Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1 (LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis

Triacylglycerols (TAGs) are major storage materials that accumulate in developing seeds and serve as carbon and energy reserves for germination and growth of the seedling. One of the critical reactions in TAG biosynthesis is activation of fatty acyl chains to fatty acyl CoAs, catalyzed by long-chain acyl CoA synthetases (LACSs). Of the nine LACSs identified in Arabidopsis, only LACS9 is known to reside in the plastid, the site of de novo fatty acid synthesis, and is considered the major LACS isoform involved in plastidial fatty acid export for TAG formation. Because the lacs9 null mutant did not show any detectable phenotype, it was hypothesized that at least one additional LACS enzyme must be active in the plastid. Expression analyses to identify potential plastid-localized LACSs involved in TAG biosynthesis revealed that, in addition to LACS9, isoforms LACS1, LACS2, LACS4 and LACS8 are transcribed in the seed. LACS8 showed the highest expression level in the embryo and a high sequence similarity with LACS9, and was therefore characterized further and shown to be associated with the ER, not the plastid. Furthermore, disruption of LACS8 in the lacs8 mutant and lacs8 lacs9 double mutant, and over-expression of LACS8, did not affect the seed fatty acid content. In contrast, 11 and 12% decreases in fatty acid content were detected in lacs1 lacs9 and lacs1 lacs8 lacs9 seeds, respectively, indicating that LACS1 and LACS9 have overlapping functions in TAG biosynthesis. This result is surprising because, unlike LACS9, LACS1 is localized in the ER and has been shown to be involved in cuticular lipid synthesis.     

Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis

Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8 / LACS1 , one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C₂₄) were elevated more than 155%. The C₁₆ cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C₁₈ monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C₂₀–C₃₀, with highest activity for C₃₀ acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C₁₆ but not C₁₈ cutin monomers are reduced in lacs1 , and C₁₆ acids are the next most preferred acid (behind C₃₀) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C₁₆ monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C₁₆) fatty acids for cutin synthesis.     

The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis

Long-chain acyl-CoA synthetase (LACS) activities are encoded by a family of at least nine genes in Arabidopsis (Arabidopsis thaliana). These enzymes have roles in lipid synthesis, fatty acid catabolism, and the transport of fatty acids between subcellular compartments. Here, we show that the LACS2 gene (At1g49430) is expressed in young, rapidly expanding tissues, and in leaves expression is limited to cells of the adaxial and abaxial epidermal layers, suggesting that the LACS2 enzyme may act in the synthesis of cutin or cuticular waxes. A lacs2 null mutant was isolated by reverse genetics. Leaves of mutant plants supported pollen germination and released chlorophyll faster than wild-type leaves when immersed in 80% ethanol, indicating a defect in the cuticular barrier. The composition of surface waxes extracted from lacs2 leaves was similar to the wild type, and the total wax load was higher than the wild type (111.4 microg/dm(2) versus 76.4 microg/dm(2), respectively). However, the thickness of the cutin layer on the abaxial surface of lacs2 leaves was only 22.3 +/- 1.7 nm compared with 33.0 +/- 2.0 nm for the wild type. In vitro assays showed that 16-hydroxypalmitate was an excellent substrate for recombinant LACS2 enzyme. We conclude that the LACS2 isozyme catalyzes the synthesis of omega-hydroxy fatty acyl-CoA intermediates in the pathway to cutin synthesis. The lacs2 phenotype, like the phenotypes of some other cutin mutants, is very pleiotropic, causing reduced leaf size and plant growth, reduced seed production, and lower rates of seedling germination and establishment. The LACS2 gene and the corresponding lacs2 mutant will help in future studies of the cutin synthesis pathway and in understanding the consequences of reduced cutin production on many aspects of plant biology.     

Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis

As the outermost layer on aerial tissues of the primary plant body, the cuticle plays important roles in plant development and physiology. The major components of the cuticle are cutin and cuticular wax, both of which are composed primarily of fatty acid derivatives synthesized in the epidermal cells. Long-chain acyl-CoA synthetases (LACS) catalyze the formation of long-chain acyl-CoAs and the Arabidopsis genome contains a family of nine genes shown to encode LACS enzymes. LACS2 is required for cutin biosynthesis, as revealed by previous investigations on lacs2 mutants. Here, we characterize lacs1 mutants of Arabidopsis that reveals a role for LACS1 in biosynthesis of cuticular wax components. lacs1 lacs2 double-mutant plants displayed pleiotropic phenotypes including organ fusion, abnormal flower development and reduced seed set; phenotypes not found in either of the parental mutants. The leaf cuticular permeability of lacs1 lacs2 was higher than that of either lacs1 or lacs2 single mutants, as determined by measurements of chlorophyll leaching from leaves immersed in 80% ethanol, staining with toluidine blue dye and direct measurements of water loss. Furthermore, lacs1 lacs2 mutant plants are highly susceptible to drought stress. Our results indicate that a deficiency in cuticular wax synthesis and a deficiency in cutin synthesis together have compounding effects on the functional integrity of the cuticular barrier, compromising the ability of the cuticle to restrict water movement, protect against drought stress and prevent organ fusion.     

Alterations in CER6, a gene identical to CUT1, differentially affect long-chain lipid content on the surface of pollen and stems

Very long chain lipids contribute to the hydrophobic cuticle on the surface of all land plants and are an essential component of the extracellular pollen coat in the Brassicaceae. Mutations in Arabidopsis CER genes eliminate very long chain lipids from the cuticle surface and, in some cases, from the pollen coat. In Arabidopsis, the loss of pollen coat lipids can disrupt interactions with the stigma, inhibiting pollen hydration and causing sterility. We have positionally cloned CER6 and demonstrate that a wild-type copy complements the cer6-2 defect. In addition, we have identified a fertile, intragenic suppressor, cer6-2R, that partially restores pollen coat lipids but does not rescue the stem wax defect, suggesting an intriguing difference in the requirements for CER6 activity on stems and the pollen coat. Importantly, analysis of this suppressor demonstrates that low amounts of very long chain lipids are sufficient for pollen hydration and germination. The predicted CER6 amino acid sequence resembles that of fatty acid-condensing enzymes, consistent with its role in the production of epicuticular and pollen coat lipids >28 carbons long. DNA sequence analysis revealed the nature of the cer6-1, cer6-2, and cer6-2R mutations, and segregation analysis showed that CER6 is identical to CUT1, a cDNA previously mapped to a different chromosome arm. Instead, we have determined that a new gene, CER60, with a high degree of nucleotide and amino acid similarity to CER6, resides at the original CUT1 locus.     

Long chain acyl CoA synthetase 4 catalyzes the first step in peroxisomal indole-3-butyric acid to IAA conversion

Indole-3-butyric acid (IBA) is an endogenous storage auxin important for maintaining appropriate indole-3-acetic acid (IAA) levels, thereby influencingprimary root elongation and lateral root development. IBA is metabolized into free IAA in peroxisomes in a multistep process similar to fatty acid β-oxidation. We identified LONG CHAIN ACYL-COA SYNTHETASE 4 (LACS4) in a screen for enhanced IBA resistance in primary root elongation in Arabidopsis thaliana. LACSs activate substrates by catalyzing the addition of CoA, the necessary first step for fatty acids to participate in β-oxidation or other metabolic pathways. Here, we describe the novel role of LACS4 in hormone metabolism and postulate that LACS4 catalyzes the addition of CoA onto IBA, the first step in its β-oxidation. lacs4 is resistant to the effects of IBA in primary root elongation and dark-grown hypocotyl elongation, and has reduced lateral root density. lacs6 also is resistant to IBA, although both lacs4 and lacs6 remain sensitive to IAA in primary root elongation, demonstrating that auxin responses are intact. LACS4 has in vitro enzymatic activity on IBA, but not IAA or IAA conjugates, and disruption of LACS4 activity reduces the amount of IBA-derived IAA in planta. We conclude that, in addition to activity on fatty acids, LACS4 and LACS6 also catalyze the addition of CoA onto IBA, the first step in IBA metabolism and a necessary step in generating IBA-derived IAA.

An enhancer mutant revealed an acyl-CoA synthetase that catalyzes CoA addition to indole-3-butryic acid, required for the β-oxidation steps necessary to generate indole-3-butryic acid-derived IAA.     

Fatty acid export from the chloroplast. Molecular characterization of a major plastidial acyl-coenzyme A synthetase from Arabidopsis

Acyl-coenzyme A (CoA) synthetases (ACSs, EC 6.2.1.3) catalyze the formation of fatty acyl-CoAs from free fatty acid, ATP, and CoA. Essentially all de novo fatty acid synthesis occurs in the plastid. Fatty acids destined for membrane glycerolipid and triacylglycerol synthesis in the endoplasmic reticulum must be first activated to acyl-CoAs via an ACS. Within a family of nine ACS genes from Arabidopsis, we identified a chloroplast isoform, LACS9. LACS9 is highly expressed in developing seeds and young rosette leaves. Both in vitro chloroplast import assays and transient expression of a green fluorescent protein fusion indicated that the LACS9 protein is localized in the plastid envelope. A T-DNA knockout mutant (lacs9-1) was identified by reverse genetics and these mutant plants were indistinguishable from wild type in growth and appearance. Analysis of leaf lipids provided no evidence for compromised export of acyl groups from chloroplasts. However, direct assays demonstrated that lacs9-1 plants contained only 10% of the chloroplast long-chain ACS activity found for wild type. The residual long-chain ACS activity in mutant chloroplasts was comparable with calculated rates of fatty acid synthesis. Although another isozyme contributes to the activation of fatty acids during their export from the chloroplast, LACS9 is a major chloroplast ACS.     

Deficiency of very long chain alkanes biosynthesis causes humidity‐sensitive male sterility via affecting pollen adhesion and hydration in rice

Pollen adhesion and hydration are the earliest events of the pollen–stigma interactions, which allow compatible pollen to fertilize egg cells, but the underlying mechanisms are still poorly understood. Rice pollen are wind dispersed, and its pollen coat contains less abundant lipids than that of insect‐pollinated plants. Here, we characterized the role of OsGL1‐4, a rice member of the Glossy family, in pollen adhesion and hydration. OsGL1‐4 is preferentially expressed in pollen and tapetal cells and is required for the synthesis of very long chain alkanes. osgl1‐4 mutant generated apparently normal pollen but displayed excessively fast dehydration at anthesis and defective adhesion and hydration under normal condition, but the defective adhesion and hydration were rescued by high humidity. Gas chromatography–mass spectrometry analysis suggested that the humidity‐sensitive male sterility of osgl1‐4 was probably due to a significant reduction in C25 and C27 alkanes. These results indicate that very long chain alkanes are components of rice pollen coat and control male fertility via affecting pollen adhesion and hydration in response to environmental humidity. Moreover, we proposed that a critical point of water content in mature pollen is required for the initiation of pollen adhesion.     

The Arabidopsis cer26 mutant,like the cer2 mutant,is specifically affected in the very long chain fatty acid elongation process

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl‐CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2‐like family: CER2, CER26 and CER26‐like . Expression pattern analysis showed that the three genes are differentially expressed in an organ‐ and tissue‐specific manner. Using individual T–DNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl‐CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity.     

Very long chain fatty acid beta-oxidation by rat liver mitochondria and peroxisomes

Crude mitochondrial fractions were isolated by differential centrifugation of rat liver homogenates. Subfractionation of these fractions on self-generating continuous Percoll gradients resulted in clearcut separation of peroxisomes from mitochondria. Hexacosanoic acid beta-oxidation was present mainly in peroxisomal fractions whereas hexacosanoyl CoA oxidation was present in the mitochondrial as well as in the peroxisomal fractions. The presence of much greater hexacosanoyl CoA synthetase activity in the purified preparations of microsomes and peroxisomes compared to mitochondria, suggests that the synthesis of coenzyme A derivatives of very long chain fatty acids (VLCFA) is limited in mitochondria. We postulate that a specific VLCFA CoA synthetase may be required to effectively convert VLCFA to VLCFA CoA in the cell. This specific synthetase activity is absent from the mitochondrial membrane, but present in the peroxisomal and the microsomal membranes. We postulate that substrate specificity and the subcellular localization of the specific VLCFA CoA synthetase directs and regulates VLCFA oxidation in the cell.     

A novel male-sterile mutant of Arabidopsis thaliana,faceless pollen-1, produces pollen with a smooth surface and an acetolysis-sensitive exine

A mutant exhibiting conditional male sterility, in which fertility was restored under conditions of high humidity, was identified in T-DNA tagged lines of Arabidopsis thaliana. Scanning electron microscopy (SEM) demonstrated that the pollen surface was almost smooth and the reticulate pattern not prominent. Thus, the mutant was named faceless pollen-1 (flp1). Transmission electron microscopy (TEM) revealed that the smooth appearance was due to tryphine filling in the exine cavities and covering the pollen surface. The lipid droplets in the tryphine of mutant pollen were smaller and more numerous than those of the wild type. SEM analysis also demonstrated that pollen exine was easily damaged by acetolysis, suggesting that a component of exine, sporopollenin, was defective in the mutant. In addition, the stems and siliques had reduced amounts of wax crystals. A predicted amino acid sequence of the cDNA that corresponded to the tagged gene, fip1, showed sequence similarity to proteins involved in wax biosynthesis. The FLP1 protein is likely to play a role in the synthesis of the components of tryphine, sporopollenin of exine and the wax of stems and siliques.     

Pollen-stigma adhesion in Arabidopsis: a species-specific interaction mediated by lipophilic molecules in the pollen exine.

To investigate the nature and role of cell adhesion in plants, we analyzed the initial step of pollination in Arabidopsis: the binding of pollen grains to female stigma cells. Here we show this interaction occurs within seconds of pollination. Because it takes place prior to pollen hydration, it also requires adhesion molecules that can act in a virtually dry environment. We developed assays that monitored adhesion of populations of pollen grains and individual cells. Adhesion between pollen and stigma cells is highly selective - Arabidopsis pollen binds with high affinity to Arabidopsis stigmas, while pollen from other species fails to adhere. Initial binding is independent of the extracellular pollen coat (tryphine), indicating that adhesion molecules reside elsewhere on the pollen surface, most likely within the exine walls. Immediately after pollination, the stigma surface becomes altered at the interface, acquiring a pattern that interlocks with the exine; this pattern is evident only with pollen from Arabidopsis and its close relatives. Purified exine fragments bind to stigma cells, and biochemical analyses indicate that this specific, rapid and anhydrous adhesion event is mediated by lipophilic interactions.     

A comparative analysis of the distribution and composition of lipidic constituents and associated enzymes in pollen and stigma of sunflower

Spatial distribution and compositional analyses of the lipidic constituents in pollen and stigma of sunflower (Helianthus annuus L. cv. Morden) were conducted using ultrastructural, histochemical, and biochemical analysis. Detection of secretions at the base of stigmatic papillae and neutral lipid accumulations on the surface of stigmatic papillae and between adjacent pseudopapillae demonstrates the semidry nature of stigma surface in sunflower. Pollen coat is richer in lipids (8%) than stigma (2.2%) on fresh weight basis. Nile Red-fluorescing neutral lipids are preferentially localized in the pollen coat. Neutral esters and triacylglycerols (TAGs) are the major lipidic constituents in pollen grains and stigma, respectively. Lignoceric acid (24:0) and cis-11-eicosenoic acid (20:1) are specifically expressed only in the pollen coat. Similar long-chain fatty acids have earlier been demonstrated to play a significant role during the initial signaling mechanism leading to hydration of pollen grains on the stigma surface. Lipase (EC 3.1.1.3) activity is expressed both in pollen grains and stigma. Stigma exhibits a better expression of acyl-ester hydrolase (EC 3.1.1.1) activity than that of observed in both the pollen fractions. Expression of two acyl-ester hydrolases (41 and 38 kDa) has been found to be specific to pollen coat. Specific expression of lignoceric acid (24:0) in pollen coat and localization of lipase in pollen and stigma have been discussed to assign possible roles that they might play during pollen–stigma interaction.     

Pollenkitt – its composition, forms and functions

Two types of sticky pollen coat material exist in angiosperms, both produced by the anther tapetum. Pollenkitt is the most common adhesive material present around pollen grains of almost all angiosperms pollinated by animals, whereas tryphine seems to be restricted only to Brassicaceae. Tapetal cell protoplasts have different patterns of development according to the products formed during their development and degeneration. If tryphine is formed, the tapetal cell protoplasts lose their individuality at the microspore stage. If pollenkitt is formed, their contents degenerate at later stages. Cell content is totally reabsorbed, when ripe pollen is not surrounded by any gluing material. Current knowledge of pollenkitt formation, deposition on pollen grains and chemical composition are reviewed and discussed. Methods for detecting this viscous fluid are also presented. The many functions of pollenkitt, deduced from personal observations and the literature, act in the period between anther opening and pollen hydration on the stigma; they are: (1) to hold pollen in the anther until dispersal; (2) to enable secondary pollen presentation; (3) to facilitate pollen dispersal; (4) to protect pollen from water loss; (5) to protect pollen from ultra-violet radiation; (6) to maintain sporophytic proteins responsible for pollen–stigma recognition inside exine cavities; (7) to protect pollen protoplasts from fungi and bacteria; (8) to keep together pollen grains during transport; (9) to protect pollen from hydrolysis and exocellular enzymes; (10) to render pollen attractive to animals; (11) to render pollen visible to animal eyes; (12) to hide pollen from animal eyes; (13) to avoid predation of pollen through smell; (14) to enable adhesion to insect bodies; (15) to enable pollen packaging by bees and to form corbicules; (16) to provide a digestible reward for pollinators; (17) to enable pollen clumps to reach the stigma; (18) to allow self-pollination; (19) to facilitate adhesion to the stigma; (20) to facilitate pollen rehydration. Depending on the developmental program of the species, these functions may act during pollen presentation, in relation to pollinators, during pollen dispersal and when pollen reaches the stigma.     

Oleosin-like proteins are not present on the surface of tobacco pollen

Pollen-stigma interactions on wet- and dry-type stigmas involve similar processes: the hydration of the pollen, followed by pollen tube growth and penetration of the stigma. Furthermore, in some species, identical molecules, namely lipids, are used to achieve this. In addition to lipids, oleosin-like proteins of the pollen coat of dry-type stigma plants have been shown to be involved in pollen-stigma interactions. However, little information is present about the proteins on the surface of pollen of wet-type stigma plants, in particular that of the Solanaceae. To analyze proteins from the surface of pollen of Nicotiana tabacum (tobacco), a solanaceous plant, we used an antiserum raised against Brassica pollen coat, a dry-type stigma plant of the Brassicaceae. In addition we used a molecular approach to identify tobacco homologues of oleosin-like genes. Our results show that no proteins similar to Brassica oleracea pollen coat proteins are present on the surface of tobacco pollen, and that oleosin-like genes are not expressed in tobacco anthers or stigmas.     

Molecular characterization of the CER1 gene of arabidopsis involved in epicuticular wax biosynthesis and pollen fertility.

The aerial parts of plants are coated with an epicuticular wax layer, which is important as a first line of defense against external influences. In Arabidopsis, the ECERIFERUM (CER) genes effect different steps of the wax biosynthesis pathway. In this article, we describe the isolation of the CER1 gene, which encodes a novel protein involved in the conversion of long chain aldehydes to alkanes, a key step in was biosynthesis. CER1 was cloned after gene tagging with the heterologous maize transposable element system Enhancer-Inhibitor, also known as Suppressor-mutator. cer1 mutants display glossy green stems and fruits and are conditionally male sterile. The similarity of the CER1 protein with a group of integral membrane enzymes, which process highly hydrophobic molecules, points to a function of the CER1 protein as a decarbonylase.     

The role of Δ6-desaturase acyl-carrier specificity in the efficient synthesis of long-chain polyunsaturated fatty acids in transgenic plants

The role of acyl‐CoA‐dependent Δ6‐desaturation in the heterologous synthesis of omega‐3 long‐chain polyunsaturated fatty acids was systematically evaluated in transgenic yeast and Arabidopsis thaliana. The acyl‐CoA Δ6‐desaturase from the picoalga Ostreococcus tauri and orthologous activities from mouse (Mus musculus) and salmon (Salmo salar) were shown to generate substantial levels of Δ6‐desaturated acyl‐CoAs, in contrast to the phospholipid‐dependent Δ6‐desaturases from higher plants that failed to modify this metabolic pool. Transgenic plants expressing the acyl‐CoA Δ6‐desaturases from either O. tauri or salmon, in conjunction with the two additional activities required for the synthesis of C20 polyunsaturated fatty acids, contained higher levels of eicosapentaenoic acid compared with plants expressing the borage phospholipid‐dependent Δ6‐desaturase. The use of acyl‐CoA‐dependent Δ6‐desaturases almost completely abolished the accumulation of unwanted biosynthetic intermediates such as γ‐linolenic acid in total seed lipids. Expression of acyl‐CoA Δ6‐desaturases resulted in increased distribution of long‐chain polyunsaturated fatty acids in the polar lipids of transgenic plants, reflecting the larger substrate pool available for acylation by enzymes of the Kennedy pathway. Expression of the O. tauriΔ6‐desaturase in transgenic Camelina sativa plants also resulted in the accumulation of high levels of Δ6‐desaturated fatty acids. This study provides evidence for the efficacy of using acyl‐CoA‐dependent Δ6‐desaturases in the efficient metabolic engineering of transgenic plants with high value traits such as the synthesis of omega‐3 LC‐PUFAs.     

The extracellular lipase EXL4 is required for efficient hydration of Arabidopsis pollen

Pollination in species with dry stigmas begins with the hydration of desiccated pollen grains on the stigma, a highly regulated process involving the proteins and lipids of the pollen coat and stigma cuticle. Self-incompatible species of the Brassicaceae block pollen hydration, and while the early signaling steps of the self-incompatibility response are well studied, the precise mechanisms controlling pollen hydration are poorly understood. Both lipids and proteins are important for hydration; loss of pollen coat lipids and proteins results in defective or delayed hydration on the stigma surface. Here, we examine the role of the pollen coat protein extracellular lipase 4 (EXL4), in the initial steps of pollination, namely hydration on the stigma. We identify a mutant allele, exl4-1, that shows a reduced rate of pollen hydration. exl4-1 pollen is normal with respect to pollen morphology and the downstream steps in pollination, including pollen tube germination, growth, and fertilization of ovules. However, owing to the delay in hydration, exl4-1 pollen is at a disadvantage when competed with wild-type pollen. EXL4 also functions in combination with GRP17 to promote the initiation of hydration. EXL4 is similar to GDSL lipases, and we show that it functions in hydrolyzing ester bonds. We report a previously unknown function for EXL4, an abundant pollen coat protein, in promoting pollen hydration on the stigma. Our results indicate that changes in lipid composition at the pollen–stigma interface, possibly mediated by EXLs, are required for efficient pollination in species with dry stigmas.