Research Progress on Calcium Ion in Gametophytic Self-Incompatibility

The effects of Ca²⁺ on pollen tube growth were investigated in a large number of studies, and it was found that Ca²⁺ was critical to pollen germination and pollen tube growth in vitro (Brewbaker & Kwack 1963; Steinhorst & Kudla 2013a). The Ca²⁺ gradient accumulation was observed for the first time in the tip of the lily (Lilium longiflorum ‘Arai’) pollen tube (Jaffe et al. 1975). It has been well proved that a typical Ca²⁺ gradient is present at the tip of all growing pollen tubes; otherwise, the pollen tube cannot grow normally (Feijó et al. 2001). The Ca²⁺ gradient in the cytoplasm of the pollen tube could not only affect the elongation of the pollen tube, but it also affects its growth direction and changes the orientation of the pollen tube tip to make it grow toward the part with high Ca²⁺ concentration (Malhó & Trewavas 1996). Cytosolic-free Ca²⁺ concentration ([Ca²⁺]_cyt) was present in the cytoplasm of the pollen tube, and the important messenger in the free Ca²⁺ signal network could regulate the directional elongation of the pollen tube (Guan et al. 2013). After the hydration of pollen grains, an obvious Ca²⁺ gradient was observed in the protruding part of the pollen tube (Iwano et al. 2004).

The presence of Ca²⁺ gradient was conducive to controlling the directional secretion, transporting and fusion of the Golgi vesicles, and constantly forming new tube wall and plasmalemma. In the formation of plasmalemma, fracture points were caused in the cell wall, making the plasmalemma loose and realizing the opening of the Ca²⁺ channel, which in turn promoted the Ca²⁺ flow and vesicles fusion, beneficial to the continuous growth of pollen tube (Feijó et al. 2001; Gao et al. 2019). The inflow part of Ca²⁺ and the highest concentration part in the Ca²⁺ gradient determined the growth direction of the pollen tube (Malhó et al. 1995; Malhó & Trewavas 1996). Pollen tube growth was maintained through the deposition of the original cell wall materials at the apex. Once deposited on the tip, the wall would experience a hardening mature process, resulting in a viscosity/elasticity gradient between the growth tip and the nonelongating tube (Hepler et al. 2013; Cosgrove 2016).

Successful pollination in angiosperms is achieved with the attachment of the pollen grains on the pistil stigma, then the germination of the pollen grains, the rapid elongation of the pollen tube in the style, and finally, the sperm cells are delivered to the ovule to complete the fertilization. Pollen tubes formed a tubular structure through polar growth (Qin & Yang 2011). Pollen germination was regulated by the interaction between pollen and stigma papilla cells. The pollen tube had a directional growth through the style, with Ca²⁺ involved in the whole process of growing into ovule and fertilization (Chen et al. 2015). A large amount of Ca²⁺ was observed at the attachment site for pollen grains on the surface of the stigma papilla cells, and a large amount of Ca²⁺ could be also observed in the style-transmitting tissues on the stigma surface, with the same direction as pollen germination and elongation of pollen tube tip (Iwano et al. 2004). The S protein secreted from the stigma of the Papaveraceae after pollination with incompatible pollen would interact with its homologous pollen's S receptor, causing a rapid increase in the cytoplasmic free Ca²⁺ ([Ca²⁺]_i) in the pollen tube. The extracellular Ca²⁺ inflow disrupted the Ca²⁺ gradient at the tip of the pollen tube, so that the tip growth would be inhibited (Fig. 1A). However, for compatible pollen, no significant changes in free Ca²⁺ concentration were observed (Franklin-Tong et al. 1993; Takayama & Isogai 2005; McClure & Franklin-Tong 2006; Chen et al. 2015; Lin et al. 2015) (Fig. 1A).

Figure 1

For Cruciferae, the compatible pollen coat contained the substances that could promote the output of Ca²⁺ from the pistil papilla cells. The attachment of mastoid cells to pollen coat could induce a Ca²⁺ signal pathway. In the signal transduction process, the Ca²⁺ pump was used to maintain a moderate level of Ca²⁺ in the cytoplasm (Patergnani et al. 2011). In stigmas after compatible pollination and treated with compatible pollen coat, it was found that the transcription of autoinhibited Ca²⁺-ATPase13 (ACA13) in the papilla cells was increased, and ACA13 was mainly located in the plasmalemma and vesicles and accumulated at the penetration site of the pollen tube. Then the ACA13 protein would output Ca²⁺ to the compatible pollen tube (Fig. 1B). If ACA13 expression at the stigma was affected and the output of pistil Ca²⁺ was decreased, the growth of compatible pollen tube would be also affected, failing to be fertilized normally (Iwano et al. 2014). A haplotype-specific interaction between S-locus protein 11/S-locus cysteine-rich protein (SP11/SCR) and S-locus receptor kinase (SRK) triggers the SSI response in the Brassicaceae that leads to incompatible pollen rejection (Sehgal & Singh 2018). Through signal transduction studies, self-pollination specifically induced an increase in cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) in papilla cells and suggested that Ca²⁺ influx mediated by a glutamate receptor-like channel (GLR) is the essential SI response leading to incompatible pollen rejection (Iwano et al. 2015) (Fig. 1B). Although the mechanism of inhibiting pollen tube growth is different between GSI and SSI, genetic boundaries between GSI and SSI may not be so rigid. Even so, there are convincing reports of the presence of a cryptic GSI system operating alongside the SSI system in the Brassicaceae and Asteraceae (Brennan et al. 2003). Therefore, we believe that calcium is involved in the recognition of pollen and stigma, which is necessary for S-RNase-based GSI response.

As a kind of ubiquitous second messenger, Ca²⁺ signals are involved in most signal processes and all aspects of cell life (Edel et al. 2017). It played a key role in the reproductive process of flowering plants. The interaction and recognition between pollen and pistils depended on the changes in Ca²⁺ concentration and distribution at the tip of the pollen tube (Steinhorst & Kudla 2013a; Gu et al. 2015). The calcium signal-sensing protein acted as a signal molecule to control the Ca²⁺ gradient and regulate the growth of the pollen tube. The intracellular Ca²⁺ signal is decoded and transmitted by a set of Ca²⁺ binding proteins that transmit this information to downstream reactions (Kudla et al. 2010; Bagur & Hajnóczky 2017). The complex structure of Ca²⁺-sensing proteins provided binding sites for Ca²⁺ and provided a reliable mechanism for complex and flexible signal processes (Gu et al. 2015; Demidchik et al. 2018).

Various Ca²⁺-related proteins play a crucial role in the growth of pollen tube tips. The calcium binding EF-hand superfamily was the most prominent among them (Konrad et al. 2011; Steinhorst & Kudla 2013b). Ca²⁺ sensory proteins regulating pollen tube growth included calmodulin (CaM), CaM-like (CML), calcium-dependent protein kinase (CDPK), and calcineurin B-like protein (CBL) (Zhou et al. 2009; Hashimoto & Kudla 2011; Steinhorst & Kudla 2013b). However, the detailed regulatory mechanism of Ca²⁺ sensory proteins is not yet clear. Ca²⁺ regulated the production of reactive oxygen species (ROS). CaM can promote Ca²⁺ inflow through the plasmalemma, increase the level of ROS at the tip, and stabilize actin filaments (Shang et al. 2005); CDPK phosphorylation and dephosphorylation could regulate the substrate activity (Cheng et al. 2002), and CBL often interacted with CBL-interacting protein kinases (CIPKs) to form a complex to regulate Ca signals (Weinl & Kudla 2009). During the SI response in apple, CBL may interact with S-RNase in the pollen tube cells to affect the combination of CBL and CIPK and destroy the signal transduction in the pollen tube, thus inhibiting the growth of the pollen tube. CBL was not only a signal response protein but also a feedback signal molecule that can regulate Ca²⁺ concentration (Gu et al. 2015).

In the SI response of Papaver rhoeas, the inflow of Ca²⁺ into the pollen tube was a key step in the early response. The cascade reaction of the calcium signal was helpful for Ca²⁺ to rapidly respond and achieve consistency with the SI response receptor and acts as a signal molecule for interaction with S proteins (Franklin-Tong et al. 2002). The S protein at an incompatible stigma interacted with the pollen S receptor in Papaver rhoeas, and then in some way, the pollen S receptor triggered the inflow of extra-cellular Ca²⁺ and the Ca²⁺ release in the cell compartment through the plasmalemma channel. Ca²⁺ inflow increased the concentration of free Ca²⁺ in the cytoplasm (Franklin-Tong et al. 1997). The increase in Ca²⁺, as a second messenger, causes a series of downstream events, such as changes in cyto-skeleton structure, ROS increase, and phosphorylation of mitogen-activated protein kinase (MAPK), so that the growth of the pollen tube was inhibited and was unable to complete normal fertilization (Eaves et al. 2014). Caruso et al. (2012) hypothesized that the proteins regulated by the Asp-rich encoding genes in Citrus clementina ‘Comune’ functioned as Ca²⁺-trap elements, which could lead to a distinct decrease in Ca²⁺ availability. Even if the concentration of Ca²⁺ in the cytoplasm increased, the degree of the cascade reaction caused thereof decreased, and the Ca²⁺ concentration gradient required for the elongation of the pollen tube was changed (Caruso et al. 2012). In the SI reaction of the gametophyte of pear, self-S-RNase also can destroy the calcium gradient at the tip of the pollen tube, but inhibit the calcium influx, thereby arresting pollen tube growth (Qu et al. 2016).

A tip-focused calcium gradient is an essential requirement for pollen tube growth. Both Ca²⁺ channel inhibitors and Ca²⁺ chelating agents could inhibit the normal growth of the pollen tube (Qu et al. 2007). Too high or too low concentration showed adverse effects on pollen germination and pollen tube growth, indicating that suitable Ca²⁺ concentration in the cytoplasm shall be maintained for the normal growth of the pollen tube (Guan et al. 2013; Gao et al. 2019). The free Ca²⁺ was distributed in a gradient manner at the tip of the pollen tube, and the factors leading to the disappearance of the Ca²⁺ gradient would cause the pollen tube to stop growing (Wu et al. 2011). A large number of Ca²⁺ influx channels may be present in the cells at the tip of a polar growing plant cell. The electrophysiological study on the plasmalemma of the pollen tube showed that hyperpolarization could activate the Ca²⁺ influx channel. With the increase of hyperpolarization activity of the plant's plasmalemma, these hyperpolarized Ca²⁺ channels would play an important role in Ca²⁺ transport (Qu et al. 2007). The Ca²⁺ in plant cells was maintained at a very low level; otherwise, it was easy to interact with plasma-lemma and karyolemma to form phosphorylated precipitates. Ca²⁺ was constantly pumped out of cells or pumped into the cell compartment, maintaining the plasmalemma-specific Ca²⁺ concentration gradient or forming transient Ca²⁺ changes (Chen et al. 2015).

Extracellular Ca²⁺ inflow was essential for maintaining the polar growth of pollen tubes. The high gradient of plasmalemma was inseparable from an efficient Ca²⁺ transport system and effective signaling mechanism (Iwano et al. 2009; Qu et al. 2012). Activation of the Ca²⁺ influx channel on the cell surface or in organelle could promote Ca²⁺ inflow and rapidly increase intracellular Ca²⁺ concentration. Even minor changes in Ca²⁺ concentration were enough to make the related enzymes sense the response regulation of downstream receptors, thus regulating a series of cellular responses (Chen et al. 2015). Intracellular calcium also played a key role in regulating actin-binding proteins (ABPs) and pollen tube growth (Staiger et al. 2010; Qu et al. 2015). The cytoplasmic Ca²⁺ gradient at the tip of pollen tube was closely related to the growth rate and morphology of elongated pollen tubes (Steinhorst & Kudla 2013b). Although ABPs were evenly distributed in the whole pollen tube cytoplasm, their activity still shall be regulated by the concentration of Ca²⁺ in the cell, and the concentration difference would affect the assembly of actin in each area of the pollen tube. Therefore, the appearance of a Ca²⁺ steady state was important for maintaining the structure and function of actin; otherwise, the tip of the pollen tube could not continue to elongate. The expression of the calreticulin gene (CRT) was necessary for maintaining the Ca²⁺ gradient as well as proper actin cytoskeleton structure and functions (Suwińska et al. 2017). However, ABP was involved in the functional mechanism of CRT in the pollen tube growth, which was required to form actin filaments at the pollen tube stem and actin kinetics at the tube tip. In addition, most of the ABP was regulated by Ca²⁺ (Fu 2010; Staiger et al. 2010; Qu et al. 2015; Hepler & Winship 2015).

In conclusion, it is believed from the existing research results that the mechanism of Ca²⁺ in SI for fruit trees among the genus Rosaceae is as follows: pollen and stigma recognize each other after pollination (Li et al. 2020). In the case of compatible pollen, the activity of the Ca²⁺ channel at the tip of the pollen tube is not affected, and a normal Ca²⁺ concentration gradient is maintained at the tip, where the vesicles are fused with the plasma-lemma to form more plasmalemma. The release of precursors for the cell wall could lead to the elongation of the pollen tube (de Win et al. 1999), and the S-RNase enveloped by the vesicles would be discharged out of the cells (Fig. 2A). However, for the incompatible pollen, the pollen tube germinates on the stigma, but some unknown substance in the style, which may be S-RNase, reduces the activity of the calcium channel at the tip of the pollen tube, inhibits the inflow of extracellular Ca²⁺, destroys the Ca²⁺ concentration gradient, and ruptures the vesicles in the pollen tube. S-RNase is released from vesicles and degrades RNA as a cytotoxin when accumulated above a threshold concentration in the pollen tube (Qu et al. 2017) (Fig. 2B). Related studies have shown that Ca²⁺ in the incompatible pollen tube is also involved in mediating other downstream events, such as programmed cell death in cells at the pollen tube tip (Franklin-Tong & Franklin 2003; Wang et al. 2010). In recent years, great progress has been made in the understanding of Ca²⁺ in pollen, with great achievements in research on SI. However, the specific regulatory mechanism of Ca²⁺ in SI is still unclear. Further studies on Ca²⁺ channel regulation and Ca²⁺ signal regulation network would help to further understand the mechanism of SI.

Figure 2