Research article
Arabinogalactan proteins: Immunolocalization in the developing ovary of a facultative apomict Fragaria x ananassa (Duch.)

https://doi.org/10.1016/j.plaphy.2017.12.003Get rights and content

Highlights

  • AGPs occur in ovule of amphimictic and apomictic plants.

  • In F. x ananassa mostly resembles the distribution in the amphimictic rather than apomictic reproduction modes.

  • AGPs presence confirms their role in megasporogenesis and embryo sac differentiation.

  • AGPs as a signaling molecules participate in female-male gametophytes interaction.

Abstract

Background and aims

Arabinogalactan proteins are present in the extracellular matrix and their occurrence is developmentally regulated. The studies were carried out to localise arabinogalactan proteins in ovary cells of Fragaria x ananassa Duchesne (strawberry) during megasporogenesis, megagametogenesis, and formation of the embryo.

Methods

The research included studies of ovary histochemistry and immunofluorescence labelling of AGP epitopes was performed with antibodies JIM13, JIM15 and MAC207. The use of the immunogold labelling method allowed specific detection of AGP epitopes at the subcellular level.

Key results

The localization of AGPs was studied in the cells of the ovary wall and elements building the developing ovule i.e. the integument, nucellus, archespore, megaspores, embryo sac, and embryo of a facultative apomict Fragaria x ananassa cv. 'Mount Everest'. For the first time the presence of AGP epitopes at the stage of a multicellular archespore was described. The occurrence of AGPs in the functional megaspore walls is related to selection of a megaspore continuing development; during later stages of development, AGPs are also evident markers of the female gametophyte. The intense fluorescence indicates the presence of AGPs in the embryo sac wall as well as in the cytoplasm compartment of the egg apparatus and around the secondary nucleus of the central cell. The localization of AGPs in the ovule of F. x ananassa resembles the distribution of these proteins in amphimictic plants.

Conclusions

Arabinogalactan proteins occur in similar parts of the ovule of amphimictic and apomictic plants. The results confirm the participation of AGPs in reproductive structures as a useful marker during development of female gametophyte.

Introduction

As integral components of plant cell surface and secretions, arabinogalactan proteins (AGPs) are widespread throughout the plant kingdom. The primary role of these cell wall elements, belonging to hydroxyproline-rich glycoproteins, is co-formation of the amorphous extracellular matrix. The variable, complex molecular structure of AGP makes them a precise cell identity marker or a signal for neighbouring cells (Knox, 1995, Cheung et al., 2000). The role of AGPs as signalling molecules is determined by the possibility of detachment of the GPI (glycosylphosphatidylinositol lipid) anchor, good solubility in aqueous solutions, and the ability to diffuse within the extracellular matrix (Showalter, 2001, Rumyantseva, 2005). Some research performed on different tissue of various species indicates that the presence of AGPs is regulated both in time and in space. Even in a single cell and in the particular stages of it development, it is possible to notice the presence of different kinds of AGPs (Majewska-Sawka and Nothnagel, 2000, Showalter, 2001, Tan et al., 2012). The heterogeneous nature of AGPs is reflected in the intensively studied variety of their functions. However, it is still impossible to determine their specific role(s) (Ellis et al., 2010). Therefore, to determine their functions precisely, the localization of AGPs at the level of tissues and cells in different plant organs can provide knowledge of their great versatility.

Arabinogalactan proteins are perceived as indispensable participants in the process of sexual plant reproduction. They are considered as a group of proteins involved in many variable stages of this process. Currently, there are available data about the localization and roles of arabinogalactan proteins in the development of pollen grains as well as their contribution during the germination and pollen tube movement in the transmission tissue at the progamic phase (Cheung and Wu, 1999, Nguema-Ona et al., 2012, Pereira et al., 2015). Moreover, authors of many studies focused on the biochemical structure of the pistil and, in particular, on megasporogenesis and the development of female gametophyte (Coimbra and Pereira, 2012, Losada and Herrero, 2012, Suárez et al., 2013, Costa et al., 2013, Pereira et al., 2014). The first report about the role of arabinogalactan proteins during megasporogenesis appeared over two decades ago. Pennell and Roberts (1990) described AGPs as markers indicating the initiation of the female gametophyte development. Since that time, different reports have discussed the role of arabinogalactan proteins as a source of nutrient materials for the growing embryo sac, their association with the pollen tube directing in the nucellus, and the possibility of signalling the location of a fertile female gametophyte based on the studies of amphimictic plants (Pereira et al., 2006, Pereira et al., 2015). As precise markers, mostly in the embryo sac cell wall, AGPs have been reported in Actinidia deliciosa (Coimbra and Duarte, 2003) or Arabidopsis thaliana (Coimbra et al., 2007, Pereira et al., 2014). The immunochemical and molecular biology methods facilitate examination of AGP functions as macromolecules determining changes in ovule cell development programs (Rafińska and Bednarska, 2011), functional megaspore (Acosta-García and Vielle-Calzada, 2004; Tucker and Koltunow, 2014), and egg apparatus (Qin and Zhao, 2006, Demesa-Arévalo and Vielle-Calzada, 2013, Lopez and Renzaglia, 2016).

Although the interest in arabinogalactan proteins has recently increased significantly, the current knowledge concerning the localization and function of AGPs has still many shortcomings. So far, no one has dealt with the distribution of AGPs in facultative apomicts. The existing many inconsistencies and gaps in the information regarding the widely understood generative reproduction of Fragaria x ananassa have prompted studies of factors influencing proper formation of embryos and viable seeds (Mudge et al., 1981, Strik and Proctor, 1988). Ariza et al. (2011) indicate the disruption of post-fertilization processes as a cause of the occurrence of non-functional achenes. Such disturbances can appear also during key periods of plants reproduction such as megasporogenesis and during the formation of the female gametophyte. All mentioned processes determine the quality of fruits, thereby affecting their macro-properties, which are important for consumers and industry. Generally, there are a few reports on the female gametophyte in economically important strawberry and they are limited to only a few cultivars. The analysis of AGPs as one of the cell wall components could be used to understand the changeable (amphimictic and apomictic) modes of Fragaria x ananassa reproduction. In our previous work (Leszczuk and Szczuka, 2017) the first, preliminary information on specific epitopes of arabinogalactan proteins in the cells of Fragaria x ananassa (Duch) ovules has been reported. Therefore, the aim of our present study was to carry out a particular analysis of AGP localizations at different stages of ovule and ovary development in the facultative apomict Fragaria x ananassa cultivar 'Mount Everest'.

Section snippets

Plant material

Fresh flowers buds of different sizes and flowers during and after anthesis were collected directly from strawberry plants (Fragaria x ananassa Duchesne cv. 'Mount Everest') during three flowering seasons from 2014 to 2016. The plants were cultivated in an experimental plot in the Botanical Garden of the MCS University in Lublin (Poland). The observations were carried out on the individual ovaries with ovules in various developmental stages. Fresh flower buds were collected and then ovaries

Immunolocalization of arabinogalactan proteins during megasporogenesis

The flower of Fragaria x ananassa cv. 'Mount Everest' has a typical structure for representatives of the Fragaria genus. Anther and pistil appendages directly associated with reproduction are located in the centre of the bisexual flower (Fig. 1A). Numerous pistils deposited on an expanded receptacle differ in the phase of development (Fig. 1B). Each note-like shaped pistil consists of the ovary, style, and stigma (Fig. 1C). Ovaries also vary in size: at the early stages of development, they are

Discussion

The diverse gene expression and localization of the various types of arabinogalactan proteins (AGPs) in stamens and carpels are clear evidence for their significant involvement and impact on various aspects of different processes of plant growth and development, including reproductive processes (Showalter, 2001, Pereira et al., 2015). In the present paper, the localization of AGPs in the ovary, usually containing only a single ovule, of the facultative apomict Fragaria x ananassa was studied

Author contribution

AL conducted the experiments, interpreted data and wrote the manuscript; ES helped in data interpretation and manuscript preparation.

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