Male Sterile2 encodes a plastid-localized fatty acyl carrier protein reductase required for pollen exine development in Arabidopsis

Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino-terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. In addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30°C), MS2 exhibits a K_m for 16:0-Acyl Carrier Protein of 23.3 ± 4.0 μm, a V_max of 38.3 ± 4.5 nmol mg⁻¹ min⁻¹, and a catalytic efficiency/K_m of 1,873 m⁻¹ s⁻¹. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis.In flowering plants, the life cycle alternates between diploid sporophyte and haploid gametophyte generations. Pollen grains play a biologically protective role for the haploid male sperm cells surrounded by the outer cell wall lipidic biopolymers called the exine (Blackmore et al., 2007; Li and Zhang, 2010; Ariizumi and Toriyama, 2011). Pollen exine protects the gametophyte against pathogen attack, dehydration, and UV irradiation as well as facilitates the pollination process, including pollen recognition and adhesion to the stigma. The highly durable exine that occurs throughout flowering plants is thought to play an essential role in land colonization by plants (Chaloner, 1976).The exine is mainly composed of the biopolymer sporopollenin and contains two sublayers, the sexine and nexine (Zinkl et al., 1999). The biochemical nature of pollen exine remains largely unknown because of the technical difficulties in purifying and obtaining large quantities of materials for analysis. In addition, sporopollenin is highly insoluble, resistant to degradation, and exceptionally stable (Brooks and Shaw, 1968; Bubert et al., 2002). Current evidence suggests that the major components of sporopollenin are derivatives of aliphatics, such as fatty acids and phenolic compounds (Bubert et al., 2002; Blackmore et al., 2007).Tapetum, the innermost sporophytic anther wall layer, is thought to play a major role in actively synthesizing and secreting sporopollenin precursors onto the microspore surface for pollen exine polymerization and patterning (Bedinger, 1992; Li and Zhang, 2010; Ariizumi and Toriyama, 2011). The model dicot plant Arabidopsis (Arabidopsis thaliana) and many other plants have a secretory-type tapetum with specialized structures such as tapetosomes in the cells, which accumulate lipidic components (Huysmans et al., 1998). The outer surface of Arabidopsis pollen grains displays elegant reticulate cavities with abundant pollen coat (tryphine) deposited inside the pollen exine.Pollen exine patterning appears to include at least three major developmental events: callose wall formation, primexine formation, and sporopollenin synthesis (Ariizumi and Toriyama, 2011). Exine formation commences after meiosis, with the accumulation of lipidic precursors onto the primexine surrounding newly formed microspores between the callose wall and the microspore plasma membrane (Paxson-Sowders et al., 2001; Blackmore et al., 2007). After the first pollen mitosis, the synthesis of the exine is almost complete; and during later stages of pollen exine formation, the pectocellulosic intine and the tryphine, called the pollen coat, are deposited onto the pollen wall (Piffanelli et al., 1998). Recent genetic and biochemical investigations showed that some genes, including MALE STERILITY1 (MS1), MS2, CER1, NO EXINE FORMATION1, FACELESS POLLEN1, CYP703A2, ACYL-COA SYNTHETASE5 (ACOS5), CYP704B1, TETRAKETIDE α-PYRONE REDUCTASE1 and -2 (TKPR1/2), LAP6/POLYKETIDE SYNTHASE A (PKSA), and LAP5/POLYKETIDE SYNTHASE B (PKSB) in Arabidopsis (Aarts et al., 1995, 1997; Wilson et al., 2001; Ariizumi et al., 2003, 2004; Morant et al., 2007; de Azevedo Souza et al., 2009; Dobritsa et al., 2009, 2010; Grienenberger et al., 2010; Kim et al., 2010b) as well as Tapetum Degeneration Retardation, Wax-Deficient Anther1, CYP704B2, C6, Postmeiotic Deficient Anther1, and Persistent Tapetal Cell1 in rice (Oryza sativa; Jung et al., 2006; Zhang et al., 2008, 2010, 2011; Hu et al., 2010; Li et al., 2006, 2010, 2011; Li and Zhang, 2010), are required for pollen exine synthesis. However, relatively little has been described on the biochemical aspects of these gene products.Fatty alcohols are widely observed in plants, animals, and algae in free forms (the component of cuticular lipids) but more frequently in esterified (wax esters) or etherified (glyceryl ethers) forms. Fatty alcohols and their derivatives are major components of the lipidic anther cuticle and pollen wall (Ahlers et al., 1999; Kunst and Samuels, 2003; Jung et al., 2006; Li et al., 2010). Previous investigations revealed that fatty acyl-CoAs are thought to be used as substrates for the production of fatty alcohols by fatty acyl-coenzyme A reductase (FAR) in garden pea (Pisum sativum), jojoba (Simmondsia chinensis), Arabidopsis, wheat (Triticum aestivum), mouse (Mus musculus), and silk moth (Bombyx mori; Aarts et al., 1997; Metz et al., 2000; Wang et al., 2002; Moto et al., 2003; Cheng and Russell, 2004; Rowland et al., 2006; Doan et al., 2009; Domergue et al., 2010).MS2 was assumed to encode a FAR-like protein that converts fatty acids to alcohols. MS2 was shown to be expressed in the tapetum shortly after the microspore was released from the tetrad (Aarts et al., 1997). ms2 mutants display abnormal pollen wall development, which is sensitive to acetolysis treatment, causing reduced pollen fertility (Aarts et al., 1997; Dobritsa et al., 2009). However, detailed biochemical characterization of the MS2 enzyme has not been performed. Recently, recombinant bacteria expressing five Arabidopsis FAR homologs were shown to produce fatty alcohols with carbon lengths of C14, C16, and C18 from endogenous bacterial fatty acids. Bacteria expressing MS2 are able to form C14:0, C16:0, and C18:1 alcohols (Doan et al., 2009). Furthermore, yeast cells expressing FAR1, FAR4, and FAR5 are able to produce alcohols using distinct but overlapping substrates with a chain length ranging from C18:0 to C24:0 (Domergue et al., 2010).In this study, we report the biochemical characterization of MS2. We show that MS2 encodes a fatty acyl-Acyl Carrier Protein (ACP) reductase, and the purified recombinant MS2 enzyme from Escherichia coli is able to convert the preferred substrate palmitoyl-ACP to C16:0 alcohol in the presence of NAD(P)H. In addition, MS2 possesses an N-terminal transit peptide that is necessary for localization to the plastid. The biological significance of MS2 subcellular localization, and the presence of conserved domains/motifs within MS2, were demonstrated by genetic complementation of ms2 mutants. This work, therefore, demonstrates the involvement of the plastid in primary fatty alcohol synthesis required for pollen wall development in Arabidopsis.