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First published online May 20, 2005; 10.1104/pp.105.063388 Plant Physiology 138:778-789 (2005) © 2005 American Society of Plant Biologists
Plantacyanin Plays a Role in Reproduction in Arabidopsis1Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, California 92521
Plantacyanins belong to the phytocyanin family of blue copper proteins. In the Arabidopsis (Arabidopsis thaliana) genome, only one gene encodes plantacyanin. The T-DNA-tagged mutant is a knockdown mutant that shows no visible phenotype. We used both promoter-  -glucuronidase transgenic plants and immunolocalization to show that Arabidopsis plantacyanin is expressed most highly in the inflorescence and, specifically, in the transmitting tract of the pistil. Protein levels show a steep gradient in expression from the stigma into the style and ovary. Overexpression plants were generated using cauliflower mosaic virus 35S, and protein levels in the pistil were examined as well as the pollination process. Seed set in these plants is highly reduced mainly due to a lack of anther dehiscence, which is caused by degeneration of the endothecium. Callose deposits occur on the pollen walls in plants that overexpress plantacyanin, and a small percentage of these pollen grains germinate in the closed anthers. When wild-type pollen was used on the overexpression stigma, seed set was still decreased compared to the control pollinations. We detected an increase in plantacyanin levels in the overexpression pistil, including the transmitting tract. Guidance of the wild-type pollen tube on the overexpression stigma is disrupted as evidenced by the growth behavior of pollen tubes after they penetrate the papillar cell. Normally, pollen tubes travel down the papilla cell and into the style. Wild-type pollen tubes on the overexpression stigma made numerous turns around the papilla cell before growing toward the style. In some rare cases, pollen tubes circled up the papilla cell away from the style and were arrested there. We propose that when plantacyanin levels in the stigma are increased, pollen tube guidance into the style is disrupted.
Pollination is a crucial step in the life cycle of flowering plants. The pistil, composed of the stigma, style, and ovary, is the female receptive organ in pollination through which the pollen tube travels to deliver the sperm cells to the egg. The stigma, as the entry into the pistil's specialized transmitting tract tissue, provides a receptive surface for compatible pollen to adhere, hydrate, germinate, and grow. Crucifers, like Arabidopsis (Arabidopsis thaliana), have dry stigmas, which do not secrete much extracellular matrix (ECM) for the pollen tube to encounter on their surface. The pollen tube must penetrate into the cell wall space of the papilla cell to enter the transmitting tract ECM. From there, the pollen tube tracks the secretory ECM to the micropyle of the ovule for fertilization. By contrast, in lily (Lilium longiflorum), pollen tubes land on a stigma with copious secretions and enter an open style to grow on an epidermal, secretory ECM, never penetrating stigma or stylar tissues. In these two extreme cases, though, pollen tubes are in contact with the ECM of the pistil transmitting tract from the stigma to the ovule for fertilization (Lord and Russell, 2002  ). For this reason, pollen tube chemotropic molecules have been sought from the ECM of pistil tissues. So far, a few such pistil ECM molecules have been implicated in pollen tube growth and directional guidance (Wu et al., 1995; Wolters-Arts et al., 1998; Kim et al., 2003; Palanivelu et al., 2003; Márton et al., 2005). Our knowledge of the proteins involved in regulation of pollen activities in the stigma, however, is very limited, except for those involved in recognition events in self-incompatibility, such as S-locus receptor kinase and S-locus glycoproteins in Brassica (Dixit and Nasrallah, 2001; Takayama et al., 2001).
Plantacyanins are ECM proteins of unknown function. They belong to the ancient, plant-specific phytocyanins, a subfamily of blue copper proteins (Ryden and Hunt, 1993
The Arabidopsis genome contains a single plantacyanin gene (protein ID At2g02850). It shows 51.9% identity and 86.8% similarity to lily chemocyanin at the amino acid level (Kim et al., 2003
Determination of the Arabidopsis plantacyanin Expression Pattern The expression patterns of Arabidopsis plantacyanin were first determined by reverse transcription (RT)-PCR. Gene-specific primers were utilized to amplify plantacyanin from the first-strand cDNA, which was reverse transcribed from total RNAs from tissues of a mature plant (6 weeks old). The highest level was found in Arabidopsis inflorescences, and moderate levels were found in the roots (Fig. 1A). Cauline leaves and stems had low levels of plantacyanin transcription and so exhibited a very low amplification in RT-PCR. We barely see an amplification using rosette leaves. Repeated (3x) RT-PCR experiments demonstrated similar expression patterns of plantacyanin in mature Arabidopsis plants.
To further characterize the Arabidopsis plantacyanin expression pattern, we generated transgenic plants harboring the -D-glucuronidase (GUS) gene driven by the plantacyanin promoter. There is only a short DNA fragment (about 800 bp) between the Arabidopsis plantacyanin (At2g02850) gene and the upstream gene (Fig. 1B). This piece of DNA was used as the promoter region of plantacyanin to drive GUS expression. In 10-d-old seedlings, GUS signals were detected in almost all tissues (cotyledons, hypocotyls, and vascular bundles of roots), except in the root apical meristem (Fig. 1C). GUS expression was not only found in mesophyll cells and vascular bundles (Fig. 1D), but also was prevalent in guard cells (Fig. 1E). plantacyanin promoter activity was detected in mature rosette and cauline leaves (data not shown), but at a very low level, consistent with the RT-PCR result.
We carefully examined the GUS signals in the inflorescences of the transgenic plants (Fig. 1F). Overall, inflorescences showed strong plantacyanin promoter activity as revealed by RT-PCR. GUS expression was found in the pistil, with the strongest level in the style (Fig. 1G). We also saw GUS expression in stigmatic papilla cells starting at flower stage 11 (stages as defined by Smyth et al., 1990
In order to investigate the subcellular expression patterns of plantacyanin in Arabidopsis flowers, we carried out an immunohistochemical analysis on wild-type tissues of the pistil. Tissues were fixed and embedded in LR White (Fig. 2, A, E, and G, are sections stained with toluidine blue after immunolocalization; Fig. 2, B–D, F, H, and I, are immunolocalizations, with C being a preimmune control). The polyclonal antibodies against Escherichia coli-expressed plantacyanin specifically recognized plantacyanin on a protein blot with total cell proteins extracted from wild-type inflorescences (Fig. 2J). A plantacyanin gradient was detected in the stigma and style, with a lower level in the stigma (Fig. 2, B and D) and a much higher level in the stylar (Fig. 2B) and ovary (Fig. 2F) transmitting tract tissues. Preimmune serum gave low to no background labeling (Fig. 2C). Plantacyanin levels were high as well in the mature embryo sac (Fig. 2H). It is evident, at a higher magnification, that plantacyanin is a cell wall protein secreted into the ECM of the transmitting tract tissues (Fig. 2I).
Genetic Approaches to Dissect the Function of Arabidopsis Plantacyanin To dissect the function of Arabidopsis plantacyanin, we obtained a T-DNA-tagged mutant line (SALK_091945) from the Arabidopsis Biological Resource Center (ABRC), with the T-DNA integrated into the intron region of the gene. Unfortunately, it proved to be a knockdown line based on the RT-PCR result (Fig. 3A), and we did not see any visible phenotype under normal growth conditions. Wild-type pollen on the mutant pistil did not exhibit the aberrant pollen tube growth we observed in the OXPs (see below). Lack of a phenotype was most likely due to residual plantacyanin expression in these plants. Immunolocalizations detected plantacyanin protein in mutant pistils, but without a detectable difference from wild type (data not shown). Thus, we resorted to the overexpression approach to determine the function of plantacyanin in reproduction.
Overexpression Lines Show Lowered Seed Set Due to Defects in Both Anther Development and Pistil Function In order to create Arabidopsis plantacyanin OXPs, we used a cauliflower mosaic virus (CaMV) 35S promoter to drive protein expression in the plant. Inflorescences of homozygous transgenic lines (T2) contain higher levels of plantacyanin, compared to wild type, on an immunoblot detected with antibodies against E. coli-expressed plantacyanin (Fig. 3B). Many fewer seeds formed in these Arabidopsis plantacyanin overexpressor siliques (Fig. 3C), which are shorter than the wild type as a result (Fig. 3D, inset). Seed formation was dramatically decreased in OXP lines (T1) when siliques were randomly picked for seed counts (Fig. 3E). In the T1 generation, 33% of OXPs (23 out of 70 lines) displayed a lower seed set.
The overexpression anthers from the most severe lines, such as OXP12 and OXP24, showed defects in anther dehiscence. Toluidine blue-stained sections of the mature indehiscent anthers showed a malformed or absent endothecium, which could contribute to the lack of dehiscence (Fig. 4, A and B). The endothecium and tapetum initiated normally at an earlier stage (data not shown), but the endothecium had degenerated by stage 14 in the overexpression anthers (Fig. 4A). At this stage, the endothecium is active in anther dehiscence in the wild type (Fig. 4B). In many overexpression anthers, we observed a low number of pollen grains (
Pollen Tubes Lose Directional Growth on the Plantacyanin Overexpression Stigma But Eventually Penetrate the Style and Enter the Ovary We further analyzed plantacyanin levels in pistils of the overexpression lines in stage 13 flowers, when pollination occurs. We found more plantacyanin protein in these overexpression pistils compared to the wild type (Fig. 5A). The overexpression pistils pollinated with wild-type pollen produced about one-half the number of seeds as the wild-type control (Fig. 5, B and C).
Pollen tube growth behavior on the stigma was visualized using scanning electron microscopy (SEM) images. We manually applied wild-type pollen on pistils from emasculated flowers of both the wild type (control) and OXP. In wild-type pistils, pollen tubes penetrate the papilla cell and normally show fewer than one encircling pattern inside the papilla cell wall before growing down to the base of the cell and into the style (Table I; Fig. 5D). On the overexpression stigmas, nearly one-half of the wild-type pollen tubes exhibited aberrant growth (Table I; Fig. 5E, left), making many turns in the papilla cell wall before growing into the style. In some severe cases (seen in about 10 out of 600 samples), pollen tubes grew around the papilla cell several times and away from the style and arrested growth at the tip of the cell (Fig. 5E, right).
To determine whether the lowered seed set in the crosses was due to failure of the wild-type pollen to penetrate the style, we did hand pollinations on overexpression and wild-type pistils, removed the stigma and style, and placed them in vitro on agar growth medium. No differences in numbers of pollen tubes emerging from the style were detected in the two samples (Fig. 5F).
We used several methods of fixation (glutaraldehyde, formaldehyde-acetic acid [FAA]), embedding (LR White, paraffin), and sectioning (3- and 8-µm-thick sections) to detect plantacyanin in the pistils of plants using antibodies to E. coli-expressed plantacyanin (Fig. 6). Overexpression results in plantacyanin localization throughout the pistil (Fig. 6A) and higher levels in the transmitting tract tissues of the stigma (Fig. 6, A–D) and a slightly higher level in the style (Fig. 6, A, B, G, and H).
Plantacyanin Localization in Tissues of the Pistil and the Effects of Overexpression on Pollination
Chemotropism is defined as directional growth determined by a chemical gradient. The chemical can be an attractant or a repellent (Lush, 1999
Using a strong promoter, CaMV 35S, we increased the expression level of Arabidopsis plantacyanin in Arabidopsis pistils. We used antibody localization to detect increased levels of plantacyanin in the transmitting tract of the pistil. We then utilized SEM to visualize the wild-type pollen tube activities on the overexpression pistils. After the pollen grains were manually deposited on the overexpression stigmas, they hydrated and germinated normally, as on the wild-type stigma, and penetrated the papilla cell wall growing underneath the stigmatic cuticle. At this stage, a striking irregularity of pollen tube growth was observed; many pollen tubes appeared to lose the ability to grow directly into the style. They made many turns around the papilla cell before penetrating from the stigma to the style. In some rare cases, pollen tubes totally lost their direction, turning away from the style and growing to the tip of the papilla cell where they arrested. By contrast, the control pollinations showed little of this behavior. In other guidance systems, when cells are presented with a uniform distribution of a chemoattractant, they show random growth or movement (Caterina and Devreotes, 1991
Our data have shown a dramatic increase of the plantacyanin expression level in overexpression pistils, but the increase in expression in the transmitting tract tissues is less dramatic, comparing the overexpression and the wild type. Plantacyanin is a putative target of one of the miRNAs in Arabidopsis (Sunkar and Zhu, 2004
Expression of Arabidopsis plantacyanin was not detected in mature anthers using the plantacyanin promoter-GUS transgenic plants, nor was it seen in pollen, which is consistent with pollen transcriptome data (Becker et al., 2003
Pollen development in the overexpression anthers does not seem to be much affected, based on the fact that it desiccates normally and gives rise to full seed set when deposited on the wild-type pistil. But we did find irregular callose formation on the pollen intine (the inner layer of the pollen wall) in the mature anthers of OXPs and a low level of precociously germinated pollen. Pollen germination in the anther has been documented in many cleistogamous species where anther proximity to the stigma is sufficient to allow normal pollination and seed set in these closed flowers (Lord, 1979
One of the most striking characteristics of plantacyanins is that they are copper-binding proteins (Nersissian et al., 1998
Degeneration of the endothecium in the Arabidopsis plantacyanin overexpression anthers may be due to plantacyanin-induced precocious programmed cell death (PCD). PCD is a normal event in endothecium development (van Doorn and Woltering, 2005
Plantacyanins have been proposed to be involved in the oxidative burst that occurs in pathogen infection and in the cross-linking and insolubilization of cell wall materials, such as lignin and callose (Nersissian et al., 1998
It is known that ROS regulates intracellular signaling pathways through the activation of Ca2+ channels on the plasma membrane in the root hair (Foreman et al., 2003 A direct connection between plantacyanin and ROS can be confirmed by testing the level of ROS in these Arabidopsis OXPs.
In this study, we provide data that support the hypothesis that Arabidopsis plantacyanin may function in pollination since it is a component of the transmitting tract ECM and its overexpression in the pistil disrupts pollen tube guidance from the stigma to the style. It appears to act as well in anther development because overexpression results in an indehiscent anther with a degenerated endothecium. This small, ECM protein is not only expressed in the vegetative and reproductive sporophytic tissues, but also in the female gametophyte. Plantacyanin is a putative target of one of the miRNAs in Arabidopsis and it appears to play several roles in the biology of this plant. Further study of this intriguing protein is warranted to better define its functions in Arabidopsis.
Plant Materials and Growth Conditions
Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col) plants were soil grown in a University of California, Riverside, growth room in Sunshine Mix No. 1 (SunGro, Bellevue, WA), supplemented with fertilizer and insecticide (Springer et al., 2000
The promoter region of Arabidopsis plantacyanin was amplified from genomic DNA with primers 5'-AGGAGCTCGAGAGTAAATGAGGATGAATTGAAG-3' and 5'-ACGAATTCTATCGAGTTCTTTCAAGTCCAC-3'. The primers contain restriction sites SacI and EcoRI to be fused with the uidA gene and a 3' octopine synthase transcription terminator and cloned into the binary T-DNA vector pCAMBIA3200. Plant transformants were selected with BASTA.
The construct for overexpression of Arabidopsis plantacyanin, OXP, was made by introducing the open reading frame of Arabidopsis plantacyanin into a binary vector pPS119 (Shuai et al., 2002
Plants were transformed using Agrobacterium tumefaciens strain GV3101 with the floral-dip method (Clough and Bent, 1998
Total RNA was extracted from different tissues of mature Arabidopsis plants using the RNeasy mini kit (Qiagen, Chatsworth, CA). For RT-PCR analysis, cDNA was synthesized from 1.2 µg of total RNA in a 20-µL RT reaction, which contains an oligo(dT) primer, SuperScript II Rnase H– reverse transcriptase (20 units), and ribonuclease inhibitor (10 units; Invitrogen, Carlsbad, CA). One microliter of each cDNA sample was used as the template for PCR amplification. The primers flanking Arabidopsis plantacyanin's two exons were 5'-TGTCTAGATCAAACCGCGGTGACTG-3' and 5'-ATGGATCCATGGCCAAGGGAAGAGG-3'. The amplifications were under the following conditions: denaturation at 94°C for 4 min, followed by 30 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C. Control reactions using the Actin2 gene-specific primers (An et al., 1996
For isolating the homozygous plants of the T-DNA-tagged mutant line (SALK_091945), PCR amplifications were carried out using genomic DNAs extracted from 10-d-old seedlings. The primers flanking the Arabidopsis plantacyanin gene are the ones used in RT-PCR, and the T-DNA left-border primer LBb1 was described at http://signal.salk.edu/tdnaprimers.html. The conditions for RT-PCR to estimate the transcript level of Arabidopsis plantacyanin were as described above, except that total RNAs were extracted from inflorescences of the homozygous T-DNA-tagged mutant.
Treated seedlings or plant tissues were incubated at 37°C in a reaction buffer containing 10 mM EDTA, 100 mM sodium phosphate, pH 7.0, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 0.1% Triton X-100 with 1 mM 5-bromo-4-chloro-
DNA fragments encoding Arabidopsis plantacyanin (At2g02850) mature protein (without the signal peptide) were amplified from the cDNA clones. The Arabidopsis plantacyanin cDNA clone U21110 was available at the ABRC (Columbus, OH). The PCR products were designed to have proper restriction sites, NcoI and EcoRI, for a multicloning site in a vector (pHIS8-3) to generate recombinant proteins fused with a His-8-tag. Protein expression was induced by the addition of IPTG (ICN Biomedicals, Irvine, CA) at a final concentration of 1 mM. Expressed protein was purified on a Ni-NTA affinity column (Qiagen) following the manufacturer's instructions. Before protein refolding, the purified protein (in 8 M urea) was incubated with the reducing reagent
Plant tissues (seedlings, inflorescences, or pistils) were ground in liquid nitrogen to a fine powder. Total proteins were prepared by vortexing a sample of tissue powder in phosphate-buffered saline (PBS; 0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4) and separated by 13% SDS-PAGE. Proteins were transferred onto nitrocellulose membrane (MSI) by electroblotting. The membranes were blocked with 5% nonfat milk and incubated with a 2,000-fold dilution of antiserum against His plantacyanin. The antibody was detected with a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG; Bio-Rad, Boston) using the western-blot Chemiluminescence Reagent Plus (Bio-Rad).
Stage 12 Arabidopsis flowers (Smyth et al., 1990
Stage 12 to 13 flowers (nonpollinated) were emasculated and pollinated with wild-type pollen. The stigma/style was then cut off from the ovary using a razor blade and transferred to an 0.5% (v/v) agarose growth medium [containing 0.1% (v/v) boric acid, 25 mM CaCl2, 25 mM Ca(NO3)2, 10 mM MgSO4, and 18% (v/v) Suc, 1 M KOH added to adjust pH value to 6.5 to 7]. Samples were kept in a humid chamber overnight at room temperature. The samples were dried in a 60°C oven, stained with Coomassie Blue, and then observed under the light microscope. Images were documented with the Spot Insight camera (Diagnostic Instruments, Sterling Heights, MI) attached to a Leica (Deerfield, IL) MZ 12 stereomicroscope.
Plant tissues were fixed in FAA containing 37% (w/v) formaldehyde, glacial acetic acid, and 70% (v/v) ethanol (5:5:90; v/v/v), overnight at 4°C or in 2.5% glutaraldehyde/PBS overnight at 4°C (Lennon et al., 1998 Anthers from stage 13 flowers were squashed to release pollen and mounted in 50% (v/v) glycerol and viewed on a Nikon microscope. Pollen in Arabidopsis plantacyanin overexpression and wild-type anthers was released by hand dissection and viewed under a compound microscope.
Plant tissues were fixed with a FAA solution or a 2.5% glutaraldehyde/PBS solution and dehydrated as described above for light microscopy and then embedded in LR White (medium grade; EMS) and polymerized in a 56°C oven for 24 h. Three-micrometer sections were cut and mounted on Poly-L-Lys-coated slides (Fisher Scientific, Loughborough, Leicestershire, UK). Arabidopsis plantacyanin expression was detected by polyclonal antibodies (1:100 dilution) against E. coli-expressed plantacyanin (made by Cocalico Biologicals) and an alkaline phosphatase-conjugated secondary antibody (goat anti-rabbit IgG; 1:200 dilution; Bio-Rad). Tissues for paraffin embedding were fixed first in 2.5% glutaraldehyde/PBS for 2 h and then transferred to FAA overnight. After serial dehydration with ethanol (30%, 50%, 70%, 95%, and 100%), tissues were soaked with a series of tert-butanol (50%, 70%, and 100%) and infiltrated with Paraplast X-TRA (Fisher Scientific). Sections (8 µm) were cut and mounted on Poly-L-Lys-coated slides. Sections were deparaffinized by soaking in CitriSolv (Fisher) for 10 min and rehydrated in an ethanol series (100%, 95%, 70%, 50%, and 30%). For immunohistochemistry, sections were incubated with the primary antibody (anti-His plantacyanin; 1:200) and an alkaline phosphatase-conjugated secondary antibody (goat anti-rabbit IgG; 1:500). The samples were visualized mounted in nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution (Bio-Rad).
Anthers containing pollen were fixed in a solution of 75% (v/v) ethanol and 25% (v/v) acetic acid for 1 h and cleared overnight in 1 M NaOH at room temperature. After the samples were washed with double-distilled water for several changes, they were stained with decolorized aniline blue for callose (O'Brien and McCully, 1981
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requester.
We thank Dr. Zhenbiao Yang for his critical reading of the manuscript, Dr. Patricia Springer for providing the vectors constructed for promoter-GUS and gene overexpression, and Kimberly Tan for general lab assistance. Received March 26, 2005; returned for revision April 4, 2005; accepted April 4, 2005.
1 This work was supported by the National Science Foundation (grant nos. 0077886 and 0420445 to E.M.L.) and the Center for Plant Cell Biology at the University of California, Riverside (microscopy and imaging grant to J.D.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.063388. * Corresponding author; e-mail lord{at}ucr.edu; fax 951–827–4437.
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ABSTRACT
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5%) germinating precociously inside the closed anthers at flower stage 12 (
-Aminobutyric acid (GABA) is proposed to be an agent of pollen tube guidance in Arabidopsis by its maintenance of a concentration gradient along the pollen tube path in the pistil. Perturbation of the GABA gradient in vivo is correlated with disrupted pollen tube guidance, but in an in vitro assay a gradient of GABA has no chemotropic effect on pollen tube growth (Palanivelu et al., 2003
3)-
