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

Abstract

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.

Figure 1.

Pollen phenotype of the ms2 mutant. Pollen grains isolated from a wild-type (WT) plant and a ms2 plant were detected by DAPI staining (A and B), I₂-KI staining (C and D), acetolysis treatment (E and F), and SEM (G and H). Arrows in D show the rough outer surface of ms2 pollen grains. Bars = 10 μm (A–F) and 5 μm (G and H).

Figure 2.

Subcellular localization analysis of MS2 in protoplasts. A, Diagrams of the constructs of the full-length MS2 cDNA with and without the sequence encoding the putative transit peptide (TP) fused with the N terminus of GFP under the control of the CaMV 35S promoter (pro35S:MS2-GFP and pro35S:MS2ΔN-GFP, respectively) and the fragment encoding the putative transit peptide of MS2 fused with YFP under the control of the CaMV 35S promoter (pro35S:TP-YFP). B to E, An Arabidopsis protoplast expressing MS2-GFP showing green fluorescent signals (B), the chlorophyll autofluorescence signals in the chloroplasts (C), blue fluorescent signals by coexpressing mitochondrion-localizing F1ATPase-γ-RFP (D), and the merged signals (E). F to I, An Arabidopsis protoplast expressing MS2ΔN-GFP showing green fluorescent signals in the cytoplasm (F), the chlorophyll autofluorescence signals (G), blue fluorescent signals by coexpressing mitochondrion-localizing F1ATPase-γ-RFP (H), and the merged signals (I). J to L, An Arabidopsis protoplast expressing TP-YFP showing yellow fluorescent signals (J), the chlorophyll autofluorescence signals in the plastids (K), and the merged signals (L). Bars = 10 μm.

Figure 3.

Localization of MS2 expression in transgenic plants. A to C, A transgenic plant line expressing full-length MS2 cDNA fused with GFP under the control of the MS2 promoter (proMS2:MS2-GFP) in the wild type, showing green fluorescent signals in the tapetal layer (A), autofluorescence signals of chlorophyll (B), and the merged signals (C). D to F, A transgenic plant line expressing MS2 cDNA without the sequence encoding the putative transit peptide fused with GFP under the control of the MS2 promoter (proMS2:MS2ΔN-GFP) showing green fluorescent signals (D), autofluorescence signals of chlorophyll (E), and the merged signals (F). Bars = 100 μm.

Figure 4.

Functional complementation of the ms2 mutant using truncated MS2 cDNA fragments. A, Diagrams of the constructs of the full-length MS2 cDNA under the control of the MS2 promoter (proMS2:MS2), MS2 cDNA without the sequence encoding the putative transit peptide (proMS2:MS2ΔN), MS2 cDNA without the sequence encoding the SD (proMS2:MS2ΔSD), MS2 cDNA without the sequence encoding the NAD-binding 4 domain (proMS2:MS2ΔNBD), MS2 cDNA without the sequence encoding the NAD(P)H-binding motif (proMS2:MS2ΔNBM), and MS2 cDNA without the sequence encoding the active site motif YX(3)K of NADP-dependent proteins (proMS2:MS2ΔASM). B to M, These constructs were transformed to the ms2 mutants individually, and pollen grains were detected by acetolysis treatment (B–G) and SEM analysis (H–M). Bars = 20 μm (B–G) and 5 μm (H–M). [See online article for color version of this figure.]

Figure 5.

Complementation analysis of the ms2 mutant using the truncated MS2 fused with the DNA fragment encoding the Arabidopsis RTP. A, Diagrams of the construct of the MS2 cDNA containing the fragment encoding the Arabidopsis RTP replacing the fragment encoding the MS2 N-terminal transit peptide under the control of the MS2 promoter (proMS2:RTP-MS2ΔN′). Ten transgenic lines could well restore the pollen phenotype of the ms2 mutant (B and E); four transgenic lines could only partly restore the pollen phenotype of the ms2 mutant (C and F); seven transgenic lines could not restore the pollen phenotype of the ms2 mutant (D and G). B to D, Analysis of acetolysis treatment. E to G, SEM analysis. Bars = 20 μm (B–D) and 5 μm (E–G). [See online article for color version of this figure.]

Figure 6.

Analysis of Ni²⁺-NTA column affinity-purified MS2. Lane 1, prestained protein markers; lane 2, 10 μL of eluted solution from the Ni²⁺-NTA affinity column incubated with the protein from control bacteria containing the empty vector pET30a; lane 3, 10 μL of purified His tag fused-MS2 protein from the Ni²⁺-NTA affinity column incubated with protein from pET30a-MS2.

Figure 7.

Substrate specificity analysis of recombinant MS2. Gas chromatograms of metabolites generated from the incubation of myristoyl-ACP (A), palmitoyl-ACP (B), palmitoleoyl-ACP (C), and stearoyl-ACP (D) with affinity-purified MS2 in the presence of NAD(P)H are shown.

Figure 8.

Catalytic activity analysis of MS2 dependent on NAD(P)H and NADH. Gas chromatograms of products generated with incubations of 20 μm palmitoyl-ACP with purified MS2 in the presence of NAD(P)H (A), NADH (B), or water (C) are shown. [See online article for color version of this figure.]

Figure 9.

pH and temperature preferences of recombinant MS2. Activity analyses of the recombinant MS2 with the pH range 5.0 to 9.0 (A) and using the optimum MES buffer (pH 6.0) incubated at 10°C, 16°C, 25°C, 30°C, 35°C, and 42°C (B) are shown. The values indicate means of four replicates ± sd. [See online article for color version of this figure.]

Figure 10.

Analysis of wax, internal lipids, and cutin in transient tobacco leaves. A, Total wax, internal lipids, and cutin amounts per unit surface area (mg cm⁻²) in tobacco leaves by transient expression of MS2 (white bars) or empty vector (black bars). B, Wax constituent amounts per unit surface area (mg cm⁻²) in tobacco leaves by transient expression of MS2 (white bars) or empty vector (black bars). C, Internal lipid amounts per unit surface area (mg cm⁻²) in tobacco leaves by transient expression of MS2 (white bars) or empty vector (black bars). D, Cutin monomer amounts per unit surface area (mg cm⁻²) in tobacco leaves by transient expression of MS2 (white bars) or empty vector (black bars). The values indicate means of five biological replicates ± sd. ** P < 0.01. Compound names are as follows: C16-1-OL, hexacosanol; C18-1-OL, octadecanol; C16-FA, hexadecanoic acid; C18-FA, octadecanoic acid; C18(2/3)-FA, linoleic and linolenic acid; C18(1)-FA, oleic acid; C20-FA, eicosanoic acid; C22-FA, docosanoic acid; C24-FA, etracosanoic acid; C16-2HFA, 2-hydroxy-hexadecanoic acid; C23-2HFA, 2-hydroxy-tricosanoic acid; C24-2HFA, 2-hydroxy-tetracosanoic acid; C25-2HFA, 2-hydroxy-pentacosanoic acid; C26-2HFA, 2-hydroxy-hexacosanoic acid; C16-ω-HFA, 16-hydroxyhexadecanoic acid; C18(1)-ω-HFA, 18-hydroxy-octadecanoic acid; C18(1)-DFA, octadecene-1,18-dioic acid; UI, unknown cutin monomer; ALK, alkane; BR, branched aliphates.