Screening of mtr-miR156a from exosomes of dairy cow blood to milk and its regulatory effect on milk protein synthesis in BMECs.

MicroRNA (miRNA) is a type of endogenous non-coding small RNA, which is abundant in living organisms. miRNAs play an important role in regulating gene expression and myriad cellular processes by binding to target messenger RNAs through complementary base pairing, and cross-species regulation mammalian cells by plant-derived xeno-miRNAs has been described. Here, we examined the miRNA species in two alfalfa (Medicago sativa, lucerne) cultivars commonly grown in Ningxia, China: cv. Zhongmu 1 and cv. Xinyan 52. Both cultivars have good salt and drought resistance. We found that the miRNA profiles were similar between the cultivars, with a slightly higher number of miRNAs present in the newer cv. Xinyan 52, which may contribute to its improved salt and drought tolerance. miRNAs were stable during drying, and some miRNAs were increased in dry versus fresh alfalfa, suggesting some miRNAs may be upregulated during drying. Alfalfa-derived miRNAs could be detected in exosomes from serum and whey collected from dairy cows, confirming the ability of the exogenous miRNAs (xeno-miRNAs) to enter the circulation and reach the mammary epithelium. In vitro studies confirmed that overexpression of mtr-miR156a could downregulate expression of Phosphatase 2 Regulatory Subunit B'gamma ( PPP2R5D) and Phosphoinositide-3-kinase Regulatory Subunit 2 (PIK3R2). Overexpression of mtr-miR156a also modulated PI3K-AKT-mTOR signaling as well as the casein content of milk produced by bovine mammary epithelial cells. Based on the known roles of PPP2R5D and PIK3R2 in regulating the PI3K-AKT-mTOR pathway as well as the effect of PI3K-AKT-mTOR on milk protein content, our findings implicate alfalfa-derived miR156a as a new cross-species regulator of milk quality in dairy cows.

Impact of Partial Replacement of Clover by Anise, Basil, Mint and Fennel Remnants in Rabbits Diet on Blood Constituents.

<b>Background and Objective:</b> The clover plant (alfalfa) is considered the primary fiber source in the rabbit diet in Mediterranean Sea countries like Egypt, so researchers are always trying to find alternatives and aromatic and medicinal plant remnants could be one of them. So, this study was designed to determine the effects of some aromatic and medicinal plant remnants on New Zealand white rabbits' blood hematological and biochemical aspects. <b>Materials and Methods:</b> A total of 108 weaned white New Zealand rabbits at five weeks of age were used to consider the effect of using remnants of mint, fennel, basil and anise with or without probiotics to replace 50% from alfalfa hay in rabbits' diets. Four remnants were obtained after etheric oil distillation and were incorporated in rabbit diets at level 17.5% without probiotics and with probiotics (replacement 50% of alfalfa hay). Rabbits were randomly assigned into nine experimental groups; the experimental period lasted eight weeks. Rabbit blood hematological and blood biochemical were analyzed. <b>Results:</b> The highest values of RBC 6.03 µL, HCT 37.13%, WBC 12.70 µL and lymph percentage were found in the basil+probiotics group. In contrast, the highest value of hemoglobin (HGB 10,50 g/dL), MCV 64.13 fl, MCH 23.27pg, MCHC 36.40 g/dL, PLT 463 µL, urea 50.33 mg/dL and creatinine 1.30 mg/dL were found in anise+probiotic group. In contrast, RDW-CV 33.17%, Mid 13.17 µL, granulocytes (Gran 7.13 µL) and PDW 16.73 in the mint group. Furthermore, RDW-SD (34.40 fl) and procalcitonin (PCT 0.35%) were found in the control group and the highest values ALT 142 IU/L and AST 77.33 IU/L were found in the fennel group. The highest albumin value (3.10 g/dL) was found in the anise group and the highest total protein (TP 5.23 g/dL) was found in the mint+probiotic group. <b>Conclusion:</b> The results proved that using these medicinal plant remnants and probiotics as substitutes for half the amount of alfalfa used in the diet of New Zealand white rabbits did not have a negative effect and improved their health condition.

Effects of protein grass hay as alternative feed resource on lamb's fattening performance and meat quality.

Protein grass hay (PGH) was used as a new feed source for lambs to study its effect on fattening performance and meat quality. Fifty-six male lambs were allotted to four experimental groups and fed for eight weeks either alfalfa hay (AH)-based diet (control) or diets in which AH was replaced with 33 %, 66 %, or 99 % PGH. The inclusion of PGH did not affect final body weight, dry matter intake, average daily gain, feed conversion ratio, or carcass weight. Moreover, substituting AH with PGH at any level did not influence the ruminal fermentation or serum biochemical parameters, meat color, water holding capacity, shear force, or amino acid profile. However, relative liver weight was increased with 66 % substitutions. Furthermore, replacing 99 % AH with PGH decreased the meat's pH at 24 h. Higher levels of C18:3n-3, C20:5n-3, and total n-3 PUFA and a lower ratio of n-6: n-3 PUFA were also observed in meat from lambs fed PGH at 99 %. These findings suggest that PGH could be incorporated into the lamb's diet up to 99 % without compromising fattening performance and body health while improving their meat n-3 PUFA deposition.

Genome-wide investigation of the TIFY transcription factors in alfalfa (Medicago sativa L.): identification, analysis, and expression.

BACKGROUND: Alfalfa (Medicago sativa L.) is an essential leguminous forage with high nutrition and strong adaptability. The TIFY family is a plant-specific transcription factor identified in many plants. However, few reports have been reported on the phylogenetic analysis and gene expression profiling of TIFY family genes in alfalfa. RESULT: A total of 84 TIFY genes belonging to 4 categories were identified in alfalfa, including 58 MsJAZs, 18 MsZMLs, 4 MsTIFYs and 4 MsPPDs, respectively. qRT-PCR data from 8 genes in different tissues revealed that most MsTIFY genes were highly expressed in roots. The expression of MsTIFY14 was up-regulated after different times in both thrips-resistant and susceptible alfalfa after thrips feeding, and the expression of the remaining MsTIFYs had a strong correlation with the time of thrips feeding. Different abiotic stresses, including drought, salt, and cold, could induce or inhibit the expression of MsTIFY genes to varying degrees. In addition, the eight genes were all significantly up-regulated by JA and/or SA. Interestingly, MsTIFY77 was induced considerably by all the biotic, abiotic, or plant hormones (JA or SA) except ABA. CONCLUSION: Our study identified members of the TIFY gene family in alfalfa and analyzed their structures and possible functions. It laid the foundation for further research on the molecular functions of TIFYs in alfalfa.

Genome-wide investigation of the PIF gene family in alfalfa (Medicago sativa L.) expression profiles during development and stress.

BACKGROUND: Phytochrome-interacting factors (PIFs) plays an important role in plants as hubs for intracellular signaling regulation. The PIF gene family has been identified and characterized in many plants, but alfalfa (Medicago sativa L.), an important perennial high-quality legume forage, has not been reported on the PIF gene family. RESULTS: In this study, we presented the identification and characterization of five MsPIF genes in alfalfa (Medicago sativa L.). Phylogenetic analysis indicated that PIFs from alfalfa and other four plant species could be divided into three groups supported by similar motif analysis. The collinearity analysis of the MsPIF gene family showed the presence of two gene pairs, and the collinearity analysis with AtPIFs showed three gene pairs, indicating that the evolutionary process of this family is relatively conservative. Analysis of cis-acting elements in promoter regions of MsPIF genes indicated that various elements were related to light, abiotic stress, and plant hormone responsiveness. Gene expression analyses demonstrated that MsPIFs were primarily expressed in the leaves and were induced by various abiotic stresses. CONCLUSION: This study conducted genome-wide identification, evolution, synteny analysis, and expression analysis of the PIFs in alfalfa. Our study lays a foundation for the study of the biological functions of the PIF gene family and provides a useful reference for improving abiotic stress resistance in alfalfa.

Forage Height and Above-Ground Biomass Estimation by Comparing UAV-Based Multispectral and RGB Imagery.

Crop height and biomass are the two important phenotyping traits to screen forage population types at local and regional scales. This study aims to compare the performances of multispectral and RGB sensors onboard drones for quantitative retrievals of forage crop height and biomass at very high resolution. We acquired the unmanned aerial vehicle (UAV) multispectral images (MSIs) at 1.67 cm spatial resolution and visible data (RGB) at 0.31 cm resolution and measured the forage height and above-ground biomass over the alfalfa (Medicago sativa L.) breeding trials in the Canadian Prairies. (1) For height estimation, the digital surface model (DSM) and digital terrain model (DTM) were extracted from MSI and RGB data, respectively. As the resolution of the DTM is five times less than that of the DSM, we applied an aggregation algorithm to the DSM to constrain the same spatial resolution between DSM and DTM. The difference between DSM and DTM was computed as the canopy height model (CHM), which was at 8.35 cm and 1.55 cm for MSI and RGB data, respectively. (2) For biomass estimation, the normalized difference vegetation index (NDVI) from MSI data and excess green (ExG) index from RGB data were analyzed and regressed in terms of ground measurements, leading to empirical models. The results indicate better performance of MSI for above-ground biomass (AGB) retrievals at 1.67 cm resolution and better performance of RGB data for canopy height retrievals at 1.55 cm. Although the retrieved height was well correlated with the ground measurements, a significant underestimation was observed. Thus, we developed a bias correction function to match the retrieval with the ground measurements. This study provides insight into the optimal selection of sensor for specific targeted vegetation growth traits in a forage crop.

RNA-seq analyses on gametogenic tissues of alfalfa (Medicago sativa) revealed plant reproduction- and ploidy-related genes.

BACKGROUND: In alfalfa (Medicago sativa), the coexistence of interfertile subspecies (i.e. sativa, falcata and coerulea) characterized by different ploidy levels (diploidy and tetraploidy) and the occurrence of meiotic mutants capable of producing unreduced (2n) gametes, have been efficiently combined for the establishment of new polyploids. The wealth of agronomic data concerning forage quality and yield provides a thorough insight into the practical benefits of polyploidization. However, many of the underlying molecular mechanisms regarding gene expression and regulation remained completely unexplored. In this study, we aimed to address this gap by examining the transcriptome profiles of leaves and reproductive tissues, corresponding to anthers and pistils, sampled at different time points from diploid and tetraploid Medicago sativa individuals belonging to progenies produced by bilateral sexual polyploidization (dBSP and tBSP, respectively) and tetraploid individuals stemmed from unilateral sexual polyploidization (tUSP). RESULTS: Considering the crucial role played by anthers and pistils in the reduced and unreduced gametes formation, we firstly analyzed the transcriptional profiles of the reproductive tissues at different stages, regardless of the ploidy level and the origin of the samples. By using and combining three different analytical methodologies, namely weighted-gene co-expression network analysis (WGCNA), tau (τ) analysis, and differentially expressed genes (DEGs) analysis, we identified a robust set of genes and transcription factors potentially involved in both male sporogenesis and gametogenesis processes, particularly in crossing-over, callose synthesis, and exine formation. Subsequently, we assessed at the same floral stage, the differences attributable to the ploidy level (tBSP vs. dBSP) or the origin (tBSP vs. tUSP) of the samples, leading to the identification of ploidy and parent-specific genes. In this way, we identified, for example, genes that are specifically upregulated and downregulated in flower buds in the comparison between tBSP and dBSP, which could explain the reduced fertility of the former compared to the latter materials. CONCLUSIONS: While this study primarily functions as an extensive investigation at the transcriptomic level, the data provided could represent not only a valuable original asset for the scientific community but also a fully exploitable genomic resource for functional analyses in alfalfa.

Multi-omics integration analysis reveals the molecular mechanisms of drought adaptation in homologous tetraploid alfalfa(Medicago sativa 'Xinjiang-Daye').

Drought stress is a predominant abiotic factor leading to decreased alfalfa yield. Genomic ploidy differences contribute to varying adaptation mechanisms of different alfalfa cultivars to drought conditions. This study employed a multi-omics approach to characterize the molecular basis of drought tolerance in a tetraploid variant of alfalfa (Medicago sativa, Xinjiang-Daye). Under drought treatment, a total of 4446 genes, 859 proteins, and 524 metabolites showed significant differences in abundance. Integrative analysis of the multi-omics data revealed that regulatory modules involved in flavonoid biosynthesis, plant hormone signalling transduction, linoleic acid metabolism, and amino acid biosynthesis play crucial roles in alfalfa adaptation to drought stress. The severity of drought led to the substantial accumulation of flavonoids, plant hormones, free fatty acids, amino acids, and their derivatives in the leaves. Genes such as PAL, 4CL, CHI, CHS, PP2C, ARF_3, and AHP_4 play pivotal regulatory roles in flavonoid biosynthesis and hormone signalling pathways. Differential expression of the LOX gene emerged as a key factor in the elevated levels of free fatty acids. Upregulation of P5CS_1 and GOT1/2 contributed significantly to the accumulation of Pro and Phe contents. ERF19 emerged as a principal positive regulator governing the synthesis of the aforementioned compounds. Furthermore, observations suggest that Xinjiang-Daye alfalfa may exhibit widespread post-transcriptional regulatory mechanisms in adapting to drought stress. The study findings unveil the critical mechanisms by which Xinjiang-Daye alfalfa adapts to drought stress, offering novel insights for the improvement of alfalfa germplasm resources.

Effects of temperature and lactic acid Bacteria additives on the quality and microbial community of wilted alfalfa silage.

This study investigated the influence of different temperatures (35℃ High temperature and average indoor ambient temperature of 25℃) and lactic acid bacterial additives (Lactiplantibacillus plantarym, Lentilactobacillus buchneri, or a combination of Lactiplantibacillus plantarym and Lentilactobacillus buchneri) on the chemical composition, fermentation quality, and microbial community of alfalfa silage feed. After a 60-day ensiling period, a significant interaction between temperature and additives was observed, affecting the dry matter (DM), crude protein (CP), acid detergent fiber (ADF), and neutral detergent fiber (NDF) of the silage feed (p < 0.05). Temperature had a highly significant impact on the pH value of the silage feed (p < 0.0001). However, the effect of temperature on lactic acid, acetic acid, propionic acid, and butyric acid was not significant (p > 0.05), while the inoculation of additives had a significant effect on lactic acid, acetic acid, and butyric acid (p > 0.05). As for the dynamic changes of microbial community after silage, the addition of three kinds of bacteria increased the abundance of lactobacillus. Among all treatment groups, the treatment group using complex bacteria had the best fermentation effect, indicating that the effect of complex lactic acid bacteria was better than that of single bacteria in high temperature fermentation. In summary, this study explained the effects of different temperatures and lactic acid bacterial additives on alfalfa fermentation quality and microbial community, and improved our understanding of the mechanism of alfalfa related silage at high temperatures.

Synergism of endophytic microbiota and plants promotes the removal of polycyclic aromatic hydrocarbons from the Alfalfa rhizosphere.

Endophytic bacteria can promote plant growth and accelerate pollutant degradation. However, it is unclear whether endophytic consortia (Consortium_E) can stabilize colonisation and degradation. We inoculated Consortium_E into the rhizosphere to enhance endophytic bacteria survival and promote pollutant degradation. Rhizosphere-inoculated Consortium_E enhanced polycyclic aromatic hydrocarbon (PAH) degradation rates by 11.5-13.1 % compared with sole bioaugmentation and plant treatments. Stable-isotope-probing (SIP) showed that the rhizosphere-inoculated Consortium_E had the largest number of degraders (8 amplicon sequence variants). Furthermore, only microbes from Consortium_E were identified among the degraders in bioaugmentation treatments, indicating that directly participated in phenanthrene metabolism. Interestingly, Consortium_E reshaped the community structure of degraders without significantly altering the rhizosphere community structure, and strengthened the core position of degraders in the network, facilitating close interactions between degraders and non-degraders in the rhizosphere, which were crucial for ensuring stable functionality. The synergistic effect between plants and Consortium_E significantly enhanced the upregulation of aromatic hydrocarbon degradation and auxiliary degradation pathways in the rhizosphere. These pathways showed a non-significant increasing trend in the uninoculated rhizosphere compared with the control, indicating that Consortium_E primarily promotes rhizosphere effects. Our results explore the Consortium_E bioaugmentation mechanism, providing a theoretical basis for the ecological restoration of contaminated soils.

Comparative proteomic discovery of salt stress response in alfalfa roots and overexpression of MsANN2 confers salt tolerance.

Soil salinity constrains growth, development and yield of alfalfa (Medicago sativa L.). To illustrate the molecular mechanisms responsible for salt tolerance, a comparative proteome analysis was explored to characterize protein profiles of alfalfa seedling roots exposed to 100 and 200 mM NaCl for three weeks. There were 52 differentially expressed proteins identified, among which the mRNA expressions of 12 were verified by Real-Time-PCR analysis. The results showed increase in abundance of ascorbate peroxidase, POD, CBS protein and PR-10 in salt-stressed alfalfa, suggesting an effectively antioxidant and defense systems. Alfalfa enhanced protein quality control system to refold or degrade abnormal proteins induced by salt stress through upregulation of unfolded protein response (UPR) marker PDIs and molecular chaperones (eg. HSP70, TCP-1, and GroES) as well as the ubiquitin-proteasome system (UPS) including ubiquitin ligase enzyme (E3) and proteasome subunits. Upregulation of proteins responsible for calcium signal transduction including calmodulin and annexin helped alfalfa adapt to salt stress. Specifically, annexin (MsANN2), a key Ca2+-binding protein, was selected for further characterization. The heterologous of the MsANN2 in Arabidopsis conferred salt tolerance. These results provide detailed information for salt-responsive root proteins and highlight the importance of MsANN2 in adapting to salt stress in alfalfa.

Comparative role of calcium oxide nanoparticles and calcium bulk fertilizer to alleviate cadmium toxicity by modulating oxidative stress, photosynthetic performance and antioxidant-defense genes expression in alfalfa.

Cadmium (Cd) toxicity poses a significant threat to soil health and sustainable food production. Its bioaccumulation in plant tissues induces phytotoxicity by affecting physiological and biochemical attributes, leading to a reduction in plant biomass and production. Recently, nanotechnology has emerged as a promising approach for addressing heavy metal toxicity in an eco-friendly manner to enhance crop production. However, the comparative role of foliar applied calcium oxide nanoparticles (CaO-NPs) and bulk calcium fertilizer under Cd stress in alfalfa remains unexplored. Herein, we studied the ameliorative role of CaO-NPs and bulk calcium (50 and 100 mg L-1) to alleviate Cd stress (30 mg kg-1) in alfalfa seedlings. Plants exposed to Cd exhibited significant decreases in morpho-physiological traits, gas exchange attributes, and pigment contents as well as increase in Cd bioaccumulation in plant tissues. Notably, exogenous application of CaO-NPs ameliorates the toxic impact of Cd by enhancing plant biomass (45%), fluorescence efficiency and gaseous exchange attributes. The maximum dose of CaO-NPs induced Cd-tolerance response accompanied by a significant increase in antioxidative enzyme activities, such as superoxide dismutase (SOD; 29%), peroxidase (POD; 41%), catalase (CAT; 36%) and ascorbate peroxidase (APX; 49%), which play positive roles in ROS scavenging. TEM examination further revealed the protective role of these NPs in averting Cd-induced damage to leaf ultrastructure and mesophyll cells. Furthermore, CaO-NPs had a substantial influence on both Cd and Ca2+ accumulation in plant tissues, while qRT‒PCR analysis demonstrated higher expression of antioxidant defense genes viz. Cu/ZnSOD (0.38 fold change (FC)), MtPOD (0.51 FC), MtCAT (0.61 FC) and MtAPX (0.79 FC) under CaO-NPs application, over Cd control. Overall, our findings suggested that exogenous CaO-NPs could be effective in alleviating the adverse effects of Cd on alfalfa seedlings to ensure food safety and support sustainable agriculture.

Plant-based hydrolysates as building blocks for cellular agriculture.

Cellular agriculture, an emerging technology, aims to produce animal-based products such as meat through scalable tissue culture methods. Traditional techniques rely on chemically undefined media using fetal bovine serum (FBS) or chemically defined media utilizing specific growth factors. To be a viable alternative to conventional meat production, cellular agriculture requires cost-effective materials with established supply chains for growth media. Here, we investigate hydrolysates from Kikuyu grass, Alfalfa grass, and cattle rearing pellets. We identified conditions that promote C2C12 myoblast cell growth in media containing 0.1% and 0% serum. These effects are more pronounced in combination with existing growth promoters such as insulin, transferrin, and selenium. Overall, the rearing pellet hydrolysates were most effective in promoting growth particularly when in combination with the growth promoters. Our findings suggest that leveraging these materials, along with known growth factors, can facilitate the development of improved, scalable, and commercially viable media for cellular agriculture.

Overexpression of protection of telomeres 1 (POT1), a single-stranded DNA-binding proteins in alfalfa (Medicago sativa), enhances seed vigor.

Extensive bodies of research are dedicated to the study of seed aging with a particular focus on the roles of reactive oxygen species (ROS), and the ensuing oxidative damage during storage, as a primary cause of seed vigor decreasing. ROS diffuse to the nucleus and damage the telomeres, resulting in a loss of genetic integrity. Protection of telomeres 1 (POT1) is a telomeric protein that binds to the telomere region, and plays an essential role in maintaining genomic stability in plants. In this study, there were totally four MsPOT1 genes obtained from alfalfa genome. Expression analysis of four MsPOT1 genes in germinated seed presented the different expressions. Four MsPOT1 genes displayed high expression levels at the early stage of seed germination, Among the four POT1 genes, it was found that MS. gene040108 was significantly up-regulated in the early germination stage of CK seeds, but down-regulated in aged seeds. RT-qPCR assays and RNA-seq data revealed that MsPOT1-X gene was significantly induced by seed aging treatment. Transgenic seeds overexpressing MsPOT1-X gene in Arabidopsis thaliana and Medicago trunctula exhibited enhanced seed vigor, telomere length, telomerase activity associated with reduced H2O2 content. These results would provide a new way to understand aging stress-responsive MsPOT1 genes for genetic improvement of seed vigor. Although a key gene regulating seed vigor was identified in this study, the specific mechanism of MsPOT1-X gene regulating seed vigor needs to be further explored.

Transcriptome-Wide Identification of Dark- and Salt-Induced Senescence-Related NAC Gene Family Members in Alfalfa.

Leaves are a key forage part for livestock, and the aging of leaves affects forage biomass and quality. Preventing or delaying premature leaf senescence leads to an increase in pasture biomass accumulation and an improvement in alfalfa quality. NAC transcription factors have been reported to affect plant growth and abiotic stress responses. In this study, 48 NAC genes potentially associated with leaf senescence were identified in alfalfa under dark or salt stress conditions. A phylogenetic analysis divided MsNACs into six subgroups based on similar gene structure and conserved motif. These MsNACs were unevenly distributed in 26 alfalfa chromosomes. The results of the collinearity analysis show that all of the MsNACs were involved in gene duplication. Some cis-acting elements related to hormones and stress were screened in the 2-kb promoter regions of MsNACs. Nine of the MsNAC genes were subjected to qRT-PCR to quantify their expression and Agrobacterium-mediated transient expression to verify their functions. The results indicate that Ms.gene031485, Ms.gene032313, Ms.gene08494, and Ms.gene77666 might be key NAC genes involved in alfalfa leaf senescence. Our findings extend the understanding of the regulatory function of MsNACs in leaf senescence.

Telomerase reverse transcriptase, a telomere length maintenance protein in alfalfa (Medicago sativa), confers Arabidopsis thaliana seeds aging tolerance via modulation of telomere length.

Numerous studies have investigated seed aging, with a particular emphasis on the involvement of reactive oxygen species. Reactive oxygen species diffuse into the nucleus and damage telomeres, resulting in loss of genetic integrity. Telomerase reverse transcriptase (TERT) plays an essential role in maintaining plant genomic stability. Genome-wide analyses of TERT genes in alfalfa (Medicago sativa) have not yet been conducted, leaving a gap in our understanding of the mechanisms underlying seed aging associated with TERT genes. In this study, four MsTERT genes were identified in the alfalfa genome. The expression profiles of the four MsTERT genes during seed germination indicated that MS. gene79077 was significantly upregulated by seed aging. Transgenic seeds overexpressing MS. gene79077 in Arabidopsis exhibited enhanced tolerance to seed aging by reducing the levels of H2O2 and increasing telomere length and telomerase activity. Furthermore, transcript profiling of aging-treated Arabidopsis wild-type and overexpressing seeds showed an aging response in genes related to glutathione-dependent detoxification and antioxidant defense pathways. These results revealed that MS. gene79077 conferred Arabidopsis seed-aging tolerance via modulation of antioxidant defense and telomere homeostasis. This study provides a new way to understand stress-responsive MsTERT genes for the potential genetic improvement of seed vigor.

Tandem mass tag (TMT)-based quantitative proteomics analysis reveals the different responses of contrasting alfalfa varieties to drought stress.

BACKGROUND: Drought stress restricts the growth, distribution and productivity of alfalfa (Medicago sativa L.). In order to study the response differences of alfalfa cultivars to drought stress, we previously carried out physiological and molecular comparative analysis on two alfalfa varieties with contrasting drought resistance (relatively drought-tolerant Longdong and drought-sensitive Algonquin). However, the differences in proteomic factors of the two varieties in response to drought stress still need to be further studied. Therefore, TMT-based quantitative proteomic analysis was performed using leaf tissues of the two alfalfa cultivars to identify and uncover differentially abundant proteins (DAPs). RESULTS: In total, 677 DAPs were identified in Algonquin and 277 in Longdong under drought stress. Subsequently, we conducted various bioinformatics analysis on these DAPs, including subcellular location, functional classification and biological pathway enrichment. The first two main COG functional categories of DAPs in both alfalfa varieties after drought stress were 'Translation, ribosomal structure and biogenesis' and 'Posttranslational modification, protein turnover, chaperones'. According to KEGG database, the DAPs of the two alfalfa cultivars after drought treatment were differentially enriched in different biological pathways. The DAPs from Algonquin were enriched in 'photosynthesis' and 'ribosome'. The pathways of 'linoleic acid metabolism', 'protein processing in endoplasmic reticulum' and 'RNA transport' in Longdong were significantly enriched. Finally, we found significant differences in DAP enrichment and expression patterns between Longdong and Algonquin in glycolysis/glycogenesis, TCA cycle, photosynthesis, protein biosynthesis, flavonoid and isoflavonoid biosynthesis, and plant-pathogen interaction pathway after drought treatment. CONCLUSIONS: The differences of DAPs involved in various metabolic pathways may explain the differences in the resistance of the two varieties to drought stress. These DAPs can be used as candidate proteins for molecular breeding of alfalfa to cultivate new germplasm with more drought tolerance to adapt to unfavorable environments.

Seasonality and alternative floral resources affect reproductive success of the alfalfa leafcutting bee, Megachile rotundata.

Background: Managed populations of the alfalfa leafcutting bee (ALCB), Megachile rotundata (F.), are often not sustainable. In addition to numerous mortality factors that contribute to this, the dense bee populations used to maximize alfalfa pollination quickly deplete floral resources available to bees later in the summer. Providing alternative floral resources as alfalfa declines may help to improve ALCB reproduction. Methods: We examined the relationship between floral resource availability and ALCB reproduction and offspring condition via (1) a field study using alfalfa plots with and without late-blooming wildflower strips to supply food beyond alfalfa bloom, and (2) a field-cage study in which we provided bees with alfalfa, wildflowers, or both as food resources. Results: In the field study, bee cell production closely followed alfalfa floral density with an initial peak followed by large declines prior to wildflower bloom. Few bees visited wildflower strips, whose presence or absence was not associated with any measure of bee reproduction. However, we found that female offspring from cells provisioned earlier in the season, when alfalfa predominated as a source of provisions, eclosed with greater body sizes and proportion body lipids relative to total body mass. For bees restricted to cages, the proportion of offspring that survived to adults was highest on pure alfalfa diets. Adding wildflowers to cages with alfalfa did not affect adult offspring production or female offspring body size and lipid content. Furthermore, although similar numbers of adults were produced on wildflowers alone as with alfalfa alone, females eclosed with smaller body sizes and lower proportion body lipids on wildflowers despite the higher protein content we estimated for wildflower pollen. We found no evidence that adding the late-season wildflower species that we chose to plant enhanced ALCB offspring numbers. Our results highlight the importance of considering multiple measures of reproductive success, including offspring body size and lipid stores, when designing and evaluating floral resource management strategies for agroecosystems.

Contribution of the alternative pathway on spring rejuvenation of alfalfa.

Alfalfa often suffers from low temperature during spring rejuvenation, so it is important to improve the cold tolerance of alfalfa leaves for its smooth rejuvenation, and the alternative pathway (AP) could effectively improve the plant's tolerance. In this study, the contribution of AP on spring rejuvenation of alfalfa was investigated in Xinmu No.4 and Gannong No.5 with different fall dormancy levels. Though the protein and AP capacity were decreased during the rejuvenation, the ratio of AP/TP were increased in two alfalfa varieties, compared to those in alfalfa before overwintering. This indicated that AP had positive response to alfalfa rejuvenation. The limitation of AP significantly affected the leaf length, leaf width and growth rate of greening alfalfa, showing that AP played an important role in alfalfa rejuvenation. Inhibition of AP resulted in a significant decrease in Pn, Ci, Gs and stomatal structure deformity, suggestion that AP affected photosynthesis by influencing stomatal development during rejuvenation. AP reduces oxidative damage to PSII core protein repair in alfalfa leaves and optimizes photosynthesis by up-regulating NADP-MDH activity, decreasing the accumulation of excess reducing power in the chloroplasts, and by increasing SOD and POD activities and decreasing the accumulation of hydrogen peroxide. The higher proportion of AP keeps it more tolerant to low temperature for rejuvenation in Xinmu No.4 with a lower fall dormancy level.

Grazing camels under semi-extensive production system: selectivity, feed intake capacity, digestion and energy expenditure.

BACKGROUND: It was proposed that camels are more effective than other livestock species in selecting plants for their nutritional value. They may self-regulate their voluntary feed intake to satisfy their nutritional needs. This study was designed to investigate camels' feeding selectivity and ability to cover nutritional requirements when grazing alfalfa (high in protein) and/ or barley (high in energy) in a desert climate. METHODS: Eighteen lactating camels were equally divided into three feeding treatments. They grazed daily on alfalfa, barley, or a mixed pasture of both, for two periods of one month each. The concentrate supplement was individually administered at 40 g/kg BW0.75, divided into two equal parts, in the morning and in afternoon. Total energy expenditure (EE) was estimated by heart rate (HR) monitors for 48 h after being calibrated by oxygen consumption using an upgraded face mask open-circuit respiratory system. RESULTS: During the first period, camels had a greater forage intake and digestibility when they grazed barley rather than alfalfa, while those grazing mixed pasture performed intermediately. In the second period, camels had a similar forage intake and digestibility among treatments due to a decline in barley intake and digestibility compared to the first period, which was expected since the preferred plant part gradually shifted from barley grains to predominantly straw as a function of time. Similar HR and EE were found across periods and treatments. As a result of greater gross and digestible energy intake in period 1, a better energy balance in period 1 was observed compared to period 2. Camels better utilize barley than alfalfa. Grazing on barley had a higher energy balance than grazing alfalfa alone or in combination with barley. However, camels grazing barley produced lower milk yield and energy than those grazing alfalfa alone or in combination with barley, with no interaction detected between period and treatment. CONCLUSIONS: Lactating camels are able to self-regulate their voluntary intake to cover their energy requirements when they are grazing barley and/or alfalfa supplemented with a concentrate supplement at 40 g/kg BW0.75. Grazing barley is better utilized by camels than alfalfa. The chemical and physical properties of plant species play an important role in the selectivity of foraging camels. It also impacts their intake and digestibility, which is negatively associated with the proportion of cell wall content consumed.