JXB Advance Access originally published online on June 27, 2005
Journal of Experimental Botany 2005 56(418):2229-2238; doi:10.1093/jxb/eri222
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RESEARCH PAPER |
A coupled yeast signal sequence trap and transient plant expression strategy to identify genes encoding secreted proteins from peach pistils
1Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA
2Center for Plant Molecular Genetics and Breeding Research, Seoul National University, Seoul 151-921, Korea
3Laboratory of Pomology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
* To whom correspondence should be addressed. Fax: +1 607 255 5407. E-mail: jr286{at}cornell.edu
Received 4 November 2004; Accepted 10 May 2005
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Abstract |
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Many developmental processes and induced plant responses have been identified that are directly or indirectly influenced by wall-localized, or apoplastic, molecular interactions and signalling pathways. The yeast-based signal sequence trap (YSST) is a potentially valuable experimental tool to characterize the proteome of the wall and apoplast, or ‘secretome’, although few studies have been performed with plants and to date this strategy has not been coupled with a subsequent analysis to confirm extracellular localization of candidate proteins in planta. This current report describes the use of the YSST, together with transient expression of a selection of identified proteins as fusions with the reporter GFP, focusing on the complex extracellular interactions between peach (Prunus persica) pollen and pistil tissues. The coupled YSST and GFP localization assay was also used to confirm the extracellular localization of a recently identified pistil-specific basic RNase protein (PA1), as has been observed with S-RNases that are involved in self-incompatibility. This pilot YSST screen of pollinated and unpollinated pistil cDNAs revealed a diverse set of predicted cell wall-localized or plasma membrane-bound proteins, several of which have not previously been described. Transient GFP-fusion assays and RNA gel blot analyses were used to confirm their subcellular localization and to provide further insights into their expression or regulation, respectively. These results demonstrated that the YSST strategy represents an effective means either to confirm the extracellular localization of a specific candidate secreted protein, as demonstrated here with PA1, or to conduct a screen for new extracellular proteins.
Key words: Extracellular proteins, non-S-RNase, peach, pollen–pistil interaction, secreted proteins, yeast signal sequence trap
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The plant cell wall, which in the broadest sense comprises the extracellular matrix and apoplast milieu, represents the interface with the biotic and abiotic environment (Hoson, 1998
; Sakurai, 1998; Lee et al., 2004). Accordingly, many developmental processes and induced plant responses have been identified that are directly or indirectly influenced by wall-localized molecular interactions and signalling pathways. Accordingly, a significant portion of the plant proteome is localized in the apoplast and the wall–plasma membrane interface, although in comparison with the protein complements of other subcellular compartments, such as the chloroplast or mitochondrion, the extracellular proteome has received far less attention and is less well defined (Lee et al., 2004). There is therefore much interest in developing and refining experimental tools to characterize the secreted component of the cellular proteome, or ‘secretome’.
A number of cell wall proteome studies that use well-established strategies to characterize protein populations, typically involving protein electrophoresis and mass spectrometry have been reported (reviewed in Lee et al., 2004). However, in addition, several complementary genome-scale screens have been developed in the last few years that either directly or indirectly reveal populations of secreted proteins (Escobar et al., 2003; Groover et al., 2003). These include a relatively high throughput approach termed the yeast-based signal sequence trap (YSST; Tashiro et al., 1993; Klein et al., 1996; Jacobs et al., 1997). Briefly, this involves ligating a library of cDNAs from the target tissue to the 5' end of the DNA sequence encoding a yeast invertase gene that has been truncated to eliminate the initiator methionine and N-terminal signal peptide. The resulting library is then transformed into an invertase-deficient yeast mutant, which is grown on a medium with sucrose as the sole carbon source. Any yeast transformed with a cDNA encoding a secreted protein has the potential to secrete an invertase fusion protein, resulting in the reconstitution of extracellular invertase activity and the rescue of the mutant. Both plasma membrane-associated and freely soluble apoplastic fusion proteins have the capacity to rescue the invertase mutants. YSST screens have only been used in a few studies in plants (Goo et al., 1999; Belanger et al., 2003; Hugot et al., 2004) and to date this strategy has not been coupled with a subsequent analysis to confirm the extracellular localization of candidate proteins in planta. This current study describes the combined use of the YSST and subsequent transient expression of a selection of identified proteins as fusions with the reporter protein GFP, focusing on the complex extracellular interactions between pollen and pistil tissue as a model system.
In flowering plants, molecular communication between the tissues of the pistil and adhering pollen grains plays a central role in regulating fertilization. Once a pollen grain attaches to the stigma, factors derived from both the pistil and pollen interact and determine whether or not pollen germination and pollen tube growth can proceed, and also guide the pollen tube to the ovule, where fertilization occurs. Since germinated pollen tubes are in intimate contact with the extracellular matrix (ECM) of the transmitting tract, there is much interest in the role of extracellular proteins in this interaction. These comprise a complex mixture of both enzymatic and structural proteins (Sanchez et al., 2004), such as arabinogalactan proteins (AGPs), hydroxyproline-rich glycoproteins (HRGPs), and extensin-like proteins. In the context of pollen–pistil interactions, some of the best-characterized examples are transmitting tissue-specific (TTS) proteins from tobacco (Cheung et al., 1993; Wang et al., 1993) that promote pollen tube elongation in vitro and in vivo (Cheung et al., 1995; Wu et al., 1995). Additional proteins that may have a similar role include other HRGPs, a galactose-rich style glycoprotein (Lind et al., 1994), a 120 kDa glycoprotein (Lind et al., 1996; Sommer-Knudsen et al., 1996) and a pistil-specific extensin-like protein (PELP; de Graaf et al., 2003). Other examples of secreted proteins that influence pollen–pistil interactions include stigma/stylar cysteine-rich adhesin (SCA), a lipid transfer protein (LTP)-like polypeptide (Mollet et al., 2000; Park and Lord, 2003) and plantacyanins (Kim et al., 2003). These reports collectively reinforce the view that extracellular pistil proteins play a crucial role in pollen–pistil interactions in the extracellular matrix of the pistil transmitting tissue (Wheeler et al., 2001; Lord and Russell, 2002).
The best characterized class of interactions between pollen and pistil are the self-incompatibility (SI) systems, which comprise genetically controlled mechanisms to promote outcrossing and prevent inbreeding depression (Kao and Tsukamoto, 2004). Members of the genus Prunus in the family Rosaceae, which include many important fruit tree crops such as peach, almond, plum, cherry, and apricot, exhibit S-RNase-based gametophytic SI (GSI). In this case, the pistil S-gene encodes a secreted S-RNase (Kao and Tsukamoto, 2004) that is taken up by the pollen, where it degrades pollen RNAs in a haplotype-specific manner (McClure et al., 1990; Luu et al., 2000). Since these species are unable to bear fruit parthenocarpically, successful pollination and fertilization are essential for fruit production. To date, S-RNases have been characterized in several Prunus species, including almond (Tao et al., 1997; Ushijima et al., 1998), apricot (Burgos et al., 1998), Japanese apricot (Tao et al., 2002), Japanese plum (Yamane et al., 1999), sour cherry (Yamane et al., 2001; Hauck et al., 2002), and sweet cherry (Tao et al., 1999). In addition to S-RNases, a recent study identified a pistil-specific RNase gene from several Prunus species, termed non-S-RNase PA1, that is closely related to S-RNase but that is not a pistil-determinant of SI (Yamane et al., 2003). The PA1 RNase has a basic pI, unlike many other well-characterized acidic non-S-RNases (Igic and Kohn, 2001). Further molecular analyses suggest that PA1 might be the ancestral form of Prunus S-RNases (Yamane et al., 2003), although its molecular function is unknown and its subcellular localization has not previously been reported. However, PA1 is predicted to contain an N-terminal signal sequence, suggesting that it is a secreted extracellular factor with a potential role in plant reproduction, such as the SI response and/or self-compatibility (SC) responses.
In this study, the utility is demonstrated of coupling the YSST technique with a plant transient expression assay both to confirm the extracellular localization of PA1 and as broad-scale screen to identify new genes encoding extracellular proteins from peach (Prunus persica) pistil tissues. Peach pistils were used since this species represents one of the most important stone fruits displaying SC and is considered to be a model plant for genetic studies in the Rosaceae (Dirlewanger et al., 2004). In addition, the subcellular localization and expression patterns of a subset of the YSST clones are described.
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Materials and methods |
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Plant materials
The peach (Prunus persica cv. ‘Shimizu Hakuto’) trees used in this study were grown at the Kyoto University experimental farm at Takatsuki, Osaka, Japan. Flowers were emasculated at 1–2 d before anthesis and pistil tissues, consisting of ovaries, styles, and stigmas, were collected 3 d or 5 d after the stigmas were self-pollinated, since pollen tubes normally reach the ovule 5–7 d after pollination (dap), although this can vary with environmental conditions (Herrero and Arbeloa, 1989
; Sanzol and Herrero, 2001). Unpollinated flowers of the same age were collected as controls. The pistils were frozen in liquid nitrogen immediately after excision, lyophilized, and stored at –80 °C until use. Petals and pollen at anthesis, mature fruits, and young leaves were similarly collected and stored from peach trees (cv. ‘Gold nine’) grown at the Geneva experimental station, Cornell University, Geneva, NY.
Construction of the yeast signal sequence trap (YSST) PA1 and S-RNase control plasmids
The predicted cleavage site of the signal sequence of S1-RNase and PA1 (Genbank accession numbers, AB028153 and AB096918, respectively), was determined using SignalP software (version 3.0; www.cbs.dtu.dk/services/SignalP). The full-length coding sequences of the PA1-cDNA, or the predicted mature protein without putative signal peptide (amino acids 1–17), were amplified by PCR with PA1ws or PA1wos (Table 1) as the forward oligonucleotide primers and with PA1anti2 (Table 1) as the reverse oligonucleotide primer. As a reference, the full-length cDNAs encoding S1-RNase or the predicted mature protein without the predicted signal peptide (amino acids 1–24) were also amplified by primer pairs, S1Rws and S1Ranti2, or S1Rwos and S1Ranti2, respectively. Forward and reverse primers contained the EcoRI and NotI restriction sites, respectively. The amplified DNA products were digested with EcoRI and NotI and ligated into the EcoRI-NotI site of the pSMASH vector (Goo et al., 1999).
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Peach YSST library construction
RNA was extracted from the peach pistils collected at the time points listed above, as described in Tao et al. (1999)
and equal amounts of total RNA from pollinated and unpollinated pistils were mixed and used for cDNA library construction. Polyadenylated RNA was isolated from the total RNA using Oligotex-dT30 (TaKaRa Shuzo Co., Shiga, Japan) and used to synthesize double-stranded cDNA with oligo-(dT)12–18 primers, which was then cloned into the Lambda ZAP II vector (Stratagene, La Jolla, CA) following the manufacturer's instructions. Reactions were packaged in vitro using the MaxPlax Packaging Extract Kit (Epicentre Technologies, Madison, WI) as in Tao et al. (1999). The resulting primary cDNA library was used without further amplification to construct a YSST library, as described below.
The cDNA library was amplified by PCR using the T3-20 oligonucleotide primer (Table 1) derived from the Lambda ZAP II vector sequence located 5' to the EcoRI site of the insert and a random hexamer primer mix (Applied Biosystems, Foster City, CA). PCR was performed using a programme of 5 cycles of 94 °C for 1 min, 38 °C for 1 min, and 72 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 48 °C for 1 min, and 72 °C for 2 min with an initial denaturing step of 94 °C for 3 min and a final extension of 72 °C for 5 min. The PCR reaction mixture contained 1x buffer (Eppendorf, Hamburg, Germany), 0.5x Taq master (Eppendorf), 200 µM dNTPs, 1 µM of each the oligonucleotide primers, 2 µl of primary cDNA library constructed as described above, and 1 U of Taq polymerase (Eppendorf) in a 50 µl reaction volume. The PCR products were fractionated by 1% agarose gel electrophoresis and those within an estimated size range of 300–1000 bp excised from the gel and purified using QIAquick gel extraction kit (Qiagen, Valencia, CA). The termini of the gel-purified PCR fragments (approximately 100 ng) were blunted using End Conversion Mix (Novagen, San Diego, CA) and ligated with NotI adapters generated with two oligonucleotides: NotI-1 and NotI-2 (Table 1). The cDNA fragments ligated with the NotI adapters were subjected to another round of PCR amplification with the E-18 and P-3 (Table 1) oligonucleotide primers. PCR was performed using a programme of 35 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min, with an initial denaturing step at 94 °C for 3 min and a final extension of 72 °C for 10 min. The amplified DNA fragments of 300–1000 bp were isolated as described above, digested with EcoRI and NotI, and ligated into the EcoRI–NotI sites of the pSMASH vector (Goo et al., 1999). Electrocompetent TOP10F' Escherichia coli cells (Invitrogen, Carlsbad, CA) were transformed with approximately 1 µg of the resulting YSST library by electroporation (MicroPulser Electroporator, Bio-Rad, Hercules, CA) and spread on 10–15 large LB plates. Plasmid DNA was isolated from a pooled sample of the resulting transformants using the Perfectprep Plasmid Midi kit (Eppendorf).
Yeast transformation, selection and sequencing
Fifty micrograms of the YSST library was transformed into the yeast (Saccharomyces cerevisiae) strain DBY
2445 (MAT, suc2
-9, lys2-801, ura3-52, ade2-101) using the YEASTMAKER Yeast Transformation System2 (BD Biosciences, San Jose, CA). Transformants were spread on YP sucrose plates (1% yeast extract, 2% peptone, 2% sucrose, 2% agar, pH 6.5), incubated at 30 °C for 4–9 d, and visible colonies were re-streaked on sucrose plate followed by incubation at 30 °C for 2–3 d. Plasmids were isolated from visible colonies as described in Hoffman and Winston (1987), transformed into XL1-blue electrocompetent E. coli, and purified using a Qiaprep kit (Qiagen). Plasmid inserts were sequenced at the Bio Resource Center, Cornell University, Ithaca, NY (http://www.brc.cornell.edu).
RACE amplification
One microgram of peach pistil total RNA was used for first strand cDNA synthesis with SuperScript II RT (Invitrogen) with an adapter primer Adp-dT (Tao et al., 1999). Oligonucleotide primers (Table 1) derived from the DNA sequences of P12 (P12-F1 and P12-F2), P16 (P16-F1 and P16-F2), and P25 (P25-F1), corresponding to the predicted signal sequences of isolated YSST clones were used in 3' RACE with the M13-20 (Table 1) primer as an adapter primer. To obtain the full-length cDNA sequences, ‘touchdown’ PCR was performed using a programme with 35 cycles of 94 °C for 1 min, 63 °C for 1 min, 72 °C for 2.5 min with the annealing temperature decreasing by 1 °C every second cycle to 60 °C, followed by final extension of 72 °C for 10 min. For P12 and P16, nested PCR was performed using primary PCR products as the template. The PCR products were subcloned into the pGEM-T Easy vector (Promega, Madison, WI). DNA sequences were determined as described above.
Construction of GFP fusion vector for transient expression
The full-length cDNAs corresponding to the ORFs of selected YSST clones, as well as PA1 (Yamane et al., 2003), were amplified by PCR with oligonucleotide primers containing the BamHI recognition site, P3-, P12-, P16-, and P25-GFP-F and -R (Table 1), using peach pistil cDNA as a template. After digestion with BamHI, the DNA products were ligated into the BamHI site located between the 35S promoter and the green fluorescent protein (GFP) coding region of the pSMGFP vector (David and Viestra, 1996), generating a series of constructs encoding fusion proteins with GFP at the C-termini.
Plant transformation and confocal microscopy
Segments of fresh supermarket onion bulbs were placed with the inside facing upwards on sterilized water in a sterile Petri dish. Plasmid DNAs encoding the GFP fusion proteins (1 µg) were introduced into the onion tissue using 1.1 µm tungsten microcarriers and a helium-driven PDS-1000 particle delivery system (Bio-Rad) operating with a vacuum of 28 inch HG, a helium pressure of 1300 psi and a 6 cm target distance. After bombardment, tissues were incubated for 16 h at 22 °C. The double staining with P3-GFP and FM4-64 (SynaptoRed C2, Sigma, St Louis, MO) was performed as described in Ueda et al. (2001). Bombarded onion cells were soaked in the MS basal liquid medium containing 50 µM FM4-64 dissolved in dimethylsulphoxide. After incubation for 15 min on ice, the cells were washed with MS basal liquid medium to remove the excess dye. The onion epidermal cell layer was peeled, transferred to glass slides and observed using a confocal laser-microscope (TCS SP2 system, Leica Microsystems, Wetzlar, Germany) with an argon laser excitation wavelength of 488 nm. The fluorescent and DIC images were captured simultaneously with 2048x2048 pixel resolution.
RNA gel blot analysis
Total RNA was isolated from several peach tissues as described above and 10 µg aliquots fractionated on formaldehyde (1%) agarose gels and blotted onto nylon membranes (Hybond-N+, GE Healthcare, Piscataway, NJ). The membranes were hybridized at 42 °C overnight with the inserts generated by the YSST screen that had been radiolabelled with 32P-dCTP (PerkinElmer, Wellesley, MA) using Ready-To-Go DNA labelling beads (GE Healthcare) according to the manufacturer's instruction, followed by purification using Probe Quant G-50 Micro Columns (GE Healthcare) in a hybridization solution containing 50% formamide, 6x SSPE, 10% Denhardt's solution, 0.5% SDS, and 125 µg ml–1 denatured salmon sperm DNA. After high stringency washes (twice with 5x SSC and 0.1% SDS for 20 min at 65 °C followed by twice with 0.5x SSC and 0.5% SDS for 20 min at 65 °C), the labelled membranes were exposed to X-ray film.
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Results |
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Subcellular localization of pistil specific basic non-S-RNase (RNase PA1)
The subcellular localization of the Prunus non-S-RNase PA1, pistil-specific non-S-RNase has not previously been reported (Yamane et al., 2003
), although the presence of a putative N-terminal signal peptide, as predicted using SignalP software (www.cbs.dtu.dk/services/SignalP), suggests that it is targeted to the secretory pathway. To confirm the localization, the full-length PA1 cDNA was cloned into the pSMASH vector (Goo et al., 1999), which contains an invertase gene (suc2) lacking the initiator methionine (Met) and signal peptide (Goo et al., 1999), thereby generating a construct encoding a PA1-SUC2 fusion protein. Similarly, the PA1 cDNA sequence without the region encoding the predicted signal sequence was cloned into the pSMASH vector creating a construct encoding PA1SP-SUC2 fusion protein. As a control, constructs were generated encoding fusion proteins corresponding to the full-length S1-RNase (Tao et al., 1999) and the truncated S-RNase without the signal sequence (termed S1-RNase-SUC2 and S1-RNaseSP-SUC2, respectively). The plasmids were transformed into the invertase-deficient yeast strain DBY2445, which is able to grow on YP sucrose medium only when transformed with a vector containing a cDNA encoding an initiator Met and a signal peptide cloned in-frame with the invertase coding sequence. Yeast transformed with plasmids encoding either PA1-SUC2 or S1-RNase-SUC2 grew on YP sucrose (Fig. 1A, C, respectively), while no growth was seen in the yeast transformed with the signal sequence-deficient PA1SP-SUC2 or S1-RNaseSP-SUC2 fusions (Fig. 1B, D, respectively). This suggests that S1-RNase and PA1 are localized in the cell wall in planta.
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To confirm the subcellular localization of PA1 further, a transient GFP fusion protein expression assay was performed in onion epidermal cells. When expressed by itself, GFP was detected in the cytosol and nucleus (Fig. 2A), while the PA1-GFP fusion protein was mainly detected in the cell wall and showed no nuclear localization (Fig. 2B), confirming the results of the YSST analysis above. GFP fluorescence was also apparent in an intracellular organelle that, given its distribution around the nucleus and the nature of the secretory pathway, was likely to be the endoplasmic reticulum (ER).
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Identification and characterization of secreted pistil proteins using a yeast signal sequence trap (YSST) screen
As a pilot study to evaluate the YSST screen strategy as a means to identify new extracellular or plasma membrane-bound pistil proteins, a peach pistil cDNA library was constructed in the pSMASH vector using mRNA from pistil tissues at different developmental stages. After making an E. coli plasmid library, extracted plasmids were transformed into DBY
2445. As shown in Table 2, nine distinct genes were identified. Several could not be assigned a function based on sequence homology (P3, P12, P16, P25) while others had homology to pathogenesis-related (PR) proteins (P33 and P306), a non-specific lipid transfer protein (P103), an arabinogalactan protein (AGP; P444) and a hydroxyproline-rich glycoprotein (HRGP; P501). EST database searches (www.ncbi.nlm.nih.gov) revealed that P3, P12, P103, and P306 are expressed in peach fruit mesocarp (corresponding to ESTs BU048869
[GenBank]
, BU047325
[GenBank]
, BU047210
[GenBank]
, and BU045012
[GenBank]
, respectively), whereas P25 more closely matched an almond pistil EST1206 (Jiang and Ma, 2003).
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Characterization of isolated YSST clones
The full-length cDNAs encoding the proteins with uncharacterized function (P3, P12, P16, and P25) were amplified by RACE and transient GFP fusion protein assays were used to verify their cell wall localization. As shown in Fig. 3, fluorescence was evident in the walls of onion cells that had been transformed with constructs encoding P3, P12, P16, or P25-GFP fusion proteins (Fig. 3C, B, F, G, respectively), while the GFP alone (Fig. 3A) showed a characteristic expression pattern in the cytosol and nucleus. Since the cytosol is often present as a thin layer around the cell periphery, multiple cells transformed with each construct were evaluated by careful focusing of the microscope through many focal planes of the cells. This allowed the distinction between the presence of GFP inside the nucleus and cytosol and GFP around the nuclear periphery, which would indicate ER localization and presence in the secretory pathway. For example, the distribution of P16-GFP (Fig. 3F) was similar to that of RNase PA1-GFP (Fig. 2B), which was reminiscent of ER localization. To examine whether the GFP fluorescence was indeed in the plasma membrane/cell wall, a double-labelling experiment was performed (Fig. 3C–E) with P3-GFP and FM4-64, a membrane-selective dye that has been widely used as an endocytic tracer (Bolte et al., 2004
). The GFP signals of P3-GFP overlapped the red fluorescence of FM4-64, indicating that P3 localized to the plasma membrane. These results suggest that these proteins are secreted in planta and that the YSST screen represents an effective means to identify new extracellular proteins.
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To gain further insight into the possible function of the isolated YSST clones, their expression patterns in different organs were examined by RNA gel blot analysis. As shown in Fig. 4, P3 and P12 were expressed in most tissues tested. P33, which encodes a PR-1 protein, showed flower-specific expression and was highly abundant in pollen (Fig. 4). By contrast, P16 was expressed in young leaves and pistils, while P25 showed pistil-specific expression (Fig. 4).
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To determine whether the expression of these genes was associated with pollination, mRNA abundance was examined in both pollinated and non-pollinated pistils at 3 d or 5 d after anthesis. Transcript levels of the pistil-specific gene P25 were greater in pollinated pistils at both 3 d and 5 d after anthesis (Fig. 4) while P3 mRNA levels were higher later in pistil development. This latter result showed some parallels with the expression of an orthologous gene from lily (DSA5), whose expression is induced by petal senescence. In pollinated pistils, P33 mRNA levels were slightly greater than in unpollinated pistils at 3 dap, but not after 5 dap. As shown in Fig. 4, P33 expression was particularly high in pollen, which is probably the basis of the difference in P33 mRNA levels between pollinated and unpollinated pistils at 3 dap. P12 and P16 mRNA levels were apparently unaffected by pollination (data not shown).
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Discussion |
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RNase PA1 is a secreted cell wall-localized protein in planta
PA1 is a basic non-S-RNase belonging to the RNase T2 family of ribonucleases (Yamane et al., 2003
). Although many acidic non-S-RNases are known to be secreted, such as the tomato RNase LE (Löffler et al., 1993), the subcellular localization of the basic non-S-RNases has not previously been described. The YSST vector proved to be an efficient and rapid means to confirm that PA1 is secreted, as is S-RNase. This was subsequently confirmed by detecting the transient expression of a PA1-GFP fusion protein in the cell wall of onion epidermal cells. The best studied secreted RNases are S-RNases that represent pistil determinants of GSI in Solanaceae, Rosaceae, and Scrophulariaceae. Secreted S-RNases from the pistil enter the cytosol of the growing pollen tube (McClure et al., 1990; Luu et al., 2000) and catalyse pollen RNA degradation, resulting in an inhibition of pollen tube growth, which, in turn, is manifested as an SI reaction. The function and mode of action of the non-S-RNase PA1 is still unknown, although there are clearly some similarities with the S-RNases, in that they are pistil-specific basic RNases (Yamane et al., 2003). It is known that some acidic non-S-RNases are induced by wounding and pathogen infection (Ye and Droste, 1996; Galiana et al., 1997; Kariu et al., 1998; Hugot et al., 2002; LeBrasseur et al., 2002); however, PA1 mRNA levels are neither induced by wounding nor by salicylic acid application (data not shown). Since PA1 is a pistil-specific protein, the suggestion is that its function is more likely to be related to reproduction, as with S-RNases (Murfett et al., 1996; Hancock et al., 2003), rather than part of a wounding or disease response.
Characterization of isolated pistil YSST clones
To demonstrate the utility of a coupled YSST screen and transient GFP-fusion expression analysis, the focus was on identifying new genes encoding proteins that are localized at the peach pistil cell surface or in the extracellular matrix, and that thus have potential roles in cell-to-cell communication during pollen–pistil interactions. However, it was anticipated that genes would also be identified that have other cell wall-associated functions, such as defence. Indeed, the isolated clones included two genes encoding PR proteins: PR-4 (P306) and PR-1 (P33). Northern blot analysis revealed that P33 is expressed at high levels in floral tissues and particularly so in pollen, in contrast with two PR-1 proteins that have been reported as being specifically expressed in pistils (van Eldik et al., 1996; Tomimoto et al., 1999). Although PR-1 proteins have been found in many plants (Selitrennikoff, 2001) and one has been shown to have antifungal activity (Niderman et al., 1995), their biochemical mechanism of action is not known (Selitrennikoff, 2001).
P103 is predicted to encode a protein with sequence homology to non-specific lipid transfer proteins (LTPs), which are thought to belong to a group of plant antimicrobial peptides, including thionins and defensins, that act as a primitive defence system with analogues that are common to all multicellular organisms (Terras et al., 1992; Cammue et al., 1995; Garcia-Olmedo et al., 1995; Broekaert et al., 1997; Selitrennikoff, 2001). Interestingly, it has been reported that SCA, a molecule that is necessary for pollen tube adhesion in the stylar matrix of lily, has substantial amino acid sequence identity with LTPs (Park et al., 2000, 2003; Lord et al., 2003). The deduced amino acid sequence of P103 contains cysteine residues with a distribution that is conserved with SCA (data not shown), suggesting that the proteins may share a similar secondary structure.
In accordance with previous suggestions that ECM glycoproteins contribute to adhesion between the growing pollen tube wall and the wall of the pistil transmitting tract (Wheeler et al., 2001; Lord and Russell, 2002), two clones, encoding an AGP (P444) and a HRGP (P501), were isolated through the YSST screen. To date, AGPs from one class, the transmitting-tissue-specific (TTS) proteins, have been shown to stimulate pollen tube growth (Cheung et al., 1993, 1995; Wang et al., 1993; Wu et al., 1995). Other than TTS proteins, class III pistil-specific extensin-like proteins (PELPIII), which are chimeric hydroxyproline-rich glycoproteins with characteristics of both extensins and AGPs, are known to interact closely with the pollen tube in the stylar canal of Nicotiana tabacum (de Graaf et al., 2003). Similar results have been described in N. alata, where a 120 kDa glycoprotein, also with extensin- and AGP-like properties, has been found to enter the pollen tube during its growth through the style (Lind et al., 1996; Schultz et al., 1997).
As expected, a number of predicted proteins (P3, P12, P16, and P25; Table 1) were identified whose function cannot be predicted based on sequence homology. These were expressed as fusion proteins with GFP in a transient assay in onion epidermal cells and the subcellular expression patterns indicated that all are targeted to the secretory pathway (Fig. 3). Three of the clones contained annotated and/or characterized structural domains. P12 has a predicted cyclase or metal-dependent hydrolase domain and is expressed in leaves, petals, pistils, and fruits (Fig. 4). P3 has sequence homology to tetraspanin proteins, which have four transmembrane domains and play roles in cell–cell communication during diverse cellular functions, including motility, metastasis, proliferation, differentiation, and cell fusion (Charrin et al., 2003; Hemler, 2003; Olmos et al., 2003; Yunta and Lazo, 2003). Interestingly, a knockout mutant of the P3 orthologue in Arabidopsis thaliana showed aberrant floral morphology and few or no seeds (Olmos et al., 2003), which may suggest a role in pollination. In addition, another gene with sequence homology to P3, DSA5, has been found to be associated with daylily petal senescence (Panavas et al., 1999), which is notable since the increase in P3 mRNA accumulation at 5 d after anthesis corresponds to a point at which pistil senescence is imminent. P16 has a predicted N-terminal DoH domain, which is considered to be a catecholamine-binding domain and may mediate a range of extracellular adhesive interactions or signalling functions (Aravind, 2001; Ponting, 2001). Catecholamines include animal neurotransmitters, such as dopamine and norepinephrine, and while the functions of these compounds in plants are not known, it is interesting that another animal neurotransmitter, 
-amino butyric acid (GABA), has been associated with pollen tube guidance in Arabidopsis (Palanivelu et al., 2003). Although P25 encodes a protein with no apparent sequence homology to functionally annotated proteins, it shares a number of predicted features with a protein (ptl1) that is specifically in the transmitting tissue of Antirrhinum pistils (Baldwin et al., 1992), including a high frequency of proline and serine, a pollination induced expression pattern (Fig. 4) and higher mRNA levels in mature than immature pistils (Baldwin et al., 1992). The increase in P25 expression in pistils after pollination suggests the existence of a signalling pathway between pollen and pistil, although the nature of the signals and the pathway through which they operate are unknown.
It was noted that genes encoding certain highly abundant secreted proteins, such as the S-RNases, were conspicuous by their absence from the screen. The reason for this is not clear, although a similar lack of genes encoding abundant cell wall proteins has been noted in YSST screens of other plant tissues (S-J Lee, JKC Rose, unpublished data). However, in conclusion, the YSST strategy represents an effective means either to confirm the extracellular localization of a specific candidate secreted protein, as demonstrated here with PA1, or to conduct a screen for new extracellular proteins.
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Acknowledgements |
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We thank Christopher Carpita for assistance with the YSST screening and Dr Robert Anderson and Dr Jay Freer (Geneva Experimental Station, Cornell University, Geneva, NY) for kindly providing plant materials. We also thank Dr OK Park (Kumho Life and Environmental Science Laboratory, Kwangju, Korea) for generously providing the pSMASH vector. This work was supported by a JSPS Postdoctoral Fellowship for Research Abroad to HY from the Japan Society for the Promotion of Science and by a grant from KOSEF to SJL and BDK of the Center for Plant Molecular Genetics and Breeding Research. Additional funding was provided to JKCR by the NSF award DBI-0431335.
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