Efficient and multiplexable genome editing using Platinum TALENs in oleaginous microalga, Nannochloropsis oceanica NIES-2145.

Algae accumulate large amounts of lipids produced by photosynthesis, and these lipids are expected to be utilized as feedstocks for sustainable new energies, known as biodiesels. Nannochloropsis species are eukaryotic microalgae that produce high levels of lipids. However, since the production costs of algal biodiesels are higher than those of fossil fuels, the improved productivity of algal lipids by molecular breeding of algae is required for practical use. In the present study, we developed a highly efficient genome-editing system involving Platinum transcription activator-like effector nucleases (TALENs) in Nannochloropsis oceanica. Platinum TALENs codon-optimized for N. oceanica were synthesized, and their DNA-binding activity was confirmed by single-strand annealing assays in human HEK293T cells. All-in-one expression vectors for Platinum TALEN targeting the nitrate reductase gene, NoNR, and acyltransferase gene, LPAT1, were transfected into Nannochloropsis species. The introduction of each Platinum TALEN revealed high genome-editing efficiency with no detectable off-target mutations at the candidate sites in N. oceanica. By simultaneously introducing TALENs targeting two genes, we obtained double mutant strains. The loss-of-function phenotype of NoNR was also confirmed. These findings will provide an essential technology for molecular breeding in Nannochloropsis species.

Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in Arabidopsis.

Chloroplast division is performed by the constriction of envelope membranes at the division site. Although constriction of a ring-like protein complex has been shown to be involved in chloroplast division, it remains unknown how membrane lipids participate in the process. Here, we show that phosphoinositides with unknown function in envelope membranes are involved in the regulation of chloroplast division in Arabidopsis thaliana. PLASTID DIVISION1 (PDV1) and PDV2 proteins interacted specifically with phosphatidylinositol 4-phosphate (PI4P). Inhibition of phosphatidylinositol 4-kinase (PI4K) decreased the level of PI4P in chloroplasts and accelerated chloroplast division. Knockout of PI4Kß2 expression or downregulation of PI4Kα1 expression resulted in decreased levels of PI4P in chloroplasts and increased chloroplast numbers. PI4Kα1 is the main contributor to PI4P synthesis in chloroplasts, and the effect of PI4K inhibition was largely abolished in the pdv1 mutant. Overexpression of DYNAMIN-RELATED PROTEIN5B (DRP5B), another component of the chloroplast division machinery, which is recruited to chloroplasts by PDV1 and PDV2, enhanced the effect of PI4K inhibition, whereas overexpression of PDV1 and PDV2 had additive effects. The amount of DRP5B that associated with chloroplasts increased upon PI4K inhibition. These findings suggest that PI4P is a regulator of chloroplast division in a PDV1- and DRP5B-dependent manner.

Expression of the nucleus-encoded chloroplast division genes and proteins regulated by the algal cell cycle.

Chloroplasts have evolved from a cyanobacterial endosymbiont and their continuity has been maintained by chloroplast division, which is performed by the constriction of a ring-like division complex at the division site. It is believed that the synchronization of the endosymbiotic and host cell division events was a critical step in establishing a permanent endosymbiotic relationship, such as is commonly seen in existing algae. In the majority of algal species, chloroplasts divide once per specific period of the host cell division cycle. In order to understand both the regulation of the timing of chloroplast division in algal cells and how the system evolved, we examined the expression of chloroplast division genes and proteins in the cell cycle of algae containing chloroplasts of cyanobacterial primary endosymbiotic origin (glaucophyte, red, green, and streptophyte algae). The results show that the nucleus-encoded chloroplast division genes and proteins of both cyanobacterial and eukaryotic host origin are expressed specifically during the S phase, except for FtsZ in one graucophyte alga. In this glaucophyte alga, FtsZ is persistently expressed throughout the cell cycle, whereas the expression of the nucleus-encoded MinD and MinE as well as FtsZ ring formation are regulated by the phases of the cell cycle. In contrast to the nucleus-encoded division genes, it has been shown that the expression of chloroplast-encoded division genes is not regulated by the host cell cycle. The endosymbiotic gene transfer of minE and minD from the chloroplast to the nuclear genome occurred independently on multiple occasions in distinct lineages, whereas the expression of nucleus-encoded MIND and MINE is regulated by the cell cycle in all lineages examined in this study. These results suggest that the timing of chloroplast division in algal cell cycle is restricted by the cell cycle-regulated expression of some but not all of the chloroplast division genes. In addition, it is suggested that the regulation of each division-related gene was established shortly after the endosymbiotic gene transfer, and this event occurred multiple times independently in distinct genes and in distinct lineages.

Genome editing with removable TALEN vectors harboring a yeast centromere and autonomous replication sequence in oleaginous microalga.

Algal lipids are expected to become a basis for sustainable fuels because of the highly efficient lipid production by photosynthesis accompanied by carbon dioxide assimilation. Molecular breeding of microalgae has been studied to improve algal lipid production, but the resultant gene-modified algae containing transgenes are rarely used for outdoor culture because the use of genetically modified organisms (GMOs) is strictly restricted under biocontainment regulations. Recently, it was reported that plasmids containing yeast centromere and autonomous replication sequence (CEN/ARS) behaved as episomes in Nannochloropsis species. We previously reported that the Platinum TALEN (PtTALEN) system exhibited high activity in Nannochloropsis oceanica. Therefore, we attempted to develop a genome editing system in which the expression vectors for PtTALEN can be removed from host cells after introduction of mutations. Using all-in-one PtTALEN plasmids containing CEN/ARS, targeted mutations and removal of all-in-one vectors were observed in N. oceanica, suggesting that our all-in-one PtTALEN vectors enable the construction of mutated N. oceanica without any transgenes. This system will be a feasible method for constructing non-GMO high-performance algae.

Factors affecting the long-term stability of orthodontic mini-implants.

INTRODUCTION: The placement and removal torques of mini-implants were evaluated as an index of implant stability. We examined factors affecting the initial and long-term stability of mini-implants. METHODS: We measured the placement and removal torques of 134 mini-implants placed in buccal posterior alveolar bone and assessed the relationships among placement and removal torques, placement period, age, sex, and cortical bone thickness. The mini-implants were machine-surfaced, 1.6 mm in diameter and 8 mm long. A torque screwdriver was used to measure the peak torque values. RESULTS AND CONCLUSIONS: The placement and removal torques averaged approximately 8 and 4 N cm, respectively. A torque of 4 N cm suggests sufficient anchorage capability for mini-implants. No significant correlation between placement and removal torques was found. Placement torque was significantly related to age and cortical bone thickness in the maxilla, whereas removal torque was not significantly related to placement period, age, sex, or cortical bone thickness.

Relationship between initial crowding and interproximal force during retention phase.

This study investigated the change in interproximal force (IPF) in mandibular anterior teeth during retention and the relationship between the irregularity index before orthodontic treatment and the IPF. The effect that an erupting third molar had on IPF was also examined. Forty treated patients (40 with extraction of four bicuspids) were followed for 18 months during the retention phase. The irregularity index was determined from initial plaster casts. The total IPF was determined by measuring the interdental frictional forces at the mandibular anterior teeth by withdrawing a metal strip. The total IPF increased during the retention phase until 18 months, and there was a positive correlation between the irregularity index and total IPF 6 to 18 months after active treatment. An erupting third molar did not affect the total IPF. An increase in the total IPF may be an indication of relapse in mandibular anterior crowding. In conclusion, orthodontists should pay special attention to potential relapse in the lower anterior teeth 6 months after active treatment in cases with severe anterior crowding before treatment.

Conservation and differences of the Min system in the chloroplast and bacterial division site placement.

Chloroplasts are descended from a cyanobacterial endosymbiont and divide by binary fission. Reminiscent of the process in their bacterial ancestor, chloroplast division involves a part of cyanobacteria-derived division machineries in addition to those acquired during chloroplast evolution.1,2 In both bacterial and chloroplast division, formation of the FtsZ ring at the mid position is required for subsequent constriction and fission at the mid division site.1-4 As in bacteria, positioning of the FtsZ ring at the mid-chloroplast is mediated by the Min system.1,2 Recently, we identified the MCD1 protein, a plant-specific component of the Min system in Arabidopsis thaliana chloroplasts.5 Unlike other division components that have been acquired after endosymbiosis and function outside of the chloroplasts (i.e., in/on the outer envelope membrane),6-9 MCD1 functions inside the chloroplast. Since we already discussed about the function and significance of MCD1 as a division component of plant origin,5 here we focus on and discuss about the diversity and evolution of the Min system.

The PLASTID DIVISION1 and 2 components of the chloroplast division machinery determine the rate of chloroplast division in land plant cell differentiation.

In most algae, the chloroplast division rate is held constant to maintain the proper number of chloroplasts per cell. By contrast, land plants evolved cell and chloroplast differentiation systems in which the size and number of chloroplasts change along with their respective cellular function by regulation of the division rate. Here, we show that PLASTID DIVISION (PDV) proteins, land plant-specific components of the division apparatus, determine the rate of chloroplast division. Overexpression of PDV proteins in the angiosperm Arabidopsis thaliana and the moss Physcomitrella patens increased the number but decreased the size of chloroplasts; reduction of PDV levels resulted in the opposite effect. The level of PDV proteins, but not other division components, decreased during leaf development, during which the chloroplast division rate also decreased. Exogenous cytokinins or overexpression of the cytokinin-responsive transcription factor CYTOKININ RESPONSE FACTOR2 increased the chloroplast division rate, where PDV proteins, but not other components of the division apparatus, were upregulated. These results suggest that the integration of PDV proteins into the division machinery enabled land plant cells to change chloroplast size and number in accord with the fate of cell differentiation.

The significance of C16 fatty acids in the sn-2 positions of glycerolipids in the photosynthetic growth of Synechocystis sp. PCC6803.

Most extant cyanobacteria contain C16 fatty acids in the sn-2 positions of glycerolipids, which are regulated by lysophosphatidic acid acyltransferase (LPAAT; EC 2.3.1.51). Synechocystis sp. PCC6803 contains sll1848, sll1752, and slr2060 as putative acyltransferase genes. sll1848 was recently reported to encode an indispensable palmitoyl-specific LPAAT; however, here we show that each of the three genes is dispensable. Delta1848 and Delta1848 Delta2060 cells had markedly higher contents of stearate (18:0), oleate (18:1), and linoleate (18:2) in place of palmitate (16:0) in the sn-2 positions, suggesting that Delta1848 Delta2060 cells incorporate 18:0 and 18:1 in the sn-2 positions. The levels of sll1752 transcripts increased in Delta1848 Delta2060 cells. This was accompanied by increased LPAAT activity toward 18:0 coenzyme A and its derivative in the membrane fraction. From these findings, together with the activity of a recombinant sll1752 protein and complementation of the Escherichia coli LPAAT mutant plsC, we conclude that sll1752 encodes a second LPAAT that prefers stearoyl and oleoyl substrates. Delta1848 Delta2060 cells grew slowly at 30 degrees C at lower cell density, and exhibited more severe damage at 20 degrees C than wild-type cells. Furthermore, Delta1848 Delta2060 cells exhibited photoinhibition more severely than wild-type cells. A phycobilisome core-membrane linker protein (slr0335) was also found to be susceptible to protein extraction under our conditions; its content decreased in the membrane fractions of Delta1848 Delta2060 cells. We conclude that C16 fatty acids in sn-2 positions are preferred in the photosynthetic growth of this cyanobacterium, despite sll1752 orthologs being conserved in most cyanobacteria. However, no sll1752 ortholog is conserved among photosynthetic eukaryotes including Cyanidioschyzon merolae.