Overexpression of MsDREB1C Modulates Growth and Improves Forage Quality in Tetraploid Alfalfa (Medicago sativa L.).

DREB has been reported to be involved in plant growth and response to environmental factors. However, the function of DREB in growth and development has not been elucidated in alfalfa (Medicago sativa L.), a perennial tetraploid forage cultivated worldwide. In this study, an ortholog of MtDREB1C was characterized from alfalfa and named MsDREB1C accordingly. MsDREB1C was significantly induced by abiotic stress. The transcription factor MsDREB1C resided in the nucleus and had self-transactivation activity. The MsDREB1C overexpression (OE) alfalfa displayed growth retardation under both long-day and short-day conditions, which was supported by decreased MsGA20ox and upregulated MsGA2ox in the OE lines. Consistently, a decrease in active gibberellin (GA) was detected, suggesting a negative effect of MsDREB1C on GA accumulation in alfalfa. Interestingly, the forage quality of the OE lines was better than that of WT lines, with higher crude protein and lower lignin content, which was supported by an increase in the leaf-stem ratio (LSR) and repression of several lignin-synthesis genes (MsNST, MsPAL1, MsC4H, and Ms4CL). Therefore, this study revealed the effects of MsDREB1C overexpression on growth and forage quality via modifying GA accumulation and lignin synthesis, respectively. Our findings provide a valuable candidate for improving the critical agronomic traits of alfalfa, such as overwintering and feeding value of the forage.

Alfalfa Responses to Intensive Soil Compaction: Effects on Plant and Root Growth, Phytohormones and Internal Gene Expression.

The perennial legume alfalfa (Medicago sativa L.) is of high value in providing cheap and high-nutritive forages. Due to a lack of tillage during the production period, the soil in which alfalfa grows prunes to become compacted through highly mechanized agriculture. Compaction deteriorates the soil's structure and fertility, leading to compromised alfalfa development and productivity. However, the way alfalfa responses to different levels of soil compaction and the underlying molecular mechanism are still unclear. In this study, we systematically evaluated the effects of gradient compacted soil on the growth of different cultivars of alfalfa, especially the root system architecture, phytohormones and internal gene expression profile alterations. The results showed that alfalfa growth was facilitated by moderate soil compaction, but drastically inhibited when compaction was intensified. The inhibition effect was universal across different cultivars, but with different severity. Transcriptomic and physiological studies revealed that the expression of a set of genes regulating the biosynthesis of lignin and flavonoids was significantly repressed in compaction treated alfalfa roots, and this might have resulted in a modified secondary cell wall and xylem vessel formation. Phytohormones, like ABA, are supposed to play pivotal roles in the regulation of the overall responses. These findings provide directions for the improvement of field soil management in alfalfa production and the molecular breeding of alfalfa germplasm with better soil compaction resilience.

Preventive Effects of Melatonin on Delirium in Intensive Care Unit Patients: A Meta-Analysis of Randomized Controlled Trials.

Aims: To investigate the preventive efficacy of melatonin on the incidence of delirium and other clinical outcomes of subjects in the intensive care unit (ICU). Methods: Randomized controlled trials concerning the effects of melatonin on delirium published from inception to July 2022 were identified from PubMed, Embase, and the Cochrane Library. The primary outcome was delirium incidence. The secondary outcome was the length of ICU stay, the duration of mechanical ventilation, and the mortality in ICU. A meta-analysis was performed. Estimates were presented as risk ratio (RR) or standard mean difference (SMD) with 95% confidence interval (CI). Results: Eleven RCTs with 2002 patients were included. The forest plots showed that the delirium incidence did not significantly decrease after melatonin administration (RR 0.85; 95% CI, 0.61~1.18, P = .32, I2=60%, P for heterogeneity = .01). The subgroup analyses confirmed that melatonin significantly reduced the incidence of delirium (RR 0.70; 95% CI, 0.56~0.89, P = .003, I2 = 32%, P for heterogeneity = .22) for the special ICU patients. Also, for ICU patients, the length of ICU stays, duration of mechanical ventilation, and mortality were not significantly decreased after melatonin treatment (all P > .05). Conclusion: Melatonin may decrease the incidence of delirium for special ICU patients. PROSPERO registration number: CRD42022354874.

LL-37 improves sepsis-induced acute lung injury by suppressing pyroptosis in alveolar epithelial cells.

BACKGROUND: LL-37 (also known as murine CRAMP) is a human antimicrobial peptide that plays a crucial role in innate immune defence against sepsis through various mechanisms. However, its involvement in sepsis-induced lung injury remains unclear. OBJECTIVES: This work investigates the impact of LL-37 on pyroptosis generated by LPS in alveolar epithelial cells. The research utilizes both in vivo and in vitro sepsis-associated acute lung injury (ALI) models to understand the underlying molecular pathways. METHODS: In vivo, an acute lung injury model induced by sepsis was established by intratracheal administration of LPS in C57BL/6J mice, which were subsequently treated with low-dose CRAMP (recombinant murine cathelicidin, 2.5 mg.kg-1) and high-dose CRAMP (5.0 mg.kg-1). In vitro, pyroptosis was induced in a human alveolar epithelial cell line (A549) by stimulation with LPS and ATP. Treatment was carried out with recombinant human LL-37, or LL-37 was knocked out in A549 cells using small interfering RNA (siRNA). Subsequently, haematoxylin and eosin staining was performed to observe the histopathological changes in lung tissues in the control group and sepsis-induced lung injury group. TUNEL and PI staining were used to observe DNA fragmentation and pyroptosis in mouse lung tissues and cells in the different groups. An lactate dehydrogenase (LDH) assay was performed to measure the cell death rate. The expression levels of NLRP3, caspase1, caspase 1 p20, GSDMD, NT-GSDMD, and CRAMP were detected in mice and cells using Western blotting, qPCR, and immunohistochemistry. ELISA was used to assess the levels of interleukin (IL)-1ß and IL-18 in mouse serum, bronchoalveolar lavage fluid (BALF) and lung tissue and cell culture supernatants. RESULTS: The expression of NLRP3, caspase1 p20, NT-GSDMD, IL 18 and IL1ß in the lung tissue of mice with septic lung injury was increased, which indicated activation of the canonical pyroptosis pathway and coincided with an increase in CRAMP expression. Treatment with recombinant CRAMP improved pyroptosis in mice with lung injury. In vitro, treatment with LPS and ATP upregulated these classic pyroptosis molecules, LL-37 knockdown exacerbated pyroptosis, and recombinant human LL-37 treatment alleviated pyroptosis in alveolar epithelial cells. CONCLUSION: These findings indicate that LL-37 protects against septic lung injury by modulating the expression of classic pyroptotic pathway components, including NLRP3, caspase1, and GSDMD and downstream inflammatory factors in alveolar epithelial cells.

Prognostic Value of Neutrophils-to-Lymphocytes Ratio and Platelets-to-Lymphocytes Ratio in Sepsis Patients With Lymphopenia.

Background: Inflammation plays a critical role in sepsis. The integration of neutrophil-to-lymphocyte ratio (NLR) and platelets-to-lymphocytes ratio (PLR) from multiple cell types offers a novel approach to rapidly assess inflammation status. However, the predictive role of NLR and PLR in sepsis with lymphopenia remains uncertain. Objectives: The purpose of this study was to explore the prognostic value of NLR and PLR in sepsis patients with lymphopenia. Design and methods: In this observational retrospective study, we included 172 sepsis patients with lymphopenia and collected clinical characteristics for analysis. Through binary logistic regression analysis, we identified independent factors. Receiver-operating characteristic curves (ROC) and areas under the curves (AUC) were employed to assess the ability to predict hospital mortality risk. Results: Our results showed a total hospital mortality rate of 53.49%. Multivariate analysis demonstrated that NLR (OR = 1.11, P < .001) and PLR (OR = 1.01, P = .003) were independent predictors associated with hospital mortality in sepsis patients with lymphopenia. The AUCs of NLR and PLR were 0.750 (95% CI: 0.634-0.788, P < .001) and 0.662 (95% CI: 0.580-0.743, P < .001), respectively. Notably, an optimal cut-off value of 18.93 for NLR displayed a sensitivity of 75.0% and specificity of 63.0% in discriminating hospital mortality in sepsis patients with lymphopenia, while the optimal cut-off value for PLR was 377.50, with a sensitivity of 67.5% and specificity of 64.1%. Conclusion: NLR and PLR serve as independent predictors of hospital mortality in sepsis patients with lymphopenia.

Antagonistic impact on cadmium stress in alfalfa supplemented with nano-zinc oxide and biochar via upregulating metal detoxification.

Eco-toxicological estimation of cadmium induced damages by morpho-physiological and cellular response could be an insightful strategy to alleviate negative impact of Cd in agricultural crops. The current study revealed novel patterns of Cd-bioaccumulation and cellular mechanism opted by alfalfa to acquire Cd tolerance under various soil applied zinc oxide nanoparticles (nZnO) doses (0, 30, 60, 90 mg kg-1), combined with 2% biochar (BC). Herein, the potential impact of these soil amendments was justified by decreased Cd and increased Zn-bioaccumulation into roots by 38 % and 48 % and shoots by 51 % and 72 % respectively, with co-exposure of nZnO with BC. As, the transmission electron microscopy (TEM) and scanning electron microscopy and energy dispersive spectroscopy (SEM-EDS) ultrastructural observations confirmed that Cd-exposure induced stomatal closure, and caused damage to roots and leaves ultrastructure as compared to the control group. On the contrary, the damages to the above-mentioned traits were reversed by a higher nZnO dose, and the impact was further aggravated by adding BC along nZnO. Furthermore, higher nZnO and BC levels efficiently alleviated the Cd-mediated reductions in alfalfa biomass, antioxidant enzymatic response, and gaseous exchange traits than control. Overall, soil application of 90 mg kg-1 nZnO with BC (2 %) was impactful in averting Cd stress damages and ensuring better plant performance. Thereby, applying soil nZnO and BC emerge as promising green remediation techniques to enhance crop tolerance in Cd-polluted soil.

Atrophie blanche complicated with lower limb infection and maggot growth: A case report.

Background: Atrophie blanche (AB) is a thrombotic vascular disease, also known as venous vasculitis or segmental hyaline vasculitis, characterized by chronic recurring painful ulcers on the lower legs, especially the ankles. AB is a clinically rare condition, affecting 1%-5% of the population, specifically middle-aged women with an average age of 45 years, and cases of AB in children are rare. Following recovery, ivory-white atrophy spots accompanied by pigmentation and telangiectasia remain in patients. One of the complications of AB is the parasitic growth of microorganisms infecting the ischemic soft tissue undergoing necrosis in the lower limbs. Furthermore, although infection combined with microbial parasitism is a type of surgical site infection, myiasis is particularly rare, which may warrant limb amputation or may even be life-threatening. Understanding the complications of AB may help in early and timely surgical debridement as well as wound repair. Summarizing the knowledge and treatment strategies of AB and formulating clinical strategies and guidelines for AB management with insights from relevant cases are important. Case summary: A 59-year-old woman was hospitalized due to repeated ulceration of the skin of the right lower leg for 3 years, aggravation, and maggot growth for 3 days. In the previous 3 years, the skin and soft tissue of the right calf had become ischemic, necrotic, and infected, but the patient did not seek any medical treatment. Subsequently, 2 years ago, she was diagnosed with AB at the dermatology department of our hospital. After hormone treatment, her right leg improved. However, 1 year ago, the skin and soft tissue of the right leg again became ischemic, necrotic, and infected. This time, the patient did not seek medical treatment and applied musk on her wound. The wound deepened, resulting in the exposure of the tendon and some bones. In addition, a large number of maggots and microorganisms grew in and infested the wound for 3 days before the patient came to our department for treatment. Debridement of the necrotizing infected site on the right lower leg combined with negative pressure vacuum sealing drainage were performed twice within 16 days after admission. Simultaneously, antibiotics were given systemically. On the 17th day after admission, the wound appeared clean, myiasis had resolved, and the growth and coverage of the granulation tissue on the wound were satisfactory. Subsequently, debridement of the infected site on the right leg, removal of skin of the right thigh, and autologous free skin grafting were performed. After 10 days, the wound was clean, all skin grafts had survived, and wound repair was satisfactory. Finally, the patient was discharged after 38 days of hospitalization. Conclusion: Although AB is rare, leukodystrophy requires specialized treatment and regular follow-up. If lower limb infection and maggot growth occur simultaneously, self-treatment should be avoided and medical attention must be sought immediately. Early implementation of wound debridement and anti-infective treatment combined with wound repair, which should be performed after cleaning the wound, is advised.

Exogenous melatonin promotes the growth of alfalfa (Medicago sativa L.) under NaCl stress through multiple pathways.

Salinity is one of the most common factors affecting alfalfa (Medicago sativa L.), and NaCl is one of the main factors of salinity stress which can cause heavy losses in agricultural production in the world. The application of exogenous melatonin (MT) plays a major role in numerous plants against various stress environments. The effects of exogenous MT on the NaCl tolerance of alfalfa treated with the control, 100 µmol L-1 MT, 150 mmol L-1 NaCl, or 150 mmol L-1 NaCl+ 100 µmol L-1 MT were investigated. The results showed that MT increased growth parameters, inhibited chlorophyll degradation and promoted photosynthetic gas exchange parameters (photosynthetic rate, conductance to H2O, and transpiration rate) and stomatal opening under NaCl stress. Osmotic regulation substances such as soluble sugar, proline and glycine betaine were the highest in the NaCl treatment and the second in the NaCl+MT treatment. Nitrogen, phosphorus, potassium, calcium and magnesium were reduced and sodium was increased by NaCl, whereas these levels were reversed by the NaCl+MT treatment. MT inhibited cell membrane imperfection, lipid peroxidation and reactive oxygen species (ROS) accumulation caused by NaCl stress. MT up-regulated the gene expression and activity of antioxidant enzymes and increased the content of antioxidant non-enzyme substances to scavenge excessive ROS in NaCl-treated plants. In addition, all indicators interacted with each other to a certain extent and could be grouped according to the relative values. All variables were divided into PC 1 (89.2 %) and PC 2 (4 %). They were clustered into two categories with opposite effects, and most of them were significant variables. Hence, these findings reveal that exogenous MT alleviates the inhibitory effects of NaCl stress on photosynthesis, stomata opening, osmotic adjustment, ion balance and redox homeostasis, enhancing tolerance and growth of alfalfa. Furthermore, it suggests that MT could be implemented to improve the NaCl tolerance of alfalfa.

Nanosized zinc oxide (n-ZnO) particles pretreatment to alfalfa seedlings alleviate heat-induced morpho-physiological and ultrastructural damages.

Global efforts are in rapid progress to tackle the emerging conundrum of climate change-induced heat stress in grassland ecosystems. Zinc oxide nanoparticles (n-ZnO) are known to play a crucial role in plants' abiotic stress regulation, but its response in alfalfa against heat stress has not been explored. This study aimed at assessing the effects of n-ZnO on alfalfa under heat stress by various morpho-physiological and cellular approaches. Five-week-old alfalfa seedlings were subjected to foliar application of n-ZnO as a pretreatment before the onset of heat stress (BHS) to evaluate its effect on heat tolerance, and as a post-treatment after heat stress (AHS) to evaluate recovery efficiency. In vitro studies on Zn release from n-ZnO by Inductively coupled plasma mass spectroscopy (ICPMS) disclosed that the particle uptake and Zn release were concentration dependent. The uptake and translocation of n-ZnO examined by transmission electron microscope (TEM) reveling showed that n-ZnO was primarily localized in the vacuoles and chloroplasts. TEM images showed that ultrastructural modifications to chloroplast, mitochondria, and cell wall were reversible by highest dose of n-ZnO applied before heat stress, and damages to these organelles were not recoverable when n-ZnO was applied after heat stress. The results further enlightened that 90 mg L-1 n-ZnO better prevented the heat stress-mediated membrane damage, lipid peroxidation and oxidative stress by stimulating antioxidant systems and enhancing osmolyte contents in both BHS and AHS. Although, application of 90 mg L-1 n-ZnO in BHS was more effective in averting heat-induced damages and maintaining better plant growth and morpho-physiological attributes compared to AHS. Conclusively, foliar application of n-ZnO can be encouraged as an effective strategy to protect alfalfa from heat stress damages while minimizing the risk of nanoparticle transmission to environmental compartments, which could happen with soil application.

Zinc Oxide Nanoparticles Interplay With Physiological and Biochemical Attributes in Terminal Heat Stress Alleviation in Mungbean (Vigna radiata L.).

Gradually rising atmospheric temperature is the vital component of the environment, which is anticipated as the riskiest abiotic stress for crop growth. Nanotechnology revolutionizing the agricultural sectors, notably, zinc oxide nanoparticles (nano-ZnO) has captured intensive research interests due to their distinctive properties and numerous applications against abiotic stresses. Mungbean (Vigna radiata L.), being a summer crop, is grown all over the world at an optimum temperature of 28-30°C. A rise in temperature above this range, particularly during the flowering stage, can jeopardize the potential performance of the plant. Hence, an outdoor study was performed to evaluate the effect of multiple suspensions of nano-ZnO (0, 15, 30, 45, and 60 mg l-1) on physicochemical attributes and yield of mungbean crop under heat stress. Heat stress was induced by fine-tuning of sowing time as: S1 is the optimal sowing time having day/night temperatures <40/25°C and S2 and S3 are late sown that were above >40/25°C during the flowering stage. In vitro studies on Zn release from nano-ZnO by inductively coupled plasma mass spectroscopy (ICPMS) disclosed that the Zn release and particles uptake from nano-ZnO were concentration-dependent. Exogenous foliar application of nano-ZnO significantly upstreamed the production of antioxidants and osmolytes to attenuate the shocks of heat stress in S2 and S3. Likewise, nano-ZnO substantially rebated the production of reactive oxygen species in both S2 and S3 that was associated with curtailment in lipid peroxidation. Adding to that, foliar-applied nano-ZnO inflates not only the chlorophyll contents and gas exchange attributes, but also the seeds per pod (SPP) and pods per plant (PPP), which results in the better grain yield under heat stress. Thus, among all the sowing dates, S1 statistically performed better than S2 and S3, although foliar exposure of nano-ZnO boosted up mungbean performance under both the no heat and heat-induced environments. Hence, foliar application of nano-ZnO can be suggested as an efficient way to protect the crop from heat stress-mediated damages with the most negligible chances of nanoparticles delivery to environmental compartments that could be possible in case of soil application.

Ascorbic acid regulates nitrogen, energy, and gas exchange metabolisms of alfalfa in response to high-nitrate stress.

The effects of exogenous ascorbic acid (AsA) on the growth parameters, nitrogen metabolism, energy status, and photosynthetic gas exchange in alfalfa under high-nitrate stress were studied. The seedlings treated with the control, 200 mmol L-1 nitrates (HN) or 200 mmol L-1 nitrate + 0.1 mmol L-1 AsA (HN + AsA), were sampled on days 0 and 10 after treatments. AsA was sprayed on the leaves, while HN was conducted by watering. Both of them were performed once every other day for three times in total. The results revealed that in the HN treatment, the growth parameters were the lowest; total phosphorus (TP), nitrogen-related enzyme activities, soluble protein (SP), adenosine triphosphate (ATP), and energy charge (EC) were reduced; and photosynthetic rate (Photo), conductance to H2O (Cond), transpiration rate (Trmmol), instantaneous water use efficiency (WUE), and apparent CO2 use efficiency (CUE) were also inhibited; and total nitrogen (TN), nitrate-nitrogen (NO3¯-N), ammonium-nitrogen (NH4+-N), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and intercellular CO2 concentration (Ci) were increased compared with the control. However, these parameters changed conversely in the HN + AsA treatment. In addition, there was a good curve regression equation relationship between TN and NO3¯-N, TN and NH4+-N, NO3¯-N and NH4+-N, respectively. It indicates that AsA improves the growth parameters, nitrogen-related enzyme activities, energy metabolism, and photosynthesis, whereas it inhibits the toxicity of excess NO3¯-N and NH4+-N accumulations, thereby promoting the growth of alfalfa under high-nitrate stress. These metabolisms are closely related to each other during the regulatory process in alfalfa. Hence, AsA has potential to be applied to improve the growth of alfalfa under high-nitrate stress.

Phytotoxic effect and molecular mechanism induced by graphene towards alfalfa (Medicago sativa L.) by integrating transcriptomic and metabolomics analysis.

Although the widespread use of nanoparticles has been reported in various fields, the toxic mechanisms of molecular regulation involved in the alfalfa treated by nanomaterials is still in the preliminary research stage. In this study, Bara 310 SC (Bara, tolerant genotype) and Gold Empress (Gold, susceptible genotype) were used to investigate how the leaves of alfalfa interpret the physiological responses to graphene stress based on metabolome and transcriptome characterizations. Herein, graphene at different concentrations (0, 1% and 2%, w/w) were selected as the analytes. Physiological results showed antioxidant defence system and photosynthesis was significantly disturbed under high environmental concentration of graphene. With Ultra high performance liquid chromatography electrospray tandem mass spectrometry (UPLC-ESI-MS/MS), 406 metabolites were detected and 62/13 and 110/58 metabolites significantly changed in the leaves of Gold/Bara under the 1% and 2%-graphene treatments (w/w), respectively. The most important metabolites which were accumulated under graphene stress includes amino acids, flavonoids, organic acids and sugars. Transcriptomic analysis reveals 1125 of core graphene-responsive genes in alfalfa that was robustly differently expressed in both genotypes. And differential expression genes (DEGs) potentially related to photosynthetic enzymes, antioxidant enzymes, amino acids metabolism, and sucrose and starch metabolic which finding was supported by the metabolome study. Gold was more disturbed by graphene stress at both transcriptional and metabolic levels, since more stress-responsive genes/metabolites were identified in Gold. A comprehensive analysis of transcriptomic and metabolomic data highlights the important role of amino acid metabolism and nicotinate and nicotinamide metabolism pathways for graphene tolerance in alfalfa. Our study provide necessary information for better understanding the phytotoxicity molecular mechanism underlying nanomaterials tolerance of plant.

Utilization of Transcriptome, Small RNA, and Degradome Sequencing to Provide Insights Into Drought Stress and Rewatering Treatment in Medicago ruthenica.

Drought is a major limiting factor in foraging grass yield and quality. Medicago ruthenica (M. ruthenica) is a high-quality forage legume with drought resistance, cold tolerance, and strong adaptability. In this study, we integrated transcriptome, small RNA, and degradome sequencing in identifying drought response genes, microRNAs (miRNAs), and key miRNA-target pairs in M. ruthenica under drought and rewatering treatment conditions. A total of 3,905 genes and 50 miRNAs (45 conserved and 5 novel miRNAs) were significantly differentially expressed in three test conditions (CK: control, DS: plants under drought stress, and RW: plants rewatering after drought stress). The degradome sequencing (AllenScore < 4) analysis revealed that 104 miRNAs (11 novel and 93 conserved miRNAs) were identified with 263 target transcripts, forming 296 miRNA-target pairs in three libraries. There were 38 differentially expressed targets from 16 miRNAs in DS vs. CK, 31 from 11 miRNAs in DS vs. RW, and 6 from 3 miRNAs in RW vs. CK; 21, 18, and 3 miRNA-target gene pairs showed reverse expression patterns in DS vs. CK, DS vs. RW, and RW vs. CK comparison groups, respectively. These findings provide valuable information for further functional characterization of genes and miRNAs in response to abiotic stress, in general, and drought stress in M. ruthenica, and potentially contribute to drought resistance breeding of forage in the future.

Integrating transcriptome and physiological analyses to elucidate the essential biological mechanisms of graphene phytotoxicity of alfalfa (Medicago sativa L.).

The phytotoxicity of nanoparticles has attracted considerable interest, given the broad applications of nanomaterials in different fields. Alfalfa (Medicago sativa L.) is a major forage crop grown worldwide with a high protein content. The molecular regulation mechanisms involved in nanomaterial-treated alfalfa were examined in this research. In our lab, 18 cDNA libraries of Golden Empress (GE) and Bara 310SC (SC) under control (CK), middle (10 g kg-1)- and high (20 g kg-1)-graphene stress treatments were constructed in 2019. All clean reads were matched to the reference Medicago_truncatula genome, the mapping ratio was higher than 50%, and a total of 3946 differentially expressed genes (DEGs) were obtained. The number of DEGs that reflect transcriptional activity is proportional to the degree of stress. For example, 1241/610 and 1794/1422 DEGs were identified as significant in the leaves of GE/SC under mid- and high-graphene treatment, respectively. Furthermore, GO analysis of the DEGs annotated in some significant biochemical process terms included 'response to abiotic stimulus', 'oxidation-reduction process', 'protein kinase activity', and 'oxidoreductase activity'. KEGG pathway analysis of the DEGs revealed strongly mediated graphene-responsive genes in alfalfa mainly linked to the 'biosynthesis of amino acids', 'isoflavonoid biosynthesis', 'linoleic acid metabolism', and 'phenylpropanoid biosynthesis' pathways. In addition, hundreds of DEGs, including photosynthetic, antioxidant enzyme, nitrogen metabolism, and metabolic sucrose and starch genes, have been identified as potentially involved in the response to graphene. Physiological findings revealed that enzymes related to the metabolism of nitrogen play a crucial role in the adaptation of graphene stress to alfalfa. Ultimately, in response to graphene stress, a preliminary regulatory mechanism was proposed for the self-protective mechanism of alfalfa, which helps to explain the phytotoxicity of the molecular mechanism of nanoparticle-treated crops.

Graphene ameliorates saline-alkaline stress-induced damage and improves growth and tolerance in alfalfa (Medicago sativa L.).

Nanoscience is leading to the production of nanotech applications to enhance growth and environmental stress tolerance of plants. However, there are few studies on the morphological and physiological regulative mechanism of coupling effects between graphene (Nano-C) and pH in alfalfa, especially both in promotion and phytotoxicity effect by Nano-C under abiotic stress. The coupling effects of Nano-C and pH on plant growth, photosynthetic parameters and enzymes of the antioxidant defence system on leaves and roots were investigated at graphene concentrations of 0, 5, 10, and 20 g kg-1 in pots trials. The result showed a significantly promoted the plant growth at treatment of 5 g kg-1 Nano-C, whereas 10-20 g kg-1 Nano-C harmed the physiological and morphological characterizations. Under salt and alkali stresses, the dry weight, fresh weight, seedling length and antioxidant enzymes activities were increased significantly at 5 g kg-1 Nano-C treatment. These findings revealed that 5 g kg-1 Nano-C enhanced alfalfa tolerance by modulating various morphological and physiological characteristics. Meanwhile, through models analyses, the equivalent coupling effects of Nano-C and pH were located at rectangular area defined by 5.43-7.80 Nano-C combining 8.59-8.96 pH value, and the final optimal application of Nano-C was at 6.57 g kg-1 when high exposure dose of Nano-C indeed resulted in phytotoxicity to the plant. Results of the model analyses also showed significant synergistic effects on the photosynthesis and antioxidant system. Overall, our findings suggested fundamental insights into carbon nanomaterials-mediated physiological adaptation in plants.

Occurrence, Distribution, and Genetic Diversity of Alfalfa (Medicago sativa L.) Viruses in Four Major Alfalfa-Producing Provinces of China.

Alfalfa (Medicago sativa L.) is one of the most widely cultivated forage crops in the world. China is the second largest producer of alfalfa in terms of the planting area worldwide, with Gansu, Henan, Inner Mongolia, and Shaanxi provinces being the production hubs. Alfalfa viruses have been reported on a small-scale survey in some of these areas, but they have not been well characterized. In the present study, seven viruses were detected in 12 fields of 10 cities/counties of the four abovementioned provinces by high-throughput sequencing and assembly of small RNA. Their incidence, distribution, and genetic diversity were analyzed by enzyme-linked immunosorbent assay, polymerase chain reaction (PCR)/reverse transcription-PCR and clone sequencing. The results showed that alfalfa mosaic virus (AMV), pea streak virus (PeSV), lucerne transient streak virus (LTSV), alfalfa dwarf virus (ADV), Medicago sativa alphapartitivirus 1 (MsAPV1), MsAPV2, and alfalfa leaf curl virus (ALCV) were the main viruses infecting alfalfa in four examined provinces. AMV and MsAPV1 had the highest incidences in all 4 provinces. SDT analysis of the 7 viruses isolated in China revealed a highly conserved among AMV, LTSV, ADV, MsAPV1, MsAPV2, and ALCV, but the sequence was a high variation between China isolates to abroad isolates in PeSV, ADV, and ALCV. To our knowledge, this is the first report of ADV in Inner Mongolia and Gansu, ALCV in Inner Mongolia, MsAPV1 and MsAPV2 in all 4 provinces, and PeSV and LTSV in China. These findings provide a basis for future research on the genetic evolution of alfalfa viruses in China and on strategies to prevent diseases in alfalfa caused by these viruses.

Plant Growth-Promoting Rhizobacteria With ACC Deaminase Activity Enhance Maternal Lateral Root and Seedling Growth in Switchgrass.

Switchgrass, a C4 plant with high potential as a bioenergy source, is unsteady in yield under sub-optimal conditions. Bacteria containing 1-aminocyclopropane-1-carboxylate synthase (ACC) deaminase can promote plant growth. We isolated bacteria containing ACC deaminase activity from switchgrass rhizosphere using an orthogonal matrix experimental design with four factors (bacterial liquid concentration, bacterial liquid treatment time, nitrogen content, and NaCl) to quantitatively investigate the effects and pairwise interactions on the seedling growth. Pseudomonas sp. Y1, isolated from the switchgrass cv. Blackwell rhizomes was selected. We optimized the inoculation bacterial concentration, treatment time, NaCl, and nitrogen concentration for the seedling growth. The optimal bacterial concentration, treatment time, NaCl, and nitrogen content was 0.5-1.25 OD at 600 nm, 3 h, 60-125 mM and 158 mg L-1, respectively. Pseudomonas sp. Y1 significantly increased the total root length, root surface, shoot length, and fresh and dry weight through an effective proliferation of the number of first-order lateral roots and root tips. This indicated that Pseudomonas sp. Y1 has a higher potential to be used as a plant growth-promoting rhizobacteria bacteria.

Seed yield can be explained by altered yield components in field-grown western wheatgrass (Pascopyrum smithii Rydb.).

Western wheatgrass (Pascopyrum smithii Rydb.) is an important cool-season forage and turfgrass. However, due to seed dormancy and poor seedling vigor, it is difficult to develop high seed yield production systems, and assessing these components in response to seed yield. Based on multifactor orthogonally designed field experimental plots under various field management regimes, the effects of numbers of fertile tillers m-2 (Y1), spikelets/fertile tiller (Y2), florets/spikelet (Y3), seed numbers/spikelet (Y4), and seed weight (Y5) on seed yield (Z) were determined over three successive years. Correlation analysis indicated that fertile tillers (Y1) was the most important seed yield component. And the biggest contribution of those five yield component is fertile tillers (Y1), followed by seed numbers/spikelet (Y4), spikelets/fertile tiller (Y2), florets/spikelet (Y3) and seed weight (Y5), respectively. By using ridge regression analysis, we have developed an accurate model of seed yield with its five components. Finally, the results of synergism and antagonism among these yield components on seed yield showed that fertile tillers and seed numbers/spikelet had an antagonistic effect on seed yield. Therefore, selection for high seed yield by direct selection for large values of fertile tillers and seed numbers/spikelet would be the most effective breeding strategy for western wheatgrass.

Phytoremediation of Cd and Pb interactive polluted soils by switchgrass (Panicum virgatum L.).

Using phytoremediation as an efficient technique to remove heavy metals from contaminated soils is a current research hotspot. This study used an orthogonal matrix experimental design with three factors (Cd, Pb, and pH) and five levels (Cd at 9.45, 30, 60, 90, and 110.46 µg/g; Pb at 195.4, 400, 700, 1000, and 1204.6 µg/g; and pH at 3, 4.1, 5.8, 7.5, and 8.6) to investigate the phytoremediation potential of Panicum virgatum L. for soils polluted with cadmium (Cd) and lead (Pb). The results indicated that there was a significant decrease in belowground biomass in plants exposed to the stresses compared to the control. Superoxide dismutase (SOD) activity, peroxidase (POD) activity, and malondialdehyde (MDA) content were affected. Interaction of Cd with Pb in the soil had an antagonistic effect on the Cd bioaccumulation factor, whereas the interaction of pH with Cd or Pb had synergistic effects on the Cd bioaccumulation factor. When exposed to the three stressors, switchgrass plants could grow in soil that had a Cd concentration of a 46.68 µg/g, Pb concentration of 568.75 µg/g and pH of 5.34, which is a mildly acidic condition. Switchgrass, used as a phytoremediation plant, was more efficient in Cd-contaminated than in Pb-contaminated soil.

Optimizing irrigation and nitrogen fertilization for seed yield in western wheatgrass [Pascopyrum smithii (Rydb.) Á. Löve] using a large multi-factorial field design.

It is crucial for agricultural production to identify the trigger that switches plants from vegetative to reproductive growth. Agricultural sustainability in semiarid regions is challenged by nitrogen (N) fertilizer overuse, inadequate soil water, and heavy carbon emissions. Previous studies focused on the short-term effects of a single application of N and water but have not investigated the long-term effects of different irrigation and N fertilizer regimens on crop yields and yield components. N application is routinely coupled with water availability, and crop yields can be maximized by optimizing both. We examined the growth of western wheatgrass [Pascopyrum smithii (Rydb.) Á. Löve], a temperate-region forage and turf grass, using multiple different combinations of N fertilizer [(NH4)2·CO3] and irrigation levels over 3 years to determine optimal field management. We conducted multifactorial, orthogonally designed field experiments with large sample sizes, and measured fertile tillers m-2 (Y1), spikelets/fertile tillers (Y2), florets/spikelet (Y3), seed numbers/spikelet (Y4), seed weight (Y5), and seed yield (Z) to study factors associated with the switch between vegetative and reproductive growth. Fertilization had a greater effect on seed yield and yield components than irrigation. Y1 had the strongest positive effect on Z, whereas Y5 had a negative effect on Z. Irrigation and fertilization affected Z, Y1, and Y5. Fertilizer concentrations were positively correlated with Z, Y1, and Y5, whereas irrigation levels were negatively correlated. The ridge regression linear model results suggested N application rate and irrigation had antagonistic effects on Y1 (X3 = 867.6-4.23×X2; R2 = 0.988, F = Infinity, P<0.0001). We conclude that the optimal amount of N fertilizer and irrigation was 156 kg ha-1 + 115 mm for seed yield, 120 kg ha-1 + 146 mm for spikelets/fertile tillers, and 108 kg ha-1 + 119 mm for seed numbers/spikelets. These results will improve yield and reduce agricultural inputs for P. smithii in semiarid and arid regions, thereby reducing fertilizer pollution and conserving water.