Probiotic Escherichia coli Nissle 1917 propelled micro-robot with pH sensitivity for hypoxia targeted intestinal tumor therapy.

Poor drug penetration in hypoxia area of solid tumor is a big challenge for intestinal tumor therapy and thus it is crucial to develop an effective strategy to overcome this challenge. Compared with other bacteria used for construction of hypoxia targeted bacteria micro-robot, the Escherichia coli Nissle 1917 (EcN) bacteria are nonpathogenic Gram-negative probiotic and can especially target and identify the signal molecules in the hypoxic region of tumor, and thus, in this study, we choose EcN to construct a bacteria propelled micro-robot for targeting intestinal tumor therapy. Firstly, the MSNs@DOX with average diameter of 200 nm were synthesized and conjugated with EcN bacteria using EDC/NHS chemical crosslinking method to construct a EcN propelled micro-robot. The motility of micro-robot was then evaluated and the motion velocity of EcN-pMSNs@DOX was 3.78 µm/s. Compared with pMSNs@DOX without EcN driven, EcN bacteria propelled micro-robot transported much more pMSNs@DOX into the inner of HCT-116 3D multicellular tumor spheroids. However, the EcN bacteria are non-intracelluar bacteria which lead to the micro-robot can not directly enter into tumor cells. Therefore, we utilized acid-labile linkers of cis-aconitic amido bone to link EcN with MSNs@DOX nanoparticles to achieve the pH sensitive separation of EcN with MSNs@DOX from the micro-robot. At 4 h of incubation, the isolated MSNs@DOX began to enter into the tumor cells through CLSM observation. In vitro live/dead staining results show that EcN-pMSNs@DOX induced much more cell death than pMSNs@DOX at 24 and 48 h of incubation with HCT-116 tumor cells in acid culture media (pH 5.3). For the validation of the therapeutic efficacy of the micro-robot for intestinal tumor, we established the HCT-116 subcutaneous transplantation tumor model. After 28 days of treatment, EcN-pMSNs@DOX dramatically inhibit tumor growth with tumor volume was around 689 mm3, induce much more tumor tissues necrosis and apoptosis. Finally, the toxicity of this micro-robot was investigated by pathological analysis the liver and heart tissues. We expect that the pH sensitive EcN propelled micro-robot here we constructed may be a safe and feasible strategy for intestinal tumor therapy.

Calcium peroxide alleviates the waterlogging stress of rapeseed by improving root growth status in a rice-rape rotation field.

Waterlogging stress has a negative influence on agricultural production, particularly for rapeseed yield in a rice-rape rotation field. To alleviate the profound impacts of waterlogging stress on rapeseed production, a new fertilization with calcium peroxide (CaO2) was proposed. In this field experiment, with the conventional rape (Brassica napus L.) variety fengyou958 (FY958) and early maturing rape variety xiangyou420 (XY420) as materials, waterlogging was imposed from the bud to flowering stage, and three supplies of CaO2 (0, C1 for the 594 kg hm-2 and C2 for the 864 kg hm-2) were added as basal fertilizer. The results showed that CaO2 significantly reduced the accumulation of fermentation products in roots and alleviated the peroxidation of leaves. The reduced waterlogging stress promoted the root vigor and agronomic characters, such as branches, plant height and stem diameter, accelerated dry matter and nutrients accumulation, and resulting in 22.7% (C1) to 232.8% (C2) higher grain yields in XY420, and 112.4% (C1) to 291.8% (C2) higher grain yields in FY958, respectively. In conclusion, 594 kg hm-2 to 864 kg hm-2 CaO2 application restored the growth of waterlogged rapeseed leaves, and reduced the anaerobic intensity of root, which enhanced the resistance of plants to waterlogging, and improved crop productivity. In a certain range, the higher CaO2 application, the more the yield. This study provides a valid method to prevent damage from flooding in crop fields.

Aus rice root architecture variation contributing to grain yield under drought suggests a key role of nodal root diameter class.

The aus rice variety group originated in stress-prone regions and is a promising source for the development of new stress-tolerant rice cultivars. In this study, an aus panel (~220 genotypes) was evaluated in field trials under well-watered and drought conditions and in the greenhouse (basket, herbicide and lysimeter studies) to investigate relationships between grain yield and root architecture, and to identify component root traits behind the composite trait of deep root growth. In the field trials, high and stable grain yield was positively related to high and stable deep root growth (r = 0.16), which may indicate response to within-season soil moisture fluctuations (i.e., plasticity). When dissecting component traits related to deep root growth (including angle, elongation and branching), the number of nodal roots classified as 'large-diameter' was positively related to deep root growth (r = 0.24), and showed the highest number of colocated genome-wide association study (GWAS) peaks with grain yield under drought. The role of large-diameter nodal roots in deep root growth may be related to their branching potential. Two candidate loci that colocated for yield and root traits were identified that showed distinct haplotype distributions between contrasting yield/stability groups and could be good candidates to contribute to rice improvement.

PDF1.5 Enhances Adaptation to Low Nitrogen Levels and Cadmium Stress.

Environmental acclimation ability plays a key role in plant growth, although the mechanism remains unclear. Here, we determined the involvement of Arabidopsis thaliana PLANT DEFENSIN 1 gene AtPDF1.5 in the adaptation to low nitrogen (LN) levels and cadmium (Cd) stress. Histochemical analysis revealed that AtPDF1.5 was mainly expressed in the nodes and carpopodium and was significantly induced in plants exposed to LN conditions and Cd stress. Subcellular localization analysis revealed that AtPDF1.5 was cell wall- and cytoplasm-localized. AtPDF1.5 overexpression significantly enhanced adaptation to LN and Cd stress and enhanced the distribution of metallic elements. The functional disruption of AtPDF1.5 reduced adaptations to LN and Cd stress and impaired metal distribution. Under LN conditions, the nitrate transporter AtNRT1.5 expression was upregulated. Nitrate transporter AtNRT1.8 expression was downregulated when AtPDF1.5 was overexpressed, resulting in enhanced transport of NO3- to shoots. In response to Cd treatment, AtPDF1.5 regulated the expression of metal transporter genes AtHMP07, AtNRAMP4, AtNRAMP1, and AtHIPP3, resulting in higher Cd accumulation in the shoots. We conclude that AtPDF1.5 is involved in the processing or transmission of signal substances and plays an important role in the remediation of Cd pollution and LN adaptation.

A comparison of the mechanisms and performances of Acorus calamus, Pontederia cordata and Alisma plantagoaquatica in removing nitrogen from farmland wastewater.

This study examined the performances of Acorus calamus, Pontederia cordata, and Alisma plantagoaquatica in removing nitrogen (N) from farmland wastewater. P. cordata showed the fastest rate of N removal, followed by A. plantagoaquatica, whereas that of A. calamus was slowest. P. cordata and A. plantagoaquatica achieving a greater rate of TN reduction in soil than that by A. calamus. A. plantagoaquatica demonstrated the highest N adsorption capacity, 32.6% and 392.1% higher than that of P. cordata and A. calamus, respectively. The higher potential nitrification and denitrification rate, and abundance of functional genes in the P. cordata microcosm resulted in a stronger process of nitrification-denitrification, which accounted for 65.99% of TN loss. Plant uptake and nitrification-denitrification were responsible for 50.06% and 49.94% of TN removed within the A. plantagoaquatica. Nitrification-denitrification accounted for 86.35% of TN loss in A. calamus. These findings helped to insight into N removal mechanisms in different plants.

Ammonium Accumulation Caused by Reduced Tonoplast V-ATPase Activity in Arabidopsis thaliana.

Plant vacuoles are unique compartments that play a critical role in plant growth and development. The vacuolar H+-ATPase (V-ATPase), together with the vacuolar H+-pyrophosphatase (V-PPase), generates the proton motive force that regulates multiple cell functions and impacts all aspects of plant life. We investigated the effect of V-ATPase activity in the vacuole on plant growth and development. We used an Arabidopsisthaliana (L.) Heynh. double mutant, vha-a2 vha-a3, which lacks two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a. The mutant is viable but exhibits impaired growth and leaf chlorosis. Nitrate assimilation led to excessive ammonium accumulation in the shoot and lower nitrogen uptake, which exacerbated growth retardation of vha-a2 vha-a3. Ion homeostasis was disturbed in plants with missing VHA-a2 and VHA-a3 genes, which might be related to limited growth. The reduced growth and excessive ammonium accumulation of the double mutant was alleviated by potassium supplementation. Our results demonstrate that plants lacking the two tonoplast-localized subunits of V-ATPase can be viable, although with defective growth caused by multiple factors, which can be alleviated by adding potassium. This study provided a new insight into the relationship between V-ATPase, growth, and ammonium accumulation, and revealed the role of potassium in mitigating ammonium toxicity.

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.

Effects of biochar-based controlled release nitrogen fertilizer on nitrogen-use efficiency of oilseed rape (Brassica napus L.).

Biochar-based controlled release nitrogen fertilizers (BCRNFs) have received increasing attention due to their ability to improve nitrogen-use efficiency (NUE) and increase crop yields. We previously developed a novel BCRNF, but its effects on soil microbes, NUE, and crop yields have not been reported. Therefore, we designed a pot experiment with five randomised treatments: CK (without urea and biochar), B (addition biochar without urea), B + U (biochar mixed urea), Urea (addition urea without biochar), and BCRNF (addition BCRNF), to investigate the effects of BCRNF on nitrifiers and denitrifiers, and how these impact nitrogen supply and NUE. Results of high-throughput sequencing revealed bacterial community groups with higher nutrient metabolic cycling ability under BCRNF treatment during harvest stage. Compared to Urea treatment, BCRNF treatment stimulated nitrification by increasing the copy number of the bacterial amoA gene and reducing nitrous oxide emission by limiting the abundance of nirS and nirK. Eventually, BCRNF successfully enhanced the yield (~ 16.6%) and NUE (~ 58.79%) of rape by slowly releasing N and modulating the abundance of functional microbes through increased soil nitrification and reduced denitrification, as compared with Urea treatment. BCRNF significantly improved soil NO3-, leading to an increase in N uptake by rape and NUE, thereby promoting rape growth and increasing grain yield.

Cell Wall Polysaccharide-Mediated Cadmium Tolerance Between Two Arabidopsis thaliana Ecotypes.

Cadmium (Cd) is a toxic metal element and the mechanism(s) underlying Cd tolerance in plants are still unclear. Increasingly more studies have been conducted on Cd binding to plant cell walls (CW) but most of them have focused on Cd fixation by CW pectin, and few studies have examined Cd binding to cellulose and hemicellulose. Here we found that Cd binding to CW pectin, cellulose, and hemicellulose was significantly higher in Tor-1, a Cd tolerant A. thaliana ecotype, than in Ph2-23, a sensitive ecotype, as were the concentrations of pectin, cellulose, and hemicellulose. Transcriptome analysis revealed that the genes regulating CW pectin, cellulose, and hemicellulose polysaccharide concentrations in Tor-1 differed significantly from those in Ph2-23. The expressions of most genes such as pectin methyl esterase inhibitors (PMEIs), pectin lyases, xyloglucan endotransglucosylase/hydrolase, expansins (EXPAs), and cellulose hydrolase were higher in Ph2-23, while the expressions of cellulose synthase-like glycosyltransferase 3 (CSLG3) and pectin ethyl esterase 4 (PAE4) were higher in Tor-1. The candidate genes identified here seem to regulate CW Cd fixation by polysaccharides. In conclusion, an increase in pectin demethylation activity, the higher concentration of cellulose and hemicellulose, regulated by related genes, in Tor-1 than in Ph2-23 are likely involved in enhanced Cd CW retention and reduce Cd toxicity.

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.

Boron mitigates cadmium toxicity to rapeseed (Brassica napus) shoots by relieving oxidative stress and enhancing cadmium chelation onto cell walls.

In southern China, Brassica napus (rapeseed) is a widely planted oilseed crop in rice-rapeseed rotation systems with characteristically high levels of cadmium (Cd) and low levels of available boron (B). Current knowledge of the ameliorative effects of B on Cd toxicity in plants mainly concerns plant growth, Cd uptake, and Cd translocation, while little attention has been paid to the role of B on plant antioxidant enzyme systems and cell wall chelation of Cd. We explored the mechanisms whereby B improves rapeseed Cd resistance. Application of B alleviated Cd-induced oxidative stress caused by reactive oxygen species (ROS) in the shoots of Cd-treated plants, by increasing the activity of the major antioxidant enzymes, superoxide dismutase, peroxidase, and catalase. Moreover, the shoots of rapeseed plants supplied with B under Cd toxicity had higher ionic soluble pectin (ISP) content, thereby providing more Cd-binding sites in pectin, as well as higher methylesterase activity. However, no changes in covalent soluble pectin were observed. In addition, B also induced higher cellulose in Cd-toxic shoots, thus promoting Cd chelation onto cell walls. Fourier infrared spectrum analysis confirmed that the addition of B increased protein, pectin, cellulose, and carbohydrate content in the cell walls of Cd-toxic leaves. In conclusion, B can mitigate Cd phytotoxicity by alleviating oxidative stress and immobilizing Cd on the ISP and cellulose of shoot cell walls, thereby playing a potential role in improving the growth potential of crops and Cd phytoremediation. The results also provide a theoretical basis for alleviating Cd toxicity in crops and development of Cd-tolerant varieties.

Boron alleviates cadmium toxicity in Brassica napus by promoting the chelation of cadmium onto the root cell wall components.

In Southern China, rice-oil rotations occur on soils with high levels of cadmium (Cd) and low levels of available boron (B). Boron can alleviate Cd toxicity, as it affects the plant cell wall structures and the components that block the entry of Cd into the cytoplasm; however, these mechanisms are not well understood. Fourier transform infrared spectroscopy (FTIR), fluorescent probe dye, electron microscope, ion abundance (inductively coupled plasma mass spectrometry), metabonomics and transcriptomics were used in the study, and we found that under Cd stress, B increased root pectin content by affecting the biosynthesis pathways and decreasing the activity of pectinase and the expression levels of related genes. The increased pectin content and pectin demethylation increased the chelation of Cd onto the cell walls and reduced the levels of Cd entering the organelles. Application of B to the roots decreased the amounts of cellulose and hemicellulose in the cell walls to normal levels and promoted the expression of genes from the expansin, xyloglucan endotransglucosylase, and α-xylosidase families. This contributed to cell wall integrity and root flexibility. Consequently, the accumulation of reactive oxygen species was inhibited and cell viability in the roots was increased, which reduced the destruction of root surface structures. These results have improved our understanding of how B participates in chelation of Cd onto cell walls and in maintaining cell wall integrity, thereby improving Cd toxicity resistance in rapeseed roots.

Encapsulation of Escherichia coli strain Nissle 1917 in a chitosan-alginate matrix by combining layer-by-layer assembly with CaCl2 cross-linking for an effective treatment of inflammatory bowel diseases.

Escherichia coli strain Nissle 1917 (EcN) has been widely shown to effectively treat inflammatory bowel diseases (IBDs). Unfortunately, after oral administration, EcN viability dramatically decreases due to severe environmental factors, including low gastric pH, temperature and osmotic pressure. To address these challenges and improve oral bio-availability, this study utilized layer-by-layer assembly (LbL) and ionic cross-linking with CaCl2 as a method of EcN encapsulation (GEcN). Upon examination, GEcN cells were shown to maintain their ability to grow and proliferate, but had a slightly longer stationary phase (10 h) relative to free EcN (4 h). When exposed to simulated gastric fluid (SGF), a higher number of GEcN cells survived up to 12 h when compared to the other groups. To assess the therapeutic effect of EcN encapsulation in vivo, a TNBS-induced colitis rat model was established. When compared with the oral administration of free EcN, GEcN exhibited a significantly enhanced anti-inflammatory effect. Furthermore, GEcN treatment showed a lower disease activity index (DAI), decreased pro-inflammatory cytokine expression (MPO, TNF-α, IL-6) and increased anti-inflammatory cytokine expression (IL-10). Additionally, rats that received GEcN had much higher ZO-1 expression levels. These results suggest that EcN encapsulation in a chitosan-alginate matrix when utilizing the LbL assembly with CaCl2 cross-linking can improve probiotic viability in a gastric environmental and thereby offer a more effective treatment for IBDs.

SPION-Decorated Exosome Delivered BAY55-9837 Targeting the Pancreas through Magnetism to Improve the Blood GLC Response.

BAY55-9837, a potential therapeutic peptide in the treatment of type 2 diabetes mellitus (T2DM), is capable of inducing glucose (GLC)-dependent insulin secretion. However, the therapeutic benefit of BAY55-9837 is limited by its short half-life, lack of targeting ability, and poor blood GLC response. How to improve the blood GLC response of BAY55-9837 is an existing problem that needs to be solved. In this study, a method for preparing BAY55-9837-loaded exosomes coupled with superparamagnetic iron oxide nanoparticle (SPIONs) with pancreas islet targeting activity and an enhanced blood GLC response with the help of an external magnetic force (MF) is demonstrated. The plasma half-life of BAY55-9837 loaded in exosome-SPION is 27-fold longer than that of BAY55-9837. The active targeting property of SIPONs enables BAY-exosomes to gain a favorable targeting property, which improves the BAY55-9837 blood GLC response capacity with the help of an external MF. In vivo studies show that BAY-loaded exosome-based vehicle delivery enhances pancreas islet targeting under an external MF and markedly increases insulin secretion, thereby leading to the alleviation of hyperglycemia. The chronic administration of BAY-exosome-SPION/MF significantly improves glycosylated hemoglobin and lipid profiles. BAY-exosome-SPION/MF maybe a promising candidate for a peptide drug carrier for T2DM with a better blood GLC response.

Cytocompatible cerium oxide-mediated antioxidative stress in inhibiting ocular inflammation-associated corneal neovascularization.

As oxidative stress is involved with inflammation and neovascularization, blocking oxidative stress may be beneficial for reducing inflammation. To investigate the potential use of cerium oxide nanoparticles (CeNPs) in treating neovascularization-related ophthalmic diseases, various CeNP samples were synthesized, and the sample with the best antioxidant efficacy was used in a rat model of inflammation-associated corneal neovascularization. This synthesized cerium oxide showed good biocompatibility and was capable of mediating a decrease in the expression levels of inflammatory factors via antioxidative stress. Additionally, in vitro tests showed that the Ce3+/Ce4+ ratio of the CeNPs directly affected the antioxidative activity, with higher ratios achieving better efficacy. The anti-inflammatory efficacy of the functional CeNPs was examined both in vitro and in vivo. Slit-lamp biomicroscopy and histological analysis revealed the gradual development of corneal neovascularization, suggesting that inflammation and neovascularization could be controlled by reducing the level of oxidative stress. CeNP-induced antioxidation could serve as a new strategy in the development of long-acting functional agents for treating ophthalmic diseases.

Low concentration of exogenous ethanol promoted biomass and nutrient accumulation in oilseed rape (Brassica napus L.).

With hydroponics culture, we monitored the response of the growth and nutrient accumulation of oilseed rape (Brassica napus L.) to five ethanol concentrations: 0 mL•L-1 (control), 0.0125 mL•L-1, 0.025 mL•L-1, 0.05 mL•L-1, and 0.25 mL•L-1, respectively. The results showed that a high concentration of exogenous ethanol (0.25 mL•L-1) significantly inhibited oilseed rape growth by 52.28%. However, the biomass of oilseed rape with a low concentration of exogenous ethanol (0.0125-0.05 mL•L-1) manipulation was raised by 16.62-44.08%. A similar result was found on the total nitrogen, phosphorus, and potassium of the oilseed rape. Results of micro-element determination showed that iron and zinc accumulation in oilseed rape was unchanged, while manganese and copper accumulation was increased first and then decreased with increasing ethanol concentration. This study provided a possibility for improving plant growth with low concentration ethanol application in oilseed rape planting.

A Biochar-Based Route for Environmentally Friendly Controlled Release of Nitrogen: Urea-Loaded Biochar and Bentonite Composite.

Biochar-based fertilizers have attracted increased attention, because biochar can improve the soil fertility, promote plant growth and crop yield. However, biochar-based controlled release nitrogen fertilizers (BCRNFs) still face problems because of the high cost, inefficient production technology, instability of nitrides, and the challenge associated with the controlled release of nutrients. In this study, we hydrothermally synthesised novel BCRNFs using urea-loaded biochar, bentonite and polyvinyl alcohol for controlled release of nutrients. Scanning electron microscopy and gas adsorption were conducted to identify the urea-loading and storage of bentonite in the inner pores of the biochar particles. X-ray diffraction, Fourier transform infrared spectroscopic and X-ray photoelectron spectroscopic studies demonstrated that strengthening the interactions among biochar, urea, and bentonite, helps control the moisture diffusion and penetration of bentonite, thereby leading to nutrient retention. The BCRNF showed significantly improved nutrient release characteristic compared with that of a mixture of biochar and urea. This urea-bentonite composite loaded with urea provides control over the release of nutrients stored in the biochar. BCRNF, especially those produced hydrothermally, can have potential applications in sustainable food security and green agriculture.

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.