INTRODUCTION
Pollen-pistil interaction is critical for the successful fertilization of flowering plants. This interaction is a crucial step in preventing inbreeding and maintaining species identity, thus contributing to angiosperm diversity. Pollen-pistil interaction consists of multiple selective steps, including pollen adhesion, hydration, germination and polarized tube growth (Bedinger, Broz, Tovar-Mendez, & McClure, 2017; Edlund, Swanson, & Preuss, 2004; Zheng, Lin, Liang, Wang, & Chen, 2018). Pollen adhesion and hydration are two earliest events in pollination among species with dry stigmas. These highly regulated processes require proteins and lipids deriving from pollen wall (Elleman & Dickinson, 1990; Safavian & Goring, 2013).
The mature pollen wall of flowering plants includes three main layers, intine, exine and pollen coat. Intine is produced by microspores and is mainly composed of pectin, cellulose and hemicellulose. Exine is constituted of sexine and nexine (Lou et al., 2014). The main composition of sexine is sporopollenin. Its precursors are synthesized by multiple enzymes regulated by a MYB transcription factor MS188 in tapetum (K. Wang et al., 2018; Z. B. Zhang et al., 2007). Pollen coat contains many sticky and heterogenous material composed of lipids, proteins, carotenoids and polysaccharides (Hernández-Pinzón, Ross, Barnes, Damant, & Murphy, 1999; Piffanelli, Ross, & Murphy, 1998), which is crucial to pollen protection from abiotic stresses, successful pollen contact, hydration and subsequent pollen germination on dry stigma (Dickinson, 1995; Elleman & Dickinson, 1990). The pollen coat contains various components including pollen coat proteins (PCP) and pollen coat lipids. PCPs are derived from tapetum after its programmed cell death (PCD) alongside gametophytically derived proteins. In Arabidopsis, several lipases and lipid-binding oleosin proteins were identified in PCPs (Mayfield, Fiebig, Johnstone, & Preuss, 2001). Male sterility1 (MS1) is a PHD transcription factor for late tapetum development (Wilson, Morroll, Dawson, Swarup, & Tighe, 2001). These sporophyte-derived PCPs are regulated by MS1 in tapetum (Lu et al., 2020). They may play roles in the early events of pollen-stigma recognition including pollen adhesion and hydration. S-locus cysteine-rich protein (SCR), SLR1-BP1/2, PCP-A1(PCP7) and PCP-Bs have been identified as gametophyte-derived PCPs (Doughty et al., 1998; Nasrallah & Nasrallah, 2014; Takayama et al., 2000; L. Wang et al., 2017). They are likely to play important roles in pollen germination and pollen tube growth.
Lipids are one of the major subcellular components, and comprise different combinations and positional distributions of fatty acids. They play important roles in plants by forming membrane structures, acting as storage lipids, signaling molecules, and surface coverings (Li-Beisson, Nakamura, & Harwood, 2016). The pollen coat lipids display a semi-solid state and may guide the water transfer from the stigma to pollen during pollen-stigma interaction (Edlund et al., 2004). In lipid metabolism, 3-ketoacyl CoA synthase (KCS) are involved in fatty acid elongation or very long chain fatty acid (VLCFA) synthesis (Haslam & Kunst, 2013; Joubès et al., 2008). Eceriferum 2 (Cer2) and cer2-like 2 (Cer2l2) encode BAHD acyltransferase and enhance the elongation of VLCFA from 28 to 30 carbon atoms catalyzed by KCS6 (Fiebig et al., 2000; Haslam et al., 2015). The kcs6 single mutant and cer2cer2l2 double mutant can produce mature pollen. However, they fail to hydrate (Fiebig et al., 2000; Haslam et al., 2015; Hulskamp, Schneitz, & Pruitt, 1995; Preuss, Lemieux, Yen, & Davis, 1993). KCS6, CER2 and CER2L2 express in endothecium suggesting that endothecium plays a role to synthesize pollen coat lipids for pollen hydration (Zhan, Xiong, Wang, & Yang, 2018). It was generally considered that pollen coat lipids are derived from tapetum (Hernández-Pinzón et al., 1999). It is not clear how tapetum contributes to pollen coat lipids synthesis.
The KCS family in Arabidopsis contains 21 KCS members (Costaglioli et al., 2005; Joubès et al., 2008). In this study, we reported KCS7, KCS15 and KCS21 were expressed in tapetum. They act downstream of MS1, a regulator for late tapetum development. The reduced pollen coat lipid in the triple mutant (kcs7/15/21 ) and defective pollen hydration demonstrate that they play redundant roles for pollen coat lipid synthesis for pollen hydration.