Kyosuke Niwa

Induction and isolation of pigmentation mutants of Porphyra yezoensis (Bangiales, Rhodophyta) by heavy-ion beam irradiation.

The present study describes the isolation of pigmentation mutants of Porphyra yezoensis Ueda induced by heavy‐ion beam irradiation for the first time. The gametophytic blades were irradiated with 12C+6 ion beams within a dose range of 25–400 Gy. From the survival rate and cell growth of the irradiated blades, it is suggested that a dose of 150 Gy or less is suitable to induce mutation for the isolation of mutants of P. yezoensis. After irradiation, red, green and deep reddish brown‐colored gametophytic blades developed from archeospores that were released from each of the mutated cell clusters of the respective different colors, and the red mutant strain (IBY‐R1) and green mutant strain (IBY‐G1) were established as a conchocelis colony in culture. Blades of the mutants were characterized by their growth and photosynthetic pigment contents compared with those of the wild‐type. From these results, it is clear that heavy‐ion beam mutagenesis will be an effective tool for genetic and breeding studies of Porphyra, and also for other algal research.

INTERSPECIFIC HYBRIDIZATION IN THE HAPLOID BLADE‐FORMING MARINE CROP PORPHYRA (BANGIALES, RHODOPHYTA): OCCURRENCE OF ALLODIPLOIDY IN SURVIVING F1 GAMETOPHYTIC BLADES1

We performed interspecific hybridization in the haploid blade‐forming marine species (nori) of the genus Porphyra, which have a heteromorphic life cycle with a haploid gametophytic blade and a diploid microscopic sporophyte called the “conchocelis phase.” The green mutant HGT‐6 of P. tenera var. tamatsuensis A. Miura was crossed with the wildtype HG‐1 of P. yezoensis f. narawaensis A. Miura; the F1 heterozygous conchocelis developed normally and released numerous conchospores. However, almost all the conchospore germlings did not survive past the four‐cell stage or thereabouts, and only a few germlings developed into gametophytic blades. These results indicate that hybrid breakdown occurred during the meiosis, while the surviving F1 gametophytic blades were considered a breakthrough in the interspecific hybridization of Porphyra. Organelle genomes (cpDNA and mtDNA) were found to be maternally inherited in the interspecific hybridization by molecular analyses of the organelle DNA. In particular, molecular analyses of nuclear DNA revealed that the surviving F1 blades were allodiploids in the haploid gametophytic phase; however, there is a possibility of the occurrence of rapid chromosomal locus elimination and rearrangement in the F1 conchocelis phase. Our findings are noteworthy to the breeding of cultivated Porphyra and will provide important information for understanding of the speciation of marine plants with high species diversity.

Cryptic species in the Pyropia yezoensis complex (Bangiales, Rhodophyta): Sympatric occurrence of two cryptic species even on same rocks

In a previous study on wild populations of Pyropia, the occurrence of two possible new species (Pyropia sp. 2 and Pyropia sp. 3) which are closely related to the two commercially important Pyropia species, P. yezoensis and P. tenera, was confirmed as the result of molecular phylogenetic analyses. To characterize the morphological features of the two wild Pyropia species, we collected Pyropia blades in a natural population in which Pyropia sp. 3 was known to occur, and carried out molecular identification before detailed morphological observations. Through the molecular identification we found, unexpectedly, that Pyropia sp. 2 blades grew sympatrically in the same site. Therefore, after molecular identification, we examined in detail the external morphology and anatomy of the two wild Pyropia species using more than 10 blades each. As a result, it is concluded that all of the blades of the two species are morphologically identical to P. yezoensis, but distinct from P. tenera. It is therefore considered that both of the two wild Pyropia species are cryptic species within the P. yezoensis complex. Furthermore, this study revealed that the two cryptic species grew sympatrically, even on the same rocks within the natural habitat.

Phenotypic differentiation in the morphology and nutrient uptake kinetics among Undaria pinnatifida cultivated at six sites in Japan

Understanding the nutrient uptake kinetics of kelp populations will contribute to an improved understanding of environmental adaptation and the breeding of new cultivars. In this study, we examined the morphological characteristics, carbon (C) and nitrogen (N) contents, and NO3−–N and NH4+–N uptake kinetics of Undaria pinnatifida sporophytes cultivated at six industrial farms throughout Japan. We detected significant differences in morphology among sites. At Matsushima Bay (northern Pacific coast of Japan), where autumnal seawater temperatures fall in concert with increasing nutrient concentrations, sporophytes were significantly larger than at other sites from December to February. The C content of the sporophytes was seasonally stable at all of the locations, but the N content of sporophytes declined after February due to a decrease in seawater nutrients. We compared the uptake kinetics of NO3−–N and NH4+–N among cultivation sites. Vmax and Ks, which are Michaelis–Menten parameters that measure adaptation to nutrient concentrations, were highest in the Seto Inland Sea and lowest in the northern sector of the Sea of Japan. The Vmax/Ks ratio is a measure of adaptation to low nutrient concentrations; the highest values were measured in the northern sector of the Sea of Japan. The parameter ranges were broader than those previously reported for invasive populations of U. pinnatifida in other parts of the world. Thus, we detected population-level adaptations to the various nutrient conditions in Japanese waters, and these results suggest the existence of ecotypes according to nutrient uptake kinetics. The different populations may be used to provide sources of genetic material that could be of value in breeding programmes by improving productivity and quality.

Speciation in the marine crop Pyropia yezoensis (Bangiales, Rhodophyta).

In the marine red alga Pyropia yezoensis, commonly known in Japan as nori, sympatric occurrence of two cryptic species Pyropia sp. 2 and Pyropia sp. 3 on the same rock in a natural habitat has been confirmed by molecular analysis and detailed morphological observations. To confirm whether Pyropia sp. 2 and Pyropia sp. 3 were reproductively isolated in the sympatric population, 170 blades that had previously been studied using a maternally inherited plastid marker were examined with a nuclear gene marker. The results suggested that Pyropia sp. 2 and Pyropia sp. 3 with identical morphological features were reproductively isolated in the sympatric population and that they were different species based on the biological species concept. Although gametophytic blades of Pyropia were usually assumed to be haploid, 18 of 170 blades possessed both of the two genotypes derived from Pyropia sp. 2 and from Pyropia sp. 3. These results inferred that allodiploid blades were generated from the interspecific hybridization between these two cryptic species. The present findings provide insights for future studies on the speciation mechanism in seaweeds, particularly for genera that contain numerous species.

Variety in excitation energy transfer processes from phycobilisomes to photosystems I and II

The light-harvesting antennas of oxygenic photosynthetic organisms capture light energy and transfer it to the reaction centers of their photosystems. The light-harvesting antennas of cyanobacteria and red algae, called phycobilisomes (PBSs), supply light energy to both photosystem I (PSI) and photosystem II (PSII). However, the excitation energy transfer processes from PBS to PSI and PSII are not understood in detail. In the present study, the energy transfer processes from PBS to PSs in various cyanobacteria and red algae were examined in vivo by selectively exciting their PSs or PBSs, and measuring the resulting picosecond to nanosecond time-resolved fluorescences. By observing the delayed fluorescence spectrum of PBS-selective excitation in Arthrospira platensis, we demonstrated that energy transfer from PBS to PSI via PSII (PBS→PSII→PSI transfer) occurs even for PSI trimers. The contribution of PBS→PSII→PSI transfer was species dependent, being largest in the wild-type of red alga Pyropia yezoensis (formerly Porphyra yezoensis) and smallest in Synechococcus sp. PCC 7002. Comparing the time-resolved fluorescence after PSs- and PBS-selective excitation, we revealed that light energy flows from CP43 to CP47 by energy transfer between the neighboring PSII monomers in PBS–PSII supercomplexes. We also suggest two pathways of energy transfer: direct energy transfer from PBS to PSI (PBS→PSI transfer) and indirect transfer through PSII (PBS→PSII→PSI transfer). We also infer that PBS→PSI transfer conveys light energy to a lower-energy red chlorophyll than PBS→PSII→PSI transfer.

Simple molecular discrimination of cultivated Porphyra species (Porphyra yezoensis and Porphyra tenera) and related wild species (Bangiales, Rhodophyta).

To discriminate between cultivated Porphyra species (Porphyra yezoensis and Porphyra tenera) and closely related wild Porphyra species, we developed a polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis of the rbcL gene using five restriction enzymes. Although our previous PCR‐RFLP analyses of internal transcribed spacer (ITS) rDNA and plastid RuBisCO spacer regions could not always discriminate wild P. yezoensis, wild P. tenera, and closely related wild species, the PCR‐RFLP profiles of the rbcL gene were useful in discriminating samples collected from natural habitats. Therefore, PCR‐RFLP analysis of the rbcL gene will help in the simple identification of a large number of samples, not only for the establishment of reliable cultures as breeding material, but also for the taxonomic investigations of species that are closely related to cultivated Porphyra.

CHIMERAS WITH MOSAIC PATTERN IN ARCHEOSPORE GERMLINGS OF PYROPIA YEZOENSIS UEDA (BANGIALES, RHODOPHYTA)(1).

In the marine crop Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi, it is known that conchospores from heterozygous conchocelis develop into sectored gametophytic blades (chimeras), but archeospores asexually released from haploid blades do not usually grow into chimeric blades. In this study, chimeras with mosaic pattern consisting of the green and wildtype colors were developed from archeospores that were released from a blade piece containing a cell cluster of green color induced by heavy‐ion beam irradiation. To make clear whether these archeospores were produced from the green‐colored cells or the wildtype‐colored cells, cell clusters of the green mutant, wildtype, and mosaic pattern were cut out from the grown chimera, and archeospores were released from each of the three blade pieces. Archeospores from the green‐mutant blade piece and from the wildtype blade piece developed into only green‐mutant blades and wildtype blades, respectively. In contrast, archeospores from the blade piece with mosaic pattern developed into green‐mutant blades, wildtype blades, and chimeric blades with mosaic pattern of the two colors, although the frequency of the chimeras was low. Because each gametophytic cell possesses a single plastid, it is difficult to explain the occurrence of the new chimeras as a mutation of the plastid DNA. Thus, the new chimeras are considered to be due to transposable elements in Pyropia.

Comparative analysis of strategies to prepare electron sinks in aquatic photoautotrophs

While subject to illumination, photosystem I (PSI) has the potential to produce reactive oxygen species (ROS) that can cause photo-oxidative damage in oxygenic photoautotrophs. The reaction center chlorophyll in PSI (P700) is kept oxidized in excess light conditions to limit over-excitation of PSI and alleviate the production of ROS. Oxidation of P700 requires a sufficient electron sink for PSI, which is responsible for flavodiiron proteins (FLV) safely dissipating electrons to O2 in cyanobacteria, green algae, and land plants except for angiosperms during short-pulse light (SP) illumination under which photosynthesis and photorespiration do not occur. This fact implies that O2 usage is essential for P700 oxidation but also raises the question why angiosperms lost FLV. Here, we first found that aquatic photoautotrophs in red plastid lineage, in which no gene for FLV has been found, could keep P700 oxidized during SP illumination alleviating the photo-oxidative damage in PSI even without O2 usage. We comprehensively assessed P700 oxidation during SP illumination in the presence and absence of O2 in cyanobacteria (Cyanophyta), green algae (Chlorophyta), angiosperms (Streptophyta), red algae (Rhodophyta), and secondary algae (Cryptophyta, Haptophyta, and Heterokontophyta). A variety of dependencies of P700 oxidation on O2 among these photoautotrophs clearly suggest that O2 usage and FLV are not universally required to oxidize P700 for protecting PSI against ROS damage. Our results expand the understanding of the diverse strategies taken by oxygenic photoautotrophs to oxidize P700 and mitigate the risks of ROS.

Possibility of polyploidy breeding using cryptic species in the marine crop Pyropia yezoensis (Bangiales, Rhodophyta)

In the marine crop Pyropia yezoensis, a cross-fertilization experiment between a cultivated strain and cryptic species was carried out to investigate the possibility of polyploidy breeding using cryptic species. To perform the cross-fertilization, we isolated a green mutant strain (IBYC-G1) of the cryptic species by heavy-ion beam irradiation. The green mutant was crossed with a wildtype strain (HG-4) of P. yezoensis f. narawaensis. The F1 heterozygous conchocelis developed normally and released numerous conchospores. However, almost all the conchospore germlings did not survive past the four-cell stage or thereabout, and a few germlings developed into gametophytic blades. These results indicated that reproductive isolation occurred during meiosis and that P. yezoensis and cryptic species were different species using the biological species concept. Almost all the archeospores that were released from the surviving F1 blades developed into normal gametophytic blades. The result of nuclear DNA marker analysis revealed that the F1 blades that developed from both conchospores and archeospores were allodiploids in the haploid gametophytic phase. Although their F1 blades were extremely slender, their blade color was a deeper reddish brown as compared with that of P. yezoensis f. narawaensis, and the rate of blade length increase was remarkably higher in the blades of F1 than in those of the cryptic species. These results suggested that polyploidy breeding in the marine crop will attract more attention as a breeding method for further development of new cultivars.