Germline recombination in a novel Cre transgenic line,Prl3b1‐Cre mouse

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1‐cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL‐2) protein in placenta along with increased expression toward the end of pregnancy. PL‐2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1‐cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1‐cre;R26GRR mice revealed that tdsRed‐positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1‐cre;R26GRR testes suggested that Cre‐mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1‐cre mice line provides a unique resource to understand testicular germ‐cell development. genesis 54:389–397, 2016. © 2016 Wiley Periodicals, Inc.     

Chemical induction of disease resistance in rice is correlated with the expression of a gene encoding a nucleotide binding site and leucine-rich repeats

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is an agricultural chemical primarily used to prevent rice blast disease. Probenazole-treated rice acquires resistance to blast fungus irrespective of the rice variety. The chemical is applied prophylactically, and is thought to induce or bolster endogenous plant defenses. However, the mechanisms underlying this effect have not been established. To understand the mode of the chemical's action, we screened for novel probenazole-responsive genes in rice by means of differential display and identified a candidate gene, RPR1. RPR1 contains a nucleotide binding site and leucine-rich repeats, thus sharing structural similarity with known disease resistance genes. The expression of RPR1 in rice can be up-regulated by treatment with chemical inducers of systemic acquired resistance (SAR) and by inoculation with pathogens. RPR1-related sequences in rice varieties seem to be varied in sequence and/or expression, indicating that RPR1 itself is not a crucial factor for induced resistance in rice. However, Southern blot analysis revealed the existence of homologous sequences in all varieties examined. While the role of RPR1 has yet to be clarified, this is the first report of the identification of a member of this gene class and its induction during the systemic expression of induced disease resistance.     

The effect of temperature,light-spectrum,desiccation and salinity gradients on the photosynthetic performance of a subtidal brown alga,Sargassum macrocarpum,from Japan

The effect of temperature, light-spectrum, desiccation and salinity gradients on the photosynthesis of a Japanese subtidal brown alga, Sargassum macrocarpum (Fucales), was determined using a pulse amplitude modulation-chlorophyll fluorometer and dissolved oxygen sensors. Temperature responses of the maximum (F_v/F_m in darkness) and effective (ΔF/F_m′ at 50 μmol photons m⁻² s⁻¹; = Φ_PSII) quantum yields during 6-day culture (4–36°C) remained high at 12–28°C, but decreased at higher temperatures. Nevertheless, ΔF/F_m′ also dropped at temperatures below 8°C, suggesting light sensitivity under chilling temperatures because F_v/F_m remained high. Photosynthesis–irradiance responses at 24°C under red (660 nm), green (525 nm), blue (450 nm) and white light (metal halide lamp) showed that maximum net photosynthesis under blue and white light was greater than under red and green light, indicating the sensitivity and photosynthetic availability of blue light in the subtidal light environment. In the desiccation experiment, samples under aerial exposure of up to 8 h under dim-light at 24°C and 50% humidity showed that ΔF/F_m′ quickly declined after more than 45 min of emersion; furthermore, ΔF/F_m′ also failed to recover to initial levels even after 1 day of rehydration in seawater. Under the emersion state, the ΔF/F_m′ remained high when the relative water content (RWC) was greater than 50%; in contrast, it quickly dropped when the RWC was less than 50%. When the RWC was reduced below 50%, ΔF/F_m′ did not return to initial levels, regardless of subsequent re-hydration, suggesting a low capacity of photosynthesis to recover from desiccation. The stenohaline response of photosynthesis under 3-day culture is evident, given that ΔF/F_m′ declined when salinity was beyond 20–40 psu. Adaptation to subtidal environments in temperate waters of Japan can be linked to these traits.     

Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme

 NADP-dependent malic enzyme (NADP-ME) is a major decarboxylating enzyme in NADP-ME-type C₄ species such as maize and Flaveria. In this study, chloroplastic NADP-ME was transferred to rice (Oryza sativa L.) using a chimeric gene composed of maize NADP-ME cDNA under the control of rice light-harvesting chlorophyll-a/b-binding protein (Cab) promoter. There was a 20- to 70-fold increase in the NADP-ME activity in leaves of transgenic rice compared to that in wild-type rice plants. Immunocytochemical studies by electron microscopy showed that maize NADP-ME was mostly localized in chloroplasts in transgenic rice plants, and that the chloroplasts were agranal without thylakoid stacking. Chlorophyll content and photosystem II activity were inversely correlated with the level of NADP-ME activity. These results suggest that aberrant chloroplasts in transgenic plants may be caused by excessive NADP-ME activity. Based on these results and the known fact that only bundle sheath cells of NADP-ME species, among all three C₄ subgroups, have agranal chloroplasts, we postulate that a high level of chloroplastic NADP-ME activity could strongly affect the development of chloroplasts. Received: 27 January 1999 / Accepted: 20 January 2000     

Japan's nationwide long‐term monitoring survey of seaweed communities known as the “Monitoring Sites 1000”: Ten‐year overview and future perspectives

“Monitoring Sites 1000” – Japan's long‐term monitoring survey was established in 2003, based on the Japanese Government policy for the conservation of biodiversity. Ecological surveys have been conducted on various types of ecosystems at approximately 1000 sites in Japan for 15 years now and are planned to be carried out for 100 years. Since 2008, seaweed communities had been monitored at six sites, featuring the kelp (e.g. Saccharina and Ecklonia; Laminariales) and Sargassum (Fucales) communities in the subarctic and temperate regions of Japan. Annual surveys were carried out during the season when these canopy‐forming seaweeds are most abundant. A non‐destructive quadrat sampling method, with permanent quadrats placed along transects perpendicular to the shoreline, was used to determine species composition, coverage, and vertical distribution of seaweeds at these sites; while destructive sampling was done every 5 years to determine biomass. The occurrence of canopy‐forming species Saccharina japonica (var. japonica) and Ecklonia cava have appeared to be stable at the Muroran (southwestern part of Hokkaido Island) and Shimoda (Pacific coast of middle Honshu Island) sites, respectively; whereas the coverage of Ecklonia radicosa (= Eckloniopsis radicosa) at the Satsuma‐Nagashima site in southern part of Kyushu Island was highly variable until its sudden disappearance from the habitat in 2016. Thalli of E. radicosa lost most of their blades through browsing by herbivorous fish, and thus, this may be one of the causes of the decline. A shift in the community structure related to environmental changes had also been observed at some other sites. Pre‐ and post‐disaster data revealed the impact of the 2011 earthquake and tsunami disasters, including a shift in the vertical distribution of Ecklonia bicyclis (= Eisenia bicyclis) to shallower depths at the Shizugawa site in the Pacific coast of northern Honshu Island, due to seafloor subsidence.     

Construction of rice cybrid plants

Summary The mitochondrial genomes of rice cells were transferred to a fertile rice variety (N8) from a cytoplasmic male sterile variety (CMS) by asymmetric protoplast fusion based on metabolic complementation. Protoplasts derived from CMS were X-irradiated (125 krad) and electrofused with protoplasts which had been treated with iodoacetamide. Metabolic complementation, presumably between nuclear and cytoplasmic compartments, enabled fused protoplasts to form colonies at high efficiency. Restriction digest analysis of mitochondrial DNA (mtDNA) indicated that hybrid cells carried mtDNA derived from both parents. Of the plants regenerated from hybrid calli, 68% carried a diploid chromosome set (2n=24) and the rest of them carried 48 chromosomes. All of them expressed the aryl acylamidase I deficient phenotype encoded by the recessive allele of the fertile N8 parent. These results indicate that the novel somatic hybrid plants regenerated were cybrids, deriving their nucleus from the iodoacetamide treated parent and their mitochondria from both parents.     

Photosynthetic activity including the thermal‐ and chilling‐light sensitivities of a temperate Japanese brown alga Sargassum macrocarpum

The effects of irradiance, temperature, thermal‐ and chilling‐light sensitivities on the photosynthesis of a temperate alga, Sargassum macrocarpum (Fucales) were determined by a pulse amplitude modulation (PAM)‐chlorophyll fluorometer and dissolved oxygen sensors. Oxygenic photosynthesis–irradiance curves at 8, 20, and 28°C revealed that the maximum net photosynthetic rates (NP _max) and saturation irradiance were highest at 28°C, and lowest at 8°C. Gross photosynthesis and dark respiration determined over a range of temperatures (8–36°C) at 300 μmol photons m^?2 s^?1 revealed that the maximum gross photosynthetic rate (GP_max) occurred at 27.8°C, which is consistent with the highest seawater temperature in the southern distributional limit of this species in Japan. Additionally, the maximum quantum yields of photosystem II (F _v/F _m) during the 72‐h temperature exposures were stable at 8–28°C, but suddenly dropped to zero at higher temperatures, indicative of PSII deactivation. Continuous exposure (12 h) to irradiance of 200 (low) and 1000 (high) μmol photons m^?2 s^?1 at 8, 20, and 28°C revealed greater declines in their effective quantum yields (Φ _PSII) under high irradiance. While Φ _PSII under low irradiance were very similar with the initial F _v/F _m under 20 and 28°C, values rapidly decreased with exposure duration at 8°C. At this temperature, F _v/F _m did not recover to initial values even after 12 h of dark acclimation. Final F _v/F _m of alga at 28°C under high irradiance treatment also did not recover, suggesting its sensitivity to photoinhibition at both low and high temperatures. These photosynthetic characteristics reflect both the adaptation of the species to the general environmental conditions, and its ability to acclimate to seasonal changes in seawater temperature within their geographical range of distribution.     

Isolation of novel types of Arabidopsis mutants with altered reactions to cadmium: cadmium-gradient agar plates are an effective screen for the heavy metal-related mutants

We are interested in elucidating the molecular mechanisms underlying plant reactions to the toxic heavy metal cadmium (Cd). To this end, we devised a new screening strategy using agar plates with a gradient of Cd concentrations, termed Cd-gradient agar plates (CGAPs), to isolate Arabidopsis mutants that displayed altered reactions to the metal. Arabidopsis M₂ seeds, derived from ethyl methanesulfonate (EMS) treated seeds, were germinated on the CGAPs such that the primary root of each seedling elongated against increasing concentrations of Cd on the surface of the plate. Under these conditions, the lengths of the primary roots reliably demonstrated the degree of Cd tolerance of individual seedlings. The use of CGAPs also allowed close observation of the root reaction of each seedling to Cd without causing lethal damage. The screen identified three mutant lines, MRC-32, MRC-22 and MRC-26, which showed distinctly different characteristics. MRC-32 plants exhibited enhanced tolerance to Cd and contained Cd at higher concentrations than wild-type (WT) plants treated with the heavy metal. The whole root system of MRC-22 plants showed a Cd-phobic response. MRC-26 plants accumulated less Cd in their aboveground tissues than WT plants, suggesting that they were defective in transporting the heavy metal from roots to aboveground tissues. We also determined the likely chromosomal location of each mutation.     

Structure,chromosomal location and expression of a rice gene encoding the microsome ω-3 fatty acid desaturase

The -3 fatty acid desaturases are membrane-bound enzymes catalyzing the conversion of linoleic acid to linolenic acid in lipids, and are located both in the microsome and plastid envelopes as two different isoforms. A cDNA encoding the microsome -3 fatty acid desaturase (OsFAD3) and the corresponding genomic clone were isolated from rice (Oryza sativa L.). The OsFAD3 gene was composed of 8 exons and 7 introns. A microsatellite was present in the second exon of the OsFAD3 gene, showing polymorphism between Indica and Japonica rice varieties. The mapping of this microsatellite showed that the OsFAD3 gene was located on chromosome 11. Expression of the OsFAD3 cDNA in tobacco hairy root tissues and subsequent analysis of fatty acid compositions demonstrated the activity of the microsome -3 fatty acid desaturase. The OsFAD3 mRNA was abundant in root tissues, but was hardly detectable in leaves. In root tissues, a high level of the OsFAD3 mRNA was observed at 15 °C and 20 °C, with its level decreasing markedly at temperatures below 10 °C. The accumulation of the OsFAD3 mRNA in leaf tissues remained at quite low levels, both at normal growth temperatures and at chilling temperatures. Similar temperature responses of the OsFAD3 gene were observed both in chilling- tolerant and in chilling-intolerant rice cultivars.