CRISPR/Cas9-mediated knockout of NYC1 gene enhances chlorophyll retention and reduces tillering in Zoysia matrella (L.) Merrill.

KEY MESSAGE: Genome editing by CRISPR/Cas9 can be applied to Z. matrella 'Wakaba', and knockout mutants of ZmNYC1 gene exhibited stay-green phenotype and reduced tillering. Zoysia matrella is a widely used C4 warm-season turfgrass for landscaping, golf courses, and sports fields. Here, we used the CRISPR/Cas9 system to target the Non-Yellow Coloring1 (ZmNYC1) gene in the highly heterozygous allotetraploid Z. matrella 'Wakaba', aiming to generate a novel stay-green variety. Of 441 Agrobacterium-infected calli, 22 (5.0%) were transformed, and 14 of these (63.6%) showed targeted mutations through cleaved amplified polymorphic sequences analysis. Sequencing analysis revealed mutations mostly consisting of 1 or 2 bp indels, occurring 2 to 4 bp upstream of the PAM sequence. Regenerated plants exhibited five ZmNYC1 target locus genotypes, including homozygous mutants with a complete knockout of all four alleles in the T0 generation. Under dark treatment, ZmNYC1-mutated plants displayed suppressed chlorophyll b (Chl b) degradation, leading to higher chlorophyll content and Chl b, with a lower chlorophyll a/chlorophyll b ratio compared to the wild type (WT). However, the ZmNYC1 mutation also inhibited plant growth in homozygous mutant genotypes, exhibiting reduced tillering compared to WT. Additionally, during winter simulation, mutant with a complete knockout retained greenness longer than the WT. This is the first successful use of CRISPR/Cas9 genome editing in zoysiagrass. The mutants of the ZmNYC1 gene would serve as valuable breeding material for developing improved zoysiagrass varieties that can maintain their green color for longer periods, even during winter dormancy.

Varietal differences in flavonoid and antioxidant activity in Japanese soybean accessions.

Soybean seeds contain many antioxidants, including flavonoids and other phytochemicals. Isoflavone is a phytoestrogen that mimics estrogenic effects on target tissues and also exerts antioxidant activity by sequestering free radicals. Despite many cultivars developed to date, varietal differences in flavonoid content and antioxidant activity in Japanese soybean accessions remain less well characterized. Here, we evaluated the seed content of isoflavones, total flavonoids, and total phenolics in 26 soybean accessions. Next, the antioxidant activity of each accession was examined using antioxidant responsive element (ARE) linked to a luciferase reporter in human HepG2 stable cells. The relative ARE luciferase activity rate of all soybean accessions varied up to 4-fold which ranged from 1.00 to 4.02; and 22 accessions exhibited significant antioxidant activities. Correlation analysis indicated that the level of total isoflavone moderately correlated with antioxidant activity.

Replacement of soybean meal with levels of inclusion of soya waste in the diet of growing goats.

An experiment was conducted to investigate the effect of replacing soybean meal with soya waste at different levels on intake, digestibility and growth in goats. Eighteen male goat kids with initial body weight (BW) of 13.0 kg were distributed equally to three dietary groups. They were fed Napier grass (Pennisetum purpureum) and concentrate mixture, and each goat was assigned to an individual pen. Soybean meal in the concentrate mixture was replaced with soya waste at 0% (T1), 50% (T2) and 100% (T3) levels in respective dietary groups. These diets were isocaloric and isonitrogenous. Results showed that animals fed T3 diet exhibited higher Napier grass intake than those fed T1 or T2 diet. There was no influence on total intakes of dry matter (DM), organic matter (OM), crude protein (CP), metabolic BW, per cent BW and metabolisable energy by the dietary groups. However, there was an increasing trend on intake and digestibility of neutral detergent fibre (NDF) with increasing levels of soya waste in the diets. Animals fed T3 diet showed higher intake and digestibility of NDF than those fed T1 diet. There was no influence of the dietary groups on digestibilities of DM, OM and CP. Similarly, there was no effect of them on the final BW, total BW gain, daily BW gain, feed conversion ratio and feed cost. Soya waste can replace 100% soybean meal in diets for growing goats, because no change was observed in nutrient intake, digestibility and growth performance; inclusion of soya waste enhanced the intake and digestibility of NDF.

Highly Efficient and Selective Oxidation of Ethanol to Acetaldehyde by a Hybrid Photocatalyst Consisting of SnO2 Nanorod and Rutile TiO2 with Heteroepitaxial Junction.

Single-crystal SnO2 nanorods were grown on rutile TiO2 with a heteroepitaxial relation of SnO2 {001}/TiO2 {001} (SnO2 -NR#TiO2 ) by a hydrothermal reaction. Resulting compressive lattice strain in the SnO2 -NR near the interface induces a continuous increase in the a-axis length extending over 60 nm to relax towards the [001] direction from the root to the tip. UV-light irradiation of the robust SnO2 -NR#TiO2 stably progresses the selective oxidation of ethanol to acetaldehyde with an external quantum yield of 25.6 % at excitation wavelength (λex )=365 nm under ambient temperature and pressure. Spectroscopic analyses and density functional theory simulation results suggested that the extremely high photocatalytic activity stems from the smooth interfacial electron transfer from TiO2 to SnO2 -NR through the high-quality junction and subsequent efficient charge separation due to the lattice strain-induced unidirectional potential gradient of the conduction band minimum in the SnO2 -NR.

Red-Light-Driven Water Splitting by Au(Core)-CdS(Shell) Half-Cut Nanoegg with Heteroepitaxial Junction.

A key material for artificial photosynthesis including water splitting is heteronanostructured (HNS) photocatalysts. The photocatalytic activity depends on the geometry and dimension, and the quality of junctions between the components. Here we present a half-cut Au(core)-CdS(shell) (HC-Au@CdS) nanoegg as a new HNS plasmonic photocatalyst for water splitting. UV-light irradiation of Au nanoparticle (NP)-loaded ZnO (Au/ZnO) at 50 °C induces the selective deposition of hexagonal CdS on the Au surface of Au/ZnO with an epitaxial (EPI) relation of CdS{0001}/Au{111}. The subsequent selective dissolution of the ZnO support at room temperature yields HC-Au@CdS with the Au NP size and EPI junction (#) retained. Red-light irradiation (λex = 640 nm) of HC-Au@#CdS gives rise to continuous stoichiometric water splitting with an unprecedentedly high external quantum yield of 0.24%.

Longitudinal Optical Phonons Modified by Organic Molecular Cation Motions in Organic-Inorganic Hybrid Perovskites.

We performed terahertz time-domain spectroscopy for methylammonium (MA) lead halide perovskite single crystals and characterized the longitudinal optical (LO) phonons directly. We found that the effective LO phonon wave number does not change in the wide temperature range between 10 and 300 K. However, the coupling between MA cation modes and the LO phonon mode derived from lead halide cages induces a mode splitting at low temperatures and a damping of the LO phonon mode at high temperatures. These results influence the interpretation of electron-LO phonon interactions in perovskite semiconductors, as well as the interpretations of mobility, carrier diffusion, and polaron formation.

Short-term grazing behavior of cattle under indoor housing for a new-bred tetraploid ruzigrass (Brachiaria ruziziensis Germain et Everard).

OBJECTIVE: The preference evaluation of cattle is an important factor for estimation and improvement of the grazing amounts of newly introduced or bred grasses or cultivars in barn. This study was performed to assess the grazing behavior (the amount of grazing and/or the grazing speed) of cattle as indirect method using newly bred Brachiaria ruziziensis tetraploid strain 'OKI-1'(BR) hay as treatment group and Cloris gayana 'Callide' (CG) hay as control group. It also compared the feasibility of using behavioral differences between two groups as one criteria for evaluating preference by Japanese black cattle in barn. METHODS: Three experiments were carried out using 12 growing Japanese Black cattle including 6 males and 6 females. In each experiment, the four Japanese Black cattle (2 males and 2 females) were placed in separated stall and allowed to graze BR and CG in manger that was separated into two portions for about 30 min. The position and behavior of the cattle were recorded, and weighed the residual of each gay at 15 and 30 minutes after experiment start. RESULTS: The BR was superior to CG in chemical composition such as protein, fibers and non-fibrous carbohydrate. The cattle, over all, tended to prefer BR over CG in the first half 15 minutes in terms of the time spent and amount of grazing. Additionally, growing cattle exhibited neophilia for BR bred newly. CONCLUSION: These findings indicated the current approach could be applied for one of criteria to evaluate the preference of hay by Japanese black cattle under indoor housing environment.

Enhanced Stomatal Conductance by a Spontaneous Arabidopsis Tetraploid, Me-0, Results from Increased Stomatal Size and Greater Stomatal Aperture.

The rate of gas exchange in plants is regulated mainly by stomatal size and density. Generally, higher densities of smaller stomata are advantageous for gas exchange; however, it is unclear what the effect of an extraordinary change in stomatal size might have on a plant's gas-exchange capacity. We investigated the stomatal responses to CO2 concentration changes among 374 Arabidopsis (Arabidopsis thaliana) ecotypes and discovered that Mechtshausen (Me-0), a natural tetraploid ecotype, has significantly larger stomata and can achieve a high stomatal conductance. We surmised that the cause of the increased stomatal conductance is tetraploidization; however, the stomatal conductance of another tetraploid accession, tetraploid Columbia (Col), was not as high as that in Me-0. One difference between these two accessions was the size of their stomatal apertures. Analyses of abscisic acid sensitivity, ion balance, and gene expression profiles suggested that physiological or genetic factors restrict the stomatal opening in tetraploid Col but not in Me-0. Our results show that Me-0 overcomes the handicap of stomatal opening that is typical for tetraploids and achieves higher stomatal conductance compared with the closely related tetraploid Col on account of larger stomatal apertures. This study provides evidence for whether larger stomatal size in tetraploids of higher plants can improve stomatal conductance.

Photocatalytic Synthesis of CdS(core)-CdSe(shell) Quantum Dots with a Heteroepitaxial Junction on TiO2 : Photoelectrochemical Hydrogen Generation from Water.

A major challenge in chemistry for the synthesis of hetero-nanostructures is to build up atomically commensurate interfaces for smooth interfacial charge transfer. Photodeposition of CdSe on a CdS-preloaded mesoporous TiO2 nanocrystalline film yields CdS(core)-CdSe(shell) quantum dots (CdS@CdSe/mp-TiO2 ) with a heteroepitaxial nanojunction at 298 K. Two-electrode quantum-dot-sensitized photoelectrochemical (QD-SPEC) cells with the structure photoanode |0.25 M Na2 S, 0.35 M Na2 SO3 (solvent=water)| cathode were fabricated. The CdS@CdSe QD-SPEC cell affords a solar-to-current efficiency (STCE) of 0.03 % without external bias under illumination of simulated sunlight (λ>430 nm, AM 1.5, one sun). By applying 0.1 V between the electrodes, the STCE increases up to 0.048 %, far surpassing the CdS/mp-TiO2 and CdSe/mp-TiO2 photoanode cells. The CdS-CdSe interfacial analysis by high-resolution transmission electron microscopy and the band energy analysis taking the size quantization and the electrolyte effect indicate that the excellent performance of the CdS@CdSe/mp-TiO2 photoanode originates from the effective charge separation due to the cascade-like band edge alignment and the heteroepitaxial junction between CdS and CdSe QDs. In addition, high surface coverage of TiO2 with QDs can contribute to the reduction in the loss of the electron transport from TiO2 to the electron collecting electrode.

Red/Far Red Light Controls Arbuscular Mycorrhizal Colonization via Jasmonic Acid and Strigolactone Signaling.

Establishment of a nitrogen-fixing symbiosis between legumes and rhizobia not only requires sufficient photosynthate, but also the sensing of the ratio of red to far red (R/FR) light. Here, we show that R/FR light sensing also positively influences the arbuscular mycorrhizal (AM) symbiosis of a legume and a non-legume through jasmonic acid (JA) and strigolactone (SL) signaling. The level of AM colonization in high R/FR light-grown tomato and Lotus japonicus significantly increased compared with that determined for low R/FR light-grown plants. Transcripts for JA-related genes were also elevated under high R/FR conditions. The root exudates derived from high R/FR light-grown plants contained more (+)-5-deoxystrigol, an AM-fungal hyphal branching inducer, than those from low R/FR light-grown plants. In summary, high R/FR light changes not only the levels of JA and SL synthesis, but also the composition of plant root exudates released into the rhizosphere, in this way augmenting the AM symbiosis.

Lotus japonicus nodulation is photomorphogenetically controlled by sensing the red/far red (R/FR) ratio through jasmonic acid (JA) signaling.

Light is critical for supplying carbon to the energetically expensive, nitrogen-fixing symbiosis between legumes and rhizobia. Here, we show that phytochrome B (phyB) is part of the monitoring system to detect suboptimal light conditions, which normally suppress Lotus japonicus nodule development after Mesorhizobium loti inoculation. We found that the number of nodules produced by L. japonicus phyB mutants is significantly reduced compared with the number produced of WT Miyakojima MG20. To explore causes other than photoassimilate production, the possibility that local control by the root genotype occurred was investigated by grafting experiments. The results showed that the shoot and not the root genotype is responsible for root nodule formation. To explore systemic control mechanisms exclusive of photoassimilation, we moved WT MG20 plants from white light to conditions that differed in their ratios of low or high red/far red (R/FR) light. In low R/FR light, the number of MG20 root nodules dramatically decreased compared with plants grown in high R/FR, although photoassimilate content was higher for plants grown under low R/FR. Also, the expression of jasmonic acid (JA) -responsive genes decreased in both low R/FR light-grown WT and white light-grown phyB mutant plants, and it correlated with decreased jasmonoyl-isoleucine content in the phyB mutant. Moreover, both infection thread formation and root nodule formation were positively influenced by JA treatment of WT plants grown in low R/FR light and white light-grown phyB mutants. Together, these results indicate that root nodule formation is photomorphogenetically controlled by sensing the R/FR ratio through JA signaling.

Assessing genetic diversity and geographical differentiation in a global collection of wild soybean (Glycine soja Sieb. et Zucc.) and assigning a mini-core collection.

Wild soybean (Glycine soja), the ancestor of the cultivated soybean (G. max), is a crucial resource for capturing the genetic diversity of soybean species. In this study, we used a set of 78 genome-wide microsatellite markers to analyse the genetic diversity and geographic differentiation patterns in a global collection of 2,050 G. soja accessions and a mini-core collection of G. max stored in two public seed banks. We observed a notable reduction in the genetic diversity of G. max compared with G. soja and identified a close phylogenetic relationship between G. max and a G. soja subpopulation located in central China. Furthermore, we revealed substantial genetic divergence between northern and southern subpopulations, accompanied by diminished genetic diversity in the northern subpopulations. Two clusters were discovered among the accessions from north-eastern China-one genetically close to those from South Korea and Southern Japan, and another close to those from Amur Oblast, Russia. Finally, 192 accessions were assigned to a mini-core collection of G. soja, retaining 73.8% of the alleles detected in the entire collection. This mini-core collection is accessible to those who need it, facilitating efficient evaluation and utilization of G. soja genetic resources in soybean breeding initiatives.

Identification and characterization of a novel kaempferol sulfotransferase from Arabidopsis thaliana.

In plants, flavonoids have been shown to be subjected to conjugation modifications such as glycosylation, methylation, and sulfation. Among these modifications, sulfation is known as an important pathway in the regulation of the levels of endogenous compounds such as steroids. Although a large variety of flavonoid sulfates also exist in plants, the detailed biochemical characterization of Arabidopsis thaliana sulfotransferases (AtSULTs) remains to be fully clarified. We report here that uncharacterized AtSULT202E1 (AGI code: At2g03770), a SULT202E subfamily member, shows the sulfating activity toward flavonoids. The general characteristics of the enzyme were studied on the optimum temperature and pH, the effect of divalent cations, and the thermal stability with kaempferol as substrate. A comparative analysis of the sulfation of flavonoids by AtSULT202E1, AtSULT202B1 and AtSULT202A1 revealed that three AtSULTs have differential substrate specificities. Surprisingly, 3-hydroxyflavone was sulfated only by AtSULT202A1 while 7-hydroxyflavone was highly sulfated by AtSULT202E1 and AtSULT202B1. These results indicate that flavonols might be sulfated in a position specific manner. In conclusion, our studies indicate that a novel AtSULT202E1 has the sulfating activity toward flavonoids together with AtSULT202B1 and AtSULT202A1. The existence of three flavonoid sulfotransferases in A. thaliana suggests that sulfation of flavonoids have an important role in regulation of their functions.

Effect of palm kernel cake as protein source in a concentrate diet on intake, digestibility and live weight gain of goats fed Napier grass.

The effects of palm kernel cake (PKC) as a protein source in a concentrate diet (comprising 35 % crushed maize, 30 % rice bran, 32 % PKC, 2 % vitamin mineral premix and 1 % salt) were examined on intake, live weight (LW) gain and digestibility in female goats (average LW of 12.4 ± 2.6 kg). Four goats were randomly allocated to each of the four treatment diets: (a) Napier grass (Pennisetum purpureum) offered ad libitum (T1), (b) T1 + concentrate at 0.5 % of LW (T2), (c) T1 + concentrate at 1.0 % of LW (T3) and (d) T1 + concentrate at 2.0 % of LW (T4). A 7-day digestibility trial and an 82-day growth experiment were conducted. No differences were observed among diets for intakes of roughage dry matter (DM), total DM, organic matter (OM) and neutral detergent fibre (NDF). The crude protein (CP) intake increased (P < 0.05) as the level of concentrate in the diets increased. Goats fed the T2, T3 and T4 diets gained 10.2, 34.1 and 52.5 g/head/day, respectively, while the control group (T1) lost weight (-12.7 g/head/day). The apparent digestibilities of DM, OM and CP were similar (P > 0.05) among treatments. The digestibility of dietary NDF decreased (P < 0.05) with increasing levels of concentrate, but there was no significant (P > 0.05) difference between T2 and T3 diets. Supplementing a basal diet of Napier grass with PKC-based concentrate improved CP intake and LW gain. The PKC-based concentrate diet can therefore be exploited for the use of local feed resources for goat production; however, further research is required to achieve the best growth response.

Quantitative trait locus analysis of symbiotic nitrogen fixation activity in the model legume Lotus japonicus.

Many legumes form nitrogen-fixing root nodules. An elevation of nitrogen fixation in such legumes would have significant implications for plant growth and biomass production in agriculture. To identify the genetic basis for the regulation of nitrogen fixation, quantitative trait locus (QTL) analysis was conducted with recombinant inbred lines derived from the cross Miyakojima MG-20 × Gifu B-129 in the model legume Lotus japonicus. This population was inoculated with Mesorhizobium loti MAFF303099 and grown for 14 days in pods containing vermiculite. Phenotypic data were collected for acetylene reduction activity (ARA) per plant (ARA/P), ARA per nodule weight (ARA/NW), ARA per nodule number (ARA/NN), NN per plant, NW per plant, stem length (SL), SL without inoculation (SLbac-), shoot dry weight without inoculation (SWbac-), root length without inoculation (RLbac-), and root dry weight (RWbac-), and finally 34 QTLs were identified. ARA/P, ARA/NN, NW, and SL showed strong correlations and QTL co-localization, suggesting that several plant characteristics important for symbiotic nitrogen fixation are controlled by the same locus. QTLs for ARA/P, ARA/NN, NW, and SL, co-localized around marker TM0832 on chromosome 4, were also co-localized with previously reported QTLs for seed mass. This is the first report of QTL analysis for symbiotic nitrogen fixation activity traits.

NBRP databases: databases of biological resources in Japan.

The National BioResource Project (NBRP) is a Japanese project that aims to establish a system for collecting, preserving and providing bioresources for use as experimental materials for life science research. It is promoted by 27 core resource facilities, each concerned with a particular group of organisms, and by one information center. The NBRP database is a product of this project. Thirty databases and an integrated database-retrieval system (BioResource World: BRW) have been created and made available through the NBRP home page (http://www.nbrp.jp). The 30 independent databases have individual features which directly reflect the data maintained by each resource facility. The BRW is designed for users who need to search across several resources without moving from one database to another. BRW provides access to a collection of 4.5-million records on bioresources including wild species, inbred lines, mutants, genetically engineered lines, DNA clones and so on. BRW supports summary browsing, keyword searching, and searching by DNA sequences or gene ontology. The results of searches provide links to online requests for distribution of research materials. A circulation system allows users to submit details of papers published on research conducted using NBRP resources.

The National BioResource Project (NBRP) Lotus and Glycine in Japan.

The objective of the National BioResource Project (NBRP) in Japan is to collect, conserve and distribute biological materials for life sciences research. The project consists of twenty-eight bioresources, including animal, plant, microorganism and DNA resources. NBRP Lotus and Glycine aims to support the development of legume research through the collection, conservation, and distribution of these bioresources. Lotus japonicus is a perennial legume that grows naturally throughout Japan and is widely used as a model plant for legumes because of such advantages as its small genome size and short life cycle. Soybean (Glycine max) has been cultivated as an important crop since ancient times, and numerous research programs have generated a large amount of basic research information and valuable bioresources for this crop. We have also developed a "LegumeBase" a specialized database for the genera Lotus and Glycine, and are maintaining this database as a part of the NBRP. In this paper we will provide an overview of the resources available from the NBRP Lotus and Glycine database site, called "LegumeBase".

Tissue Culture and Somatic Embryogenesis in Warm-Season Grasses-Current Status and Its Applications: A Review.

Warm-season grasses are C4 plants and have a high capacity for biomass productivity. These grasses are utilized in many agricultural production systems with their greatest value as feeds for livestock, bioethanol, and turf. However, many important warm-season perennial grasses multiply either by vegetative propagation or form their seeds by an asexual mode of reproduction called apomixis. Therefore, the improvement of these grasses by conventional breeding is difficult and is dependent on the availability of natural genetic variation and its manipulation through breeding and selection. Recent studies have indicated that plant tissue culture system through somatic embryogenesis complements and could further develop conventional breeding programs by micropropagation, somaclonal variation, somatic hybridization, genetic transformation, and genome editing. This review summarizes the tissue culture and somatic embryogenesis in warm-season grasses and focus on current status and above applications including the author's progress.

Rhamnogalacturonan-I as a nematode chemoattractant from Lotus corniculatus L. super-growing root culture.

Introduction: The soil houses a tremendous amount of micro-organisms, many of which are plant parasites and pathogens by feeding off plant roots for sustenance. Such root pathogens and parasites often rely on plant-secreted signaling molecules in the rhizosphere as host guidance cues. Here we describe the isolation and characterization of a chemoattractant of plant-parasitic root-knot nematodes (Meloidogyne incognita, RKN). Methods: The Super-growing Root (SR) culture, consisting of excised roots from the legume species Lotus corniculatus L., was found to strongly attract infective RKN juveniles and actively secrete chemoattractants into the liquid culture media. The chemo-attractant in the culture media supernatant was purified using hydrophobicity and anion exchange chromatography, and found to be enriched in carbohydrates. Results: Monosaccharide analyses suggest the chemo-attractant contains a wide array of sugars, but is enriched in arabinose, galactose and galacturonic acid. This purified chemoattractant was shown to contain pectin, specifically anti-rhamnogalacturonan-I and anti-arabinogalactan protein epitopes but not anti-homogalacturonan epitopes. More importantly, the arabinose and galactose sidechain groups were found to be essential for RKN-attracting activities. This chemo-attractant appears to be specific to M. incognita, as it wasn't effective in attracting other Meloidogyne species nor Caenorhabditis elegans. Discussion: This is the first report to identify the nematode attractant purified from root exudate of L corniculatus L. Our findings re-enforce pectic carbohydrates as important chemicals mediating micro-organism chemotaxis in the soil, and also highlight the unexpected utilities of the SR culture system in root pathogen research.

Construction of prediction models for growth traits of soybean cultivars based on phenotyping in diverse genotype and environment combinations.

As soybean cultivars are adapted to a relatively narrow range of latitude, the effects of climate changes are estimated to be severe. To address this issue, it is important to improve our understanding of the effects of climate change by applying the simulation model including both genetic and environmental factors with their interactions (G×E). To achieve this goal, we conducted the field experiments for soybean core collections using multiple sowing times in multi-latitudinal fields. Sowing time shifts altered the flowering time (FT) and growth phenotypes, and resulted in increasing the combinations of genotypes and environments. Genome-wide association studies for the obtained phenotypes revealed the effects of field and sowing time to the significance of detected alleles, indicating the presence of G×E. By using accumulated phenotypic and environmental data in 2018 and 2019, we constructed multiple regression models for FT and growth pattern. Applicability of the constructed models was evaluated by the field experiments in 2020 including a novel field, and high correlation between the predicted and measured values was observed, suggesting the robustness of the models. The models presented here would allow us to predict the phenotype of the core collections in a given environment.