Cloning of PsMYB62 and analysis of cadmium resistant in Potentilla sericea

Potentilla sericea is a heavy metal hyperaccumulator landscaping plant.MYB transcription factors play an important role in regulating plant stress response to adversity.However,there are few studies on MYB transcription factors in stress tolerance in Potentilla sericea.In this study,the PsMYB62 gene was successfully cloned from Potentilla sericea.Methods:Bioinformatic analysis and real-time quantitative PCR(qPCR)methods were used to evaluate this gene.The transgenic A.thaliana were obtained by flower dipping and the gene function was identified by determining physiological indicators under cadmium stress.Results:The open reading frame of PsMYB62 is 942 bp,which encodes 313 amino acids(aa)and belongs to the R2R3 MYB transcription factor.The plant overexpression vector PBI121-PsMYB62-GFP was constructed and successfully transferred into A.thaliana.The relative expression level of PsMYB62 was significantly increased by CdCl_(2),NaCl,ABA,and mannitol treatments.The germination rate of transgenic seeds was higher than those of wild type(WT)and empty vector(EV)under different concentrations of cadmium treatment.Upon treatment with 100μmol·L^(−1)of CdCl_(2)·2.5H_(2)O,the activities of superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT)in the transgenic plants were significantly higher than those in the WT and EV.The contents of H_(2)O_(2),O_(2)·−and malondialdehyde(MDA)in transgenic lines were increased,but lower than those in WT and EV.The expression levels of AtGSH,AtPCS,and AtNAS4 that were related to the regulation of cadmium were increased,but the expression levels of transgenic lines were higher than those of WT and EV.Conclusion:The above results showed that PsMYB62 could be induced by cadmium and could improve the cadmium resistance of plants.

Effect of Cadmium Stress on the Growth, Antioxidative Enzymes and Lipid Peroxidation in Two Kenaf (Hibiscus cannabinus L.) Plant Seedlings

The effects of cadmium stress on the growth, antioxidative enzymes and lipid peroxidation in two kenaf plants, Fuhong 991 and ZM412, were analysed under control （0.5-strength Hoagland＇s nutrient solution） or five levels of cadmium stress （0.5- strength Hoagland＇s nutrient solution containing different concentrations of Cd2＋）. The leaves and roots of control and cadmium-stressed plants were harvested after 3 wk. At the same Cd concentration, the Cd tolerance index of Fuhong 991 was higher than that of ZM412, indicating that Fuhong 991 may be more tolerant to Cd than ZM412. The superoxide dismutase （SOD）, catalase activity （CAT） and peroxidase （POD） activities fluctuated in the leaves of the Cd-stressed plants compared to the control, whereas the glutathione reductase activity （GR） was much larger than the control for Fuhong 991, ensuring that sufficient quantities of GSH were available to respond to the cadmium stress. In comparison to the control, the dynamic tendency of the SOD, CAT and POD activities in roots of the Cd-stressed plants all increased and then declined, but the POD activity of Fuhong 991 remained nearly unchanged at all of the stress levels. The increase in the enzyme activities demonstrated that Fuhong 991 was more tolerant to cadmium than ZM 412. The lipid peroxidation was enhanced only in the leaves of Cd-stressed ZM 412. These findings indicated that antioxidative activities may play important roles in Cd-stressed Fuhong 991 and ZM 412 and that the leaf and root cell membranes of Fuhong 991 have a greater stability than those of ZM 412. For pollution monitoring purposes, the GR activity in the roots and leaves may serve as a biomarker of Cd for Fuhong 991, whereas lipid peroxidation may serve as biomarker for ZM 412.

Response of Glutathione and Glutathione S-transferase in Rice Seedlings Exposed to Cadmium Stress

A hydroponic culture experiment was done to investigate the effect of Cd stress on glutathione content （GSH） and glutathione S-transferase （GST, EC 2.5.1,18） activity in rice seedlings. The rice growth was severely inhibited when Cd level in the solution was higher than 10 mg/L. In rice shoots, GSH content and GST activity increased with the increasing Cd level, while in roots, GST was obviously inhibited by Cd treatments, Compared with shoots, the rice roots had higher GSH content and GST activity, indicating the ability of Cd detoxification was much higher in roots than in shoots. There was a significant correlation between Cd level and GSH content or GST activity, suggesting that both parameters may be used as biomarkers of Cd stress in rice.

Molecular cloning, sequence analysis, and cadmium stress-rated expression changes of BTG1 in freshwater pearl mussel(Hyriopsis schlegelii)

The B cells translocation gene 1 （BTG1） is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, for the first time we cloned the full-length eDNA sequence of Hyriopsis schlegelii （Hs-BTG1）, an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of eDNA ends （RACE） together with splicing the EST sequence from a haemocyte eDNA library, we found that Hs-BTG1 contains a 525 bp open reading frame （ORF） encoding a 174 amino-acid polypeptide, a 306 bp 5＇ untranslated region （5＇ UTR）, and a 571 bp 3＇ UTR with a Poly（A） tail as well as a transcription termination signal （AATAAA）. Homologne searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the invertebrate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas （2.03-fold）, mantle （2.07-fold）, kidney （2.2-fold） and haemocyte （2.5-fold） were enhanced by cadmium （Cd2＋） stress, suggesting that Hs-BTG 1 may have played a significant role in H, schlegelii adaptation to adverse environmental conditions.

Effect of Biochar on Relieving Cadmium Stress and Reducing Accumulation in Super japonica Rice

It is of great importance to solve the threats induced by cadmium pollution on crops. This paper examined the effect of biochar on cadmium accumulation in japonica rice and revealed the mechanism underlying the response of protective enzyme system to cadmium stress. Biochar derived from rice straw was applied at two application rates under three cadmium concentrations. Shennong 265, super japonica rice variety, was selected as the test crop. The results indicated that cadmium content in above-ground biomass of rice increased with increasing soil cadmium concentrations, but the biochar application could suppress the accumulation of cadmium to some extent. Under high concentrations of cadmium, content of free proline and MDA （malondialdehyde） were high, so did the SOD （superoxide dismutase）, POD （peroxidase） and CAT （catalase） activity in the flag leaf of rice. However, the protective enzyme activities remained at low level when biochar was added.

Purification and Identification of Glutathione S-transferase in Rice Root under Cadmium Stress

Cadmium （Cd） contamination in paddy soils poses a serious threat to the production and quality of rice. Among various biochemical processes related to Cd detoxification in rice, glutathione S-transferase （GST） plays an important role, catalyzing Cd complexation with glutathione （GSH） and scavenging reactive oxygen species （ROS） in cells. In this study, a hydroponic experiment was conducted to investigate the response of GST isozymes in rice roots upon Cd exposure. Results showed that the GST activity in rice roots was clearly enhanced by 50 pmol/L Cd treatment for 7 d. The GST isozymes were purified by ammonium sulphate precipitation, gel filtration chromatography and affinity chromatography. After being separated by SDS-PAGE and visualized by silver staining, GSTU6 was identified by in-gel digestion, MALDI-TOF-MS analysis and peptide mass fingerprint. The results confirm the vital function of tau class rice GST in Cd detoxification.

Effects of Melatonin on Growth,Physiology and Gene Expression in Rice Seedlings Under Cadmium Stress

Melatonin(MLT)is a hormonal substance found in many organisms and can improve plant stress resistance.In this study,the japonica rice variety Y32 and indica rice variety NJ6 were cultivated in hydroponics under different concentrations of CdCl2 at the two-leaf stage.The growth,physiological and biochemical responses of the seedlings and the expression of cadmium(Cd)-related genes under exogenous melatonin(MLT)treatment were assessed.The results indicated that Cd stress destroyed the dynamic balance between reactive oxygen species(ROS)production and removal,resulting in ROS accumulation,membrane lipid peroxidation,and impaired growth and development.Following the application of exogenous MLT to rice seedlings,increases in plant biomass including both underground and above-ground areas were observed.MLT also scavenged the inhibition of superoxide dismutase(SOD)and peroxidase(POD)in a concentration dependent manner in response to Cd stress.Catalase(CAT)activity and malondialdehyde(MDA)expression also decreased following MLT treatment.Amongst the six Cd-related genes assessed,five genes were downregulated and one was up-regulated in response to MLT treatment.Taken together,these data demonstrate that MLT improves the resilience of rice seedlings at the biochemical,physiological,and molecular levels,and diminishes the damage caused by Cd stress.

Oxidative stress responses to cadmium in the seedlings of a commercial seaweed Sargassum fusiforme

Cadmium(Cd)is a common heavy metal pollutant in the aquatic environment,generally toxic to plant growth and leading to growth inhibition and biomass reduction.To study the oxidation resistance in Sargassum fusiforme seedlings in response to inorganic Cd stress,we cultured the seedlings under two different Cd levels:natural seawater and high Cd stress.High Cd stress significantly inhibited the seedlings growth,and darkened the thalli color.Additionally,the pigment contents,growth rate,peroxidase(POD)activity,dehydroascorbic acid(DHA)content,and glutathione reductase(GR)activity in S.fusiforme were significantly reduced by high Cd treatment.Contrarily,the Cd accumulation,Cd2+absorption rate,dark respiration/net photosynthetic rate(Rd/Pn),ascorbic acid(Vc)content,soluble protein(SP)content,glutathione(GSH),and the activities of superoxide dismutase(SOD)and catalase(CAT)of S.fusiforme under Cd treatment significantly increased compared to the control group.The decrease of malondialdehyde(MDA)was not significant.Although S.fusiforme seedlings increased the antioxidant activities of POD,SOD,Vc,and the AsA-GSH cycle to disseminate H2O2 and maintain healthy metabolism,high Cd stress caused Cd accumulation in the stem and leaves of S.fusiforme seedlings.The excessive Cd significantly restricted photosynthesis and reduced photosynthetic pigments in the seedlings,resulting in growth inhibition and deep morphological color,especially of the stems.High levels of Cd in seawater had toxic effects on commercial S.fusiforme seedlings,and risked this edible seaweed for human food.

Role of phosphorus on the biogeochemical behavior of cadmium in the contaminated soil under leaching and pot experiments

Phosphorus(P)is involved in various biochemical reactions in plant growth,so it is beneficial to plants growing in soils contaminated by metals,including cadmium(Cd).However,few studies have reported on the mechanistic roles of P in mitigating Cd toxicity to ryegrass root,and especially in alleviating the disruption of the mitochondrial function of living cells.In this study,the physiological and biochemical mechanisms associated with ryegrass growth under various Cd and P treatments were investigated using leaching and pot systems.The concentration of Cd in soil leachates showed a significantly positive relationship with redox potential(P<0.05),but negative relationship(P<0.05)with leachate pH values and dissolved organic carbon(DOC),indicating that exogenous P addition(as H_(2)PO_(4)*)may decrease Cd leaching from contaminated soil.Compared to the control(without P addition),the cumulative Cd content was reduced by 53.3%and 64.5%in the soil leachate with exogenous P application(20 mg/L and 80 mg/L),respectively.Notably,application of P decreased the Cd concentrations in the symplastic fractions and increased the Cd concentrations in the apoplastic fractions in root tips,which may help to alleviate Cd stress to the protoplast.Moreover,exogenous P was found to play a positive role in mitochondrial function and Ca^(2+)variation in root cells under Cd stress,which provides novel insights into the mechanisms of exogenous P in alleviating plant Cd injury.

Cadmium induced a non-coding RNA microRNA535 mediates Cd accumulation in rice

Identifying key regulators related to cadmium(Cd)tolerance and accumulation is the main factor for genetic engineering to improve plants for bioremediation and ensure crop food safety.MicroRNAs(miRNAs),as fine-tuning regulators of genes,participate in various abiotic stress processes.MiR535 is an ancient conserved non-coding small RNA in land plants,positively responding to Cd stress.We investigated the effects of knocking out(mir535)and overexpressing miR535(mir535 and OE535)under Cd stress in rice plants in this study.The mir535 plants showed better Cd tolerance than wild type(WT),whereas the OE535 showed the opposite effect.Cd accumulated approximately 71.9%and 127%in the roots of mir535 and OE535 plants,respectively,compared to WT,after exposure to 2μmol/L Cd.In brown rice,the total Cd accumulation of OE535 and mir535 was about 78%greater and 35%lower than WT.When growing in 2 mg/kg Cd of soil,the Cd concentration was significantly lower in mir535 and higher in OE535 than in the WT;afterward,we further revealed the most possible target gene SQUAMOSA promoter binding-like transcription factor 7(SPL7)and it negatively regulates Nramp5 expression,which in turn regulates Cd metabolism.Therefore,the CRISPR/Cas9 technology may be a valuable strategy for creating new rice varieties to ensure food safety.

The transcription factor NAC102 confers cadmium tolerance by regulating WAKL11 expression and cell wall pectin metabolism in Arabidopsis

Cadmium(Cd) toxicity severely limits plant growth and development. Moreover, Cd accumulation in vegetables, fruits, and food crops poses health risks to animals and humans. Although the root cell wall has been implicated in Cd stress in plants, whether Cd binding by cell wall polysaccharides contributes to tolerance remains controversial, and the mechanism underlying transcriptional regulation of cell wall polysaccharide biosynthesis in response to Cd stress is unknown. Here, we functionally characterized an Arabidopsis thaliana NAC-type transcription factor, NAC102, revealing its role in Cd stress responses. Cd stress rapidly induced accumulation of NAC102.1, the major transcript encoding functional NAC102, especially in the root apex. Compared to wild type(WT) plants, a nac102 mutant exhibited enhanced Cd sensitivity, whereas NAC102.1-overexpressing plants displayed the opposite phenotype. Furthermore, NAC102 localizes to the nucleus, binds directly to the promoter of WALL-ASSOCIATED KINASE-LIKE PROTEIN11(WAKL11), and induces transcription, thereby facilitating pectin degradation and decreasing Cd binding by pectin. Moreover, WAKL11 overexpression restored Cd tolerance in nac102mutants to the WT levels, which was correlated with a lower pectin content and lower levels of pectin-bound Cd. Taken together, our work shows that the NAC102-WAKL11 module regulates cell wall pectin metabolism and Cd binding, thus conferring Cd tolerance in Arabidopsis.

Hydrogen Peroxide-Mediated Growth of the Root System Occurs via Auxin Signaling Modification and Variations in the Expression of Cell-Cycle Genes in Rice Seedlings Exposed to Cadmium Stress

The link between root growth, H2O2, auxin signaling, and the ceil cycle in cadmium （Cd）-stressed rice （Oryza sativa L. cv. Zhonghua No. 11） was analyzed in this study. Exposure to Cd induced a significant accumulation of Cd, but caused a decrease in zinc （Zn） content which resulted from the decreased expression of OsHMA9 and OsZIP. Analysis using a Cd-specific probe showed that Cd was mainly localized in the meristematic zone and vascular tissues. Formation and elongation of the root system were significantly promoted by 3-amino-l,2,4-triazole （AT）, but were markedly inhibited by N,N＇. dimethylthiourea （DMTU） under Cd stress. The effect of H2O2 on Cd-stressed root growth was further confirmed by examining a gain-of-function rice mutant （carrying catalasel and glutathione-S-transferase） in the presence or absence of diphenylene iodonium. DR5-GUS staining revealed close associations between H2O2 and the concentration and distribution of auxin. H2O2 affected the expression of key genes, including OsYUCCA, OsPIN, OsARF, and OslAA, in the auxin signaling pathway in Cd-treated plants. These results suggest that H2O2 functions upstream of the auxin signaling pathway. Furthermore, H2O2 modified the expression of cell-cycle genes in Cd-treated roots. The effects of H2O2 on root system growth are therefore linked to auxin signal modification and to variations in the expression of cell-cycle genes in Cd-stressed rice. A working model for the effects of H2O2 on Cd-stressed root system growth is thus proposed and discussed in this paper.

Different Responses of Plant Growth and Antioxidant System to the Combination of Cadmium and Heat Stress in Transgenic and Non-transgenic Rice

A comparative study of just cadmium （Cd） or heat and their combination treatments on some physiological parameters and the antioxidant systems in transgenic rice （Oryza sativa L. cv. Zhonghua No.11） carrying glutathione-S-transferase （GST, EC. 2.5.1.18） and catalasel （CAT1, EC. 1.11.1.6） and non-transgenics was conducted. The results revealed improved resistance in the transgenics to Cd and the combined Cd and heat stress than non-transgenics. Data showed that the activities of CAT, GST, superoxide dismutase （EC.1.15.1.1） and all components of the ascorbate-glutatbione cycle measured in the stressed transgenics shoots are significantly different from those of non-transgenics. Results indicated that co-expression of GST and CAT1 had an important effect on the antioxidant system, in particular, the whole ascorbate-glutathione cycle. The less oxidative damage induced by Cd and the stress combination in the transgenics resulted not only from the GST and CAT1 transgene but also from the coordination of the whole ascorbate-glutathione cycle.

bHLH104 confers tolerance to cadmium stress in Arabidopsis thaliana

Cd is a non-essential heavy metal that is toxic to both plants and animals. Here, we reveal that the transcription factor bHLH104 positively regulates Cd tolerance in Arabidopsis thaliana. We show that Fe deficiency-responsive genes were induced by Cd treat- ment, and that their upregulation was suppressed in bhlh104 loss-of-function mutants, but enhanced upon overexpression of bHLH104. Correspondingly, the bhlh104 mutants displayed sensitivity to Cd stress, whereas plants overexpressing bHLH104 exhibited enhanced Cd tolerance. Further analysis suggested that bHLH104 positivelyregulates four heavy metal detoxification-associated genes, IREG2, A4TP3, HA4A3 and NAS4, which play roles in Cd sequestration and tolerance. The bHLH104 overexpres- sion plants accumulated high levels of Cd in the root but low levels of Cd in the shoot, which might contribute to the Cd tolerance in those lines. The present study thus points to bHLH104 as a potentially useful tool for genetic engineering of plants with enhanced Cd tolerance.

A novel aldo-keto reductase gene, IbAKR, from sweet potato confers higher tolerance to cadmium stress in tobacco

High concentrations of Cd can inhibit growth and reduce the activity of the photosynthetic apparatus in plants. In several plant species, aldo-keto reductases(AKRs) have been shown to enhance tolerance to various abiotic stresses by scavenging cytotoxic aldehydes;however, few AKRs have been reported to enhance Cd stress tolerance. In this study, the gene Ib AKR was isolated from sweet potato. The relative expression levels of Ib AKR increased significantly(approximately 3-fold) after exposure to 200 mmol$L–1 Cd Cl2 or 10 mmol$L–1 H2 O2. A subcellular localization assay showed that Ib AKR is predominantly located in the nucleus and cytoplasm.Ib AKR-overexpressing tobacco plants showed higher tolerance to Cd stress than wild-type(WT). Transgenic lines showed a significant ability to scavenge malondialdehyde(MDA) and methylglyoxal(MG). In addition,proline content and superoxide dismutase activity were significantly higher and H2 O2 levels were significantly lower in the transgenic plants than in the WT. Quantitative real-time PCR analysis showed that the reactive oxygen species(ROS) scavenging genes encoding guaiacol peroxidase(GPX), ascorbate peroxidase(APX), monodehydroascorbate reductase(MDHAR) and peroxidase(POD) were significantly upregulated in transgenic plants compared to WT under Cd stress. These findings suggest that overexpressing Ib AKR enhances tolerance to Cd stress via the scavenging of cytotoxic aldehydes and the activation of the ROS scavenging system.

Root cell walls and phytochelatins in low-cadmium cultivar of Brassica parachinensis

‘Lvbao-701’ is a cultivar of Chinese flowering cabbage(Brassica parachinensis) that exhibits low cadmium(Cd) accumulation and high Cd tolerance.In this study, this cultivar was compared with a high-Cd accumulating cultivar, ‘Chixin-4’, to characterize the mechanisms influencing Cd accumulation in B. parachinensis. Root cell walls were isolated by dissolving the cytoplasm with an organic solvent, and root Cd and phytochelatin(PC) contents were analyzed. In addition, a PC synthase gene fragment was cloned and expressed under Cd stress conditions. The proportions of Cd bound to root cell walls were higher in the ‘Lvbao-701’ plants(68.32%–76.80%) than in the ‘Chixin-4’ plants(35.36%–54.18%) after exposure to Cd stress. The proportions of Cd bound to root cell walls measured using cell walls isolated with a non-destructive method were higher than those obtained using a conventional method that required grinding and centrifugation. Exposure to Cd stress induced the PC production and resulted in higher PC contents in the ‘Lvbao-701’ plants than in the ‘Chixin-4’ plants. Cloning and expression analysis of a B. parachinensis PC synthase cDNA fragment indicated that PC synthase gene expression was induced by Cd and occurred mainly in the roots of both ‘Lvbao-701’ and ‘Chixin-4’ plants. However, the PC synthase gene expression level was higher in the‘Lvbao-701’ roots than in the ‘Chixin-4’ roots. Therefore, a higher abundance of Cd in the root cell walls of ‘Lvbao-701’ and up-regulated PC production in the roots are probably the main reasons why ‘Lvbao-701’ exhibits lower Cd translocation to the shoots and higher tolerance to Cd than ‘Chixin-4’.