Molecular Plant Advance Access originally published online on June 23, 2008
Molecular Plant 2008 1(4):634-644; doi:10.1093/mp/ssn018
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New microsome-associated HT-family proteins from Nicotiana respond to pollination and define an HT/NOD-24 protein family
Department of Biochemistry, Interdisciplinary Plant Group, and the Christopher S. Bond Life Sciences Center, University of Missouri, MI, USA
* To whom correspondence should be addressed: E-mail mcclureb{at}missouri.edu
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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HT-family proteins have been identified in Nicotiana, Solanum, and Petunia. HT-B-type proteins are implicated in S-RNase-based self-incompatibility, but the functions of other family members are unknown. Screening for cDNA sequences with an expression pattern similar to HT-B in Nicotiana alata revealed a new group of small HT-family proteins, designated HT-M. HT-M proteins resemble HT-B in several respects: their pistil-specific expression pattern is indistinguishable from HT-B, they pellet with a microsome fraction, and their abundance decreases after pollination. Unlike HT-B, there is no S-specificity to this response, and RNAi experiments show that HT-M proteins are not necessary for self-incompatibility. Identification of a third group of pistil-specific HT-family proteins helps better define the characteristics of the family and allowed identification of putative new family members. By searching the databases with only the most conserved HT-family sequence elements, the signal sequence and cysteine motifs, we identified nodulin-24-like proteins and several small glycine-rich proteins as putative HT-family members. Like HT-M and HT-B, nodulin-24 is membrane associated. We propose that the conserved features in HT-family proteins are important for targeting or modification and refer to the broader family that includes both HT- and nodulin-24-like proteins as the HT/NOD-24-family.
Received for publication February 24, 2008. Accepted for publication March 25, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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HT-family proteins were first identified in Nicotiana alata as part of studies of S-RNase-based self-incompatibility (SI). In SI, self-pollen and pollen from close relatives are rejected (de Nettancourt, 2001). The specificity of pollen rejection is controlled by the S-locus: pollen is rejected if its S-haplotype is the same as either of the two S-haplotypes expressed in the diploid pistil. S-specificity genes are expressed in both the pollen and the pistil. S-RNase is produced in transmitting tract cells and secreted into the stylar extracellular matrix (Anderson et al., 1986; McClure et al., 1989). S-locus F-box (SLF/SFB) proteins are expressed in pollen (Lai et al., 2002; Entani et al., 2003; Ushijima et al., 2003; Sijacic et al., 2004). Interaction between these proteins determines compatibility; for example, S1-RNase interacting with SLF/SFB-1 results in incompatibility, while any non-self combination results in compatibility (McClure, 2004). Although S-RNase and SLF/SFB determine pollination specificity, other factors are required for SI. This has been known almost since the earliest studies of SI (Anderson and de Winton, 1931), but the requirement for other proteins (i.e., products of genes that are not linked to the S-locus) became especially apparent from studies of S-RNase expression in transgenic plants. For example, SA2-RNase from N. alata does not cause S-specific pollen rejection when expressed in Nicotiana plumbaginifolia, but this function is restored if the transgene is expressed in (N. plumbaginifoliaxN. alata) hybrids (Murfett et al., 1996; Murfett and McClure, 1998). HT-B was the first non-S-specific protein implicated in SI. It was identified by screening for sequences present in N. alata pistils but not present in N. plumbaginifolia pistils (McClure et al., 1999).
HT-B from N. alata is a small asparagine-rich protein. It is expressed only in the pistil, and the timing of expression coincides with the onset of S-specific pollen rejection. It also is a secreted protein; the experimentally determined N-terminus, RDMVDPSISL, corresponds to the cleavage site predicted by the SignalP algorithm (McClure et al., 1999). The mature protein consists of 78 amino acids with a predicted molecular weight of 8.6 kDa, although it shows an apparent molecular weight of 13–18 kDa in SDS PAGE, depending on gel conditions. N. alata HT-B contains a string of 20 asparagines and aspartate residues (the ND domain) near the C-terminus. The ND domain is flanked by two cysteine motifs, CAACKC and CQTVCC in N. alata HT-B (McClure et al., 1999).
An antisense experiment implicated HT-B in SI in Nicotiana. The antisense construct was transformed into N. plumbaginifolia, and pollination phenotypes were determined after crossing with N. alata to form (N. plumbaginifoliaxN. alata) hybrids. Hybrids with HT-B protein levels suppressed below the limit of detection failed to show S-specific pollen rejection, while untransformed hybrids showed normal S-specific responses (McClure et al., 1999). HT-B (as well as a very similar protein called HT-A) has also been described in Solanum species (Kondo et al., 2002a, b; O'Brien et al., 2002). An RNAi experiment confirmed a requirement for HT-B in S-specific pollen rejection in S. chacoense (O'Brien et al., 2002).
HT-B protein is produced in pistil cells and degraded in pollen tubes. We performed immunolocalization experiments to follow the fate of HT-B, S-RNase, and the 120 kDa glycoprotein (i.e., another non-S-specific SI factor, 120K), during compatible and incompatible pollinations. The results showed that S-RNase and 120K are taken up by pollen tubes and associate with vacuoles (Goldraij et al., 2006). In compatible pollinations, S-RNase remains stably sequestered in the vacuole where it cannot inhibit pollen tube growth. However, the distorted slow growth characteristic of incompatible pollinations is associated with breakdown of the pollen vacuole and release of S-RNase. Immunolocalization experiments using an anti-HT-B antibody showed HT-B protein in incompatible pollen tubes and little or none in compatible pollen tubes. Protein blot analysis showed that stylar HT-B levels decrease after both compatible and incompatible pollination, but the effect is more pronounced after compatible pollination (i.e., 75–97% decrease in compatible pollinations compared to unpollinated controls). Although the exact function of HT-B is still unknown, the antisense and RNAi experiments clearly show that HT-B must be present for a normal S-specific pollen rejection response. Thus, selective degradation of HT-B in compatible pollen tubes could protect them from rejection. Moreover, a portion of the HT-B protein in the pistil pellets with a microsome fraction. We speculate that it is associated with lipids taken up by pollen tubes and it may play a role in trafficking of pistil proteins or in destabilizing the pollen tube vacuole in which S-RNase is sequestered.
New HT-family proteins were recently identified in Petunia inflata. These proteins, designated HT-like A and B (PiHTL-A and PiHTL-B), are also specifically expressed in the mature pistil (Sassa and Hirano, 2006). The PiHTL-A and -B transcripts are derived from a common gene by alternative splicing. Their signal sequences, which are derived from a common exon, are very similar to HT-A and HT-B proteins. The PiHTL proteins also possess a cysteine motif (CXXCXCCXXXCXXXC) that is similar to both cysteine motifs in HT-A and HT-B. Neither PiHTL protein, however, possesses an ND domain. Although the expression pattern of the PiHTL proteins is consistent with a role in pollination, RNA silencing experiments showed no effect on SI.
In this paper, we identify another new group of pistil-specific HT-family proteins. These proteins, designated HT-Ms (the M stands for mijikai, which is Japanese for ‘short’), are similar to HT-B in many respects. Their structure is similar, and they display the same expression pattern as HT-B. Also like HT-B, HT-M levels decrease after pollination. However, unlike HT-B, this change in HT-M levels is not S-specific; it occurs after both compatible and incompatible pollination. The availability of a third group of HT-family proteins allows the characteristics of the family to be better defined. A database search using conserved HT-family signal sequences identified several glycine-rich proteins (GRPs), including nodulin-24, that could be regarded as HT-family proteins. We refer to the broader protein family including both pistil-specific HT-family proteins and nodulin-24-like GRPs as the HT/NOD-24 protein family.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Identification and Sequence Analysis of HT-family Genes from Nicotiana Pistils
New HT-family genes were obtained by a differential screen for sequences with an expression pattern similar to HT-B and S-RNase. The screen identified abundant cDNAs from mature SI N. alata pistils that were not expressed in either mature SC N. plumbaginifolia or in immature SI N. alata pistils. The most abundant class of sequences recovered (apart from HT-B and S-RNase) was a group of cDNAs with a clear similarity to HT-B, with the exception that they were smaller in size. These genes were designated HT-M (M = mijikai, or ‘short’ in Japanese). Figure 1 is an alignment of the deduced amino acid sequences of the three N. alata HT-M proteins (HT-M1, M3, and M4) with HT-B and PiHTL-A and -B from P. inflata. The three HT-M proteins are 38–41% identical to HT-B and 34–47% identical to the petunia HT-like sequences (Figure 1). All HT-family proteins possess very similar signal sequences and cysteine motifs similar to CXXCXC near the C-terminus. The signal sequence and cysteine motifs are separated by a more variable core sequence. HT-B proteins, as well as the very similar HT-A sequences found in Solanum, include a signal sequence, a core sequence of
50 amino acids, and an asparagine-aspartate rich ND-domain flanked by two cysteine motifs: CXXCXC and CXXXCC (Hancock et al., 2003). Nicotiana HT-M and petunia HTL proteins lack ND-domains and contain a single cysteine motif followed by a 2–10 residue tail (Figure 1). The core sequences of HT-M and PiHTL proteins are less similar than their signal sequences, and the petunia proteins possess a longer cysteine motif (CXXCXCCXXXCXXXC) that is similar to both motifs in HT-B.
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Diverse Nicotiana HT-M genes are expressed in different species. RT-PCR was performed using cDNA from mature pistils of SI accessions in N. bonariensis, N. forgetiana, and N. langsdorfii as well as SC accessions in N. plumbaginifolia, N. longiflora, and N. tabacum. Representative sequences from each species are aligned in Figure 2A. The amino acid alignment shows that the HT-M signal sequences, core sequences, and cysteine motifs are very highly conserved and that there is a striking variable region of 12–15 residues near the C-terminus that is highly divergent. Although the surrounding regions contain many invariant residues, there are no invariant residues at all in the variable region. These variable regions define different classes of HT-M proteins. The classes are not species specific, but different species often express a different set of HT-Ms. Figure 2B shows a phylogenetic tree including representative HT-family proteins along with HTL, HT-A, and HT-B proteins from P. inflata, S. peruvianum, and S. chacoense, respectively. The HT-Ms clearly form a separate group; the five classes of HT-M proteins are largely distinguished by the variable region.
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We investigated HT-M1
4 class genes, those expressed in species in section Alatae, in more detail to test for similarities to HT-B. Figure 2C shows RT-PCR results using pistil cDNA and primers specific for HT-B and HT-M14. HT-B is strongly expressed in SI accessions of N. alata, N. forgetiana, N. bonariensis, and N. langsdorfii but not expressed in SC N. plumbaginifolia, as reported previously (McClure et al., 1999). HT-M1 and HT-M4 class genes show the same pattern. HT-M3 is similar, but it is not expressed in N. forgetiana. HT-M2 class genes were abundant in N. bonariensis and N. plumbaginifolia, and expressed at such low levels in N. forgetiana and N. langsdorfii that no bands are visible in Figure 2C. Thus, expression of HT-M genes across species is variable, but the HT-M1, HT-M3, and HT-M4 class genes resemble HT-B most closely.
HT-M Genes are Expressed in Mature Pistils
RT-PCR was used to determine the temporal and organ-specific gene expression patterns for these three HT-M genes (i.e., HT-M1, HT-M3, HT-M4) in N. alata. Figure 3 shows that, like HT-B, the HT-M genes are expressed in stigmas and styles but not in pollen or in the non-sexual organs tested (petal, sepal, stem, leaf). To determine the temporal pattern of transcript accumulation, anthers and pistils (i.e., stigma plus style) were isolated from buds at different developmental stages, and total RNA was isolated for RT-PCR. As expected, neither HT-B nor any of the HT-M genes is expressed in developing anthers. Previously, we showed that HT-B expression is timed precisely with the onset of S-specific pollen rejection in SI N. alata pistils (McClure et al., 1999). HT-M genes are similar; the RT-PCR results in Figure 3 (right) show little or no detectable HT-M transcript in the early stages of pistil development and rising levels as the pistil matures.
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HT-Ms are not Required for S-specific Pollen Rejection
Expression studies show that HT-M genes display the same accumulation pattern as HT-B: both are expressed when the pistil can support pollen tube growth, and accumulation coincides with the onset of SI. Because of this similarity, we tested whether HT-M is required for S-specific pollen rejection. Figure 4A shows the RNAi strategy for suppressing all HT-M genes in N. alata. A highly conserved 205 bp segment corresponding to the 5'UTR, the signal sequence, and part of the core sequence was cloned as an inverted repeat that flanks the intron in pHANNIBAL (Wesley et al., 2001).
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Eleven independent N. plumbaginifolia transformants were crossed with SI N. alata S105S105 to obtain (N. plumbaginifoliaxN. alata S105S105) hybrid progeny for analysis (Murfett et al., 1996; Murfett and McClure, 1998; Beecher and McClure, 1999). Results are presented for progeny of six independent transformants displaying a range of expression levels (Figure 5). Transcript levels were investigated by RT-PCR using total pistil RNA and gene-specific primers. Figure 5A shows that an untransformed control hybrid expresses normal levels of all three HT-M genes as well as HT-B. The RNAi construct suppressed HT-M transcript levels in transformed hybrids with no effect on HT-B. Among the six RNAi hybrids chosen for further analysis, A4-3 shows nearly normal transcript levels, A5-1 shows a trace of expression, and hybrids A3-2, A10-1, A14-1, and A16-1 show no detectable HT-M transcript. An affinity purified anti-peptide antibody was raised against a part of the core sequence in HT-M proteins. The antibody specifically detects polypeptides with apparent molecular weights of
9.7 kDa that are present in N. alata and not in N. plumbaginifolia. This antibody was used to test for HT-M protein expression in both control and transgenic hybrids. Figure 5B shows that HT-M polypeptides are present in the control and the non-suppressed hybrid A4-3 and are not detectable in the suppressed hybrids (A3-2, A5-1, A10-1, A14-1, A16-1).
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Pollination phenotypes were assessed by staining style squashes with decolorized aniline blue (Kho and Baer, 1968). The (N. plumbaginifoliaxN. alata) hybrids do not set seed, so S-specific pollen rejection is best determined by directly observing pollen tube growth (Murfett and McClure, 1998; Beecher and McClure, 1999). The pollination results for the HT-M RNAi hybrids are summarized in Figure 5B, and sample style squashes are shown in Figure 5C. The untransformed control hybrid shows a normal S-specific pollen rejection response: few N. alata S105-pollen tubes have penetrated to the base of the style, while numerous SC10-pollen pollen tubes are evident. Suppressed hybrids showing no detectable HT-M protein also show normal, or near normal, S-specific pollen rejection (Figure 5C). Thus, HT-M proteins are not essential for SI.
HT-Ms are Present in a Microsomal Fraction and are Degraded after Pollination
We hypothesized that the conserved secretion signals and cysteine motifs in HT-M and HT-B lead to similar processing and targeting. We previously showed that HT-B pellets with microsomes and is preferentially degraded after compatible pollinations (Goldraij et al., 2006). Differential centrifugation experiments were performed to determine whether HT-M proteins behave similarly. Extracts were prepared from N. alata SA2SA2 pistils that were emasculated or pollinated with incompatible (SA2-) or compatible (S105-) pollen. Homogenized samples were centrifuged at 10,000 xg, producing a supernatant (S10). The S10 was subjected to ultracentrifugation at 156,000 xg, producing supernatant (S156) and microsome pellet (P156) fractions.
Figure 6 shows that HT-M proteins are present exclusively in the microsome fraction and appear to be degraded after both compatible and incompatible pollination. Consistent with previous results, half the HT-B pellets with the microsome fraction. Although the levels of HT-B in both the S156 and P156 fractions drop after compatible pollination, this effect is much more pronounced in the microsomal P156 fraction. HT-M proteins behave differently than HT-B proteins and are present only in the P156 microsomal pellet fraction. HT-M levels consistently drop after pollination, but the final levels in compatible and incompatible pollinations are variable. The HT-M bands from compatible pollinations are somewhat darker than the bands from incompatible pollination, but the results of five experiments showed no consistent S-specific effect, as is seen with HT-B.
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Newly Identified Putative HT-family Proteins
Database searches with the conserved HT-family signal sequence and cysteine motifs revealed homologous proteins in several species. These sequence elements were used in BLASTP and PATMATCH searches against the GenBank protein database. A BLASTP search using the HT-B signal sequence returned several members of a GRP family (pfam07172) and a nodulin that has similar signal sequences and that display cysteine motifs similar to HT-family proteins. Figure 7 shows the conserved features of six representative proteins. The secretion signals from major groups of pistil-specific HT-family proteins (PiHTL-A and HT-M1 and HT-B) are aligned with two GRPs (Arabidopsis At2g05540 and tomato tyrosine- lysine-rich protein, TLRP (Domingo et al., 1994)) and soybean nodulin-24 (Cheon et al., 1994). The regions surrounding the experimentally determined N-terminal arginines of HT-B and nodulin-24 (Cheon et al., 1994; McClure, 1999) are the most conserved and are always similar to SEVAAR (Figure 7, arrow). When the pattern <MX{17,21}SEVJAR was used to query the Green Plant GB all database (The Arabidopsis Information Resource (TAIR), http://www.arabidopsis.org/cgi-bin/patmatch/nph-patmatch.pl, on www.arabidopsis.org, 20 February 2008), 27 hits were returned. Thirteen of these hits are unique sequences not previously identified as pistil-specific HT-family protein homologs, and 12 of the 13 possess cysteine motifs near their C-terminus that are similar to the HT-B motifs (Figure 7, Supplementary Figure 1). Other patterns of cysteine residues are also present near the C-termini of these proteins. Although the GRPs are not well characterized, nodulin-24 is known to be associated with the symbiosome membrane (Cheon et al., 1994). Like HT-B and HT-M, nodulin-24 does not possess transmembrane domains, and its membrane association is thought to be driven by post-translational modification. We suggest that the conserved sequences identified here may be important for modification.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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HT-M proteins resemble previously identified HT-family proteins but also possess unique characteristics. Sequence analyses show that HT-M proteins have very similar signal sequences and cysteine motifs to HT-B and PiHTL proteins. HT-M core sequences are not similar to HT-B, nor do HT-M proteins possess the ND domains characteristic of HT-A and HT-B proteins (Figure 1). The HT-M core sequences show limited similarity to PiHTL proteins (i.e., 13/51 identical positions when NaHT-M1, NaHT-M3, NaHT-M4, PiHTL-A, and PiHTL-B are compared), but the latter proteins possess a more extended cysteine motif than HT-M proteins. The high similarity among HT-M proteins (25/50 identical positions in the core sequences including the variable region or 24/31 if the variable region is excluded) suggests that they comprise a distinct group (Figures 1 and 2A). Moreover, the possibility that HT-M and HTL proteins form a single functional group cannot be excluded since significant sequence drift will have occurred since Petunia and Nicotiana species shared a common ancestor. Nevertheless, based on sequence analysis conducted to date, we conclude that there are three distinct groups of pistil-specific HT-family genes: HT-M, HTL, and HT-A/B (Figure 2B). It is likely that more pistil-specific HT-family genes remain to be discovered in other species, but we believe that we have found all the genes in N. alata.
The variable region near the HT-M protein's C-terminus is striking. We identified five classes of HT-M genes (i.e., 19 total genes) in seven Nicotiana species. Overall, HT-M protein sequences are highly conserved; this sequence conservation extends across the signal sequences, core sequences, and cysteine motifs (Figure 2A). However, the variable regions are drastically different between different classes of HT-M proteins (Figure 2A and B). The variable regions are also sharply defined, being flanked by a PIKPQ motif present in 18 of 19 sequences and the perfectly conserved cysteine motif. Most HT-M classes were found in more than one species. The three classes found in SI N. alata (HT-M1, -M3, and -M4) were also expressed in SI N. langsdorfii accession 28B. However, all other species examined possess unique sets of HT-M genes. The function of this region is not known, but such a sharply defined variable domain could function in recognition. The variability between species is intriguing, but there is no obvious correlation between HT-M expression and crossability of the species examined.
HT-M cDNAs were identified in a screen for sequences with expression patterns consistent with a role in pollination. Our results confirm that HT-M gene expression is pistil-specific and that the expression pattern is indistinguishable from HT-B (Figures 3 and 5A). We, therefore, used RNAi to test whether HT-M genes are required for SI. The 205 bp sequence, chosen for its similarity among HT-M1, -M3, and -M4, was effective and specific. Transgenic hybrids were obtained showing little or no HT-M expression at either the transcript or protein levels, but HT-B expression was not affected. All plants tested showed normal pollen tube growth and normal S-specific pollen rejection. In contrast, suppression of HT-B changes pollination phenotype in both Nicotiana and Solanum. Previously, we used antisense HT-B, S-RNase, and 120K RNAi constructs to suppress expression in the same types of (N. plumbaginifoliaxN. alata) hybrids shown in Figure 5 (Murfett et al., 1995; Murfett et al., 1996; Beecher et al., 1998; Murfett and McClure, 1998; McClure, 1999; Beecher and McClure, 2001; Hancock et al., 2005). Hybrids with suppressed expression of these SI factors showed clear changes in pollination phenotype and lost S-specific pollen rejection. O'Brien et al. (2002) reported similar results in S. chacoense. Thus, if HT-M proteins were essential for SI, we would expect to see a change in pollination phenotype; no such change was observed. Moreover, HT-Ms do not appear to have an essential role in supporting pollen tube growth, since suppression did not interfere with compatible pollen tube growth. Sassa and Hirano (2006) reported similar results with PiHTL-A and -B; like HT-B and HT-M, these petunia proteins also show pistil-specific accumulation. However, RNAi suppression of PiHTL did not affect SI in P. inflata even when the PiHTL-A and -B transcripts were reduced to undetectable levels. Thus, some pistil-specific HT-family proteins (i.e., PiHTL and HT-M) clearly have functions outside SI.
HT-M proteins are microsome-associated and decrease in abundance after pollination. We previously showed that HT-B protein partitions between the 156,000 xg supernatant and pellet fractions (S156 and P156, respectively). Moreover, microsomal P156 fraction may be the most susceptible to S-specific degradation (Goldraij et al. 2006). HT-M proteins differ in that they do not partition between the S156 and P156 fractions; essentially all the HT-M protein pellets with the microsome fraction (Figure 6). Furthermore, unlike HT-B, degradation of HT-M proteins is not S-specific. The potential for S-specific degradation and microsome association of the petunia HTL proteins has not been investigated (Sassa and Hirano, 2006).
With three groups of pistil-specific HT-proteins identified (Figure 2B), it is possible to better define the family as a whole and identify homologous proteins. Pistil-specific HT-family proteins possess a conserved signal sequence that has a sequence similar to SEVAAR forming the cleavage site, a variable core sequence, and conserved CXXCXC and/or CXXXCC motifs near the C-terminus. When these conserved sequence elements were used to query protein databases, several new putative HT-family members were identified. A BLASTP search identified many proteins in the GRP family (pfam07172:GRP) with signal sequences that include a sequence similar to SEVAAR, but not all of these proteins possess a C-terminal cysteine motif that would identify them as HT-family proteins. However, twelve of thirteen sequences identified in a PATMATCH search (i.e., using the signal sequence alone as query) also proved to possess C-terminal cysteine motifs. These include nodulin-24-like GRPs. The GRPs are a diverse class of plant proteins that have been recovered in screens for tissue specific markers and genes induced by hormones, stress, and environmental perturbation (Sachetto-Martins et al., 2000). The glycine residues in GRPs usually occur in repeats such as GGGX, GGXXXGG, or GXGX that may account for 70% of the protein. Most GRP proteins include a signal sequence, although this is not universal. The presence of a signal sequence and the repetitive nature of GRPs suggest a structural function. Indeed, GRP 1.8 from French bean has been directly shown to be present in the extracellular matrix, or cell wall, by EM-level immunolocalization (Ryser and Keller, 1992). However, ptGRP1 from petunia is not localized to the cell wall proper but to the membrane-wall interface (Condit, 1993). Interestingly, cells expressing GRP1.8 or ptGRP1 appear to secrete at least a portion of the protein for incorporation into the walls of adjacent cells, rather than their own wall.
Nodulin-24-like proteins comprise a sub-group of GRPs found in diverse plants. These proteins possess conserved signal sequences and cysteine motifs near the C-terminus (Sachetto-Martins et al., 2000). During root nodule development, soybean nodulin-24 is synthesized and inserted into the plant-derived symbiosome membrane that surrounds the bacteroids (Cheon et al., 1994). However, the nodulin-24 processing pathway is unusual. Fusion of its signal sequence to β-glucuronidase (GUS) resulted in translocation without cleavage; proper cleavage only occurs when the entire nodulin-24 sequence is fused to GUS (Cheon et al., 1994). Furthermore, like HT-B, nodulin-24 does not possess a membrane spanning domain, and it is not known how it associates with the symbiosome membrane. It has been suggested that nodulin-24 is post-translationally modified in the endoplasmic reticulum for membrane association (Cheon et al., 1994). It is noteworthy that the expected size of nodulin-24 is 24 kDa but that the plant protein has an apparent molecular weight of 33 kDa (Cheon et al., 1994). A similar discrepancy is observed in HT-family proteins. For example, the calculated molecular weights of HT-M proteins are near 6 kDa, but the apparent molecular weight is near 10 kDa (Figure 6) Such differences are consistent with post-translational modification, but this has not been directly demonstrated.
Based on the similarities between the pistil-specific HT-family proteins and nodulin-24-like GRPs, we propose to refer to the broader group as HT/NOD-24-family proteins. The characteristics of the family include a conserved signal sequence, a variable core sequence that may or may not be glycine-rich, and one or more cysteine motifs similar to CXXXC, CXXXCC, or CXXCXC near the C-terminus. Our hypothesis is that the conserved sequences in the HT/NOD-24 protein family are important for modification or targeting. The variable core sequences are so divergent that the overall similarities of the HT/NOD-24 family did not stand out in previous analyses. Of course, the GRP core sequences are repetitive and glycine rich. Although it is not true of the N. alata HT-B protein, it is worth mentioning that HT-A and HT-B proteins from Solanum contain runs of four to six glycine residues (Kondo et al., 2002a). While the functions of the HT/NOD-24-family core sequences are not known, it is likely that these sequences play roles in diverse processes.
Targeting of the broad HT/NOD-24-family of proteins to membranes (Figure 7; Goldraij et al., 2006; Cheon et al., 1994) and the extracellular matrix, or cell wall, may be a widespread characteristic of the family. Some members of this family such as the tomato tyrosine and lysine-rich protein (TLRP, Figure 7) may be covalently attached to the cell wall, or extracellular matrix (Domingo et al., 1994). HT-B and HT-M proteins are not rich in tyrosine and appear to be soluble components of the extracellular matrix. It is intriguing that both the pistil-specific HT-family and nodulin-24 are positioned for some form of intercellular communication; nodulin-24 is present in the symbiosome membrane, where it could have a role in communication between the plant cell and the bacteroid, and HT-B, which is involved in pollen-pistil signaling, is produced in transmitting tract cells and then taken up and degraded in pollen tubes. While it is not known whether HT-M proteins interact with pollen tubes, their apparent degradation in response to pollination makes this a possibility. It will be interesting to learn whether other family members also are positioned where they could participate in intercellular communication.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Plant Materials
The N. alata, N. tabacum, and N. plumbaginifolia plant materials have been previously described (Murfett et al., 1996; Beecher et al., 1998). N. bonariensis (TW28, accession 11), N. forgetiana (TW50, accession 21A), and N. langsdorfii (TW75, accession 28B) were obtained from the US Tobacco Germplasm Collection (Crops Research Laboratory, Oxford, NC, USA). N. longiflora (accession 4C15) was collected by Dr Tim Holtsford, Division of Biological Sciences, University of Missouri-Columbia; a voucher specimen is lodged in the University of Missouri Herbarium (UMO 189123).
HT-M Sequencing and RT-PCR Analysis
Total RNA was extracted from mature pistils (i.e., stigma plus style) and used to prepare cDNA using the SMART cDNA library construction kit (BD Biosciences, Palo Alto, CA, USA). HT-M1, -M3, and -M4 were isolated in a differential screen for cDNAs that were abundant in mature N. alata pistils but rare or absent in immature N. alata pistils and in SC N. plumbaginifolia pistils. HT-M1 and HT-M2 cDNAs were amplified from N. plumbaginifolia, N. forgetiana, N. bonariensis, N. longiflora, and N. tabacum using general sequence primers (HTn(Met): 5'- GAGAGTAATACGACTCACTATAGGGATGGTT-3’; HTN3’: 5'- GATGTTTCCTTTATTTAGACTCGTAGC-3’). Gene specific primers were used for HT-M3 and -M4. Gene-specific amplification of HT-M genes was accomplished using a single sense primer derived from conserved sequences in the 5’ UTR and adjacent sequences (5'-TCAAAAAATGGTTTT CAAGTCAAAT-3’) and gene-specific antisense primers (n1-3ter: 5'- ATCTGCTACTATCGGTATCAKGGCTTG-3’; n2-3ter: 5'- GTAGCTATCGTTCCG GCGAAATTCCACC-3’; n3-c: 5'- GAACCATCACTTTCTACTTTTCTC-3’; n4.2-c: 5'-TGTATGAACATTACGTTCCACTC-3’) derived from the variable regions or 3'-UTRs of HT-M14, respectively. These gene-specific primers generate specific PCR products of 336, 211, 360, and 358 bp for HT-M14, respectively. Amplification specificity was checked by sequencing PCR products to ensure that they were derived from the correct gene. Amplification was performed using first-strand cDNA synthesized from mature pistil RNA as a template and ExTaq DNA polymerase (Takara Bio USA, Madison, WI, USA). PCR was performed for 30 cycles (95°C, 30 sec; 60°C to 62°C, 60 s; and 72°C, 60 s). PCR products were separated in 1.5% agarose gels and visualized by ethidium bromide staining. Actin sequences were amplified using primers (act-5: 5'- TGGAGAAGATATGGCATCATAC-3’ and act-3: 5'-CTGGAAGGTGCTGAGGGAAG-3’).
Amino acid sequence alignments were generated using DNASIS-Mac (HITACHI Software Engineering Co. Ltd, Japan). The phylogenetic tree in Figure 2B was prepared using TreeView (Page, 1996) and a Clustal W (Thompson et al., 1994) alignment implemented at DDBJ (DNA Data Bank of Japan, http://www.ddbj.nig.ac.jp).
Plant Transformation
The HT-M1 sequences shown in Figure 4 were amplified and cloned in pHANNIBAL (CSIRO Plant Industry, Canberra, Australia) (Wesley et al., 2001) in the sense and antisense orientations to produce a construct capable of suppressing multiple HT-M genes. N. plumbaginifolia was transformed and transgenic hybrids were produced by crossing primary transformants with SI N. alata S105S105, as described (Hancock et al., 2005). Pollination phenotypes were determined by examining style squashes (Murfett et al., 1996; Beecher et al., 1998; Murfett and McClure, 1998; McClure, 1999; Beecher and McClure, 2001; Hancock et al., 2005). Pistils were harvested 48 h after pollination, fixed in ethanol acetic acid (3:1) for 1 h, autoclaved in 10% sodium sulfite, and stained with decolorized aniline blue (Kho and Baer, 1968). Style squashes were viewed and photographed with a Leica MZFLIII stereomicroscope using an ultraviolet filter.
Protein Electrophoresis
Pistils were collected at maturity, frozen in liquid nitrogen, and stored at –80°C until needed. Extracts were prepared in 0.1 M Tris-HCl (pH 7.8), 10 mM EDTA, 1.5 % (w/v) ascorbic acid, and 2 % (w/v) polyvinylpyrrolidone from material powdered under liquid nitrogen, cleared by centrifugation at 10,000 xg for 20 min, and stored at –20°C. Protein concentration was determined using bovine serum albumin as a standard (BioRad, Richmond, CA, USA). Proteins were separated in 15% Tris-Tricine SDS-PAGE gels (Schägger and von Jagow, 1987) and blotted onto polyvinylidene difluoride (PVDF) membranes. An affinity purified HT-M antibody was prepared to the sequence CQPSRPLLKSNEAQST (Bethyl Laboratories, Inc. Montgomery, TX, USA), and protein blots were immunostained as described (Hancock et al., 2005).
Subcellular Fractionation
SI N. alata SA2SA2 flowers were emasculated and pollinated with SA2- (incompatible) or S105-pollen (compatible) pollen. Thirty pistils were collected after 48 h, frozen, and ground under liquid nitrogen. The samples were homogenized in 5 ml of 50 mM Tris-HCl (pH 7.5), 0.25 M mannitol, 2 mM EDTA, 5 mM ascorbic acid, 1 mM PMSF, and 2% (v/v) protein inhibitor cocktail (Sigma, St Louis, MO, USA) with a PowerGen 700 homogenizer (Fisher Scientific, St Louis, MO, USA) at speed level 2 for 30 s. After centrifugation at 10,000 xg (20 min, 4°C, HB-6 rotor, Sorvall, Asheville, NC, USA), the supernatant (S10) was subjected to differential centrifugation (1 h, 4°C, TLA 100.3 rotor, Beckman Coulter, Inc. Fullerton, CA, USA) to obtain 156,000 xg pellet (P156), and supernatant (S156) fractions. HT-B and vPPase were detected after SDS PAGE, blotting, and immunostaining according to standard methods McClure et al., 1999). Lipids interfere with detection of HT-M proteins and fractions were purified with methanol-chloroform prior to analysis (Wessel and Flugge, 1984). Briefly, 100 µl (1µg/µl) samples were treated by sequential addition of 400 µl methanol, 100 µl chloroform, and 300 µl water with vortexing after each addition. After centrifugation, the aqueous phase was recovered and precipitated with 300 µl methanol. Proteins were pelleted and resuspended in loading buffer for separation by SDS-PAGE, blotting, and immunostaining with anti-HT-M.
Accession Numbers
The new sequences reported here have been deposited in GenBank. The accession numbers are as follows: AB385644
[GenBank]
, N. alata HT-M1; AB385645
[GenBank]
, N. alata HT-M3; AB385646
[GenBank]
, N. alata HT-M4; AB385647
[GenBank]
, N. bonariensis HT-M1; AB385648
[GenBank]
, N. bonariensis HT-M2; AB385649
[GenBank]
, N. bonariensis HT-M3; AB385650
[GenBank]
, N. bonariensis HT-M4; AB385651
[GenBank]
, N. forgetiana HT-M1; AB385652
[GenBank]
, N. forgetiana HT-M2; AB385653
[GenBank]
, N. forgetiana HT-M4; AB385654
[GenBank]
, N. langsdorfii HT-M1; AB385655
[GenBank]
, N. langsdorfii HT-M3; AB385656
[GenBank]
, N. langsdorfii HT-M4; AB385657
[GenBank]
, N. longiflora 4C15 HT-M1; AB385658
[GenBank]
, N. longiflora 4C15 HT-M2; AB385659
[GenBank]
, N. longiflora 4C15 HT-M3; AB385660
[GenBank]
, N. plumbaginifolia HT-M2; AB385661
[GenBank]
, N. tabacum HT-M5; and AB385662
[GenBank]
, N. tabacum HT-M6.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Supplementary data associated with this paper can be viewed at Molecular Plant Online.
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FUNDING |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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National Science Foundation (IOB 0614962 to BM). MU-Monsanto plant research program grant to BM.
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Acknowledgements |
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We thank Chris Lee and Dr Sunran Kim in the McClure lab for helpful discussions and comments on early versions of this manuscript. We thank Melody Kroll for editorial assistance. No conflict of interest is declared.
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