International Journal of Molecular Sciences Article Spatio-Temporal Distribution of Cell Wall Components in the Placentas, Ovules and Female Gametophytes of Utricularia during Pollination Bartosz Jan Płachno 1,* , Małgorzata Kapusta 2 , Piotr Swi´ ˛atek 3 , Krzysztof Bana´s 4, Vitor F. O. Miranda 5 and Anna Bogucka-Kocka 6 1 Department of Plant Cytology and Embryology, Faculty of Biology, Institute of Botany, Jagiellonian University in Kraków, 9 Gronostajowa St., 30-387 Kraków, Poland 2 Department of Plant Cytology and Embryology, Faculty of Biology, University of Gda´nsk,59 Wita Stwosza St., 80-308 Gda´nsk,Poland; [email protected] 3 Faculty of Natural Sciences, Biotechnology and Environmental Protection, Institute of Biology, University of Silesia in Katowice, 9 Bankowa St., 40-007 Katowice, Poland; [email protected] 4 Department of Plant Ecology, Faculty of Biology, University of Gda´nsk,59 Wita Stwosza St., 80-308 Gda´nsk,Poland; [email protected] 5 Laboratory of Plant Systematics, School of Agricultural and Veterinarian Sciences, São Paulo State University (Unesp), Jaboticabal CEP 14884-900, SP, Brazil; [email protected] 6 Chair and Department of Biology and Genetics, Medical University of Lublin, 20-093 Lublin, Poland; [email protected] * Correspondence: [email protected] Abstract: In most angiosperms, the female gametophyte is hidden in the mother tissues and the Citation: Płachno, B.J.; Kapusta, M.; pollen tube enters the ovule via a micropylar canal. The mother tissues play an essential role in Swi´ ˛atek,P.; Bana´s,K.; Miranda, the pollen tube guidance. However, in Utricularia, the female gametophyte surpasses the entire V.F.O.; Bogucka-Kocka, A. micropylar canal and extends beyond the limit of the integument. The female gametophyte then Spatio-Temporal Distribution of Cell invades the placenta and a part of the central cell has direct contact with the ovary chamber. To date, Wall Components in the Placentas, information about the role of the placenta and integument in pollen tube guidance in Utricularia, Ovules and Female Gametophytes of which have extra-ovular female gametophytes, has been lacking. The aim of this study was to Utricularia Int. J. during Pollination. evaluate the role of the placenta, central cell and integument in pollen tube pollen tube guidance in Mol. Sci. 2021, 22, 5622. https:// Utricularia nelumbifolia Gardner and Utricularia humboldtii R.H. Schomb. by studying the production doi.org/10.3390/ijms22115622 of arabinogalactan proteins. It was also determined whether the production of the arabinogalactan Utricularia Academic Editor: Ryszard Lobinski proteins is dependent on pollination in . In both of the examined species, arabinogalactan proteins (AGPs) were observed in the placenta (epidermis and nutritive tissue), ovule (integument, Received: 3 April 2021 chalaza), and female gametophyte of both pollinated and unpollinated flowers, which means that the Accepted: 23 May 2021 production of AGPs is independent of pollination; however, the production of some AGPs was lower Published: 25 May 2021 after fertilization. There were some differences in the production of AGPs between the examined species. The occurrence of AGPs in the placental epidermis and nutritive tissue suggests that they Publisher’s Note: MDPI stays neutral function as an obturator. The production of some AGPs in the ovular tissues (nucellus, integument) with regard to jurisdictional claims in was independent of the presence of a mature embryo sac. published maps and institutional affil- iations. Keywords: arabinogalactan proteins; glycoproteins; AGPs; carnivorous plants; embryo sac; integu- ment; Lentibulariaceae; ovule; pollen tube guidance Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article In typical angiosperm plants, the female gametophyte (embryo sac) is hidden in the distributed under the terms and ovule and is covered by the nucellus and one or two integuments [1]. Angiosperm ovules conditions of the Creative Commons are incredibly diverse in the terms of their size, the degree of ovule curvature, nucellus Attribution (CC BY) license (https:// thickness, number of integuments and their thickness, the formation of the micropyle, the creativecommons.org/licenses/by/ length of the funiculus, the degree of the vascularization of the ovule and the presence of 4.0/). Int. J. Mol. Sci. 2021, 22, 5622. https://doi.org/10.3390/ijms22115622 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 5622 2 of 16 structures such as an endothecium, hypostase and postament [2]. Sporophytic tissues not only protect the female gametophyte and transport nutrients to it, but also guide the male gametophyte to the female gametophyte e.g., [3–7]. The great success of angiosperms is associated with the diverse structure of their styles and ovules and the sites of the pollen tube pathway [8–11]. During pollen tube growth, there is a dialogue between the male gametophytes and sporophytic tissues [12], but there is also competition between the pollen tubes, i.e., gametophytic competition [9,13]. In most cases, the pollen tube meets the female gametophyte only when it grows into the micropylar canal and the first elements of the female gametophyte that it meets are the synergids. However, there are some exceptions. In Plumbago and Plumbagella in which synergids are absent, the egg cell takes over the functions of a synergid [14]. Another evolutionary experiment is an extra-ovular female gametophyte, which extends beyond the limit of the integument. These specific partially “naked” female gametophytes have evolved in the different lineages of angiosperms, such as: Santalales: Thesium; Gentianales: Galium; Cornales: Philadelphus; Lamiales: Torenia, Lindernia and Utricularia [15–18]. Unfortunately, apart from Torenia and Lindernia, detailed knowledge about the interactions of an extra-ovular female gametophyte with the sporophytic tissues and pollen tubes is quite limited. The model species is Torenia fournieri Linden ex E. Fourn. [19,20], which has the extra-ovular egg apparatus and was used to prove that the synergids are responsible for attracting the pollen tubes in angiosperms [21–23]. In Torenia, the synergids produce small proteins (LUREs), which attract the pollen tubes [24]. However, the ovular arabinogalactan sugar chain (AMOR) is needed to make the pollen tubes fully competent for LURE peptides, and therefore the sporophytic tissues use arabinogalactan proteins to guide the gametophytic pollen tube [20,25]. Arabinogalactan proteins (AGPs) are important signaling molecules during cell–cell communication, which are spatio-temporally distributed in the plant generative organs e.g., [26–28] but have been also identified in vegetative organs (in mesophyll cell, epidermal cells, xylem, root cap cells, etc.) [29]. AGPs have been recorded in the styles, ovules and female gametophytes of various plant species. Their occurrence is connected with the specific stages of development, a specific tissue, organ and/or pollen tube growth e.g., [7,28,30–33]. In some species, the production of AGPs in ovules is triggered by pollen tube passage [6,33,34], while in others, their production is independent of pollination [7]. However, there is still a lack of information about the AGPs in ovules of Utricularia. In some species of this genus, the central cell is hypertrophied at the micropylar pole and invades the placenta. Moreover, a part of the central cell has direct contact with the ovary chamber [16,18,35]. According to Khan [36], in Utricularia, the pollen tube meets the female gametophyte in the ovarian cavity (exogamous fertilization). Although the occurrences of AGPs in the ovules and female gametophytes have been extensively studied in porogamous species, only one study has addressed this in a plant with a different mode of pollen tube growth, the chalazogamous species Mangifera indica L. [37]. Therefore, the aim of this study was to evaluate the role of the placenta (the epidermis and the special “nutritive tissue” that is situated below the embryo sac [38]), the ovu- lar tissues and hypertrophied central cell and the integument in pollen tube guidance in Utricularia nelumbifolia Gardner and Utricularia humboldtii R.H.Schomb. by studying the pro- duction of arabinogalactan proteins. It was also determined whether the arabinogalactan proteins in Utricularia are dependent on pollination. 2. Results 2.1. Arabinogalactan Proteins in the Placenta and Ovules before Pollination In both species, there were mature embryo sacs in most of the ovules during anthesis (Figure1A–C and Figure2A–C). Only some of the ovules did not contain a mature embryo sac (e.g., there was one nucleate embryo sac that had arrested in development). In Utricu- laria nelumbifolia and U. humboldtii, there was an intense accumulation of AGPs (labeled with JIM13) in the embryo sac, especially in the micropylar enlarged part of the central cell (Figure2D–H). Moreover, these AGPs were also detected in the synergids and egg cell Int. J. Mol. Sci. 2021, 22, 5622 3 of 16 Int. J. Mol. Sci. 2021, 22, 5622 3 of 16 nelumbifolia and U. humboldtii, there was an intense accumulation of AGPs (labeled with JIM13) in the embryo sac, especially in the micropylar enlarged part of the central cell (Figure 2D–H). Moreover, these AGPs were also detected in the synergids and egg cell (Figure 22E–G).E–G). InIn U. humboldtii , there was an intense accumulation of AGPs (labeled with JIM13) in the chalaza cells and integument cells at the micropylar pole (Figure 22G).G). OnlyOnly some of the placenta cells had these AGPs. Th Theseese cells were located near the micropylar part of the embryo sac. There were no AGPs (labeled with JIM13) in the chalazal ovular cells in Utricularia nelumbifolia ,, although although they they did did occur in a few of thethe integumentintegument cells that adhered to the embryo embryo sa sacc (Figure 22D).D). However,However, theythey onlyonly occurredoccurred inin thethe placentalplacental epidermal cells cells that that had had contact contact with with central central cell cell (Figure (Figure 2E).2E).