Distribution of phenanthrene in the ospho2 reveals the involvement of phosphate on phenanthrene translocation and accumulation in rice.

The intricate mechanisms involved in the acquisition and translocation of polycyclic aromatic hydrocarbons (PAHs) in plants have not been elucidated. Phosphate (Pi) is the bioavailable form of essential macronutrient phosphorus, which is acquired and subsequently assimilated for plant optimal growth and development. Rice phosphate overaccumulator 2 (OsPHO2) is a central constituent of the regulation of Pi homeostasis in rice. In the present study, the role of OsPHO2 in regulating the translocation and accumulation of phenanthrene (Phe) and the involvement of Pi in this process were investigated. The temporal study (1 d-35 d) revealed a significant and gradual increase of Phe accumulation in Pi-deprived roots of wild-type (WT) seedlings. Compared with the WT, the concentrations of Phe were significantly higher in the shoots of ospho2 (OsPHO2 mutant) grown hydroponically with Phe (1.5 mg/L) under +Pi (200 µM) and -Pi (10 µM) conditions. The sap experiment clearly showed the significant increases in levels of Phe in the xylem sap of ospho2 than the WT grown hydroponically with Phe and +Pi. Further, the concentrations of both Phe and P were coordinately higher in the culms and flag leaves of the mutants than WT at maturity in potting soil with LPhe (6 mg/kg) and HPhe (60 mg/kg). However, the concentrations of Phe in the seeds were comparable in the WT and mutants, suggesting a pivotal of OsPHO2 in attenuating Phe toxicity in the seed. In +Phe WT, the relative expression level of OsPHO2 in the shoots was significantly lower, while those of Pi transporters (PTs) OsPT4 and OsPT8 were significantly higher in the roots compared with -Phe. Together, the results provided evidence towards the involvement of Pi in OsPHO2-regulated translocation and accumulation of Phe in rice.

Essentiality for rice fertility and alternative splicing of OsSUT1.

Sucrose-proton symporters play important roles in carbohydrate transport during plant growth and development. Their physiological functions have only been partly characterized and their regulation mechanism is largely unclear. Here we report that the knockout of a sucrose transporter gene, OsSUT1, by CRISPR-Cas9 mediated gene editing resulted in a slightly dwarf size and complete infertility of the gene's homozygous mutants. Observation of caryopsis development revealed that the endosperm of OsSUT1 mutants failed to cellularize and did not show any sign of seed-filling. Consistently, OsSUT1 was identified to express strongly in developing caryopsis of wild-type rice, particularly in the nucellar epidermis and aleurone which are critical for the uptake of nutrients into the endosperm. These results indicate that OsSUT1 is indispensable during the rice reproductive stage particularly for caryopsis development. Interestingly, OsSUT1 possesses at least 6 alternative splicing transcripts, including the 4 transcripts deposited previously and the other two identified by us. The differences among these transcripts primarily lie in their coding region of the 3' end and 3' UTR region. Real-time PCR showed that 4 of the 6 transcripts had different expressional patterns during rice vegetative and reproductive growth stages. Given the versatility of the gene, addressing its alternative splicing mechanism may expand our understanding of SUT's function substantially.

Reducing phenanthrene uptake and translocation, and accumulation in the seeds by overexpressing OsNRT2.3b in rice.

The uptake and accumulation of polycyclic aromatic hydrocarbons (PAHs) in crops have gained much attention due to their toxicity to humans. Nitrogen (N) is an essential element for plant growth and has also been implicated in the acquisition and acropetal translocation of PAHs. OsNRT2.3b encodes a nitrate (NO3-) transporter that is involved in the acquisition and mobilization of N in rice. Here, we investigated whether overexpression of OsNRT2.3b would exert any mitigating influence on the uptake and translocation of phenanthrene (Phe, a model PAH) in transgenic rice (Oryza sativa). The wild-type seedlings exhibited a reduction in plant height, primary root length, and shoot biomass when grown hydroponically in a medium supplemented with Phe. Acquisition of Phe by the roots and its subsequent translocation to shoots increased concomitantly with an increase in Phe concentration in the medium and duration of the treatment. OsNRT2.3b-overexpressing lines (Ox-6 and Ox-8) were generated independently. Compared with the wild-type, the concentration of Phe in Ox-6 and Ox-8 were significantly lower in the roots (47%-54%) and shoots (22%-31%) grown hydroponically with Phe (1 mg/L). Further, the wild-type and Ox lines were grown to maturity in a pot soil under Phe conditions and the concentrations of Phe and total N were assayed in the culms and flag leaves. Compared with the wild-type, in Ox lines the concentration of total N significantly increased in the culms (288%-366%) and flag leaves (12%-25%), while that of Phe significantly reduced in the culms (25%-28%) and flag leaves (18%-21%). The results revealed an antagonistic correlation between the concentration of total N and Phe. The concentration of Phe was also significantly lower (29%-38%) in the seeds of Ox lines than the wild-type. The study highlighted the efficacy of overexpressing OsNRT2.3b in mitigating the Phe toxicity by attenuating its acquisition, mobilization, and allocation to the seeds.

Knockdown of OsSAE1a affects the growth and development and phosphate homeostasis in rice.

SUMOylation is a post-translational modification process that comprises a tandem enzymatic cascade, i.e., maturation, activation, conjugation, and ligation of a small ubiquitin-like modifier, which triggers the modulated activities and transport of the cellular proteins to other areas of the cell. In Oryza sativa (rice), OsSIZ1/2 encoding E3 SUMO ligase exerts regulatory influences on Pi homeostasis and developmental responses. However, the role of OsSAE1a, SUMO E1 activating enzyme, in regulating phosphate (Pi) utilization and/or growth and development is not known in rice and was thus investigated in this study. The qRT-PCR assay revealed a constitutive and variable spatiotemporal expression pattern of OsSAE1a in the vegetative and reproductive tissues and was comparable in the root and shoot grown under different Pi regimes. RNAi-mediated suppression of OsSAE1a exerted variable effects on the concentrations of Pi and total P in different tissues, uptake and distribution of 32Pi, and relative expression levels of several genes that play pivotal roles in the maintenance of Pi homeostasis. The effects of the mutation in OsSAE1a were also evident in the vegetative and reproductive traits of rice during growth in a hydroponic system and pot soil, respectively. Overall, these results suggest a broad-spectrum role of OsSAE1a in the maintenance of Pi homeostasis and regulating growth and development.

The ferroxidase LPR5 functions in the maintenance of phosphate homeostasis and is required for normal growth and development of rice.

Members of the Low Phosphate Root (LPR) family have been identified in rice (Oryza sativa) and expression analyses have been conducted. Here, we investigated the functions of one of the five members in rice, LPR5. qRT-PCR and promoter-GUS reporter analyses indicated that under Pi-sufficient conditions OsLPR5 was highly expressed in the roots, and specific expression occurred in the leaf collars and nodes, and its expression was increased under Pi-deficient conditions. In vitro analysis of the purified OsLPR5 protein showed that it exhibited ferroxidase activity. Overexpression of OsLPR5 triggered higher ferroxidase activity, and elevated concentrations of Fe(III) in the xylem sap and of total Fe in the roots and shoots. Transient expression of OsLPR5 in Nicotiana benthamiana provided evidence of its subcellular localization to the cell wall and endoplasmic reticulum. Knockout mutation in OsLPR5 by means of CRISPR-Cas9 resulted in adverse effects on Pi translocation, on the relative expression of Cis-NATOsPHO1;2, and on several morphological traits, including root development and yield potential. Our results indicate that ferroxidase-dependent OsLPR5 has both a broad-spectrum influence on growth and development in rice as well as affecting a subset of physiological and molecular traits that govern Pi homeostasis.

OsSQD1 at the crossroads of phosphate and sulfur metabolism affects plant morphology and lipid composition in response to phosphate deprivation.

In phosphate (Pi)-deprived Arabidopsis (Arabidopsis thaliana), phosphatidylglycerol (PG) is substituted by sulfolipid for maintaining Pi homeostasis. Sulfoquinovosyl diacylglycerol1 (AtSQD1) encodes a protein, which catalyzes uridine diphosphate glucose (UDPG) and sulfite (SO32- ) to UDP-sulfoquinovose, which is a key component in the sulfolipid biosynthetic pathway. In this study, a reverse genetics approach was employed to decipher the function of the AtSQD1 homolog OsSQD1 in rice. Differential expressions of OsSQD1 in different tissue and response to -P and -S also detected, respectively. The in vitro protein assay and analysis suggests that OsSQD1 is a UDP-sulfoquinovose synthase. Transient expression analysis showed that OsSQD1 is located in the chloroplast. The analyses of the knockout (ossqd1) and knockdown (Ri1 and Ri2) mutants demonstrated reductions in Pi and total P concentrations, 32 Pi uptake rate, expression levels of Pi transporters and altered developmental responses of root traits, which were accentuated during Pi deficiency. The inhibitory effects of the OsSQD1 mutation were also evident in the development of reproductive tissue. Furthermore, OsSQD1 differently affects lipid composition under different Pi regime affects sulfur (S) homeostasis. Together, the study revealed that OsSQD1 affects Pi and S homeostasis, and lipid composition in response to Pi deprivation.

Spatial-temporal characteristics of nitrogen degradation in typical Rivers of Taihu Lake Basin, China.

In this study, we focus on the measurement of different nitrogen (N) forms and investigate the spatial-temporal variability of degradation coefficient in river channels. We aim to provide a new approach of deriving in-situ degradation coefficients of different N forms, and highlight factors that determine the spatial-temporal variability of degradation coefficients. Our results are based on a two-year field survey in 34 channels around the Taihu Lake Basin, eastern China. The derived degradation coefficients of different N forms based our newly-developed experimental device are: degradation coefficients of TN, NH4+-N and NO3-N range from 0.006-0.449 d-1, 0.022-1.175 d-1 and -0.096-2.402 d-1, respectively. The degradation coefficients of N show strong dependence on N concentration and water temperature. The seasonal difference of water temperature and N concentration leads to spatial-temporal variability of degradation coefficients. The derived degradation coefficients of N are further verified through one-dimensional water quality model simulations. The degradation coefficient obtained in this study and the influencing factors of its spatial-temporal variability provide invaluable reference for studies in aquatic environment.

Spatio-Temporal Differences in Nitrogen Reduction Rates under Biotic and Abiotic Processes in River Water of the Taihu Basin, China.

Understanding spatio-temporal differences in nitrogen (N) transformation, transport and reduction rates in water bodies is critical to achieve effective mitigation of river eutrophication. We performed culture experiments in six rivers in the Taihu Basin using a custom made in-situ experimental apparatus. We investigated spatio-temporal differences in reduce processes and rates of different N forms and assessed the contribution of biological processes to dissolved inorganic N (DIN) reduce. Results showed that biological processes played a major role in N reduction in summer, while non-microbial processes were dominant in winter. We observed significant spatial and temporal differences in the studied mechanisms, with reduction rates of different N compounds being significantly higher in summer and autumn than spring and winter. Reduction rates ranged from 105.4 ± 25.3 to 1458.8 ± 98.4 mg·(m³·d)-1 for total N, 33.1 ± 12.3 to 440.9 ± 33.1 mg·(m³·d)-1 for ammonium, 56.3 ± 22.7 to 332.1 ± 61.9 mg·(m³·d)-1 for nitrate and 0.4 ± 0.3 to 31.8 ± 9.0 mg·(m³·d)-1 for nitrite across four seasons. Mean DIN reduction rates with and without microbial activity were 96.0 ± 46.4 mg·(m³·d)-1 and 288.1 ± 67.8 mg·(m³·d)-1, respectively, with microbial activity rates accounting for 29.7% of the DIN load and 2.2% of the N load. Results of correlation and principal component analysis showed that the main factors influencing N processing were the concentrations of different N forms and multiple environmental factors in spring, N concentrations, DO and pH in summer, N concentrations and water velocity in autumn and N concentrations in winter.

Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis.

Dental plaque is a biofilm of water-soluble and water-insoluble polysaccharides, produced primarily by Streptococcus mutans. Dextranase can inhibit biofilm formation. Here, a dextranase gene from the marine microorganism Arthrobacter oxydans KQ11-1 is described, and cloned and expressed using E. coli DH5α competent cells. The recombinant enzyme was then purified and its properties were characterized. The optimal temperature and pH were determined to be 60°C and 6.5, respectively. High-performance liquid chromatography data show that the final hydrolysis products were glucose, maltose, maltotriose, and maltotetraose. Thus, dextranase can inhibit the adhesive ability of S. mutans. The minimum biofilm inhibition and reduction concentrations (MBIC50 and MBRC50) of dextranase were 2 U ml-1 and 5 U ml-1, respectively. Scanning electron microscopy and confocal laser scanning microscope (CLSM) observations confirmed that dextranase inhibited biofilm formation and removed previously formed biofilms.

Multiple NUCLEAR FACTOR Y transcription factors respond to abiotic stress in Brassica napus L.

Members of the plant NUCLEAR FACTOR Y (NF-Y) family are composed of the NF-YA, NF-YB, and NF-YC subunits. In Brassica napus (canola), each of these subunits forms a multimember subfamily. Plant NF-Ys were reported to be involved in several abiotic stresses. In this study, we demonstrated that multiple members of thirty three BnNF-Ys responded rapidly to salinity, drought, or ABA treatments. Transcripts of five BnNF-YAs, seven BnNF-YBs, and two BnNF-YCs were up-regulated by salinity stress, whereas the expression of thirteen BnNF-YAs, ten BnNF-YBs, and four BnNF-YCs were induced by drought stress. Under NaCl treatments, the expression of one BnNF-YA10 and four NF-YBs (BnNF-YB3, BnNF-YB7, BnNF-YB10, and BnNF-YB14) were greatly increased. Under PEG treatments, the expression levels of four NF-YAs (BnNF-YA9, BnNF-YA10, BnNF-YA11, and BnNF-YA12) and five NF-YBs (BnNF-YB1, BnNF-YB8, BnNF-YB10, BnNF-YB13, and BnNF-YB14) were greatly induced. The expression profiles of 20 of the 27 salinity- or drought-induced BnNF-Ys were also affected by ABA treatment. The expression levels of six NF-YAs (BnNF-YA1, BnNF-YA7, BnNF-YA8, BnNF-YA9, BnNF-YA10, and BnNF-YA12) and seven BnNF-YB members (BnNF-YB2, BnNF-YB3, BnNF-YB7, BnNF-YB10, BnNF-YB11, BnNF-YB13, and BnNF-YB14) and two NF-YC members (BnNF-YC2 and BnNF-YC3) were greatly up-regulated by ABA treatments. Only a few BnNF-Ys were inhibited by the above three treatments. Several NF-Y subfamily members exhibited collinear expression patterns. The promoters of all stress-responsive BnNF-Ys harbored at least two types of stress-related cis-elements, such as ABRE, DRE, MYB, or MYC. The cis-element organization of BnNF-Ys was similar to that of Arabidopsis thaliana, and the promoter regions exhibited higher levels of nucleotide sequence identity with Brassica rapa than with Brassica oleracea. This work represents an entry point for investigating the roles of canola NF-Y proteins during abiotic stress responses and provides insight into the genetic evolution of Brassica NF-Ys.

The atmospheric and room-temperature plasma (ARTP) method on the dextranase activity and structure.

A novel atmospheric and room-temperature plasma (ARTP) method was used to breed high-yielding mutations of Arthrobacter KQ11. Mutagenesis produced two mutations, 4-1 and 4-13, which increased enzyme activity by 19 and 30%, respectively. Dents on the cell envelope were observed under scanning electron microscopy (SEM). The optimal temperature and pH of the wild strain were 45°C and 5.5 and those of the mutant strains were 45°C, pH 6.0 (4-1) and 50°C, pH 6.0 (4-13). Under optimal enzyme production conditions of the wild and mutant strains, the dextranase activity of 4-13 was 50% higher than that of the wild strain. Through amino acid alignment, several nucleotides of the mutant strains were found to have changed. Experiments performed in vitro suggested that this endo-dextranase may inhibit biofilm formation by Streptococcus mutans.

Identification and characterization of NF-Y transcription factor families in Canola (Brassica napus L.).

NF-Y (NUCLEAR FACTOR-Y), a heterotrimeric transcription factor, is composed of NF-YA, NF-YB, and NF-YC proteins in yeast, animal, and plant systems. In plants, each of the NF-YA/B/C subunit forms a multi-member family. NF-Ys are key regulators with important roles in many physiological processes, such as drought tolerance, flowering time, and seed development. In this study, we identified, annotated, and further characterized 14 NF-YA, 14 NF-YB, and 5 NF-YC proteins in Brassica napus (canola). Phylogenetic analysis revealed that the NF-YA/B/C subunits were more closely clustered with the Arabidopsis thaliana (Arabidopsis) homologs than with rice OsHAP2/3/5 subunits. Analyses of the conserved domain indicated that the BnNF-YA/B/C subfamilies, respectively, shared the same conserved domains with those in other organisms, including Homo sapiens, Saccharomyces cerevisiae, Arabidopsis, and Oryza sativa (rice). An examination of exon/intron structures revealed that most gene structures of BnNF-Y were similar to their homologs in Arabidopsis, a model dicot plant, but different from those in the model monocot plant rice, suggesting that plant NF-Ys diverged before monocot and dicot plants differentiated. Spatial-tempo expression patterns, as determined by qRT-PCR, showed that most BnNF-Ys were widely expressed in different tissues throughout the canola life cycle and that several closely related BnNF-Y subunits had similar expression profiles. Based on these findings, we predict that BnNF-Y proteins have functions that are conserved in the homologous proteins in other plants. This study provides the first extensive evaluation of the BnNF-Y family, and provides a useful foundation for dissecting the functions of BnNF-Y.