Anther Endothecium-Derived Very-Long-Chain Fatty Acids Facilitate Pollen Hydration in Arabidopsis

Pollen hydration is a prerequisite for pollen germination and subsequent pollen tube growth. As a result of two decades of biochemical studies and genetic identification of the eceriferum (cer) male-sterile mutants, the very-long-chain fatty acid (VLCFA) lipids in the pollen coat have been well known to play a critical role in pollen hydration (Preuss et al., 1993Preuss D. Lemieux B. Yen G. Davis R.W. A conditional sterile mutation eliminates surface components from Arabidopsis pollen and disrupts cell signaling during fertilization.Genes Dev. 1993; 7: 974-985Crossref PubMed Scopus (295) Google Scholar, Fiebig et al., 2000Fiebig A. Mayfield J.A. Miley N.L. Chau S. Fischer R.L. Preuss D. Alterations in CER6, a gene identical to CUT1, differentially affect long-chain lipid content on the surface of pollen and stems.Plant Cell. 2000; 12: 2001-2008Crossref PubMed Scopus (267) Google Scholar, Hiscock and Allen, 2008Hiscock S.J. Allen A.M. Diverse cell signalling pathways regulate pollen-stigma interactions: the search for consensus.New Phytol. 2008; 179: 286-317Crossref PubMed Scopus (184) Google Scholar). VLCFAs are referred to as fatty acids with 20 or more carbons, which are synthesized through four sequential reactions catalyzed by 3-ketoacyl-CoA synthase (KCS), 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase, and enoyl reductase (ECR). Among all the reactions, KCS-mediated condensation is the rate-limiting step and determines the length of the final acyl-CoA products (Haslam and Kunst, 2013Haslam T.M. Kunst L. Extending the story of very-long-chain fatty acid elongation.Plant Sci. 2013; 210: 93-107Crossref PubMed Scopus (184) Google Scholar). KCS6 (also known as CER6, POP1, and CUT1) catalyzes the production of C28 fatty acyl-CoAs and is essential for pollen hydration (Fiebig et al., 2000Fiebig A. Mayfield J.A. Miley N.L. Chau S. Fischer R.L. Preuss D. Alterations in CER6, a gene identical to CUT1, differentially affect long-chain lipid content on the surface of pollen and stems.Plant Cell. 2000; 12: 2001-2008Crossref PubMed Scopus (267) Google Scholar, Haslam and Kunst, 2013Haslam T.M. Kunst L. Extending the story of very-long-chain fatty acid elongation.Plant Sci. 2013; 210: 93-107Crossref PubMed Scopus (184) Google Scholar). In addition, BAHD acyltransferase CER2/CER2-like proteins are required for VLCFAs synthesis (Xia et al., 1997Xia Y. Nikolau B.J. Schnable P.S. Developmental and hormonal regulation of the Arabidopsis CER2 gene that codes for a nuclear-localized protein required for the normal accumulation of cuticular waxes.Plant Physiol. 1997; 115: 925-937Crossref PubMed Scopus (60) Google Scholar, Haslam et al., 2012Haslam T.M. Manas-Fernandez A. Zhao L. Kunst L. Arabidopsis ECERIFERUM2 is a component of the fatty acid elongation machinery required for fatty acid extension to exceptional lengths.Plant Physiol. 2012; 160: 1164-1174Crossref PubMed Scopus (109) Google Scholar, Haslam et al., 2015Haslam T.M. Haslam R. Thoraval D. Pascal S. Delude C. Domergue F. Fernandez A.M. Beaudoin F. Napier J.A. Kunst L. et al.ECERIFERUM2-LIKE proteins have unique biochemical and physiological functions in very-long-chain fatty acid elongation.Plant Physiol. 2015; 167: 682-692Crossref PubMed Scopus (75) Google Scholar). It was proposed that CER2/CER2-like proteins function as novel components of the VLCFA elongation machinery through association with the specific KCS enzyme (Haslam and Kunst, 2013Haslam T.M. Kunst L. Extending the story of very-long-chain fatty acid elongation.Plant Sci. 2013; 210: 93-107Crossref PubMed Scopus (184) Google Scholar). CER2 acts redundantly with CER2-like2 (CER2L2) to affect male fertility under low-humidity environments (Haslam et al., 2015Haslam T.M. Haslam R. Thoraval D. Pascal S. Delude C. Domergue F. Fernandez A.M. Beaudoin F. Napier J.A. Kunst L. et al.ECERIFERUM2-LIKE proteins have unique biochemical and physiological functions in very-long-chain fatty acid elongation.Plant Physiol. 2015; 167: 682-692Crossref PubMed Scopus (75) Google Scholar), indicating that CER2 and CER2L2 are likely involved in pollen hydration. However, the cytological and genetic evidence of CER2 and CER2L2 in male sterility remains elusive. To investigate the role of VLCFAs in pollen hydration, we obtained and studied T-DNA insertion alleles of cer2 (SALK_084443, At4g24510) and cer2l2 (SAIL_3_F11, At3g23840) from Nottingham Arabidopsis Stock Centre. We found that the cer2 cer2l2 double mutant showed male sterility as described (Haslam et al., 2015Haslam T.M. Haslam R. Thoraval D. Pascal S. Delude C. Domergue F. Fernandez A.M. Beaudoin F. Napier J.A. Kunst L. et al.ECERIFERUM2-LIKE proteins have unique biochemical and physiological functions in very-long-chain fatty acid elongation.Plant Physiol. 2015; 167: 682-692Crossref PubMed Scopus (75) Google Scholar), whereas the CER2/cer2;cer2l2/cer2l2 plants displayed reduced fertility while cer2/cer2;CER2L2/cer2l2 was fertile (Supplemental Figure 1), indicating that CER2L2 plays a major role in plant fertility. Genotypically, the self-crossed progeny of CER2/cer2;cer2l2/cer2l2 and cer2/cer2;CER2L2/cer2l2 segregated at a ratio close to 1:2:1 (Supplemental Table 1). This genetic analysis indicates that CER2 and CER2L2 function in the sporophytic cell layer of anther, rather than the gametophytic microspore/pollen. Although cer2 cer2l2 is male-sterile, Alexander's staining and scanning electron microscopy revealed that its pollen viability and pollen morphology have no obvious difference from that of wild type (Figure 1A and Supplemental Figure 2). Also, the stamen elongation and anthesis of cer2 cer2l2 appeared normal (Supplemental Figure 2). Hence, the formation and release of cer2 cer2l2 pollen proceeded normally. To figure out the cause of male sterility of cer2 cer2l2, a pollination assay was performed. After landing on stigma, the wild-type pollen began to absorb water and became spherical in a few minutes. However, a similar expansion was not detected for cer2 cer2l2 pollen, even when observations were extended to 1 h after pollination (Figure 1B). Nevertheless, in vitro germination of cer2 cer2l2 pollen was indistinguishable from the wild-type pollen (Supplemental Figure 3). The fertility of the cer2 cer2l2 double-mutant plant was restored by exposure to high-humidity conditions (Supplemental Figure 4). The content of C30 VLCFA lipids is dramatically reduced in cer2 mutant (Haslam et al., 2012Haslam T.M. Manas-Fernandez A. Zhao L. Kunst L. Arabidopsis ECERIFERUM2 is a component of the fatty acid elongation machinery required for fatty acid extension to exceptional lengths.Plant Physiol. 2012; 160: 1164-1174Crossref PubMed Scopus (109) Google Scholar). Consistently, exogenous addition of melissic acid (C30:0) rescued the fertility (Supplemental Figure 4). Taken together, these results demonstrated that shortage of VLCFAs in the pollen coat of cer2 cer2l2 double mutant causes the defective hydration, leading to male sterility. A co-pollination assay was then performed to investigate the role of VLCFAs in pollen hydration and its species specificity. Co-pollination with the mixed cer2 cer2l2 and wild-type pollen led to normal siliques. Examination of the offspring plants revealed the successful transmission of cer2 cer2l2 to the next generation (Supplemental Figure 5). When cer2 cer2l2 stigma was used as the pollen receptor in co-pollination, approximately 25.6% offspring plants (n = 74) were found to be sterile and homozygous for cer2 cer2l2 (Supplemental Figure 5). These indicate that the hydration defect of cer2 cer2l2 pollen was rescued by contact with normal Arabidopsis pollen. Pollen from plants of the family Cruciferae (Brassica pekinensis, Brassica chinensis, and Raphanus sativus), Liliaceae (Lilium brownii), and Solanaceae (Nicotiana benthamiana) was also used for co-pollination (Figure 1C and Supplemental Figure 6). Pollination of Arabidopsis stigma with these heterospecific pollens could not result in fertilization and seed set. However, pollen of Brassica pekinensis, Brassica chinensis, and Raphanus sativus got hydrated on Arabidopsis stigma and rescued the defect of cer2 cer2l2 pollen (Figure 1C and 1D). Although Lilium pollen failed to be hydrated on Arabidopsis stigma, it restored the fertility of cer2 cer2l2 when co-pollinated with cer2 cer2l2 pollen (Figure 1C and 1D). This observation ruled out the possibility that water from the hydrated pollen is directly transferred to cer2 cer2l2 pollen. Although Lilium is a wet stigma plant, lipids are present in the pollen coat (Heslop-Harrison, 1968Heslop-Harrison J. Tapetal origin of pollen-coat substances in Lilium pollen.New Phytol. 1968; 67: 779-786Crossref Scopus (109) Google Scholar). A reasonable explanation is that the pollen coat VLCFA lipids were dispersed from Lilium pollen to cer2 cer2l2. The large size of Lilium pollen likely leads to its failure of hydration on Arabidopsis stigma (Figure 1D and Supplemental Figure 6). These results suggest that the pollen coat VLCFA lipids required for pollen hydration are not strictly species-specific. Nicotiana benthamiana is a plant with wet stigma lacking the typical structure of pollen coat (Figure 1D and Supplemental Figure 6). N. benthamiana pollen was not hydrated on Arabidopsis stigma and failed to rescue the hydration defect of cer2 cer2l2 (Figure 1C and 1D). In addition to CER2/CER2L2, KCS6 was reported to be essential for pollen hydration (Preuss et al., 1993Preuss D. Lemieux B. Yen G. Davis R.W. A conditional sterile mutation eliminates surface components from Arabidopsis pollen and disrupts cell signaling during fertilization.Genes Dev. 1993; 7: 974-985Crossref PubMed Scopus (295) Google Scholar, Fiebig et al., 2000Fiebig A. Mayfield J.A. Miley N.L. Chau S. Fischer R.L. Preuss D. Alterations in CER6, a gene identical to CUT1, differentially affect long-chain lipid content on the surface of pollen and stems.Plant Cell. 2000; 12: 2001-2008Crossref PubMed Scopus (267) Google Scholar). The catalytic activity of KCS6 is enhanced by co-expression with CER2 in yeast (Haslam et al., 2012Haslam T.M. Manas-Fernandez A. Zhao L. Kunst L. Arabidopsis ECERIFERUM2 is a component of the fatty acid elongation machinery required for fatty acid extension to exceptional lengths.Plant Physiol. 2012; 160: 1164-1174Crossref PubMed Scopus (109) Google Scholar). All three proteins are localized in the endoplasmic reticulum (Supplemental Figure 7; Haslam et al., 2012Haslam T.M. Manas-Fernandez A. Zhao L. Kunst L. Arabidopsis ECERIFERUM2 is a component of the fatty acid elongation machinery required for fatty acid extension to exceptional lengths.Plant Physiol. 2012; 160: 1164-1174Crossref PubMed Scopus (109) Google Scholar, Haslam and Kunst, 2013Haslam T.M. Kunst L. Extending the story of very-long-chain fatty acid elongation.Plant Sci. 2013; 210: 93-107Crossref PubMed Scopus (184) Google Scholar). By split-ubiquitin-based yeast two-hybrid assays we showed that both CER2 and CER2L2 could interact with KCS6 in yeast (Figure 1E). Their interactions were further verified in plant cells by the split-luciferase (LUC) complementation imaging assay. Co-injection of KCS6-nLUC with cLUC-CER2 or cLUC-CER2L2 into N. benthamiana leaves led to strong fluorescence signals (Figure 1F), suggesting that CER2/CER2L2 and KCS6 form a complex in planta. These results also support that CER2/CER2-like protein functions as novel components of the VLCFA elongation machinery by associating with the KCS condensing enzyme. GUS staining and in situ hybridization showed that CER2, CER2L2 and KCS6 are expressed in anther (Xia et al., 1997Xia Y. Nikolau B.J. Schnable P.S. Developmental and hormonal regulation of the Arabidopsis CER2 gene that codes for a nuclear-localized protein required for the normal accumulation of cuticular waxes.Plant Physiol. 1997; 115: 925-937Crossref PubMed Scopus (60) Google Scholar, Haslam et al., 2015Haslam T.M. Haslam R. Thoraval D. Pascal S. Delude C. Domergue F. Fernandez A.M. Beaudoin F. Napier J.A. Kunst L. et al.ECERIFERUM2-LIKE proteins have unique biochemical and physiological functions in very-long-chain fatty acid elongation.Plant Physiol. 2015; 167: 682-692Crossref PubMed Scopus (75) Google Scholar). However, their exact expression patterns in anther remain unknown. We generated three constructs, CER2-GFP, CER2L2-GFP and KCS6-GFP, using their genomic sequences fused to GFP gene driven by their native promoters (Supplemental Figure 8). Expression of CER2-GFP or CER2L2-GFP rescued the sterility of cer2 cer2l2 (Supplemental Figure 9), validating that they are biologically functional. For CER2-GFP, fluorescence was predominantly localized in the epidermis of anther, with a relatively weak GFP signal in endothecium. It was reported that CER2L2 is expressed in the young bud, and the expression decreased at stages 9 and 10, with no expression at stage 11 (Haslam et al., 2015Haslam T.M. Haslam R. Thoraval D. Pascal S. Delude C. Domergue F. Fernandez A.M. Beaudoin F. Napier J.A. Kunst L. et al.ECERIFERUM2-LIKE proteins have unique biochemical and physiological functions in very-long-chain fatty acid elongation.Plant Physiol. 2015; 167: 682-692Crossref PubMed Scopus (75) Google Scholar). In the CER2L2-GFP lines, a GFP signal was specifically detected in endothecium at stages 8–11 (Figure 1G and Supplemental Figure 8). The endothecial expression of CER2 and CER2L2 is consistent with their sporophytic effect on pollen formation. Expression of CER2L2 in endothecium is stronger than that of CER2. This is in agreement with the genetic data that CER2L2 plays the major role in synthesizing pollen coat VLCFAs. Likewise, dense fluorescence of KCS6-GFP was also observed in endothecium (Figure 1G and Supplemental Figure 8). The endothecial co-distribution of CER2, CER2L2, and KCS6 not only enables their physical interaction but, more importantly, confers an essential role of endothecium in the synthesis of pollen coat VLCFAs in Arabidopsis. In flowering plants, anther is typically composed of four distinct somatic cell layers from the exterior to the interior: epidermis, endothecium, middle layer, and tapetum. The epidermis serves as a barrier to protect the anther. The endothecium is responsible for anther dehiscence to release pollen (Wilson et al., 2011Wilson Z.A. Song J. Taylor B. Yang C. The final split: the regulation of anther dehiscence.J. Exp. Bot. 2011; 62: 1633-1649Crossref PubMed Scopus (168) Google Scholar). The middle layer is located between the endothecium and tapetum, and undergoes programmed cell death (PCD) before tapetum. The tapetum directly contacts with the microspores and provides nutrients, metabolites, and enzymes for pollen development (Wilson et al., 2011Wilson Z.A. Song J. Taylor B. Yang C. The final split: the regulation of anther dehiscence.J. Exp. Bot. 2011; 62: 1633-1649Crossref PubMed Scopus (168) Google Scholar). Upon apoptosis, the remnants of the tapetal cells, containing many classes of lipids and proteins, fill the exine cavity to form the pollen coat (Levesque-Lemay et al., 2016Levesque-Lemay M. Chabot D. Hubbard K. Chan J.K. Miller S. Robert L.S. Tapetal oleosins play an essential role in tapetosome formation and protein relocation to the pollen coat.New Phytol. 2016; 209: 691-704Crossref PubMed Scopus (17) Google Scholar). The pollen coat plays vital roles in protecting the pollen from desiccation and ultraviolet radiation, adhering the pollen to the stigma, and aiding hydration (Levesque-Lemay et al., 2016Levesque-Lemay M. Chabot D. Hubbard K. Chan J.K. Miller S. Robert L.S. Tapetal oleosins play an essential role in tapetosome formation and protein relocation to the pollen coat.New Phytol. 2016; 209: 691-704Crossref PubMed Scopus (17) Google Scholar). The VLCFAs in pollen coat are essential for pollen hydration. It is generally thought that VLCFAs are derived from tapetum. However, the interaction between CER2/CER2L2 and KCS6 and their endothecial localization demonstrated here indicate that the endothecium plays an essential role in the synthesis of pollen coat VLCFAs for pollen hydration (Figure 1E–1G). During anther development, tapetum undergoes PCD at stage 10 and is completely degraded at stage 12 (Wilson et al., 2011Wilson Z.A. Song J. Taylor B. Yang C. The final split: the regulation of anther dehiscence.J. Exp. Bot. 2011; 62: 1633-1649Crossref PubMed Scopus (168) Google Scholar). It is likely that the material synthesis in tapetum is inhibited or stopped since stage 10. The fluorescence of CER2L2-GFP and KCS6-GFP was intensely observed in endothecium at stage 10 and remained detectable at stage 11 (Supplemental Figure 8), suggesting that the endothecium likely continues to maintain the synthesis of pollen coat VLCFAs after tapetal PCD, providing the VLCFAs accumulated on the surface of mature pollen for pollen hydration. During the process of pollen–stigma interaction, the components of pollen coat mobilize to the site of contact between the pollen and papilla cell, forming a foot between the two surfaces (Edlund et al., 2004Edlund A.F. Swanson R. Preuss D. Pollen and stigma structure and function: the role of diversity in pollination.Plant Cell. 2004; 16: S84-S97Crossref PubMed Scopus (417) Google Scholar). VLCFAs, as an important type of pollen coat lipids, may play a biophysical role in establishing a gradient of water potential between the pollen and stigma, which guides water transfer from papilla cell to pollen grain (Hiscock and Allen, 2008Hiscock S.J. Allen A.M. Diverse cell signalling pathways regulate pollen-stigma interactions: the search for consensus.New Phytol. 2008; 179: 286-317Crossref PubMed Scopus (184) Google Scholar). Noteworthy, increasing evidence suggests that VLCFAs and/or their derivatives also act as signal messengers to modulate a variety of developmental processes in plants (Shang et al., 2016Shang B. Xu C. Zhang X. Cao H. Xin W. Hu Y. Very-long-chain fatty acids restrict regeneration capacity by confining pericycle competence for callus formation in Arabidopsis.Proc. Natl. Acad. Sci. USA. 2016; 113: 5101-5106Crossref PubMed Scopus (75) Google Scholar). It can be hypothesized that the VLCFAs and/or their derivatives in pollen coat function as signaling molecules to activate water transfer from papilla cell to pollen during pollen hydration. In conclusion, our results demonstrated that the BAHD acyltransferases CER2 and CER2L2 interact with KCS6 in endothecium to modulate the biosynthesis of pollen coat VLCFAs for pollen hydration in Arabidopsis. Anther endothecium is not only responsible for mature pollen release but also plays a vital role for pollen hydration during pollen–stigma recognition. This work was supported by grants from the National Natural Science Foundation of China (31700277 and 31670314) and the National Key Research and Development Program of China (2016YFD0100902).