Potential Antidiabetic, Antioxidative and Antiproliferative Properties of Functional Wheat Flour Muffins Enriched with White Clover Flowers (Trifolium repens L.).

The aim of the study was to evaluate the functional properties of muffins fortified with white clover flowers (Trifolium repens L.), which were added to the dough in the following amounts: (i) 0% (control); (ii) 2.5%; (iii) 5.5%; (iv) 7.5%; and (v) 10%. The organoleptic properties were assessed by a panel of consumers. Additionally, the following parameters were also tested: basic chemical composition, total polyphenols, the antioxidant activity together with antiproliferative effects on the A375 melanoma cell line, starch nutritional fractions and the in vitro glycemic index. As a result, replacing wheat flour with white clover flour significantly affected the color, aroma and taste of the muffins. The content of proteins, fats, total ash, dietary fiber, resistant starch (RS), slowly digestible starch (SDS),total polyphenols and antioxidant activity increased statistically significantly with the elevated amount of white clover flour added to the dough. At the same time, the content of free glucose (FG), rapidly available glucose (RAG) and rapidly digestible starch (RDS), the value of the in vitro glycemic index and the viability of melanoma cancer cells decreased significantly. The muffins enriched with white clover flowers might constitute an interesting proposition and extension of the existing assortment of confectionery products.

Physiological and comprehensive transcriptome analysis reveals distinct regulatory mechanisms for aluminum tolerance of Trifolium repens.

It is estimated that up to 50 % of arable lands worldwide are acidic, and most crops are severely inhibited due to the high active aluminum (Al). Trifolium repens is an excellent legume forage with a certain acid tolerance, although it is affected by Al toxicity in acidic soil. In this study, physiological and transcriptomic responses of different white clover varieties were analyzed when exposed to a high-level of Al stress. The results revealed that Trifolium repens had a high level of Al toxicity tolerance, and accumulated nearly 70 % of Al3+ in its roots. Al toxicity significantly inhibited the root length and root activity, decreased the chlorophyll (Chl) content and photosynthetic pigments, while significantly increased the intercellular CO2 concentration (Ci). The content of malondialdehyde (MDA), electrolyte leakage (EL), proline and reactive oxygen species (ROS) were significantly accumulated under Al stress. Furthermore, a total of 27,480 differentially expressed genes (DEGs) were identified after the treatment. Gene ontology (GO) and Kyoto encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that most Al-responsive genes enriched to chloroplast thylakoid membrane, chloroplast stroma and photosynthesis in Haifa leaf while in MAG leaf highly enriched in response to regulation of defense response, which could induce the different tolerance of the two cultivars to Al stress. Besides, pectin methylesterase (PME), glycosyl transferases (GT1) and chalcone synthase genes associated with cell wall biosynthesis may improve the Al accumulation and enhance tolerance of Al toxicity. The results established here would help to understand the morphological structure, physiological and biochemical response, and molecular mechanism of white clover under Al tolerance.

De novo genome assembly of white clover (Trifolium repens L.) reveals the role of copy number variation in rapid environmental adaptation.

BACKGROUND: White clover (Trifolium repens) is a globally important perennial forage legume. This species also serves as an eco-evolutionary model system for studying within-species chemical defense variation; it features a well-studied polymorphism for cyanogenesis (HCN release following tissue damage), with higher frequencies of cyanogenic plants favored in warmer locations worldwide. Using a newly generated haplotype-resolved genome and two other long-read assemblies, we tested the hypothesis that copy number variants (CNVs) at cyanogenesis genes play a role in the ability of white clover to rapidly adapt to local environments. We also examined questions on subgenome evolution in this recently evolved allotetraploid species and on chromosomal rearrangements in the broader IRLC legume clade. RESULTS: Integration of PacBio HiFi, Omni-C, Illumina, and linkage map data yielded a completely de novo genome assembly for white clover (created without a priori sequence assignment to subgenomes). We find that white clover has undergone extensive transposon diversification since its origin but otherwise shows highly conserved genome organization and composition with its diploid progenitors. Unlike some other clover species, its chromosomal structure is conserved with other IRLC legumes. We further find extensive evidence of CNVs at the major cyanogenesis loci; these contribute to quantitative variation in the cyanogenic phenotype and to local adaptation across wild North American populations. CONCLUSIONS: This work provides a case study documenting the role of CNVs in local adaptation in a plant species, and it highlights the value of pan-genome data for identifying contributions of structural variants to adaptation in nature.

Genetics and plasticity of white leaf mark variegation in white clover (Trifolium repens L.).

BACKGROUND AND AIMS: Leaf variegation is common in plants and confers diverse adaptive functions. However, its genetic underpinnings remain largely unresolved; this is particularly true for variegation that arises through modified leaf tissue structure that affects light reflection. White clover is naturally polymorphic for structure-based white leaf mark variegation. It therefore provides a useful system to examine the genetic basis of this phenotype, and to assess potential costs to photosynthetic efficiency resulting from modified leaf structures. This study sought to map the loci controlling the white leaf mark in white clover and evaluate the relationship between white leaf mark, leaf thickness, and photosynthetic efficiency. METHODS: We generated a high-density genetic linkage map from an F3 mapping population, employing reference genome-based SNP markers. White leaf mark was quantified through detailed phenotypic evaluations alongside leaf thickness to test how tissue thickness may affect the variegation phenotype. Quantitative trait locus (QTL) mapping was performed to characterize their genetic bases. Photosynthetic efficiency measurements were used to test for physiological trade-offs between variegation and photosynthetic output. KEY RESULTS: The V locus, a major gene responsible for the white leaf mark polymorphism, was mapped to the distal end of chromosome 5, and several modifier loci were also mapped that contribute additively to variegation intensity. The presence and intensity of white leaf mark was associated with greater leaf thickness; however, increased variegation did not detectably affect photosynthetic efficiency. CONCLUSIONS: We have successfully mapped the major locus governing the white leaf mark in white clover, along with several modifier loci, revealing a complex basis for this structure-based variegation. The apparent absence of compromised photosynthesis in variegated leaves challenges the notion that variegation creates fitness trade-offs between photosynthetic efficiency and other adaptive functions. This finding suggests that other factors may maintain the white leaf mark polymorphism in white clover.

Beyond cyanogenesis: Temperature gradients drive environmental adaptation in North American white clover (Trifolium repens L.).

Species that repeatedly evolve phenotypic clines across environmental gradients have been highlighted as ideal systems for characterizing the genomic basis of local environmental adaptation. However, few studies have assessed the importance of observed phenotypic clines for local adaptation: conspicuous traits that vary clinally may not necessarily be the most critical in determining local fitness. The present study was designed to fill this gap, using a plant species characterized by repeatedly evolved adaptive phenotypic clines. White clover is naturally polymorphic for its chemical defence cyanogenesis (HCN release with tissue damage); climate-associated cyanogenesis clines have evolved throughout its native and introduced range worldwide. We performed landscape genomic analyses on 415 wild genotypes from 43 locations spanning much of the North American species range to assess the relative importance of cyanogenesis loci vs. other genomic factors in local climatic adaptation. We find clear evidence of local adaptation, with temperature-related climatic variables best describing genome-wide differentiation between sampling locations. The same climatic variables are also strongly correlated with cyanogenesis frequencies and gene copy number variations (CNVs) at cyanogenesis loci. However, landscape genomic analyses indicate no significant contribution of cyanogenesis loci to local adaptation. Instead, several genomic regions containing promising candidate genes for plant response to seasonal cues are identified - some of which are shared with previously identified QTLs for locally adaptive fitness traits in North American white clover. Our findings suggest that local adaptation in white clover is likely determined primarily by genes controlling the timing of growth and flowering in response to local seasonal cues. More generally, this work suggests that caution is warranted when considering the importance of conspicuous phenotypic clines as primary determinants of local adaptation.

Evaluating foliar insecticides and economic thresholds for Tychius picirostris (Coleoptera: Curculionidae) management in Oregon white clover seed production.

The clover seed weevil, Tychius picirostris Fabricius (Coleoptera: Curculionidae), is a major pest in Oregon white clover seed crops. Reliance on synthetic pyrethroid insecticides and limited availability of diverse modes of action (MoAs) has increased insecticide resistance selection in regional T. picirostris populations, emphasizing the need to evaluate novel chemistries and rotational strategies for effective insecticide resistance management (IRM). The efficacy of 8 foliar insecticide formulations for managing T. picirostris adult and larval life stages was determined in small and large-plot field trials across 2 crop years. In both years, bifenthrin (Brigade 2EC), the grower's standard, showed negligible adult and larval suppression. Insecticide formulations with isocycloseram and cyantraniliprole active ingredients reduced adult and larval populations when applied at BBCH 59-60 (prebloom) and BBCH 65-66 (full bloom) growth stages, respectively. While differences in T. picirostris abundance were observed among insecticide treatments, seed yield differences were not detected in large-plot trials. Larval abundance was correlated with reduced seed yield, and an economic threshold of ≥3 larvae per 30 inflorescences was determined as a conservative larval threshold to justify foliar applications of diamide insecticides. Additional commercial white clover seed fields were surveyed to compare larval scouting techniques, including a standard Berlese funnel and a grower's do-it-yourself funnel. Both larval extraction techniques were correlated and provided similar estimates of larval abundance. These findings demonstrate new MoAs, optimal insecticide application timing, and larval monitoring methods that can be incorporated into an effective T. picirostris IRM program in white clover seed crops.

The pollen quality of woody and herbaceous plants from the Chernobyl exclusion zone.

Pollen abnormalities frequency of Scots pine and pollen sterility of white clover, common columbine, and greater celandine growing in the Bryansk and Gomel' regions radioactively contaminated after the accident at the Chernobyl nuclear power plant (NPP) were assessed. The frequency of abnormal pine pollen at the most contaminated plots significantly exceeded the control level and positively correlated with the total absorbed dose and the absorbed dose from ß-radiation. No sustainable significant changes in pollen sterility of the herbaceous plants studied were found in the exclusion zone of the Chernobyl NPP.

Trifolium repens L. recruits root-associated Microbacterium species to adapt to heavy metal stress in an abandoned Pb-Zn mining area.

Root-associated microbiota provide great fitness to hosts under environmental stress. However, the underlying microecological mechanisms controlling the interaction between heavy metal-stressed plants and the microbiota are poorly understood. In this study, we screened and isolated representative amplicon sequence variants (strain M4) from rhizosphere soil samples of Trifolium repens L. growing in areas with high concentrations of heavy metals. To investigate the microecological mechanisms by which T. repens adapts to heavy metal stress in abandoned mining areas, we conducted potting experiments, bacterial growth promotion experiments, biofilm formation experiments, and chemotaxis experiments. The results showed that high concentrations of heavy metals significantly altered the rhizosphere bacterial community structure of T. repens and significantly enriched Microbacterium sp. Strain M4 was demonstrated to significantly increased the biomass and root length of T. repens under heavy metal stress. Additionally, L-proline and stigmasterol could promote bacterial growth and biofilm formation and induce chemotaxis for strain M4, suggesting that they are key rhizosphere secretions of T. repens for Microbacterium sp. recruitment. Our results suggested that T. repens adapted the heavy metal stress by reshaping rhizosphere secretions to modify the rhizosphere microbiota.

Invasion of Trifolium repens L. aggravated by biodegradable plastics: adjustable strategy for foraging N and P.

The invasion of alien plant and the pollution caused by soil microplastics have emerged as significant ecological threats. Recent studies have demonstrated aggravating effect of non-biodegradable microplastics on plant invasion. However, the impact of biodegradable microplastics (BMPs) on plant invasion remains unclear. Therefore, it is imperative to explore the impact of BMPs on plant invasion. In this study, a 30-day potting experiment with Trifolium repens L. (an invasive plant) and Oxalis corniculata L. (a native plant) was conducted to evaluate the influence of BMPs on T. repens's invasion. The findings revealed that BMPs results in a reduction in available N and P contents, thereby facilitating the colonization of arbuscular mycorrhizal fungi on T. repens 's roots. Consequently, T. repens adjusted its N and P foraging strategy by increasing P absorption ratio, and enhancing the accumulation of N and P in leaves. This ultimately led to the decrease of relative neighbor effect index of T. repens, indicating an aggravated invasion by T. repens. This study significantly enhances and expands the understanding of mechanisms by which microplastics aggravate plant invasion.

Can the Inclusion of Forage Chicory in the Diet of Lactating Dairy Cattle Alter Milk Production and Milk Fatty Acid Composition? Findings of a Multilevel Meta-Analysis.

In traditional ryegrass/white clover (Lolium perenne L./Trifolium repens L.) pastoral systems, forage herbs such as chicory (Cichorium intybus L.) present an opportunity to fill feed deficits during late spring and summer. Although multiple research publications have evaluated the efficacy of chicory for enhancing milk production and milk fatty acid (FA) profile, no publication has quantitatively synthesised the body of research. This systematic review and meta-analysis examined the effect of chicory on milk production and composition, as well as on the milk fatty acid composition of dairy cattle. A total of 29 comparisons from 15 unique research publications involving 597 dairy cattle were used to develop a dataset for analysis. Three-level random-effect and robust variance estimator models were used to account for the hierarchical structure of the data and the dependency of effect sizes within publications. Chicory inclusion increased milk yield when compared to grass-based diets {weighted mean difference (WMD) = 1.07 (95% CI 0.54-1.60) kg/cow/d, p < 0.001}, but it provided a similar milk yield when compared to other forages such as legumes and herbs {dicots; WMD = -0.30, (95% CI -89-0.29) kg/cow/day, p = 0.312}. Increases in milk yield were congruent with differences in DM intake (p = 0.09) and ME intakes (p = 0.003), being similar in chicory-fed and dicot-fed cows but higher than grass-fed cows. Chicory feeding's effect on milk solids was twice as high during mid lactation {154 days in milk; WMD = 0.13, (95% 0.081-0.175) kg/cow/day, p < 0.001} as during late lactation {219 days in milk; WMD = 0.06, (95% 0.003-0.13) kg/cow/day, p = 0.041}. In line with milk yield, greater and more significant effect sizes were found for alpha linolenic acid {ALA; WMD = 0.20 (95% CI 0.06-0.35) g/100 g FA, p = 0.011} when chicory was compared to grass species only. Comparing chicory with dicots suggests that chicory inclusion did not impact ALA concentrations {WMD = 0.001 (95% CI -0.02-0.2) g/100 g FA, p = 0.99}. There were no differences in conjugated linoleic acid concentration in the milk of cows fed chicory or control diets. The study provides empirical evidence of chicory's efficacy for improved milk production and milk fatty acid composition.

Investigation of Trifolium repens L. from the Indian Himalayan region as a phyto-therapeutic agent.

Trifolium repens is a well-known herbaceous, perennial herb and has been extensively used in the traditional medicine system over the years. Various parts of the plant are traditionally used as a curative agent against several health ailments such as skin problems, wound healing, stomach disorders, sedative, fever, antiseptic, analgesic, expectorant, psoriasis and eczema. To maximise the plant's potential for usage in the future, the review also aims to update information about its significant pharmacological properties. The ethnomedicinal benefits of T. repens have been well studied; however, the facets of the plant have not been explored yet. The current review outlines several bioactive compounds quantified from T. repens and a few of them namely quercetin, kaempferol, myricetin, acacetin and linamarin, have been reported to have biological activities such as antibacterial, antifungal, antileishmanial, anti-inflammatory, antiaging and anti-hepatotoxic activities. A significant number of in vitro studies have been done on the plant extract, but little is known about the isolation and efficacy of the potent natural bioactive compounds of T. repens. The bioactive compounds in T. repens can be used for advanced drug development against various health disorders.

Field-evolved pyrethroid resistance in Tychius picirostris (Coleoptera: Curculionidae) populations in Oregon white clover seed crops.

The clover seed weevil, Tychius picirostris Fabricius, a serious pest of white clover, Trifolium repens L., grown for seed in western Oregon, causing feeding damage to flowers and developing seeds. Since 2017, white clover seed producers have anecdotally reported T. picirostris control failures using foliar pyrethroid insecticide applications. This mode of action (MoA) is an important chemical control option for T. picirostris management. To evaluate insecticide resistance selection to pyrethroids (bifenthrin) and other MoAs labeled for T. picirostris management (malathion and chlorantraniliprole), adult populations were collected from 8 commercial white clover grown for seed fields in the Willamette Valley, OR, in 2022 and 2023. Among collected Oregon populations, very high resistance ratios (RR50 = 178.00-725.67) were observed to technical grade bifenthrin and low to high resistance ratios (RR50 = 7.80-32.80) to malathion in surface contact assays compared to a susceptible Canadian field population. Moreover, >2.73 times the labeled rate of formulated product containing bifenthrin as the sole MoA was required to kill >50% of T. picirostris in topical assays. Synergistic assays with a mixed-function oxidase inhibitor, an esterase inhibitor, and a glutathione-S-transferase inhibitor revealed phase I and II detoxification enzymes are present in Oregon T. picirostris populations and confer metabolic resistance to bifenthrin. This is the first report of T. picirostris insecticide resistance selection to pyrethroid and organophosphate insecticides. Results will inform continued monitoring and insecticide resistance management strategies to slow the evolution of T. picirostris insecticide resistance selection in Oregon's white clover seed production.

Co-culture between Miscanthus x giganteus and Trifolium repens L. to enhance microbial activity, biomass and density in a PAH contaminated technosol.

Phytoremediation is a biological soil remediation technique using plants and their associated microorganisms to clean-up contaminated soils and improve soils' quality. We tested whether a co-culture between Miscanthus x giganteus (MxG) and Trifolium repens L. would enhance the soil biological quality. The objective was to determine the influence of MxG in mono- and in co-culture with white clover on the soil microbial activity, biomass and density. MxG was tested in mono- and in co-culture with white clover in a mesocosm over 148 days. The microbial respiration (CO2 production), the microbial biomass and the microbial density of the technosol were measured. Results showed that MxG induced an increase in microbial activity in the technosol compared to the non-planted condition with the co-culture having a greater impact. Regarding the bacterial density, MxG in mono- and in co-culture significantly increased the 16S rDNA gene copy number. The co-culture increased the microbial biomass, the fungal density and stimulated the degrading bacterial population, contrary to the monoculture and the non-planted condition. We can conclude the co-culture between MxG and white clover was more interesting than MxG monoculture in regards to the technosol biological quality and its potential for PAH remediation improvement.

Our precedent results have shown the benefits of using Miscanthus x giganteus in association with Trifolium repens L. to improve polycyclic aromatic hydrocarbons dissipation and decrease soil ecotoxicity compared to monocultures. In this study we focused on the plant species' influence on the soil's biological quality to improve MxG biomass productivity in the long term and phytoremediation. Many bioindicators were used such as microbial activity, microbial biomass as well as bacteria, fungi and PAH-degrading bacteria density.We showed it was more beneficial to use co-culture instead of MxG monoculture to improve biological technosol quality and in particular microbial activity and biomass as well as fungi and PAH-degrading bacteria density.

Identification and characterization of Colletotrichum truncatum and C. destructivum causing stem rot of white clover in China.

White clover (Trifolium repens L.) is an excellent quality forage legume species with superior planting efficiency, which reduces the cost of artificial weeding and nitrogen fertilizer inputs and has feeding and economic value. However, from June to September 2022, severe stem rot affected the yield and quality of white clover crops in Harbin, Heilongjiang Province, China. The aim of this study was to identify the causative agents of the disease. Overall, Colletotrichum truncatum (6 isolates) and C. destructivum (10 isolates) were obtained from rotten white clover stems and identified according to morpho-molecular characteristics and phylogenetic analyses. Pathogenicity tests of the isolates revealed that C. destructivum had a higher pathogenicity to white clover than C. truncatum. In addition, all isolates were highly pathogenic to broad bean, fodder soybean, soybean, pak choi, and chickpea, pathogenic to mint, and did not infect corn, wheat, or cilantro. C. destructivum and C. truncatum isolates were very sensitive to tebuconazole and pyraclostrobin, with EC50 values of 0.54 to 0.70 µg/ml and 0.42 to 0.62 µg/ml, respectively, efficacies ranging between 93.2 to 94.9% and 90.2 to 95.2% at 600 µg/ml and 450 µg/ml, respectively, and EC90 values of 1.88 to 13.36 µg/ml and 1.32 to 23.39 µg/ml, respectively. Therefore, intercropping of host and non-host plants and chemicals can be considered to control stem rot in white clover. These results provide a basis for controlling C. destructivum and C. truncatum in white clover in China.

Variable expression of cyanide detoxification and tolerance genes in cyanogenic and acyanogenic white clover (Trifolium repens).

PREMISE: ß-Cyanoalanine synthase (ß-CAS) and alternative oxidase (AOX) play important roles in the ability of plants to detoxify and tolerate hydrogen cyanide (HCN). These functions are critical for all plants because HCN is produced at low levels during basic metabolic processes, and especially for cyanogenic species, which release high levels of HCN following tissue damage. However, expression of ß-CAS and Aox genes has not been examined in cyanogenic species, nor compared between cyanogenic and acyanogenic genotypes within a species. METHODS: We used a natural polymorphism for cyanogenesis in white clover to examine ß-CAS and Aox gene expression in relation to cyanogenesis-associated HCN exposure. We identified all ß-CAS and Aox gene copies present in the genome, including members of the Aox1, Aox2a, and Aox2d subfamilies previously reported in legumes. Expression levels were compared between cyanogenic and acyanogenic genotypes and between damaged and undamaged leaf tissue. RESULTS: ß-CAS and Aox2a expression was differentially elevated in cyanogenic genotypes, and tissue damage was not required to induce this increased expression. Aox2d, in contrast, appeared to be upregulated as a generalized wounding response. CONCLUSIONS: These findings suggest a heightened constitutive role for HCN detoxification (via elevated ß-CAS expression) and HCN-toxicity mitigation (via elevated Aox2a expression) in plants that are capable of cyanogenesis. As such, freezing-induced cyanide autotoxicity is unlikely to be the primary selective factor in the evolution of climate-associated cyanogenesis clines.

High-quality chromosome-level de novo assembly of the Trifolium repens.

BACKGROUND: White clover (Trifolium repens L.), an excellent perennial legume forage, is an allotetraploid native to southeastern Europe and southern Asia. It has high nutritional, ecological, genetic breeding, and medicinal values and exhibits excellent resistance to cold, drought, trample, and weed infestation. Thus, white clover is widely planted in Europe, America, and China; however, the lack of reference genome limits its breeding and cultivation. This study generated a white clover de novo genome assembly at the chromosomal level and annotated its components. RESULTS: The PacBio third-generation Hi-Fi assembly and sequencing methods generated a 1096 Mb genome size of T. repens, with contigs of N50 = 14 Mb, scaffolds of N50 = 65 Mb, and BUSCO value of 98.5%. The newly assembled genome has better continuity and integrity than the previously reported white clover reference genome; thus provides important resources for the molecular breeding and evolution of white clover and other forage. Additionally, we annotated 90,128 high-confidence gene models from the genome. White clover was closely related to Trifolium pratense and Trifolium medium but distantly related to Glycine max, Vigna radiata, Medicago truncatula, and Cicer arietinum. The expansion, contraction, and GO functional enrichment analysis of the gene families showed that T. repens gene families were associated with biological processes, molecular function, cellular components, and environmental resistance, which explained its excellent agronomic traits. CONCLUSIONS: This study reports a high-quality de novo assembly of white clover genome obtained at the chromosomal level using PacBio Hi-Fi sequencing, a third-generation sequencing. The generated high-quality genome assembly of white clover provides a key basis for accelerating the research and molecular breeding of this important forage crop. The genome is also valuable for future studies on legume forage biology, evolution, and genome-wide mapping of quantitative trait loci associated with the relevant agronomic traits.

White clover from the exclusion zone of the Chernobyl NPP: Morphological, biochemical, and genetic characteristics.

A comprehensive study of the biological effects of chronic radiation exposure (8 µGy/h) in populations of white clover (Trifolium repens L.) from the Chernobyl exclusion zone was carried out. White clover is one of the most important pasture legumes, having many agricultural applications. Studies at two reference and three radioactively contaminated plots showed no stable morphological effects in white clover at this level of radiation exposure. Increased activities of catalase and peroxidases were found in some impacted plots. Auxin concentration was enhanced in the radioactively contaminated plots. Genes involved in the maintenance of water homeostasis and photosynthetic processes (TIP1 and CAB1) were upregulated at radioactively contaminated plots.

Trifolium repens and biochar addition affecting soil nutrients and bacteria community.

Biochar has wide application prospects as a good soil conditioner, leguminous plants can fix nitrogen and improve soil available nutrients. However, it is not clear how adding biochar when planting leguminous plants affects soil bacterial community and soil available nutrients. This study investigates the effects of biochar addition on the content of ammonia nitrogen, Olsen-P, and available potassium in northeastern farmland soils under the plantation of Trifolium repens and then compared with the application of organic fertilizer. A 90-day incubation experiment was conducted to compare the changes in the structure and relative abundance of soil microflora under varied biochar additions. It was found that the addition of biochar could affect the structure of the microflora and the available nutrients in the soil. When compared with soil planted with T. repens without the addition of biochar, with the application of 3% biochar increased the content of ammonia nitrogen, Olsen-P, and available potassium in the soil by 31.71%, 21.40%, and 11.51%, respectively. High throughput sequencing revealed that the relative abundance of functional bacteria such as azotobacter, rhizobacteria, and phosphorus solubilizing bacteria in the soil increased with the addition of biochar. Furthermore, the effect was more obvious with the addition of organic fertilizers. The addition of biochar improved the microbial community structure and increased the relative abundance of functional bacteria and the content of available nutrients in the soil. This is expected to reduce the application of chemical fertilizers, thereby protecting the environment and conserving natural resources.

Differential gene expression in chronically irradiated herbaceous species from the Chernobyl exclusion zone.

PURPOSE: Transcriptional activity of genes related to ionizing radiation responses in chronically irradiated plant populations at radioactively contaminated territories can be a cost-effective and precise approach for stress response evaluation. However, there are limits to studying non-model plants in field conditions. The work studies the transcriptional activity of candidate genes of adaptation to chronic radiation exposure in plant populations from radioactively contaminated territories of the Chernobyl. MATERIALS AND METHODS: In this work, we studied plant species with different sensitivity to acute irradiation: Trifolium repens L., Taraxacum officinale Wigg., and Dactylis glomerata L., sampled in the Chernobyl exclusion zone. The differential expression of several candidate genes of adaptation to chronic radiation exposure in the leaves of these species was analyzed, including homologs of Arabidopsis thaliana genes SLAC1, APX1, GPX2, CAB1, NTRB, PP2-B11, RBOH-F, HY5, SnRK2.4, PDS1, CIPK20, SIP1, PIP1, TIP1. RESULTS AND CONCLUSIONS: All studied species were characterized by upregulation of the CAB1 homolog, encoding chlorophyll a/b binding protein, at radioactively contaminated plots. An increase in the expression of genes associated with water and hydrogen peroxide transport, intensity of photosynthesis, and stress responses (homolog of aquaporin TIP1 for T. repens; homologs of aquaporin PIP1 and transcription factor HY5 for D. glomerata; homolog of CBL-interacting serine/threonine protein kinase CIPK20 for T. officinale) was revealed. The methodological approach for studying gene expression in non-model plant species is described, which may allow large-scale screening studies of candidate genes in various plant species abundant in radioactively contaminated areas.

A Heat Shock Transcription Factor TrHSFB2a of White Clover Negatively Regulates Drought, Heat and Salt Stress Tolerance in Transgenic Arabidopsis.

Heat shock transcription factors (HSF) are divided into classes A, B and C. Class A transcription factors are generally recognized as transcriptional activators, while functional characterization of class B and C heat shock transcription factors have not been fully developed in most plant species. We isolated and characterized a novel HSF transcription factor gene, TrHSFB2a (a class B HSF) gene, from the drought stress-sensitive forage crop species, white clover (Trifolium repens). TrHSFB2a was highly homologous to MtHSFB2b, CarHSFB2a, AtHSFB2b and AtHSFB2a. The expression of TrHSFB2a was strongly induced by drought (PEG6000 15% w/v), high temperature (35 °C) and salt stresses (200 mM L-1 NaCl) in white clover, while subcellular localization analysis showed that it is a nuclear protein. Overexpression of the white clover gene TrHSFB2a in Arabidopsis significantly reduced fresh and dry weight, relative water contents (RWC), maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PIABS), while it promoted leaf senescence, relative electrical conductivity (REC) and the contents of malondialdehyde (MDA) compared to a wild type under drought, heat and salt stress conditions of Arabidopsis plants. The silencing of its native homolog (AtHSFB2a) by RNA interference in Arabidopsis thaliana showed opposite trends by significantly increasing fresh and dry weights, RWC, maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PIABS) and reducing REC and MDA contents under drought, heat and salt stress conditions compared to wild type Arabidopsis plants. These phenotypic and physiological indicators suggested that the TrHSFB2a of white clover functions as a negative regulator of heat, salt and drought tolerance. The bioinformatics analysis showed that TrHSFB2a contained the core B3 repression domain (BRD) that has been reported as a repressor activator domain in other plant species that might repress the activation of the heat shock-inducible genes required in the stress tolerance process in plants. The present study explores one of the potential causes of drought and heat sensitivity in white clover that can be overcome to some extent by silencing the TrHSFB2a gene in white clover.