PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability.

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores

Developmental timelines for <i>pabB4</i>, the untransformed control strain, and <i>ascl-2</i> sporophytes and spores.

<p>Photomicrographs of the typical morphologies of sporophytes and spores of control (A–N) and <i>ascl-2</i> (a–p) at successive developmental stages. The number of days after irrigation of the cultures is shown with the names assigned to each sporophytic stage. Some stages have been subdivided to allow more detailed description of changes in spore development. White arrowheads denote the outlines of the spore masses within capsules. No spores are seen during the initial growth and expanding capsule stages. The control did not reach the brown sporophytic stage during the observation period. Sporophyte scale bars = 500 μm; Spore scale bars = 10 μm.</p

Strategy for targeted knockout of <i>PpASCL</i> and genotyping of the resulting stable transformants by PCR.

<p>(a) Schematic diagram of insertion of the linear knockout construct into the <i>PpASCL</i> locus via double homologous recombination. 35S-P, CaMV 35S promoter; <i>nptII</i>, neomycin phosphotransferase II gene; 35S-T, CaMV 35S transcription termination signal. (b) Schematic diagram of recombined gene locus after successful insertion. Expected PCR product sizes based on sequence information are shown. Single-headed arrows denote the locations of primers specific to <i>PpASCL</i> (Primers 1 and 2, which bind to genomic DNA sequences located outside the locus-specific regions used for homologous recombination) or to the <i>nptII</i> resistance cassette (Primers 3 and 4) used in the PCR analyses. Primer 1, ASCL-gDNA-F; 2, ASCL-gDNA-R; 3, pTN182-5ʹ-R; 4, pTN182-3ʹ-F. Primer sequences are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146817#pone.0146817.s005" target="_blank">S1 Table</a>. (c) PCR products using locus-specific primers 1 and 2 with DNA from untransformed control and each of three stable putative <i>PpASCL</i> knockout lines: <i>ascl-1</i>, <i>-2</i> and <i>-3</i>. (d) PCR products, indicative of 5′ and 3′ recombination between the knockout vector and homologous DNA in the <i>PpASCL</i> locus, using primers 1 plus 3 (5ʹ recombination) and primers 2 plus 4 (3ʹ recombination). Amplified DNA products were resolved electrophoretically on 1.2% agarose gels and visualized by ethidium bromide fluorescence.</p

Photomicrographs of cryosectioned <i>pabB4</i> control and <i>ascl-2</i> sporophytes.

<p>Cross sections of control (20 μm, a–e) and <i>ascl-2</i> (30 μm, f–j) sporophytes were taken at the yellow (a,f) and orange (b–e, g–j) stages. Images were saved before (a,b,f,g) and after toluidine blue O staining (c–e, h–j). Orbicules present in locules of the air-space and on the tapetum wall surface are indicated with red arrows in (e) and (j). Sections (e) and (j) are magnified images of red-boxed areas of (d) and (i), respectively. Co, columella; E, epidermis; Lo, locule; T, tapetum. Scale bars = 100 μm.</p

Reaction sequence for the biosynthesis of hydroxylated alkylpyrones as sporopollenin building blocks.

<p>Medium- to long-chain fatty acids are produced in plastids and then translocated out to be used for the consecutive action of enzymes in sporopollenin biosynthesis. This proposed pathway produces sporopollenin building blocks that are polymerized along with fatty alcohols and phenylpropanoid acids on the surface of the spore or pollen wall by the formation of ester and ether linkages. Enzymes are listed to the right of the arrows with their corresponding reactions on the left. ACOS, acyl-CoA synthetase; ASCL, anther-specific chalcone synthase-like enzyme; MS2, Male Sterility 2; TKPR, tetraketide α-pyrone reductase.</p

Transmission electron micrographs of <i>pabB4</i> control and ascl-2 spores.

<p>Cross sections of spores from mature orange control (a) and <i>ascl-2</i> (b) sporophytes were examined with TEM. An amorphous layer found below much of the perine is indicated by an arrow in (b). Ex, exine; In, intine; Pe, perine; PM, plasma membrane. Scale bars = 500 nm.</p

RT-PCR analysis of (a) <i>PpASCL</i> expression in <i>Physcomitrella</i> at successive developmental stages and (b) <i>PpASCL</i> promoter activity in <i>ascl-2</i>.

<p>(a) The primers, ASCL-RT-F and ASCL-RT-R (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146817#pone.0146817.s005" target="_blank">S1 Table</a>), were used to amplify 1115 nucleotides, including 1108 nts from the protein coding region of the <i>PpASCL</i> transcript. In the untransformed <i>pabB4</i> control, <i>PpASCL</i> expression was detected in sporophytes at the expanding (E) and green (G) capsule stages, but not in the mature orange (O) stage nor in protonemata and gametophores. RT-PCR using <i>ascl-2</i> sporophytes, comprising a minority at the expanding and a majority at the green capsule stages respectively (E+G), failed to yield the 1115 bp amplicon thus providing additional evidence that <i>PpASCL</i> has been knocked out in this mutant. (b) <i>PpASCL</i> promoter activity was examined in <i>pabB4</i> E sporophytes and in <i>ascl-2</i> E+G sporophytes. The primer pair, ASCL-RT-F and 5'R-ASCL-ClaI (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146817#pone.0146817.s005" target="_blank">S1 Table</a>), was used to amplify a 5′ region predicted to be 332 nucleotides long in mature <i>PpASCL</i> transcripts. Similarly, the primer pair, 3'F-ASCL-NdeI and ASCL-RT-F, was used to amplify a 3′ region predicted to be 536 nucleotides long in mature <i>PpASCL</i> transcripts. Both expected amplicons were generated with sporophytes of both strains. An additional 5ʹ amplicon (upper band) was produced with <i>ascl-2</i> that, based on its size, we attribute to inefficient splicing of intron 1 from the mutant pre-mRNA. Expression of the <i>Physcomitrella actin3</i> gene (<i>act</i>) was used as a reference.</p

Morphological comparison of orange stage <i>pabB4</i> control and <i>ascl-2</i> spores.

<p>Images of typical spores isolated from mature orange sporophytes were acquired using light microscopy (a,e) and SEM (b,c,f,g). Spores from the control (a–c) and <i>ascl-2</i> (e–g) are shown. Light microscopy images of control (d) and <i>ascl-2</i> (h) spores after treatment with simplified Alexander’s stain are also shown. Scale bars = 10 μm.</p