Functional divergence of FTL9 and FTL10 in flowering control in rice.

BACKGROUND: Floral transition in cereals is a critical phenomenon influenced by exogenous and endogenous signals, determining crop yield and reproduction. Flowering Locus T-like (FT-like) genes encode a mobile florigen, the main signaling molecule for flowering. RESULTS: In this study, we characterized two FT-like genes, FTL9 and FTL10, to study their functional diversity in flowering control in rice. We compared independent mutant lines of ftl10 with WT and observed negligible differences in the flowering phenotype, or agronomic traits implying potentially redundant roles of FTL10 loss-of-function in flowering control in rice. Nevertheless, we found that overexpression of FTL10, but not FTL9, substantially accelerated flowering, indicating the flowering-promoting role of FTL10 and the divergent functions between FTL9 and FTL10 in flowering. Besides flowering, additive agronomic roles were observed for FTL10-OE regulating the number of effective panicles per plant, the number of primary branches per panicle, and spikelets per panicle without regulating seed size. Mechanistically, our Y2H and BiFC analyses demonstrate that FTL10, in contrast to FTL9, can interact with FD1 and GF14c, forming a flowering activation complex and thereby regulating flowering. CONCLUSION: Altogether, our results elucidate the regulatory roles of FTL9 and FTL10 in flowering control, unveiling the molecular basis of functional divergence between FTL10 and FTL9, which provides mechanistic insights into shaping the dynamics of flowering time regulation in rice.

Non-targeted GC-MS metabolomics-based differences in Indica rice seeds of different varieties.

Rice seeds of different varieties exhibited distinct metabolic profiles in our study. We analyzed the metabolites in seeds of six rice varieties (CH, HM, NX, YX, HY, and MX) using non-targeted GC-MS. Our findings revealed that amino acids, sugars, and organic acids were predominant in all varieties, with significant differences observed in CH compared to the others. Specifically phenylalanine and glycine content differed notably in NX and YX, respectively. Additionally, 1,5-anhydroglucitol content in NX, and glutamate, aspartate, and lactulose in NX, YX, HM, HY, and MX were up-regulated. Due to the biological functions of these amino acids and sugars, these indicated that compared to CH, rice of NX were more conducive to metabolism of carbohydrate and fat, and healthy growth maintenance in the human body, but mightThese variations suggest that NX rice may be more beneficial for carbohydrate and fat metabolism and overall health maintenance compared to CH. However, it may not be suitable for diabetic patients. YX rice may not be an ideal glycine supplement, rice ofwhile HM, HY, and MX rice could serve as potential lactulose sources. Furthermore, NX and YX rice exhibited higher levels of main storage proteins compared to CH. This study offers valuable insights into the metabolic differences among various rice varieties.

Comparative assessment on ammonia nitrogen adsorption onto a saline soil-groundwater environment: distribution, multi-factor interaction, and optimization using response surface methodology and artificial neural network.

The adsorption of soil can reduce the leaching of NH4+-N from the external environment into groundwater. The adsorption of NH4+-N is affected by many factors. It is critical to use statistical model to quantitatively describe the effects of interaction between two or more factors on the system response. In this study, HJ-Biplot was used to analyze the correlation characteristics of soil water, salt, and nitrogen, and the response surface methodology and artificial neural network were used to statistically visualize the interaction between factors, including concentration, total dissolved solids (TDS), temperature, and pH. The results showed that the study soil was a typical saline soil, with maximum soil NH4+-N content of 85.45 mg/kg. For the adsorption experiments of NH4+-N on saline soils, the effects of factors on the adsorption capacity were assessed using the RSM model. The RSM model was coupled with an ANN to predict the adsorption of NH4+-N by saline soils. The NH4+-N concentration and water pH were both significant at a linear level (p < 0.0001). The interaction between NH4+-N concentration and pH was also more significant (p < 0.01). Under optimal conditions (concentration: 800 mg/L; temperature: 24 °C; TDS: 637 mg/L; pH: 7.83), the NH4+-N adsorption capacity was 1650.2 ug/g, which was in general agreement with the calculated values from the Box-Behnken and RSM model. In addition, a statistical error criterion for the model showed that the RSM-ANN model had greater predictive ability than RSM model.

A base substitution in OsphyC disturbs its Interaction with OsphyB and affects flowering time and chlorophyll synthesis in rice.

BACKGROUND: Phytochromes are important photoreceptors in plants, and play essential roles in photomorphogenesis. The functions of PhyA and PhyB in plants have been fully analyzed, while those of PhyC in plant are not well understood. RESULTS: A rice mutant, late heading date 3 (lhd3), was characterized, and the gene LHD3 was identified with a map-based cloning strategy. LHD3 encodes phytochrome C in rice. Animo acid substitution in OsphyC disrupted its interaction with OsphyB or itself, restraining functional forms of homodimer or heterodimer formation. Compared with wild-type plants, the lhd3 mutant exhibited delayed flowering under both LD (long-day) and SD (short-day) conditions, and delayed flowering time was positively associated with the day length via the Ehd1 pathway. In addition, lhd3 showed a pale-green-leaf phenotype and a slower chlorophyll synthesis rate during the greening process. The transcription patterns of many key genes involved in photoperiod-mediated flowering and chlorophyll synthesis were altered in lhd3. CONCLUSION: The dimerization of OsPhyC is important for its functions in the regulation of chlorophyll synthesis and heading. Our findings will facilitate efforts to further elucidate the function and mechanism of OsphyC and during light signal transduction in rice.

Adsorption of benzene on soils under different influential factors: an experimental investigation, importance order and prediction using artificial neural network.

The adsorption of benzene on soils is specifically associated with its migration and transformation. Although previous studies have proved that the adsorption of benzene is affected by various factors, studies simultaneously considering the effects of multiple factors are rare. This study aimed to identify the qualitative and quantitative relationships between multiple influential factors and the adsorption capacity of benzene (BC). Batch adsorption experiments considering different influential factors, including initial concentration (IC), pH, temperature (T), ion strength (IS) and organic matter content (OMC), were conducted in three kinds of soils collected in a chemical industry park. The correlation analysis between different influential factors and BC was carried out based on the experimental data. The artificial neural network (ANN) was applied to predict BC. The results showed that BC increased with the increase of T. As the pH increased, BCs on silty loam and loam increased, while that on sandy loam decreased. Besides, BCs on silty loam and loam raised with increasing OMC, while that on sandy loam remained unchanged. BCs on all three kinds of soils attained their peaks when IS was small and then become stable with an increase in IS. The sequence of correlation between BC and influential factors is listed as IC > OMC > T > IS > pH for silty loam, OMC > IC > T > IS > pH for loam and IC > T > IS > pH > OMC for sandy loam. ANN analysis showed satisfactory accuracy in predicting BC under different influential factors. These results help us understand the important factors affecting benzene adsorption and provide a tool to get the adsorption information easily in complex site conditions.

Identification of cold tolerance QTLs at the bud burst stage in 211 rice landraces by GWAS.

BACKGROUND: Rice is a crop that is very sensitive to low temperature, and its morphological development and production are greatly affected by low temperature. Therefore, understanding the genetic basis of cold tolerance in rice is of great significance for mining favorable genes and cultivating excellent rice varieties. However, there have been limited studies focusing on cold tolerance at the bud burst stage; therefore, considerable attention should be given to the genetic basis of cold tolerance at this stage. RESULTS: In this study, a natural population consisting of 211 rice landraces collected from 15 provinces in China and other countries was used for the first time to evaluate cold tolerance at the bud burst stage. Population structure analysis showed that this population was divided into two groups and was rich in genetic diversity. Our evaluation results confirmed that japonica rice was more tolerant to cold at the bud burst stage than indica rice. A genome-wide association study (GWAS) was performed with the phenotypic data of 211 rice landraces and a 36,727 SNP dataset under a mixed linear model. Twelve QTLs (P < 0.0001) were identified for the seedling survival rate (SR) after treatment at 4 °C, in which there were five QTLs (qSR2-2, qSR3-1, qSR3-2, qSR3-3 and qSR9) that were colocalized with those from previous studies and seven QTLs (qSR2-1, qSR3-4, qSR3-5, qSR3-6, qSR3-7, qSR4 and qSR7) that were reported for the first time. Among these QTLs, qSR9, harboring the most significant SNP, explained the most phenotypic variation. Through bioinformatics analysis, five genes (LOC_Os09g12440, LOC_Os09g12470, LOC_Os09g12520, LOC_Os09g12580 and LOC_Os09g12720) were identified as candidates for qSR9. CONCLUSION: This natural population consisting of 211 rice landraces combined with high-density SNPs will serve as a better choice for identifying rice QTLs/genes in the future, and the detected QTLs associated with cold tolerance at the bud burst stage in rice will be conducive to further mining favorable genes and breeding rice varieties under cold stress.

Health risk assessment of groundwater nitrogen pollution in Songnen Plain.

Access to safe drinking water is one of the fundamental human rights and an important part of healthy living. This study considered various land use methods, used geostatistical analysis, and triangular random model to explore nitrogen pollution and estimate its potential risk to human health for local populations in Songnen Plain of Northeast China and recognize parameter uncertainties. Nitrate concentrations in groundwater ranged from 0.01 to 523.45 mg/L, more than 72.35% of the samples exceeded Grade III threshold (20 mg/L of N) as per China's standard, and nitrate nitrogen content is greater than 20 mg/L accounted for around 60% of the research area, mainly distributed in the eastern and central high plain area. The nitrate-nitrogen content of groundwater in the town land was significantly higher than that of agricultural land, and the ammonia nitrogen content was conversely. The townland's risk value was two times that of agricultural land, considering different land use methods would avoid overestimating or underestimating regional risk value. Non-carcinogenic risks (HI) of two land use were above the safety level (i.e., HI > 1), suggesting that groundwater nitrate would have significant health effects on the age groups, and further threaten children. There was a wide range of fluctuations in the uncertainty of nitrogen concentration and model evaluation parameters; triangular random model was more sensitive to data changes, which could reduce the uncertainty. The contribution rate of nitrate-nitrogen concentration to risk was above 90%, which explained the need for random sampling to improve the evaluation results reliability. The findings in this paper will provide new insight for solving uncertainties in water safety management.

Combined proteomics, metabolomics and physiological analyses of rice growth and grain yield with heavy nitrogen application before and after drought.

BACKGROUND: Nitrogen application can effectively mitigate the damage to crop growth and yield caused by drought. However, the efficiency of heavy nitrogen application before drought (NBD) and heavy nitrogen application after drought (NAD) to regulate rice response to drought stress remains controversial. In this study, we profiled physiology, proteomics and metabolomics in rice variety Wufengyou 286 of two nitrogen management modes (NBD and NAD) to investigate their yield formation and the mechanism of nitrogen regulation for drought resistance. RESULTS: Results revealed that the yield of NBD and NAD decreased significantly when it was subjected to drought stress at the stage of young panicle differentiation, while the yield of NBD was 33.85 and 36.33% higher than that of NAD in 2017 and 2018, reaching significant levels. Under drought conditions, NBD increased chlorophyll content and net photosynthetic rate in leaves, significantly improved the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase and catalase, and decreased malondialdehyde (MDA) content compared with NAD. NBD promoted nitrogen assimilation in leaves, which was characterized by increased activities of nitrate reductase (NR) and glutamine synthetase (GS). In addition, NBD significantly increased the contents of osmotic regulatory substances such as soluble sugar, soluble protein and free proline. Gene ontology and KEGG enrichment analysis of 234 differentially expressed proteins and 518 differential metabolites showed that different nitrogen management induced strong changes in photosynthesis pathway, energy metabolism pathway, nitrogen metabolism and oxidation-reduction pathways. CONCLUSION: Different nitrogen management methods have significant differences in drought resistance of rice. These results suggest that heavy nitrogen application before drought may be an important pathway to improve the yield and stress resistance of rice, and provide a new ecological perspective on nitrogen regulation in rice.

Genetic mapping and identification of new major loci for tolerance to low phosphorus stress in rice.

Phosphorus (P) is one of the essential macronutrients for rice. In this study, we used 120 rice backcross recombinant inbred lines (BRILs) derived from a cross indica cv. Changhui 891 and japonica cv. 02428. To elucidate the genetic control of P deficiency tolerance in rice, we have used high quality SNPs bin markers to identify some important loci underlying phosphorus deficiency. The bin map was generated which includes 3057 bins covering distance of 1266.5 cM with an average of 0.41 cM between markers. Based on this map, 50 loci, including four novel loci, qSL-3, qRL-11, qSDW-1, qRDW-1 with phenotypic variance 23.26%, 12.06%, 9.89% associated with P deficiency-related seedling traits were identified. No significant QTLs was found for root length under P+, shoot fresh weight P- and root length, shoot fresh weight for P+, P- and their ratio respectively. Root fresh weight, and root dry weight were strongly correlated to each other, and QTLs for these variables were located on the same chromosome 1 at the same region. Notably, 3 pleiotropic regions is the pioneer of our study, and these regions would facilitate map-based cloning to expedite the MAS selection for developing low phosphorous tolerant varieties. This study not only improves our knowledge about molecular processes associated with P deficiency, but also provides useful information to understand the genetic architecture of low phosphorous tolerance.

Pesticide application has little influence on coding and non-coding gene expressions in rice.

BACKGROUND: Agricultural insects are one of the major threats to crop yield. It is a known fact that pesticide application is an extensive approach to eliminate insect pests, and has severe adverse effects on environment and ecosystem; however, there is lack of knowledge whether it could influence the physiology and metabolic processes in plants. RESULTS: Here, we systemically analyzed the transcriptomic changes in rice after a spray of two commercial pesticides, Abamectin (ABM) and Thiamethoxam (TXM). We found only a limited number of genes (0.91%) and (1.24%) were altered by ABM and TXM respectively, indicating that these pesticides cannot dramatically affect the performance of rice. Nevertheless, we characterized 1140 Differentially Expressed Genes (DEGs) interacting with 105 long non-coding RNAs (lncRNAs) that can be impacted by the two pesticides, suggesting their certain involvement in response to farm chemicals. Moreover, we detected 274 alternative splicing (AS) alterations accompanied by host genes expressions, elucidating a potential role of AS in control of gene transcription during insecticide spraying. Finally, we identified 488 transposons that were significantly changed with pesticides treatment, leading to a variation in adjacent coding or non-coding transcripts. CONCLUSION: Altogether, our results provide valuable insights into pest management through appropriate timing and balanced mixture, these pesticides have no harmful effects on crop physiology over sustainable application of field drugs.

Comprehensive metabolomic, proteomic and physiological analyses of grain yield reduction in rice under abrupt drought-flood alternation stress.

Abrupt drought-flood alternation (T1) is a meteorological disaster that frequently occurs during summer in southern China and the Yangtze river basin, often causing a significant loss of rice production. In this study, the response mechanism of yield decline under abrupt drought-flood alternation stress at the panicle differentiation stage was analyzed by looking at the metabolome, proteome as well as yield and physiological and biochemical indexes. The results showed that drought and flood stress caused a decrease in the yield of rice at the panicle differentiation stage, and abrupt drought-flood alternation stress created a synergistic effect for the reduction of yield. The main reason for the decrease of yield per plant under abrupt drought-flood alternation was the decrease of seed setting rate. Compared with CK0 (no drought and no flood), the net photosynthetic rate and soluble sugar content of T1 decreased significantly and its hydrogen peroxidase, superoxide dismutase, peroxidase activity increased significantly. The identified differential metabolites and differentially expressed proteins indicated that photosynthesis metabolism, energy metabolism pathway and reactive oxygen species response have changed strongly under abrupt drought-flood alteration stress, which are factors that leads to the rice grain yield reduction.

Genome-wide analysis of long non-coding RNAs affecting roots development at an early stage in the rice response to cadmium stress.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been found to play a vital role in several gene regulatory networks involved in the various biological processes in plants related to stress response. However, systematic analyses of lncRNAs expressed in rice Cadmium (Cd) stress are seldom studied. Thus, we presented the characterization and expression of lncRNAs in rice root development at an early stage in response to Cd stress. RESULTS: The lncRNA deep sequencing revealed differentially expressed lncRNAs among Cd stress and normal condition. In the Cd stress group, 69 lncRNAs were up-regulated and 75 lncRNAs were down-regulated. Furthermore, 386 matched lncRNA-mRNA pairs were detected for 120 differentially expressed lncRNAs and 362 differentially expressed genes in cis, and target gene-related pathway analyses exhibited significant variations in cysteine and methionine metabolism pathway-related genes. For the genes in trans, overall, 28,276 interaction relationships for 144 lncRNAs and differentially expressed protein-coding genes were detected. The pathway analyses found that secondary metabolites, such as phenylpropanoids and phenylalanine, and photosynthesis pathway-related genes were significantly altered by Cd stress. All of these results indicate that lncRNAs may regulate genes of cysteine-rich peptide metabolism in cis, as well as secondary metabolites and photosynthesis in trans, to activate various physiological and biochemical reactions to respond to excessive Cd. CONCLUSION: The present study could provide a valuable resource for lncRNA studies in response to Cd treatment in rice. It also expands our knowledge about lncRNA biological function and contributes to the annotation of the rice genome.

Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival.

Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of ß-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote ß-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF.

Hydrochemical characteristics and quality assessment of groundwater for irrigation use in central and eastern Songnen Plain, Northeast China.

The hydrochemical characteristics of groundwater in Songnen Plain's agricultural area were analyzed based on aquifer types and topography classification to evaluate irrigation suitability and factors influencing groundwater quality. Samples of different groundwater types and topographical conditions within the research area were collected and chemical indices, such as sodium adsorption ratio, %Na+, residual sodium carbonate, and magnesium hazard values, were calculated to assess the groundwater suitability for irrigation. The results indicated that groundwater was generally neutral, with low total dissolved solids and slightly high hardness; the dominant anion in groundwater was HCO3-, while Ca2+ was the relatively stable primary cation found in water samples from the high plain and river valley plain. The nitrate in groundwater significantly exceeded WHO drinking water standards, especially in the unconfined water of the high plain, which was due to the large-scale agricultural production activities in the eastern regions. The main reactions in the groundwater system were weathering and dissolution of carbonates and sulfates and ion-exchange reactions. Horizontal zoning in water chemical characteristics was prominent; from the high plain to river valley plain and low plain, the hydrochemistry gradually transitioned from HCO3-Ca-Na to HCO3-Na-Ca and HCO3-Na. Based on the chemical indices, the majority of samples were suitable for agricultural irrigation except for some in the western area with high salinity and sodium hazards. Treatment measures to groundwater and soil should be taken to reduce the possibility of soil salinization and promote crop growth in these latter regions.

Genome-wide identification and phylogenetic analysis of the chalcone synthase gene family in rice.

The enzymes of the chalcone synthase family are also known as type III polyketide synthases (PKS), and produce a series of secondary metabolites in bacteria, fungi and plants. In a number of plants, genes encoding PKS comprise a large multigene family. Currently, detailed reports on rice (Oryza sativa) PKS (OsPKS) family genes and tissue expression profiling are limited. Here, 27 candidate OsPKS genes were identified in the rice genome,and 23 gene structures were confirmed by EST and cDNA sequencing; phylogenetic analysis has indicated that these 23 OsPKS members could be clustered into three groups (I-III). Comparative analysis has shown OsPKS08 and OsPKS26 could be classified with the CHS genes of other species. Two members OsPKS10 and OsPKS21 were grouped into anther specific chalcone synthase-like (ASCL) clade. Intron/exon structure analysis revealed that nearly all of the OsPKS members contained one phase-1 intron at a conserved Cys. Analysis of chromosomal localization and genome distribution showed that some of the members were distributed on a chromosome as a cluster. Expression data exhibited widespread distribution of the rice OsPKS gene family within plant tissues, suggesting functional diversification of the OsPKS genes. Our results will contribute to future study of the complexity of the OsPKS gene family in rice.

Inhibition of perillyl alcohol on cell invasion and migration depends on the Notch signaling pathway in hepatoma cells.

Cell metastasis, especially the process of invasion and migration, is considered as the main cause for the high mortality rate of hepatocellular carcinoma (HCC), which has become the sixth most common cancer worldwide and the third leading cause of cancer death. In this present study, we aimed to exploit the effects of perillyl alcohol on cell invasion and migration and the underlying molecular mechanisms in HCC. According to the transwell assays, cell invasiveness and migratory capacity were markedly higher in hepatoma cells (HepG2, SMMC-7721 and MHCC97H) than those in normal liver cells (HL-7702), and then significantly suppressed by perillyl alcohol treatment (P < 0.05). Meanwhile, the mRNA levels of Notch signaling pathway downstream target genes, HES1, HES5, and HEY1, were notably higher in hepatoma cells detected with real-time reverse transcription polymerase chain reaction (RT-PCR) (P < 0.05). After treated with perillyl alcohol, these mRNA levels were significantly decreased in hepatoma cells (P < 0.05). In addition, compared with the normal liver cells, the protein expression levels of Notch1 intracellular domain (N1ICD) and Snail were significantly increased, while E-cadherin protein expression was significantly decreased in hepatoma cells (P < 0.05). However, perillyl alcohol treatment significantly decreased N1ICD and Snail protein expressions and increased E-cadherin protein expression in hepatoma cells (P < 0.05). In conclusion, perillyl alcohol might play an important role in the process of hepatoma cell invasion and migration via decreasing the activity of Notch signaling pathway and increasing E-cadherin expression regulated by Snail.

RNA-seq reveals differentially expressed genes of rice (Oryza sativa) spikelet in response to temperature interacting with nitrogen at meiosis stage.

BACKGROUND: Rice (Oryza sativa) is one of the most important cereal crops, providing food for more than half of the world's population. However, grain yields are challenged by various abiotic stresses such as drought, fertilizer, heat, and their interaction. Rice at reproductive stage is much more sensitive to environmental temperatures, and little is known about molecular mechanisms of rice spikelet in response to high temperature interacting with nitrogen (N). RESULTS: Here we reported the transcriptional profiling analysis of rice spikelet at meiosis stage using RNA sequencing (RNA-seq) as an attempt to gain insights into molecular events associated with temperature and nitrogen. This study received four treatments: 1) NN: normal nitrogen level (165 kg ha(-1)) with natural temperature (30 °C); 2) HH: high nitrogen level (330 kg ha(-1)) with high temperature (37 °C); 3) NH: normal nitrogen level and high temperature; and 4) HN: high nitrogen level and natural temperature, respectively. The de novo assembly generated 52,553,536 clean reads aligned with 72,667 unigenes. About 10 M reads were identified from each treatment. In these differentially expressed genes (DEGs), we found 151 and 323 temperature-responsive DEGs in NN-vs-NH and HN-vs-HH, and 114 DEGs were co-expressed. Meanwhile, 203 and 144 nitrogen-responsive DEGs were focused in NN-vs-HN and NH-vs-HH, and 111 DEGs were co-expressed. The temperature-responsive genes were principally associated with calcium-dependent protein, cytochrome, flavonoid, heat shock protein, peroxidase, ubiquitin, and transcription factor while the nitrogen-responsive genes were mainly involved in glutamine synthetase, transcription factor, anthocyanin, amino acid transporter, leucine zipper protein, and hormone. It is noted that, rice spikelet fertility was significantly decreased under high temperature, but it was more reduced under higher nitrogen. Accordingly, numerous spikelet genes involved in pollen development, pollen tube growth, pollen germination, especially sporopollenin biosynthetic process, and pollen exine formation were mainly down-regulated under high temperature. Moreover, the expression levels of co-expressed DEGs including 5 sporopollenin biosynthetic process and 7 pollen exine formation genes of NN-vs-NH were lower than that of HN-vs-HH. Therefore, these spikelet genes may play important roles in response to high temperature with high nitrogen and may be good candidates for crop improvement. CONCLUSIONS: This RNA-seq study will help elucidate the molecular mechanisms of rice spikelet defense response to high temperature interacting with high nitrogen level.

[Cryopreservation of microencapsulated human hepatocytes].

OBJECTIVE: To establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for therapeutic usage in hepatocyte transplantation and bioartificial liver support systems for the treatment of acute and chronic liver diseases,and for experimental usage as an in vitro model of the liver. METHODS: Adult hepatocytes from 20 human donors undergoing partial hepatectomy were isolated using a two-step extracoporeal collagenase perfusion technique.Seven preincubation time points (2h,6h,12h,24h,36h,48h and 72h) were selected for optimization.After pre-incubation at 4 degrees C for 12-24h in HepatoZYME-SFM (the optimal condition),hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules,transferred to a complete medium containing 10% dimethyl sulphoxide and immediately placed into an isopropanol progressive freezing container for overnight freezing at -80 degrees C followed by immersion in liquid nitrogen the next day.During the post-thawing culture period,the cells were tested for albumin secretion,urea synthesis,cell cycling,transcription and protein synthesis (measuring mRNA and protein levels),and the morphological structure and pathology,for comparison with the features from before microencapsulated cryopreservation (PMC). RESULTS: The viability and plating efficiency of the hepatocytes isolated using the two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0%,respectively.The pre-incubation times of 12h and 24h (viability:61.4+/-4.8% and 62.0+/-5.6%; plating efficiency:3.2+/-5.8% and 62.6+/-3.6%,respectively) showed significantly higher albumin secretion than all other time points tested (F =40.3,all P less than 0.05).Compared with the immediate cryopreservation (immediately frozen control) hepatocytes,the PMC hepatocytes showed significantly better transcription and protein synthesis and higher albumin secretion and urea levels.The PMC group did not show a significantly different level of albumin production from the directly cultured hepatocytes (culture day 2:ll9.2ng/ml vs.131.36ng/ml,P =0.051; day 3:110ng/ml vs.120.4ng/ml,P=0.063; day 4:98.2ng/ml vs.109.8ng/ml,P more than 0.05).However,over culturing days 2,3 and 4,comparison of the PMC hepatocytes to the immediate cryopreservation hepatoeytes showed the former to have significantly higher secretion of albumin (119.2ng/ml vs.101.2ng/ml,110.0ng/ml vs.87.6ng/ml and 98.2ng/ml vs.73.8ng/ml; all P less than 0.05) and urea level (7.83 mug/ml vs.6.79 mug/ml,6.83 mug/ml vs.5.89 mug/ml and 5.85 mug/ml vs.4.83 mug/ml; all P less than 0.05).The post-thawed PMC hepatoeytes showed preservation of the morphological structure,while the immediate cryopreservation hepatocytes did not. CONCLUSION: The two-step extracorporeal collagenase perfusion technique after partial hepatectomy is a novel,simple,and reliable method for hepatocyte isolation.Pre-incubation at 4 degrees C for 12-24h before the microencapsulation cryopreservation allows for efficient recovery of functional and morphological integrity after thawing and provides viable hepatoeytes that may be useful for clinical applications in pharmacotoxicology,bioartificial liver therapy and cell therapy in humans.

Uncovering Novel QTLs and Candidate Genes for Salt Tolerance at the Bud Burst Stage in Rice through Genome-Wide Association Study.

Salt stress is one of the most important factors limiting rice growth and yield increase. Salt tolerance of rice at the bud burst (STB) stage determines whether germinated seeds can grow normally under salt stress, which is very important for direct seeding. However, reports on quantitative trait loci (QTLs) and candidate genes for STB in rice are very limited. In this study, a natural population of 130 indica and 81 japonica rice accessions was used to identify STB-related QTLs and candidate genes using a genome-wide association study (GWAS). Nine QTLs, including five for relative shoot length (RSL), two for relative root length (RRL), and two for relative root number (RRN), were identified. Five of these STB-related QTLs are located at the same site as the characterized salt tolerance genes, such as OsMDH1, OsSRFP1, and OsCDPK7. However, an important QTL related to RSL, qRSL1-2, has not been previously identified and was detected on chromosome 1. The candidate region for qRSL1-2 was identified by linkage disequilibrium analysis, 18 genes were found to have altered expression levels under salt stress through the RNA-seq database, and 10 of them were found to be highly expressed in the shoot. It was also found that, eight candidate genes (LOC_Os01g62980, LOC_Os01g63190, LOC_Os01g63230, LOC_Os01g63280, LOC_Os01g63400, LOC_Os01g63460, and LOC_Os01g63580) for qRSL1-2 carry different haplotypes between indica and japonica rice, which exactly corresponds to the significant difference in RSL values between indica and japonica rice in this study. Most of the accessions with elite haplotypes were indica rice, which had higher RSL values. These genes with indica-japonica specific haplotypes were identified as candidate genes. Rice accessions with elite haplotypes could be used as important resources for direct seeding. This study also provides new insights into the genetic mechanism of STB.

CIP2A expression is associated with altered expression of epithelial-mesenchymal transition markers and predictive of poor prognosis in pancreatic ductal adenocarcinoma.

CIP2A has been regarded as a novel potential therapeutic target for multiple cancers. The aim of this study was to detect CIP2A expression in pancreatic ductal adenocarcinoma (PDA) and to analyze its association with prognosis of PDA patients. The expression of CIP2A and three epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin, and vimentin) was examined in 96 PDA tissue samples by immunohistochemistry. Fifty-four cases (56.3 %) were defined as positive for CIP2A expression. Immunohistochemistry showed that CIP2A expression was correlated with poor tumor differentiation, TNM stage, and lymph node metastasis. Kaplan-Meier survival analysis showed that patients with CIP2A-positive expression showed lower overall survival rate than those with CIP2A-negative expression. Multivariate analysis showed that CIP2A expression was an independent prognostic factor for PDA patients. Furthermore, positive expression of CIP2A was strongly associated with loss of the epithelial marker E-cadherin and acquisition of the expression of the mesenchymal markers N-cadherin and vimentin. These findings suggest that CIP2A might promote EMT and progression in PDA, and thus may be a potential therapeutic target for patients with PDA.