The Expression of the StNRAMP2 Gene Determined the Accumulation of Cadmium in Different Tissues of Potato.

Cadmium (Cd) is a toxic metal that threatens human health when enriched in crops. NRAMPs are a family of natural macrophage proteins reported to play a key role in Cd transport in plants. In order to explore the gene regulation mechanism of potato under Cd stress and the role of NRAMPs family in it, this study analyzed the gene expression differences of two different Cd accumulation levels in potato after 7 days of 50 mg/kg Cd stress and screened out the key genes that may play a major role in the differential accumulation of Cd in different varieties. Additionally, StNRAMP2 was selected for verification. Further verification showed that the StNRAMP2 gene plays an important role in the accumulation of Cd in potato. Interestingly, silencing StNRAMP2 increased Cd accumulation in tubers but significantly decreased Cd accumulation in other sites, suggesting a critical role of StNRAMP2 in Cd uptake and transport in potatoes. To further confirm this conclusion, we performed heterologous expression experiments in which overexpression of StNRAMP2 gene in tomato resulted in a threefold increase in Cd content, which further confirmed the important role of StNRAMP2 in the process of Cd accumulation compared with wild-type plants. In addition, we found that the addition of Cd to the soil increased the activity of the plant antioxidant enzyme system, and silencing StNRAMP2 partially reversed this effect. This suggests that the StNRAMP2 gene plays an important role in plant stress tolerance, and future studies could further explore the role of this gene in other environmental stresses. In conclusion, the results of this study improve the understanding of the mechanism of Cd accumulation in potato and provide experimental basis for remediation of Cd pollution.

Unraveling the Mechanism of StWRKY6 in Potato (Solanum tuberosum)'s Cadmium Tolerance for Ensuring Food Safety.

The WRKY transcription factor plays a crucial role in plant stress adaptation. Our research has found that WRKY6 in Solanum tuberosum (potatoes) is closely related to cadmium (Cd) tolerance. Therefore, investigating the mechanism of StWRKY6 in plant resistance to Cd toxicity is of great scientific importance for food safety. This research further analyzed the gene structure and functional regions of the nuclear transcription factor WRKY6 in potatoes, discovering that StWRKY6 contains W box, GB/box, ABRE, and other elements that can act as a nuclear transcription regulatory factor to execute multiple functional regulations. The results of the heterologous expression of StWRKY6 in Arabidopsis under Cd stress showed that the overexpression line (StWRKY6-OE) had significantly higher SAPD values and content of reactive oxygen species scavenging enzymes than the wild type, indicating that StWRKY6 plays a crucial role in protecting the photosynthetic system and promoting carbohydrate synthesis. Transcriptome analysis also revealed that the Cd-induced expression of StWRKY6 up-regulated many potential gene targets, including APR2, DFRA, ABCG1, VSP2, ERF013, SAUR64/67, and BBX20, which are involved in Cd chelation (APR2, DFRA), plant defense (VSP2, PDF1.4), toxic substance efflux (ABCG1), light morphology development (BBX20), and auxin signal (SAUR64/67). These genes coordinate the regulation of Cd tolerance in the StWRKY6 overexpression line. In summary, this study identified a potential gene set of the co-expression module of StWRKY6, providing useful evidence for the remediation of Cd-contaminated soil and the genetic breeding of low Cd-accumulating crops, thereby ensuring food safety.

Synergistic Effects of Water Management and Silicon Foliar Spraying on the Uptake and Transport Efficiency of Cadmium in Rice (Oryza sativa L.).

To study the synergistic effects of water management and silicon (Si) foliar spraying on the uptake and transport of cadmium (Cd) in rice, we designed four treatments: conventional intermittent flooding + no Si foliar spraying (CK), continuous flooding throughout the growth stage + no Si foliar spraying (W), conventional intermittent flooding + Si foliar spraying (Si) and continuous flooding throughout the growth stage + Si foliar spraying (WSi). The results show that WSi treatment reduced the uptake and translocation of Cd by rice and significantly reduced the brown rice Cd content, with no effect on rice yield. Compared with CK, the Si treatment increased the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) of rice by 6.5-9.4%, 10.0-16.6% and 2.1-16.8%, respectively. The W treatment decreased these parameters by 20.5-27.9%, 8.6-26.8% and 13.3-23.3%, respectively, and the WSi treatment decreased them by 13.1-21.2%, 3.7-22.3% and 2.2-13.7%, respectively. The superoxide dismutase (SOD) and peroxidase (POD) activity decreased by 6.7-20.6% and 6.5-9.5%, respectively, following the W treatment. Following the Si treatment, SOD and POD activity increased by 10.2-41.1% and 9.3-25.1%, respectively, and following the WSi treatment, they increased by 6.5-18.1% and 2.6-22.4%, respectively. Si foliar spraying ameliorated the detrimental effects of continuous flooding throughout the growth stage on photosynthesis and antioxidant enzyme activity. We conclude that synergistic continuous flooding throughout the growth stage, combined with Si foliar spraying, can significantly block Cd uptake and translocation and is therefore an effective means of reducing the accumulation of Cd in brown rice.

Sensitive or tolerant functional microorganisms under cadmium stress: suggesting potential specific interaction network characteristics in the rhizosphere system of karst potato.

The heavy metal cadmium (Cd) pollution in Chinese karst soils threatens food security, and microorganisms play an important role in regulating the migration and transformation of Cd in the soil-plant system. Nevertheless, the interaction characteristics between key microbial communities and environmental factors in response to Cd stress in specific crop environmental systems need to be explored. In this study, the soil (ferralsols)-microbe-crop (potato) system was taken as the object to explore the potato rhizosphere microbiome, using toxicology and molecular biology approaches, to explore the potato rhizosphere soil properties, microbial stress characteristics, and important microbial taxa under Cd stress. We hypothesized that different members of fungal and bacterial microbiome would regulate the resilience of potato rhizosphere and plants to Cd stress in the soil environment. Meanwhile, individual taxa will have different roles in the contaminated rhizosphere ecosystem. We found that soil pH was the main environmental factor affecting fungal community structure; urea-decomposing and nitrate-reducing functional bacteria as well as endosymbiotic and saprophytic functional fungi gradually decreased. In particular, Basidiomycota may play a key role in preventing the migration of Cd from the soil to plants (potato). These findings provide important candidates for screening the cascade of Cd inhibition (detoxification/regulation) from soil to microorganisms to plants. Our work provides an important foundation and research insights for the application of microbial remediation technology in the karst cadmium-contaminated farmland.

Effect of amendments on bioavailability of cadmium in soil-rice system: a field experiment study.

The field experiment study investigated the effect of lime (L), manure compost (M), combination of lime and manure (LM), and combinations of lime with four kinds of passivators (LP1, LP2, LP3, and LP4) on the bioavailability of cadmium (Cd) in soil and Cd accumulation in rice plants. These four passivating products were composed of organic and inorganic compounds such as silicon-sulfhydryl group, CaO, SiO2, and so on. The results indicated that the application of these amendments improved soil pH, organic matter content, and cation exchange capacity (CEC) by 0.19-0.73 unit, 0.6-8.2%, and 5.7-38.9%, respectively; meanwhile, decreased soil acid-extractable Cd by 4.0-13.9% compared with before remediation. Alleviating Cd stress to rice also resulted in a significant increase in rice grains yield, whereas the LP4 showed an increment of 15.8-27.6%. Among these amendments, LP4 had a relatively high effectiveness, it promoted the decrease of extractable Cd by 13.9% and the increase of residual Cd by 8.1%; meanwhile, the bioconcentration factor of rice grain in LP4 decreased by 71.3%. The high pH, CEC, and rich functional groups in amendments might cause soil Cd transform from mobile fraction to residual fraction, and the cation ions in amendments also competed with Cd ions due to the antagonism. Taken all of these effects, the amendments alleviated Cd pollution in soil-rice system, decreasing Cd migration from soil to grain. In future, the long-term field experiment will need to be done for verify the long-term effect of soil amendments.

Discovery of cadmium-tolerant biomacromolecule (StCAX1/4 transportproteins) in potato and its potential regulatory relationship with WRKY transcription factors.

The cation/H+ exchanger (CAX) involved in Ca2+, Mg2+ and Mn2+ transport is a special class of vacuolar transporters that play an important role in maintaining ion homeostasis in plant cells. However, it has been rarely reported whether CAX proteins have unique tolerance to cadmium stress. In our research, the cadmium-resistant potato variety "Yunshu 505" was taken as the object, through biological etc. methods, explored 1: response mode of StCAXs to cadmium stress; 2: the evolutionary characteristics and Cd ion binding sites of StCAXs; and 3: possible upstream regulatory pathways of StCAXs. The results showed that cadmium stress significantly induced the expression of StCAX1/4, and there were specific mutations in the evolution process, thus the possible main binding site of Cd ion (EDEE/DH/GxxxxxS/EEEE) was speculated. StCAX1/4 interacts with several proteins, and be regulated by transcription factors, especially the WRKY6. This synergistic regulation through WRKY6 may be an important pathway through which StCAX1/4 imparts high cadmium tolerance to potato. These results provide certain support for understanding the binding sites and specific evolutionary mechanisms of key amino acid residues of cadmium ion in StCAXs, also provide new clues for the identification and regulatory model of potato CAX key positive stress-responsive proteins under cadmium stress.

Identification of the Major Effector StSROs in Potato: A Potential StWRKY-SRO6 Regulatory Pathway Enhances Plant Tolerance to Cadmium Stress.

SIMILAR TO RCD-ONE (SRO) family members and transcription factors (TFs) often improve plant antioxidant capacity through interaction and co-regulation and participate in plant resistance to drought and high-salt stress. However, whether SROs are involved in the response to heavy metal stress, especially SRO genes with a specific response and tolerance characteristics to cadmium (Cd) stress, remains unclear. We first identified six SRO genes in the potato genome by PARP and RST domains. Special and conserved StSROs were found, and the spatio temporal tissue-specific expression patterns and co-expression network diagrams of StSROs under the stress of 5 heavy metals were constructed. Second, we identified StSRO6 as a major effector gene (StSRO6-MEG) and StSRO5 as a secondary effector gene (StSRO5-SEG) through a comprehensive analysis. Interestingly, they may hold true for various physiological or stress responses in plants. In addition, using systematic genomics and comparative omics techniques, the key gene StSRO6 that affects the difference in Cd accumulation was discovered, cloned in the low-Cd accumulation "Yunshu 505", and transformed into the yeast mutant ycf1 for overexpression. The results proved that StSRO6 could confer Cd tolerance. Finally, through transient expression and in vitro culture tests, we hypothesized that StSROs 5/6 are regulated by the transcription factor StWRKY6 and mediates the reactive oxygen species (ROS) system to confer Cd tolerance. These findings offer a new perspective for understanding the mechanisms underlying Cd tolerance in plants, and simultaneously provide clues for the development of biological agents for preventing and controlling Cd migration and transformation.

Removal of organochlorine pesticides and metagenomic analysis by multi-stage constructed wetland treating landfill leachate.

Constructed wetlands (CWs) can effectively treat landfill leachate (LL). However, there is limited research on the removal of organochlorine pesticides (OCPs) refractory organics during LL treatment in CWs. In this study, multi-stage subsurface flow CWs was used to treat LL, and the removal fate of hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) in CWs was investigated. The structural differences between plant roots and substrate microbial communities were compared and the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway of organic matter was analyzed based on metagenomic analysis. The results showed that substrate adsorption (50.55%-72.74%) and microbial degradation (20.38%-27.89%) were the main ways to remove OCPs. The Proteobacteria occupied a dominant position in the CWs system, among which Betaproteobacteria (34.37%-35.90%) were contained in the substrate, and Alphaproteobacteria (21.19%-23.84%) was a more dominant microorganism in plant roots. Formaldehyde assimilation and serine pathway were the main pathways of methane metabolism. This study provides a reference for the removal mechanism of OCPs to promote the application of CWs technology in LL treatment.

Metalloprotein-Specific or Critical Amino Acid Residues: Perspectives on Plant-Precise Detoxification and Recognition Mechanisms under Cadmium Stress.

Cadmium (Cd) pollution in cultivated land is caused by irresistible geological factors and human activities; intense diffusion and migration have seriously affected the safety of food crops. Plants have evolved mechanisms to control excessive influx of Cd in the environment, such as directional transport, chelation and detoxification. This is done by some specific metalloproteins, whose key amino acid motifs have been investigated by scientists one by one. The application of powerful cell biology, crystal structure science, and molecular probe targeted labeling technology has identified a series of protein families involved in the influx, transport and detoxification of the heavy metal Cd. This review summarizes them as influx proteins (NRAMP, ZIP), chelating proteins (MT, PDF), vacuolar proteins (CAX, ABCC, MTP), long-distance transport proteins (OPT, HMA) and efflux proteins (PCR, ABCG). We selected representative proteins from each family, and compared their amino acid sequence, motif structure, subcellular location, tissue specific distribution and other characteristics of differences and common points, so as to summarize the key residues of the Cd binding target. Then, we explain its special mechanism of action from the molecular structure. In conclusion, this review is expected to provide a reference for the exploration of key amino acid targets of Cd, and lay a foundation for the intelligent design and breeding of crops with high/low Cd accumulation.

Comparing the effects of calcium and magnesium ions on accumulation and translocation of cadmium in rice.

Rice (Oryza sativa L.) is one of China's most important food crops, and it is considered the primary source of human exposure to cadmium (Cd) pollution. Adding calcium (Ca) and magnesium (Mg) to the plant nutrient solutions reduces the accumulation of Cd in the rice, but under the same condition, which one has the better effect remains unclear. Thus, hydroponic experiments were performed to compare the effects of Ca and Mg ions with concentration gradients (0.10, 0.25, and 0.50 g/L, respectively) on the absorption, distribution, and translocation of Cd in rice. The Cd contents of roots, stems, leaves, panicles, husks, and grains in different growth stages were determined. The results revealed that the supplementation of both Ca and Mg influenced the Cd accumulation and translocation in rice tissues. The Cd concentrations of different patterns were in the following order: roots > stems > leaves ≈ panicles ≈ husks > grains. Both of Ca and Mg had an apparent antagonism with Cd in different parts of the rice plant, and the antagonism was more obvious in the high Cd stress treatments. With the addition of 0.1 g/L Ca2+ and Mg2+ ions, the grain Cd contents increased, while the application of 0.25 and 0.5 g/L Ca2+ and Mg2+ ions reduced grains Cd by 19.08-38.99%, with the average value of 26.75%. Under the same concentrations, the grain Cd content of Ca treatments was lower than that of Mg treatments by 8.74%. In the Ca (Mg)-deficient and Ca (Mg)-sufficient conditions, the husks and panicles accumulated Cd to hinder Cd translocation, respectively. Altogether, the results of this study indicated that Ca had a greater effect for decreasing rice Cd accumulation and translocation than Mg, and the panicle and husk were the important parts for reducing Cd translocation to grain, and these might be a focal point for the future research. It was possible to plant and grow rice in Cd-polluted soil and that the accumulation and translocation of Cd in rice plants could be reduced by optimizing soil nutrient elements.

Identification of StAP2/ERF genes of potato (Solanum tuberosum) and their multiple functions in detoxification and accumulation of cadmium in yest: Implication for Genetic-based phytoremediation.

The discovery of genes responsible for the tolerance to heavy metals is critical for genome-based phytotechnologies. In this study, we exposed potato (Solanum tuberosum L.) to Cd/Pb/Zn/Ni/Cu as an approach to explore the potential genes associated with stress tolerance. Using genome-wide analysis, we identified 181 potential StAP2/ERF genes that were classified into three subgroups. These StAP2/ERF genes were significantly related to heavy metal stress and are more specifically related to Cd tolerance in yeast. Yeast complementation tests showed that the StAP2/ERF129/139 genes (Subgroup 1) decreased Cd accumulation (Cd reduction-type), whilst the StAP2/ERF044/180 genes (Subgroup 2) promoted Cd accumulation in yeast which showed inhibited growth (Cd accumulation-type). The StAP2/ERF075/077/126 genes (Subgroup 3) promoted Cd accumulation and yeast growth (Cd detoxification-type). We used phylogenetic analysis to classify the 181 genes into three Cd tolerant types defined above in which the numbers of Cd reduction, accumulation, and detoxification type genes were 81, 65 and 35 respectively. Also, we performed tandem duplication, phylogenetic, and conserved motifs analysis to characterization the StAP2/ERF genes and results supported their functions in Cd tolerance. Our study showed that StAP2/ERFs is indispensable in Cd uptake and tolerance, and may be useful towards designing gene-modified plants with improved Cd tolerances.

Genome-wide analysis of the NRAMP gene family in potato (Solanum tuberosum): Identification, expression analysis and response to five heavy metals stress.

NRAMP family genes participate in the absorption and transport of heavy metals such as cadmium (Cd), zinc (Zn), copper (Cu), lead (Pb), iron (Fe) and manganese (Mn) and play an important role in the response to heavy metal stress. There is an abundance of research on these genes in bacteria, plants and fungi, although not in S. tuberosum. A total of 48 members(potato(5), Arabidopsis(7), Tomato(9), pepper(9), rice(12) and tobacco(6)) were identified from 6 species (potato (Solanum tuberosum), Arabidopsis thaliana, Tomato (Solanum lycopersicum), pepper (Capsicum annuum), rice (Oryza sativa) and tobacco (Nicotiana attenuate)) and were classified into four subgroups. Across NRAMP gene family members, there are 15 highly conserved motifs that have similar genetic structures and characteristics. In addition, a total of 16 pairs of colinear genes were found in eight species. Analysis of cis-elements indicated that, in response to abiotic stress, NRAMPs are mainly regulated by phytohormones and transcription factors. In addition, analysis of expression profiles indicated that StNRAMP4 is mainly expressed in the roots. According to a qRT-PCR-based analysis of the StNRAMP family, with the exception of Pb2+ stress, StNRAMPs positively responded to stress from Cu2+, Cd2+, Zn2+ and Ni2+ and The expression patterns is similar of StNRAMP2, under Pb2+, and Cu2+ treatment, the relative expression peaked at 24 h. the relative expression peaked at 12 h and was upregulated 428-fold in the roots under Ni2+ stress. Under Cd2+ stress, StNRAMP3 was upregulated 28-fold in the leaves. StNRAMP1, StNRAMP4 and StNRAMP5 showed significant upregulation under Cu2+, Cd2+ and Zn2+ stress, respectively. Expression of StNRAMPs could be specifically induced by heavy metals, implying their possible role in the transport and absorption of heavy metals. This research explains the colinear characteristics of NRAMPs in several food crop species, which is useful for providing important genetic resources for cultivating food crop that accumulate low amounts of heavy metals and for explaining the biological functions of NRAMPs in plants.