NaHS immersion alleviates the stress effect of chromium(III) on alfalfa seeds by affecting active oxygen metabolism.

OBJECTIVE: This study aimed to investigate the regulatory effects of exogenous hydrogen sulfide (H2S) on seed germination, seedling growth, and reactive oxygen species (ROS) homeostasis in alfalfa under chromium (Cr) ion (III) stress. METHODS: The effects of 0-4 mM Cr(III) on the germination and seedling growth of alfalfa were first assessed. Subsequently, following seed NaHS immersion, the influence of H2S on alfalfa seed germination and seedling growth under 2 mM Cr(III) stress was investigated, and the substance contents and enzyme activities associated with ROS metabolism were quantified. RESULTS: Compared to the control group, alfalfa plant germination was delayed under 2 mM Cr(III) stress for up to 48 h (p < 0.05). At 120 h, the total seedling length was approximately halved, and the root length was roughly one-third of the control. Treatment with 0.02-0.1 mM NaHS alleviated the delay in germination and root growth inhibition caused by 2 mM Cr(III) stress, resulting in an increased ratio of root length to hypocotyl length from 0.57 to 1 above. Additionally, immersion in 0.05 mM NaHS reduced hydrogen peroxide (H2O2) and oxygen-free radicals (O2· -) levels (p < 0.05), boosted glutathione (GSH) levels (p < 0.05), and notably enhanced catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) activities (p < 0.05) compared to the 2 mM Cr(III) stress treatment group. CONCLUSION: Seed immersion in NaHS mitigated the delay in germination and inhibition of root elongation under 2 mM Cr(III) stress. This effect is likely attributed to the regulation of intracellular ROS homeostasis and redox balance through enzymatic and non-enzymatic systems; thus, providing a potential mechanism for combating oxidative stress.

Interactive effects of ammonium sulfate and lead on alfalfa in rare earth tailings: Physiological responses and toxicity thresholds.

Ion-adsorption rare earth ore contains significant levels of leaching agents and heavy metals, leading to substantial co-contamination. This presents significant challenges for ecological rehabilitation, yet there is limited understanding of the toxicity thresholds associated with the co-contamination of ammonium sulfate (AS) and lead (Pb) on pioneer plants. Here, we investigated the toxicity thresholds of various aspects of alfalfa, including growth, ultrastructural changes, metabolism, antioxidant system response, and Pb accumulation. The results indicated that the co-contamination of AS-Pb decreased the dry weight of shoot and root by 26 %-77 % and 18 %-92 %, respectively, leading to irregular root cell morphology and nucleus disintegration. The high concentration and combined exposures to AS and Pb induced oxidative stress on alfalfa, which stimulated the defense of the antioxidative system and resulted in an increase in proline levels and a decrease in soluble sugars. Structural equation modeling analysis and integrated biomarker response elucidated that the soluble sugars, proline, and POD were the key physiological indicators of alfalfa under stresses and indicated that co-exposure induced more severe oxidative stress in alfalfa. The toxicity thresholds under single exposure were 496 (EC5), 566 (EC10), 719 (EC25), 940 (EC50) mg kg-1 for AS and 505 (EC5), 539 (EC10), 605 (EC25), 678 (EC50) mg kg-1 for Pb. This study showed that AS-Pb pollution notably influenced plant growth performance and had negative impacts on the growth processes, metabolite levels, and the antioxidant system in plants. Our findings contribute to a theoretical foundation and research necessity for evaluating ecological risks in mining areas and assessing the suitability of ecological restoration strategies.

Harnessing plant-beneficial bacterial encapsulation: A sustainable strategy for facilitating cadmium bioaccumulation in Medicago sativa.

Plant-beneficial bacteria (PBB) have emerged as a promising approach for assisting phytoremediation of heavy metal (HM)-contaminated soils. However, their colonization efficiency is often challenged by complex soil environments. In this study, we screened one rhizobacterium (Klebsiella variicola Y38) and one endophytic bacterium (Serratia surfactantfaciens Y15) isolated from HM-contaminated soils and plants for their high resistance to Cd and strong growth-promoting abilities. These strains were encapsulated individually or in combination with alginate and applied with Medicago sativa in Cd-contaminated soil pot experiments. The effectiveness of different bacterial formulations in promoting plant growth and enhancing Cd bioconcentration in M. sativa was evaluated. Results showed that PBB application enhanced plant growth and antioxidant capacity while reducing oxidative damage. Encapsulated formulations outperformed unencapsulated ones, with combined formulations yielding superior results to individual applications. Quantitative PCR indicated enhanced PBB colonization in Cd-contaminated soils with alginate encapsulation, potentially explaining the higher efficacy of alginate-encapsulated PBB. Additionally, the bacterial agents modified Cd speciation in soils, resulting in increased Cd bioaccumulation in M. sativa by 217-337 %. The alginate-encapsulated mixed bacterial agent demonstrated optimal effectiveness, increasing the Cd transfer coefficient by 3.2-fold. Structural equation modeling and correlation analysis elucidated that K. variicola Y38 promoted Cd bioaccumulation in M. sativa roots by reducing oxidative damage and enhancing root growth, while S. surfactantfaciens Y15 facilitated Cd translocation to shoots, promoting shoot growth. The combined application of these bacteria leveraged the benefits of both strains. These findings contribute to diversifying strategies for effectively and sustainably remediating Cd-contaminated soils, while laying a foundation for future investigations into bacteria-assisted phytoremediation.

Performance and mechanistic study of biochar and magnesium-enhanced phytoremediation in cadmium-contaminated soil by alfalfa.

Recently, phytoremediation has been regarded as a green and environment friendly technique to treat metals contaminated soils. Thus, in this study, pot experiments were designed to investigate the combine effects of biochar and magnesium (MPs) to purify cadmium (Cd)-contaminated soils by Medicago sativa L. (alfalfa). The results showed that the combined use of biochar and Mg significantly increased the accumulation of Cd and promoted the transport of Cd from root to shoot in alfalfa, simultaneously. Importantly, the combined use of biochar and Mg could increase the accumulation of Cd in shoot and whole plant (shoot + root) of alfalfa up-to 59.1% and 23.1%, respectively. Moreover, the enhancement mechanism can be analyzed from several aspects. Firstly, the photosynthesis was enhanced, which was beneficial to plant growth. The product of photosynthesis provided energy for uptake and transport of Cd. Meanwhile, its transport in phloem could promote the transport of Cd. Secondly, the enhancement of antioxidant capacity of alfalfa effectively protected the membrane structure of alfalfa, which indicated that Cd could enter alfalfa from the channel on the cell membrane. Lastly, the chemical form of Cd and microbial community structure in soil were changed. Overall, these changes reduced the Cd toxicity in soil, enhanced the resistance capability of alfalfa, increased the Cd uptake by alfalfa and promoted the growth of alfalfa. Thus, the obtained results suggested that the combined use of biochar and Mg is an effective approach to enhance phytoremediation performance for purifying Cd-contaminated soils.

Environmental risks of mask wastes binding pollutants: Phytotoxicity, microbial community, nitrogen and carbon cycles.

The increasing contamination of mask wastes presents a significant global challenge to ecological health. However, there is a lack of comprehensive understanding regarding the environmental risks that mask wastes pose to soil. In this study, a total of 12 mask wastes were collected from landfills. Mask wastes exhibited negligible morphological changes, and bound eight metals and four types of organic pollutants. Masks combined with pollutants inhibited the growth of alfalfa and Elymus nutans, reducing underground biomass by 84.6 %. Mask wastes decreased the Chao1 index and the relative abundances (RAs) of functional bacteria (Micrococcales, Gemmatimonadales, and Sphingomonadales). Metagenomic analysis showed that mask wastes diminished the RAs of functional genes associated with nitrification (amoABC and HAO), denitrification (nirKS and nosZ), glycolysis (gap2), and TCA cycle (aclAB and mdh), thereby inhibiting the nitrogen transformation and ATP production. Furthermore, some pathogenic viruses (Herpesviridae and Tunggulvirus) were also found on the mask wastes. Structural equation models demonstrated that mask wastes restrained soil enzyme activities, ultimately affecting nitrogen and carbon cycles. Collectively, these evidences indicate that mask wastes contribute to soil health and metabolic function disturbances. This study offers a new perspective on the potential environmental risks associated with the improper disposal of masks.

Environmental microplastic interact with heavy metal in polluted soil from mine site in the North of Tunisia: Effects on heavy metal accumulation, growth, photosynthetic activities, and biochemical responses of alfalfa plants (Medicago saliva L.).

As emerging persistent pollutants, microplastic (MPs) pollution attracted increasing attention worldwide since it is posing several environmental concerns. MPs interact with heavy metals in soil and may provoke damages on soil properties and ultimately impaired plants and human health. The present study aims to evaluate alfalfa plants (Medicago sativa) response after exposure to heavy metal polluted soils from mine site in the North of Tunisia in presence of environmental microplastic. For that, soils were sampled from two sites of Jebel Ressass mine in addition to a control soil. Plants were exposed to the three soils in presence of two increasing rates of microplastics D1 (1 mg/kg of soil) and D2 (100 mg/kg of soil) for 60 days. After harvest, agronomic parameters, chlorophyll content as well as heavy metal accumulation in plants were analyzed. Furthermore, oxidative status was evaluated in terms of malondialdehyde accumulation (MDA), catalase (CAT) activities and glutathion-S-transferase (GST). Overall, our finding highlights that MPs disrupted agronomic parameters and the photosynthetic activities of alfalfa plants. Additionally, our results revealed that the presence of MPs in polluted soils cause an increase on heavy metal accumulation in alfalfa shoots. Biochemical analyses demonstrated that the combined exposure to MPs and heavy metal induced oxidative stress in alfalfa plants by increasing CAT activity and MDA accumulation. The present investigation highlights the ecological risks of microplastics in terrestrial environment.

Genome-Wide Analysis of BBX Gene Family in Three Medicago Species Provides Insights into Expression Patterns under Hormonal and Salt Stresses.

BBX protein is a class of zinc finger transcription factors that have B-box domains at the N-terminus, and some of these proteins contain a CCT domain at the C-terminus. It plays an important role in plant growth, development, and metabolism. However, the expression pattern of BBX genes in alfalfa under hormonal and salt stresses is still unclear. In this study, we identified a total of 125 BBX gene family members by the available Medicago reference genome in diploid alfalfa (Medicago sativa spp. Caerulea), a model plant (M. truncatula), and tetraploid alfalfa (M. sativa), and divided these members into five subfamilies. We found that the conserved motifs of BBXs of the same subfamily reveal similarities. We analyzed the collinearity relationship and duplication mode of these BBX genes and found that the expression pattern of BBX genes is specific in different tissues. Analysis of the available transcriptome data suggests that some members of the BBX gene family are involved in multiple abiotic stress responses, and the highly expressed genes are often clustered together. Furthermore, we identified different expression patterns of some BBX genes under salt, ethylene, salt and ethylene, salicylic acid, and salt and salicylic acid treatments, verified by qRT-PCR, and analyzed the subcellular localization of MsBBX2, MsBBX17, and MsBBX32 using transient expression in tobacco. The results showed that BBX genes were localized in the nucleus. This study systematically analyzed the BBX gene family in Medicago plants, which provides a basis for the study of BBX gene family tolerance to abiotic stresses.

An integration of physiology, transcriptomics, and proteomics reveals carbon and nitrogen metabolism responses in alfalfa (Medicago sativa L.) exposed to titanium dioxide nanoparticles.

Nanoparticle (NP) pollution has negative impacts and is a major global environmental problem. However, the molecular response of alfalfa (Medicago sativa L.) to titanium dioxide nanoparticles (TiO2 NPs) is limited. Herein, the dual effects of TiO2 NPs (0-1000 mg L-1) on carbon (C) and nitrogen (N) metabolisms in alfalfa were investigated. The results showed that 500 mg L-1 TiO2 NPs (Ti-500) had the highest phytotoxicity in the C/N metabolizing enzymes; and it significantly increased total soluble sugar, starch, sucrose, and sucrose-phosphate synthase. Furthermore, obvious photosynthesis responses were found in alfalfa exposed to Ti-500. By contrast, 100 mg L-1 TiO2 NPs (Ti-100) enhanced N metabolizing enzymes. RNA-seq analyses showed 4265 and 2121 differentially expressed genes (DEGs) in Ti-100 and Ti-500, respectively. A total of 904 and 844 differentially expressed proteins (DEPs) were identified in Ti-100 and Ti-500, respectively. Through the physiological, transcriptional, and proteomic analyses, the DEGs and DEPs related to C/N metabolism, photosynthesis, chlorophyll synthesis, starch and sucrose metabolism, and C fixation in photosynthetic organisms were observed. Overall, TiO2 NPs at low doses improve photosynthesis and C/N regulation, but high doses can cause toxicity. It is valuable for the safe application of NPs in agriculture.

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.

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.

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.

Biochar enhances the growth and physiological characteristics of Medicago sativa, Amaranthus caudatus and Zea mays in saline soils.

Biochar is a promising solution to alleviate the negative impacts of salinity stress on agricultural production. Biochar derived from food waste effect was investigated on three plant species, Medicago sativa, Amaranthus caudatus, and Zea mays, under saline environments. The results showed that biochar improved significantly the height by 30%, fresh weight of shoot by 35% and root by 45% of all three species compared to control (saline soil without biochar adding), as well as enhanced their photosynthetic pigments and enzyme activities in soil. This positive effect varied significantly between the 3 plants highlighting the importance of the plant-biochar interactions. Thus, the application of biochar is a promising solution to enhance the growth, root morphology, and physiological characteristics of plants under salt-induced stress.

Biological nitrogen fixation maintains carbon/nitrogen balance and photosynthesis at elevated CO2.

Understanding crop responses to elevated CO2 is necessary to meet increasing agricultural demands. Crops may not achieve maximum potential yields at high CO2 due to photosynthetic downregulation, often associated with nitrogen limitation. Legumes have been proposed to have an advantage at elevated CO2 due to their ability to exchange carbon for nitrogen. Here, the effects of biological nitrogen fixation (BNF) on the physiological and gene expression responses to elevated CO2 were examined at multiple nitrogen levels by comparing alfalfa mutants incapable of nitrogen fixation to wild-type. Elemental analysis revealed a role for BNF in maintaining shoot carbon/nitrogen (C/N) balance under all nitrogen treatments at elevated CO2, whereas the effect of BNF on biomass was only observed at elevated CO2 and the lowest nitrogen dose. Lower photosynthetic rates at were associated with the imbalance in shoot C/N. Genome-wide transcriptional responses were used to identify carbon and nitrogen metabolism genes underlying the traits. Transcription factors important to C/N signalling were identified from inferred regulatory networks. This work supports the hypothesis that maintenance of C/N homoeostasis at elevated CO2 can be achieved in plants capable of BNF and revealed important regulators in the underlying networks including an alfalfa (Golden2-like) GLK ortholog.

PECTIN ACETYLESTERASE12 regulates shoot branching via acetic acid and auxin accumulation in alfalfa shoots.

Shoot branching is an important biological trait affecting alfalfa (Medicago sativa L.) production, but its development is complicated and the mechanism is not fully clear. In the present study, pectin acetylesterase 12 (MsPAE12) and NAM/ATAF/CUC-domain transcription factor gene (MsNAC73) were isolated from alfalfa. MsPAE12 was highly expressed in shoot apexes, and MsNAC73 was found to be a key transcriptional repressor of MsPAE12 by directly binding to salicylic acid (SA) and jasmonic acid (JA) elements in the MsPAE12 promoter. The biological functions of MsPAE12 and MsNAC73 were studied through overexpression (OE) and down-expression (RNAi) of the 2 genes in alfalfa. The numbers of shoot branches increased in MsPAE12-OE lines but decreased in MsPAE12-RNAi and MsNAC73-OE plants, which was negatively related to their indole-3-acetic acid (IAA) accumulation in shoot apexes. Furthermore, the contents of acetic acid (AA) in shoot apexes decreased in MsPAE12-OE plants but increased in MsPAE12-RNAi and MsNAC73-OE plants. The changes of AA contents were positively related to the expression of TRYPTOPHAN AMINOTRANSFERASE 1 (MsTAA1), TRYPTOPHAN AMINOTRANSFERASE-RELATED 2 (MsTAR2), and YUCCA flavin monooxygenase (MsYUCC4) and the contents of tryptophan (Trp), indole-3-pyruvic acid (IPA), and IAA in shoot apexes of MsPAE12-OE, MsPAE12-RNAi, and MsNAC73-OE plants. Exogenous application of AA to wild type (WT) and MsPAE12-OE plants increased Trp, IPA, and IAA contents and decreased branch number. Exogenous IAA suppressed shoot branching in MsPAE12-OE plants, but exogenous IAA inhibitors increased shoot branching in MsPAE12-RNAi plants. These results indicate that the MsNAC73-MsPAE12 module regulates auxin-modulated shoot branching via affecting AA accumulation in shoot apexes of alfalfa.

Generation of a multi-herbicide-tolerant alfalfa by using base editing.

KEYMESSAGE: We present the first report on base editing in alfalfa. Specifically, we showed edited alfalfa with tolerance to both sulfonylurea- and imidazolinone-type herbicides.

Exogenous proanthocyanidins improve tolerance of Cu-toxicity by amelioration of oxidative damage and re-programming of gene expression in Medicago sativa.

Excess copper (Cu) in soil due to industrial and agricultural practices can result in reduced plant growth. Excess Cu resulted in severely retarded root growth with severe discoloration of Alfalfa (Medicago sativa) and Medicago truncatula. Growth in the presence of hydrogen peroxide resulted in similar symptoms that could be partially recovered by the addition of the reductant ascorbic acid revealing damage was likely due to oxidative stress. The addition of proanthocyanidins (PAs) in the presence of Cu prevented much of the damage, including plant growth and restoration of lignin synthesis which was inhibited in the presence of excess Cu. Transcriptome analyses of the impact of excess Cu and the amelioration after PAs treatment revealed that changes were enriched in functions associated with the cell wall and extracellular processes, indicating that inhibition of cell wall synthesis was likely the reason for retarded growth. Excess Cu appeared to induce a strong defense response, along with alterations in the expression of a number of genes encoding transcription factors, notably related to ethylene signaling. The addition of PAs greatly reduced this response, and also induced novel genes that likely help ameliorate the effects of excess Cu. These included induction of genes involved in the last step of ascorbic acid biosynthesis and of enzymes involved in cell wall synthesis. Combined, these results show that excess Cu causes severe oxidative stress damage and inhibition of cell wall synthesis, which can be relieved by the addition of PAs.

Phytotoxic Activity of Alkaloids in the Desert Plant Sophora alopecuroides.

Sophora alopecuroides is known to produce relatively large amounts of alkaloids; however, their ecological consequences remain unclear. In this study, we evaluated the allelopathic potential of the main alkaloids, including aloperine, matrine, oxymatrine, oxysophocarpine, sophocarpine, sophoridine, as well as their mixture both in distilled H2O and in the soil matrix. Our results revealed that all the alkaloids possessed inhibitory activity on four receiver species, i.e., Amaranthus retroflexus, Medicago sativa, Lolium perenne and Setaria viridis. The strength of the phytotoxicity of the alkaloids was in the following order: sophocarpine > aloperine > mixture > sophoridine > matrine > oxysophocarpine > oxymatrine (in Petri dish assays), and matrine > mixture > sophocarpine > oxymatrine > oxysophocarpine > sophoridine > aloperine (in pot experiments). In addition, the mixture of the alkaloids was found to significantly increase the IAA content, MDA content and POD activity of M. sativa seedlings, whereas CTK content, ABA content, SOD activity and CAT activity of M. sativa seedlings decreased markedly. Our results suggest S. alopecuroides might produce allelopathic alkaloids to improve its competitiveness and thus facilitate the establishment of its dominance; the potential value of these alkaloids as environmentally friendly herbicides is also discussed.

Dehydrin MsDHN1 improves aluminum tolerance of alfalfa (Medicago sativa L.) by affecting oxalate exudation from root tips.

A SK3 -type dehydrin MsDHN1 was cloned from alfalfa (Medicago sativa L.). Its function and gene regulatory pathways were studied via overexpression and suppression of MsDHN1 in alfalfa seedlings or hairy roots. The results showed that MsDHN1 is a typical intrinsically disordered protein that exists in the form of monomers and homodimers in alfalfa. The plant growth rates increased as a result of MsDHN1 overexpression (MsDHN1-OE) and decreased upon MsDHN1 suppression (MsDHN1-RNAi) in seedlings or hairy roots of alfalfa compared with the wild-type or the vector line under Al stress. MsDHN1 interacting with aquaporin (AQP) MsPIP2;1 and MsTIP1;1 positively affected oxalate secretion from root tips and Al accumulation in root tips. MsABF2 was proven to be an upstream transcription factor of MsDHN1 and activated MsDHN1 expression by binding to the ABRE element of the MsDHN1 promoter. The transcriptional regulation of MsABF2 on MsDHN1 was dependent on the abscisic acid signaling pathway. These results indicate that MsDHN1 can increase alfalfa tolerance to Al stress via increasing oxalate secretion from root tips, which may involve in the interaction of MsDHN1 with two AQP.