Plant Signaling & Behavior
ISSN: (Print) 1559-2324 (Online) Journal homepage: http://www.tandfonline.com/loi/kpsb20
Localization of arabinogalactan protein 6 fused with Sirius ultramarine fluorescent protein in Arabidopsis pollen and pollen tubes Luís Gustavo Pereira, Mário Costa & Sílvia Coimbra To cite this article: Luís Gustavo Pereira, Mário Costa & Sílvia Coimbra (2013) Localization of arabinogalactan protein 6 fused with Sirius ultramarine fluorescent protein in Arabidopsis pollen and pollen tubes, Plant Signaling & Behavior, 8:10, e25998, DOI: 10.4161/psb.25998 To link to this article: http://dx.doi.org/10.4161/psb.25998
Published online: 20 Aug 2013.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kpsb20 Download by: [Stockholm University Library]
Date: 13 September 2015, At: 07:35
Short Communication
Short Communication
Plant Signaling & Behavior 8:10, e25998; October 2013; © 2013 Landes Bioscience
Localization of arabinogalactan protein 6 fused with Sirius ultramarine fluorescent protein in Arabidopsis pollen and pollen tubes Luís Gustavo Pereira1,2, Mário Costa1,2, and Sílvia CoimbrB1,2,* Departamento de Biologia; Faculdade de Ciências; Universidade do Porto; Porto, Portugal; 2BioFIG; Center for Biodiversity; Functional and Integrative Genomics; Porto, Portugal
1
Downloaded by [Stockholm University Library] at 07:35 13 September 2015
Keywords: Arabidopsis, arabinogalactan proteins, AGP6, pollen tube, Sirius fluorescent protein
Following studies on the transcriptome of pollen tubes of an agp6 agp11 Arabidopsis double null mutant, together with the knowledge that arabinogalactan protein (AGP) 6 is important for male gametogenesis and pollen germination, we sought to know whether AGP6 could be present in the vegetative cell wall or in the generative cell wall or in both. To that end, Arabidopsis plants were transformed with AGP6 gene sequence fused with Sirius fluorescent protein. Fluorescent blue light emission could be detected in the vegetative cell wall only. This result supports the model by which AGP6 and other pollen AGPs are probably important signaling molecules at the pollen tube apex.
Arabinogalactan proteins (AGPs) are massively glycosylated plant proteins whose polysaccharide chains are composed predominantly but not exclusively of arabinose and galactose, and are O-linked to Hyp residues of the polypeptide chain. An added distinguishing feature of this class of proteins is the presence of a complex lipid anchor, the glycosylphosphatidylinositol (GPI) anchor, which holds these molecules to the exoplasmic side of the plasma membrane. AGPs can be found in virtually every cell throughout the plant kingdom. Their cytological localization, besides the plasma membrane, is mainly the cell wall, extracellular space, and sometimes they are also found abundantly in secretions. They have been functionally correlated with several fundamental biological phenomena, such as cell expansion and division, cell death, plant defense, pollen tube growth, and guidance, among others, but a definitive mode of action has not yet been established.1 As a result of studies in our group on Arabidopsis pistil and pollen development with anti-AGP monoclonal antibodies,2,3 it has become evident that AGPs could be used as fine molecular markers of gametophytic cell differentiation. For instance, JIM8, an anti-AGP monoclonal antibody, was found to label the generative cell only, with high specificity and precision, in bicellular pollen.3 Anti-AGP monoclonal antibodies, however, bind to sugar epitopes present in different AGP molecules, and consequently an analysis of the role of individual arabinogalactan gene products in a particular cell type or developmental stage is not possible by such approach. AGP6 and AGP11 are Arabidopsis pollen-specific AGPs, which, among others, have been a major interest in our
laboratory. These proteins were found to be important for male gametophytic viability and timely germination.4,5 We were therefore keen on knowing whether AGP6 epitopes were present in the generative cell wall or in the vegetative cell wall only. To that end, a custom-made synthetic construct based on a fusion between AGP6 and Sirius fluorescent protein6 was purchased from GenScript USA Inc. The construct included, from 5' to 3' direction: 1) AGP6 promoter region plus 5' untranslated region (771 nt in total), 2) start codon followed by the predicted signal peptide of AGP6 (63 nt), 3) the Sirius fluorescent protein coding sequence with codon optimization for plant expression, 4) a 6 amino acid spacer (ASGGGA), 5) the AGP6 mature peptide coding sequence (393 nt). The whole DNA sequence was flanked by Gateway attL sites for insertion into a suitable plant destination vector. A shorter version of the sequence described but without the promoter region, was isolated by PCR and the resultant DNA fragment was inserted into a pET30a plasmid, for IPTG-induced protein expression in E. coli. Bacterial colonies were bright blue under the fluorescence microscope indicating conformational viability of the Sirius::AGP6 fusion polypeptide. A Gateway expression vector containing the whole DNA sequence was introduced into wild type Arabidopsis by the floral dip method. Seeds from basta-resistant plants were subsequently grown and the respective pollen was collected and analyzed under the confocal fluorescence microscope. Blue fluorescence was detected in approximately half of the inspected pollen grains, as would be expected for hemizygous individuals (Fig. 1C and D). Higher magnifications of germinated pollen
*Correspondence to: Name: Sílvia Coimbra; Email: [email protected] Submitted: 07/25/13; Accepted: 08/01/13 Citation: Pereira LG, Costa M, Coimbra S. Localization of arabinogalactan protein 6 fused with Sirius ultramarine fluorescent protein in Arabidopsis pollen and pollen tubes. Plant Signaling & Behavior 2013; 8:e25998; http://dx.doi.org/10.4161/psb.25998; http://dx.doi.org/10.4161/jrn.25998 www.landesbioscience.com
Plant Signaling & Behavior e25998-1
Downloaded by [Stockholm University Library] at 07:35 13 September 2015
showed blue fluorescence along the vegetative cell wall, with no indications whatsoever of blue light emission associated with the generative cell (Fig. 1E). Very recently, we performed microarrays with the double mutant agp6 agp11 pollen tube as well as yeast two-hybrid assays with the AGP6 and AGP11.7 Efforts to understand the biological function of AGPs have been persistent because many members of the class seem to regulate many important aspects of plant growth, development, and resistance to different types of stress. The results obtained in those studies provided possible interactors for these AGPs that have reinforced their roles as important structural proteins for pollen grain and pollen tube, knowing that AGPs are complex supramolecular scaffolds which can contribute to the reinforcement of cell walls. We also propose that AGP6 and AGP11 are most likely important signaling molecules at the pollen tube apex, being recycled back again to the apex as the pollen tube grows, by a clathrin mediated endocytosis pathway. Besides, GPI-anchored proteins are widespread cell sensors in mammals, especially during egg-sperm communication. References 1. Seifert GJ, Roberts K. The biology of arabinogalactan proteins. Annu Rev Plant Biol 2007; 58:137-61; http://dx.doi.org/10.1146/annurev. arplant.58.032806.103801; PMID:17201686 2. Pereira LG, Coimbra S, Oliveira H, Monteiro L, Sottomayor M. Expression of arabinogalactan protein genes in pollen tubes of Arabidopsis thaliana. Planta 2006; 223:374-80; http://dx.doi.org/10.1007/s00425005-0137-4; PMID:16228244 3. Coimbra S, Almeida J, Junqueira V, Costa ML, Pereira LG. Arabinogalactan proteins as molecular markers in Arabidopsis thaliana sexual reproduction. J Exp Bot 2007; 58:4027-35; http://dx.doi.org/10.1093/jxb/ erm259; PMID:18039740
e25998-2
4.
5.
6.
The choice of Sirius fluorescent protein, a GFP variant obtained after random mutagenesis of the amino acids surrounding the chromophore,6 was used in the present work for its alleged pH insensitivity, and as far as we know was used for the first time in plant cells or tissues. This should potentially be advantageous in studies of the plant cell wall, which is known to be an acidic environment. However, a major drawback for its wider use is its extremely low absorption peak (355 nm), which is not compatible with most light sources of confocal fluorescence microscopes. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
This work was supported by FEDER funds through the COMPETE program and by FCT (Fundação para a Ciência e Tecnologia, Portugal) national funds within the project PTDC/ AGR-GPL/115358/2009.
Coimbra S, Costa ML, Jones B, Mendes MA, Pereira LG. Pollen grain development is compromised in Arabidopsis agp6 agp11 null mutants. J Exp Bot 2009; 60:3133-42; http://dx.doi.org/10.1093/jxb/erp148; PMID:19433479 Coimbra S, Costa ML, Mendes MA, Pereira AM, Pinto J, Pereira LG. Early germination of Arabidopsis pollen in a double null mutant for the arabinogalactan protein genes AGP6 and AGP11. Sex Plant Reprod 2010; 23:199-205; http://dx.doi.org/10.1007/s00497-0100136-x; PMID:20162305 Tomosugi W, Matsuda T, Tani T, Nemoto T, Kotera I, Saito K, Horikawa K, Nagai T. An ultramarine fluorescent protein with increased photostability and pH insensitivity. Nat Methods 2009; 6:351-3; http:// dx.doi.org/10.1038/nmeth.1317; PMID:19349978
Plant Signaling & Behavior
7.
Costa M, Nobre S, Becker J, Masiero S, Amorim MI, Pereira LG, Coimbra S. On hand, putative ligands for arabinogalactan proteins in Arabidopsis pollen development. BMC Plant Biol 2013; 13:7; http://dx.doi. org/10.1186/1471-2229-13-7; PMID:23297674
Volume 8 Issue 10
Downloaded by [Stockholm University Library] at 07:35 13 September 2015
Figure 1. Localization of AGP6 in Arabidopsis pollen (A-D) and pollen tube cell wall (E), using a sirius fluorescence protein-based fusion (F). Blue fluorescent light was found associated with the vegetative cell wall only. (A) Bright field microscopy image; (B and E) Confocal optical microscopy images; (C) Bright field and confocal microscopy images combined; (D) Higher magnification of combined image; attL – Gateway recombination sequences; CDS – coding sequence; SP – signal peptide; UTR – untranslated sequence. Scale bars in (A), (B), and (C), 150 μm, in (D), 80 μm, and in (E), 40 μm.
www.landesbioscience.com
Plant Signaling & Behavior e25998-3
ISSN: (Print) 1559-2324 (Online) Journal homepage: http://www.tandfonline.com/loi/kpsb20
Localization of arabinogalactan protein 6 fused with Sirius ultramarine fluorescent protein in Arabidopsis pollen and pollen tubes Luís Gustavo Pereira, Mário Costa & Sílvia Coimbra To cite this article: Luís Gustavo Pereira, Mário Costa & Sílvia Coimbra (2013) Localization of arabinogalactan protein 6 fused with Sirius ultramarine fluorescent protein in Arabidopsis pollen and pollen tubes, Plant Signaling & Behavior, 8:10, e25998, DOI: 10.4161/psb.25998 To link to this article: http://dx.doi.org/10.4161/psb.25998
Published online: 20 Aug 2013.
Submit your article to this journal
Article views: 63
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kpsb20 Download by: [Stockholm University Library]
Date: 13 September 2015, At: 07:35
Short Communication
Short Communication
Plant Signaling & Behavior 8:10, e25998; October 2013; © 2013 Landes Bioscience
Localization of arabinogalactan protein 6 fused with Sirius ultramarine fluorescent protein in Arabidopsis pollen and pollen tubes Luís Gustavo Pereira1,2, Mário Costa1,2, and Sílvia CoimbrB1,2,* Departamento de Biologia; Faculdade de Ciências; Universidade do Porto; Porto, Portugal; 2BioFIG; Center for Biodiversity; Functional and Integrative Genomics; Porto, Portugal
1
Downloaded by [Stockholm University Library] at 07:35 13 September 2015
Keywords: Arabidopsis, arabinogalactan proteins, AGP6, pollen tube, Sirius fluorescent protein
Following studies on the transcriptome of pollen tubes of an agp6 agp11 Arabidopsis double null mutant, together with the knowledge that arabinogalactan protein (AGP) 6 is important for male gametogenesis and pollen germination, we sought to know whether AGP6 could be present in the vegetative cell wall or in the generative cell wall or in both. To that end, Arabidopsis plants were transformed with AGP6 gene sequence fused with Sirius fluorescent protein. Fluorescent blue light emission could be detected in the vegetative cell wall only. This result supports the model by which AGP6 and other pollen AGPs are probably important signaling molecules at the pollen tube apex.
Arabinogalactan proteins (AGPs) are massively glycosylated plant proteins whose polysaccharide chains are composed predominantly but not exclusively of arabinose and galactose, and are O-linked to Hyp residues of the polypeptide chain. An added distinguishing feature of this class of proteins is the presence of a complex lipid anchor, the glycosylphosphatidylinositol (GPI) anchor, which holds these molecules to the exoplasmic side of the plasma membrane. AGPs can be found in virtually every cell throughout the plant kingdom. Their cytological localization, besides the plasma membrane, is mainly the cell wall, extracellular space, and sometimes they are also found abundantly in secretions. They have been functionally correlated with several fundamental biological phenomena, such as cell expansion and division, cell death, plant defense, pollen tube growth, and guidance, among others, but a definitive mode of action has not yet been established.1 As a result of studies in our group on Arabidopsis pistil and pollen development with anti-AGP monoclonal antibodies,2,3 it has become evident that AGPs could be used as fine molecular markers of gametophytic cell differentiation. For instance, JIM8, an anti-AGP monoclonal antibody, was found to label the generative cell only, with high specificity and precision, in bicellular pollen.3 Anti-AGP monoclonal antibodies, however, bind to sugar epitopes present in different AGP molecules, and consequently an analysis of the role of individual arabinogalactan gene products in a particular cell type or developmental stage is not possible by such approach. AGP6 and AGP11 are Arabidopsis pollen-specific AGPs, which, among others, have been a major interest in our
laboratory. These proteins were found to be important for male gametophytic viability and timely germination.4,5 We were therefore keen on knowing whether AGP6 epitopes were present in the generative cell wall or in the vegetative cell wall only. To that end, a custom-made synthetic construct based on a fusion between AGP6 and Sirius fluorescent protein6 was purchased from GenScript USA Inc. The construct included, from 5' to 3' direction: 1) AGP6 promoter region plus 5' untranslated region (771 nt in total), 2) start codon followed by the predicted signal peptide of AGP6 (63 nt), 3) the Sirius fluorescent protein coding sequence with codon optimization for plant expression, 4) a 6 amino acid spacer (ASGGGA), 5) the AGP6 mature peptide coding sequence (393 nt). The whole DNA sequence was flanked by Gateway attL sites for insertion into a suitable plant destination vector. A shorter version of the sequence described but without the promoter region, was isolated by PCR and the resultant DNA fragment was inserted into a pET30a plasmid, for IPTG-induced protein expression in E. coli. Bacterial colonies were bright blue under the fluorescence microscope indicating conformational viability of the Sirius::AGP6 fusion polypeptide. A Gateway expression vector containing the whole DNA sequence was introduced into wild type Arabidopsis by the floral dip method. Seeds from basta-resistant plants were subsequently grown and the respective pollen was collected and analyzed under the confocal fluorescence microscope. Blue fluorescence was detected in approximately half of the inspected pollen grains, as would be expected for hemizygous individuals (Fig. 1C and D). Higher magnifications of germinated pollen
*Correspondence to: Name: Sílvia Coimbra; Email: [email protected] Submitted: 07/25/13; Accepted: 08/01/13 Citation: Pereira LG, Costa M, Coimbra S. Localization of arabinogalactan protein 6 fused with Sirius ultramarine fluorescent protein in Arabidopsis pollen and pollen tubes. Plant Signaling & Behavior 2013; 8:e25998; http://dx.doi.org/10.4161/psb.25998; http://dx.doi.org/10.4161/jrn.25998 www.landesbioscience.com
Plant Signaling & Behavior e25998-1
Downloaded by [Stockholm University Library] at 07:35 13 September 2015
showed blue fluorescence along the vegetative cell wall, with no indications whatsoever of blue light emission associated with the generative cell (Fig. 1E). Very recently, we performed microarrays with the double mutant agp6 agp11 pollen tube as well as yeast two-hybrid assays with the AGP6 and AGP11.7 Efforts to understand the biological function of AGPs have been persistent because many members of the class seem to regulate many important aspects of plant growth, development, and resistance to different types of stress. The results obtained in those studies provided possible interactors for these AGPs that have reinforced their roles as important structural proteins for pollen grain and pollen tube, knowing that AGPs are complex supramolecular scaffolds which can contribute to the reinforcement of cell walls. We also propose that AGP6 and AGP11 are most likely important signaling molecules at the pollen tube apex, being recycled back again to the apex as the pollen tube grows, by a clathrin mediated endocytosis pathway. Besides, GPI-anchored proteins are widespread cell sensors in mammals, especially during egg-sperm communication. References 1. Seifert GJ, Roberts K. The biology of arabinogalactan proteins. Annu Rev Plant Biol 2007; 58:137-61; http://dx.doi.org/10.1146/annurev. arplant.58.032806.103801; PMID:17201686 2. Pereira LG, Coimbra S, Oliveira H, Monteiro L, Sottomayor M. Expression of arabinogalactan protein genes in pollen tubes of Arabidopsis thaliana. Planta 2006; 223:374-80; http://dx.doi.org/10.1007/s00425005-0137-4; PMID:16228244 3. Coimbra S, Almeida J, Junqueira V, Costa ML, Pereira LG. Arabinogalactan proteins as molecular markers in Arabidopsis thaliana sexual reproduction. J Exp Bot 2007; 58:4027-35; http://dx.doi.org/10.1093/jxb/ erm259; PMID:18039740
e25998-2
4.
5.
6.
The choice of Sirius fluorescent protein, a GFP variant obtained after random mutagenesis of the amino acids surrounding the chromophore,6 was used in the present work for its alleged pH insensitivity, and as far as we know was used for the first time in plant cells or tissues. This should potentially be advantageous in studies of the plant cell wall, which is known to be an acidic environment. However, a major drawback for its wider use is its extremely low absorption peak (355 nm), which is not compatible with most light sources of confocal fluorescence microscopes. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
This work was supported by FEDER funds through the COMPETE program and by FCT (Fundação para a Ciência e Tecnologia, Portugal) national funds within the project PTDC/ AGR-GPL/115358/2009.
Coimbra S, Costa ML, Jones B, Mendes MA, Pereira LG. Pollen grain development is compromised in Arabidopsis agp6 agp11 null mutants. J Exp Bot 2009; 60:3133-42; http://dx.doi.org/10.1093/jxb/erp148; PMID:19433479 Coimbra S, Costa ML, Mendes MA, Pereira AM, Pinto J, Pereira LG. Early germination of Arabidopsis pollen in a double null mutant for the arabinogalactan protein genes AGP6 and AGP11. Sex Plant Reprod 2010; 23:199-205; http://dx.doi.org/10.1007/s00497-0100136-x; PMID:20162305 Tomosugi W, Matsuda T, Tani T, Nemoto T, Kotera I, Saito K, Horikawa K, Nagai T. An ultramarine fluorescent protein with increased photostability and pH insensitivity. Nat Methods 2009; 6:351-3; http:// dx.doi.org/10.1038/nmeth.1317; PMID:19349978
Plant Signaling & Behavior
7.
Costa M, Nobre S, Becker J, Masiero S, Amorim MI, Pereira LG, Coimbra S. On hand, putative ligands for arabinogalactan proteins in Arabidopsis pollen development. BMC Plant Biol 2013; 13:7; http://dx.doi. org/10.1186/1471-2229-13-7; PMID:23297674
Volume 8 Issue 10
Downloaded by [Stockholm University Library] at 07:35 13 September 2015
Figure 1. Localization of AGP6 in Arabidopsis pollen (A-D) and pollen tube cell wall (E), using a sirius fluorescence protein-based fusion (F). Blue fluorescent light was found associated with the vegetative cell wall only. (A) Bright field microscopy image; (B and E) Confocal optical microscopy images; (C) Bright field and confocal microscopy images combined; (D) Higher magnification of combined image; attL – Gateway recombination sequences; CDS – coding sequence; SP – signal peptide; UTR – untranslated sequence. Scale bars in (A), (B), and (C), 150 μm, in (D), 80 μm, and in (E), 40 μm.
www.landesbioscience.com
Plant Signaling & Behavior e25998-3
