Characterization Of Male Sterile 2 Biochemical Function In Pollen Exine Synthesis In Arabidopsis

Pollen exine formation is necessary for pollen maturation. 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 exine is mainly composed of sporopollenin, which is thought to consist primarily of polymerized hydroxylated fatty acids and phenylpropanoids.Male Sterile 2 (MS2), which encodes a FAR-like protein that converts fatty acids to alcohols, is required for pollen wall development in Arabidopsis thaliana. MS2 was shown to be expressed in the tapetum shortly after the microspore released from tetrad. However, detailed biochemical characterization of the MS2 enzyme has not been investigated. We report here the subcellular localization and functional analysis of MS2 in pollen exine synthesis. Our main research results are as follows:1. The localization of MS2 in plastids is required for its function: Sequence analysis using TargetP 1.1 , ChloroP 1.1, and MitoProt II-v1.101 suggested that the MS2 protein contained a putative 46-amino acid plastid (or mitochondria) targeting signal at the N-terminus, a conserved NAD(P)H binding domain(NBD), and a sterile domain(SD). In order to determine the subcellular localization of MS2, we constructed pro35S:MS2-GFP with full length MS2 cDNA fused with GFP, pro35S:MS2△N-GFP in which MS2 deleted for the N-terminal transit peptide was fused with GFP, and pro35S:TP-YFP with the N-terminal transit peptide of MS2 fused to YFP. Using Arabidopsis leaf protoplasts and Nicotiana benthamiana leaves, we confirmed that the transit peptide of MS2 is able to target MS2 to chloroplasts.The biological significance of MS2's localization to plastids was confirmed by the observation that proMS2:MS2△N could not complement the defective pollen wall development in ms2 homozygous mutants, whereas proMS2:MS2 rescued the defect in pollen wall development in ms2. Furthermore, we made a construct proMS2:RTP-MS2△N', in which the N-terminal transit peptide of MS2 was replaced by the transit peptide of the small subunit of the Rubisco protein (RTP). Analysis of pollen grains from transgenic plants expressing the construct showed that the phenotype of pollen exine formation was partially rescued in ms2. These results confirmed that the localization of MS2 to plastids is critical to its function in anther development.2. NBD and SD domains are essential for the function of MS2: To examine whether these domains are essential for MS2's function, we made several constructs containing mutated MS2 fragments driven by the MS2 promoter. MS2 deleted for SD (proMS2:MS2△SD) or NBD (proMS2:MS2△NBD) could not rescue the defective development of pollen exine in the ms2 background, suggesting an essential role of both SD and NBD domains in MS2 function.3. Purified recombinant MS2 has fatty acyl ACP reductase activity. Although MS2 was predicted to be a fatty acid reductase involved in fatty alcohol formation, and recombinant bacteria expressing MS2 can produce C14:0, C16:0 and C18:1 alcohols (Aarts, 1997; Doan, 2009), a comprehensive biochemical characterization of this enzyme was needed. To this end, we cloned the full length MS2 cDNA into the expression vector pET30a and introduced the construct into E. coli BL21 (DE3). We showed that the purified recombinant MS2 has a strong preference for converting palmitoyl-ACP to hexadecanol, and the reaction is dependent on the electron donor NAD(P)H versus NADH. Furthermore, we showed that the optimal pH value is 6.0 and the optimal working temperature is 30°C for the conversion of palmitoyl-ACP to C16:0 alcohol of MS2. Under the optimum pH and temperature, the recombinant MS2 has a Km of 23.3±4.0μM for palmitoyl-ACP and a Vmax of 38.3±4.5 nmol mg^-1 min^-1.To demonstrate the physiological role of MS2 in producing fatty acyl alcohols, we transiently expressed the MS2 protein in tobacco leaves, and analyzed the lipidic compositions of chloroform-extractable cuticular waxes, cutin monomers and total soluble lipids in the transgenic lines using gas chromatography–mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID). Fatty alcohols were produced in tobacco leaves transiently expressing MS2 but no in lines carrying the empty vector pEAQ, suggesting that MS2 can synthesize fatty alcohols in plants.In conclusion, we have characterized the Arabidopsis plastid-localized fatty acyl-ACP reductase-MS2, which is able to reduce palmiltoyl-ACP to hexadecanol and essential for pollen exine synthesis. Moreover, the N-terminal transit peptide-mediated plastid localization, NAD(P)H binding domain (NBD), sterile domain (SD), and the active site motif YX(3)K are required for MS2's function in anther development. This work has revealed a key and conserved step of primary fatty alcohol synthesis in pollen wall biosynthesis in Arabidopsis.