Genome-Wide Differential DNA Methylation and miRNA Expression Profiling Reveals Epigenetic Regulatory Mechanisms Underlying Nitrogen-Limitation-Triggered Adaptation and Use Efficiency Enhancement in Allotetraploid Rapeseed.

Improving crop nitrogen (N) limitation adaptation (NLA) is a core approach to enhance N use efficiency (NUE) and reduce N fertilizer application. Rapeseed has a high demand for N nutrients for optimal plant growth and seed production, but it exhibits low NUE. Epigenetic modification, such as DNA methylation and modification from small RNAs, is key to plant adaptive responses to various stresses. However, epigenetic regulatory mechanisms underlying NLA and NUE remain elusive in allotetraploid B. napus. In this study, we identified overaccumulated carbohydrate, and improved primary and lateral roots in rapeseed plants under N limitation, which resulted in decreased plant nitrate concentrations, enhanced root-to-shoot N translocation, and increased NUE. Transcriptomics and RT-qPCR assays revealed that N limitation induced the expression of NRT1.1, NRT1.5, NRT1.7, NRT2.1/NAR2.1, and Gln1;1, and repressed the transcriptional levels of CLCa, NRT1.8, and NIA1. High-resolution whole genome bisulfite sequencing characterized 5094 differentially methylated genes involving ubiquitin-mediated proteolysis, N recycling, and phytohormone metabolism under N limitation. Hypermethylation/hypomethylation in promoter regions or gene bodies of some key N-metabolism genes might be involved in their transcriptional regulation by N limitation. Genome-wide miRNA sequencing identified 224 N limitation-responsive differentially expressed miRNAs regulating leaf development, amino acid metabolism, and plant hormone signal transduction. Furthermore, degradome sequencing and RT-qPCR assays revealed the miR827-NLA pathway regulating limited N-induced leaf senescence as well as the miR171-SCL6 and miR160-ARF17 pathways regulating root growth under N deficiency. Our study provides a comprehensive insight into the epigenetic regulatory mechanisms underlying rapeseed NLA, and it will be helpful for genetic engineering of NUE in crop species through epigenetic modification of some N metabolism-associated genes.

Integrated Transcriptional and Proteomic Profiling Reveals Potential Amino Acid Transporters Targeted by Nitrogen Limitation Adaptation.

Nitrogen (N) is essential for plant growth and crop productivity. Organic N is a major form of remobilized N in plants' response to N limitation. It is necessary to understand the regulatory role of N limitation adaption (NLA) in organic N remobilization for this adaptive response. Transcriptional and proteomic analyses were integrated to investigate differential responses of wild-type (WT) and nla mutant plants to N limitation and to identify the core organic N transporters targeted by NLA. Under N limitation, the nla mutant presented an early senescence with faster chlorophyll loss and less anthocyanin accumulation than the WT, and more N was transported out of the aging leaves in the form of amino acids. High-throughput transcriptomic and proteomic analyses revealed that N limitation repressed genes involved in photosynthesis and protein synthesis, and promoted proteolysis; these changes were higher in the nla mutant than in the WT. Both transcriptional and proteomic profiling demonstrated that LHT1, responsible for amino acid remobilization, were only significantly upregulated in the nla mutant under N limitation. These findings indicate that NLA might target LHT1 and regulate organic N remobilization, thereby improving our understanding of the regulatory role of NLA on N remobilization under N limitation.

A multiomics approach reveals the pivotal role of subcellular reallocation in determining rapeseed resistance to cadmium toxicity.

Oilseed rape (Brassica napus) has great potential for phytoremediation of cadmium (Cd)-polluted soils due to its large plant biomass production and strong metal accumulation. Enhanced plant Cd resistance (PCR) is a crucial prerequisite for phytoremediation through hyper-accumulation of excess Cd. However, the complexity of the allotetraploid genome of rapeseed hinders our understanding of PCR. To explore rapeseed Cd-resistance mechanisms, we examined two genotypes, 'ZS11' (Cd-resistant) and 'W10' (Cd-sensitive), that exhibit contrasting PCR while having similar tissue Cd concentrations, and characterized their different fingerprints in terms of plant morphophysiology (electron microscopy), ion abundance (inductively coupled plasma mass spectrometry), DNA variation (whole-genome resequencing), transcriptomics (high-throughput mRNA sequencing), and metabolomics (ultra-high performance liquid chromatography-mass spectrometry). Fine isolation of cell components combined with ionomics revealed that more Cd accumulated in the shoot vacuoles and root pectins of the resistant genotype than in the sensitive one. Genome and transcriptome sequencing identified numerous DNA variants and differentially expressed genes involved in pectin modification, ion binding, and compartmentalization. Transcriptomics-assisted gene co-expression networks characterized BnaCn.ABCC3 and BnaA8.PME3 as the central members involved in the determination of rapeseed PCR. High-resolution metabolic profiles revealed greater accumulation of shoot Cd chelates, and stronger biosynthesis and higher demethylation of root pectins in the resistant genotype than in the sensitive one. Our comprehensive examination using a multiomics approach has greatly improved our understanding of the role of subcellular reallocation of Cd in the determination of PCR.

Balance between nitrogen use efficiency and cadmium tolerance in Brassica napus and Arabidopsis thaliana.

The transmembrane transport of NO3- and Cd2+ into plant cell vacuoles relies on the energy from their tonoplast proton pumps, V-ATPase and V-PPase. If the activity of these pumps is reduced, it results in less NO3- and Cd2+ being transported into the vacuoles, which contributes to better nitrogen use efficiency (NUE) and lower Cd2+ tolerance in plants. The physiological mechanisms that regulate the balance between NUE and Cd2+ tolerance remain unknown. In our study, two Brassica napus genotypes with differential NUEs, xiangyou 15 and 814, and Atclca-2 mutant and AtCAX4 over-expression line (AtCAX4-OE) of Arabidopsis thaliana, were used to investigate Cd2+ stress responses. We found that the Brassica napus genotype, with higher NUE, was more sensitive to Cd2+ stress. The AtCAX4-OE mutant, with higher Cd2+ vacuolar sequestration capacity (VSC), limited NO3- sequestration into root vacuoles and promoted NUE. Atclca-2 mutants, with decreased NO3- VSC, enhanced Cd2+ sequestration into root vacuoles and conferred greater Cd2+ tolerance than the WT. This may be due to the competition between Cd2+ andNO3- in the vacuoles for the energy provided by V-ATPase and V-PPase. Regulating the balance between Cd2+ and NO3- vacuolar accumulation by inhibiting the activity of CLCa transporter and increasing the activity of CAX4 transporter will simultaneously enhance both the NUE and Cd2+ tolerance of Brassica napus, essential for improving its Cd2+ phytoremediation potential.

Integrated physiologic, genomic and transcriptomic strategies involving the adaptation of allotetraploid rapeseed to nitrogen limitation.

BACKGROUND: Nitrogen (N) is a macronutrient that is essential for optimal plant growth and seed yield. Allotetraploid rapeseed (AnAnCnCn, 2n = 4x = 38) has a higher requirement for N fertilizers whereas exhibiting a lower N use efficiency (NUE) than cereal crops. N limitation adaptation (NLA) is pivotal for enhancing crop NUE and reducing N fertilizer use in yield production. Therefore, revealing the genetic and molecular mechanisms underlying NLA is urgent for the genetic improvement of NUE in rapeseed and other crop species with complex genomes. RESULTS: In this study, we integrated physiologic, genomic and transcriptomic analyses to comprehensively characterize the adaptive strategies of oilseed rape to N limitation stresses. Under N limitations, we detected accumulated anthocyanin, reduced nitrate (NO3-) and total N concentrations, and enhanced glutamine synthetase activity in the N-starved rapeseed plants. High-throughput transcriptomics revealed that the pathways associated with N metabolism and carbon fixation were highly over-represented. The expression of the genes that were involved in efficient N uptake, translocation, remobilization and assimilation was significantly altered. Genome-wide identification and molecular characterization of the microR827-NLA1-NRT1.7 regulatory circuit indicated the crucial role of the ubiquitin-mediated post-translational pathway in the regulation of rapeseed NLA. Transcriptional analysis of the module genes revealed their significant functional divergence in response to N limitations between allotetraploid rapeseed and the model Arabidopsis. Association analysis in a rapeseed panel comprising 102 genotypes revealed that BnaC5.NLA1 expression was closely correlated with the rapeseed low-N tolerance. CONCLUSIONS: We identified the physiologic and genome-wide transcriptional responses of oilseed rape to N limitation stresses, and characterized the global members of the BnamiR827-BnaNLA1s-BnaNRT1.7s regulatory circuit. The transcriptomics-assisted gene co-expression network analysis accelerates the rapid identification of central members within large gene families of plant species with complex genomes. These findings would enhance our comprehensive understanding of the physiologic responses, genomic adaptation and transcriptomic alterations of oilseed rape to N limitations and provide central gene resources for the genetic improvement of crop NLA and NUE.

Genome-scale characterization of the vacuole nitrate transporter Chloride Channel (CLC) genes and their transcriptional responses to diverse nutrient stresses in allotetraploid rapeseed.

The Chloride Channel (CLC) gene family is reported to be involved in vacuolar nitrate (NO3-) transport. Nitrate distribution to the cytoplasm is beneficial for enhancing NO3- assimilation and plays an important role in the regulation of nitrogen (N) use efficiency (NUE). In this study, genomic information, high-throughput transcriptional profiles, and gene co-expression analysis were integrated to identify the CLCs (BnaCLCs) in Brassica napus. The decreased NO3- concentration in the clca-2 mutant up-regulated the activities of nitrate reductase and glutamine synthetase, contributing to increase N assimilation and higher NUE in Arabidopsis thaliana. The genome-wide identification of 22BnaCLC genes experienced strong purifying selection. Segmental duplication was the major driving force in the expansion of the BnaCLC gene family. The most abundant cis-acting regulatory elements in the gene promoters, including DNA-binding One Zinc Finger, W-box, MYB, and GATA-box, might be involved in the transcriptional regulation of BnaCLCs expression. High-throughput transcriptional profiles and quantitative real-time PCR results showed that BnaCLCs responded differentially to distinct NO3- regimes. Transcriptomics-assisted gene co-expression network analysis identified BnaA7.CLCa-3 as the core member of the BnaCLC family, and this gene might play a central role in vacuolar NO3- transport in crops. The BnaCLC members also showed distinct expression patterns under phosphate depletion and cadmium toxicity. Taken together, our results provide comprehensive insights into the vacuolar BnaCLCs and establish baseline information for future studies on BnaCLCs-mediated vacuolar NO3- storage and its effect on NUE.

Genomics-Assisted Identification and Characterization of the Genetic Variants Underlying Differential Nitrogen Use Efficiencies in Allotetraploid Rapeseed Genotypes.

Nitrogen (N) is a non-mineral macronutrient essential for plant growth and development. Oilseed rape (AnAnCnCn, 2n = 4x = 38) has a high requirement for N nutrients whereas showing the lowest N use efficiency (NUE) among crops. The mechanisms underlying NUE regulation in Brassica napus remain unclear because of genome complexity. In this study, we performed high-depth and -coverage whole-genome re-sequencing (WGS) of an N-efficient (higher NUE) genotype "XY15" and an N-inefficient (lower NUE) genotype "814" of rapeseed. More than 687 million 150-bp paired-end reads were generated, which provided about 93% coverage and 50× depth of the rapeseed genome. Applying stringent parameters, we identified a total of 1,449,157 single-nucleotide polymorphisms (SNPs), 335,228 InDels, 175,602 structure variations (SVs) and 86,280 copy number variations (CNVs) between the N-efficient and -inefficient genotypes. The largest proportion of various DNA polymorphisms occurred in the inter-genic regions. Unlike CNVs, the SNP/InDel and SV polymorphisms showed variation bias of the An and Cn subgenomes, respectively. Gene ontology analysis showed the genetic variants were mapped onto the genes involving N compound transport and ATPase complex metabolism, but not including N assimilation-related genes. On basis of identification of N-starvation responsive genes through high-throughput expression profiling, we also mapped these variants onto some key NUE-regulating genes, and validated their significantly differential expression between the N-efficient and -inefficient genotypes through qRT-PCR assays. Our data provide genome-wide high resolution DNA variants underlying NUE divergence in allotetraploid rapeseed genotypes, which would expedite the effective identification and functional validation of key NUE-regulating genes through genomics-assisted improvement of crop nutrient efficiency.

Effects of a controlled-release fertilizer on yield, nutrient uptake, and fertilizer usage efficiency in early ripening rapeseed (Brassica napus L.).

BACKGROUND: Nitrogen (N), phosphorous (P), and potassium (K) are critical nutrient elements necessary for crop plant growth and development. However, excessive inputs will lead to inefficient usage and cause excessive nutrient losses in the field environment, and also adversely affect the soil, water and air quality, human health, and biodiversity. METHODS: Field experiments were conducted to study the effects of controlled-release fertilizer (CRF) on seed yield, plant growth, nutrient uptake, and fertilizer usage efficiency for early ripening rapeseed (Xiangzayou 1613) in the red-yellow soil of southern China during 2011-2013. It was grown using a soluble fertilizer (SF) and the same amounts of CRF, such as SF1/CRF1 (3750 kg/hm2), SF2/CRF2 (3000 kg/hm2), SF3/CRF3 (2250 kg/hm2), SF4/CRF4 (1500 kg/hm2), SF5/CRF5 (750 kg/hm2), and also using no fertilizer (CK). RESULTS: CRF gave higher seed yields than SF in both seasons by 14.51%. CRF4 and SF3 in each group achieved maximum seed yield (2066.97 and 1844.50 kg/hm2, respectively), followed by CRF3 (1929.97 kg/hm2) and SF4 (1839.40 kg/hm2). There were no significant differences in seed yield among CK, SF1, and CRF1 (P>0.05). CRF4 had the highest profit (7126.4 CNY/hm2) and showed an increase of 12.37% in seed yield, and it decreased by 11.01% in unit fertilizer rate compared with SF4. The branch number, pod number, and dry matter weight compared with SF increased significantly under the fertilization of CRF (P<0.05). The pod number per plant was the major contributor to seed yield. On the other hand, the N, P, and K uptakes increased at first and then decreased with increasing the fertilizer rate at maturity, and the N, P, and K usage efficiency decreased with increasing the fertilizer rate. The N, P, and K uptakes and usage efficiencies of the CRF were significantly higher than those of SF (P<0.05). The N accumulation and N usage efficiency of CRF increased by an average of 13.66% and 9.74 percentage points, respectively, compared to SF. In conclusion, CRF significantly promoted the growth of rapeseed with using total N as the base fertilizer, by providing sufficient N in the later growth stages, and last by reducing the residual N in the soil and increasing the N accumulation and N usage efficiency.

Nitrogen Use Efficiency Is Mediated by Vacuolar Nitrate Sequestration Capacity in Roots of Brassica napus.

Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the environment. In Arabidopsis (Arabidopsis thaliana), allocation of more NO3 (-) to shoots was associated with higher NUE; however, the commonality of this process across plant species have not been sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE. We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE genotype (814); and consequently, less vacuolar NO3 (-) was retained in roots of Xiangyou15. Moreover, NO3 (-) concentration in xylem sap, [(15)N] shoot:root (S:R) and [NO3 (-)] S:R ratios were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity, vacuolar sequestration capacity (VSC) of NO3 (-) in roots substantially decreased. Expression of NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO3 (-) long-distance transport from roots to shoots. NUE in Arabidopsis mutants impaired in proton pumps was also significantly higher than in the wild type col-0. Taken together, these data suggest that decrease in VSC of NO3 (-) in roots will enhance transport to shoot and essentially contribute to higher NUE by promoting NO3 (-) allocation to aerial parts, likely through coordinated regulation of NRT1.5 and NRT1.8.

[Effect of atmospheric CO2 concentration and nitrogen application level on absorption and transportation of nutrient elements in oilseed rape].

Effect of elevated atmospheric-CO2 (780 µmol . mol-1) on the absorption and transportation of secondary nutrient elements (calcium, magnesium, sulphur) and micronutrient elements (iron, manganese, zinc, molybdenum and boron) in oilseed rape at the stem elongation stage were studied by greenhouse simulated method. Compared with the ambient CO2 condition, the content of Zn in stem was increased and the contents of other nutrient elements were decreased under the elevated atmospheric-CO2 with no nitrogen (N) application; the contents of Ca, S, B and Zn were increased, and the contents of Mg, Mn, Mo and Fe were decreased under the elevated atmospheric CO2 with N application (0.2 g N . kg-1 soil); except the content of Mo in leaf was increased, the contents of other nutrient elements were decreased under the elevated atmospheric-CO2 with two levels of N application. Compared with the ambient CO2 condition, the amounts of Ca and S relative to the total amount of secondary nutrient elements in stem and the amounts of B and Zn relative to the total amount of micronutrient elements in stem were increased under the elevated-CO2 treatment with both levels of N application, and the corresponding values of Mg, Fe, Mn and Mo were decreased; no-N application treatment increased the proportion of Ca distributed into the leaves, and the proportion of Mg distributed into leaves was increased by the normal-N application level; the proportions of Mn, Zn and Mo distributed into the leaves were increased at both N application levels. Without N application, the elevation of atmospheric CO2 increased the transport coefficients of SFe, Mo and SS,B, but decreased the transport coefficients of SMg,Fe, SMg, Mn and SS,Fe, indicating the proportions of Mo, S transported into the upper part of plant tissues was higher than that of Fe, and the corresponding value of B was higher than that observed for S, the corresponding value of Mg was higher than that of Fe and Mn. Under normal-N application, the elevation of atmospheric CO2 increased the transport coefficients of SMg,Fe, SMg,Mn and SS,B, but decreased the transport coefficients of SCa, Mg, SFe,Mo and SS,Fe indicating the proportions of Fe, Mn and Ca transported into the upper part of plant tissues was higher than that of Mg; the corresponding value of B was higher than that observed for S, the corresponding value of Fe was higher than that of Mo, and the corresponding value of S was higher than that of Fe.

Snail regulated by PKC/GSK-3ß pathway is crucial for EGF-induced epithelial-mesenchymal transition (EMT) of cancer cells.

Cancer metastasis is considered a major challenge in cancer therapy. Recently, epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling has been shown to induce epithelial-mesenchymal transition (EMT) and thereby to promote cancer metastasis. However, the underlying mechanism has not been fully elucidated. We demonstrate that EGF can induce EMT in human prostate and lung cancer cells and thus promote invasion and migration. EGF-induced EMT has been characterized by the cells acquiring mesenchymal spindle-like morphology and increasing their expression of N-cadherin and fibronectin, with a concomitant decrease of E-cadherin. Both protein and mRNA expression of transcription factor Snail rapidly increases after EGF treatment. The knockdown of Snail significantly attenuates EGF-induced EMT, suggesting that Snail is crucial for this process. To determine the way that Snail is accumulated, we demonstrate (1) that EGF promotes the stability of Snail via inhibiting the activity of glycogen synthase kinase 3 beta (GSK-3ß), (2) that protein kinase C (PKC) rather than the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is responsible for GSK-3ß inhibition and (3) that GSK-3ß inhibition promotes the transcription of Snail. Taken together, these results reveal that the PKC/GSK-3ß signaling pathway controls both the stability and transcription of Snail, which is crucial for EMT induced by EGF in PC-3 and A549 cells. Our study suggests a novel signaling pathway for Snail regulation and provides a better understanding of growth-factor-induced tumor EMT and metastasis.

[Efficacy of treatment with siRNA targeting Bcl-2 in combination with HCPT against transplanted H22 hepatoma in mice].

OBJECTIVE: To investigate the efficacy of treatment with siRNA targeting Bcl-2 in combination with HCPT against H22 hepatoma transplanted in mice. METHODS: siRNA targeting Bcl-2 mRNA was successfully designed and synthesized. Then, the Bcl-2 siRNA was transfected into H22 hepatoma transplanted in mice in combination with HCPT for treatment. The changes of tumor volume, body weight and survival rate were observed. Tumor tissues were processed into paraffin blocks and sections were stained with hematoxylin and eosin (HE) to investigate the morphological changes of the tumor cells. RT-polymerase chain reaction (PT-PCR) was used to assess the expression of Bcl-2 mRNA in tumors and cells. Cell cycle and apoptosis of H22 hepatoma cells transplanted in mice were further determined by flow cytometry. RESULTS: After treatment for 21 days, the tumor volume was around (571.47 ± 67.31)mm³ in the group of siRNA in combination with HCPT, which was significant smaller than that of the groups of HCPT [(880.47 ± 107.31) mm³, P < 0.05], siRNA interfere [(1119.55 ± 158.60)mm³, P < 0.01] and saline (1357.64 ± 197.92)mm³, P < 0.01]. The median survival time of the group receiving siRNA in combination with HCPT treatment was 26 days, which was significantly longer than that of the group receiving HCPT (14 day, P < 0.05), siRNA interfere (21 day, P < 0.05) and saline (12 day, P < 0.05). Larger necrotic area, lower expression of Bcl-2 mRNA, less cells at S phase and more apoptotic cells could be obviously seen in tumor tissues in the group of siRNA in combination with HCPT treatment. CONCLUSION: Bcl-2 siRNA in combination with HCPT has good synergetic antitumor efficacy in H22 hepatoma-bearing mice.

[Changes of heavy metals form during aerobic high temperature composting of pig manure and the effects of passivators].

Sequential extraction method was employed to study the heavy metals concentration and form change during aerobic high temperature compositing of pig manure, and the effects of amendment with different proportion of passivators on the concentration and form change. During the composting process, the concentrations of total As, Cu, and Zn in the manure all increased to some extent. As for the form change of the heavy metals, the exchangeable As and Zn decreased while the residual As and Zn increased, indicating that the availability of As and Zn declined through the composting process. On the other hand, the exchangeable and residual Cu decreased while the carbonate-, Fe/Mn-, and organic bound Cu increased, suggesting the potential environmental risk of the future application of the compost. Among the passivators amended, 5.0% of sepiolite and 2.5% of bentonite had the best effect in reducing the availability of As and Zn, with the residual form of As and Zn after composting increased by 79.8% and 158.6%, respectively, and 7.5% of sepiolite induced the least decrement (39.3%) of residual Cu, compared with the control. Therefore, amendment with appropriate proportion of passivator during pig manure composting could decrease the availability of heavy metals in the manure, and reduce the environmental risk of applying the compost to farmland.

Growth inhibition of K562 cells by cyclin E gene-specific small interfering RNA.

OBJECTIVE: To study the inhibitory effect of small interference RNA(siRNA) of cyclin E gene on the growth of K562 cells. METHODS: siRNA targeting the 940 bp site of the cyclin E mRNA were designed and generated by PCR amplification. The PCR products containing U6 promoter and the siRNA were then transfected into K562 cells via Lipofectamine2000. The cells transfected with non-functional siRNA served as the negative control group and those only treated with serum-free RPMI1640 as the blank control group. Cell counting, reverse transcriptase (RT)-PCR and flow cytometry were employed to evaluate the effect of RNA interference. RESULTS: Compared with the negative and blank control groups, the viable cell count in the interference group was decreased by approximately 80%, the ratio of G(1)-phase cells increased by nearly 30%, and growth arrest was observed. Cyclin E mRNA expression in the cells of the interference group was significantly lowered by about 70%; as compared with that of the negative and blank control groups, whereas the latter two groups had similar expression levels. CONCLUSION: RNA interference induces obvious inhibition of cyclin E gene expression, which consequently affects the proliferation of K562 cells.