Cell wall characteristics during sexual reproduction of Mougeotia sp. (Zygnematophyceae) revealed by electron microscopy, glycan microarrays and RAMAN spectroscopy

Abstract

Mougeotia spp. collected from field samples were investigated for their conjugation morphology by light-, fluorescence-, scanning- and transmission electron microscopy. During a scalarifom conjugation, the extragametangial zygospores were initially surrounded by a thin cell wall that developed into a multi-layered zygospore wall. Maturing zygospores turned dark brown and were filled with storage compounds such as lipids and starch. While M. parvula had a smooth surface, M. disjuncta had a punctated surface structure and a prominent suture. The zygospore wall consisted of a polysaccharide rich endospore, followed by a thin layer with a lipid-like appaerance, a massive electron dense mesospore and a very thin exospore composed of polysaccharides. Glycan microarray analysis of zygospores of different developmental stages revealed the occurrence of pectins and hemicelluloses, mostly composed of homogalacturonan (HG), xyloglucans, xylans, arabino-galactan proteins and extensins. In situ localization by the probe OG7-13^{AF 488} labelled HG in young zygospore walls, vegetative filaments and most prominently in conjugation tubes and cross walls. Raman imaging showed the distribution of proteins, lipids, carbohydrates and aromatic components of the mature zygospore with a spatial resolution of ~ 250 nm. The carbohydrate nature of the endo- and exospore was confirmed and in-between an enrichment of lipids and aromatic components, probably algaenan or a sporopollenin-like material. Taken together, these results indicate that during zygospore formation, reorganizations of the cell walls occured, leading to a resistant and protective structure.

Keywords: Cell wall; Conjugation; Mougeotia; Sexual reproduction; Streptophyte; Zygospore.

Fig. 1

Conjugation stages and zygospores of Mougeotia spp.. M. disjuncta (a-h, m-s), M. parvula (i-l), conjugation process leading to zygospore formation (a-f), 4-month maturation process (i-p). (a) formation of a papilla, (b) formation of conjugation tube, (c) beginning gamete fusion in conjugation tube, (d) gamete fusion, (e) zygospore formation final stage (suspensor, formed from closed conjugation tube is indicated by an arrow), (f) freshly formed zygospore, (g) sheath around zygospores inaccesible for India Ink staining, (h) zygospore encased in a gelatinous sheath visualized with India Ink staining, (i, m) 0 d, (j, n) 40 d, (k, o) 80 d, (l, p) 120 d, (q) zygospore wall split in half along suture line, (r) germinating zygospore, (s) freshly formed filament released from its zygospore. Abbreviation: papilla. Scale bars (a-f, h–s) 20 µm, (g) 100 µm

Fig. 2

Scanning electron micrographs of Mougeotia spp.. (a) M. parvula, (b) M. disjuncta zygospore with a micropunctate surface structure, (c) M. disjuncta zygospore displaying a suture. Abbreviation: Su suture. Scale bars 5 µm

Fig. 3

Transmission electron micrographs of Mougeotia spp.. M. parvula (a-c), M. disjuncta (d-g). (a) young zygospore with single layered cell wall, (b) maturing zygospore showing the incipient development of a multilayered cell wall, (c) detail view of mature zygospore wall, (d) zygospore with lipid bodies spread through cell lumen, (e) detail view of mature zygospore wall, (f) detail view of zygospore wall with lipid bodies accumulated at the periphery of the cytoplasm, (g) pried open zygospore releasing a newly formed filament. Abbreviations: C chloroplast , CP cytoplasma, L lipid body, S starch grain, En endospore, Ex exospore, Me mesospore, arrow indicates a lipid-like fourth layer. Scale bars (a, b, d) 1 µm, (c, e–g) 500 nm

Fig. 4

Determining cell wall epitopes in Mougeotia disjuncta using carbohydrate array profiling. > 40 cell wall probes were incubated with CDTA and NaOH extracts prepared from Mougeotia AIR. The heatmap colour intensity represents the strength of the probe binding and is directly correlated to the numerical value. The strongest signal was assigned value 100 and the cut‐off signal set to 5. Probe codes are in bold and epitopes in brackets. Abbreviations: Ara arabinose, DE degree of esterification, Fuc fucose, Gal galactose, GalA galacturonic acid, GalNAc N-acetylgalactosamin, GlcA glucuronic acid, HG homogalacturonan, RGI rhamnogalacturonan I, Rha rhamnose, AGP arabinogalactan protein. Oligosaccharide nomenclature for xyloglucan probes see Fry et al. (1993)

Fig. 5

In situ labelling of Mougeotia disjuncta cell walls. Fluorescent images (green) are shown on the left and corresponding bright field images on the right (a-c, f–h, j) or were merged (d, e, i). Images were taken with an epifluorescence microscope (a-c, g) or confocal microscope (d-f, h-i). (a) LM19 (recognises HG with low DE) binding to filaments (arrows: suspensor) and zygospores. ~ 50% of zygospores with weak/no signal (asterisks), (b) OG7-13^AF488 (demethylated HG) staining with similar results as in (a): strong staining of suspensor (arrows) and weak signal in some zygospores (asterisks), (c) OG7-13^AF488 staining of cell wall remnants (arrows) after HG removal via PL and EPG, (d) INRA-RU1 (RGI) binding to outer sheath (arrowhead) with fibrous structure (inlet) and in inner areas (arrow), (e) Some staining in outer sheath after CDTA extraction (arrowhead) close to staining spots in inner wall layer (arrow), (f) INRA-RUI (RGI) signal mainly restricted to cross cell walls (arrowheads); occasionally, whole cell wall areas with signal (arrow), (g) 400–2 (callose) signal in suspensor, (h) LM25 (xyloglucan) signal in cross cell walls, (i) JIM13 (AGP) binding in outer zygospore wall with fibrous appearance, (j) JIM20 (extensin) binding in areas around cross cell walls. Abbreviations: DE degree of esterification, EPG endo-polygalacturonase, HG homogalacturonan, PL pectate lyase, RGI rhamnogalacturonan I, AGP arabinogalactan protein. Scale bars 10 µm

Fig. 6

Raman images of Mougeotia disjuncta reveal aromatic compounds merged with a lipid layer in the mesospore. (a) Merged image of four NMF endmembers including carbohydrates (cyan), lipids (yellow), aromatics (red), and proteins (blue), (b) Detailed NMF anlysis of the multi-layered cell wall visualizes lipids, aromatics and carbohydrates, (c) Corresponding endmember spectra show respective bands of the three component classes. Inlay represents the overlay of the aromatics and lipids in the mesospore as a schematic drawing and a section of the images from c as an overlay. Scale bars 5 µm