Soil co-contamination with cadmium (Cd) and salinity imposes combined stress on plants in China. This study screened 40 alfalfa varieties, identifying a Cd-tolerant (’Concept’) and a Cd-sensitive (’SR4030’) cultivar, to elucidate their adaptive responses. Under combined Cd-salt stress (100 μM CdCl₂ + 150 mM NaCl), SR4030 exhibited more severe growth inhibition (e.g., biomass reduction up to 25%) and cellular damage than Concept, indicating synergistic stress effects. The cultivars showed distinct ion profiles: Concept accumulated less Cd²⁺ in shoots and had a lower translocation factor (TF) than SR4030. Salt stress increased shoot Cd²⁺ TF by approximately 7-fold in both cultivars. Although antioxidant enzymes were activated, elevated ROS and MDA indicated oxidative damage, exacerbated under combined stress. Transcriptome analysis revealed 1,401 and 1,378 more differentially expressed genes (DEGs) under combined stress in Concept at 12 h and 24 h, respectively, primarily enriched in plant hormone signaling and MAPK pathways. Concept specifically upregulated salicylic acid (SA)-related genes, whereas SR4030 downregulated jasmonic acid pathway genes. Heterologous expression confirmed MsCIPK enhanced yeast tolerance to both Cd and salt stress. Exogenous SA alleviated damage by reducing shoot Cd²⁺ content (by nearly 50% in SR4030) and Na⁺ accumulation, enhancing antioxidant activities, and regulating osmotic substances. These findings demonstrate the roles of MsCIPK and SA in mitigating combined stress, providing a theoretical basis for breeding tolerant alfalfa and managing co-contaminated soils.

Caucasian clover (Trifolium ambiguum M. Bieb.) is a valuable perennial legume with a robust root system. HECT-type E3 ubiquitin ligases (UPLs) are known regulators of plant growth, but their functions in root development, particularly in forage legumes, remain largely unexplored. Here, we identified 32 TaUPL genes in Caucasian clover and found that TaUPL21 is highly expressed in rhizome buds. Overexpression of TaUPL21 significantly enhanced root growth, including increases in root length, volume, and tip number. Strikingly, DNA affinity purification sequencing (DAP-Seq) revealed an unexpected DNA-binding capacity of TaUPL21. We further identified the MADS-box transcription factor TaAGL29 as a direct downstream target. Yeast one-hybrid (Y1H) and dual-luciferase (d-Luc) assays confirmed that TaUPL21 directly binds to the promoter of TaAGL29 and functions as a transcriptional activator. Our results unveil a novel, non-canonical function for a HECT E3 ubiquitin ligase, providing fresh insights into the functional versatility of E3 ubiquitin ligases and present a valuable genetic resource for improving root architecture in plants.

Microplastic pollution is increasingly serious worldwide, threatening human and animal health. The cow rumen is a key organ for nutrient digestion and absorption, and its fermentation is closely related to rumen microorganisms. Here, we investigated how polystyrene microplastics (PS-MPs) with varying particle sizes and concentrations affect rumen fermentation and the biodegradability of PS-MPs by rumen fermentation. The results reveal that exposure to PS-MPs lowered gas production and gas concentrations, as well as volatile fatty acid content, and these decreases were positively correlated with PS-MP concentration. However, higher PS-MP concentration and larger particle size increased the activity of carboxymethyl cellulose, β-glucosidase, and xylanase. Furthermore, PS-MP exposure reduced the abundance of certain rumen microorganisms and altered metabolic pathways and metabolites linked to PS-MP biodegradation. It was also found that PS-MP content decreased significantly after 24 h fermentation. Therefore, PS-MPs can inhibit rumen fermentation by affecting the rumen microbiome, and rumen microorganisms and their secreted enzymes can biodegrade PS-MPs to produce styrene and derivatives; such small molecules may further disrupt rumen homeostasis, thereby affecting lactation performance. In addition, rumen microbial degradation of PS-MPs provides a new idea to resolve future microplastic contamination challenges.

Red clover (Trifolium pratense), a high-quality forage plant, faces significant threats from anthracnose in northeastern China, but the pathogen responsible remains unidentified. The phyllosphere microbiota is crucial in plantpathogen interactions, yet its role in the resistance of red clover to anthracnose is poorly understood. Using morphological, molecular, and multigene phylogenetic analyses, we identified Colletotrichum americae-borealis (Cab) as the pathogen that causes anthracnose in red clover in China. We also investigated changes in the phyllosphere microbiomes of highly resistant (XJ) and susceptible (SC) red clover materials after Cab infection, via 16S rRNA gene sequencing. The results revealed significant differences in bacterial α- and β-diversity, with novel microbial taxa and a complex microbial network emerging postinfection. Notably, after Cab inoculation, the Shannon diversity index in XJ exhibited more pronounced changes, manifested as an increase in the abundance of beneficial microorganisms such as Bacillus, Pantoea, and Pseudomonas. Network analysis revealed that the XJ microbiome was more complex and stable than the SC microbiome was, regardless of infection status. Bacillus J2, the dominant bacterium, significantly inhibited Cab growth in vitro and reduced the disease index by 33.4-47.7 % when it was reapplied to the leaf surface, suggesting its role in enhancing disease resistance. This study is the first to report that C. americae-borealis causes anthracnose in red clover in China, and demonstrates the potential of the beneficial bacterium J2 in enhancing disease resistance, providing insights into disease resistance mechanisms and the role of the phyllosphere microbiome in pathogen challenge.

Alfalfa (Medicago sativa L.) is an outstanding species used for the remediation of heavy metal-contaminated soil, and our previous research has shown that PGPR can promote plant growth under high-concentration lead stress. This discovery has forced scientists to search for PGPR strains compatible with alfalfa to develop an innovative bioremediation strategy for the remediation of lead-contaminated soil. This study used lead-tolerant rhizosphere soil of red clover as experimental material; cultured, isolated, and screened 52 excellent lead-tolerant bacteria that promote rhizosphere growth; and then inoculated them into alfalfa. Marked differences existed in the secretion of auxin, protease, and ACC deaminase among these strains. The results indicated that Pseudomonas spp. (strain Y2), Pseudomonas spp. (strain Y22), and Bacillus spp. (strain Y23) exhibited a strong growth-promoting ability in alfalfa, and there was no antagonistic reaction among the three strains, enabling their coexistence. The pot experiment manifested that strains Y2, Y22, Y23, and YH (a mixture of Y2, Y22, and Y23) could increase the plant height, root length, fresh and dry weight above ground, and fresh and dry weight below ground of alfalfa. They could all significantly raise the chlorophyll content and antioxidant enzyme activity in alfalfa (p < 0.05) and the content of malondialdehyde (MDA) in alfalfa. Furthermore, the concurrent inoculation of three distinct types of plant growth-promoting rhizobacteria (PGPR) significantly diminished lead (Pb) concentrations in rhizosphere soil, enhanced the levels of available potassium (AK) and available phosphorus (AP), and augmented the capacity of plants to absorb Pb. The results imply that PGPR can be employed to facilitate plant growth and microbial-assisted remediation of lead and other heavy metal-contaminated soil and establish a basis for further research on the growth-promoting mechanism of PGPR in plants.

Objective The study aimed to assess effects of supplemented co-fermented edible plants and probiotics (AEPP) on growth performance, disease resistance, plasma and rumen metabolites, and bacterial communities in the rumen and feces of pre-weaned calves. Methods Twenty female Holstein calves (7±0.50 d, 41.65±6.20 kg) were randomly assigned to one of two treatments: the control group or the treatment group (30 g/head/day AEPP supplementation). Growth performance, blood, and fecal samples were measured on regular basis. On day 30 of the trial, rumen fluid and fecal samples were collected for multi-omics analysis. Results Dietary supplementation with AEPP enhanced calf growth and improved disease resistance, as evidenced by a reduced incidence of respiratory disease and diarrhea and a decreased frequency of antibiotic therapy (p<0.05). The treatment group exhibited enrichment of rumen microorganisms Prevotella, Ruminococcus, and Xylanibacter (linear discriminant analysis>2, p<0.05), along with increased activity in beneficial metabolites such as indoleacetic acid, which activated starch and sucrose metabolism and tryptophan metabolism pathway. This intervetion significantly improved average daily gain, feed efficiency, immunoglobulin G, total superoxide dismutase, and glutathione peroxidase activities, as well as significantly reduced levels of tumor necrosis factor-alpha and interleukin-6 (p<0.05), promoting calf growth and health. The elevated abundance of fecal microorganisms, Subdoligranulum and Bifidobacterium, in the treatment group altered fecal pH, short-chain fatty acids, and butyrate proportions (p<0.05). Conclusion Feeding AEPP improved growth performance, disease resistance, and antioxidant function. It altered the bacterial communities and metabolic profiles in the rumen and feces of preweaning dairy calves, providing a data reference for the use of AEPP in young ruminant production.

Hepatic oxidative stress is a key driver in liver injury pathogenesis, with D-galactose (D-gal) modeling serving as an established inducer of accelerated oxidative damage. Silibinin (SLB), a flavonolignan from milk thistle, shows therapeutic promise through potent antioxidant activity and gut-liver axis modulation. This study investigated whether the hepatoprotective effect of SLB against oxidative stress depends on gut microbiota regulation. Using mouse models with gut microbiota ablation by oral antibiotics or direct oxidative stress induction by D-gal (150 mg/kg), SLB treatment (200 mg/kg) was administered. The protective mechanisms were evaluated through the Nrf2/ARE pathway, target gene expression, gut microbiota profiling, and cecal metabolomics. Results demonstrated that SLB significantly alleviated D-gal-induced hepatic oxidative stress (e.g., reduced MDA by 33.3%), but this protection was markedly weakened after antibiotic-induced microbiota depletion (e.g., a loss of efficacy exceeding 50%). Integrated omics revealed that antibiotics caused a severe reduction in unclassified_Muribaculaceae (a butyrate producer, decreased by 80%), impairing butyrate-mediated Nrf2/Keap1 activation. Simultaneously, the absence of Parabacteroides led to accumulated primary bile acids and inhibited secondary bile acid production (e.g., taurochenodeoxycholate reduced by 75%), further disrupting redox homeostasis. Conclusion: Silibinin's mitigation of hepatic oxidative stress is gut microbiota-dependent, highlighting the therapeutic potential of microbiota-targeted antioxidant strategies for oxidative stress-related pathologies.

The huge milk thistle meal (MTM) production requires proper processing. The study utilized response surface methodology to optimize fermentation conditions and assessed complex probiotics and cellulase impacts on nutrients, fermentation, digestibility, and in vitro ruminal features of fermented MTM (FMTM) for advancing clean bioresource technologies and feed utilization. Optimal solid-state fermentation conditions for maximum neutral detergent fiber reduction (14.25 %) were: 1.0 g/kg complex probiotics, 7.1 g/kg enzyme, 38 degrees C, 456.9 g/kg moisture, and 96 h. Under fermentation, the contents of crude protein (254.0 vs. 268.8 g/kg DM; P = 0.002), total amino acid (201.7 vs. 249.2 g/kg DM; P < 0.001), ammonia-N (9.92 vs. 19.2 g/kg TN; P < 0.001), and in situ degradation rates of DM (371.0 vs. 410.9 g/kg; P = 0.006) and crude protein (576.7 vs. 614.2 g/kg; P < 0.001) of FMTM were elevated. In vitro gas production experiments have shown that compared with TMR including MTM, TMR involved FMTM had lower methane concentration (200.40 vs. 182.54 g/kg; P = 0.095), higher ammonia-N concentration (40.06 vs. 44.18 mg/dL; P < 0.001), total volatile fatty acid production (79.72 vs. 83.63 mmol/L; P = 0.0031), and molar ratios of propionate (24.41 vs. 24.96 %; P = 0.0002) in the rumen. Moreover, TMR containing FMTM increased microbial diversity, and enrichment of norank_f__F082, Succiniclasticum, Christensenellaceae_R_7_group, Ruminococcus, NK4A214_group, and unclassified_f__Lachnospiraceae. In conclusion, solid-state fermentation using complex probiotics and cellulose enables sustainable MTM utilization, making it a high-quality protein source for ruminants and providing a reference for other Chinese herb residue applications.

Trifolium repens L. (T. repens) is considered a potential phytoremediation species due to its large biomass and ability to accumulate and tolerate heavy metals. Lead (Pb) is an important heavy metal pollutant that can affect plant growth, photosynthesis, and enzyme activity. However, response mechanism of microorganisms in three root niches of metal tolerant plants to Pb is not completely understood. Therefore, in this study, a Pb poisoning model of T. repens was established with a Pb gradient (0, 1000 mg/kg, 2000 mg/kg, and 3000 mg/kg), and was used to evaluate growth and physiological responses, as well as enrichment and transport coefficients in T. repens, and explore the characteristics of rhizosphere soil and microbial composition of three root niches. We found that Pb stress caused oxidative injury, and inhibited photosynthesis in T. repens. 16S rDNA sequencing analysis showed that the richness of microbial communities in bulk soil was higher than that in rhizosphere soil both under Pb stress and Pb nonstress conditions. Moreover, Proteobacteria was dominant phylum in bulk and rhizosphere soils, and Proteobacteria and Cyanobacteria were dominant phylum in endophytic bacteria. For the first time, we systematically investigated the response of Pb from bulk soil to plant leaves. The results showed that microbial interaction existed between bulk and rhizosphere soil. Rhizosphere bacterium Haliangium was positively correlated with urease activity and soil nutrients. Endophytic bacterium Pseudomonas was positively correlated with plant biomass and played an important role in Pb tolerance of T. repens. In addition, endophytic bacteria formed complex correlation networks with growth and physiological indexes of both root and shoot, moreover the network in root was more complicated. Taken together, Pb stress dose-dependently inhibited the growth of plants. This study provided a theoretical basis for the further development of microbial cooperation with plant remediation of heavy metal contaminated soil.

Background The NAC TF family is widely involved in plant responses to various types of stress. Red clover ( Trifolium pratense ) is a high-quality legume, and the study of NAC genes in red clover has not been comprehensive. The aim of this study was to analyze the NAC gene family of red clover at the whole-genome level and explore its potential role in the Pb stress response. Results In this study, 72 TpNAC genes were identified from red clover; collinearity analysis showed that there were 5 pairs of large fragment replicators of TpNAC genes, and red clover was found to be closely related to Medicago truncatula . Interestingly, the TpNAC genes have more homologs in Arabidopsis thaliana than in soybean ( Glycine max ). There are many elements in the TpNAC genes promoters that respond to stress. Gene expression analysis showed that all the TpNAC genes responded to Pb stress. qRT-PCR showed that the expression levels of TpNAC29 and TpNAC42 were significantly decreased after Pb stress. Protein interaction network analysis showed that 21 TpNACs and 23 other genes participated in the interaction. In addition, the TpNAC proteins had three possible 3D structures, and the secondary structure of these proteins were mainly of other types. These results indicated that most TpNAC members were involved in the regulation of Pb stress in red clover. Conclusion These results suggest that most TpNAC members are involved in the regulation of Pb stress in red clover. TpNAC members play an important role in the response of red clover to Pb stress.

Biochar is an inexpensive and effective material that can immobilize heavy metals. However, the impact of varying levels of biochar application on red clover cultivated in soil contaminated with Pb remains unclear. In this study, five different doses of biochar (0, 2.5, 5, 10, and 15%) were applied to soils containing two levels of Pb (LPb, 1000 mg/kg; HPb, 5000 mg/kg) to assess the growth, physiological characteristics, and heavy metal enrichment capacity of red clover. Pb stress dramatically slowed down plant development, decreased the amount of photosynthetic pigment, and caused oxidative damage, according to the results of pot tests. However, the addition of biochar mitigated Pb toxicity in red clover by effectively adsorbing Pb from soil and reducing Pb translocation to plant tissues. Specifically, red clover growth was significantly enhanced by the addition of 5% biochar to soil contaminated with either low- or high-level Pb. This resulted in an increase in plant biomass, stimulated the upregulation of LHCA1 and CCS genes, and facilitated the production of photosynthetic pigments. Furthermore, it increased antioxidant enzyme activity while reducing proline accumulation. This work offers a theoretical foundation for the remediation of Pb-contaminated soils using plants and biochar.

Caucasian clover (Trifolium ambiguum M. Bieb.) is an excellent perennial plant in the legume family Fabaceae, with a well-developed rhizome and strong clonal growth. Auxin is one of the most important phytohormones in plants and plays an important role in plant growth and development. Auxin response factor (ARF) can regulate the expression of auxin-responsive genes, thus participating in multiple pathways of auxin transduction signaling in a synergistic manner. No genomic database has been established for Caucasian clover. In this study, 71 TaARF genes were identified through a transcriptomic database of Caucasian clover rhizome development. Phylogenetic analysis grouped the TaARFs into six (1–6) clades. Thirty TaARFs contained a complete ARF structure, including three relatively conserved regions. Physical and chemical property analysis revealed that TaARFs are unstable and hydrophilic proteins. We also analyzed the expression pattern of TaARFs in different tissues (taproot, horizontal rhizome, swelling of taproot, rhizome bud and rhizome bud tip). Quantitative real–time RT–PCR revealed that all TaARFs were responsive to phytohormones (indole-3-acetic acid, gibberellic acid, abscisic acid and methyl jasmonate) in roots, stems and leaves. These results helped elucidate the role of ARFs in responses to different hormone treatments in 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.

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.

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.

4-octylphenol (4-OP), a toxic estrogenic environmental pollutant, can threaten aquatic animal and human health. However, toxic effect of 4-OP on fish has not been reported. To investigate molecular mechanism of gill poisoning caused by 4-OP exposure, a carp 4-OP poisoning model was established, and then blood and gills were collected on day 60. The results demonstrated that gill was a target organ attacked by 4-OP, and exposure to 4-OP caused carp gill inflammatory injury. There were 1605 differentially expressed genes (DEGs, including 898 up-regulated DEGs and 707 down-regulated DEGs). KEGG and GO were used to further analyze obtained 1605 DEGs, indicating that complement activation, immune response, and inflammatory response participated in the mechanism of 4-OP-caused carp gill inflammatory injury. Our data at transcription level further revealed that 4-OP caused complement activation through triggering complement component 3a/complement component 3a receptor (C3a/C3aR) axis and complement component 5a/complement component 5a receptor 1 (C5a/C5aR1) axis, induced immunosuppression through the imbalances of T helper (Th) 1/Th2 cells and regulatory T (Treg)/Th17 cells, as well as caused inflammatory injury via toll like receptor 7/inhibitor kappa B alpha/nuclear factor-kappa B (TLR7/IκBα/NF-κB) pathway. Taken together, immunosuppression participated in complement activation-mediated inflammatory damage in carp gills after 4-OP treatment. The findings of this study will provide pioneering information and theoretical support for the mechanism of 4-OP poisoning, and will provide reference for the assessment of estrogenic environmental pollution risk.

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.

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.

为获得高加索三叶草转录组特征,对高加索三叶草的根茎系统进行三代转录组测序,检测到79424条Unigene,序列总长为167351.88kb,进一步分析得到48235个SSR位点,平均3.47 kb出现一个SSR.高加索三叶草转录组的SSR类型丰富,其中单核苷酸和三核苷酸重复SSR数量最多,分别占SSR总数的45.8％和22.6％,其次是复杂SSR序列占比为15.2％、二核苷酸重复占比为12.8％、四核苷酸重复占比为2.1％、六核苷酸重复占比为0.9％和五核苷酸重复占比为0.6％.本研究对高加索三叶草转录组数据SSR位点信息进行分析,为三叶草属种质资源鉴定和分子标记辅助育种提供更多的标记资源.

The forage species Caucasian clover ( Trifolium ambiguum M. Bieb.), a groundcover plant, is resistant to both cold and drought. However, reference genes for qRT-PCR-based analysis of Caucasian clover are lacking. In this study, 12 reference genes were selected on the basis of transcriptomic data. These genes were used to determine the most stably expressed genes in various organs of Caucasian clover under cold, salt and drought stress for qRT-PCR-based analysis. Reference gene stability was analyzed by geNorm, NormFinder, BestKeeper, the ∆Ct method and RefFinder. Under salt stress, RCD1 and PPIL3 were the most stable reference genes in the leaves, and NLI1 and RCD1 were the most stable references genes in the roots. Under low-temperature stress, APA and EFTu-GTP were the most stable reference genes in the leaves, and the RCD1 and NLI2 genes were highly stable in the roots. Under 10% PEG-6000 stress, NLI1 and NLI2 were highly stable in the leaves, and RCD1 and PPIL3 were the most stable in the roots. Overall, RCD1 and NLI2 were the most stable reference genes in organs under normal conditions and across all samples. The most and least stable reference genes were validated by assessing their appropriateness for normalization via WRKY genes.