Abstract
Based on the morphology of gametes, sexual reproduction in brown algae is usually classified into three types: isogamy, anisogamy, and oogamy. In isogamy, chloroplasts and chloroplast DNA (chlDNA) in the sporophyte cells are inherited biparentally, while mitochondria (or mitochondrial DNA, mtDNA) is inherited maternally. In oogamy, chloroplasts and mitochondria are inherited maternally. However, the patterns of mitochondrial and chloroplast inheritance in anisogamy have not been clarified. Here, we examined derivation of mtDNA and chlDNA in the zygotes through strain-specific PCR analysis using primers based on single nucleotide polymorphism in the anisogamous brown alga Mutimo cylindricus. In 20-day-old sporophytes after fertilization, mtDNA and chlDNA derived from female gametes were detected, thus confirming the maternal inheritance of both organelles. Additionally, the behavior of mitochondria and chloroplasts in the zygotes was analyzed by examining the consecutive serial sections using transmission electron microscopy. Male mitochondria were isolated or compartmentalized by a double-membrane and then completely digested into a multivesicular structure 2 h after fertilization. Meanwhile, male chloroplasts with eyespots were observed even in 4-day-old, seven-celled sporophytes. The final fate of male chloroplasts could not be traced. Organelle DNA copy number was also examined in female and male gametes. The DNA copy number per chloroplast and mitochondria in male gametes was lower compared with female organelles. The degree of difference is bigger in mtDNA. Thus, changes in different morphology and DNA amount indicate that maternal inheritance of mitochondria and chloroplasts in this species may be based on different processes and timing after fertilization.
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Acknowledgments
We are grateful to Prof. Shigeo Kawaguchi, Faculty of Agriculture, Kyushu University for providing the material, Fukuoka-strain of M. cylindricus.
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This work was supported by the Sumitomo Foundation (grant number 190311).
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Yuan Shen and Chikako Nagasato designed the experiment and maintained the strains of M. cylindricus. Toyoki Iwao collected the fresh samples in the field. Taizo Motomura analyzed the data and critically reviewed this manuscript. All authors wrote and edited this manuscript.
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Fig S1
Gametes and development of a zygote in Mie-strain. a Female gamete. b Male gamete. c-g Development of a single zygote. c Six-hour-old zygote, where two eyespots (arrowheads) can be detected in the zygote. d Two-day-old sporophyte, two eyespots (arrowheads) exist in each daughter cell. e Four-day-old sporophyte, two eyespots (arrowheads) can still be detected. f Eight-day-old sporophyte, only one eyespot (arrowhead) is observed. g 20-day-old crustose sporophyte. AF, anterior flagellum; PF, posterior flagellum. Scale bars: 10 μm (a-f); 20 μm (g). (PNG 215 kb)
Fig S2
Ultrastructure of mitochondria and chloroplasts in female and male gametes a A female gamete. b A male gamete. c The enlarged view of mitochondrion (m1 in a). d The enlarged view of mitochondrion (m2 in b). The black linear material (arrowhead) is found in tubular cristae of mitochondrion in c and d. e The enlarged view of a chloroplast (c1 in a) with an eyespot (es). f The enlarged view of a chloroplast (c2 in b) with an eyespot (es). Arrow and arrowhead in chloroplast of e and f indicate the girdle lamella and the thylakoid lamellae, respectively. es, eyespot; n, nucleus. Scale bars: 1 μm (a, e); 500 nm (b, c, d, f). (PNG 1311 kb)
Fig. S3
Ultrastructure of a zygote at two hours after fertilization. a-l Zygote nucleus just after karyogamy. Twenty-seven mitochondria are numbered, and nine chloroplasts are indicated by serial number with C. Mitochondrion numbered as 21 is excluded to show in m-p with high magnification. g-j A female gamete chloroplast with eyespots (c7) is seen. h Male nuclear part with an enriched heterochromatin region that is found in the right side of nucleus. Besides the male nucleus, a chloroplast (c9) of male gamete with eyespots exists. m-p Degrading mitochondrion (numbered as 21) near a male chloroplast (c9). C, centriole; es, eyespot; n, nucleus. Scale bars: 500 nm. (PNG 1729 kb)
Fig. S4
Digestion of male mitochondria in a two-hour-old zygote before karyogamy. a Whole cell image. Female nucleus (Fn) and male nucleus (Mn) before karyogamy are observed. b-h Partial serial sections of a magnified view from the white rectangular region in a. Mitochondria labeled with m1-m3 are isolated by double-membranes and inside different destruction degrees of mitochondria can be observed. e, f Male chloroplast with eyespot tightly attaches with male nucleus. Scale bars: 1 μm (a); 200 nm (b-h). (PNG 1819 kb)
Fig S5
(PNG 1.66 mb)
Fig. S6
Ultrastructure of zygote at six hours after fertilization. a-l Twenty-nine mitochondria numbered, and ten chloroplasts are numbered with C. a, b Male chloroplast with eyespots (c5). d-h Female chloroplast with an eyespot (c7). es, eyespot; n, nucleus. Scale bars: 500 nm. (TIF 3877 kb) (PNG 645 kb)
Fig. S7
Ultrastructure of a seven-celled crustose sporophyte. a, b The whole image of a 7-celled crustose sporophyte in two different sections. c The enlarged view from the white rectangular region in a. d The enlarged view from the white rectangular region in b. Note that the size of eyespots is diminished (c1 and c2). es, eyespot; m, mitochondrion; n, nucleus; v, vacuole. Scale bars: 2 μm (a, b); 500 nm (c, d). ((PNG 184 kb)
Fig S8
Diagrams of primer locations in the PCR assay. a Primer location of nrDNA ITS1 region. b mtDNA cox1 gene. c chlDNA psaA gene. Predicted sizes are shown between the primer pairs. (PNG 127 kb)
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Shen, Y., Iwao, T., Motomura, T. et al. Cytoplasmic inheritance of mitochondria and chloroplasts in the anisogamous brown alga Mutimo cylindricus (Phaeophyceae). Protoplasma 258, 19–32 (2021). https://doi.org/10.1007/s00709-020-01540-x
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DOI: https://doi.org/10.1007/s00709-020-01540-x