, Denmark
3Department of Applied Science, 575 ETAS Building, University of Arkansas at Little Rock, 2801 S University Ave, Little Rock, Arkansas 72204-1099, USA
* To whom correspondence should be addressed. Fax: +45 35 28 33 65. E-mail: als{at}kvl.dk
Received 12 April 2005; Accepted 31 August 2005
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Abstract |
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Serpins are unique inhibitors of serine proteases that are located in various plant tissues and organs. An orthologue of the pumpkin (Cucurbita maxima) phloem serpin CmPS-1 was amplified from cucumber (Cucumis sativus) RNA by RT-PCR, cloned, and designated as CsPS-1 (GenBank accession no. AJ866989). Alternative amino acid sequences in the reactive centre loop suggest distinct inhibitory specificity between CmPS-1 and CsPS-1. A difference in the electrophoretic mobility of these serpins was used in heterografts to establish that serpins are phloem-mobile. Immuno light microscopy revealed that the phloem serpins are localized exclusively to sieve elements (SE), while the phloem filament protein CmPP1, used as a reference, is localized to both SEs and companion cells (CCs). Similar to CmPS-1, CsPS-1 accumulates over time in phloem exudates, indicating that serpins differ from other phloem-mobile proteins whose concentrations appear to be stable in phloem exudates. These differences could reflect alternative mechanisms regulating protein turnover and/or inaccessibility of protein degradation. The functionality of the pore/plasmodesma units connecting SEs and CCs was tested with graft-transmitted CmPP1 as a transport marker. The occurrence of CmPP1 in the CCs of the Cucumis graft partner shows that translocated 88 kDa phloem filament protein monomers can symplasmically exit the SE and accumulate in the CC. By contrast, serial sections probed with the serpin antibody demonstrate that the 43 kDa serpin does not enter CCs. Collectively, these data indicate that CCs play a decisive role in homeostasis of exudate proteins; proteins not accessing the CCs accumulate in SEs and display a time-dependent increase in concentration.
Key words: Cucumis sativus, Cucurbita maxima, long-distance transport, phloem exudate, phloem protein, proteinase inhibitor, serpin
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Introduction |
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The phloem of higher plants distributes carbohydrates, amino acids, and other nutrients from source to sink tissues. Recent studies have shown that hormones, mRNAs, and proteins are also transported within the phloem and that some of these transported molecules play pivotal roles in the communication between organs to co-ordinate plant development and physiology (Thompson and Schulz, 1999
; Citovsky and Zambryski, 2000; Lucas et al., 2001; van Bel et al., 2002). The sieve elements (SEs) within the phloem are specialized for long-distance transport and, at functional maturity, lack the cellular machinery required for protein synthesis and processing. The closely associated companion cells (CCs) are thought to maintain the enucleated SE by symplasmically trafficking macromolecules and other compounds accessing the SEs through sieve pore/plasmodesma units (PPUs) (Oparka and Turgeon, 1999; van Bel and Knoblauch, 2000). Thus, proteins found in mature SEs can either be synthesized in the immature (nucleate) SEs or imported via PPUs from the transcriptionally and translationally active CCs.
Since the early 1990s, a variety of analytical and collection techniques has been used to identify soluble proteins from vascular exudates. The phloem sap seems to be particularly rich in proteinase inhibitors, even though target proteinases have yet to be identified. The complement of proteinase inhibitors detected in phloem exudate includes a number of low molecular weight (<15 kDa) reversible proteinase inhibitors belonging to different mechanistic classes (Murray and Christeller, 1995; Xu et al., 2001; Christeller et al., 1998; Kehr et al., 1999; Schobert et al., 1998; Dannenhoffer et al., 2001). In addition to the low molecular weight inhibitors, a 43 kDa serpin (serine proteinase inhibitor) has been identified in the phloem exudates from Cucurbita maxima (CmPS-1; Yoo et al., 2000). Interestingly, the amount of CmPS-1 present in phloem exudates appears in the plants between 10 d and 14 d after germination and increases thereafter (Yoo et al., 2000), when levels of other phloem exudate proteins have stabilized (Dannenhoffer et al., 1997; Golecki et al., 1998).
Serpins employ a unique irreversible mechanism of suicide-substrate inhibition. Serpins contain an exposed reactive centre loop (RCL) acting as bait for the target proteinase. During inhibition, a peptide bond at the reactive centre (P1-P1') is cleaved and the serpin is bound covalently to the active site of the proteinase through an ester bond. Only after many hours or days is the intact proteinase released from the now inactivated serpin (for details about the serpin inhibitory mechanism, including the conformational changes during inhibition, see Gettins, 2002). Serpins are widespread in the plant kingdom, but only the properties of cereal serpins, abundant in the grains, have been characterized in detail (Dahl et al., 1996a, b; Østergaard et al., 2000; Hejgaard, 2001; Hejgaard and Hauge, 2002). Roles in defence against pathogens and/or pests have been suggested, but the physiological functions of these serpins are unknown. The serpin identified in the phloem exudate of Cucurbita maxima (CmPS-1) was shown to be an inhibitor of pancreas elastase, a non-physiological target enzyme. In vivo feeding experiments suggested a role in defence against piercing-sucking aphids, but in vitro feeding with the purified serpin failed to demonstrate a direct effect on aphid survival (Yoo et al., 2000). Serpins in mammals have evolved to act as specific regulatory inhibitors of many complex intra- as well as extracellular proteolytic systems, including complement activation and blood coagulation (Gettins, 2002). Thus, it is reasonable to hypothesize that serpins could have regulatory functions in phloem transport.
An accurate analysis of phloem-mobile compounds is dependent upon the collection technique. Exudates collected from cut plant segments contain contaminants from other vascular and non-vascular cells due to the sudden pressure release that dislocates cellular contents (Lehmann, 1981). Even the elegant aphid stylectomy technique leads to small pressure changes and, thereby, potentially to local contamination. Therefore, the occurrence of a protein or mRNA in the phloem exudate neither establishes its cellular location in the intact phloem system nor its participation in long-distance transport. The cellular location can be derived from immunolocalization and in situ hybridization, although fixation-induced dislocations have in any case to be considered. Long-distance transport of macromolecules in the phloem can be tested by grafting experiments, where the formation of phloem bridges allows transport of macromolecules from one graft partner to the other (Tiedemann and Carstens-Behrens, 1994; Golecki et al., 1998). Heterografts between pumpkin and cucumber have been used to demonstrate the long-distance transport of several phloem proteins (PPI, PP2, and PFTI; Golecki et al., 1998, 1999; Dannenhoffer et al., 2001) and mRNAs (CmNACP, CmGAIP, and CmPP16; Ruiz-Medrano et al., 1999; Xoconostle-Cázares et al., 1999). A serpin has been immunolocalized to the phloem of barley, but the exact cellular localization was not determined (Roberts et al., 2003). Similarly, the cellular localization of the pumpkin phloem serpin has not been determined.
The goals of the present study were to evaluate the divergence between orthologous phloem serpins by cloning a cucumber serpin cDNA, to establish the cellular localization of serpins in the phloem by immunocytochemistry, and to determine whether serpins are phloem-mobile in pumpkin–cucumber heterografts. The results indicate that CCs play a key role in phloem protein homeostasis and assigns a significant role to the PPUs that allow selected compounds of the phloem sap access to the CCs.
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Materials and methods |
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Plant material
Seeds from pumpkin (Cucurbita maxima Duch., cv. Gelber Zentner) and cucumber (Cucumis sativus L., cv. Hoffmanns Produkta) were germinated on wet filter-paper and transferred to wet vermiculite in 12 cm pots and then fertilized weekly with conventional soluble greenhouse fertilizer. The plants were grown in walk-in growth chambers at 25 °C at a light intensity of 150 µE with a day–night cycle of 12/12 h. Seven to ten days after germination grafts between Cucurbita maxima and Cucumis sativus were made using the approach graft technique (Golecki et al., 1999
). In the figures, the heterografts are identified by letter codes, for example, Cs/Cm designates that Cucumis sativus is the scion and Cucurbita maxima the rootstock and bold indicates the sampled grafting partner (Cm).
RT-PCR and sequencing
Petioles from the last-formed leaf of 22-d-old Cucumis sativus plants were ground in liquid nitrogen and RNA extracted from 600 mg of tissue using the Trizol method (Invitrogen, Taastrup, Denmark; www.powow.com/akatlab/tigr.html). The reverse primer 5'-CCTCGAAGTGTTAGCTAG-3', designed against the 3'UTR of CmPS-1, was used for first-strand cDNA synthesis from 1 µg of total RNA using the Invitrogen SuperscriptTM First-strand Synthesis System. From this reaction, 1 µl was PCR-amplified from the N-terminal primer 5'-GCTGCCGGAAATGGACATC-3' (containing the start codon) and the C-terminal primer 5'-ATCCACAAGAGGGTTTAACAC-3' (without the stop codon). PCR conditions were 98 °C for 30 s, followed by 35 cycles at 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 20 s, and, finally, 72 °C for 7 min using HF phusion DNA polymerase (Medinova, Glostrup, Denmark). An 1177 bp amplicon was cloned into the pCR 2.1-TOPO vector (Invitrogen, Taastrup, Denmark) and sequenced (MWG Biotech, Eberswalde, Germany). A control PCR with these primers and total RNA from Cucurbita maxima petioles resulted in an amplicon of 1177 bp identical to CmPS-1.
SDS-PAGE and immunoblotting
Twenty-five days after grafting, SE exudate was collected in 2x Laemmli buffer (Laemmli, 1970). Samples from ungrafted plants were collected just below the last-formed leaf (
1 cm in length). Samples from grafting partners were collected just above the graft union. Samples were heated to 95 °C for 5 min and stored at –20 °C. The protein concentration was measured by the Lowry method with BSA as a standard, and 30 µg of protein per sample were separated by SDS–PAGE (15% gel) and then transferred to a nitrocellulose membrane by semi-dry electroblotting. Serpins were immunodetected with a protein A-purified, polyclonal antibody (R360) raised in rabbits against recombinant barley serpin BSZx (dilution 1:5000) as the primary antibody and alkaline phosphatase conjugated goat-anti-rabbit Ig (DAKO, Glostrup, Denmark) as the secondary antibody (dilution 1:5000). Blocking, washing, and antibody incubations were performed in PBS, pH 7.4, containing 0.2% casein and 0.1% Tween-20. Goat normal serum (10%) was added to the blocking and primary antibody solutions. Proteins were visualized by chemiluminescence (CDP-star, Tropix, Applied Biosystems) or with NBT/BCIP (Sigma).
Immunohistochemistry
Tissue was collected 25 d after grafting. Sections (less than 2 mm on the thinnest side) from the root neck, root tips, and adventitious roots were fixed in 3.7% paraformaldehyde, 0.1% glutaraldehyde, 0.05 M cacodylate buffer for 2–2.5 h, washed 3x20 min each in 0.05 M cacodylate buffer, and dehydrated in a graded isopropanol series. Tissues from ungrafted plants were sampled at 42 DAG and identically fixed. Samples were infiltrated with paraplast and 7 µm sections were mounted on superfrost+ slides (Menzel-Gläser, Braunschweig, Germany) then allowed to dry overnight at 43 °C. Sections were de-paraffinized with histoclear, rehydrated in a graded IPA series, and transferred to TBS (10 mM TRIS, 150 mM NaCl, pH 7.5). To reduce non-specific labelling, sections were blocked with 1% BSA and 10% goat normal serum in TBS for 1 h. After blocking, the sections were briefly rinsed and protein A-purified R360 or protein A-purified non-immune serum (controls) was applied (dilution 1:600 in TBS buffer). Sections were incubated overnight at room temperature in a humid chamber. Slides were washed 3x10 min each in TBS, incubated for 1 h in the secondary antibody diluted in TBS, washed as described above, and rinsed in ddH2O for 3x5 min each. Silver-enhancement (IntenSE M, Amersham Biosciences, Hillerød, Denmark) was applied for 12–15 min according to the manufacturer's instructions. Sections were subsequently rinsed in ddH2O, 2x10 min each and observed with a Zeiss Photomikroscop II (Oberkochen, Germany) equipped with a CCD camera. For comparison of the CmPP1 and CmPS-1 labelling patterns, four serial sections were incubated with the serpin antibody on sections 1 and 3 and with the CmPP1 antibody on sections 2 and 4. Antibody concentrations were as described above. Control sections were incubated with protein A-purified non-immune serum (1:400) and ungrafted Cucumis sativus served as a negative control for CmPP1 labelling.
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Results |
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To determine if an orthologue of the Cucurbita maxima phloem serpin was expressed in Cucumis sativus, RT-PCR was performed on total RNA extracted from cucumber petioles using primers designed against the phloem serpin CmPS-1 from Cucurbita maxima (Genbank accession no. AF284038; Yoo et al., 2000
). A 1177 bp amplicon (CsPS-1; GenBank accession no. AJ866989) obtained from Cucumis sativus petiole tissue matched CmPS-1 in size. The nucleotide sequence translated into a sequence of 389 amino acids with a calculated molecular mass of 42601.86 Da. The amino acid sequence was compared with those of previously characterized inhibitory plant serpins and of putative serpins for which full-length sequences have been obtained (not shown). The two related Cucurbitaceae serpins, CsPS-1 and CmPS-1, have 70% identical amino acid sequences, but CsPS-1 was also similar to serpins from other dicot plant families, including putative serpins from Citrus paradisi, Malus domestica, Glycine max, Gossypium hirsutum, and Arabidopsis thaliana (61–69% amino acid identity). Sequence identity with serpins from monocots, including Triticum aestivum, Hordeum vulgare, Zea mays, and Oryza sativa was in the range of 51–55%. Most plants for which one or more serpin RCL sequences have been established, mainly based on EST analyses (not shown), contain a serpin expressed in various vegetative tissues with a conserved reactive centre sequence P2–
Leu-Arg-Ser/Gly (Hejgaard et al., 2005). This sequence, which is the major determinant of the inhibitory specificity, is also present in CsPS-1 (Fig. 1). In addition, the RCL sequence immediately preceding the reactive centre is also highly conserved in these serpins, as illustrated by the close similarity of the P8-P1' sequences in serpins from Cucumis, Arabidopsis, Malus, Nicotiana, and Hordeum representing five different angiosperm orders. A related serpin mRNA was detected in a phloem-specific library from Picea glauca (Fig. 1). These observations suggest that the RCL structures and, therefore, the inhibitory specificity of these plant serpins, has been highly conserved during plant evolution. Interestingly, the related and previously characterized pumpkin serpin CmPS-1 has conserved the neutral, mainly hydrophobic P7-P2 sequence, but not the basic P1 Arg (Fig. 1).
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Immunoblot analysis of phloem exudate proteins collected at 42 d after germination (42 DAG) using the barley BSZx antibody confirmed the presence of cross-reacting 43 kDa serpins in Cucurbita maxima and Cucumis sativus (Fig. 2, lanes 2 and 3). Similar to CmPS-1, CsPS-1 accumulated over time in the phloem exudate (Fig. 3A, B), but at different time intervals reflecting the difference in the growth rates of the two plants. The serpins could already be detected in the exudate between 10 and 14 DAG in Cucurbita maxima, whereas in CS, the proteinase inhibitor appeared between 14 and 21 DAG. In relation to the total protein content of the exudate, serpin is less concentrated in Cucumis sativus than in Cucurbita maxima, provided that the antibody binds to both serpins with the same affinity (compare Fig. 3A and B).
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Phloem transport of CmPS-1
In order to test for long-distance mobility of CmPS-1 in the phloem, Cucumis sativus was grafted upon Cucurbita maxima and 25 d later, the presence of the two serpins was tested by immunoblotting of SE exudates collected separately from stock and scion. A small difference in electrophoretic mobility between CmPS-1 and CsPS-1 (Fig. 2, lanes 2, 3) allowed for the specific detection of the two serpins (Fig. 3C). As a positive control for protein transport across the graft union, a replicate protein blot was incubated with an antibody against CmPP2, known to be phloem-mobile (Golecki et al., 1999
). In both grafts (Fig. 3C: graft a and graft b), a protein with immunoreactivity and the electrophoretic mobility of CmPS-1 appeared in the Cucumis sativus grafting partner. The phloem transport capacity varied considerably between heterografts, but the ratio between translocated CmPS-1 and CmPP2 was fairly constant, as confirmed by densitometry (compare graft a and b in Fig. 3C, D). Grafts that were negative for translocation of CmPP2 were also negative for CmPS-1 (results not shown). These results confirm that CmPS-1 belongs to the group of phloem-mobile proteins (Tiedeman and Carstens-Behrens, 1994; Golecki et al., 1998).
Tissue localization of serpin
In order to determine whether the serpin is restricted to the phloem tissue, serpins in different regions of Cucurbita maxima or Cucumis sativus plants, including the leaf petiole, tendril (Cucumis sativus only), stem, root–shoot transition region, primary root, and adventitious root (Cucurbita maxima only), were immunolocalized. Serpin was only found in the phloem independent of the origin of the tissue. Strong labelling appeared in cells close to the cambium and in the bundle-associated phloem in the root–shoot transition region in both Cucurbita maxima and Cucumis sativus (Fig. 4A, B). Using serial sections, the localization of serpin was compared with that of another phloem exudate protein, the phloem filament protein CmPP1 (Fig. 4C). Interestingly, the localization patterns were quite different. While serpin was present in similar concentrations in all regions of the phloem, i.e. primary and secondary phloem, CmPP1 was prominent in bundle-associated phloem, present in primary bundle phloem, and least abundant in young secondary phloem (i.e. the phloem close to the cambium; compare Fig. 4A and C, arrows). As expected, controls with non-immune serum did not show any silver labelling, and the species-specific antibody against CmPP1 did not cross-react with any phloem protein in the ungrafted cucumber control in serial sections (Fig. 4D and E, respectively).
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This localization pattern suggests that serpin is primarily present in the young secondary phloem and CmPP1 in older parts of the vascular bundle. This result was confirmed by immunolocalization of longitudinal sections from the root–shoot transition region, again showing that serpin was localized to cells close to the cambium in the area where SEs are differentiating (Fig. 4F). By contrast, CmPP1 was localized to cells present at the periphery of the vascular bundle (Fig. 4G). The late detection of serpins in the phloem exudate led to the investigation in which seedling stage serpins start to appear. CmPS-1 transcript was detected in seedlings as early as 1 d after germination, and the protein was immunolocalized to the phloem of very young vascular bundles 2 d after germination of both Cucurbita maxima and Cucumis sativus seedlings (data not shown). This shows that serpin is already present in young sieve tubes and increases in concentration, reaching the detection limit in the exudate about 14 DAG.
Serpins immunolocalize to SEs
The different phloem localization patterns of serpins and CmPP1 led to the identification of the specific cell-types that accumulate serpin within the phloem. A cross-section of the external phloem in a vascular bundle within the root–shoot transition region (Fig. 5A, B) depicts a high number of serpin-positive cells in both Cucurbita maxima and Cucumis sativus (Fig. 5A, B). Under higher magnification and with aniline blue counterstaining, the serpin was shown to be SE-specific (Fig. 5C, D, arrows). The strong labelling at the sieve plates that are identified by aniline blue staining (Fig. 5E, F, arrowheads) does not necessarily reflect the in vivo distribution of serpin, but merely suggests that CmPS-1 accumulates on sieve plates during sample preparation.
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The accumulation in the exudate over development characteristic of serpins (Fig. 3) raised the question whether serpin is detectable in functional SEs. In order to test this, reciprocal graft combinations between Cucumis sativus and Cucurbita maxima were used (Cm/Cs; Fig. 6E) and the Cucurbita-specific protein CmPP1 was chosen as a molecular marker for functional SEs. The presence of CmPP1 will identify those SEs in Cucumis sativus that participate in long-distance transport of sugars and mobile proteins from scion to stock. Serial cross-sections of the root neck of the stock Cucumis sativus were incubated with antibodies against serpin or CmPP1 (Fig. 6A, B, respectively). Comparison of details (Fig. 6C, D) reveals that most SEs positive for serpin also contained CmPP1, confirming that the serpin occurs in functional SEs.
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Thorough analysis of different plant organs demonstrated that serpin exclusively occurs in SEs in both control and grafted plants, whereas, the adjacent CCs were unlabelled (Fig. 7A, C). By contrast, the graft-transmitted CmPP1 was present both within the SEs and the CCs, the latter showing very intense labelling (Fig. 7B, C). This implies that CmPP1 was able to pass the sieve PPUs and accumulate in the CCs. The CmPP1 present within the SEs was shown to be associated with the P-protein bodies (Fig. 7D, arrows) indicating that it had become a part of the structural P-proteins present in the SEs of the Cucumis sativus grafting partner.
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Discussion |
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Serpins belong to the emerging group of phloem-localized proteinase inhibitors in cucurbits that include low molecular weight serine proteinase inhibitors of several families, cystatins, and aspartic proteinase inhibitors (Murray and Christeller, 1995
; Christeller et al., 1998; Walz et al., 2004). The overall similarity between the serpins in phloem exudates of cucumber (CsPS-1) and pumpkin (CmPS-1) suggests a common evolutionary origin, but their divergence within the RCL strongly suggests that they have evolved in different directions with respect to their target proteinases. Closely related proteinase inhibitors often exhibit different substrate specificity. For example, members of the highly conserved group of PFTIs show individual specificity for either trypsin or chymotrypsin (Murray and Christeller, 1995). Collectively, proteinase inhibitors within the phloem provide a broad spectrum of substrate specificity as well as alternative mechanisms to inactivate proteinases within the assimilate transport stream or to serve as a defence against phloem-associated pathogens or insects.
Long-distance transport of serpins in sieve elements
The concept of ‘group transfer’ of phloem mobile proteins between graft partners (Tiedemann and Carstens-Behrens, 1994; Golecki et al., 1998) implies that the transfer of one soluble protein within the assimilate stream is indicative of the transfer of all other soluble proteins. The movement of CmPS-1 from the pumpkin to the cucumber grafting partner concomitant with the phloem mobile marker proteins CmPP1 and CmPP2, as well as the conserved ratio between the amounts of CmPS-1 and CmPP2 that are transported across the graft border (Fig. 3) agree with this concept.
As expected from their movement over long distances in the phloem, serpins were shown to occur in the conducting SEs in Cucurbita maxima and Cucumis sativus. This localization pattern is similar that of CmPP16, which predominantly localizes to sieve elements (Xoconostle-Cázares et al., 1999). By contrast, most other phloem-mobile proteins accumulate in both SEs and CCs. The site of synthesis for the phloem serpins is unknown, but the proteinases could be synthesized in either immature SEs or in the CCs at undetectable levels and rapidly trafficked into the SEs through PPUs. In general, differences in the occurrence of phloem proteins in SEs and CCs might indicate a difference in the release of proteins synthesized in the CCs towards the SEs, or a difference in the rate of protein degradation in the CCs wherever recycling involves the return of the protein in question into the CC (Fisher et al., 1992).
Serpin synthesis is not restricted to secondary phloem as the late appearance in the phloem exudate might suggest. The serpin was immunolocalized to the primary and secondary phloem (Fig. 4) and was detected in the protophloem as early as 2 d after germination; this is temporally similar to the initial detection of the pumpkin fruit trypsin inhibitor PFTI in the protophloem of pumpkin hypocotyls (Dannenhoffer et al., 2001). In phloem exudates, PFTIs could be detected at these early developmental stages and rapidly reached steady-state levels that remained stable throughout the subsequent development, as did the other phloem-mobile proteins except for the serpins (Clark et al., 1997; Dannenhoffer et al., 1997). Serpins seem to accumulate gradually over an extended period of time reaching a detectable level in the exudate considerably later than the other phloem-mobile proteins mentioned. The results of grafting studies appear to shed some light on a possible explanation for the differential accumulation of proteinases in the phloem.
The standard marker proteins for phloem mobility, PP1 and PP2, are synthesized in CCs and then transported through PPUs into the SEs (Bostwick et al., 1992; Clark et al., 1997). Grafting studies have shown that both proteins enter the assimilate stream where they are transported across the graft union and can subsequently be detected in both SEs and CCs (Golecki et al., 1999). Although it was unclear from the initial studies performed by Golecki and coworkers, the transport experiments reported here clearly document that the 88 kDa PP1 (Leineweber et al., 2000) exits the assimilate stream in SEs and enters the CCs of the transport phloem. Thus, PPUs between SEs and CCs appear to regulate the transport of high molecular weight macromolecules in either direction. The consistent accumulation of serpins in SEs and their conspicuous absence from CCs in the transport phloem of both ungrafted and grafted plants indicate, that unlike the marker proteins, serpins cannot exit the SEs by trafficking through the PPUs linking SEs with their CCs. In general, phloem protein homeostasis in the transport phloem is most easily explained by the ability of CCs both to synthesize and to degrade translocated proteins (Fisher et al., 1992; Thompson and Schulz, 1999). Exclusion from transfer into CCs can explain the time-dependent accumulation observed for serpins (data presented here and in Yoo et al., 2000). The lack of degradation eventually leads to an accumulation of this protein within the phloem of a plant. The search for a CC targeting sequence for any long-distance transported phloem protein has yet to prove successful. The plasmodesma trafficking machinery could require a specific structural motif in combination with plasmodesma-gating regulatory proteins (Lee et al., 2003). Therefore, the absence of serpin from the CCs could be due to the lack of either the motif or a specific regulatory protein, gating PPUs to traffic serpins from the SEs into the CCs.
Function of serpin in the long-distance transport system
The phloem mobility of serpin and its presence within the SEs suggests that it could function in long-distance signalling and/or be involved in the protection of the long-distance transport pathway. In animals, serpins have been shown to possess distinct functions in different tissues (Silvermann et al., 2001). The presence of serpins in both phloem tissue and seed tissues of the same plant suggests that this could also be the case in plants (Roberts et al., 2003). One serpin with RCL-properties similar to those of CsPS-1, the barley serpin BSZx, has been characterized in detail (Dahl et al., 1996a, b). BSZx is an efficient irreversible inhibitor at P1 Arg of trypsin and several proteinases of the blood coagulation system with trypsin-like specificity. However, it is also an inhibitor of chymotrypsin and cathepsin G at the overlapping site P2 (Leu), and thus inhibits proteinases with specificity for basic as well as large hydrophobic residues at P1 in the protein substrates cleaved. By contrast, CmPS-1 (Yoo et al., 2000) has specificity for elastase-like proteinases with valine at the P1 position resulting in a preference for small hydrophobic residues at P1 in their substrates (Yoo et al., 2000).
The role of the phloem serpins remains unclear as long as exogenous or endogenous target proteases are not identified. The gut of the phloem-feeding insect Nilaparvata lugens (rice brown plant hopper) has both cathepsin b-like and trypsin-like protease activity that appear to contribute to digestive proteolysis of the phloem sap (Foissac et al., 2002). This and other observations have led investigators to hypothesize that the primary function of phloem proteinase inhibitors is defensive. It was suggested that CmPS-1 functions as a defence protein directed against the proteases from insects (Yoo et al., 2000), but no direct interaction with proteases from Myzus persicae was demonstrated. Plants do have endogenous serine proteases (Vierstra, 1996; Beer et al., 2004), but none have been identified that can be inhibited by serpins. BLAST searches of the A. thaliana database using the trypsin sequence failed to identify homologous proteases (Silvermann et al., 2001), but such a search does not exclude the presence of target proteases in plants.
These results indicate that the active form of serpin is enriched in SEs during development and that it is not subjected to protein degradation in CCs. The rapid accumulation of low molecular weight proteinase inhibitors like PFT1 and possibly also of other phloem protease inhibitors (Habu et al., 1996) protects the developing phloem, whereas serpins take over and protect against proteolytic activity at later developmental stages. This will inhibit the damaging activity of exogenous pest proteases and down-regulate the activity of endogenous target proteases. Future investigations into the role of phloem serpins will involve the search for target proteases and the silencing of serpin in selected cucurbits.
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Acknowledgements |
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This work was supported by the VELUX Visiting Professor Programme, The Villum Kann Rasmussen Fund, Denmark to GAT, and by the Danish Research Agency to AS.
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Footnotes |
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Abbreviations: CC, companion cell; CmPP1, Cucurbita maxima phloem filament protein; CmPP2, Cucurbita maxima phloem lectin; DAG, days after germination; SE, sieve element; PPU, pore/plasmodesma unit; RCL, reactive centre loop.
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References |
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