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Maintenance of ion homeostasis in plant cells is an essential physiological requirement for sustainable growth, development, and yield of crops. Plants respond to high levels of heavy metals such as copper (Cu) and cadmium (Cd) to avoid irreversible damage at the structural, physiological and molecular levels. Our previous study found that rice germin-like proteins (OsGLPs) are a type of Cu-responsive proteins. The deletion of 10 tandem OsGLP genes on chromosome 8 led to more severe heavy metal toxicity in rice. In this study, we show that rice WRKY transcription factor OsWRKY72 negatively regulates OsGLP8-7 transcription. Overexpression of OsWRKY72 weakens the Cu/Cd tolerance of rice when exposed to Cu and Cd. OsWRKY72 suppressed expression of OsGLP8-7 and lignin synthesis genes, resulting in reduced lignin polymerization and consequently lower lignin accumulation in cell walls, thereby increasing the Cu and Cd accumulation. In addition, OsWRKY53 bound to OsWRKY72 to alleviate the transcriptional inhibition of OsGLP8-7. These results revealed that OsWRKY72-OsGLP8-7 is an important module response of rice to heavy metal stress, and that transcription factor OsWRKY72 acts upstream of OsGLP8-7 to regulate Cu/Cd toxicity.

Context and backgroundMicroplastics (MPs) and heavy metals (HMs) coexist in the farmland of China.MotivationIt still remains unclear the extent of their exposure and distribution in sweet potato fields.HypothesisPolyethylene (PE) or polyamide (PA) is the main MP pollutant in contaminated sweet potato sites, and the MP abundance in low-latitude and eastern areas is highest. Methods: In this study, saturated NaCl solution, a stereo microscope, a Fourier transform infrared spectrometer, and an electrothermal digester are used for the extraction, observation, identification of MPs, and analysis of Cd elements in soil, respectively.MethodsIn this study, saturated NaCl solution, a stereo microscope, a Fourier transform infrared spectrometer, and an electrothermal digester are used for the extraction, observation, identification of MPs, and analysis of Cd elements in soil, respectively.ResultsHere, we found an average MP level of 112,400 items/kg in 30 sweet potato field sites based on the items in 5 g soil and the magnification (200x), and the maximum abundance was 197,153 items/kg in Laiyang city, Shandong province, by field survey. The distribution characteristics of MPs are middle-latitude areas < low-latitude areas, and eastern areas > central areas. Most MPs are of the fragment and film shape, which account for 47.96 and 40.22%, respectively. In order to detect MP polymers in three cities named “Liancheng,” “Huanggang,” and “Laiyang” with different degrees of development, a laser infrared imaging system was used as a novel instrument to explore the MPs larger than 10 μm.ConclusionThe results showed that PA is the main MP pollutant in contaminated sweet potato sites, and soil texture, planting time, and urbanization processes may be the main factors affecting MP distribution. The average cadmium (Cd) concentration in 215 field sites is 0.15 mg/kg, and the local Cd pollution is existing, but the overall pollution is low. In addition, Cd concentration was negatively correlated with MP abundance. This study reveals the status of MP and also Cd pollution in sweet potato fields, which provides a theoretical basis for the safe production and utilization of sweet potato fields.

Quinoa (Chenopodium quinoa Willd) sprouts are rich in bioactive compounds that offer numerous health benefits. However, limited research exists on their cultivation, nutritional value, and processing potential. This study compared the nutritional composition and antioxidant activity of quinoa sprouts from different varieties at various time points. Results showed a general increase in most nutrients over time. At the 24 h mark, JQ-W3 exhibited a 17.77% increase in leucine, 1.68 times higher than in eggs, along with a 6.11-fold elevation in GABA content. JQ-B1 exhibited the preeminent antioxidant potency composite (APC) score. Saponins, known for their bitter taste, decreased at 12 h but returned to original levels by 24 h. Based on nutritional components and saponin content, 24 h sprouted black quinoa JQ-B1 and white quinoa JQ-W3 were selected, providing a basis for quinoa sprout development in the food industry. These findings contribute to the understanding and utilization of quinoa sprouts.

Background: An evident association between mood disorders and TMJ dysfunction has been demonstrated in previous studies. This study observed both the behavioral changes and the pathological changes in hippocampal tissue of rats in an animal model of TMJ-OA by injecting MIA into TMJ. Methods: Eighteen SD rats were randomly assigned to the NC group and the MIA groups. A TMJ-OA model was established to assess the HWT in the TMJ region, and the rats were subjected to the OFT and EPM. HE, O-fast green staining, qRT-PCR and immunofluorescence were used to detect condylar damage. Serum and hippocampal oxidative stress levels were detected. Functions of genes obtained by RNA-Seq were investigated using H2O2, ZnCl2 and transfection of siRNA on HT22 cells. Results: Injection of MIA resulted in disorganization of the chondrocyte layer on the condylar surface of rats, with reduced synthesis and increased degradation of the condylar cartilage matrix and reduced HWT. The results of the OFT and EPM showed that the rats in the MIA group developed anxiety-like behavior during the sixth week of MIA injection. Increased Nox4 expression, decreased SOD2 expression, elevated MDA level, and reduced GSH level were detected in serum and hippocampal neurons in the MIA group, with nuclear pyknosis and reduced Nissl bodies observed in neurons. The expression of Slc39a12 in hippocampal neurons of rats in the MIA group decreased. Slc39a12 knockdown in HT22 cells induced increased Nox4 expression, decreased SOD2 expression, increased MDA level, and reduced GSH and intracellular Zn2+. Oxidative stress in HT22 cells after transfection and H2O2 stimulation was reversed when ZnCl2 was added. Conclusion: Loss of Slc39a12 in hippocampal neurons results in cellular oxidative stress, further leading to neuronal damage. This may potentially explain how TMJ-OA triggers anxiety-like behavior in rats.

Quinoa (Chenopodium quinoa Willd.) is an ancient crop with perfect nutritional composition and antioxidants substances. However, the current research on the nutritional quality of quinoa is limited to a small number of varieties or a single origin. In this study, we aimed at providing a detailed evaluation of abundant nutrients of quinoa seeds from thirty varieties with different color in different origins, including soluble protein, soluble sugar, amino acid, vitamin, fatty acid and saponin. Results showed that there were significant differences in the contents of γ-aminobutyric acid (6.67–78.67 mg/100 g DW) and vitamin C (11.675–105.135 mg/100 g DW) in quinoa seeds. Here, we scored thirty quinoa seeds using a weighted average score system first time and identified four varieties, black quinoa JQ-00145, red quinoa JQ-00125 and two white quinoa JQ-00005/JQ-00077, with superior nutritional quality and oxidation resistance. The results of this study will provide theoretical guidance for consumption of quinoa.

Abstract Background Soil microbiomes are considered a cornerstone of the next green revolution, and plant growth-promoting bacteria (PGPB) are critical for microbiome engineering. However, taking plant-beneficial microorganisms from discovery to agricultural application remains challenging, as the mechanisms underlying the interactions between beneficial strains and plants in native soils are still largely unknown. Increasing numbers of studies have indicated that strains introduced to manipulate microbiomes are usually eliminated in soils, while others have reported that application of PGPB as inocula significantly improves plant growth. This contradiction suggests the need for a deeper understanding of the mechanisms underlying microbe-induced growth promotion. Results We showed PGPB-induced long-term plant growth promotion after elimination of the PGPB inoculum in soils and explored the three-way interactions among the exogenous inoculum, indigenous microbiome, and plant, which were key elements of the plant growth-promoting process. We found the rhizosphere microbiome assembly was mainly driven by plant development and root recruitments greatly attenuated the influence of inocula on the rhizosphere microbiome. Neither changes in the rhizosphere microbiome nor colonization of inocula in roots was necessary for plant growth promotion. In roots, modification of DNA methylation in response to inoculation affects gene expression related to PGPB-induced growth promotion, and disruptions of the inoculation-induced DNA methylation patterns greatly weakened the plant growth promotion. Together, our results showed PGPB-induced DNA methylation modifications in roots mediated the promotion process and these modifications remained functional after elimination of the inoculum from the microbiome. Conclusion This study suggests a new mechanism in which PGPB affect DNA methylation in roots to promote plant growth, which provides important insights into microbiome–plant interactions and offers new strategies for plant microbiome engineering beyond the perspective of maintaining inoculum persistence in soils. Video abstract Graphical abstract

Microplastics (MPs) and heavy metals (HMs) co-exist in sweet potato fields of China. As the main component of agricultural field mulch and one of the most polluting and harmful HMs, the effects of polyethylene microplastics (PE MPs) and cadmium (Cd) on sweet potato and soil environment are remains unclear. Here, pot and hydroponic experiments are used to explore the effects of original and weathered PE MPs on growth and Cd uptake of sweet potatoes. The results of pot experiments reveal that compared with the control (0%), 5% of weathered PE MPs can significantly increase soil electrical conductivity (EC); both 5% of the original PE MPs and weathered PE MPs can significantly reduce the concentration of Olsen phosphorus (P) and Olsen potassium (K) in soil, inhibit plant growth, but significantly increase Cd accumulation and glutathione (GSH) level in tissues of sweet potatoes, and also induce membrane lipid peroxidation. In addition, compared to 5% weathered PE MPs, 5% original PE MPs significantly reduce soil EC, growth and peroxidase level of sweet potatoes, but significantly increase Cd concentration in leaves and stems. The results of hydroponic experiment show that original PE MPs significantly increase the P, K, and Cd adsorption compared with weathered PE MPs, and Cd increases the original PE MPs accumulation in the root cortex but decrease PE MPs accumulation in shoots. To sum up, our study investigates the differences and reasons of the effects of original and weathered PE MPs on growth and Cd absorption of sweet potatoes.

Copper (Cu) and cadmium (Cd) are common heavy metal pollutants. When Cd and excessive Cu accumulate in plants, plant growth is reduced. Our previous study showed that Germin-like proteins (GLPs), which exist in tandem on chromosomes, are a class of soluble glycoproteins that respond to Cu stress. In this study, hydroponic cultures were carried out to investigate the effect of GLP on Cd and Cu tolerance and accumulation in rice. The results showed that knockout of a single OsGLP8-2 gene or ten OsGLP genes (OsGLP8-2 to OsGLP8-11) resulted in a similar sensitivity to Cd and Cu toxicity. When subjected to Cu and Cd stress, the glp8-2 and glp8-(2-11) mutants displayed a more sensitive phenotype based on the plant height, root length, and dry biomass of the rice seedlings. Correspondingly, Cu and Cd concentrations in the glp8-2 and glp8-(2-11) mutants were significantly higher than those in the wild-type (WT) and OsGLP8-2-overexpressing line. However, Cu and Cd accumulation in the cell wall was the opposite. Furthermore, we determined lignin accumulation. The overexpressing-OsGLP8-2 line had a higher lignin accumulation in the shoot and root cell walls than those of the WT, glp8-2, and glp8-(2-11). The expression of lignin synthesis genes in the OsGLP8-2-overexpressing line was significantly higher than that in the WT, glp8-2, and glp8-(2-11). The SOD activity of OsGLP8-2, Diaminobe-nzidine (DAB), propidium iodide (PI) staining, and Malondialdehyde (MDA) content determination suggested that OsGLP8-2 is involved in heavy metal-induced antioxidant defense in rice. Our findings clearly suggest that OsGLPs participate in responses to heavy metal stress by lignin deposition and antioxidant defense capacity in rice, and OsGLP8-2 may play a major role in the tandem repeat gene clusters of chromosome 8 under heavy metal stress conditions.

It has been shown that multidrug and toxic compound extrusion/detoxification (MATE/DTX) family transporters are involved in the regulation of plant development and stress response. Here, we characterized the mini body1 (mib1) mutants in mungbean, which gave rise to increased branches, pentafoliate compound leaves, and shortened pods. Map-based cloning revealed that MIB1 encoded a MATE/DTX family protein in mungbean. qRT-PCR analysis showed that MIB1 was expressed in all tissues of mungbean, with the highest expression level in the young inflorescence. Complementation assays in Escherichia coli revealed that MIB1 potentially acted as a MATE/DTX transporter in mungbean. It was found that overexpression of the MIB1 gene partially rescued the shortened pod phenotype of the Arabidopsis dtx54 mutant. Transcriptomic analysis of the shoot buds and young pods revealed that the expression levels of several genes involved in the phytohormone pathway and developmental regulators were altered in the mib1 mutants. Our results suggested that MIB1 plays a key role in the control of plant architecture establishment in mungbean.

Assimilatory-type nitrate reductase (NR) and nitrite reductase (NiR) are the key enzymes that involve in nitrate assimilation and nitrogen cycling in microorganisms. NR and NiR with NADH or NADPH and FMN or FAD domains could be coupled to the reduction process of hexavalent chromium [Cr(VI)] in microorganisms. A new assimilatory-type NR gene (named niaD) and a new assimilatory-type NiR gene (named niiA) are cloned, identified, and functionally characterized by 5′ and 3′ RACE, alignment, annotation, phylogenetic tree, and yeast mutant complementation analyses from Pisolithus sp.1, a dominant symbiotic ectomycorrhizal fungi (EMF) that can assist in phytoremediation. Assimilatory-type niaD and niiA were 2,754 bp and 3,468 bp and encode a polypeptide with 917 and 1,155 amino acid residues, respectively. The isoelectric points of NR (Pisolithus sp.1 NR) and NiR (Pisolithus sp.1 NiR) of Pisolithus sp.1 are 6.07 and 6.38, respectively. The calculated molecular mass of Pisolithus sp.1 NR and Pisolithus sp.1 NiR is 102.065 and 126.914 kDa, respectively. Yeast mutant complementation analysis, protein purification, and activities of NR and NiR under Cr treatment suggest that Pisolithus sp.1 NR is a functional NR that mediates Cr(VI) tolerance and reduction. The multiple alignment demonstrates that Pisolithus sp.1 NR is potentially a nicotinamide adenine dinucleotide phosphate-dependent flavin mononucleotide reductase and also Class II chromate reductase. Our results suggest that Pisolithus sp.1 NR plays a key role in Cr(VI) reduction in the EMF Pisolithus sp.1.

Heavy metals (HMs) have become a major environmental pollutant threatening ecosystems and human health. Although hyperaccumulators provide a viable alternative for the bioremediation of HMs, the potential of phytoremediation is often limited by the small biomass and slow growth rate of hyperaccumulators and HM toxicity to plants. Here, plant growth-promoting bacteria (PGPB)-assisted phytoremediation was used to enhance the phytoremediation of HM-contaminated soils. A PGPB with HM-tolerant (HMT-PGPB), Bacillus sp. PGP15 was isolated from the rhizosphere of a cadmium (Cd) hyperaccumulator, Solanum nigrum. Pot experiments demonstrated that inoculation with strain PGP15 could significantly increase the growth of S. nigrum. More importantly, strain PGP15 markedly improved Cd accumulation in S. nigrum while alleviating Cd-induced stress in S. nigrum. Specifically, PGP15 inoculation significantly decreased the contents of H2O2, MDA, and O2·− in S. nigrum, while the activities (per gram plant fresh weight) of SOD, APX, and CAT were significantly increased in the PGP15-inoculated plants compared with the control sample. These results suggested that the interactions between strain PGP15 and S. nigrum could overcome the limits of phytoremediation alone and highlighted the promising application potential of the PGPB-hyperaccumulator collaborative pattern in the bioremediation of HM-contaminated soils. Furthermore, the PGP15 genome was sequenced and compared with other strains to explore the mechanisms underlying plant growth promotion by HMT-PGPB. The results showed that core genes that define the fundamental metabolic capabilities of strain PGP15 might not be necessary for plant growth promotion. Meanwhile, PGP15-specific genes, including many transposable elements, played a crucial role in the adaptive evolution of HM resistance. Overall, our results improve the understanding of interactions between HMT-PGPB and plants and facilitate the application of HMT-PGPB in the phytoremediation of HM-contaminated soils.

DNA methylation is a rapid response strategy promoting plant survival under heavy metal (HM) stress. However, the roles of DNA methylation underlying plant adaptation to HM stress remain largely unknown. Here, we used pokeweed, a hyperaccumulator of manganese (Mn) and cadmium (Cd), to explore responses of plant to HM stress at phenotypic, transcriptional and DNA methylation levels. Mn- and Cd-specific response patterns were detected in pokeweed. The growth of pokeweed was both inhibited with exposure to excess Mn/Cd, but pokeweed distinguished Mn and Cd with different subcellular distributions, ROS scavenging systems, transcriptional patterns including genes involved in DNA methylation, and differentially methylated loci (DML). The number of DML between Mn/Cd treated and untreated samples increased with increased Mn/Cd concentrations. Meanwhile, pretreatment with NADPH oxidase inhibitors prior to HM exposure markedly reduced HM-induced reactive oxygen species (ROS), which caused reductions in expressions of DNA methylase and demethylase in pretreated samples. The increased levels of HM-induced demethylation were suppressed with alleviated ROS stress, and a series of HM-related methylated loci were also ROS-related. Taken together, our study demonstrates that different HMs affect different DNA methylation sites in a dose-dependent manner and changes in DNA methylation under Mn/Cd stress are partly mediated by HM-induced ROS.

The retention of copper (Cu) in saline–alkali soil (SAS) during long-term application of biogas residue (BR) with a high concentration of Cu raises concerns. In this work, the fate of Cu was detected using adsorption isotherms, scanning electron microscope—energy dispersive spectrometer, Fourier transform infrared spectrometer, X-ray diffraction, isothermal titration calorimetry, X-ray photoelectron spectroscopy, and microzone X-ray fluorescence spectrometer. The results showed that the main groups for Cu adsorption by SAS and BR were carboxyl, hydroxyl, amide and amine. The adsorption of Cu by the carboxyl group was entropy–enthalpy co-driven (|ΔH| < |TΔS|, ΔH < 0). The adsorption of Cu by the amine group was entropy-driven (|ΔH| > |TΔS|, ΔH > 0). The adsorption of Cu on the SAS and BR was achieved by organic matter rather than minerals. The degradation of BR in the SAS increases the content of Cu adsorption groups such as carboxyl and amine groups, and Cu was adsorbed on the surface or inside SAS through organic groups. This study provides further theoretical support for the application of BR in SAS.

Genetics: Leaf architecture gene evolved early in legume plants A legume gene involved in determining the spatial pattern of leaflets seems to have evolved this function earlier than previously thought. Xin Li from Nanjing Agricultural University, China, and coworkers studied a mutant variety of mungbean in which the plant’s leaves display a simple architecture instead of the usual compound arrangement, with several leaflets joined to a single stem. They discovered a mutation in the mungbean version of a gene called LEAFY. This gene, together with others, proved pivotal in compound leaf development in mungbeans. The results run counter to prior data showing that, outside of a small group of related legume species, LEAFY plays only to play a minor role in compound leaf development in these agriculturally important plants. The findings could thus inform breeding efforts in legumes generally.

Aspergillus includes both plant pathogenic and beneficial fungi. Although endophytes beneficial to plants have high potential for plant growth promotion and improving stress tolerance, studies on endophytic lifestyles and endophyte-plant interactions are still limited. Here, three endophytes belonging to Aspergillus, AS31, AS33, and AS42, were isolated. They could successfully colonize rice roots and significantly improved rice growth. The genomes of strains AS31, AS33, and AS42 were sequenced and compared with other Aspergillus species covering both pathogens and endophytes. The genomes of AS31, AS33, and AS42 were 36.8, 34.8, and 35.3 Mb, respectively. The endophytic genomes had more genes encoding carbohydrate-active enzymes (CAZymes) and small secreted proteins (SSPs) and secondary metabolism gene clusters involved in indole metabolism than the pathogens. In addition, these endophytes were able to improve Pi (phosphorus) accumulation and transport in rice by inducing the expression of Pi transport genes in rice. Specifically, inoculation with endophytes significantly increased Pi contents in roots at the early stage, while the Pi contents in inoculated shoots were significantly increased at the late stage. Our results not only provide important insights into endophyte-plant interactions but also provide strain and genome resources, paving the way for the agricultural application of Aspergillus endophytes.

Currently, the research and application of biochar in the remediation of heavy metal contaminated soil has become a hotspot, especially regarding the remediation of agricultural land. Biochar has been proved to be effective in reducing the content of available heavy metals in the soil as well as the heavy metals in plants. However, the long-term effectiveness of biochar immobilization has not been widely studied. In this review, retrospective search was carried out on the published literature results concerning remediation effects of biochar on different areas of heavy metal contaminated soil in the recent years, its application in field remediation (several years), and some potential abiotic and biotic factors that may weaken the immobilization effects of biochar. This results indicate that: (1) biochar is widely used in the remediation of heavy metal contaminated soil in different areas and has excellent immobilization effect. (2) Most of the research demonstrate that the immobilization effect of biochar is effective for 2–3 years or according to few results even for 5 years. However, there have been various reports claiming that the immobilization effect of biochar decreases with time. (3) Abiotic factors such as acid rain, flooded environment, changes in soil condition (pH, redox and dissolved organic matter) and changes in biochar (Cl− and alkali leaching) can significantly weaken the immobilization effect of biochar. (4) Biotic factors such as plant roots, earthworms and soil microorganisms can also significantly reduce the immobilization effect of biochar. Therefore, field experiments having longer time span with biochar need to be further carried out, and the developmental research of modified biochar with a more stable immobilization effect also needs further attention.

Phytotoxicity and accumulation of Cu in mature and young leaves of submerged macrophyte Hydrilla verticillata (L.f.) Royle were investigated by analyzing the chlorophyll contents, chloroplast ultrastructure and leaf surface structure under different Cu treatments (0, 0.01, 0.05 and 0.1 f mg L−1). The results showed that 0.05 and 0.1 mg L−1 Cu treatment decreased the contents of Chl a and Chl b, and caused damage on leaf surface structure and chloroplast ultrastructure compared with control (0 mg L−1 Cu treatment). Higher concentration of Cu induced Chlorophyll decreases and the damages on the leaf surface structure and chloroplast ultrastructure were more pronounced in mature than in young leaves. It was observed that leaf Cu concentration increased almost linearly with exposure time and majority of the Cu accumulated in the cell walls. Among different cell wall fractions, the majority of Cu accumulated in cell walls was bound to the hemicellulose 1 and cellulose, followed by the pectin, hemicellulose 2. Mature leaves had significantly higher the concentrations of total Cu and bound-Cu in cell walls due to higher uronic acid content in their cell wall fractions (pectin, hemicellulose 1 and cellulose) than young leaves. Distinct cell wall composition might partially contribute to the different Cu toxicity and accumulation between mature and young leaves of submerged macrophyte H. verticillata. Our results show that mature leaves are more efficient in the uptake and accumulation of Cu than young leaves, which might explain why mature leaves sustain more severe damage.

Within the domain of phytoremediation research, the proper disposal of harvestable plant parts, that remove pollutants from contaminated soil, has been attracted extensive attention. Here, the bioenergy generation capability of trace metals (Cu, Pb, Zn, Cd, Mn, and As) polluted plants was assessed. The biogas production potential of accumulators or hyperaccumulator plants, Elsholtzia haichowensis, Sedum alfredii, Solanum nigrum, Phytolacca americana and Pteris vittata were 259.2 ± 1.9, 238.7 ± 4.2, 135.9 ± 0.9, 129.5 ± 2.9 and 106.8 ± 2.1 mL/g, respectively. The presence of Cu (at approximately 1000 mg/kg) increased the cumulative biogas production, the daily methane production and the methane yield of E. haichowensis. For S. alfredii, the presence of Zn (≥500 mg/kg) showed a significant negative impact on the methane content in biogas, and the daily methane production, which decreased the biogas and methane yield. The biogas production potential increased when the content of Mn was at 5 000–10,000 mg/kg, subsequently, decreased when the value of Mn at 20,000 mg/kg. However, Cd (1–200 mg/kg), Pb (125–2000 mg/kg) and As (1250–10,000 mg/kg) showed no distinctive change in the cumulative biogas production of S. nigrum, S. alfredii and P. vittata, respectively. The methane yield showed a strong positive correlation (R2 =0.9704) with cumulative biogas production, and the energy potential of the plant residues were at 415–985 kWh/ton. Thus, the anaerobic digestion has bright potential for the disposal of trace metal contaminated plants, and has promising prospects for the use in energy production.

Epigenetic variation may contribute to traits that are important in domestication, but how patterns of genetic and epigenetic variation differ between cultivated and wild plants remains poorly understood. In particular, we know little about how selection may shape epigenetic variation in natural and cultivated populations. In this study, we investigated 11 natural populations and 6 major cultivated populations using amplified fragment length polymorphism (AFLP) and methylation-sensitive AFLP (MS-AFLP or MSAP) markers to identify patterns of genetic and epigenetic diversity among Corydalis yanhusuo populations. We further explored correlations among genetic, epigenetic, alkaloidal, and climatic factors in natural and cultivated C. yanhusuo. We found support for a single origin for all cultivated populations, from a natural population which was differentiated from the other natural populations. The magnitude of FST based on AFLP was significantly correlated with that for MSAP in pairwise comparisons in both natural and cultivated populations, suggesting a relationship between genetic and epigenetic variation in C. yanhusuo. This relationship was further supported by dbRDA (distance-based redundancy analyses) where some of the epigenetic variation could be explained by genetic variation in natural and cultivated populations. Genetic variation was slightly higher in natural than cultivated populations, and exceeded epigenetic variation in both types of populations. However, epigenetic differentiation exceeded that of genetic differentiation among cultivated populations, while the reverse was observed among natural populations. The differences between wild and cultivated plants may be partly due to processes inherent to cultivation and in particular the differences in mode of reproduction. The importance of epigenetic compared to genetic modifications is thought to vary depending on reproductive strategies, and C. yanhusuo usually reproduces sexually in natural environments, while the cultivated C. yanhusuo are propagated clonally. In addition, alkaloid content of C. yanhusuo varied across cultivated populations, and alkaloid content was significantly correlated to climatic variation, but also to genetic (6.89%) and even more so to epigenetic (14.09%) variation in cultivated populations. Our study demonstrates that epigenetic variation could be important in cultivation of C. yanhusuo and serve as a source of variation for response to environmental conditions.

Abstract Background Hydrogen gas (H2) is the most abundant element in the universe, and has been reported to act as a novel beneficial gaseous molecule in plant adaptive responses. Radish sprouts are popular because they contain substantial amounts of antioxidants and health-promoting compounds, such as anthocyanin and glucosinolates. Although radish sprouts accumulated more anthocyanin under UV-A after treatment with hydrogen-rich water (HRW), the molecular mechanism responsible is still elusive. To explore these mechanisms, RNA-seq analysis was used. Results Four cDNA libraries from radish sprout hypocotyls were constructed, and a total of 14,564 differentially expressed genes (DEGs) were identified through pairwise comparisons. By Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, these unigenes were found to be implicated in light signal perception and transduction, starch and sucrose metabolism, photosynthesis, nitrogen metabolism and biosynthesis of secondary metabolites. The MYB-bHLH-WD40 complex accounted for the majority of the transcription factors found to be involved in anthocyanin biosynthesis, and levels of transcripts for this complex were in accordance with the anthocyanin concentrations observed. In addition, other transcription factors (such as NAC, bZIP and TCP) might participate in HRW-promoted anthocyanin biosynthesis. Furthermore, the signaling processes of plant hormones, MAPKs and Ca2+ might be involved in HRW-promoted anthocyanin biosynthesis under UV-A. The expression patterns of 16 selected genes were confirmed using qRT-PCR analysis. Conclusions Taken together, the results of this study may expand our understanding of HRW-promoted anthocyanin accumulation under UV-A in radish sprouts.

Phytochelatins (PCs) catalyzed by phytochelatin synthases (PCS) are important for the detoxification of metals in plants and other living organisms. In this study, we isolated a PCS gene (VsPCS1) from Vicia sativa and investigated its role in regulating cadmium (Cd) tolerance. Expression of VsPCS1 was induced in roots of V. sativa under Cd stress. Analysis of subcellular localization showed that VsPCS1 was localized in the cytoplasm of mesophyll protoplasts of V. sativa. Overexpression of VsPCS1 (35S::VsPCS1, in wild-type background) in Arabidopsis thaliana could complement the defects of Cd tolerance of AtPCS1-deficent mutant (atpcs1). Compared with atpcs1 mutants, 35S::VsPCS1/atpcs1 (in AtPCS1-deficent mutant background) transgenic plants significantly lowered Cd-fluorescence intensity in mesophyll cytoplasm, accompanied with enhanced Cd-fluorescence intensity in the vacuoles, demonstrating that the increased Cd tolerance may be attributed to the increased PC-based sequestration of Cd into the vacuole. Furthermore, overexpressing VsPCS1 could enhance the Cd tolerance in 35S::VsPCS1, but have no effect on Cd accumulation and distribution, showing the same level of Cd-fluorescence intensity between 35S::VsPCS1 and wild-type (WT) plants. Further analysis indicated this increased tolerance in 35S::VsPCS1 was possibly due to the increased PCs-chelated Cd in cytosol. Taken together, a functional PCS1 homolog from V. sativa was identified, which hold a strong catalyzed property for the synthesis of high-order PCs that retained Cd in the cytosol rather the vacuole. These findings enrich the original model of Cd detoxification mediated by PCS in higher plants.

Copper (Cu) is an essential micronutrient for plants, but excess Cu can inactivate and disturb the protein function due to unavoidable binding to proteins at the cellular level. As a redox-active metal, Cu toxicity is mediated by the formation of reactive oxygen species (ROS). Cu-binding structural motifs may alleviate Cu-induced damage by decreasing free Cu2+ activity in cytoplasm or scavenging ROS. The identification of Cu-binding proteins involved in the response of plants to Cu or ROS toxicity may increase our understanding the mechanisms of metal toxicity and tolerance in plants. This study investigated change of Cu-binding proteins in radicles of germinating rice seeds under excess Cu and oxidative stress using immobilized Cu2+ affinity chromatography, two-dimensional electrophoresis, and mass spectra analysis. Quantitative image analysis revealed that 26 protein spots showed more than a 1.5-fold difference in abundances under Cu or H2O2 treatment compared to the control. The identified Cu-binding proteins were involved in anti-oxidative defense, stress response and detoxification, protein synthesis, protein modification, and metabolism regulation. The present results revealed that 17 out of 24 identified Cu-binding proteins have a similar response to low concentration Cu and H2O2 stress, and 5 out of 24 were increased under low and high concentration Cu but unaffected under H2O2 stress, which hint Cu ions can regulate Cu-binding proteins accumulation by H2O2 or no H2O2 pathway to cope with excess Cu in cell. The change pattern of these Cu-binding proteins and their function analysis warrant to further study the roles of Cu ions in these Cu-binding proteins of plant cells.

To better understand the mechanisms involved in the heavy metal stress response and tolerance in plants, a proteomic approach was used to investigate the differences in Cu-binding protein expression in Cu-tolerant and Cu-sensitive rice varieties. Cu-binding proteins from Cu-treated rice roots were separated using a new IMAC method in which an IDA-sepharose column was applied prior to the Cu-IMAC column to remove metal ions from protein samples. More than 300 protein spots were reproducibly detected in the 2D gel. Thirty-five protein spots exhibited changes greater than 1.5-fold in intensity compared to the control. Twenty-four proteins contained one or more of nine putative metal-binding motifs reported by Smith et al., and 19 proteins (spots) contained one to three of the top six motifs reported by Kung et al. The intensities of seven protein spots were increased in the Cu-tolerant variety B1139 compared to the Cu-sensitive variety B1195 (p

Hydrogen gas (H2) was recently proposed as a therapeutic antioxidant and signaling molecule in clinical trials. However, the underlying physiological roles of H2 in plants remain unclear. In the present study, hydrogen-rich water (HRW) was used to characterize the physiological roles of H2 in enhancing the tolerance of Brassica campestris against cadmium (Cd). The results showed that both 50 μM CdCl2 and 50%-saturated HRW induced an increase of endogenous H2 in Brassica campestris seedlings, and HRW alleviated Cd toxicity related to growth inhibition and oxidative damage. Seedlings supplied with HRW exhibited increased root length and reduced lipid peroxidation, similar to plants receiving GSH post-treatment. Additionally, seedlings post-treated with HRW accumulated higher levels of reduced glutathione (GSH) and ascorbic acid (AsA) and showed increased GST and GPX activities in roots. Molecular evidence illustrated that the expression of genes such as GS, GR1 and GR2, which were down-regulated following the addition of Cd, GSH or BSO, could be reversed to varying degrees by the addition of HRW. Based on these results, it could be proposed that H2 might be an important regulator for enhancing the tolerance of Brassica campestris seedlings against Cd, mainly by governing reduced glutathione homeostasis.

Differences in copper (Cu) absorption and transport, physiological responses and structural characteristics between two types of Cu-resistant plants, Oenothera glazioviana (Cu-exclusion type) and Elsholtzia haichowensis (Cu-enrichment type), were investigated in the present study. The results indicated the following: (1) After 50 μM Cu treatment, the Cu ratio in the xylem vessels of E. haichowensis increased by 60%. A Cu adsorption experiment indicated that O. glazioviana exhibited greater resistance to Cu, and Cu absorption and the shoot/root ratio of Cu were significantly lower in O. glazioviana than in E. haichowensis. (2) An analysis of the endogenous abscisic acid (ABA) variance and exogenous ABA treatment demonstrated that the ABA levels of both plants did not differ; exogenous ABA treatment clearly reduced Cu accumulation in both plants. (3) The leaf stomatal density of O. glazioviana was significantly less than that of E. haichowensis. Guard cells in E. haichowensis plants were covered with a thick cuticle layer, the epidermal hair was more numerous and longer, and the number of xylem conduits in the root was small. (4) The transpiration rate and the stomatal conductance of O. glazioviana were both significantly lower than those of E. haichowensis, regardless of whether the plants were treated with Cu. Taken together, these results indicate that the differences in the structural characteristics between these two plant species, particularly in the characteristics related to plant transpiration, are important factors that govern whether plants acquire or exclude Cu.