Effects of selenium enrichment on fermentation characteristics, selenium content and microbial community of alfalfa silage.

BACKGROUND: Selenium is essential for livestock and human health. The traditional way of adding selenium to livestock diets has limitations, and there is a growing trend to provide livestock with a safe and efficient source of selenium through selenium-enriched pasture. Therefore, this study was conducted to investigate the effects of selenium enrichment on fermentation characteristics, selenium content, selenium morphology, microbial community and in vitro digestion of silage alfalfa by using unenriched (CK) and selenium-enriched (Se) alfalfa as raw material for silage. RESULTS: In this study, selenium enrichment significantly increased crude protein, soluble carbohydrate, total selenium, and organic selenium contents of alfalfa silage fresh and post-silage samples, and it significantly decreased neutral detergent fiber and acid detergent fiber contents (p < 0.05). Selenium enrichment altered the form of selenium in plants, mainly in the form of SeMet and SeMeCys, which were significantly higher than that of CK (p < 0.05). Selenium enrichment could significantly increase the lactic acid content, reduce the pH value, change the diversity of bacterial community, promote the growth of beneficial bacteria such as Lactiplantibacillus and inhibit the growth of harmful bacteria such as Pantoea, so as to improve the fermentation quality of silage. The in vitro digestibility of dry matter (IVDMD), in vitro digestibility of acid detergent fibers (IVADFD) and in vitro digestibility of acid detergent fibers (IVNDFD) of silage after selenium enrichment were significantly higher than those of CK (p < 0.05). CONCLUSION: This study showed that the presence of selenium could regulate the structure of the alfalfa silage bacterial community and improve alfalfa silage fermentation quality. Selenium enrichment measures can change the morphology of selenium in alfalfa silage products, thus promoting the conversion of organic selenium.

A genome-wide association study reveals novel loci and candidate genes associated with plant height variation in Medicago sativa.

BACKGROUND: Plant height (PH) is an important agronomic trait influenced by a complex genetic network. However, the genetic basis for the variation in PH in Medicago sativa remains largely unknown. In this study, a comprehensive genome-wide association analysis was performed to identify genomic regions associated with PH using a diverse panel of 220 accessions of M. sativa worldwide. RESULTS: Our study identified eight novel single nucleotide polymorphisms (SNPs) significantly associated with PH evaluated in five environments, explaining 8.59-12.27% of the phenotypic variance. Among these SNPs, the favorable genotype of chr6__31716285 had a low frequency of 16.4%. Msa0882400, located proximal to this SNP, was annotated as phosphate transporter 3;1, and its role in regulating alfalfa PH was supported by transcriptome and candidate gene association analysis. In addition, 21 candidate genes were annotated within the associated regions that are involved in various biological processes related to plant growth and development. CONCLUSIONS: Our findings provide new molecular markers for marker-assisted selection in M. sativa breeding programs. Furthermore, this study enhances our understanding of the underlying genetic and molecular mechanisms governing PH variations in M. sativa.

In silico analysis identified bZIP transcription factors genes responsive to abiotic stress in Alfalfa (Medicago sativa L.).

BACKGROUND: Alfalfa (Medicago sativa L.) is the most cultivated forage legume around the world. Under a variety of growing conditions, forage yield in alfalfa is stymied by biotic and abiotic stresses including heat, salt, drought, and disease. Given the sessile nature of plants, they use strategies including, but not limited to, differential gene expression to respond to environmental cues. Transcription factors control the expression of genes that contribute to or enable tolerance and survival during periods of stress. Basic-leucine zipper (bZIP) transcription factors have been demonstrated to play a critical role in regulating plant growth and development as well as mediate the responses to abiotic stress in several species, including Arabidopsis thaliana, Oryza sativa, Lotus japonicus and Medicago truncatula. However, there is little information about bZIP transcription factors in cultivated alfalfa. RESULT: In the present study, 237 bZIP genes were identified in alfalfa from publicly available sequencing data. Multiple sequence alignments showed the presence of intact bZIP motifs in the identified sequences. Based on previous phylogenetic analyses in A. thaliana, alfalfa bZIPs were similarly divided and fell into 10 groups. The physico-chemical properties, motif analysis and phylogenetic study of the alfalfa bZIPs revealed high specificity within groups. The differential expression of alfalfa bZIPs in a suite of tissues indicates that bZIP genes are specifically expressed at different developmental stages in alfalfa. Similarly, expression analysis in response to ABA, cold, drought and salt stresses, indicates that a subset of bZIP genes are also differentially expressed and likely play a role in abiotic stress signaling and/or tolerance. RT-qPCR analysis on selected genes further verified these differential expression patterns. CONCLUSIONS: Taken together, this work provides a framework for the future study of bZIPs in alfalfa and presents candidate bZIPs involved in stress-response signaling.

Effects of replacing of alfalfa hay with Plantago lanceolata hay on digestibility, methane production and microbial protein production of total mixed diet.

The aim of current experiment was to determine the effect of replacement of alfalfa hay with ribwort plantain (Plantago lanceolata) hay in ruminant diets on the fermentation parameters such as gas production, methane (CH4) production, true digestible dry matter (TDDM), true digestibility (TD), partitioning factor, microbial protein, and efficiency of microbial protein using in vitro gas production technique. The alfalfa hay was replaced with P. lanceolata hay in a diets isocaloric (2650 kcal/kg DM) and nitrogenic (17% CP kg DM) at the ratio of 0, 5, 10 and 15%. Partial substitution of alfalfa hay with P. lanceolata hay had no significant effect on gas and methane (ml/incubated substrate or %) production whereas the partial substitution had a significant effect on TDDM, TD, gas (ml/digested DM), CH4 (ml ml/digested DM) and microbial MP of diets. The replacement of alfalfa hay with ribwort plantain hay shifted the fermentation pattern from gas and methane production to microbial protein production. Therefore alfalfa hay can be replaced with ribwort plantain hay with high digestibility and anti-methanogenic potential in ruminant diets up to 15% to decrease methane production and improve microbial protein production. However further in vivo experiments are required to determine the effect of replacement on feed intake and animal production.

Transcriptomic analysis provides insights into the molecular mechanism of melatonin-mediated cadmium tolerance in Medicago sativa L.

Cadmium (Cd), a toxic element, often makes a serious threat to plant growth and development. Previous studies found that melatonin (Mel) reduced Cd accumulation and reestablished the redox balance to alleviate Cd stress in Medicago sativa L., however, the complex molecular mechanisms are still elusive. Here, comparative transcriptome analysis and biochemical experiments were conducted to explore the molecular mechanisms of Mel in enhancing Cd tolerance. Results showed that 7237 differentially expressed genes (DEGs) were regulated by Mel pretreatment to Cd stress compared to the control condition in roots of Medicago sativa L. Besides, in comparison with Cd stress alone, Mel upregulated 1081 DEGs, and downregulated 1085 DEGs. These DEGs were mainly involved in the transcription and translation of genes and folding, sorting and degradation of proteins, carbohydrate metabolism, and hormone signal network. Application of Mel regulated the expression of several genes encoding ribosomal protein and E3 ubiquitin-protein ligase involved in folding, sorting and degradation of proteins. Moreover, transcriptomic analyse suggested that Mel might regulate the expression of genes encoding pectin lyase, UDP-glucose dehydrogenase, sucrose-phosphate synthase, hexokinase-1, and protein phosphorylation in the sugar metabolism. Therefore, these could promote sucrose accumulation and subsequently alleviate the Cd damage. In conclusion, above findings provided the mining of important genes and molecular basis of Mel in mitigating Cd tolerance and genetic cultivation of Medicago sativa L.

MsWRKY44 regulates Mg-K homeostasis of shoots and promotes alfalfa sensitivities to acid and Al stresses.

Mg-K homeostasis is essential for plant response to abiotic stress, but its regulation remains largely unknown. MsWRKY44 cloned from alfalfa was highly expressed in leaves and petioles. Overexpression of it inhibited alfalfa growth, and promoted leaf senescence and alfalfa sensitivities to acid and Al stresses. The leaf tips, margins and interveins of old leaves occurred yellow spots in MsWRKY44-OE plants under pH4.5 and pH4.5 +Al conditions. Meanwhile, Mg-K homeostasis was substantially changed with reduction of K accumulation and increases of Mg as well as Al accumulation in shoots of MsWRKY44-OE plants. Further, MsWRKY44 was found to directly bind to the promoters of MsMGT7 and MsCIPK23, and positively activated their expression. Transiently overexpressed MsMGT7 and MsCIPK23 in tobacco leaves increased the Mg and Al accumulations but decreased K accumulation. These results revealed a novel regulatory module MsWRKY44-MsMGT7/MsCIPK23, which affects the transport and accumulation of Mg and K in shoots, and promotes alfalfa sensitivities to acid and Al stresses.

Genotype-by-genotype interkingdom cross-talk between symbiotic nitrogen fixing Sinorhizobium meliloti strains and Trichoderma species.

In the understanding of the molecular interaction between plants and their microbiome, a key point is to identify simplified models of the microbiome including relevant bacterial and fungal partners which could also be effective in plant growth promotion. Here, as proof-of-concept, we aim to identify the possible molecular interactions between symbiotic nitrogen-fixing rhizobia and soil fungi (Trichoderma spp.), hence shed light on synergistic roles rhizospheric fungi could have in the biology of symbiotic nitrogen fixation bacteria. We selected 4 strains of the model rhizobium Sinorhizobium meliloti and 4 Trichoderma species (T. velutinum, T. tomentosum, T. gamsii and T. harzianum). In an experimental scheme of 4 ×4 strains x species combinations, we investigated the rhizobia physiological and transcriptomic responses elicited by fungal spent media, as well as spent media effects on rhizobia-host legume plant (alfalfa, Medicago sativa L.) symbiosis. Fungal spent media had large effects on rhizobia, specific for each fungal species and rhizobial strains combination, indicating a generalized rhizobia genotype x fungal genotype interaction, including synergistic, neutral and antagonistic effects on alfalfa symbiotic phenotypes. Differential expression of a high number of genes was shown in rhizobia strains with up to 25% of total genes differentially expressed upon treatment of cultures with fungal spent media. Percentages over total genes and type of genes differentially expressed changed according to both fungal species and rhizobial strain. To support the hypothesis of a relevant rhizobia genotype x fungal genotype interaction, a nested Likelihood Ratio Test indicated that the model considering the fungus-rhizobium interaction explained 23.4% of differentially expressed genes. Our results provide insights into molecular interactions involving nitrogen-fixing rhizobia and rhizospheric fungi, highlighting the panoply of genes and genotypic interactions (fungus, rhizobium, host plant) which may concur to plant symbiosis.

Degradation of petroleum hydrocarbon contaminants by Rhodococcus erythropolis KB1 synergistic with alfalfa (Medicago sativa L.).

Petroleum hydrocarbons are a stubborn pollutant that is difficult to degrade globally, and plant-microbial degradation is the main way to solve this type of pollutant. In this study, the physiological and ecological responses of alfalfa to petroleum hydrocarbons in different concentrations of petroleum hydrocarbon-contaminated soil with KB1 (Rhodococcus erythropolis) were analyzed and determined by laboratory potting techniques. The growth of alfalfa (CK) and alfalfa with KB1 (JZ) in different concentrations of petroleum hydrocarbons contaminated soil was compared and analyzed. The results of the CK group showed that petroleum hydrocarbons could significantly affect the activity of alfalfa antioxidant enzyme system, inhibit the development of alfalfa roots and the normal growth of plants, especially in the high-concentration group. KB1 strain had the ability to produce IAA, form biofilm, fix nitrogen, produce betaine and ACC deaminase, and the addition of KB1 could improve the growth traits of alfalfa in the soil contaminated with different concentrations of petroleum hydrocarbons, the content of soluble sugars in roots, and the stress resistance and antioxidant enzyme activities of alfalfa. In addition, the degradation kinetics of the strain showed that the degradation rate of petroleum could reach 75.2% after soaking with KB1. Furthermore, KB1 can efficiently degrade petroleum hydrocarbons in advance and significantly alleviate the damage of high concentration of petroleum hydrocarbons to plant roots. The results showed that KB1 strains and alfalfa plants could effectively enhance the degradation of petroleum hydrocarbons, which provided new ideas for improving bioremediation strategies.

Carbon sequestration potential and its main drivers in soils under alfalfa (Medicago sativa L.).

As a perennial forage crop, alfalfa (Medicago sativa L.) has been extensively utilized for the vegetation restoration of degraded soil and provides feedstock for forage. Its high usage can be attributed to its high yield potential and the increasing soil organic carbon (SOC) sequestration of alfalfa cultivation. However, the impact of land conversion to alfalfa on SOC content and its underlying drivers remain unclear. We performed a meta-analysis at the global scale to explore the quantified effects of alfalfa cultivation on SOC content and identify its controlling factors. We employed 1699 pairwise data points from 90 publications based on cropland/abandoned land conversion to alfalfa. Globally, cropland (cropland-alfalfa) and abandoned land (abandoned land-alfalfa) conversion to alfalfa enhanced SOC content by 12.1 % and 13.7 %, respectively. Alfalfa exhibited greater SOC content benefits in the surface soils (0-20 cm) with a lower level of initial SOC (<16 g kg-1), regardless of the land conversion type. Cropland-alfalfa was observed to increase SOC content with fertilization, irrigation, and conventional tillage in the long term (>5 years). Furthermore, abandoned land-alfalfa enhanced SOC content in the absence of alfalfa biomass removal and for longer cultivation durations (>5 years). Boosted regression tree analyses indicated variations in soil properties (75 % for cropland-alfalfa and 65 % for abandoned land-alfalfa) as the primary factors driving changes in SOC content. The dominant drivers were determined as the soil layer (51.6 %), cultivation duration (13.1 %), and initial SOC (12.9 %) for cropland-alfalfa, and initial SOC (43.7 %), soil layer (24.6 %) and cultivation duration (17.1 %) for abandoned land-alfalfa. Land conversion to alfalfa has great potential for SOC sequestration, particularly in low-fertility soils. Therefore, alfalfa cultivation is highly recommended for degraded lands due to its SOC sequestration benefits in vegetation restoration.

Starch and sucrose metabolism plays an important role in the stem development in Medicago sativa.

The forage quality of alfalfa (Medicago sativa ) stems is greater than the leaves. Sucrose hydrolysis provides energy for stem development, with starch being enzymatically converted into sucrose to maintain energy homeostasis. To understand the physiological and molecular networks controlling stem development, morphological characteristics and transcriptome profiles in the stems of two alfalfa cultivars (Zhungeer and WL168) were investigated. Based on transcriptome data, we analysed starch and sugar contents, and enzyme activity related to starch-sugar interconversion. Zhungeer stems were shorter and sturdier than WL168, resulting in significantly higher mechanical strength. Transcriptome analysis showed that starch and sucrose metabolism were significant enriched in the differentially expressed genes of stems development in both cultivars. Genes encoding INV , bglX , HK , TPS and glgC downregulated with the development of stems, while the gene encoding was AMY upregulated. Weighted gene co-expression network analysis revealed that the gene encoding glgC was pivotal in determining the variations in starch and sucrose contents between the two cultivars. Soluble carbohydrate, sucrose, and starch content of WL168 were higher than Zhungeer. Enzyme activities related to sucrose synthesis and hydrolysis (INV, bglX, HK, TPS) showed a downward trend. The change trend of enzyme activity was consistent with gene expression. WL168 stems had higher carbohydrate content than Zhungeer, which accounted for more rapid growth and taller plants. WL168 formed hollow stems were formed during rapid growth, which may be related to the redistribution of carbohydrates in the pith tissue. These results indicated that starch and sucrose metabolism play important roles in the stem development in alfalfa.

Genome-Wide Identification, Phylogenetic and Expression Analysis of Expansin Gene Family in Medicago sativa L.

Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa (Medicago sativa L.) due to the complexity of its tetraploidy. Based on the alfalfa (cultivar "XinjiangDaye") reference genome, we identified 168 Expansin members (MsEXPs). Phylogenetic analysis showed that MsEXPs consist of four subfamilies: MsEXPAs (123), MsEXPBs (25), MsEXLAs (2), and MsEXLBs (18). MsEXPAs, which account for 73.2% of MsEXPs, and are divided into twelve groups (EXPA-I-EXPA-XII). Of these, EXPA-XI members are specific to Medicago trunctula and alfalfa. Gene composition analysis revealed that the members of each individual subfamily shared a similar structure. Interestingly, about 56.3% of the cis-acting elements were predicted to be associated with abiotic stress, and the majority were MYB- and MYC-binding motifs, accounting for 33.9% and 36.0%, respectively. Our short-term treatment (≤24 h) with NaCl (200 mM) or PEG (polyethylene glycol, 15%) showed that the transcriptional levels of 12 MsEXPs in seedlings were significantly altered at the tested time point(s), indicating that MsEXPs are osmotic-responsive. These findings imply the potential functions of MsEXPs in alfalfa adaptation to high salinity and/or drought. Future studies on MsEXP expression profiles under long-term (>24 h) stress treatment would provide valuable information on their involvement in the response of alfalfa to abiotic stress.

Genome-wide characterization, transcriptome profiling, and functional analysis of the ALMT gene family in Medicago for aluminum resistance.

Aluminum (Al) is the major limiting factor affecting plant productivity in acidic soils. Al3+ ions exhibit increased solubility at a pH below 5, leading to plant root tip toxicity. Alternatively, plants can perceive very low concentrations of Al3+, and Al triggers downstream signaling even at pH 5.7 without causing Al toxicity. The ALUMINUM-ACTIVATED-MALATE-TRANSPORTER (ALMT) family members act as anion channels, with some regulating the secretion of malate from root apices to chelate Al, which is a crucial mechanism for plant Al resistance. To date, the role of the ALMT gene family within the legume Medicago species has not been fully characterized. In this study, we investigated the ALMT gene family in M. sativa and M. truncatula and identified 68 MsALMTs and 18 MtALMTs, respectively. Phylogenetic analysis classified these genes into five clades, and synteny analysis uncovered genuine paralogs and orthologs. The real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that MtALMT8, MtALMT9, and MtALMT15 in clade 2-2b are expressed in both roots and root nodules, and MtALMT8 and MtALMT9 are significantly upregulated by Al in root tips. We also observed that MtALMT8 and MtALMT9 can partially restore the Al sensitivity of Atalmt1 in Arabidopsis. Moreover, transcriptome analysis examined the expression patterns of these genes in M. sativa in response to Al at both pH 5.7 and pH 4.6, as well as to protons, and found that Al and protons can independently induce some Al-resistance genes. Overall, our findings indicate that MtALMT8 and MtALMT9 may play a role in Al resistance, and highlight the resemblance between the ALMT genes in Medicago species and those in Arabidopsis.

The Effect of Genome Parametrization and SNP Marker Subsetting on Genomic Selection in Autotetraploid Alfalfa.

BACKGROUND: Alfalfa, the most economically important forage legume worldwide, features modest genetic progress due to long selection cycles and the extent of the non-additive genetic variance associated with its autotetraploid genome. METHODS: To improve the efficiency of genomic selection in alfalfa, we explored the effects of genome parametrization (as tetraploid and diploid dosages, plus allele ratios) and SNP marker subsetting (all available SNPs, only genic regions, and only non-genic regions) on genomic regressions, together with various levels of filtering on reading depth and missing rates. We used genotyping by sequencing-generated data and focused on traits of different genetic complexity, i.e., dry biomass yield in moisture-favorable (FE) and drought stress (SE) environments, leaf size, and the onset of flowering, which were assessed in 143 genotyped plants from a genetically broad European reference population and their phenotyped half-sib progenies. RESULTS: On average, the allele ratio improved the predictive ability compared with other genome parametrizations (+7.9% vs. tetraploid dosage, +12.6% vs. diploid dosage), while using all the SNPs offered an advantage compared with any specific SNP subsetting (+3.7% vs. genic regions, +7.6% vs. non-genic regions). However, when focusing on specific traits, different combinations of genome parametrization and subsetting achieved better performances. We also released Legpipe2, an SNP calling pipeline tailored for reduced representation (GBS, RAD) in medium-sized genotyping experiments.

Evolutionary genomics of climatic adaptation and resilience to climate change in alfalfa.

Given the escalating impact of climate change on agriculture and food security, gaining insights into the evolutionary dynamics of climatic adaptation and uncovering climate-adapted variation can empower the breeding of climate-resilient crops to face future climate change. Alfalfa (Medicago sativa subsp. sativa), the queen of forages, shows remarkable adaptability across diverse global environments, making it an excellent model for investigating species responses to climate change. In this study, we performed population genomic analyses using genome resequencing data from 702 accessions of 24 Medicago species to unravel alfalfa's climatic adaptation and genetic susceptibility to future climate change. We found that interspecific genetic exchange has contributed to the gene pool of alfalfa, particularly enriching defense and stress-response genes. Intersubspecific introgression between M. sativa subsp. falcata (subsp. falcata) and alfalfa not only aids alfalfa's climatic adaptation but also introduces genetic burden. A total of 1671 genes were associated with climatic adaptation, and 5.7% of them were introgressions from subsp. falcata. By integrating climate-associated variants and climate data, we identified populations that are vulnerable to future climate change, particularly in higher latitudes of the Northern Hemisphere. These findings serve as a clarion call for targeted conservation initiatives and breeding efforts. We also identified pre-adaptive populations that demonstrate heightened resilience to climate fluctuations, illuminating a pathway for future breeding strategies. Collectively, this study enhances our understanding about the local adaptation mechanisms of alfalfa and facilitates the breeding of climate-resilient alfalfa cultivars, contributing to effective agricultural strategies for facing future climate change.

Effects of water access time and unlimited access to alfalfa straw on litter quality, performance, and behavior of breeder pullets.

Between 3 and 20 wk of age (WOA), the effects of water access time and access to alfalfa during the rearing phase on the litter conditions, performance, and behavior of broiler breeder pullets was studied. A total of 480 female one-day-old chicks (Ross 308) were randomly assigned to 24 floor pens (20 pullets/pen) within a 3 × 2 factorial completely randomized block design. Between 3 and 20 WOA, pullets received water 1) between 07:30 am and 10:30 pm h (3HR), 2) in 2 periods between 07:30 am and 11:00 pm h and between 14:00 pm and 15:30 pm h (5HR), or 3) during the entire light period (8HR). Half of the pens had unlimited access to alfalfa straw (ALF+) or not (ALF-). Higher water use and water-to-feed ratios were observed in the 5HR and 8HR pullets compared to the 3HR pullets (P < 0.001), with no effect observed from unlimited alfalfa. Clear differences in water use throughout the day were observed for the different water strategies. The dry matter (DM) content in the litter was lower, and the litter friability and moisture scores were higher in the 5HR and 8HR than the 3HR pens (P < 0.001), with no differences in fresh feces DM. Alfalfa straw had no effect on litter DM content, fresh feces DM content, litter friability score, or litter moisture score. Feather cover score and feather and footpad contamination score were higher in 5HR and 8HR pullets than in 3HR pullets (P < 0.05), with no differences between the ALF+ and ALF- pullets. The 5HR and 8HR pullets showed increased pecking at alfalfa straw and drinking nipples, along with decreased foraging and perching than the 3HR pullets (P < 0.05). Additionally, ALF+ pullets showed a tendency for less object pecking behavior (P = 0.066) than ALF- pullets. In conclusion, the study demonstrated that extended access to water in breeder pullets increased water use, resulting in inferior litter quality, decreased feather cover, and decreased feather cover and footpad contamination. Moreover, unlimited access to alfalfa straw decreased object pecking behavior.

Insights into the Impact of Trans-Zeatin Overproduction-Engineered Sinorhizobium meliloti on Alfalfa (Medicago sativa L.) Tolerance to Drought Stress.

Plant growth-promoting rhizobacteria have been shown to enhance plant tolerance to drought stress through various mechanisms. However, there is limited research on improving drought resistance in alfalfa by genetically modifying PGPR to produce increased levels of cytokinins. Herein, we employed synthetic biology approaches to engineer two novel strains of Sinorhizobium meliloti capable of overproducing trans-Zeatin and investigated their potential in enhancing drought tolerance in alfalfa. Our results demonstrate that alfalfa plants inoculated with these engineered S. meliloti strains exhibited reduced wilting and yellowing while maintaining higher relative water content under drought conditions. The engineered S. meliloti-induced tZ activated the activity of antioxidant enzymes and the accumulation of osmolytes. Additionally, the increased endogenous tZ content in plants alleviated the impact of drought stress on the alfalfa photosynthetic rate. However, under nondrought conditions, inoculation with the engineered S. meliloti strains had no significant effect on alfalfa biomass and nodule formation.

Heavy fuel oil-contaminated soil remediation by individual and bioaugmentation-assisted phytoremediation with Medicago sativa and with cold plasma-treated M. sativa.

Developing an optimal environmentally friendly bioremediation strategy for petroleum products is of high interest. This study investigated heavy fuel oil (HFO)-contaminated soil (4 and 6 g kg-1) remediation by individual and combined bioaugmentation-assisted phytoremediation with alfalfa (Medicago sativa L.) and with cold plasma (CP)-treated M. sativa. After 14 weeks of remediation, HFO removal efficiency was in the range between 61 and 80% depending on HFO concentration and remediation technique. Natural attenuation had the lowest HFO removal rate. As demonstrated by growth rate and biomass acquisition, M. sativa showed good tolerance to HFO contamination. Cultivation of M. sativa enhanced HFO degradation and soil quality improvement. Bioaugmentation-assisted phytoremediation was up to 18% more efficient in HFO removal through alleviated HFO stress to plants, stimulated plant growth, and biomass acquisition. Cold plasma seed treatment enhanced HFO removal by M. sativa at low HFO contamination and in combination with bioaugmentation it resulted in up to 14% better HFO removal compared to remediation with CP non-treated and non-bioaugmented M. sativa. Our results show that the combination of different remediation techniques is an effective soil rehabilitation strategy to remove HFO and improve soil quality. CP plant seed treatment could be a promising option in soil clean-up and valorization.

Genome-wide identification and expression pattern analysis of the Aux/IAA (auxin/indole-3-acetic acid) gene family in alfalfa (Medicago sativa) and the potential functions under drought stress.

BACKGROUND: Auxin/induced-3-acetic acid (Aux/IAA) is an important plant hormone that affects plant growth and resistance to abiotic stresses. Drought stress is a vital factor in reducing plant biomass yield and production quality. Alfalfa (Medicago sativa L.) is the most widely planted leguminous forage and one of the most economically valuable crops in the world. Aux/IAA is one of the early responsive gene families of auxin, playing a crucial role in response to drought stress. However, the characteristics of the Aux/IAA gene family in alfalfa and its potential function in response to drought stress are still unknown. RESULT: A total of 41 Aux/IAA gene members were identified in alfalfa genome. The physicochemical, peptide structure, secondary and tertiary structure analysis of proteins encoded by these genes revealed functional diversity of the MsIAA gene. A phylogenetic analysis classified the MsIAA genes into I-X classes in two subgroups. And according to the gene domain structure, these genes were classified into typical MsIAA and atypical MsIAA. Gene structure analysis showed that the MsIAA genes contained 1-4 related motifs, and except for the third chromosome without MsIAAs, they were all located on 7 chromosomes. The gene duplication analysis revealed that segmental duplication and tandem duplication greatly affected the amplification of the MsIAA genes. Analysis of the Ka/Ks ratio of duplicated MsAux/IAA genes suggested purification selection pressure was high and functional differences were limited. In addition, identification and classification of promoter cis-elements elucidated that MsIAA genes contained numerous elements associated to phytohormone response and abiotic stress response. The prediction protein-protein interaction network showed that there was a complex interaction between the MsAux/IAA genes. Gene expression profiles were tissue-specific, and MsAux/IAA had a broad response to both common abiotic stress (ABA, salt, drought and cold) and heavy metal stress (Al and Pb). Furthermore, the expression patterns analysis of 41 Aux/IAA genes by the quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that Aux/IAA genes can act as positive or negative factors to regulate the drought resistance in alfalfa. CONCLUSION: This study provides useful information for the alfalfa auxin signaling gene families and candidate evidence for further investigation on the role of Aux/IAA under drought stress. Future studies could further elucidate the functional mechanism of the MsIAA genes response to drought stress.

Low-concentration repeated exposure and high-concentration single exposure of cyanamide suppressed the growth of redroot pigweed in the alfalfa field.

The inclination toward natural products has led to the onset of the discovery of new bioactive metabolites that could be targeted for specific therapeutic or agronomic applications. Despite increasing knowledge coming to light of plant-derived materials as leads for new herbicides, relatively little is known about the mode of action on herbicide-resistant weeds. Cyanamide (CA) is a naturally occurring herbicide synthesized by hairy vetch (Vicia villosa Roth.). However, it has not been experimentally verified whether CA suppresses target plants via sustained discharge at low concentrations, as is often the case with most plant-derived materials. This study aimed to detect the toxicity and the mode of action of CA to alfalfa (Medicago sativa L.) and redroot pigweed (Amaranthus retroflexus L.). The toxicity of CA toward the alfalfa and redroot pigweed by three different exposure patterns was compared: low-concentration repeated exposure with 0.3 g/L CA (LRE), high-concentration single exposure with 1.2 g/L CA (HSE), and distilled water spray as control. The results showed that CA had a stronger inhibitory effect on redroot pigweed growth compared to alfalfa under both LRE and HSE exposure modes, with leaves gradually turning yellow and finally wilting. Beyond that, field trials were conducted to corroborate the toxicity of CA to alfalfa and redroot pigweed. The results have also shown that CA could inhibit the growth of redroot pigweed without significant adverse effects on alfalfa. The outcomes concerning electrolyte permeability, root activity, and malondialdehyde (MDA) content indicated that CA suppressed the growth of redroot pigweed by interfering with the structure of the cell membrane and impacting cellular osmotic potential. CA could destroy the cell membrane structure to inhibit the growth of the redroot pigweed by both LRE and HSE exposure modes, which provides a theoretical basis for preventing and controlling redroot pigweed in alfalfa fields.

Effects of Selenium on the Lignin Deposition Pattern and Stem Mechanical Properties of Alfalfa (Medicago sativa L.).

Lignin provides structural support to plants; however, it reduces their utilization rate. According to our previous studies, selenium (Se) reduces lignin accumulation in alfalfa, but the specific mechanism involved remains unclear. Therefore, at the seedling stage, four root irrigation treatments using 2.5, 50, and 5 µmol/L sodium selenite (S-RI), selenomethionine (SS-RI), Se nanoparticles (SSS-RI), and deionized water (CK-RI) were performed. At the branching stage, four treatments of foliar spraying with the three Se fertilizers described above at a concentration of 0.5 mmol/L (S-FS, SS-FS, and SSS-FS) and deionized water (CK-FS) were administered. The results revealed that all Se treatments chiefly reduced the level of deposition of syringyl (S) lignin in the first internode of alfalfa stems. SS-FS and SSS-FS treatments mainly reduced the deposition of S and guaiacyl (G) lignins in the sixth internode of alfalfa stems, respectively, while S-FS treatment only slightly reduced the deposition of G lignin. S, SS, and SSS-RI treatments reduced the level of deposition of S and G lignins in the sixth internode of alfalfa stems. Se application increased plant height, stem diameter, epidermis (cortex) thickness, primary xylem vessel number (diameter), and pith diameter of alfalfa but decreased primary xylem area and pith parenchyma cell wall thickness of the first internode, and SS(SSS)-FS treatment reduced the mechanical strength of alfalfa stems. Therefore, Se application could decrease lignin accumulation by regulating the organizational structure parameters of alfalfa stems and the deposition pattern of the lignin monomers.