The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack

Phytochemistry xxx (2014) xxx–xxx

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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Review

The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack Teagen D. Quilichini a, Etienne Grienenberger b, Carl J. Douglas a,⇑ a b

Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Pollen wall Sporopollenin Exine Polyketide Fatty alcohol Male gametophyte Tapetum

a b s t r a c t The formation of the durable outer pollen wall, largely composed of sporopollenin, is essential for the protection of the male gametophyte and plant reproduction. Despite its apparent strict conservation amongst land plants, the composition of sporopollenin and the biosynthetic pathway(s) yielding this recalcitrant biopolymer remain elusive. Recent molecular genetic studies in Arabidopsis thaliana (Arabidopsis) and rice have, however, identified key genes involved in sporopollenin formation, allowing a better understanding of the biochemistry and cell biology underlying sporopollenin biosynthesis and pollen wall development. Herein, current knowledge of the biochemical composition of the outer pollen wall is reviewed, with an emphasis on enzymes with characterized biochemical activities in sporopollenin and pollen coat biosynthesis. The tapetum, which forms the innermost sporophytic cell layer of the anther and envelops developing pollen, plays an essential role in sporopollenin and pollen coat formation. Recent studies show that several tapetum-expressed genes encode enzymes that metabolize fatty acid derived compounds to form putative sporopollenin precursors, including tetraketides derived from fatty acyl-CoA starter molecules, but analysis of mutants defective in pollen wall development indicate that other components are also incorporated into sporopollenin. Also highlighted are the many uncertainties remaining in the development of a sporopollenin-fortified pollen wall, particularly in relation to the mechanisms of sporopollenin precursor transport and assembly into the patterned form of the pollen wall. A working model for sporopollenin biosynthesis is proposed based on the data obtained largely from studies of Arabidopsis, and future challenges to complete our understanding of pollen wall biology are outlined. Ó 2014 Elsevier Ltd. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sporopollenin synthesis and deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Priming for sporopollenin deposition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Sporopollenin composition: insights provided by biochemical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Sporopollenin composition: insights provided by genetic analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. The conservation of sporopollenin composition among land plant lineages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Model for sporopollenin biosynthesis and the sporopollenin metabolon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. The sporophytic origin of the exine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Translocation of sporopollenin precursors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Sporopollenin assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⇑ Corresponding author. Tel.: +1 604 822 2618; fax: +1 604 822 6089. E-mail address: [email protected] (C.J. Douglas). http://dx.doi.org/10.1016/j.phytochem.2014.05.002 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Quilichini, T.D., et al. The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.05.002

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1. Introduction For the evolution of plant life on land, a number of adaptations were required to support terrestrial life and its inherent stresses (Cronk, 2009; Kenrick and Crane, 1997). Among these adaptations, external barriers encasing the dominant plant body and its reproductive offshoots were forefront in preventing desiccation and ensuring the transmission of genetic information (Wallace et al., 2011). The cycling between the sporophytic and gametophytic generations characteristic of land plants is made possible by the reproductive cells they produce, namely the haploid spores produced by the sporophyte through meiosis and the haploid gametes produced by the gametophytes through mitosis. However, these reproductive cells often require prolonged survival as independent entities, necessitating the fortification of their cell walls with polymers capable of withstanding terrestrial stresses. Sporopollenin, a structurally robust biopolymer, served this critical function in the evolution of land plants by enveloping and protecting the spores of early non-seed-bearing plant lineages and the pollen grains (microgametophytes) formed by the male reproductive organ, the anther, of seed-producing plants (Wallace et al., 2011). Sporopollenin appears to be conserved in its properties and to have been critical in the evolution of land plants. Sporopollenin fortifies the outer wall (exine) of pollen grains, forming a durable casing around the male gametophyte. The surfaces of pollen grains often appear intricately decorated due to species-specific sculpturing of sporopollenin present in their outer walls, commonly assuming a regular hexagonal pattern, as in Arabidopsis thaliana (Arabidopsis) (Fig. 1A and B). Apertures or colpi intercept the outer wall of pollen in species-specific locations and frequencies, and serve as the typical sites of pollen tube emergence (Fig. 1A). Pollen surface features have facilitated plant identification in the fossil record, aiding our understanding of the evolution and distribution of land plants over geological time. However, our understanding of the function of these features in plant reproductive success are limited. In some species, such outer wall features are presumably adaptations that aid pollen dispersal or hold additional wall materials such as tapetum-derived pollen coat constituents that are important for the physiology of the pollen in its interaction with the stigma. Despite the great diversity of pollen surface structure, spore/ pollen walls exhibit common structural features observed in cross-sections of developing and mature spores and pollen grains using transmission electron microscopy (TEM). These features typically include an inner intine composed of pectin, cellulose, and hemicellulose, and an outer exine composed of sporopollenin (Heslop-Harrison, 1968a). The intine appears as a light band by TEM, directly external to the pollen plasma membrane (Fig. 1C and D). The cellulosic intine maintains the structural integrity of pollen grains, as Arabidopsis plants with mutations in primary cell wall cellulose synthases produce collapsed or malformed pollen grains with aberrant pollen walls that lack or have uneven intine cellulose (Persson et al., 2007). Sporopollenin provides the rigid and sculptured framework of the exine, which serves to encapsulate and protect the contents of spores/pollen, and to assist in stigmatic capture (Fig. 1). For many species, including Arabidopsis, this structured backbone is additionally covered by a heterogeneous pollen coat (also called tryphine), which primarily serves in pollen stigmatic adhesion, recognition, and hydration and can be extracted from the underlying sporopollenin with organic solvents (Edlund, 2004; Murphy, 2006; Piffanelli et al., 1998). After pollen tube emergence from the exine shell, the intine serves as the only cell wall encasing the growing pollen tube, and is rapidly remodeled to assist growth while preventing premature rupture (Chebli et al., 2012).

Fig. 1. Surface structure and cross-section morphology of the mature Arabidopsis pollen wall. Scanning electron micrographs (A, B) and transmission electron micrographs (C, D) of wild-type pollen at maturity. (A) Critical point dried pollen grain with reticulate exine and one visible aperture (of three). (B) Pollen wall surface structure. (C) High-pressure frozen and freeze substituted pollen grain in cross-section with dense cytoplasmic contents encased by a pollen wall. Region boxed in panel C is magnified in panel D. (D) Stratified pollen wall with electronlucent intine, electron-dense sporopollenin and heterogeneous pollen coat. The plasma membrane separates wall components from pollen cytoplasm. (E) Diagrammatic representation of the pollen wall from panel D. Arrows indicate the broad categorization of wall layers into intine (light grey) and exine. Within the exine, the structured sporopollenin (black) of the wall appears homogeneous, in contrast to the heterogeneous pollen coat (mottled grey). Bars = 2 lm (A, C) and 0.2 lm (B, D).

Current understanding of pollen wall biogenesis is that components of the intine are generated by the microspore vegetative cell (Hess, 1993), while components of the exine are synthesized by the surrounding sporophytic tapetal cells and deposited on the surface of developing microspores within the locule (Ariizumi and Toriyama, 2011). Tapetal cells form the innermost cell layer of the sporophytic anther wall, and their direct proximity to developing microspores and loss of their cell walls at maturity facilitates their nutritive role in pollen development (Goldberg et al., 1993; Owen and Makaroff, 1995). Tapeta in spermatophytes are broadly grouped into the secretory (or parietal or glandular) type or the amoeboid (or periplasmodial or invasive) type, differing primarily in the extent of their intrusion into the locule during microspore development (Pacini, 2010). Amoeboid tapeta intrude into the locule, encasing microspores and providing direct nutrition to

Please cite this article in press as: Quilichini, T.D., et al. The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.05.002

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microspores, while secretory tapeta, such as in Arabidopsis, retain their shape at the locule periphery, requiring the transport of materials to microspores through locule fluid (Pacini, 2010). The importance of the tapetum as a secretory entity specifically involved in the formation of the pollen wall has also been of long-standing interest to pollen scientists (Heslop-Harrison and Mackenzie, 1967; Taylor, 1959; Ubisch, 1927). Numerous studies, particularly employing genetic and molecular approaches to characterize genes required for pollen wall formation, have demonstrated the central function of the tapetum in the synthesis of sporopollenin and pollen coat constituents (Ariizumi and Toriyama, 2011). Although both sporopollenin and pollen coat constituents of the exine arise in tapetal cells, the timing of their deposition and release differ; sporopollenin components are exported from intact tapeta, while the pollen coat is deposited as tapetal cells undergo programmed cell death and release their contents. Thus, studies of sporopollenin biosynthesis require analysis of immature anther sporophytic tissues, often by embedding and sectioning anthers for observation by TEM (Fig. 2A), or by cryo-fracturing immature anthers to reveal inner locules (Fig. 2B), to allow the ultrastructural features of tapetal cells and developing microspores to be examined. Challenged by the extreme recalcitrance of sporopollenin to chemical degradation, much of the recent progress in unraveling the biosynthesis and composition of sporopollenin has come from molecular genetic studies in which enzymes and proteins required for sporopollenin formation have been functionally characterized based on loss-of-function mutants in model plants such as Arabidopsis and Oryza sativa (rice). This article reviews the biochemistry of sporopollenin, as uncovered by chemical and genetic analyses, and considers the biochemical input of the tapetum to sporopollenin and pollen coat production. Remaining gaps in understanding of sporopollenin, particularly in processes governing its transport from tapetal cells, anchoring to microspores, and polymerization are discussed.

2. Sporopollenin synthesis and deposition 2.1. Priming for sporopollenin deposition The sculptured exine pattern evident in mature pollen grains (Fig. 1) is based on the pattern of sporopollenin deposition over the course of microspore development, which in turn requires

A

the establishment of a primexine wall early on in microspore development. Early in microspore development, a transient callose wall serves to separate the primary microspore cell wall from the microspore mother cell plasma membrane, enabling the formation of the primexine wall on microspores (Nishikawa et al., 2005; Heslop-Harrison, 1968b). Based primarily on staining experiments, the primexine appears to be predominantly cellulosic in composition and forms on the surface of microspores at the tetrad stage (Heslop-Harrison, 1968b; Paxson-Sowders et al., 1997), where it is believed to provide the anchoring site for sporopollenin (Ariizumi and Toriyama, 2011; Heslop-Harrison, 1968b). Within the primexine matrix, structural features known as probaculae and protecta emerge at regular intervals along an undulating microspore plasma membrane prior to callose dissolution. In the subsequent stage of anther development, free microspores rapidly form a sporopollenin-based outer wall, guided by probaculae and protecta within the primexine that take the shape of structured pillars (baculae) and caps (tecta) (Paxson-Sowders et al., 1997). Together, callose production, primexine formation and microspore plasma membrane undulation appear to be intimately linked with sporopollenin anchoring and exine patterning (Dong et al., 2005; Nishikawa et al., 2005; Suzuki et al., 2008). While these observations provide a framework for understanding the morphological events in pollen wall and exine formation, the corresponding biochemistry has long remained elusive.

2.2. Sporopollenin composition: insights provided by biochemical analyses Despite the importance of the sporopollenin biopolymer in plant life, an understanding of its composition, structure, biosynthesis, transport and polymerization has remained largely incomplete. In the late 1960s, the predominant model for sporopollenin proposed a monomeric backbone of polymerized carotenoids, based primarily on their known accumulation in anthers correlated with pollen formation, and their ability to polymerize in vitro into insoluble polymers resembling sporopollenin (Brooks and Shaw, 1968). One of the first studies to indicate that the carotenoid model for sporopollenin composition required revision found no substantial decrease in sporopollenin accumulation after the application of the carotenoid synthesis inhibitors norflurazon and Sandoz 9789, to developing Curcubita pepo anthers (Prahl et al., 1986, 1985).

B

M Lo

Lo

Ex

T

T

Lo

T

V

M N

T

Ep

M

M M

En

Ex

ML

T

Ep

En

ML

T

Ex

Fig. 2. The immature Arabidopsis anther with all cell types present. Anther containing developing microspores within the four sporophytic cell layers of the anther wall: epidermis (outermost), endothecium, middle layer, and tapetum (innermost). (A) Transmission electron micrograph of high-pressure frozen/freeze substituted anther revealing developing microspores in the uninucleate stage of pollen development. (B) Cryo-scanning electron micrograph of a cryo-fractured anther. The sporophytic tissues surround a locule filled with microspores at the late uninucleate stage of development. Ultrastructural features visible in these microspores include a large vacuole, nucleus with nucleolus, and exine radiating from each microspore plasma membrane. En, endothecium; Ep, epidermis; Ex, exine; Lo, locule; M, microspore; ML, middle layer; N, nucleus; T, tapetum; V, microspore vacuole. Bars = 5 lm.

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Additionally, labeling studies, primarily in developing Tulipa sp. anthers, found a substantial portion of radioactive phenylalanine incorporated in sporopollenin, indicating a role for phenolics in building this biopolymer (Gubatz et al., 1992, 1993; Herminghaus et al., 1988; Prahl et al., 1986). Since then, lipids have also been recognized for their role in sporopollenin formation, as treatment of Zea mays with a thiocarbamate herbicide inhibiting chain elongation during long-chain (C > 18) fatty acid biosynthesis altered sporopollenin chemistry, as determined by Fourier transform infrared spectroscopy (Wilmesmeier and Wiermann, 1995). Since these and other exploratory studies highlighted substantial uncertainty in the composition of sporopollenin, new analytical techniques to study sporopollenin have continued to emerge, and models for the composition of this fascinating organic compound continue to be revised. The challenge in elucidating the composition of sporopollenin lies primarily in its recalcitrance to solubilization, which requires analysis of sporopollenin components remaining after acetic anhydride treatment, extended ozonolysis, nitrobenzene oxidation, potash fusion or aluminum iodide treatment (Kawase and Takahashi, 1995; Schulze Osthoff and Wiermann, 1987; Southworth, 1974). Gas–liquid chromatography coupled to mass spectrometry (MS) found fatty acids and oxygenated cinnamic acid derivatives to be the main components of sporopollenin, however, some argue that impurities have falsely been identified as components of sporopollenin (Van Bergen et al., 2004, 1995). Using pyrolysis-MS, ferulic acid and 4-coumaric acid constituents have been identified in sporopollenin (Rozema et al., 2001; Wehling et al., 1989). Inhibitor treatments, radiolabeling, and chemical breakdown studies have yielded complementary information on the constituents present in sporopollenin, which is now typically accepted to contain phenolics and polyhydroxylated aliphatics, covalently coupled by ether and ester bonds (Ahlers et al., 2000; Guilford et al., 1988; Scott, 1994; Wiermann et al., 2005). Because the limited solubility of sporopollenin poses challenges to many chemical analyses, the application and optimization of polymer analyses could prove to be informative alternatives. Through the application of solid-state nuclear magnetic resonance (NMR), aromatic and aliphatic moieties as well as oxygen functionalities have been identified in the sporopollenin biopolymer (Ahlers et al., 2000, 2003, 1999; Guilford et al., 1988; Hemsley et al., 1992). The application of matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF) for sporopollenin structure elucidation achieved limited success and will require matrix optimization (Moore et al., 2006). Altogether, data from chemical analyses have provided some insight into the composition of sporopollenin and the cross-linking that exists within this biopolymer. However, as analyses employing harsh treatments to sporopollenin increase the likelihood of modified fragment release from the original biopolymer, our understanding of the biochemistry of intact sporopollenin requires further investigation. 2.3. Sporopollenin composition: insights provided by genetic analyses The clearly established progression of cellular events in Arabidopsis anther and pollen development and the crucial role of pollen in sexual plant reproduction have facilitated the identification of Arabidopsis mutants with phenotypic changes in pollen wall development and fertility. These mutants have provided powerful new tools for understanding the biochemistry of sporopollenin biosynthesis and mechanisms of deposition. Specifically, numerous gene products have been implicated in sporopollenin biosynthesis and deposition, since mutations in the corresponding genes produce plants with defects in pollen wall formation immediately following tetrad release (at the time of high sporopollenin flux from the tapetum), often impairing male fertility. Among these genes,

a subset encodes transcriptional regulators with tapetum-preferential expression, which has clarified the importance of regulatory networks required for exine development. MALE STERILITY 1 (MS1/ HKM), encoding a plant PHD-type zinc finger putative transcription factor, is required for tapetum development and exine formation through the apparent direct or indirect regulation of over 260 genes, primarily thought to function in exine component synthesis (Ito et al., 2007; Ito and Shinozaki, 2002; Wilson et al., 2001; Yang et al., 2007). The ABORTED MICROSPORES (AMS) gene encodes a basic helix-loop-helix transcription factor that plays a role similar to MS1 in tapetum and post-meiotic microspore development, with ams mutants displaying altered expression in over 500 genes associated with pollen wall development and tapetum function (Xu et al., 2010). Additionally, MYB80 (formerly MYB103/MS188), an R2R3-type MYB transcription factor, is required for callose wall and exine formation as well as tapetum maturation in Arabidopsis (Phan et al., 2011; Zhang et al., 2007). MS1, AMS and MYB80 are expressed in tapetal cells at a time surrounding sporopollenin synthesis and deposition, and appear to be important regulatory points in the formation of the pollen wall. These regulators have been and will continue to serve as important tools in the identification of genes involved in processes related to sporopollenin synthesis, traffic and assembly. Reverse genetic screens of candidate genes involved in anther and pollen development have identified a number of Arabidopsis genes required for sporopollenin biosynthesis, encoding enzymes whose catalytic activities have been biochemically characterized. Molecular-genetic studies on two cytochrome P450 (CYP450) enzymes capable of hydroxylating the fatty acid constituents of predicted sporopollenin precursors have been implicated in sporopollenin biosynthesis. These cytochrome P450 enzymes, CYP703A2 and CYP704B1, catalyze the in-chain and x-hydroxylation of fatty acids, with substrate chain lengths of C10–C14 and C16–18, respectively (Table 1; Dobritsa et al., 2009b; Morant et al., 2007). In support of the predicted roles of CYP703A2 and CYP704B1 in sporopollenin formation, cyp703a2/(or dex2, for DEFECTIVE IN EXINE PATTERNING2) mutants exhibit strong reductions in male fertility with pollen grains that lack exine (Morant et al., 2007), and cyp704b1 mutant pollen lack normal exine (Dobritsa et al., 2009b), though it is not sterile. Both genes are specifically expressed in developing anthers. The involvement of these two fatty acid hydroxylases in sporopollenin biosynthesis is in good agreement with the highly oxygenated nature of sporopollenin, as evidenced by the abundant ether (and to a lesser extent ester) linkages (Ahlers et al., 2000) and supports the prediction that high levels of covalent coupling among subunits results in the characteristic recalcitrance of sporopollenin. ACYL-COA SYNTHETASE5 (ACOS5) has been identified as being critical for pollen development and sporopollenin biosynthesis. ACOS5 belongs to a novel class of enzymes related to, but functionally distinct from the phenylpropanoid enzyme 4-coumarate:CoA ligase (4CL). It was originally identified as a 4CL-like gene encoding an acyl-CoA synthetase of unknown function specifically expressed in developing anthers of Arabidopsis and with putative orthologs in other species (Souza et al., 2008). In vitro assays indicated the surprising preference of recombinant ACOS5 enzyme for non-phenolic substrates, delineating a distinct function for this enzyme from true 4CLs involved in phenylpropanoid metabolism. Recombinant ACOS5 has broad activity toward medium and long-chain fatty acids, including hydroxylated forms (Table 1), producing the corresponding acyl-CoA ester in vitro (Kienow et al., 2008; de Azevedo Souza et al., 2009). The acos5 mutant is male sterile and developing microspores show defects upon release from tetrads, failing to form exine and aborting shortly after the start of the free microspore stage (stage 9 of anther development). Furthermore, ACOS5 is specifically expressed in tapetal cells at a time

Please cite this article in press as: Quilichini, T.D., et al. The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.05.002

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Table 1 Expression of genes involved in sporopollenin biosynthesis and subcellular localization and characterized enzymatic activity of the corresponding proteins. The column on the right shows the accepted substrates accepted in vitro by the enzymes. Substrates in bold have been shown to be preferentially metabolized by the enzymes based on in vitro assays and competition assays. Enzymes from Arabidopsis (prefix At), rice (Oryza sativa, prefix Os) and tobacco (Nicotiana tabacum, prefix Nt) are represented.

corresponding to the appearance of sporopollenin on free microspores (de Azevedo Souza et al., 2009), consistent with a critical role of the fatty acyl-CoA esters thought to be produced by ACOS5 in the sporopollenin biosynthesis pathway. The identification of ACOS5 as a key enzyme in exine formation facilitated the identification of additional genes encoding enzymes with putative functions in sporopollenin biosynthesis, primarily through in silico co-expression analyses. Two such co-expressed CHALCONE SYNTHASE-like genes were found to function in exine formation, as mutations in these genes result in an abnormal exine pattern (Dobritsa et al., 2010; Mizuuchi et al., 2008). These enzymes had been previously shown to catalyze the in vitro

condensation of two or three malonyl-CoAs with fatty acyl-CoAs of varying chain lengths (from C4 to C20), producing primarily tri- and tetraketide a-pyrones (Table 1; Dobritsa et al., 2010; Mizuuchi et al., 2008). These enzymes were named POLYKETIDE SYNTHASE A (PKSA) and PKSB (also named ‘‘Less Adherent Pollen 5 (LAP5) and LAP6, Dobritsa et al., 2010). Subsequent biochemical studies found similar in vitro activities for PKSA and PKSB toward medium- to long-chain fatty acyl-CoA, as well as activity towards p-coumaroyl-CoA (Dobritsa et al., 2010; Mizuuchi et al., 2008; Kim et al., 2010). Interestingly, based on in vitro competition assays, these enzymes exhibited a clear preference for long-chain hydroxylated fatty acyl-CoA esters (16-hydroxy palmitoyl-CoA or

Please cite this article in press as: Quilichini, T.D., et al. The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.05.002

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12-hydroxy stearoyl-CoA) (Kim et al., 2010), which is in agreement with the involvement of CYP703A2 and CYP704B1 in the same biosynthetic pathway. Also in support of their putative role in sporopollenin precursor synthesis, PKSA and PKSB are expressed specifically in the tapetum at a time surrounding sporopollenin synthesis and deposition, and reverse genetic analysis showed that pksa pksb double mutants are male sterile and completely lack the formation of exine after tetrad release (Dobritsa et al., 2010; Kim et al., 2010). Also co-expressed with ACOS5 were two oxidoreductase genes, DIHYROFLAVONOL REDUCTATASE LIKE1 (DRL1) and DRL2 (Tang et al., 2009; Grienenberger et al., 2010) with DRL2 previously annotated as CINNAMOYL-COA REDUCTASE LIKE6 (CCRL6; Hamberger et al., 2007). The ability of the corresponding recombinant proteins to reduce the ketone function of tetraketide a-pyrones (produced by PKSA and PKSB) in the presence of NADPH suggested that these enzymes work downstream of PKSA and PKSB, and they were thus renamed TETRAKETIDE a-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (Grienenberger et al., 2010). Consistent with their hypothesized roles in sporopollenin biosynthesis, a strong male-sterile phenotype was observed for mutant alleles of TKPR1, (Grienenberger et al., 2010; Tang et al., 2009), which lacked or had severely perturbed exine. However, loss-of-function TKPR2 mutants had only minor perturbations in their exine walls and produced fertile plants (Grienenberger et al., 2010). Thus, despite the nearly identical substrate preferences observed for recombinant TKPR1 and TKPR2 in vitro, their in vivo functions likely differ, possibly due to different timing of expression in tapetal cells over the course of microspore development (Grienenberger et al., 2010), or differences in subcellular localization as TKPR1 co-localized with PKSA and PKSB in the ER membrane, while TKPR2 is a cytoplasmic enzyme (Grienenberger et al., 2010). As summarized in Table 1, these data suggested that, in Arabidopsis, tapetum-synthesized polyhydroxylated long-chain tetraketide a-pyrones, generated by the sequential actions of ACOS5, PKSA/PKSB, TKPR1, and including fatty acid hydroxylations carried out by CYP703A2 and CYP704B1 generate key aliphatic constituents of sporopollenin. Along with tetraketide a-pyrones, genetic and biochemical analyses suggest that fatty alcohols may also form an additional key subset of the aliphatic constituents of sporopollenin. MALE STERILITY2 (MS2) was first identified as a gene required for exine formation in Arabidopsis, based on the severe reduction in male fertility and loss of exine formation observed in ms2 mutants (Aarts et al., 1997). Despite contrasting reports on the fertility of ms2 mutants with different insertion alleles, all ms2 mutants characterized to date exhibit severe exine defects (Aarts et al., 1997; Dobritsa et al., 2009b). Based on sequence homology with characterized fatty acyl reductases (FARs), MS2 was predicted to function in the production of sporopollenin fatty alcohols by reducing fatty acyl-CoA substrates. Escherichia coli strains expressing MS2 form C14:0, C16:0, and C18:1 alcohols from an unknown endogenous bacterial precursor(s) (Doan et al., 2009). In a subsequent study, Chen et al. (2011) found the MS2 enzyme localized to the plastid and in vitro assays using purified recombinant MS2, demonstrated its reducing activity toward long-chain fatty acids esterified to acyl carrier protein (ACP), with a strong preference for C16:0-ACP (or palmitoyl-ACP), producing C16:0 alcohol (Chen et al., 2011). Interestingly, these assays found no MS2 activity against acyl-CoA substrates, including C16 acyl-CoA, which serve as the preferred substrates for other known fatty acid reductases. These data suggest that MS2 functions as a plastid-localized fatty acyl-ACP reductase and its fatty alcohol products form sporopollenin constituents or precursors in Arabidopsis (Table 1). ECERIFERUM 3 (CER3/WAX2/YRE), commonly called FACELESS POLLEN1 (FLP1) in the pollen literature, encodes a protein involved in alkane biosynthesis, although its specific biochemical function is

not known (Bernard and Joubès, 2012). CER3 functions in alkane biosynthesis in stem and silique epidermal cells, as well as in the synthesis of pollen lipids (Ariizumi et al., 2003; Chen et al., 2003; Kurata et al., 2003; Rowland et al., 2007). Specifically, CER3 appears to have multiple functions in pollen exine lipid production as cer3 mutant pollen exhibit increased coat covering with altered lipid body morphology and abundance, and acetolysis-sensitive sporopollenin. Thus, alkanes may form additional aliphatic components of sporopollenin in Arabidopsis. Beyond the aliphatic constituents of sporopollenin, one or more phenolic components in this polymer have been documented. These phenolics appear to be derived from hydroxycinnamic acids of the phenylpropanoid pathway, since mutants such as the reduced epidermal fluorescence 3-2 mutant in CINNAMATE-4HYDROXYLASE, which encodes a key entry point enzyme into phenylpropanoid metabolism, fail to develop pollen, and wall fluorescence in such mutants is abolished (Schilmiller et al., 2009). The importance of the phenylpropanoids in pollen exine biosynthesis is further evident from the male sterile phenotype of a transgenic Arabidopsis line over-expressing FERULATE 5-HYDROXYLASE in a CAFFEIC ACID O-METHYLTRANSFERASE mutant (comt) background that produces high levels of an unusual 5-hydroxyguaiacyl (5H)rich lignin (Weng et al., 2010). These 5H-rich plants produced pollen that appeared devoid of exine, suggesting that manipulation of the monolignol pathway could alter the flux of select phenolics into sporopollenin synthesis. Based on these data, the authors suggest that reductions in available p-coumaroyl-CoA and feruloylCoA, substrates for a spermidine hydroxycinnamoyl transferase (SHT) involved in the synthesis of pollen coat phenolamide constituents, could cause the observed sporopollenin deficiencies (Weng et al., 2010). However, reductions of hydroxycinnamoyl spermidines, as in sht mutants, do not have fertility defects or major exine deficiencies (Grienenberger et al., 2009), consistent with the possibility that other coumaroyl-CoA and feruloyl-CoA-derived phenylpropanoids are incorporated into sporopollenin. In contrast to the characterized gene products described above, a number of genes encoding proteins of unknown function are also required for normal exine formation in Arabidopsis, and the proteins encoded by these genes may participate in sporopollenin biosynthesis or deposition in Arabidopsis. These include DEFECTIVE IN EXINE PATTERNING 1 (DEX1), NO EXINE FORMATION1 (NEF1), LESS ADHERENT/ADHESIVE POLLEN 3 (LAP3), and TRANSIENT DEFECTIVE EXINE (TDE1/DET2) (Ariizumi et al., 2004, 2008; Dobritsa et al., 2009a; Paxson-Sowders et al., 2001; Rowland et al., 2007). For extensive reviews on the putative roles of these genes in sporopollenin synthesis and/or deposition, the reader is referred to recent reviews (Ariizumi and Toriyama, 2011; Blackmore et al., 2007). Additionally, two genetic screens employed in Arabidopsis with the goal of finding novel genes required for pollen wall formation identified 12 KAONASHI genes (meaning ‘‘faceless’’ in Japanese), and 14 genes required for exine formation (Dobritsa et al., 2011; Suzuki et al., 2008). The application of forward and reverse genetics, molecular biology and biochemical analyses has enabled the identification and characterization of numerous genes, encoding proteins and enzymes required for exine formation. Together, these studies have provided clues regarding the mechanism of sporopollenin biosynthesis, particularly pertaining to its aliphatic constituents, and have defined a key role for the tapetum in pollen wall formation. 2.4. The conservation of sporopollenin composition among land plant lineages Despite the limited understanding of sporopollenin composition, structure and biosynthesis, this extracellular matrix is thought to be conserved in its composition among all land plants

Please cite this article in press as: Quilichini, T.D., et al. The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.05.002

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whether it is present in the spores of early diverging land plants, or in the pollen of seed-bearing species. Comparative phylogenetic and genomic analyses support this hypothesis, as putative orthologs of ACOS5, PKSA, PKSB, CYP703A2 and CYP704B1 are broadly distributed in flowering plants, and in the moss Physcomitrella patens (de Azevedo Souza et al., 2009; Kim et al., 2010; Grienenberger et al., 2010; Dobritsa et al., 2010; Colpitts et al., 2011; Wang et al., 2013). These genes are apparently absent from the green algal lineage, as represented by Chlamydomonas reinhardtii, supporting a key role for acquisition of these genes in the progression of plant life onto land (Colpitts et al., 2011; Dobritsa et al., 2009b; Koduri et al., 2010; Morant et al., 2007; Ríos et al., 2013; de Azevedo Souza et al., 2009). The hypothesis that a highly conserved biochemical pathway leading to sporopollenin biosynthesis exists among land plants has been further supported by genetic and biochemical approaches used to study a variety of plant species. In particular, Physcomitrella patens ASCL encodes an enzyme with in vitro preference for hydroxy fatty acyl-CoA esters that is capable of hydroxyalkylpyrone synthase activity, suggesting that PpASCL is a functional ortholog of Arabidopsis PKSA and that the pathway to sporopollenin may be conserved among land plants (Colpitts et al., 2011). In rice, OsCYP704B2 encodes a long-chain fatty acid xhydroxylase capable of metabolizing very similar substrates in vitro as CYP704B1 from Arabidopsis (Table 1; Li et al., 2010). Furthermore, mutation of OsCYP704B2 leads to male sterility and aborted pollen grains without detectable exine (Li et al., 2010). Another example comes from the rice defective pollen wall (dpw) mutant, which is male sterile with pollen grains exhibiting an irregular exine (Shi et al., 2011). OsDPW encodes a fatty acyl-ACP reductase with a strong preference for the C16:0-ACP, but weak reductase activity towards the C16:0-CoA (Shi et al., 2011). The similar functions and enzymatic activities of Arabidopsis MS2 and rice DPW, as well as the ability of OsDPW to rescue wild-type exine in the Arabidopsis ms2 mutant background, support the conservation of sporopollenin synthesis among monocotyledonous and dicotyledonous plants (Shi et al., 2011). Similarly, recombinant proteins of putative orthologs of ACOS5, PKSA and TKPR1 in rice and tobacco (Nicotiana tabacum) have been shown to catalyze very similar reactions as the corresponding Arabidopsis enzymes, using identical or similar substrates (Table 1; Wang et al., 2013). Intriguingly, both cyp704b2 and dpw rice mutants also exhibit anther cuticle defects (Li et al., 2010; Shi et al., 2011) suggesting an additional function for these genes in rice. 2.5. Model for sporopollenin biosynthesis and the sporopollenin metabolon With the insights provided by many of the molecular genetic and biochemical studies described above, it is likely that key components of sporopollenin include polyhydroxylated tetraketide apyrones, or related polyketides, based on the synthesis of these components by the successive actions of recombinant sporopollenin biosynthetic enzymes ACOS5, PKSA, PKSB, and TKPR1 (Grienenberger et al., 2010). Fig. 3 presents a proposed model based on these findings. According to this model, medium- and long-chain fatty acyl-ACP, synthesized by the Fatty Acid Synthase (FAS) complex in the plastid can be hydrolyzed by Fatty Acyl Thioesterase B (FATB) to release free fatty acids that are exported from the plastid (Li-Beisson et al., 2010) prior to their subsequent esterification by ACOS5. ACOS5-derived fatty acyl-CoAs are central precursors required for the synthesis of sporopollenin constituents in the tapetum, and serve as the substrate for ER-localized PKSA and PKSB, which generate tetraketide a-pyrones (Fig. 3, route C) that are subsequently reduced by TKPR1. Based on in vitro biochemical data, CYP703A2 and CYP704B1 catalyze the in-chain and xhydroxylations of free fatty acids, respectively. This would require

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the action of an unknown thioesterase prior to the action of these CYPs, followed by re-esterification with CoA, possibly catalyzed by ACOS5 (Fig. 3, route A). However, it is possible that CYP703A2 and CYP704B1 directly hydroxylate the fatty acyl-CoA esters (Fig. 3, route B), but this hypothesis requires experimental testing. With this hypothesis, PKSA and PKSB could metabolize polyhydroxylated fatty acyl-CoA esters to form the corresponding tetraketide apyrones (Fig. 3, route D), in agreement with their preferential activity toward hydroxylated fatty acyl-CoAs. It is also possible that CYP703A2 and/or CYP704B1 hydroxylate the aliphatic chain of the tetraketides produced by PKSA and PKSB (Fig. 3, route E). However, it is likely that the sequential action of ACOS5, CYP703A2, CYP704B1 PKSA, PKSB, and TKPR1 result in the formation of polyhydroxylated tetraketide a-pyrones that are then exported into the anther locule, possibly by the ABCG26 transporter (Quilichini et al., 2010) and/or lipid transfer proteins (LTPs), for polymerization into the pollen exine or further modified by uncharacterized enzymes (such as LAP3), prior to its export. In support of this model, ACOS5, PKSA, PKSB and TKPR1 interact in pairwise combinations and co-localize at the ER, and may form the core of a sporopollenin metabolon, facilitating efficient sporopollenin synthesis in Arabidopsis tapetal cells (Lallemand et al., 2013). In the model for sporopollenin biosynthesis, the plastid-localized MS2 reduces the C16:0 fatty acyl-ACP to produce the corresponding fatty alcohol in the tapetum. This product could then be exported to the anther locule by an unknown mechanism where it polymerizes at the surface of the microspore, or be further modified by uncharacterized enzymes (Fig. 3, route F). Alternatively, the fatty alcohol could be oxygenated by CYP450s or other enzymes to generate cytoplasmic fatty acids that could then be esterified by ACOS5 (Fig. 3, route G). In this hypothetical route, MS2 is part of the polyketide pathway, acting upstream of ACOS5, PKSA, PKSB and TKPR1 to produce a unique sporopollenin constituent. However, additional biochemical and in vivo experiments are needed to determine the exact step in the pathway where the different characterized enzymes play a role. While enzyme assays using ACOS5, PKSA, PKSB and TKPR1 sequentially produce tetraketide a-pyrones in vitro (Grienenberger et al., 2010), and enzymes of this pathway are localized to tapetal cells, the in planta sporopollenin precursor(s) exported from the tapetum to the locule and developing microspores is not known. Although the understanding of the function of a-pyrones in plants is limited, the possibility that the a-pyrones produced by the sporopollenin metabolon represent the in planta products of this pathway should be seriously considered, as it has been demonstrated that plants are capable of a-pyrone synthesis (Eckermann et al., 1998; Weng et al., 2012). However, it is also possible that the a-pyrones observed previously are by-products of in vitro assays (Abe et al., 2004; Akiyama et al., 1999; HeslopHarrison and Mackenzie, 1967; Taylor, 1959; Ubisch, 1927; Yamaguchi et al., 1999). In particular, type III polyketide synthases, such as PKSA and PKSB, are known to vary in their polyketide product cyclizations, with different intramolecular or heterocyclic condensations capable of producing aromatic rings, lactones, or lacking cyclization activity altogether, while a-pyrones represent potential derailment products generated by the enzymes in vitro (Ariizumi and Toriyama, 2011; Austin and Noel, 2003; Funa et al., 2006; Tropf et al., 1994). It is also possible that modification of the PKSA and/or PKSB products may arise from an accessory protein that alters the cyclization of polyketides as they are synthesized, as in the production of olivetolic acid in Cannabis (Gagne et al., 2012). The tetraketide product of the sporopollenin-related polyketide pathway may be further modified or cyclized prior to export from the tapetum. Future experiments aimed at identifying possible tapetum localized accessory proteins capable of polyketide cyclization activity could help clarify this point.

Please cite this article in press as: Quilichini, T.D., et al. The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.05.002

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Fig. 3. Proposed sporopollenin biosynthetic pathway. Fatty acyl-ACP esters, synthesized de novo in the plastid by the FAS complex are reduced by MS2 to produce fatty alcohols or hydrolyzed by FATB to produce free fatty acids. Fatty acids are exported from the plastid prior esterification to CoA by ACOS5. Fatty acyl-CoA esters are then condensed with malonyl-CoAs by the ER-localized PKSA and PKSB to produce tetraketide a-pyrones. Condensations by PKSs could take place either directly (route C) or after hydroxylations by CYP703A2 and CYP704B1 (routes A or B). CYP450s could hydroxylate free fatty acids, requiring the hydrolysis of the CoA esters by a putative thioesterase and regeneration of the CoA esters by ACOS5 upstream and downstream of the hydroxylation step, respectively (route A). Alternatively, CYP450s could hydroxylate fatty acylCoA esters directly (route B). Hydroxylation of the aliphatic chain might also occur at the tetraketide level (route E). Polyhydroxylated tetraketides are then reduced by TKPR1 to produce tetraketide a-pyrones with multiple hydroxy functions that could be exported to the anther locule by the ABCG26 transporter and/or LTPs, to be polymerized at the surface of developing microspores or be further processed by unknown enzymes. Alternatively, fatty alcohols produced by MS2 might putatively be hydroxylated by CYP450s or other enzymes to produce fatty acids that could subsequently be esterified by ACOS5 (route F). Question marks (?) indicate information not supported by experimental data. FAS: Fatty Acid Synthetase, FATB, Fatty Acyl Thioesterase B.

2.6. The sporophytic origin of the exine It has long been assumed that the barrier formed by callose prevents cell wall component transfer between the tapetum and developing microspores, and that thus, primexine components must be provided solely by the microspores during microsporogenesis, while tapetum-derived sporopollenin components are deposited on the microspore after callose dissolution. This view has been challenged by genetic studies, as homozygous mutants with primexine abnormalities show no defects in their respective heterozygous states, suggesting that the sporophytic tapetum functions in primexine formation (reviewed by Ariizumi and Toriyama, 2011). However, the inheritance of factors or information for primexine biosynthesis from sporophytic microspore mother cells during meiosis provides an alternate explanation and requires further investigation. Further, there are conflicting reports regarding the first appearance of sporopollenin on the surface of microspores. According to Heslop-Harrison (1968a,b), early primexine is acetolysis-sensitive, while late primexine bearing developed probaculae in Lilium longiflorum becomes acetolysis-resistant, suggesting the presence of sporopollenin in the latter (Heslop-Harrison, 1968b). However, as the material fortifying the late-primexine walls of microspores in tetrads did not stain in a manner similar to mature sporopollenin, it was considered to differ from sporopollenin, and was termed protosporopollenin. In support of these findings in Lilium, tapetumexpressed genes encoding enzymes or proteins required for sporopollenin synthesis or accumulation in Arabidopsis, such as ACOS5,

PKSA, PKSB, TKPR1 and ABCG26 (introduced below), show no apparent primexine defects, suggesting that tapetum-supplied sporopollenin is not present around callose-encased tetrads. However, a method to remove callose from Brassica rapa tetrads was recently developed, allowing the early wall around microspores to be examined by scanning electron microscopy (Kirkpatrick and Owen, 2013). After callose digestion by cellulase, pectolyase and cytohelicase, the persistence of patterned microspore walls suggested that sporopollenin may already be present on tetrad-stage microspores (Kirkpatrick and Owen, 2013). Future studies on the formation and composition of the primexine, particularly regarding the location of enzymes and proteins involved in its synthesis, will provide valuable insight into the timing of sporopollenin appearance on microspores. While it is commonly accepted based on the extensive literature discussed above that sporopollenin biosynthesis is largely restricted to sporophytic tapetal cells, there is uncertainty surrounding the gametophytic contribution to sporopollenin production. Although most genes encoding enzymes or proteins implicated in outer pollen wall formation have expression patterns that appear specific to the sporophytic generation, some genes required for sporopollenin formation appear to have dual expression in the tapetum and microspore (Reviewed by Wallace et al., 2011). For example, while ACOS5, PKSA, PKSB, TKPR1, TKPR2, and MS2 transcripts are specifically detected in tapetal cells by in situ hybridization, and PKSA and PKSB proteins are present in the tapetum at comparable developmental stages as demonstrated by

Please cite this article in press as: Quilichini, T.D., et al. The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.05.002

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immunolocalization (Aarts et al., 1997; Grienenberger et al., 2010; Kim et al., 2010; de Azevedo Souza et al., 2009), a CYP703A2 promoter-ß-glucuronidase (GUS) fusion was expressed both in the microspore and tapetum (Morant et al., 2007). Interestingly, the tapetum-localized expression of these genes appears highly restricted to the stages surrounding sporopollenin deposition on microspores, suggesting that mechanisms for high efflux from intact tapetal cells are in place at the early stages of uninucleate microspore development. Similarly, characterized mutations in KAONASHI (KNS) 4-10, CER3/FLP1/YRE/WAX2, CYP704B1, and NEF1 are recessive, and plants heterozygous for mutations in these genes produce 100% wild-type appearing microspores, suggesting that these sporopollenin-related genes function in sporophytic cells and that loss-of-function mutations in the haploid gametophyte do not affect sporopollenin deposition (Ariizumi et al., 2004; Suzuki et al., 2008). Despite the tapetum-specific expression of most genes required for sporopollenin formation and genetic evidence for a sporophytic origin, many models for pollen development maintain that the microspore secretes sporopollenin constituents (e.g., Wallace et al., 2011), and further evidence is required to substantiate these claims. In addition to sporopollenin, the pollen coat represents a second major component of the outer pollen wall in Arabidopsis that is dependent on tapetal cells. In contrast to the poorly understood composition of sporopollenin, the pollen coat layer of many pollen exines has been more thoroughly characterized, facilitated by its ease of extraction with non-polar organic solvents (Doughty et al., 1993). Analyses on Brassicaceae pollen coats have identified a mixture of primarily neutral-ester lipids, free fatty acids, verylong-chain wax esters, volatile lipid derivatives, flavonoids, alkanes, and hydroxycinnamoyl spermidines (Dobson, 1988; Grienenberger et al., 2009; Hsieh and Huang, 2007; Piffanelli et al., 1998; Preuss et al., 1993; Wu et al., 1997, 1999), as well as a number of proteins, the majority of which are classified as either processed oleosin proteins or self-incompatibility proteins (Kim, 2002; Kim and Huang, 2003; Mayfield, 2001; Piffanelli and Murphy, 1998). Many of these pollen coat components accumulate in tapetum cells within specialized organelles called tapetosomes and elaioplasts. Tapetosomes are ER-derived storage organelles that produce a plethora of metabolites, including alkanes and triacylglycerol (TAG)-rich oil droplets, which are structurally maintained by oleosins and surrounded by vesicles associated with flavonoids (Hsieh and Huang, 2007, 2005). Elaioplasts are specialized plastids bound by an outer membrane and filled with numerous steryl ester-rich globuli maintained by plastid lipid-associated proteins (PAPs) (Wu et al., 1999). In contrast to sporopollenin constituents, which are assembled on the microspore prior to tapetum death, the pollen coat is deposited in the final stage of tapetum development, as programmed cell death releases tapetum-stored materials into the locule. Interestingly, not all lipids or proteins detected in the tapetum are present in the pollen coat after expulsion from tapetum elaioplasts and tapetosomes (Ting et al., 1998). For example, tapetosome-derived oleosins, although present in the final pollen coat, are cleaved such that the C-terminus is present in the coat, while the abundant tapetosome TAGs appear absent, or minimally present in the pollen coat (Murphy and Ross, 1998; Wu et al., 1997). In summary, the sporopollenin and pollen coat layers of the exine move from their site of synthesis in tapetal cells to the surface of microspores/pollen grains by poorly understood mechanisms of active export or release by cell rupture, respectively. 2.7. Translocation of sporopollenin precursors As tapetal cells represent the major site of sporopollenin synthesis, there is a need for the rapid and efficient export of

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sporopollenin constituents upon the release of free microspores from callose. However, the underlying molecular mechanisms supporting this high and rapid efflux are poorly understood. A number of proteins required for exine formation have been hypothesized to function in sporopollenin precursor export from the tapetum, or in the translocation of sporopollenin constituents from secretory tapeta through the locule to the microspore surface. The ATP-BINDING CASSETTE26 (ABCG26) gene encoding an ABC half transporter protein is of particular interest in pollen exine assembly, due to its preferential expression in tapetal cells during early exine formation and its proposed role in the transport of sporopollenin components from their site of synthesis in tapetal cells to the anther locule (Quilichini et al., 2010), analogous to the roles of ABCG11 and ABCG12 in wax export from epidermal cells (Bird et al., 2007; Pighin et al., 2004). Furthermore, in support of their possible functions in male reproductive development and/or trafficking of aliphatic sporopollenin precursors within the locule, a large number of lipid transfer proteins (LTPs) have been noted for their anther- or tapetum-specific expression patterns (Eckermann et al., 1998; Huang et al., 2009; Weng et al., 2012). OsC6, encoding a type VII LTP in rice, is preferentially expressed in the tapetum and is required for normal exine and orbicule formation (Boutrot et al., 2008; Zhang et al., 2010). However, as with a number of rice proteins required for sporopollenin synthesis or deposition, OsC6 function appears to extend to anther cuticle formation (Zhang et al., 2010). In Arabidopsis, ARABIDOPSIS THALIANA ANTHER7 (ATA7/LTPH1) exhibits tapetum-specific expression in the free microspore stage by in situ hybridization (Rubinelli et al., 1998). In addition, the expression of three genes encoding type III LTPs (LTPC6, LTPC9, and LTPC14) is restricted to the tapetum, and plants carrying the dual knockdown of LTPC6 and LTPC14 exhibited abnormal intine and dehydration-sensitive pollen (Huang et al., 2013). Interestingly, the OsC6 and AtC6, C9 and C14 LTPs exhibit different localizations from their encoding transcripts, as they were localized to the locule and anther epidermis in rice, or in the locule and in association with the pollen exine in Arabidopsis (Huang et al., 2013; Zhang et al., 2010). Altogether, a number of genes encoding annotated transport proteins appear to exhibit spatially and developmentally restricted expression patterns within anthers, and some have roles that support their function in pollen wall or sporopollenin constituent trafficking. Structures associated with the tapetum that occur in some species, such as viscin threads (or strands bridging tapetal cells and microspores) and orbicules, also have proposed roles in the trafficking of sporopollenin constituents, although their transport capabilities have yet to be demonstrated. Orbicules (also called Ubisch bodies) are small, typically spherical granules, often with a diameter of less than 5 lm, which are of particular interest in sporopollenin translocation studies, as they appear to arise in tapetal cells, and have been proposed to carry sporopollenin components from the tapetum to developing microspores (Wang et al., 2003). In support of their proposed sporopollenin precursor-trafficking function, orbicules are abundant outside the locule-facing edge of secretory-type tapeta, typically appear in coordination with sporopollenin synthesis and accumulation on microspores, and contain acetolysis-resistant sporopollenin (Huysmans et al., 2010; Vinckier et al., 2005). However, their absence in many species, including Arabidopsis, and persistence after sporopollenin formation is complete suggest they may have other functions (Huysmans et al., 1998, 2000; Rowley and Morbelli, 2009). In summary, the export and translocation of sporopollenin components from tapetal cells is poorly understood, intensified by our lack of full understanding of the biochemical nature of sporopollenin components.

Please cite this article in press as: Quilichini, T.D., et al. The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.05.002

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2.8. Sporopollenin assembly While we are just beginning to understand the mechanisms governing sporopollenin traffic out of tapetal cells and through the locule, arguably even less is known about the processes and proteins governing sporopollenin assembly into the intricately patterned and sculptured pollen exine. However, genetic approaches to this problem have yielded some insights. Over the last decade, a number of Arabidopsis exine patterning or deposition mutants have been characterized, shedding some light on the proteins involved in pollen outer wall assembly. DEFECTIVE IN EXINE FORMATION1 (DEX1) encodes a protein of unknown function that is required for Arabidopsis exine patterning (Paxson-Sowders et al., 2001). The dex1 mutant lacks the hallmark plasma membrane invaginations of callose-encased tetrads associated with the early stages of exine patterning, and upon their release, dex1 microspores exhibit random sporopollenin-like deposits and abort prematurely. A protein of unknown function, NO EXINE FORMATION1 (NEF1), has a proposed role in the deposition of sporopollenin, as nef1 mutants exhibit primexine abnormalities and sporopolleninlike aggregates in the locule (Ariizumi et al., 2004). NEF1 encodes an integral membrane protein of plastids, and disruption of this gene alters lipid accumulation in the tapetum plastids, although the link between this predicted plastid membrane protein’s function in tapetum lipid metabolism and its function in sporopollenin anchoring requires further investigation. The tomato tapetum-produced GLYCINE-RICH PROTEIN92 (LeGRP92) also has proposed sporopollenin deposition capabilities, as its down-regulation results in uneven exine deposition and reduced pollen viability (McNeil and Smith, 2009). The broad localization of LeGRP92 in the callose wall of microspore mother cells, tetrads, microspore exine and orbicules suggests that this protein may function as an intermediary between primexine and sporopollenin precursors, guiding exine deposition (McNeil and Smith, 2009). Two putative glycosyl transferases encoded by At1g27600/SPG2/IRX9-like and At1g33430/UPEX1/UPEX2 were found to function in Arabidopsis exine patterning, possibly in the polymerization or anchoring of sporopollenin to the microspore primexine (Dobritsa et al., 2011). Although the evidence for protein-assisted sporopollenin assembly is growing, the rapid formation of a characteristic and intricate exine pattern on the microspore surface, apparently based on sporopollenin polymerization, suggests that properties enabling self-assembly of the biopolymer may contribute to exine wall formation (Hemsley et al., 1996). Despite the broad range of speciesspecific patterning, an underlying reticulate, hexagonal arrangement of bacula is a uniting feature of pollen exine and spore walls in most species, suggesting that sporopollenin self-assembly may in part account for the complex form of the exine (Hemsley et al., 1996; Scott, 1994). To test the contribution of self-assembly in exine patterning, spore wall construction has been examined in vitro by removing the leading role of the genetically encoded information (i.e. genomic information specifying primexine or glycocalyx formation, prior to sporopollenin formation) and applying sporopollenin-mimicking substances (fatty acids) under different conditions (Gabarayeva and Grigorjeva, 2013). Some sporopollenin-like patterns were observed in these and other wall-forming simulations, provide preliminary evidence for the contribution of self-assembly in the elaborate outer wall formed by pollen and spores.

3. Conclusion Recent genetic and molecular studies have highlighted the predominantly lipidic nature of sporopollenin precursors, confirming previous biochemical analyses performed on pollen walls.

Particularly, characterization of enzymatic activities of proteins required for sporopollenin formation has provided a means for uncovering putative sporopollenin biosynthetic pathways, its chemical nature and the basis of its outstanding recalcitrance. However, numerous questions remain to be answered to fully understand the processes leading to pollen wall formation. Particularly, further experiments are needed to characterize the exact role and relationships among characterized enzymes in the sporopollenin pathway as well as the physiological substrates of these enzymes. In planta identification of the intermediates and final products of the sporopollenin pathway, using for example metabolomic approaches, would be of great interest. Additionally, several enzyme-encoding genes believed to be involved in these processes require biochemical characterization and would certainly improve our understanding of the pathway producing sporopollenin. The expanding knowledge of sporopollenin composition and the processes governing its synthesis in the tapetum are in stark contrast to the modest understanding other important processes leading to pollen wall formation. Particularly, the questions of the mechanisms involved in the export of sporopollenin units from tapetal cells and its traffic through the locule to early microspores, the mechanisms driving the polymerization of sporopollenin units in a spatially and temporally specific manner, and the basis of the precisely regulated exine pattern remain to be answered. Forward genetic screens have identified many genes putatively involved in these processes. Their functional characterization would certainly help answering these important questions. Acknowledgements Work in the authors’ labs was supported by Natural Sciences and Engineering Research Council of Canada NSERC) Discovery Grants to CJD, and by the NSERC CREATE Grant ‘‘Working on Walls’’. References Aarts, M.G.M., Hodge, R., Kalantidis, K., et al., 1997. The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes. Plant J. 12, 615–623. Abe, I., Watanabe, T., Noguchi, H., 2004. Enzymatic formation of long-chain polyketide pyrones by plant type III polyketide synthases. Phytochemistry 65, 2447–2453. Ahlers, F., Thom, I., Lambert, J., et al., 1999. 1H NMR analysis of sporopollenin from Typha angustifolia. Phytochemistry 50, 1095–1098. Ahlers, F., Bubert, H., Steuernagel, S., Wiermann, R., 2000. The nature of oxygen in sporopollenin from the pollen of Typha angustifolia L. Z. Naturforsch., C: J. Biosci. 55, 129–136. Ahlers, F., Lambert, J., Wiermann, R., 2003. Acetylation and silylation of piperidine solubilized sporopollenin from pollen of Typha angustifolia L. Z. Naturforsch., C: J. Biosci. 58, 807–811. Akiyama, T., Shibuya, M., Liu, H.-M., Ebizuka, Y., 1999. P-Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii. Eur. J. Biochem. 263, 834–839. Ariizumi, T., Toriyama, K., 2011. Genetic regulation of sporopollenin synthesis and pollen exine development. Annu. Rev. Plant Biol. 62, 437–460. Ariizumi, T., Hatakeyama, K., Hinata, K., et al., 2003. A novel male-sterile mutant of Arabidopsis thaliana, faceless pollen-1, produces pollen with a smooth surface and an acetolysis-sensitive exine. Plant Mol. Biol. 53, 107–116. Ariizumi, T., Hatakeyama, K., Hinata, K., et al., 2004. Disruption of the novel plant protein NEF1 affects lipid accumulation in the plastids of the tapetum and exine formation of pollen, resulting in male sterility in Arabidopsis thaliana. Plant J. 39, 170–181. Ariizumi, T., Kawanabe, T., Hatakeyama, K., et al., 2008. Ultrastructural characterization of exine development of the transient defective exine 1 mutant suggests the existence of a factor involved in constructing reticulate exine architecture from sporopollenin aggregates. Plant Cell Physiol. 49, 58–67. Austin, M.B., Noel, J.P., 2003. The chalcone synthase superfamily of type III polyketide synthases. Nat. Prod. Rep. 20, 79–110. Bernard, A., Joubès, J., 2012. Arabidopsis cuticular waxes: advances in synthesis, export and regulation. Prog. Lipid Res. 52, 110–129. Bird, D.A., Beisson, F., Brigham, A., et al., 2007. Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion. Plant J. 52, 485–498.

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Teagen D. Quilichini completed her PhD in the Department of Botany at the University of British Columbia in February 2014. She is currently a postdoctoral fellow in the UBC Department of Botany.

Please cite this article in press as: Quilichini, T.D., et al. The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.05.002

T.D. Quilichini et al. / Phytochemistry xxx (2014) xxx–xxx Etienne Grienenberger received his Ph.D. at the University of Strasbourg in 2010, and performed postdoctoral research in the Department of Botany at the University of British Columbia from 2010-2013, when he moved to the University of California, Berkeley to take up a postdoctoral position in the Department of Plant and Microbial Biology.

13 Carl J. Douglas is a Professor in the Department of Botany at the University of British Columbia. His lab studies plant cell walls, with emphasis on the regulation of secondary cell wall biosynthesis, natural diversity in cell walls, and the pollen wall, using Arabidopsis and poplar as model systems.

Please cite this article in press as: Quilichini, T.D., et al. The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.05.002