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First published online February 3, 2006; 10.1104/pp.105.074922 Plant Physiology 140:1374-1383 (2006) © 2006 American Society of Plant Biologists An Ankyrin Repeat-Containing Protein, Characterized as a Ubiquitin Ligase, Is Closely Associated with Membrane-Enclosed Organelles and Required for Pollen Germination and Pollen Tube Growth in Lily1,[W]Protein Science Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnologies, Tsinghua University, Beijing 100084, China (J.H., F.C., M.S., S.Y., J.P., Y.L.); Dipartimento di Scienze Ambientali, Università di Siena, via Mattioli 4, 53100 Siena, Italy (C.D.C., A.A., M.C.); and Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China (H.S., C.W.)
Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth.
In higher plants, mature pollen grains land on the stigma and protrude tubes, which travel a long distance in the style. Eventually, one tube deposits two sperm cells into the ovule to achieve fertilization. Probably the fastest growth of plant cells, pollen tube growth is under tight control and elaborately modulated (Taylor and Hepler, 1997  ; Yang, 1998; Hepler et al., 2001; Feijó et al., 2004). The crucial players in this extreme type of polarized growth include Ca2+ (Franklin-Tong, 1999), Rop/Rac GTPase (Zheng and Yang, 2000; Fu et al., 2001; Gu et al., 2004), and phosphoinositide (Franklin-Tong et al., 1996). Recently, new molecules such as  -amino butyric acid (Palanivelu et al., 2003) and nitric oxide (Prado et al., 2004) have emerged as important actors. Moreover, pollen growth responds to other factors that affect the pollen cell wall (Li et al., 1996, 2002; Zhou et al., 2004; Bosch et al., 2005). The cytoskeleton and its related proteins play essential roles in pollen tube growth (Vidali et al., 2001; Chen et al., 2003), and it is most likely that the majority of the signaling pathways regulate tip growth by directly or indirectly connecting to and remodeling the cytoskeleton (Feijó et al., 2004). It is noteworthy that protein turnover during pollen elongation is also important and the ubiquitin/proteasome pathway has been implicated as a major regulator of tip growth (Speranza et al., 2001; Scoccianti et al., 2003).
Decades of intensive investigation have given deep insight into pollen physiology, but the complex mechanisms underlying this tip growth are far from being clear. However, an overall understanding of the process could be accelerated significantly with the advent of high-throughput technologies such as gene expression profiling (Schena et al., 1995 Here we report the application of a 1,536-cDNA microarray to profile the gene expression during lily pollen germination and tube growth. Sequencing of a subset of cDNA clones led to the identification of a number of unannotated genes. Through characterization of one of them, lily ankyrin repeat-containing protein (LlANK), we show that this protein is a ubiquitin ligase closely associated with membrane-enclosed organelles and required for pollen germination and pollen tube growth.
Lily Pollen cDNA Microarray A 1,536-cDNA microarray was prepared to profile the gene expression from germinated and ungerminated lily pollen. The data have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through Gene Expression Omnibus Series accession number GSE2648. We then sequenced 100 cDNA clones that exhibited the highest germinated/ungerminated ratios and matched the data against GenBank. The result is available as supplemental information (Supplemental Table I).
Of the 100 sequenced cDNA clones, 60 were identified homologous to known genes in GenBank. In these sequences, three groups of genes are highly represented: pectin methylesterases, small GTPases, and proteases including ubiquitination-proteasome pathway components (Table I
). Though the differential expression needs to be verified individually, the prominence of these genes supported their critical roles in pollen germination and pollen tube growth as characterized previously (Li et al., 1996
LlANK Protein Contains Five Ankyrin Repeats and One RING Zinc-Finger Domain
The original LlANK cDNA was a truncated form lacking its 5' end coding region. Using 5' RACE, we obtained an additional 522-bp upstream cDNA sequence. The new longer cDNA could be readily retrieved by reverse transcription (RT)-PCR from pollen at all stages of in vitro growth. Sequence analysis indicated that LlANK cDNA contains a complete coding sequence for a 516-amino acid protein in which the SMART program (http://smart.embl-heidelberg.de/) confidently predicted five tandem ankyrin repeats and a canonical C3HC4-type RING zinc-finger domain (Fig. 1
). Both ankyrin repeat and RING zinc finger are common protein-protein interaction motifs. Since ankyrin repeat appears conserved in structure rather than in function (Bork, 1993
LlANK Possesses Ubiquitin Ligase Activity in Vitro
Conjugating of ubiquitin to a target protein requires at least three kinds of enzymes: Ubiquitin-activating enzyme (E1) creates an activated ubiquitin that is transferred to ubiquitin-conjugating enzyme (E2) and subsequently, depending on ligase enzyme (E3), to a target protein. The activities and specificities of E2 are controlled by E3, which binds to substrate (Pickart, 2001
Expression of LlANK Is Up-Regulated during Pollen Germination and Pollen Tube Growth
Since the gene expression profile of cold-stored pollen differs from that of fresh pollen because of selected protein/mRNA degradation during long-term storage (Wang et al., 2004
LlANK Protein Is Associated with Membrane-Enclosed Organelles in Pollen
To determine the cellular localization of LlANK, we transiently expressed LlANK-green fluorescent protein (GFP) fusion protein in lily pollen via particle bombardment, a method that has been successfully applied in pollen (Chen et al., 2002
Transiently expressed control GFP was evenly distributed throughout the cytoplasm of tubes, while LlANK-GFP fusion protein was prominently associated with distinct structures that appeared to be membrane-enclosed organelles. One of the frequently observed phenomena was the punctate or granular dots in pollen tubes (Fig. 4B). In grains, such dots were also observed (data not shown). However, they were never found at the apical region of pollen tube, the so-called clear zone (Fig. 4C). The most notable structure with which LlANK-GFP was associated, however, was generative cell, which from a certain respect can be viewed as the largest membrane-enclosed organelle in the bicellular pollen (Fig. 4D). Furthermore, neither cell wall nor plasma membrane was found labeled with LlANK-GFP, indicating that LlANK was not targeted to these places. Observations above together led us to conclude that LlANK protein is associated with membrane-enclosed organelles in lily pollen.
Zm13 is a pollen-specific gene from maize (Zea mays; Hamilton et al., 1989
Unlike their double-CaMV-35S-driven counterparts, the Zm13-driven constructs were capable of producing a detectable signal using DNA as little as 1 µg per bombardment. Increasing the DNA amount enhanced GFP intensity, but no further improvement was observed when using more than 5 µg DNA per bombardment, presumably due to the coating limitation of gold particles. Overexpression of the control GFP invariably displayed uniform distribution throughout pollen tubes (Fig. 5B) and no apparent effect was found on germination and tube growth. In the case of Zm13::LlANK-GFP transformation, the typical punctate or granular structures were present (Fig. 5C), while in a small fraction of tubes an excess of overexpressed LlANK-GFP was found dispersed into the cytosol and morphological abnormalities were usually observed when using a large amount of DNA (Fig. 5D). These abnormalities included budding (Fig. 5E), membrane invagination (Fig. 5F), tip ballooning (Fig. 5G), and tube swelling (Fig. 5H). We also measured the average length of transformed but normal tubes as well as that of the untransformed ones, but no significant difference was found (data not shown). These observations indicated that LlANK is involved in pollen tube growth but its overexpression is prone to interrupt the fine coordinance of the regulation and to induce abnormal tube growth.
RNA-induced gene silencing is a powerful tool available for plant biologists in recent years (Burch-Smith et al., 2003
Quantitative comparative real-time PCR results demonstrated that the LlANK expression was down-regulated in pollen transformed with ihpLlANK (Fig. 6C). The calculated germination percentage for transformed pollens was 9.6%, which was significantly lower than the 42.6% in untransformed pollens from the same bombarded population (Fig. 6D). When it came to tube growth, the average length of all transformed tubes was 183 µm, much shorter than the 381 µm of the equal top proportion of tubes that received no DNA (Fig. 6E). Meanwhile, the control experiment using ihp demonstrated that bombardment as well as the subsequent exogenous expression had little effect on pollen germination and pollen tube growth (Fig. 6, D–E, right, respectively). Taken together, the results above indicated that down-regulation of LlANK impaired pollen germination and pollen tube growth. The inhibitory effect coming after LlANK down-regulation suggested the essential role that LlANK plays, directly or indirectly, in this polarized tip growth. However, it should be noted that no morphological abnormality was observed in the tubes of transformed pollens.
GFP fusion protein localization demonstrated that LlANK is associated with membrane-enclosed organelles. The nature of these organelles, however, remains to be determined. We tried a range of specific fluorescent probes such as Rhodamine 123 (for mitochondria) and ER-Tracer Blue-White DPX (for endoplasmic reticulum), but there was no convincing conclusion (data not shown). The appearance of these highlighted structures resembles that of Golgi bodies (Cheung et al., 2002 ), dispersed vacuoles (Hicks et al., 2004), and the so-called membrane-enclosed organelles (Cai et al., 2000), but the identity of these organelles awaits determination. Observations suggested that the association of LlANK with organelle is selective. For example, it seemed that not all the vesicles were fluorescently labeled (Fig. 4B). The tip area, where secretory vesicles accumulate (Derkson et al., 1999), was free of LlANK distribution (Fig. 4C). Moreover, vegetative nuclei were never found fluorescently labeled, though they were absolutely membrane-enclosed structures. Since analysis of LlANK amino acid sequence revealed no features characteristic of transmembrane region, the association selectivity may be attributed to specific interactions of LlANK, presumably via its ankyrin repeats, with certain membrane proteins that are unique to a subset of organelles.
Our data provided the biochemical evidence that LlANK possesses E3 ubiquitin ligase activity in vitro. We also showed that down-regulation of LlANK reduced pollen tube emergence and growth. This finding is consistent with the observation that ubiquitin/proteasome pathway has a direct role in regulating pollen tube emergence in kiwifruit (Actinidia deliciosa; Speranza et al., 2001
Based on the results obtained and the implications mentioned above, it is logical to launch into isolating and identifying the protein(s) that LlANK recognizes and binds to presumably via its ankyrin repeat domain. In such an attempt, we expressed recombinant GST-LlANK and used the fusion protein as a bait to pull down (Simpson, 2002
We have shown that LlANK is closely associated with membrane-enclosed organelles and required for pollen germination and tube growth in lily. Of the proteins homologous to LlANK (Table II), XBAT32 from Arabidopsis is the only one that has been comprehensively studied (Nodzon et al., 2004
Plant Material and Culture Condition Plants of lily (Lilium longiflorum) were grown in a green house under natural day length. Anther was harvested from young flowers, induced to dehisce by light, and dehydrated in a desiccator. Pollen was then collected and, if not to be used immediately, stored at –20°C. Prior to use, cold-stored pollen was brought gradually to room temperature. After that, like fresh pollen, the stored pollen was rehydrated in a moisture chamber for 3 h. Rehydrated pollen was then resuspended in Dickenson medium [0.03% Ca(NO3)2·4H2O, 0.01% KNO3, 0.001% H3BO4, and 10% Suc] at a density of 4 to 5 mg mL–1 with gentle shaking at 25°C. Generally, pollen bulk germinated and took on active tube growth 1 and 3 h after culture initiation, respectively. For practical convenience, in vitro germination and tube growth were divided into the following stages: (1) dehydrated pollen freshly collected from desiccated anther, (2) rehydrated pollen (culture initiation), (3) pregermination pollen (no visible tube), (4) germinating pollen (tube protruding), and (5) tube growth (>100 µm in length). Aliquot of pollen suspension can be withdrawn for microscopic observation at any time point as desired. For RNA/protein extraction, a large number of pollen grains/tubes were harvested from the culture medium using 10-µm nylon membrane.
Total RNA was isolated from microspores, ungerminated, and germinated pollen using Qiagen RNAeasy plant mini kit. mRNA was isolated from total RNA with FastTrack 2.0 mRNA isolation kit (Invitrogen). From the pooled mRNAs, a cDNA library was constructed using PCR cDNA library construction kit (Stratagene). As soon as it was produced, the cDNA library was used for microarray preparation. Since then, the work was done by Genetix and is only briefly described here. A total of 1,536 clones were picked randomly from the cDNA library and the cDNA inserts were PCR amplified directly from clones in culture using the M13 universal primers. PCR products were purified with Montage PCR96 cleanup kit (Millipore), transferred to 384-well microtitres, and vacuum dried. After redissolved, the PCR products were spotted (diameter: 250 µm) onto microscope glass slides in adjacent duplicate using Genetix Qarrayer, UV cross-linked, blocked, and denatured. For preparation of the labeled cDNA targets, RNA samples were extracted from the pollen harvested at culture initiation (ungerminated) as well as after 3 h (germinated) as described above, labeled with Cy3-dUTP or Cy5-dUTP, respectively, according to previously reported protocols (Hedge et al., 2000
5'-RACE was performed by following the standard strategy (Sambrook and Russell, 2001
Standard methods (Sambrook and Russell, 2001
Recombinant plasmid constructs based on GST Gene Fusion system (GE Healthcare) were used for expression and purification of GST-LlANK and GST-AtUBC8 from bacteria, as instructed by the manufacturer. Ubiquitination reactions were done as described (Hardtke et al., 2002
Pollen was cultured as described above. For particle bombardment, pollen grain suspended in Dickenson medium was applied evenly onto filter paper put in a 90-mm petri dish. Enough medium was supplied to keep the paper wet but not to immerse pollen. Plasmid DNA was purified by resin (BioDev). For each bombardment, 0.5 mg gold particles (1.0 µm) were coated with 5 µg double-CaMV-35S::(ANK-)GFP DNA for cellular localization or 1 to 5 µg Zm13::(ANK-)GFP DNA for overexpression as described in the "Results" section. In silencing experiments, either Zm13::ihpLlANK (effector) or Zm13::ihp (control) was combined 1:1 (w/w) with Zm13::GFP (indicator) and 5 µg DNA mixture used for each bombardment. Bombardment was performed with model PDS-1000/He Biolistic Particle Delivery system (Bio-Rad) as instructed by the manufacturer. Settings: 29-inch Hg vacuum, 4-cm gap distance, and 7-cm particle flight distance. To each sample, three consecutive bombardments were performed to increase transformation frequency. After bombardments, pollen was rinsed down from filter paper with the medium, cultured at a density of 4 to 5 mg mL–1 with gentle shaking at 25°C. Generally, GFP signal was detectable after 3 h. In cellular localization, over-, and down-expression experiments, pollen was observed 4 h after culture initiation. Pollen culture was applied on a microslide without any fixation. For nucleic acid staining, pollen culture was gently mixed 1:1 (v/v) with a mount solution, which was prepared by combining phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) 3:7 (v/v) with glycerol. Hoechst 33258 (Invitrogen) was supplemented to a final concentration of 100 ng mL–1 and incubated for 10 min at room temperature. Conventional fluorescence observation (filter: B-2A and UV-2A for GFP483/510 and Hoechst 33258352/461, respectively) and laser-scanning confocal microscopic observation (using the instrumental default settings for enhanced GFP) were performed on Nikon Eclipse E400 (Nikon Instech) and Olympus FluoView FV1000, respectively, and recorded with the software packaged therein. Pollen tube lengths were measured using Photoshop 7.0 software (Adobe); a layer was created above an original image, lines were drawn along pollen tubes and bars with the same width, pixel numbers were recorded, data was exported to Excel software (Microsoft), and eventually they were converted to tube lengths.
Performances from single pollen isolation to PCR product detection were conducted consecutively as described below. Sixty microliters of pollen culture suspension was diluted in 1 mL Dickenson medium in one 35-mm cell culture dish (Corning), sited open on one microslide on Nikon Eclipse E400, and observed with only 10x objective lens. Monitored in the microscopic field, single pollen was carefully pipetted out with an Eppendorf 2.5 µL pipette, and immediately put into 0.5 mL TRIzol Reagent. To each single-pollen RNA extraction system, approximately 10 pg synthetic tetracyclin resistant gene (tet) poly(A)+ mRNA (1.4 kb, in vitro transcribed from tet of plasmid pBR322; TaKaRa) was added as a normalizing standard. RNA extraction using TRIzol Reagent was carried out as instructed, with 20 µg glycogen (Sigma) added as carrier prior to precipitating RNA with isopropyl alcohol. During RNA washing in 70% ethanol, a master mix of cDNA synthesis reactions was prepared that contained 20 nM oligo(dT)15 primer, 0.3 units µL–1 of moloney murine leukemia virus reverse transcriptase (Promega), and other standard components but with RNA template omitted. At the end of RNA extraction procedure, RNA pellet was directly dissolved in 30 µL aliquot of the cDNA synthesis master mix and incubated at 42°C for 1 h. In the meantime, PCR master mixes for LlANK, actin, and tet, respectively, were prepared. The master mixes contained respective primers, EX Taq, and other standard components but omitted deoxynucleotide triphosphates and cDNA template. When the cDNA synthesis was completed, the reaction was divided into three 10-µL aliquots, each of which was subsequently combined with a 10-µL aliquot of one of the three PCR master mixes and amplification was performed on a TGRADIENT thermocycler (Biometra). Three pairs of 25-mer primers were designed to amplify the 500-bp regions of 3' portion of the three gene transcripts, respectively. Thermal profile settings were 94°C 1 min, and then 94°C 15 s, 59°C 20 s, 72°C 20 s, for 40 cycles. For comparison, conventional PCR products of three genes were separated and stained in the same agarose gel. Quantitative real-time PCR using SYBR Green I Dye (Bio-V) was performed in ABgene microtubes with ultra clear cap (ABgene) on Mx3000P (Stratagene). To minimize the formation of nonspecific amplification products, total primer concentrations for LlANK, actin, and tet were empirically optimized to 800 nM, 400 nM, and 400 nM, respectively. Amplification efficiencies of three amplicons were confirmed similar using 2– Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DN985072 to DN985171 (sequenced 100 cDNA clones after microarray), AY950617 (LlANK cDNA), and M15239 (tobacco etch virus translational enhancement element).
We thank Dr. Alice Y. Cheung (Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA) for her precious advice in experimental design and critical reviewing of the manuscript. The Zmc13 promoter was originally a gift from Prof. Joseph Mascarenhas (Department of Biological Sciences, State University of New York, Albany, NY). We thank Prof. Xingwang Deng (Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT) and Renfeng Li (Department of Biological Sciences and Biotechnologies, Tsinghua University, Beijng, China) for kindly providing the recombinant E2 AtUBC8 plasmid and recombinant human E1 enzyme protein, respectively. We also thank Prof. Naoki Sakurai (Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan) for help in optimizing particle bombardment. Received November 30, 2005; returned for revision January 4, 2006; accepted January 19, 2006.
1 This work was supported by the National Natural Science Foundation of China (grant no. 30370134). 
2 These authors contributed equally to the paper. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yiqin Li (liyq{at}mail.tsinghua.edu.cn).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.074922. * Corresponding author; e-mail liyq{at}mail.tsinghua.edu.cn; fax 86–10–62772678.
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RESULTS
-32P]CTP-labeled RNA (Riboprobe system, Promega) at 68°C in aqueous solution overnight, washed, and autographed as described (Sambrook and Russell, 2001
-mercaptoethanol, and 0.2% [w/v] bromphenol blue). Western blots using either anti-ubiquitin (Sigma) or anti-GST (Santa Cruz) antibody were performed by following standard protocols (Sambrook and Russell, 2001
