OsJRL negatively regulates rice cold tolerance via interfering phenylalanine metabolism and flavonoid biosynthesis.

The identification of new genes involved in regulating cold tolerance in rice is urgent because low temperatures repress plant growth and reduce yields. Cold tolerance is controlled by multiple loci and involves a complex regulatory network. Here, we show that rice jacalin-related lectin (OsJRL) modulates cold tolerance in rice. The loss of OsJRL gene functions increased phenylalanine metabolism and flavonoid biosynthesis under cold stress. The OsJRL knock-out (KO) lines had higher phenylalanine ammonia-lyase (PAL) activity and greater flavonoid accumulation than the wild-type rice, Nipponbare (NIP), under cold stress. The leaves had lower levels of reactive oxygen species (ROS) and showed significantly enhanced cold tolerance compared to NIP. In contrast, the OsJRL overexpression (OE) lines had higher levels of ROS accumulation and showed lower cold tolerance than NIP. Additionally, the OsJRL KO lines accumulated more abscisic acid (ABA) and jasmonic acid (JA) under cold stress than NIP. The OsJRL OE lines showed increased sensitivity to ABA compared to NIP. We conclude that OsJRL negatively regulates the cold tolerance of rice via modulation of phenylalanine metabolism and flavonoid biosynthesis.

Inactivation of SSIIIa enhances the RS content through altering starch structure and accumulating C18:2 in japonica rice.

SSIIIa was the key gene responsible for RS formation in rice endosperm. The higher RS content in ssIIIa mutant has been proposed to be majorly due to the increased amylose-lipid complexes (RS5). However, the formation of RS5 elicited by ssIIIa mutation and the importance of RS5 for total RS content in rice are still unclear. With japonica ssIIIa loss-of-function mutants created by CRISPR/Cas9 gene editing, the effects of SSIIIa mutation on RS5 were furtherly evaluated through investigating the transcriptome and metabolites. Inactivation of SSIIIa caused significant enhancement in amylose and RS content but without depletion in starch reserves. SSIIIa mutation modulated the genes involved in carbohydrate and lipid metabolisms and the redistribution of substances, led to accumulated protein, glucose, fructose, and C18:2. Besides the increased amylose content and altered amylopectin structure, the increased C18:2 contributed greatly to the enhancement in RS content in japonica ssIIIa mutants through complexing with amylose to form RS5, while the existence of lipid counted against the enhancement of RS content in indica rice. RS5 showed discrepant contributions for the total RS in rice with different genetic background. Inactivation of SSIIIa has great potential in improving RS5 content in japonica rice without great yield loss.

Morpho-histology, endogenous hormone dynamics, and transcriptome profiling in Dacrydium pectinatum during female cone development.

Dacrydium pectinatum de Laubenfels is a perennial dioeciously gymnosperm species dominant in tropical montane rain forests. Due to deforestation, natural disasters, long infancy, and poor natural regeneration ability, the population of this species has been significantly reduced and listed as an endangered protected plant. To better understand the female cone development in D. pectinatum, we examined the morphological and anatomical changes, analyzed the endogenous hormone dynamics, and profiled gene expression. The female reproductive structures were first observed in January. The morpho-histological observations suggest that the development of the D. pectinatum megaspore can be largely divided into six stages: early flower bud differentiation, bract primordium differentiation, ovule primordium differentiation, dormancy, ovule maturity, and seed maturity. The levels of gibberellins (GA), auxin (IAA), abscisic acid (ABA), and cytokinin (CTK) fluctuate during the process of female cone development. The female cones of D. pectinatum need to maintain a low level of GA3-IAA-ABA steady state to promote seed germination. The first transcriptome database for female D. pectinatum was generated, revealing 310,621 unigenes. Differential expression analyses revealed several floral (MADS2, AGL62, and LFY) and hormone biosynthesis and signal transduction (CKX, KO, KAO, ABA4, ACO, etc.) genes that could be critical for female cone development. Our study provides new insights into the cone development in D. pectinatum and the foundation for female cone induction with hormones.

Toxicokinetics of mono-(2-ethylhexyl) phthalate with low-dose exposure applying fluorescence tracing technique.

Di(2-ethylhexyl) phthalate (DEHP) belongs to environmental endocrine disrupting chemicals (EEDCs) and can be rapidly hydrolyzed into the ultimate toxicant mono-2-ethylhexyl phthalate (MEHP). In this study, we used 5-aminofluorescein modified MEHP (MEHP-AF) as a fluorescence tracer to explore the toxicokinetics, including toxicokinetic parameters, absorption and transport across the intestinal mucosal barrier, distribution and pathological changes of organs. While the dose was as lower than 10 mg/kg by intragastric administration, the toxicokinetic parameters obtained by fluorescence microplate method were similar to those with the literatures by chromatography. MEHP-AF can be rapidly absorbed through the intestinal mucosal barrier in rats. In situ organ distribution in mice showed that MEHP-AF was mainly concentrated in the liver, kidney and testis. Our results suggested that the fluorescence tracing technique had the advantages with easy processing, less time-consuming, higher sensitivity for the quantitative determination, In addition, this technology also avoids the interference of exogenous or endogenous DEHP and MEHP in the experimental system. It also can be utilized to the visualization detection of MEHP in situ localization in the absorption organ and the toxic target organ. The results show that this may be a more feasible MEHP toxicological research method.

Transcriptome Analysis Reveals the Senescence Process Controlling the Flower Opening and Closure Rhythm in the Waterlilies (Nymphaea L.).

Most waterlily flowers open at dawn and close after noon usually for three to four days, and thereafter wilt. The short lifespan of flowers restricts the development of the flower postharvest industry. The termination of flower movements is a key event during flower aging process. However, it is still unclear when the senescence process initiates and how it terminates the movement rhythm. In this study, we observed that the opening diameter of flowers was the smallest on the fourth (last) flowering day. Subsequent transcriptome profiles generated from petals at different flowering stages showed that the multiple signaling pathways were activated at the last closure stage (Time 3, T3) of the flowers, including Ca2+, reactive oxygen species and far red light signaling pathways, as well as auxin, ethylene and jasmonic acid signaling pathways. Moreover, In terms of cell metabolism regulation, the genes related to hydrolase (protease, phospholipase, nuclease) were upregulated at T3 stage, indicating that petals entered the senescence stage at that time; and the genes related to water transport and cell wall modification were also differentially regulated at T3 stage, which would affect the ability of cell expand and contract, and eventually lead to petal not open after the fourth day. Collectively, our data provided a new insight into the termination of flower opening in the waterlilies, and a global understanding of the senescence process of those opening-closure rhythm flowers.

In Situ Fluorescence Tracking Toxic Metabolite Mono-2-ethylhexyl phthalate (MEHP) of Di-(2-ethylhexyl) phthalate (DEHP) in HeLa Cells.

In this study, we synthesized a small molecule fluorescent probe for detecting mono-2-ethylhexyl phthalate (MEHP) named MEHP-AF, which formed by MEHP cross-linked with 5-aminofluorescein (5-AF) through amide bond. MEHP-AF had been purified based on the different physicochemical properties of 5-AF with MEHP. MEHP-AF showed fluorescence characteristics coming from 5-AF and liposoluble property coming from MEHP. After physicochemical characterization, a series of biological studies of its action in cells were carried out. The results indicated that MEHP-AF was a fluorescent probe with strong specificity and high sensitivity. It can visibly track the location of MEHP in HeLa cell or subcellular levels under confocal laser scanning microscopy in situ. This novel fluorescent probe is expected to use for studying its intracellular behavior at the cell level, especially for investigating the interaction between MEHP and cellular molecules.

Co-delivery nanoparticles of doxorubicin and chloroquine for improving the anti-cancer effect in vitro.

To increase the efficacy of small molecule chemotherapeutic drug (SMCD) and reduce its toxic and side effects, we selected two model drugs doxorubicin (DOX) and chloroquine (CQ). DOX is a SMCD and CQis a chemosensitizer with autophagy inhibition. Poly(lactic-co-glycolic acid) (PLGA) and alpha-tocopherol polyethylene glycol 1000 succinate were chosen as delivery carriers to design and prepare a novel type of drug co-delivery single-nanoparticles by emulsification-solvent volatilisation, named NPDOX+CQ. The physicochemical properties of NPDOX+CQ were characterised. Then A549 cells and A549/Taxol cells were used for the in vitro anti-cancer effect study. At the same time, cellular uptake, intracellular migration and anti-cancer mechanism of nanoparticles were studied. The NPs showed a uniform spherical shape with good dispersibility, and both drugs had good encapsulation efficiency and loading capacity. In all formulations, NPDOX+CQ showed the highest in vitro cytotoxicity. The results showed that NPs could protect drugs from being recognised and excluded by P-glycoprotein (P-gp). Moreover, the results of the mechanistic study demonstrated that NPs were transported by autophagy process after being taken up by the cells. Therefore, during the migration of NPDOX+CQ, CQ could exert its efficacy and block autophagy so that DOX would not be hit by autophagy. Western Blot results showed that NPDOX+CQ had the best inhibition effect of autophagy. It can be concluded that the system can prevent the drug from being recognised and excluded by P-gp, and CQ blocks the process of autophagy so that the DOX is protected and more distributed to the nucleus of multidrug resistance (MDR) cell. The NPDOX+CQ constructed in this study provides a feasible strategy for reversing MDR in tumour cells.

Two New Cytotoxic Steroidal Alkaloids from Sarcococca Hookeriana.

Two new steroidal alkaloids, named hookerianine A (1) and hookerianine B (2) were isolated from the stems and roots of Sarcococca hookeriana Baill., along with two known compounds, sarcorucinine G (3) and epipachysamine D (4). On the basis of spectroscopic methods and by comparison with literature data, their structures were determined. As well as X-ray crystallography was performed to confirm compound 4. To identify novel antitumor inhibitors, all compounds were performed a CCK-8 assay against five human cancer cell lines SW480, SMMC-7721, PC3, MCF-7 and K562 in vitro. Compound 2 exhibited moderate cytotoxic activities to all cell lines with IC50 values in the range of 5.97⁻19.44 µM. Compound 3 was the most effective one against SW480 and K562 cell lines with IC50 values of 5.77 and 6.29 µM, respectively.

Three New Cytotoxic Steroidal Alkaloids from Sarcococca hookeriana.

Three new steroidal alkaloids with an unusual 3α tigloylamide group, named sarchookloides A⁻C (1⁻3), were isolated along with four known compounds (4⁻7) from the roots of Sarcococca hookeriana. Their structures and relative configuration were elucidated on the basis of spectroscopic methods including MS, UV, IR, 1D, and 2D NMR data. The isolated compounds were evaluated for their cytotoxicity against five human cancer cell lines: Hela, A549, MCF-7, SW480, and CEM in vitro. All three amide substituted steroidal alkaloids exhibited significant cytotoxic activities with IC50 values of 1.05⁻31.83 µM.