Combined analysis of Polygonum cuspidatum transcriptome and metabolome revealed that PcMYB62, a transcription factor, responds to methyl jasmonate and inhibits resveratrol biosynthesis.

A comparative transcriptomic and metabolomic analysis of Polygonum cuspidatum leaves treated with MeJA was carried out to investigate the regulatory mechanisms of its active compounds. A total of 692 metabolites and 77,198 unigenes were obtained, including 200 differentially accumulated metabolites and 6819 differentially expressed genes. We screened potential regulatory transcription factors involved in resveratrol and flavonoids biosynthesis, and successfully identified an MYB transcription factor, PcMYB62, which could significantly decrease the resveratrol content in P. cuspidatum leaves when over-expressed. PcMYB62 could directly bind to the MBS motifs in the promoter region of stilbene synthase (PcSTS) gene and repress its expression. Besides, PcMYB62 could also repress PcSTS expression and resveratrol biosynthesis in transgenic Arabidopsis thaliana. Our results provide abundant candidate genes for further investigation, and the new finding of the inhibitory role of PcMYB62 on the resveratrol biosynthesis could also potentially be used in metabolic engineering of resveratrol in P. cuspidatum.

Chlorella pyrenoidosa as a potential bioremediator: Its tolerance and molecular responses to cadmium and lead.

Heavy metal contamination negatively affects plants and animals in water as well as soils. Some microalgae can remove heavy metal contaminants from wastewater. The aim of this study was to screen green microalgae (GM) to identify those that tolerate high concentrations of toxic heavy metals in water as possible candidates for phytoremediation. Analyses of the tolerance, physiological parameters, ultrastructure, and transcriptomes of GM under Cd/Pb treatments were conducted. Compared with the other GM, Chlorella pyrenoidosa showed stronger tolerance to high concentrations of Cd/Pb. The reduced glutathione content and peroxidase activity were higher in C. pyrenoidosa than those in the other GM. Ultrastructural observations showed that, compared with other GM, C. pyrenoidosa had less damage to the cell surface and interior under Cd/Pb toxicity. Transcriptome analyses indicated that the "peroxisome" and "sulfur metabolism" pathways were enriched with differentially expressed genes under Cd/Pb treatments, and that CpSAT, CpSBP, CpKAT2, Cp2HPCL, CpACOX, CpACOX2, and CpACOX4, all of which encode antioxidant enzymes, were up-regulated under Cd/Pb treatments. These results show that C. pyrenoidosa has potential applications in the remediation of polluted water, and indicate that antioxidant enzymes contribute to Cd/Pb detoxification. These findings will be useful for producing algal strains for the purpose of bioremediation in water contamination.

De novo assembly and comparative analysis of the mitochondrial genome of Reynoutria japonica.

Reynoutria japonica Houtt. is an important medical plant with a long history of thousands of years in China, however, its mitochondrial genome (mitogenome) has not been reported yet. In this work, we reported and analyzed the R. japonica mitogenome. The main results include: The R. japonica mitogenome was 302,229 bp in length and encoded 48 genes, including 27 protein-coding genes (PCGs), 3 rRNA genes, and 18 tRNA genes. Repeat sequence analysis revealed that there were 54 repeat sequences ranging from 193 bp to 1,983 bp in the R. japonica mitogenome. Relative synonymous codon usage (RSCU) analysis showed that leucine (900, 11.01%) and serine (732, 8.96%) were the two most abundant amino acids, and the codons with RSCU values showed the preference of A or T ending when greater than 1. The RNA editing sites of PCGs in the R. japonica mitogenome were characterized, and 299 RNA editing sites were found. Extensive sequences transfer between mitochondrion and chloroplast were found in R. japonica, where 11 complete plastid-derived tRNA genes stayed intact in the R. japonica mitogenome. Three genes (ccmFC, cox1, and nad1) were seen to play essential roles in the evolution through selection pressure analysis. The phylogenetic analysis showed that Fallopia multiflora was the closest species with R. japonica, in consistency with the results of chloroplast genome. Overall, the current work presents the first mitogenome of R. japonica and could contribute to the phylogenetic analysis of the family Polygonaceae.

Plant cadmium resistance 10 enhances tolerance to toxic heavy metals in poplar.

Toxic heavy metals originating from human activities have caused irreversible harm to the environment. Toxic heavy metal ions absorbed by crop plants can seriously threaten human health. Therefore, decreasing heavy metal contents in crop plants is an urgent need. The plant cadmium resistance protein (PCR) is a heavy metal ion transporter. In this study, PePCR10 was cloned from Populus euphratica. Bioinformatics analyses revealed its transmembrane structure and gene sequence motifs. The transcript profile of PePCR10 was analyzed by RT-qPCR, and its transcript levels increased under toxic heavy metal (cadmium, lead, aluminum) treatments. Subcellular localization analyses in tobacco cells revealed that PePCR10 localizes at the plasma membrane. Compared with wild type (WT), PePCR10-overexpressing lines showed significantly higher values for plant height, root length, fresh weight, and dry weight under heavy metal stress. Electrolyte leakage, nitroblue tetrazolium staining, and chlorophyll fluorescence analyses indicated that Cd/Al tolerance in PePCR10-overexpressing lines was stronger than that in WT. The Cd/Al contents were lower in the PePCR10-overexpressing lines than in WT under Cd/Al stress. Our results show that PePCR10 can reduce the heavy metal content in poplar and enhance its Cd/Al tolerance. Hence, PePCR10 is a candidate genetic resource for effectively reducing heavy metal accumulation in crops.

New potential transporter CIPAS8 enhances cadmium hypersensitivity and cobalt tolerance.

KEY MESSAGE: CIPAS8 is a novel Cd-influx and Co-efflux transporters, and Ser86 and Cys128 might play a decisive role in Co-binding and translocation. Cadmium (Cd) is among the most toxic heavy metals and is a widespread environmental pollutant. Cobalt (Co) is a mineral nutrient that is essential for plant growth and development, but high concentrations may be toxic. Cadmium-induced protein AS8 (CIPAS8) is widely distributed among plant species and might be induced by heavy metals, but its function has not been studied previously. In this study, Populus euphratica PeCIPAS8 and Salix linearistipularis SlCIPAS8 were investigated. The transcription of both genes was significantly enhanced under Cd and Co stresses. PeCIPAS8 and SlCIPAS8 conferred sensitivity to Cd in transgenic yeast, allowing higher quantities of Cd to accumulate within the cells, whereas SlCIPAS8 also conferred tolerance to Co and reduced Co accumulation. The determinants of substrate selectivity of the SlCIPAS8 protein were examined by site mutagenesis, which indicated that the Ser at 86th (S86) substituted for Arg (R) [S86R] and Cys at 128th (C128) substituted for Ser [C128S] mutations limited the protein's capability for Co translocation. These results suggested that PeCIPAS8 and SlCIPAS8 may be involved in Cd uptake into the plant cell. SlCIPAS8 can reduce excess Co accumulation to maintain intracellular Co homeostasis, and the site mutations S86R and C128S were essential for Co transport. These findings provide insight into the function of CIPAS8 and highlight its potential for utilization in phytoremediation applications.

An endoplasmic reticulum-associated degradation-related E2-E3 enzyme pair controls grain size and weight through the brassinosteroid signaling pathway in rice.

Grain size is an important agronomic trait, but our knowledge about grain size determination in crops is still limited. Endoplasmic reticulum (ER)-associated degradation (ERAD) is a special ubiquitin proteasome system that is involved in degrading misfolded or incompletely folded proteins in the ER. Here, we report that SMALL GRAIN 3 (SMG3) and DECREASED GRAIN SIZE 1 (DGS1), an ERAD-related E2-E3 enzyme pair, regulate grain size and weight through the brassinosteroid (BR) signaling pathway in rice (Oryza sativa). SMG3 encodes a homolog of Arabidopsis (Arabidopsis thaliana) UBIQUITIN CONJUGATING ENZYME 32, which is a conserved ERAD-associated E2 ubiquitin conjugating enzyme. SMG3 interacts with another grain size regulator, DGS1. Loss of function of SMG3 or DGS1 results in small grains, while overexpression of SMG3 or DGS1 leads to long grains. Further analyses showed that DGS1 is an active E3 ubiquitin ligase and colocates with SMG3 in the ER. SMG3 and DGS1 are involved in BR signaling. DGS1 ubiquitinates the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) and affects its accumulation. Genetic analysis suggests that SMG3, DGS1, and BRI1 act together to regulate grain size and weight. In summary, our findings identify an ERAD-related E2-E3 pair that regulates grain size and weight, which gives insight into the function of ERAD in grain size control and BR signaling.

A Comparative Phylogenetic Analysis on the Chloroplast Genome in Different Reynoutria japonica Populations.

Reynoutria japonica Houtt., a traditional medicine herb of the Polygonaceae family, has been used since ancient times in China due to its various pharmacological effects. Chloroplast genomes are conservative and play an essential role in population diversity analysis. However, there are few studies on the chloroplast genome of R. japonica. In this study, the complete chloroplast genomes of three R. japonica from different regions were performed by next-generation sequencing technology. The results revealed that the lengths of the three chloroplast genomes are between 163,371~163,372 bp, and they have a highly conserved structure with a pair of inverted repeat (IR) regions (31,121 bp), a large single-copy (LSC) region (87,571~87,572 bp), and a small single-copy (SSC) region (13,558 bp). In total, 132 genes were annotated, including 8 rRNA genes, 37 tRNA genes, and 87 protein-coding genes. The phylogenetic analysis strongly revealed that 13 populations of R. japonica form a monophyly, and Fallopia multiflora (Polygonaceae) is its closest species. The two species diverged at ~20.47 million years ago, and R. japonica in China could be further divided into two major groups based on genetic structure analysis. In addition, several potential loci with suitable polymorphism were identified as molecular markers. Our study provides important genetic resources for further development and utilization of R. japonica germplasm, as well as some new insights into the evolutionary characteristics of this medicinal plant.

Requirement and functional redundancy of two large ribonucleotide reductase subunit genes for cell cycle, chloroplast biogenesis and photosynthesis in tomato.

BACKGROUND AND AIMS: Ribonucleotide reductase (RNR), functioning in the de novo synthesis of deoxyribonucleoside triphosphates (dNTPs), is crucial for DNA replication and cell cycle progression. In most plants, the large subunits of RNR have more than one homologous gene. However, the different functions of these homologous genes in plant development remain unknown. In this study, we obtained the mutants of two large subunits of RNR in tomato and studied their functions. METHODS: The mutant ylc1 was obtained by ethyl methyl sulfonate (EMS) treatment. Through map-based cloning, complementation and knock-out experiments, it was confirmed that YLC1 encodes a large subunit of RNR (SlRNRL1). The expression level of the genes related to cell cycle progression, chloroplast biogenesis and photosynthesis was assessed by RNA-sequencing. In addition, we knocked out SlRNRL2 (a SlRNRL1 homologue) using CRISPR-Cas9 technology in the tomato genome, and we down-regulated SlRNRL2 expression in the genetic background of slrnrl1-1 using a tobacco rattle virus-induced gene silencing (VIGS) system. KEY RESULTS: The mutant slrnrl1 exhibited dwarf stature, chlorotic young leaves and smaller fruits. Physiological and transcriptomic analyses indicated that SlRNRL1 plays a crucial role in the regulation of cell cycle progression, chloroplast biogenesis and photosynthesis in tomato. The slrnrl2 mutant did not exhibit any visible phenotype. SlRNRL2 has a redundant function with SlRNRL1, and the double mutant slrnrl1slrnrl2 is lethal. CONCLUSIONS: SlRNRL1 is essential for cell cycle progression, chloroplast biogenesis and photosynthesis. In addition, SlRNRL1 and SlRNRL2 possess redundant functions and at least one of these RNRLs is required for tomato survival, growth and development.

Crosstalk Between Iron and Sulfur Homeostasis Networks in Arabidopsis.

The widespread deficiency of iron (Fe) and sulfur (S) is becoming a global concern. The underlying mechanisms regulating Fe and S sensing and signaling have not been well understood. We investigated the crosstalk between Fe and S using mutants impaired in Fe homeostasis, sulfate assimilation, and glutathione (GSH) biosynthesis. We showed that chlorosis symptoms induced by Fe deficiency were not directly related to the endogenous GSH levels. We found dynamic crosstalk between Fe and S networks and more interestingly observed that the upregulated expression of IRT1 and FRO2 under S deficiency in Col-0 was missing in the cad2-1 mutant background, which suggests that under S deficiency, the expression of IRT1 and FRO2 was directly or indirectly dependent on GSH. Interestingly, the bottleneck in sulfite reduction led to a constitutively higher IRT1 expression in the sir1-1 mutant. While the high-affinity sulfate transporter (Sultr1;2) was upregulated under Fe deficiency in the roots, the low-affinity sulfate transporters (Sultr2;1, and Sultr2;2) were down-regulated in the shoots of Col-0 seedlings. Moreover, the expression analysis of some of the key players in the Fe-S cluster assembly revealed that the expression of the so-called Fe donor in mitochondria (AtFH) and S mobilizer of group II cysteine desulfurase in plastids (AtNFS2) were upregulated under Fe deficiency in Col-0. Our qPCR data and ChIP-qPCR experiments suggested that the expression of AtFH is likely under the transcriptional regulation of the central transcription factor FIT.

MTP8 from Triticum urartu Is Primarily Responsible for Manganese Tolerance.

Mineral nutrients, such as manganese (Mn) and iron (Fe), play essential roles in many biological processes in plants but their over-enrichment is harmful for the metabolism. Metal tolerance proteins (MTPs) are involved in cellular Mn and Fe homeostasis. However, the transporter responsible for the transport of Mn in wheat is unknown. In our study, TuMTP8, a Mn-CDF transporter from diploid wheat (Triticum urartu), was identified. Expression of TuMTP8 in yeast strains of Δccc1 and Δsmf1 and Arabidopsis conferred tolerance to elevated Mn and Fe, but not to other metals (zinc, cobalt, copper, nickel, or cadmium). Compared with TuVIT1 (vacuole Fe transporter), TuMTP8 shows a significantly higher proportion in Mn transport and a smaller proportion in Fe transport. The transient analysis in tobacco epidermal cells revealed that TuMTP8 localizes to vacuolar membrane. The highest transcript levels of TuMTP8 were in the sheath of the oldest leaf and the awn, suggesting that TuMTP8 sequesters excess Mn into the vacuole in these organs, away from more sensitive tissues. These findings indicate that TuMTP8, a tonoplast-localized Mn/Fe transporter, functions as a primary balancer to regulate Mn distribution in T. urartu under elevated Mn conditions and participates in the intracellular transport and storage of excess Mn as a detoxification mechanism, thereby conferring Mn tolerance.

PcWRKY11, an II-d WRKY Transcription Factor from Polygonum cuspidatum, Enhances Salt Tolerance in Transgenic Arabidopsis thaliana.

Being an invasive plant, Polygonum cuspidatum is highly resilient and can survive in unfavorable environments for long periods; however, its molecular mechanisms associated with such environmental resistance are largely unknown. In this study, a WRKY transcription factor (TF) gene, PcWRKY11, was identified from P. cuspidatum by analyzing methyl jasmonate (MeJA)-treated transcriptome data. It showed a high degree of homology with WRKY11 from Arabidopsis thaliana, containing a WRKY domain and a zinc finger structure and II-d WRKY characteristic domains of HARF, a calmodulin-binding domain (C-motif), and a putative nuclear localization signal (NLS) through sequence alignment and functional element mining. qPCR analysis showed that the expression of PcWRKY11 can be induced by NaCl, osmotic stress, and UV-C. In this study, we also found that overexpression of PcWRKY11 in A. thaliana could significantly increase salt tolerance. To explore its possible molecular mechanism, further investigations showed that compared with the wild type (WT), under salt stress, the transgenic plants showed a lower malondialdehyde (MDA) content, higher expression of ascorbate peroxidase (APX) and superoxide dismutase (SOD), and higher enzyme activity of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). Moreover, the transgenic plants also showed higher expression of Δ1-pyrroline-5-carboxylate synthase (AtP5CS), and higher contents of proline and soluble sugar. Taken together, these results indicate that PcWRKY11 may have a positive role in plants' adaptation to salinity conditions by reducing reactive oxygen species (ROS) levels and increasing osmosis substance synthesis.

Transient expression of a TaGRF4-TaGIF1 complex stimulates wheat regeneration and improves genome editing.

Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1 (TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex (mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of mTaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, mTaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.

Physiological and Gene Expression Responses of Six Annual Ryegrass Cultivars to Cobalt, Lead, and Nickel Stresses.

Heavy metals negatively affect soil quality and crop growth. In this study, we compared the tolerance of six ryegrass cultivars to cobalt (Co2+), lead (Pb2+), and nickel (Ni2+) stresses by analyzing their physiological indexes and transcript levels of genes encoding metal transporters. Compared with the other cultivars, the cultivar Lm1 showed higher germination rates and better growth under Co2+, Pb2+, or Ni2+ treatments. After 48 h of Co2+ treatment, the total antioxidant capacity of all six ryegrass cultivars was significantly increased, especially that of Lm1. In contrast, under Pb2+ stress, total antioxidant capacity of five cultivars was significantly decreased, but that of Lm1 was unaffected at 24 h. Staining with Evans blue dye showed that the roots of Lm1 were less injured than were roots of the other five ryegrass cultivars by Co2+, Pb2+, and Ni2+. Lm1 translocated and accumulated lesser Co2+, Pb2+, and Ni2+ than other cultivars. In Lm1, genes encoding heavy metal transporters were differentially expressed between the shoots and roots in response to Co2+, Pb2+, and Ni2+. The aim of these researches could help find potential resource for phytoremediation of heavy metal contamination soil. The identified genes related to resistance will be useful targets for molecular breeding.

Characterization of the gene family encoding metal tolerance proteins in Triticum urartu: Phylogenetic, transcriptional, and functional analyses.

Homeostasis of microelements in organisms is vital for normal metabolism. In plants, the cation diffusion facilitator (CDF) protein family, also known as metal tolerance proteins (MTPs), play critical roles in maintaining trace metal homeostasis. However, little is known about these proteins in wheat. In this study, we characterized the MTP family of Triticum urartu, the donor of 'A' genome of Triticum aestivum, and analysed their phylogenetic relationships, sequence signatures, spatial expression patterns in the diploid wheat, and their transport activity when heterologously expressed. Nine MTPs were identified in the T. urartu genome database, and were classified and designated based on their sequence similarity to Arabidopsis thaliana (Arabidopsis) and Oryza sativa MTPs. Phylogenetic and sequence analyses indicated that the triticum urartu metal tolerance protein (TuMTP)s comprise three Zn-CDFs, two Fe/Zn-CDFs, and four Mn-CDFs; and can be further classified into six subgroups. Among the TuMTPs, there are no MTP2-5 and MTP9-10 counterparts but two MTP1/8/11 orthologs in relation to AtMTPs. It was also shown that members of the same cluster share similar sequence characteristic, i.e. number of introns, predicted transmembrane domains, and motifs. When expressed in yeast, TuMTP1 and TuMTP1.1 conferred tolerance to Zn and Co but not to other metal ions; while TuMTP8, TuMTP8.1, TuMTP11, and TuMTP11.1 conferred tolerance to Mn. When expressed in Arabidopsis, TuMTP1 localized to the tonoplast and significantly enhanced Zn and Co tolerance. TuMTPs showed diverse tissue-specific expression patterns. Taken together, the closely clustered TuMTPs share structural features and metal specificity but play diverse roles in the homeostasis of microelements in plant cells.

Tissue-specific transcriptome analyses reveal candidate genes for stilbene, flavonoid and anthraquinone biosynthesis in the medicinal plant Polygonum cuspidatum.

BACKGROUND: Polygonum cuspidatum Sieb. et Zucc. is a well-known medicinal plant whose pharmacological effects derive mainly from its stilbenes, anthraquinones, and flavonoids. These compounds accumulate differentially in the root, stem, and leaf; however, the molecular basis of such tissue-specific accumulation remains poorly understood. Because tissue-specific accumulation of compounds is usually associated with tissue-specific expression of the related biosynthetic enzyme genes and regulators, we aimed to clarify and compare the transcripts expressed in different tissues of P. cuspidatum in this study. RESULTS: High-throughput RNA sequencing was performed using three different tissues (the leaf, stem, and root) of P. cuspidatum. In total, 80,981 unigenes were obtained, of which 40,729 were annotated, and 21,235 differentially expressed genes were identified. Fifty-four candidate synthetase genes and 12 transcription factors associated with stilbene, flavonoid, and anthraquinone biosynthetic pathways were identified, and their expression levels in the three different tissues were analyzed. Phylogenetic analysis of polyketide synthase gene families revealed two novel CHS genes in P. cuspidatum. Most phenylpropanoid pathway genes were predominantly expressed in the root and stem, while methylerythritol 4-phosphate and isochorismate pathways for anthraquinone biosynthesis were dominant in the leaf. The expression patterns of synthase genes were almost in accordance with metabolite profiling in different tissues of P. cuspidatum as measured by high-performance liquid chromatography or ultraviolet spectrophotometry. All predicted transcription factors associated with regulation of the phenylpropanoid pathway were expressed at lower levels in the stem than in the leaf and root, but no consistent trend in their expression was observed between the leaf and the root. CONCLUSIONS: The molecular knowledge of key genes involved in the biosynthesis of P. cuspidatum stilbenes, flavonoids, and anthraquinones is poor. This study offers some novel insights into the biosynthetic regulation of bioactive compounds in different P. cuspidatum tissues and provides valuable resources for the potential metabolic engineering of this important medicinal plant.

Octaketide Synthase from Polygonum cuspidatum Implements Emodin Biosynthesis in Arabidopsis thaliana.

Plant anthranoids are medicinally used for their purgative properties. Their scaffold was believed to be formed by octaketide synthase (OKS), a member of the superfamily of type III polyketide synthase (PKS) enzymes. Here, a cDNA encoding OKS of Polygonum cuspidatum was isolated using a homology-based cloning strategy. When produced in Escherichia coli, P. cuspidatum octaketide synthase (PcOKS) catalyzed the condensation of eight molecules of malonyl-CoA to yield a mixture of unphysiologically folded aromatic octaketides. However, when the ORF for PcOKS was expressed in Arabidopsis thaliana, the anthranoid emodin was detected in the roots of transgenic lines. No emodin was found in the roots of wild-type A. thaliana. This result indicated that OKS is the key enzyme of plant anthranoids biosynthesis. In addition, the root growth of the transgenic A. thaliana lines was inhibited to an extent that resembled the inhibitory effect of exogenous emodin on the root growth of wild-type A. thaliana. Immunochemical studies of P. cuspidatum plants detected PcOKS mainly in roots and rhizome, in which anthranoids accumulate. Co-incubation of E. coli - produced PcOKS and cell-free extract of wild-type A. thaliana roots did not form a new product, suggesting an alternative, physiological folding of PcOKS and its possible interaction with additional factors needed for anthranoids assembling in transgenic A. thaliana. Thus, transgenic A. thaliana plants producing PcOKS provide an interesting system for elucidating the route of plant anthranoid biosynthesis.

Rhizosphere Soil Microbial Properties on Tetraena mongolica in the Arid and Semi-Arid Regions, China.

Tetraena mongolica is a rare and endangered species unique to China. The total number and density of Tetraena mongolica shrubs in desertification areas have experienced a sharp decrease with increases in coal mining activities. However, available information on the T. mongolica rhizosphere soil quality and microbial properties is scarce. Here, we investigated the effect of coal mining on the soil bacterial community and its response to the soil environment in the T. mongolica region. The results showed that the closer to the coal mining area, the lower the vegetation coverage and species diversity. The electrical conductivity (EC) in the contaminated area increased, while the total nitrogen (TN), available phosphorus (AP), available potassium (AK), and soil organic carbon (SOC) decreased. The activity of ß-glucosidase, urease, alkaline phosphatase, and catalase further decreased. In addition, the mining area could alter the soil's bacterial abundance and diversity. The organic pollutant degradation bacteria such as Sphingomonas, Gemmatimonas, Nocardioides, and Gaiella were enriched in the soil, and the carbon-nitrogen cycle was changed. Canonical correspondence analysis (CCA) and Pearson's correlation coefficients showed that the change in the bacterial community structure was mainly caused by environmental factors such as water content (SWC) and EC. Taken together, these results suggested that open pit mining led to the salinization of the soil, reduction the soil nutrient content and enzyme activity, shifting the rhizosphere soil microbial community structure, and altering the carbon-nitrogen cycle, and the soil quality declined and the growth of T. mongolica was affected in the end. Therefore, the development of green coal mining technology is of great significance to protect the growth of T. mongolica.

Shortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1) to nick the target DNA strand.

The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9 (SpCas9) and its variants. The detailed mechanism remains unknown. Here, based on in vitro cleavage assays and base editing analysis, we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1). We also show that these nicks are made on the target DNA strand. These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets. This system provides a novel tool for achieving trait stacking in plants.