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First published online July 2, 2004; 10.1104/pp.103.037747 Plant Physiology 135:1480-1490 (2004) © 2004 American Society of Plant Biologists The Putative Arabidopsis Homolog of Yeast Vps52p Is Required for Pollen Tube Elongation, Localizes to Golgi, and Might Be Involved in Vesicle Trafficking1Institut National de la Recherche Agronomique, Station de Génétique et d'Amélioration des Plantes, Institut Jean-Pierre Bourgin, Centre de Versailles-Grignon, 78026 Versailles cedex, France (E.L., A.G., M.F., G.P., S.B.); and Department of Biology, University of Leicester, Leicester LE1 7RH, United Kingdom (D.T.)
The screening of the Versailles collection of Arabidopsis T-DNA transformants allowed us to identify several male gametophytic mutants, including poky pollen tube (pok). The pok mutant, which could only be isolated as a hemizygous line, exhibits very short pollen tubes, explaining the male-specific transmission defect observed in this line. We show that the POK gene is duplicated in the Arabidopsis genome and that the predicted POK protein sequence is highly conserved from lower to higher eukaryotes. The putative POK homolog in yeast (Saccharomyces cerevisiae), referred to as Vps52p/SAC2, has been shown to be located at the late Golgi and to function in a complex with other proteins, Vps53p, Vps54p, and Vps51p. This complex is involved in retrograde trafficking of vesicles between the early endosomal compartment and the trans-Golgi network. We present the expression patterns of the POK gene and its duplicate P2 in Arabidopsis, and of the putative Arabidopsis homologs of VPS53 and VPS54 of yeast. We show that a POK::GFP fusion protein localizes to Golgi in plant cells, supporting the possibility of a conserved function for Vps52p and POK proteins. These results, together with the expression pattern of the POK::GUS fusion and the lack of plants homozygous for the pok mutation, suggest a more general role for POK in polar growth beyond the pollen tube elongation process.
Pollen tube growth is a vital process during male gametophyte development, since it allows male gametes to reach the ovules and achieve fertilization (Preuss, 1995  ). The elongation of the pollen tube, as well as that of animal axons, plant root hairs, fungal hyphae, and moss protonema, is achieved by a polarized mode of growth, termed tip growth (Franklin-Tong, 1999; Palanivelu and Preuss, 2000; Hepler et al., 2001), which involves the tip-localized exocytosis of Golgi-derived vesicles containing cell wall precursors (Franklin-Tong, 1999). The tip of the pollen tube is devoid of organelles, but contains almost exclusively Golgi-derived vesicles (Geitmann and Emons, 2000). In the shank of the tubes, an inverse fountain pattern of cytoplasmic streaming is observed; organelles and vesicles are transported toward the apex in the cortical region and basipetally in the central cytoplasm (Pierson et al., 1990). Actin filaments and microtubules, which are organized in longitudinal arrays more or less parallel to the elongation axis, act as tracks for cytoplasmic streaming and allow delivery of vesicles to the tip (Pierson et al., 1990; Vidali and Hepler, 2001). The use of membrane structure dyes, such as FM1-43 or FM4-64 (Parton et al., 2001; Camacho and Malhó, 2003), has revealed dynamic endo/exocytosis processes at the tip of the pollen tube, but the molecular events underlying these processes are still poorly understood. The recent identification of the Golgi-associated tobacco (Nicotiana tabacum), Rab GTPase NtRab2, predominantly expressed in pollen, suggests that tip growth and vesicle trafficking could be tightly linked (Cheung et al., 2002). Moreover, a GTP-bound dominant negative form of NtRab2 blocks endoplasmic reticulum to Golgi trafficking and inhibits pollen tube growth (Cheung et al., 2002).
The functions of numerous vesicle-trafficking proteins and several different protein-sorting pathways are well described in yeast (Saccharomyces cerevisiae; for review, see Kucharczyk and Rytka, 2001
Using insertional mutagenesis, we identified several putative male gametophytic mutants (Bonhomme et al., 1998a
The pok Phenotype Affects Pollen Tube Tip Growth In order to define the pok phenotype in situ, aniline blue staining was performed after limited pollination of wild-type pistils with pollen grains, either from wild-type or from hemizygous pok plants. Sixteen hours after pollination, all wild-type pollen grains had germinated and completely elongated their pollen tubes, reaching the base of the ovary (Fig. 1a ). The number of pollen tubes counted in the style was similar to the number of pollen grains deposited (Fig. 1a, n = 7). On the contrary, pollen from pok plants produced both elongated and short pollen tubes (Fig. 1b, five pollen grains on stigma, three tubes in the ovary). When only one or two grains were deposited, we commonly observed short, poky pollen tubes 16 h after pollination, which are presumed to carry the T-DNA construct (Fig. 1c). This observation is consistent with a role for the POK gene in pollen tube growth in vivo.
Nonetheless, some pollen tubes were still able to elongate sufficiently to reach ovules located in the upper part of the ovary (Fig. 1c). This would explain the residual male gametophytic T-DNA transmission (0.46%) in the pok mutant line (Bonhomme et al., 1998a , 1998b). Despite this residual transmission, numerous screens to isolate homozygous pok plants remained unsuccessful, suggesting that homozygous pok mutations lead to precocious lethality.
Genomic regions flanking the single T-DNA construct inserted in the pok mutant line were cloned (Bonhomme et al., 1998a
The POK and P2 genes (Fig. 2a) are composed of 20 and 19 exons, respectively (with an additional 5' exon in POK). The exon-intron structure (Fig. 2a) is strictly conserved and exon sequences share 90% identity, whereas intron sequences are largely divergent and shorter in P2 than in POK. Pok hemizygous mutant plants were complemented with an 11.2-kb genomic clone overlapping the POK gene, but excluding P2. The complementation construct was linked to the hpt gene, whereas the pok T-DNA insertion was marked with the nptII gene, conferring hygromycin (H) and kanamycin (K) resistance, respectively, to the transformed plants. Among the progeny of infiltrated mutant plants, five plants were selected that, when selfed, showed 2:1 segregation for kanamycin resistance, instead of 1:1 observed in the pok mutant. Furthermore, these five plants showed 5:1 segregation for hygromycin resistance, as expected in the case of independent segregation of both T-DNA insertions. The self progeny of one of these potentially complemented plants was analyzed. Descendants homozygous for hygromycin resistance either were also homozygous for kanamycin resistance (H/H, K/K), or showed a 3:1 ratio of kanamycin-resistant to kanamycin-sensitive progeny (H/H, K/-). Crossing these latter plants as male in with wild type confirmed that T-DNA transmission was restored. Moreover, descendants that had lost the complementing construct (showing a fully hygromycin-sensitive progeny) showed 1:1 segregation for kanamycin resistance. Finally, when germinating pollen from a complemented descendant (K/-, H/H) in vitro, the same proportion of elongated pollen tubes (78%, out of 348 counted pollen grains) was obtained as for wild-type pollen (77% of elongated tubes, out of 340 pollen grains), showing phenotypic restoration for this plant. In the control hemizygous pok line, 45% of elongated pollen tubes were counted out of 325 pollen grains. To rule out potential lesions in the linked P2 gene, the P2 genomic region was completely sequenced in the pok mutant and no mutations were observed. Taken together, these data confirm that the T-DNA insertion in the POK gene alone is responsible for the observed tip growth defect in pok plants.
Northern-blotting experiments using exon 10 of the POK gene as a probe (Fig. 2b) revealed a transcript of the expected size (2.2 kb) at every developmental stage tested and in almost all plant tissues. However, there was some variation in transcript abundance; very low levels of transcripts were detected in seeds, whole inflorescence, and mature flowers, while higher levels were observed in roots and flower buds less than 1 mm in size (i.e. before pollen mitosis [PM] I). Due to their strong identity, the POK and P2 transcripts could not be distinguished in hybridization experiments. However, oligonucleotides specific for each cDNA were designed (Fig. 2, c and d) and reverse transcription (RT)-PCR analysis was used to characterize the expression pattern of both transcripts independently. For the POK gene, RT-PCR results, although not quantitative, were qualitatively similar to those obtained by northern blotting, with the POK cDNA detected in all tissues, although at very low levels in seeds (Fig. 2e). For the P2 cDNA, no RT-PCR products were detected in any sample (Fig. 2f), even after two rounds of nested amplification (not shown). Taken together, the weak, or absent, expression of P2 versus POK and the presence of the POK cDNA in all RT-PCR samples argue that the northern-blot analysis shown in Figure 2b faithfully reflects POK mRNA distribution only.
Pollen and root cDNA libraries were screened with a probe corresponding to the T-DNA insertion region and overlapping the predicted exon 10 of the POK gene. Two types of clones were obtained from the pollen cDNA library: a POK partial cDNA clone (1,793 nt long) and a P2 cDNA clone (2,096 nt long). The screening of the root cDNA library allowed the isolation of a longer POK cDNA clone (2,217 nt); however, no cDNA clone for the P2 gene was found. Putative initiator ATGs for both transcripts (NetStart prediction software: http://www.cbs.dtu.dk/services/NetStart/), located in exon 1 (Fig. 2a), predict a total length for POK and P2 coding sequences of 2,088 and 2,106 nt, respectively. 5'RACE experiments using POK-specific primers indicated a 5'UTR of 156 to 158 nt, located 31 to 33 nt upstream of the 5' terminus of the root cDNA.
The POK and P2 predicted proteins are 696 and 702 amino acids, respectively, with a predicted molecular mass of approximately 80 kD. Both proteins share 88% identity and 93% similarity. According to BLAST software, POK shows 23% identity and 43% similarity with the yeast SAC2/Vps52p protein (Kölling et al., 1994
The AtVPS53 and AtVPS54 Genes
Conibear and Stevens (2000) AtVps53 and AtVps54 cDNA clones were isolated from the pollen cDNA library. They are both 2.7 kb long and contain 24 and 18 exons, respectively (Fig. 4, a and d ). The predicted proteins contain 829 and 1,001 amino acids, respectively, compared to 822 and 889 amino acids for Vps53p and Vps54p. A predicted translation start for the AtVps53 transcript is located in the middle of exon 2 (Fig. 4a, black arrow). For AtVps54, GENSCAN software (Chris Burge, Stanford University) predicts a long exon 1 (Fig. 4d, gray arrow), but RT-PCR experiments did not allow amplification of this region (not shown). The first in frame ATG of the isolated AtVps54 pollen cDNA is located at the end of exon 1 (Fig. 4d, black arrow), which would give rise to a 784-amino acid protein.
Expression of these two vps-like genes was studied by northern-blot analysis (Fig. 4, b, c, e, and f). As observed for POK, both AtVps53 and AtVps54 are ubiquitously expressed. Their basal expression level is low, although higher for AtVps53 compared to AtVps54 in all vegetative tissues (Fig. 4, b and e), and higher for AtVps54 in roots versus other tissues (Fig. 4e). In reproductive tissues, both transcripts are more abundant in buds less than 1 mm in size compared with later stages (Fig. 4, c and f).
Since an active translational fusion between the first 10 exons of the POK gene and the uidA gene carried by the T-DNA occurred in the pok mutant, GUS staining allowed us to determine a preliminary expression pattern for the POK protein. In 8- to 10-d-old plantlets, strong blue staining was detected in the root apex (Fig. 5a ), with enhanced intensity in the elongation zone (Fig. 5b), in emerging lateral root primordia (Fig. 5c), in very young leaves (Fig. 5d), and sometimes in stipules (Fig. 5a).
In reproductive tissues, GUS staining was observed at different stages of floral development (Fig. 5, e–j). Blue coloration was detected from the earliest stages of flower development, before meiosis and gametogenesis (bud size, 0.1 mm; Fig. 5e), and maintained later (Fig. 5, f–j). As flower bud size reached 1.6 to 1.7 mm (i.e. after PMII), expression of the fusion protein became strictly gametophytic in the male reproductive tissues, whereas it was clearly sporophytic in the female tissues, since all ovules were stained (Fig. 5h). At maturity, half of the pollen grains within the anthers were blue (Fig. 5j), as expected for hemizygous plants. At later postpollination stages, all developing seeds were stained (Fig. 5i). However, no blue staining was observed in the pistil below the stigma surface (Fig. 5i), compared to a control line (T-DNA insertion line; A. Guyon, A. Lécureuil, P. Guerche, and S. Bonhomme, unpublished data) expressing GUS in pollen and for which a blue coloration was observed in the style, corresponding to elongated pollen tubes (Fig. 5k; note that developing seeds are not stained). The absence of staining in the style of the pok mutant could be the result of lower GUS expression in this line compared to our control line, as well as of lack of effective pok pollen tube growth.
The subcellular localization of the POK protein was investigated using a POK::GFP fusion construct containing the complete open reading frame of the POK gene fused with the GFP coding sequence. Both cDNAs were cloned downstream of 2.2 kb of the predicted POK promoter region. Onion (Allium sativum) epidermal cells were bombarded with this POK::GFP construct. The collected signal exhibited a cytoplasmic dispersed punctate pattern (Fig. 6a
) characteristic of the previously described Golgi distribution pattern in plant cells (Boevink et al., 1998
POK and Pollen Tube Elongation: Slowly to the Goal
pok can be classed a late gametophytic mutant, since microsporogenesis and gametogenesis show no phenotypic differences compared to wild type. Moreover, we showed that pollen tube elongation is impaired in pok. Contrary to the raring-to-go Arabidopsis mutant (Johnson and McCormick, 2001
Although weak T-DNA transmission through the male gametophyte has been observed, suggesting incomplete penetrance of the mutation, no plants homozygous for the pok mutation were isolated. This suggests that the POK function is not restricted to reproduction, but could also be essential in early developmental events. This hypothesis is reinforced by the expression pattern of the POK gene, which is clearly not restricted to the reproductive organs, but extends to other plant tissues. Indeed, northern-blot experiments showed that, apart from reproductive tissues (see below), POK expression is high in elongating tissues, such as roots or young seedlings. Moreover, intense GUS staining is detected in the elongation zone of the root apex and in the emerging lateral roots of the pok mutant.
Even though the POK gene basal expression level is quite low, we detected higher levels of POK transcript in young flower buds (i.e. before the uninucleate microspore stage) compared with later stages of floral development. POK gene expression thus seems to be restricted to early steps of microgametogenesis, allowing us to classify it with the early class of pollen-expressed genes, which are transcribed soon after meiosis and whose transcripts accumulate until PMII and then decrease (Mascarenhas, 1990
The transcript level of putative homologs for VPS53 and VPS54 in Arabidopsis, named AtVps53 and AtVps54, was studied. Expression levels of both genes were revealed to be quite weak (particularly for AtVps54); however, expression was higher in roots (mainly for AtVps54). For both AtVps53 and AtVps54, an expression pattern comparable to that of POK was observed in reproductive tissues (i.e. higher expression in young flower buds), suggesting that they also could be part of the early class of pollen-expressed genes. It has been shown that, for a given gene, mRNA stability can be different in pollen and in somatic cells (Ylstra and McCormick, 1999
A second mRNA, sharing 90% identity with POK and named P2, is also expressed in pollen grains, although at a much lower level, as it was undetectable using RT-PCR. Similar to 17% of the full complement of Arabidopsis genes (Blanc et al., 2000
The deduced protein sequences of both POK and P2 share 23% to 35% identity with several eukaryotic proteins, all of which seem to be orthologous to one another, including the SAC2/Vps52p protein of yeast. The VPS52 gene, as well as VPS53 and VPS54, were identified by Conibear and Stevens (2000)
Proteins shown to be components of the vesicle-transport machinery are usually well conserved among eukaryotic species from yeast to human. As observed for POK, most of these genes have been shown to be ubiquitously expressed, but to higher levels in roots (Ahmed et al., 1997
Using POK::GFP and ST::DsRed fusion constructs in transient assays, we demonstrated that POK shares the same localization in plants as Vps52p does in yeast. Since tip-growing cells, such as pollen tubes, ensure rapid and polarized elongation through active apical exocytosis of Golgi vesicles (Hepler et al., 2001
Plant Material
The T-DNA transmission defect lines of Arabidopsis (L.) Heynh. ecotype Wassilewskija were isolated from the Versailles collection of T-DNA insertion mutants as described in Bonhomme et al. (1998b)
In vitro growth of pollen tubes was tested as described in Procissi et al. (2001)
Histochemical GUS assays were performed as described in Procissi et al. (2003)
A XbaI/SalI 11.2-kb fragment corresponding to the POK gene was subcloned from a genomic phage clone (Bonhomme et al., 1998a
The pollen cDNA library was constructed from poly(A+) RNA isolated from mature pollen of Arabidopsis ecotype Landsberg erecta. Mature pollen was isolated as described previously (Honys and Twell, 2003 Root cDNA library was cloned into lambda gt10 (kind gift from M. Salanoubat). Both cDNA libraries were screened with a probe corresponding to exon 10 of the POK gene, with probes corresponding to exons 10 and 22 for the AtVps53 gene and with exons 11 and 15 for the AtVps54 gene.
Seed RNA was extracted following Downing (1992)
cDNA synthesis was performed with Superscript RT (Life Technologies, Carlsbad, CA) from total RNA (3 µg) treated with DNAse I, following the manufacturer's protocol. cDNAs were amplified with 27 cycles of PCR. Primers used were: POK gene, POK-1 (5'-GGTGTTAAGTGCACATTTTCGTG-3') and POK-2 (5'-TCACCAAAGAAATCATCACAAAA-3'); P2 gene, P2-1 (5'-TGCTAAGTGCACATTTTCAGT-3') and P2-2 (5'-TCACCAAAGAAATCATCACAAAA-3'); AtVps53 gene, 53 to 1 (5'-AAAATCAGAAAATACATGGGAC-3') and 53 to 2 (5'-TCAAGAAATATGCCGTGAC-3'); and AtVps54 gene, 54 to 1 (5'-ATGCGTATCTCCATACATGA-3') and 54 to 2 (5'-AAAAATCAGATTCGAGCGAT-3').
5' RACE experiments were performed using a Life Technologies system on roots and flower bud RNA. Successively used primers were specific for exon 5 (5'-GTCTGAACTTATAGAACCAATC-3'), the end of exon 4 (5'-CTGAAATCCACTGAGAAGAGT-3'), and the beginning of exon 4 (5'-GTCACTTTCCTTTATGTAATCC-3'). RACE products were cloned into pGEM-T (Promega, Madison, WI).
The POK promoter region (2.2 kb) was amplified from Wassilewskija DNA using the primers 5'-TTTCTAGACGGCAGAGCTTCAGT-3' and 5'-TCCCCCATGGTTTGGCCCTA-3'. The resulting fragment was cloned and sequenced into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA). In parallel, the POK cDNA was amplified using the primers 5'-CTAATGGCGGACTCAAACTGATT-3' and 5'-GCTACCATGGCGAAAGTCTTGGAGTATTTCCT-3', allowing replacement of the stop codon by an NcoI restriction site at the 3' end. The resulting PCR product was cloned and sequenced in the pCRBluntII-TOPO (Invitrogen). An XbaI/BglII promoter fragment and a BglII/NcoI cDNA fragment were isolated from the constructs described above and cloned together into the pTrc99A vector (Amann et al., 1988
As described by Weigel and Glazebrook (2002) Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers bankit481797 (POK), bankit481801 (P2), AAB64912, AAH32108, AAF52254, and AAA68727.
We thank F. Brandizzi (Oxford Brookes University) for providing us with the ST::DsRed construct, R. Berthomé for help with bombardments, V. Carpentier and A. Lécureuil for the GUS assays, and C. Horlow and F. Nolent for their contribution to this work. We are also very grateful to C. Mézard and F. Nogué for critical reading of the manuscript and stimulating discussions. Received December 15, 2003; returned for revision March 17, 2004; accepted April 3, 2004.
1 This work was supported by INRA-DGAP (doctoral fellowship to E.L.). 
2 Present address: Laboratory of Molecular Biology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.037747. * Corresponding author; email bonhomme{at}versailles.inra.fr; fax 33–1–30–83–33–19.
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ABSTRACT
RESULTS
-2,6-sialyltransferase gene was used as a late Golgi marker. Both GFP (
