A unique life cycle transition in the red seaweed Pyropia yezoensis depends on apospory

Abstract

Plant life cycles consist of two temporally separated stages: a haploid gametophyte and a diploid sporophyte. In plants employing a haploid-diploid sexual life cycle, the transition from sporophyte to gametophyte generally depends on meiosis. However, previous work has shown that in the red seaweed Pyropia yezoensis, this transition is independent of meiosis, though how and when it occurs is unknown. Here, we explored this question using transcriptomic profiling of P. yezoensis gametophytes, sporophytes, and conchosporangia parasitically produced on sporophytes. We identify a knotted-like homeobox gene that is predominately expressed in the conchosporangium and may determine its identity. We also find that spore-like single cells isolated from the conchosporangium develop directly into gametophytes, indicating that the gametophyte identity is established before the release of conchospores and prior to the onset of meiosis. Based on our findings, we propose a triphasic life cycle for P. yezoensis involving production of gametophytes by apospory.

Keywords: Plant physiology; Reproductive biology.

Fig. 1

Temporal differences between establishment of gametophytic identity and meiosis in the heteromorphic haploid–diploid life cycle of P. yezoensis. a In the marine red seaweed P. yezoensis, the development of the thallus (T) starts from a conchospore that is released from the conchosporangium (CS) parasitically produced on the conchocelis (CC). Since conchospores develop into thalli and meiosis occurs during germination of conchospores, the transition from sporophyte to gametophyte is not associated with meiosis. By contrast, fertilization of male and female gametes is linked with the gametophyte–sporophyte transition in P. yezoensis as is observed in other eukaryotes. The figure is a modification of Fig. 1 in Shimizu et al.. Scale bars: conchocelis and conchosporangium, 50 μm; thallus, 0.5 cm; conchospore and its germinating organisms, 15 μm. b Expression of previously reported thallus- and conchocelis-specific genes in the conchosporangium was examined by qRT-PCR. In box plots, values on the Y axis represent the fold change of relative quantification of each gene in T, CS, or CC. The 18S rRNA transcript was quantified as an internal reference. Letters denote significant differences in expression level among the three life cycle stages from triplicate independent replicates as defined by the Tukey test (p < 0.05) in one-way ANOVA

Fig. 2

Transcriptional changes among three different life cycle stages. a Venn diagram displaying the number of unique and overlapping unigenes expressed in the thallus (green), conchosporangium (blue), and conchocelis (pink). b Comparisons of numbers of differentially upregulated (red bars) and downregulated (blue bars) unigenes between three combinations of three life cycle stages (CS vs. T, CC vs. CS, and CC vs. T). T, thallus; CS, conchosporangium; CC, conchocelis. c Heatmap representation of a cluster analysis of the gene expression patterns of the DEGs among three life cycle stages. The FPKM change patterns of three combinations of two life cycle stages (CC vs. CS, CC vs. T, and CS vs. T) were clustered. Left panel, analysis for intersection of sets; right panel, analysis for union of sets. d Box plots showing quantitative RT-PCR validation analysis of expression of selected thallus-biased (GC5 and GC6) and conchosporangium-specific (CC4) unigenes. The ratio values on the Y axis refer to the relative expression levels of genes selected from Supplementary Table 2 among three life cycle stages, T, CS, and CC. The 18S rRNA transcript was quantified as an internal reference. Letters denote significant differences in expression level among three life cycle stages from triplicated experiments as defined by the Tukey test (p < 0.05) in one-way ANOVA

Fig. 3

Conchosporangium-predominant expression of the PyKNOX gene during the life cycle of P. yezoensis. a Phylogenetic classification of three TALE homeobox genes from P. yezoensis. Amino acid sequences of the conserved TALE-HD domains from Bangiales and the land green plants Arabidopsis thaliana and P. patens were used for construction of neighbor-joining-based unrooted phylogenetic tree with the ClustalW and MEGA 8.0 programs. The bootstrap values with 1000 replicates over 50% are indicated at the nodes of the tree. The TALE homeobox proteins from P. yezoensis are boxed. Accession numbers: P. patens, PpMKN1 BAF285148, PpMKN2 BAF96739, PpMKN4 BAF96740, PpMKN5 BAF96741, PpMKN6 XM_001765523, PpBELL1 XP_001779432, PpBELL2 XP_001777380, PpBELL3 XM_001769443, PpBELL4 XP_001762111; Arabidopsis thaliana, AtKNAT3 X92392; Chondrus crispus, CcBELL1 XP_005711328; Cyanidioschyzon merolae, CmKNOX (CMR153C) XP_005538442, CmBELL1 (CMH049C) XP_005536034, CmBELL2 (CMR176C) XP_005538457. PopKNOX (esisotig02479) and PouKNOX (esisotig04347) are KNOX homologs of Porphyra purpurea and Porphyra umbilicalis, respectively, which were derived from NoriBLAST (http://dbdata.rutgers.edu/nori/). Gene IDs derived from our transcriptome analysis: P. yezoensis, PyKNOX CL1448 (Accession no. MK629536), PyBELL1 CL1176 (Accession no. MN070241), PyBELL2 Unigene19722 (Accession no. MN070242); Bangia fuscopurpurea, BfBELL1 Unigene3027. b Box plots showing qPCR validation of PyKNOX gene expression. The ratio on the Y axis refers to the relative expression levels of the PyKNOX gene among three life cycle stages, thallus (T), conchosporangia (CS) and conchocelis (CC). The 18S rRNA transcript was quantified as an internal reference. Letters denote significant differences in expression levels among the three life cycle stages from triplicated experiments as defined by the Tukey test (p < 0.05) in one-way ANOVA

Fig. 4

Spore-like single cells isolated from conchosporangia develop as gametophytes. Fragmented conchosporangia were isolated and cultured to observe early development. Representative images are shown. a Spore-like single cell just after release from the conchosporangium from the cell wall. After release, this cell moved autonomously before adhering to the base of the culture dish and thus became separated from the cell wall skeleton. b, c Two- and four-cell stages of the resulting germling, demonstrating the gametophytic development of the spore-like single cell from the conchosporangium. Scale bar: 20 μm

Fig. 5

The triphasic hypothesis for the heteromorphic sexual life cycle of the Bangiales. We propose the presence of three different generations in the Bangiales life cycle and the renaming of the conchosporangium to the conchosporophyte, as an independent generation in the life cycle. The positions of the generation switches in the diphase concept are ambiguous, especially for the sporophyte–gametophyte transition (see Fig. 1). By contrast, the triphasic hypothesis clarifies the events of the generation transitions; the fertilization of male and female gametes for the gametophyte–sporophyte transition, the swelling of the conchocelis tip cell for the sporophyte–conchosporophyte transition, and apospory for the conchosporophyte–gametophyte transition that is independent of meiosis. Scale bars: sporophyte and conchosporophyte, 50 μm; thallus, 0.5 cm; conchospore and its germinating organisms, 15 μm