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Abiotic stress affects metabolic processes in plants and restricts plant growth and development. In this experiment, Caucasian clover (Trifolium ambiguum M. Bieb.) was used as a material, and the CDS of TaMYC2, which is involved in regulating the response to abiotic stress, was cloned. The CDS of TaMYC2 was 726 bp in length and encoded 241 amino acids. The protein encoded by TaMYC2 was determined to be unstable, be highly hydrophilic, and contain 23 phosphorylation sites. Subcellular localization results showed that TaMYC2 was localized in the nucleus. TaMYC2 responded to salt, alkali, cold, and drought stress and could be induced by IAA, GA3, and MeJA. By analyzing the gene expression and antioxidant enzyme activity in plants before and after stress, we found that drought and cold stress could induce the expression of TaMYC2 and increase the antioxidant enzyme activity. TaMYC2 could also induce the expression of ROS scavenging-related and stress-responsive genes and increase the activity of antioxidant enzymes, thus improving the ability of plants to resist stress. The results of this experiment provide references for subsequent in-depth exploration of both the function of TaMYC2 in and the molecular mechanism underlying the resistance of Caucasian clover.

Amending soil with biochar can reduce the toxic effects of heavy metals (HM) on plants and the soil. However, the effects of different concentrations of biochar on the properties and microbial activities in lead (Pb)-contaminated soils are unclear. In this study, two Pb concentrations were set (low, 1000 mg/kg; high, 5000 mg/kg), and five corn straw biochar (CSB) concentrations (0, 2.5, 5, 10 and 15%) were used to determine the response of the growth and rhizosphere of red clover (Trifolium pretense L.) (in terms of soil properties and bacteria) to CSB and Pb application. The results showed that 5% CSB better alleviated the toxicity of Pb on the shoot length of red clover, the biomass increased by 74.55 and 197.76% respectively and reduced the enrichment factor (BCF) and transport factor (TF) of red clover. Pb toxicity reduced soil nutrients, catalase (CAT), acid phosphatase (ACP) and urease activity, while the addition of CSB increased soil pH, soil organic matter (SOM) content and soil enzyme activity. 16S rDNA amplicon sequencing analysis showed that Pb toxicity reduced the diversity of rhizosphere bacteria in red clover and reduced the relative abundance of plant growth-promoting rhizobacteria such as Gemmatimonas, Devosia and Bryobacter. Spearman correlation analysis showed that the addition of alkaline CSB restored the relative abundance of rhizobacteria positively correlated with pH, such as Chitinophaga, Sphingomonas, Devosia and Pseudomonas, and thus restored the rhizosphere soil environment. This study demonstrates that 5% CSB can better alleviate the toxicity of Pb to red clover and soil. We also provide a theoretical basis for the subsequent use of beneficial bacteria to regulate the repair efficiency of red clover.

Low temperature (LT) is an important threat to the normal growth of plants. In this study, based on the full-length transcriptome sequencing results, the cold resistance genes were cloned from Caucasian clover with strong cold resistance. We cloned the CDS of TaeRF1, which is 1311 bp in length and encodes 436 amino acids. The molecular weight of the protein is 48.97 kDa, which had no transmembrane structure, and its isoelectric point (pI) was 5.42. We predicted the structure of TaeRF1 and found 29 phosphorylation sites. Subcellular localization showed that TaeRF1 was localized and expressed in cell membrane and chloroplasts. The TaeRF1 gene was induced by stress due to cold, salt, alkali and drought and its expression level was higher in roots and it was more sensitive to LT. Analysis of transgenic A. thaliana plants before and after LT treatment showed that the TaeRF1 gene enhanced the removal of excess H2O2, and increased the activity of antioxidant enzymes, thus improving the plant’s ability to resist stress. Additionally, the OE lines showed increased cold tolerance by upregulating the transcription level of cold-responsive genes (CBF1, CBF2, COR15B, COR47, ICE1, and RD29A). This study demonstrates that TaeRF1 is actively involved in the responses of plants to LT stress. We also provide a theoretical basis for breeding and a potential mechanism underlying the responses of Caucasian clover to abiotic stress.