Fig 1.
Reaction sequence for the biosynthesis of hydroxylated alkylpyrones as sporopollenin building blocks.
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.
Fig 2.
Strategy for targeted knockout of PpASCL and genotyping of the resulting stable transformants by PCR.
(a) Schematic diagram of insertion of the linear knockout construct into the PpASCL locus via double homologous recombination. 35S-P, CaMV 35S promoter; nptII, 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 PpASCL (Primers 1 and 2, which bind to genomic DNA sequences located outside the locus-specific regions used for homologous recombination) or to the nptII 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 S1 Table. (c) PCR products using locus-specific primers 1 and 2 with DNA from untransformed control and each of three stable putative PpASCL knockout lines: ascl-1, -2 and -3. (d) PCR products, indicative of 5′ and 3′ recombination between the knockout vector and homologous DNA in the PpASCL 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.
Fig 3.
RT-PCR analysis of (a) PpASCL expression in Physcomitrella at successive developmental stages and (b) PpASCL promoter activity in ascl-2.
(a) The primers, ASCL-RT-F and ASCL-RT-R (S1 Table), were used to amplify 1115 nucleotides, including 1108 nts from the protein coding region of the PpASCL transcript. In the untransformed pabB4 control, PpASCL 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 ascl-2 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 PpASCL has been knocked out in this mutant. (b) PpASCL promoter activity was examined in pabB4 E sporophytes and in ascl-2 E+G sporophytes. The primer pair, ASCL-RT-F and 5'R-ASCL-ClaI (S1 Table), was used to amplify a 5′ region predicted to be 332 nucleotides long in mature PpASCL 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 PpASCL transcripts. Both expected amplicons were generated with sporophytes of both strains. An additional 5ʹ amplicon (upper band) was produced with ascl-2 that, based on its size, we attribute to inefficient splicing of intron 1 from the mutant pre-mRNA. Expression of the Physcomitrella actin3 gene (act) was used as a reference.
Fig 4.
Developmental timelines for pabB4, the untransformed control strain, and ascl-2 sporophytes and spores.
Photomicrographs of the typical morphologies of sporophytes and spores of control (A–N) and ascl-2 (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.
Fig 5.
Photomicrographs of cryosectioned pabB4 control and ascl-2 sporophytes.
Cross sections of control (20 μm, a–e) and ascl-2 (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.
Fig 6.
Morphological comparison of orange stage pabB4 control and ascl-2 spores.
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 ascl-2 (e–g) are shown. Light microscopy images of control (d) and ascl-2 (h) spores after treatment with simplified Alexander’s stain are also shown. Scale bars = 10 μm.
Fig 7.
Transmission electron micrographs of pabB4 control and ascl-2 spores.
Cross sections of spores from mature orange control (a) and ascl-2 (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.
Fig 8.
Light microscopy and SEM images of acetolysed spores.
Photomicrographs of spores of pabB4 control (a) and ascl-2 (f) and SEM images of gold coated spores from control (b–e) and ascl-2 (g–j) after acetolysis. Spores were isolated from the orange sporophytic developmental stage. Triradiate ridges are visible in some collapsed control spores (b,c). Scale bars (a,b,d,f,g,i) = 10 μm; (c,e,h,j) = 5 μm.