The complete plastid genome sequence of the enigmatic moss, Takakia lepidozioides (Takakiopsida, Bryophyta): evolutionary perspectives on the largest collection of genes in mosses and the intensive RNA editing.

KEY MESSAGE: Complete chloroplast genome sequence of a moss, Takakia lepidozioides (Takakiopsida) is reported. The largest collection of genes in mosses and the intensive RNA editing were discussed from evolutionary perspectives. We assembled the entire plastid genome sequence of Takakia lepidozioides (Takakiopsida), emerging from the first phylogenetic split among extant mosses. The genome sequences were assembled into a circular molecule 149,016 bp in length, with a quadripartite structure comprising a large and a small single-copy region separated by inverted repeats. It contained 88 genes coding for proteins, 32 for tRNA, four for rRNA, two open reading frames, and at least one pseudogene (tufA). This is the largest number of genes of all sequenced plastid genomes in mosses and Takakia is the only moss that retains the seven coding genes ccsA, cysA, cysT, petN rpoA, rps16 and trnPGGG. Parsimonious interpretation of gene loss suggests that the last common ancestor of bryophytes had all seven genes and that mosses lost at least three of them during their diversification. Analyses of the plastid transcriptome identified the extraordinary frequency of RNA editing with more than 1100 sites. We indicated a close correlation between the monoplastidy of vegetative tissue and the intensive RNA editing sites in the plastid genome in land plant lineages. Here, we proposed a hypothesis that the small population size of plastids in each vegetative cell of some early diverging land plants, including Takakia, might cause the frequent fixation of mutations in plastid genome through the intracellular genetic drift and that deleterious mutations might be continuously compensated by RNA editing during or following transcription.

WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens.

Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.

Class 1 KNOX genes are not involved in shoot development in the moss Physcomitrella patens but do function in sporophyte development.

Although the number and form of metazoan organs are determined in the embryo, plants continuously form organs via pluripotent stem cells contained within the meristem. Flowering plants have an indeterminate meristem in their diploid generation, whereas the common ancestor of land plants is inferred to have formed an indeterminate meristem in its haploid generation, as observed in the extant basal land plants, bryophytes, including mosses. It is hypothesized that the underlying gene networks for the diploid meristem were initially present in the haploid generation of the basal land plants and were eventually co-opted for expression in the diploid generation. In flowering plants, the class 1 KNOTTED1-LIKE HOMEOBOX (KNOX) transcription factors are essential for the function of the indeterminate apical meristem. Here, we show that the class 1 KNOX orthologs function in the diploid organ, with determinate growth in the moss Physcomitrella patens, but do not function in the haploid indeterminate meristem. We propose that the genetic networks governing the indeterminate meristem in land plants are variable, and the networks governing the diploid indeterminate meristem with the class 1 KNOX genes likely evolved de novo in the flowering plant lineage.

Convergent evolution of shoots in land plants: lack of auxin polar transport in moss shoots.

The shoot is a repeated structure made up of stems and leaves and is the basic body plan in land plants. Vascular plants form a shoot in the diploid generation, whereas nonvascular plants such as mosses form a shoot in the haploid generation. It is not clear whether all land plants use similar molecular mechanisms in shoot development or how the genetic networks for shoot development evolved. The control of auxin distribution, especially by polar auxin transport, is essential for shoot development in flowering plants. We did not detect polar auxin transport in the gametophytic shoots of several mosses, but did detect it in the sporophytes of mosses without shoot structure. Treatment with auxin transport inhibitors resulted in abnormal embryo development, as in flowering plants, but did not cause any morphological changes in the haploid shoots. We fused the soybean auxin-inducible promoter GH3 with a GUS reporter gene and used it to indirectly detect auxin distribution in the moss Physcomitrella patens. An auxin transport inhibitor NPA did not cause any changes in the putative distribution of auxin in the haploid shoot. These results indicate that polar auxin transport is not involved in haploid shoot development in mosses and that shoots in vascular plants and mosses are most likely regulated differently during development.

Radiocesium contamination of the moss Hypnum plumaeforme caused by the Fukushima Dai-ichi Nuclear Power Plant accident.

We investigated 134Cs and 137Cs activity concentrations in the common Japanese moss species Hypnum plumaeforme collected from 32 sites within ca. 100 km radius of the Fukushima Dai-ichi Nuclear Power Plant. A total of 32 samples of H. plumaeforme were collected during the field surveys from November 2013 to September 2014. The maximum radiocesium activity concentrations in H. plumaeforme were 60.9 ±â€¯1.8 kBq kg-1 for 134Cs and 123 ±â€¯2.3 kBq kg-1 for 137Cs. The mean value for the 134Cs/137Cs was 1.17 ±â€¯0.05, and the mean Tag value was 0.09 ±â€¯0.13. Positive correlations were obtained between total 134Cs + 137Cs activity concentrations in H. plumaeforme and the air dose rate with a correlation coefficient (r) of 0.55 (P = 0.001), and between 137Cs activity concentration in H. plumaeforme and 137Cs deposition density on soil with r of 0.55 (P = 0.001). These results suggest that the perennial moss species H. plumaeforme could be more suitable and useful as a qualitative indicator for the radiocesium pollution compared to vascular plants spreading over the lowlands including human habitation in Fukushima Prefecture.

Geographical origin of Leucobryum boninense Sull. & Lesq. (Leucobryaceae, Musci) endemic to the Bonin Islands, Japan.

Leucobryum boninense is endemic to the Bonin Islands, Japan, and its related species are widely distributed in Asia and the Pacific. We aimed to clarify the phylogenetic relationships among Leucobryum species and infer the origin of L. boninense. We also describe the utility of the chloroplast trnK intron including matK for resolving the phylogenetic relationships among Leucobryum species, as phylogenetic analyses using trnK intron and/or matK have not been performed well in bryophytes to date. Fifty samples containing 15 species of Leucobryum from Asia and the Pacific were examined for six chloroplast DNA regions including rbcL, rps4, partial 5' trnK intron, matK, partial 3' trnK intron, and trnL-F intergenic spacer plus one nuclear DNA region including ITS. A molecular phylogenetic tree showed that L. boninense made a clade with L. scabrum from Japan, Taiwan and, Hong Kong; L. javense which is widely distributed in East and Southeast Asia, and L. pachyphyllum and L. seemannii restricted to the Hawaii Islands, as well as with L. scaberulum from the Ryukyus, Japan, Taiwan, and southeastern China. Leucobryum boninense from various islands of the Bonin Islands made a monophylic group that was closely related to L. scabrum and L. javense from Japan. Therefore, L. boninense may have evolved from L. scabrum from Japan, Taiwan, or Hong Kong, or L. javense from Japan. We also described the utility of trnK intron including matK. A percentage of the parsimony-informative characters in trnK intron sequence data (5.8%) was significantly higher than that from other chloroplast regions, rbcL (2.4%) and rps4 (3.2%) sequence data. Nucleotide sequence data of the trnK intron including matK are more informative than other chloroplast DNA regions for identifying the phylogenetic relationships among Leucobryum species.

KNOX2 genes regulate the haploid-to-diploid morphological transition in land plants.

Unlike animals, land plants undergo an alternation of generations, producing multicellular bodies in both haploid (1n: gametophyte) and diploid (2n: sporophyte) generations. Plant body plans in each generation are regulated by distinct developmental programs initiated at either meiosis or fertilization, respectively. In mosses, the haploid gametophyte generation is dominant, whereas in vascular plants-including ferns, gymnosperms, and angiosperms-the diploid sporophyte generation is dominant. Deletion of the class 2 KNOTTED1-LIKE HOMEOBOX (KNOX2) transcription factors in the moss Physcomitrella patens results in the development of gametophyte bodies from diploid embryos without meiosis. Thus, KNOX2 acts to prevent the haploid-specific body plan from developing in the diploid plant body, indicating a critical role for the evolution of KNOX2 in establishing an alternation of generations in land plants.

Airborne sperm of Conocephalum conicum (Conocephalaceae).

We have obtained the first momentary photographs of sperms just as they are discharged from the antheridium of a liverwort, Conocephalum conicum, and have succeeded in monitoring the airborne sperms of bryophytes under field conditions. Airborne sperm of liverworts seems to be an effective strategy for raising the efficiency of fertilization between male and female plants separated in a drought environment.

Gamma-tubulin in basal land plants: characterization, localization, and implication in the evolution of acentriolar microtubule organizing centers.

Although seed plants have gamma-tubulin, a ubiquitous component of centrosomes associated with microtubule nucleation in algal and animal cells, they do not have discrete microtubule organizing centers (MTOCs) comparable to animal centrosomes, and the organization of microtubule arrays in plants has remained enigmatic. Spindle development in basal land plants has revealed a surprising variety of MTOCs that may represent milestones in the evolution of the typical diffuse acentrosomal plant spindle. We have isolated and characterized the gamma-tubulin gene from a liverwort, one of the extant basal land plants. Sequence similarity to the gamma-tubulin gene of higher plants suggests that the gamma-tubulin gene is highly conserved in land plants. The G9 antibody to fission yeast gamma-tubulin recognized a single band of 55 kD in immunoblots from bryophytes. Immunohistochemistry with the G9 antibody clearly documented the association of gamma-tubulin with various MTOC sites in basal land plants (e.g., discrete centrosomes with and without centrioles and the plastid surface in monoplastidic meiosis of bryophytes). Changes in the distribution of gamma-tubulin occur in a cell cycle-specific manner during monoplastidic meiosis in the liverwort Dumortiera hirsuta. gamma-Tubulin changes its localization from the plastid surface in prophase I to the spindle, from the spindle to phragmoplasts and the nuclear envelope in telophase I, and back to the plastid surfaces in prophase II. In vitro experiments show that gamma-tubulin is detectable on the surface of isolated plastids and nuclei of D. hirsuta, and microtubules can be repolymerized from the isolated plastids. gamma-Tubulin localization patterns on plastid and nuclear surfaces are not affected by the destruction of microtubules by oryzalin. We conclude that gamma-tubulin is a highly conserved protein associated with microtubule nucleation in basal land plants and that it has a cell cycle-dependent distribution essential for the orderly succession of microtubule arrays.