Reproductive Strategies of the Female Gametophyte

*Xiaoyan Liu and Ryushiro D. Kasahara*

## **Abstract**

Reproduction refers to the biological process of producing offspring. Sexual reproduction in angiosperms is a complex and precise process of regulation, which requires the mutual recognition of male and female gametes. The pollen tube, as a medium for transmission of male gametes, is attracted by chemoattractant derived from synergid cells in a target ovule. We first identified that MYB98 plays an important role in pollen tube guidance by regulating the downstream LURE peptides. Moreover, we discovered that if the first pollen tube penetrates the ovule but fertilization fails, the second pollen tube is attracted by another synergid cell to increase the chances of fertilization as a "wise" back-up system (Fertilization Recovery System). Similar feedback mechanisms also occur to seed development after the pollen tube guidance. We further identified a phenomenon, the pollen tube-dependent ovule enlargement morphology (POEM), that the pollen tube contents (PTCs) enlarge ovules and initiate seed coat formation without fertilization. Furthermore, we identified the POEM in rice by knocked-out GCS1 genes in rice genome, which led to fertilization failure and to produce enlarged sugar grain. In this chapter, we discuss from the reproductive strategies of the plants to the agricultural application based on our previous discoveries.

**Keywords:** pollen tube guidance, MYB98, LUREs, fertilization recovery system, POEM, sugar rice

## **1. Introduction**

Seed plants, also known as higher vascular plants, include gymnosperms and angiosperms, which have evolved a unique life cycle: flowering, pollination, fertilization, and seed formation. Double fertilization is a flowering plant mechanism whereby two immotile sperm cells fertilize two different female gametes. In order to achieve the fusion of sexual gametes (fertilization), the mature pollen (male gametophyte) is dispersed from the anther to the stigmas that undergo the long journey to the ovule (inside the ovule is the female gametophyte), which requires the mutual recognition of male and female gametes (**Figure 1**). In this chapter, we will discuss from the pollen tube guidance, fertilization, the induction of seed development, and to the agricultural application based on our previous discoveries.

#### **Figure 1.**

*The Arabidopsis male and female gametophyte. A. The flower of Arabidopsis. B. Anther of Arabidopsis thaliana, Anthers dehiscent, exposing pollen grains. C. Stigmas bearing pollen grains. D. Pollen tube stained by aniline blue, WT self-cross 8HAP. E. Pollens fall onto the stigma to grow pollen tubes. F. Male gametophyte (pollen grain), with one vegetative cell and two sperm cells inside. G. Female gametophyte, consists of seven cells and four different cell types: three antipodal cells, two synergid cells, one egg cell, and one central cell.*

## **2. Development of gametophytes**

During the angiosperm life cycle, the angiosperm gametophytes develop within sporophytic tissues that constitute the sexual organs of the flower. The sporophyte produces two types of spores, microspores and megaspores, that give rise to male gametophytes and female gametophytes, respectively [1]. Female gametophyte development goes through two processes referred to as megasporogenesis and megagametogenesis. The diploid megaspore mother cell undergoes meiosis and gives rise to four haploid megaspores. After that, three megaspores go through cell death, and the only megaspore left goes through three rounds of mitosis without cytokinesis, resulting in a multinucleate coenocyte. Subsequently, cell walls form around these nuclei, forming the cellular female gametophyte (**Figure 1G**), which consists of seven cells and four different cell types: three antipodal cells, two synergid cells, one egg cell, and one central cell [2]. The male gametophyte (**Figure 1F**), also referred to as the pollen grain or microgametophyte, develops within the anther (**Figure 1B**) and is composed of two sperm cells encased within a vegetative cell.

## **3. Pollen adhesion**

Once a pollen grain adheres to the stigma, compatible pollen must be distinguished from potential pathogens (fungal spores or bacteria). In response to compatible pollen, stigmas initiate a basal response pathway that transfers water to the desiccated pollen grain for pollen hydration and germination [3].

*Reproductive Strategies of the Female Gametophyte DOI: http://dx.doi.org/10.5772/intechopen.109805*

In self-incompatible plants of the genus Brassica, self-related pollen grains are recognized and prevented to germinate by interaction with the epidermal cells of the stigma (**Figure 2B**). The self-incompatibility (SI) phenomenon is genetically controlled by a complex and polymorphic locus [4]. Among the genes, S locus is a pair of sequence-related genes, the cell wall localized S-locus glycoprotein (*SLG*) gene and the plasma membrane spanning receptor protein kinase (*SRK*) gene, both of which are expressed specifically in the stigma epidermal cells. The pollen coat localized S-locus cysteine-rich/S-locus protein 11 (SCR/SP11) ligands interacts with S-receptor kinase (SRK), thereby inducing its self-activation during the self-pollen recognition process [5–7]. However, for compatible pollen, due to the absence of pollen coat protein, self-activation of SRK is inhibited by thioredoxin H-like1 (THL1), resulting pollen hydration and germination [8, 9]. Upon phosphorylation and activation, SRK forms a complex with M-locus protein kinase (MLPK, a plasma-membrane-localized receptor-like cytoplasmic kinase protein), which in turn interacts with arm-repeatcontaining protein 1 (ARC1, a pistil specific E3 ubiquitin ligase protein) [10, 11]. The activated ARC1 is directly involved in the ubiquitination of Exo70A1, a plasma membrane-localized peptide, which is involved in the secretory vesicle delivery to the papillae surface [12–14]. The study shows that in the absence of functional Exo70A1, the exocyst complex-derived stigmatic secretion failed [15]. In addition, studies indicate that pollen-specific plasma-membrane-localized aquaporins (NIP4;1,

#### **Figure 2.**

*Pollen landing on stigma to germinate pollen tube. A. Pollen adhesion, hydration, and germination. B. SRK recognizes pollen of the same plant by interacting with SCR/SP11 thereby phosphorylating ARC1 which, in turn, interacts with Exo70A1 and evokes self-incompatibility by hindering stigmatic secretion. In the absence of SRK interaction with pollen-specific SCR/SP11, it interacts with stigma-specific THL1 and ARC1 phosphorylation is hindered. Hence, normal stigmatic secretion proceeds and pollen gets hydrated. C. Model of compatible pollen recognition. Before pollination, RALF23/33 induces ROS production in the stigmatic papilla cells through an ANJ-FER-ROP2-RBOHD pathway. After pollination with compatible pollen, PCP-Bs from the pollen coat compete with RALF23/33 for interaction with the ANJ-FER complex, repressing ROS production and initiating stigmatic responses.*

NIP4;2, TIP1;3, TIP5;1) are potential macromolecules involved in pollen hydration by rendering pollen PM permeable to water and other solutes [16, 17].

Although the main mechanisms of action of different types of self-incompatibility systems have been clarified, in contrast, relatively little attention has been paid to compatible pollen recognition and the corresponding cellular responses in the stigmatic papillae. In *Arabidopsis thaliana*, the pollen coat PCP-Bs are excellent candidates for the pollen ligand to initiate the basal compatible pollen acceptance pathway in the papilla. The loss of PCP-Bs notably slows pollen hydration and germination [18]. Recently, a stigmatic gatekeeper, the ANJEA–FERONIA (ANJ–FER) receptor kinase complex, perceives the RAPID ALKALINIZATION FACTOR peptides RALF23 and RALF33 to induce reactive oxygen species (ROS) production in the stigma papillae, whereas pollination reduces stigmatic ROS, allowing pollen hydration. Upon pollination, PCP-Bs compete with RALF23/33 for binding to the ANJ-FER complex, leading to a decline of stigmatic ROS that facilitates pollen hydration (**Figure 1C**) [19].

## **4. Pollen tube guidance at the micropylar region**

Several studies have reported that the pollen tubes fail to grow onto ovules containing abnormal female gametophytes, suggesting that the embryo sac provides a guiding cue for the pollen tubes [20–22]. The study on Oenothera showed that PT directly enters synergid cell, which is where it bursts and releases their contents along with two sperm cells. Later studies further demonstrated the synergid cell as being the only source of short-distance pollen tube attractants at the micropylar region [23, 24]. The synergid cells have structural specializations that facilitate the fertilization process. Each synergid cell consists of large vacuole at its chalazal end, and a nucleus and stack of ER, along with Golgi complexes, at its micropylar end [25]. The micropylar tip of each synergid cell wall is extensively invaginated, forming a structure referred to as the filiform apparatus [26]. MYB98, a synergid cell-specific R2R3 transcription factor, affects the development of filiform apparatus and is crucially important for micropylar PT guidance [27].

A later study showed that MYB98 affects the expression of arrays of SC-specific genes encoding defensin-like cysteine-rich proteins (CRPs), which are secreted into the filiform apparatus and are involved in PT guidance [28]. LURE1 and LURE2, two of the synergid cell produced CRPs, are directly involved in micropylar pollen tube guidance [29, 30]. Recent studies have shown that these proteins interact with receptors such as PRK6 [31] and MIDIS1-MIK [32] that are produced by the PTs and guide it toward the micropylar. The pollen tube tip-specific LIP1 and LIP2, members of RLKs, also regulate micropylar PT guidance and affect PT attraction toward LURE1 (**Figure 3**A). The study in *Torenia fournieri* showed that AMOR, an arabinogalactan polysaccharide secreted by mature ovules, also plays a positive role in rendering the growing PT competent to interact with LURE attractants [33]. The AtLURE1/ PRK6-mediated signaling pathway thus guarantees a strong and biased precedence for own pollen tubes and contributes to prezygotic reproductive isolation in the genus Arabidopsis. Except LUREs, another group of CRPs, XIUQIU1–4 (**Figure 3A**), attracts PTs without any species bias [34, 35]. However, the interaction of XIUQIU to as yet unknown PT-receptor/s is likely conserved across Brassicaceae members as it can effectively attract PTs of *Arabidopsis lyrata* and Ca*ps*ella *rubella*.

In addition to the important role of synergid cell in pollen tube guidance, central cells also seem to influence pollen tube guidance. A central cell-specific Central Cell Guidance *Reproductive Strategies of the Female Gametophyte DOI: http://dx.doi.org/10.5772/intechopen.109805*

#### **Figure 3.**

*Pollen tube guidance and double fertilization. A. Micropylar guidance is regulated by LURE-PRK6/MDIS1- MIK interaction, TIC2, XIUQIU, AMOR. Central cell localized CCG and synergid cell localized MYB98 affect CRP biosynthesis. B. HAP2/GCS1 interacts with DMP8 and DMP9, which are required for the EC1-induced translocation of HAP2/GCS1 from internal storage vesicle to the sperm plasma membrane to ensure successful fertilization.*

(CCG), encoding a nuclear protein with an N-terminal conserved zinc b-ribbon domain, has also been reported to affect micropylar pollen tube guidance [36, 37]. Interestingly, in *ccg* mutant, MYB98 and other CRP genes, including LURE1, are downregulated [37]. This is most likely due to the changes in MYB98 expression, or MYB98 along with CCG and CBP1, co-regulate the expression of those CRPs. However, if they were to co-regulate along with MYB98, additional evidence and explanations are required [38].

## **5. Discharge of sperm cells from the pollen tube tip to fertilization**

Pollen tube growth arrests within a synergid and the pollen tube tip bursts, releasing the PT contents, including the two sperm cells [39]. This process is accompanied by the degeneration of the receptive synergid and is rapidly followed by the fusion of one sperm with the egg and one sperm with the central cell to give rise to the zygote and endosperm, respectively [40, 41].

Double fertilization proceeds through the recognition, attachment, and membrane fusion of male and female gametes, and these processes are directly regulated by proteins on the gamete surface (**Figure 3B**) [42]. Male gametic membrane proteins HAP2/GCS1, GEX2, DMP8, and DMP9 have been identified as fertilization regulators affecting male-female gamete fusion [43–46]. The first gamete fusogen identified was HAP2/GCS1, which share a similar structure with two distinct families of exoplasmic fusogens: the somatic Fusion Family (FF) and class II viral glycoproteins [47]. The egg cell-specific EC1, which was reported to be crucial for sperm cell activation [48], also plays an important role in sperm cell attachment to the plasma membrane of both the egg cell and central cell [46]. EC1 proteins accumulate in storage vesicles of the egg cell. Upon sperm arrival, EC1-containing vesicles are exocytosed. The sperm endomembrane system responds to exogenously applied EC1 peptides by redistributing the potential gamete fusogen HAP2/GCS1 to the cell surface. These findings provide evidence that mutual gamete activation, regulated exocytosis, and sperm plasma membrane modifications govern flowering plant gamete interactions. However, it is yet unclear whether the EC1 acts solely or in coordination with sperm cell-specific signal during the process. Recent study reported that two sperm DUF679

membrane proteins DMP8 and DMP9 interact with HAP2/GCS1, which are required for the EC1-induced translocation of HAP2/GCS1 from internal storage vesicle to the sperm plasma membrane to ensure successful fertilization [49].

Once these two cells fuse at fertilization, their nuclei must then navigate toward each other and fuse. When an animal egg cell is fertilized, cable-like protein filaments called microtubules guide the two nuclei into contact. These microtubules are organized by a cellular structure called a centrosome [50, 51]. However, flowering plants do not have centrosomes [52]. Kawashima et al. [53] found that the fertilization requires an intact F-actin network. The sperm nucleus becomes surrounded by a star-shaped structure of F-actin cables, and that this F-actin structure migrates together with the sperm nucleus. The F-actin network constantly moves inward, from the edges of the cell toward the nucleus, prior to fertilization. ROP8 is a female gamete-specific Rho-GTPase that regulates F-actin dynamics. Previous research has shown that the Wiskott-Aldrich syndrome protein family verprolin-homologous and suppressor of the cAMP receptor (WAVE/SCAR) family are effector proteins that directly interact with ROPs and promote actin nucleation [54, 55]. The WAVE/SCAR complex is the main activator of the F-actin regulatory ACTIN RELATED PROTEIN 2/3 (ARP2/3) protein complex [56, 57]. However, further study demonstrated that the F-actin regulator, SCAR2, but not the ARP2/3 protein complex, regulates F-actin dynamics in female gametophytic cells for fertilization. In addition, the class XI myosin XI-G controls active F-actin movement in the Arabidopsis central cell [58].

## **6. Fertilization recovery system and polytubey blocking**

In normal conditions, although hundreds of pollens may land on the stigma and growing pollen tubes into the transmitting tract of a pistil, usually only a single tube, in response to attractants, emerges from the septum in the vicinity of each ovule to target the ovule [59, 60]. Even the attraction of more than one pollen tube has been observed in different gametophytic mutants [61–65]. The mechanism regulating pollen tube number remains unclear. As we discussed earlier, the synergid plays a role in attracting the pollen tube. In Arabidopsis, pollen tube growth arrests within a synergid cell and accompanied by the degeneration of the receptive synergid. Thus, the second pollen tube is attracted by another synergid. Kasahara et al. [66, 67] investigated the mechanisms underlying this phenomenon in Arabidopsis upon frequently observing ovules that accepted two pollen tubes in the fertilization defective *hap2–1* (allelic to *gcs1*) mutant. They observed that the fertility rate of the ovules pollinated with pollen from male gametophytic mutants was not around 50%, as expected, it is 60–70%. This result was revealed once fertilization failed with the first pollen tube, attracted a second one through a second synergid cell, so increasing the fertility rate. This phenomenon is called fertilization recovery system (**Figure 4A** and **B**).

In addition to the fertilization recovery system, plants, like animals, also have an important task of avoiding polyspermy. In Arabidopsis, the block to polyspermy is facilitated by a mechanism that prevents polytubey. How exactly do plants control the number of pollen tubes they attract? Three block mechanisms have been discovered recently, occurring in the septum, the micropyle, and after fertilization. The first polytubey block is located at the septum, FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube-specific RALF6, 7, 16, 36, and 37 peptide ligands to establish polytubey block (**Figure 4D**) [68]. In addition, Duan et al. [69] demonstrated that pollen tube arrival at *Reproductive Strategies of the Female Gametophyte DOI: http://dx.doi.org/10.5772/intechopen.109805*

#### **Figure 4.**

*Fertilization recovery system and polytubey. A. One pollen is being directed toward the ovule. B. The first pollen tube penetrates the ovule but fertilization fails, the second pollen tube is attracted by another synergid cell to increase the chances of fertilization as a "wise" back-up system (Fertilization Recovery System). C. Fertilization signals (ECS1 and ECS2) block polytubey. ECS1 and ESC2 specifically cleave the pollen tube attractor LURE1. D. FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube-specific RALF6, 7, 16, 36, and 37 peptide ligands to establish polytubey block. In addition, NO affects LURE1 and suppresses pollen tube attraction at micropylar.*

the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE11, respectively, blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Fertilization signals can also block polytubey. After successful fertilization, the aspartic endopeptidases ECS1 and ECS2 are secreted to the extracellular space from a cortical network located at the apical domain of the Arabidopsis egg cell. ECS1 and ESC2 specifically cleave the pollen tube attractor LURE1 (**Figure 4C**). In consequence, polytubey is frequent in *ecs1 ecs2* double mutants. These findings demonstrate that plant egg cells sense successful fertilization and elucidate a mechanism as to how a relatively fast post-fertilization block to polytubey is established by fertilization-induced degradation of attraction factors [70].

## **7. Pollen tube-dependent ovule enlargement morphology (POEM)**

As discussed earlier, once two sperm cells fused to egg cell and central cell, to give rise to embryo and endosperm, which is the start of seed development. However, nobody knows the mechanism of initiation of seed development, except double fertilization. The discovery of pollen tube-dependent ovule enlargement

morphology (POEM) brings a turning point, interesting in that the PTC itself is enough to initiate the ovule enlargement (**Figure 5**) even though it may not grow to the regular seed size [71]. As a preparation for fertilization, PTC initiated the developmental activity of various cells in the ovule. While studies have suggested that fertilization is a prerequisite for seed coat development [72, 73]. The studies demonstrated that just PTC release into the ovule is enough to initiate seed coat and endosperm development [74, 75]. In addition, the expression of numerous genes is responsible for cell expansion, cell division, and seed coat development in *gcs1/gcs1* pollinated ovules. As a preparation for fertilization, PTC initiates the developmental activity of various cells in the ovule. After mobilization, the ovules are waiting for the signal from double fertilization to continue the seed development, in addition to the phenotype of ovule size and seed coat formation.

#### **Figure 5.**

*Pollen tube-dependent ovule enlarged morphology (POEM). After the pollen tube arrests within a synergid, the pollen tube bursts and releases its contents with two sperm cells. Double fertilization is accomplished by these sperm cells fertilizing egg cell and central cell to give rise to the zygote and endosperm, respectively. However, if the ovule gets gcs1 mutant pollen tube, gcs1 sperm cells fail to fertilize. The ovule will be enlarged and initiate seed coat formation without fertilization.*

*Reproductive Strategies of the Female Gametophyte DOI: http://dx.doi.org/10.5772/intechopen.109805*

Discovery of the POEM phenomenon showed its potential applications in crop breeding for seed size increment and apomixis induction. However, POEM has only been reported in Arabidopsis. There are great differences between mono and dicotyledon plants. To investigate if this phenomenon is conserved in monocot as well, Honma et al. [76] developed genome-edited rice plants by knocking-out homologs of rice *GCS1* using the CRISPR/Cas9 technology, which led to fertilization failure and pollen tube-dependent ovule enlargement morphology (POEM) phenomenon. Apparently, the POEMed-like rice ovule can grow near-normal seed size unlike earlier observations in Arabidopsis in which *gcs1* ovules were aborted quite early. The POEMed-like rice ovules contained 10–20% sugar, with extremely high sucrose content (98%). Transcriptomic analysis revealed that the osgcs1 ovules had downregulation of starch biosynthetic genes, which would otherwise have converted sucrose to starch. Overall, this study shows that pollen tube content release is sufficient to trigger sucrose unloading at rice ovules. However, successful fertilization is indispensable to trigger sucrose-starch conversion. These findings are expected to pave the way for developing novel sugar-producing crops suited for diverse climatic regions.

## **8. Summary**

This chapter discusses the journey of the pollen from the stigma to fertilization as well as the POEM phenomenon. These processes are achieved through the involvement of various male-female interactions. The molecular mechanisms underlying pollen tube guidance from the funiculus to the female gametophyte are well known in Arabidopsis, because the pollen tube attractants AtLURE1 peptides had previously been identified downstream of the master synergid cell regulator MYB98. Recently, more and more factors related to fertilization journey have been elucidated. During the final step after pollen tube bursting, HAP2/ GCS1, GEX2, DMP8, and DMP9 have been identified as direct male-related key fertilization factors. The F-actin network is also required for sperm nucleus migration. The fertilization recovery system is to increase the chances of fertilization as a "wise" back-up system. Combining fertilization recovery system with recent research on polytubey blocking, the process of how ovule attracts pollen tube is becoming clear. Finally, very few factors related to new plant phenomena and POEM have been identified. New insights into the underlying molecular mechanisms are anticipated.

## **Acknowledgements**

These works were supported by the Precursory Research for Embryonic Science and Technology (13416724, Kasahara Sakigake Project), Japan Science and Technology Agency. These works were also supported by a grant-in-aid (25840106) from the Japanese Society for the promotion of Science (JSPS). These works were also supported by FAFU-UCR Joint Center and Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University.

## **Conflict of interest**

The authors declare no conflict of interest.

*Plant Physiology Annual Volume 2023*

## **Author details**

Xiaoyan Liu1,2\* and Ryushiro D. Kasahara1,2,3

1 School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China

2 FAFU-UCR Joint Center and Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, China

3 Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan

\*Address all correspondence to: liuxy@fafu.edu.cn

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Reproductive Strategies of the Female Gametophyte DOI: http://dx.doi.org/10.5772/intechopen.109805*

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## **Chapter 7**
