Yoneda, K. | bioRxiv Science

bioRxiv2024

Genome-wide mapping of autonomously replicating sequences in the marine diatom Phaeodactylum tricornutum

Autonomously replicating sequences (ARSs) are important accessories in episomal vectors that allow them to be replicated and stably maintained within transformants. Despite their importance, no information on ARSs in diatoms have been reported. Therefore, we attempted to identify ARS candidates in the model diatom, Phaeodactylum tricornutum, via chromatin immunoprecipitation sequencing. In this study, subunits of the origin recognition complex (ORC), ORC2 and ORC4, were used to screen for ARS candidates. ORC2 and ORC4 bound to 355 sites on the P. tricornutum genome, of which 69 were constantly screened after multiple attempts. The screened ARS candidates had an AT-richness of approximately 50% (44.39-52.92%) and did not have conserved sequences. In addition, ARS candidates were distributed randomly but had a dense distribution pattern at several sites. Their positions tended to overlap with those of the genetic region (73.91%). Compared to the ARSs of several other eukaryotic organisms, the characteristics of the screened ARS candidates are complex. Thus, our findings suggest that the diatom has a distinct and unique native ARSs.

molecular biology↗

bioRxiv2024

Different functions of Lhcx isoforms in photoprotective mechanism in the marine diatom Thalassiosira pseudonana

Photosynthesis needs light energy, but that exceeding the maximal capacity of photosynthesis enhances formation of reactive oxygen species, which potentially causes photodamages. Therefore, light-harvesting complexes (Lhc) in phototrophs harbor various proteins and pigments to function in both light capture and energy dissipation. Diatom Lhcx proteins are reported to be a critical component for thermal dissipation of excess light energy, but the molecular mechanism of photoprotection is still not fully understood and the functions of each Lhcx isoform are not yet differentiated. Here, we focused on two types of Lhcx isoforms in Thalassiosira pseudonana: TpLhcx1/2, putative major components for energy-dependent fluorescence quenching (qE); and TpLhcx6_1, functionally unknown isoform uniquely conserved in Thalassiosirales. TpLhcx1/2 proteins accumulated more under high light than under low light, while the TpLhcx6_1 protein level was constitutive irrespective of light intensities and CO2 concentrations. High-light induced photodamage of photosystem II was increased in the genome-editing transformants of these Lhcx isoforms relative to the wild-type. Transformants lacking TpLhcx1/2 showed significantly lowered qE capacities, strongly suggesting that these proteins are important for the fast thermal energy dissipation. While in contrast, genome-editing transformants lacking the TpLhcx6_1 protein rather increased the qE capacity. TpLhcx6_1 transformants were further evaluated by the low-temperature time-resolved chlorophyll fluorescence measurement, showing the longer fluorescence lifetime in transformants than that in the wild type cells even at the dark-acclimated state of these cells. These results suggest that TpLhcx6_1 functions in photoprotection through non-photochemical energy dissipation in the different way from qE. One sentence summaryThe marine diatom Thalassiosira pseudonana dissipates excess light energy for photoprotection via two types of mechanisms supported by different Lhc isofoms.

plant biology↗