Effects of different concentrations of biochar amendments and Pb toxicity on rhizosphere soil characteristics and bacterial community of red clover (Trifolium pretense L.).

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.

Integrated physiological, transcriptomic and metabolomic analysis of the response of Trifolium pratense L. to Pb toxicity.

Lead (Pb) interferes with plant gene expression, alters metabolite contents and affects plant growth. However, the molecular mechanism underlying the plant response to Pb is not completely understood. In the present study, Trifolium pratense L. was exposed to Pb concentrations of 0 (Pb0), 500 (Pb500), 1000 (Pb1000), 2000 (Pb2000) and 3000 (Pb3000) mg/kg in soils. Pb stress affected the ability of T. pratense to accumulate and transport Pb, increased the activity of peroxidase (POD) and the contents of malondialdehyde (MDA) and proline, decreased the amount of photosynthetic pigments and soluble proteins, and led to changes in growth and biomass. Transcriptomic and metabolomic analyses showed that Pb mainly affected eight pathways, and LHC, flavonoids, organic acids, amino acids and carbohydrates were upregulated or downregulated. Moreover, Pb500 induced the upregulation of serA, promoted the synthesis of citric acid, maintained photosynthetic pigment levels, and ultimately promoted an increase in stem length. Pb3000 induced the upregulation of ARF, GH3 and SAUR genes, but the saccharide contents and stem length decreased in response to Pb stress. We used a variety of methods to provide a molecular perspective on the mechanism underlying the response of T. pratense to Pb stress.

Identification and responding to exogenous hormone of HB-KNOX family based on transcriptome data of Caucasian clover.

Caucasian clover (Trifolium ambiguum M. Bieb.) is a strongly rhizomatous, low-crowned perennial leguminous and ground-covering grass. The species is resistant to cold, arid temperatures and grazing due to a well-developed underground rhizome system and a strong clonal reproduction capacity. KNOTTED1-LIKE HOMEOBOX (KNOX) genes are a family of plant-specific homeobox transcription factors with important roles in plant development. Preliminary transcriptome analysis enabled us to understand the gene expression in five different tissues, which helped us to screen the predetermined genes of the HB-KNOX family genes for the rhizome growth and development of Caucasian clover. The study identified 41 TaKNOX genes from the Caucasian clover transcriptome database. Gene length, MW and pl of TaKNOX family transcription factors varied, but the gene structure and motifs were relatively conserved in bioinformatics analysis. Phylogenetic analyses of Arabidopsis thaliana, soybean, Medicago truncatula and Caucasian clover were performed to study the evolutionary and functional relationships in various species. Prediction and verification of the subcellular localizations revealed the diverse subcellular localization of these 41 TaKNOX proteins. The expression profile of exogenous hormones showed that the TaKNOX gene showed multiple expression regulation patterns, and was involved in 6-BA, IAA and KT signaling pathways. Our results reveal the characteristics of the TaKNOX gene family, thus laying a foundation for further functional analysis of the KNOX family in Caucasian clover.

The Caucasian Clover Gene TaMYC2 Responds to Abiotic Stress and Improves Tolerance by Increasing the Activity of Antioxidant Enzymes.

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.

Cloning of TaeRF1 gene from Caucasian clover and its functional analysis responding to low-temperature stress.

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.