The Physiological Basis of Alfalfa Plant Height Establishment.

Plant height plays an important role in crop yield, product quality, and cultivation management. However, the physiological mechanisms that regulate the establishment of plant height in alfalfa plants remain unclear. Herein, we measured plant height traits, leaf characteristics, photosynthetic physiology, cell wall composition, and endogenous hormone contents of tall- and short-stalked alfalfa materials at different reproductive periods. We analyzed the physiology responsible for differences in plant height. The results demonstrated that the number of internodes in tall- and short-stalked alfalfa materials tended to converge with the advancement of the fertility period. Meanwhile, the average internode length (IL) of tall-stalked materials was significantly higher than that of short-stalked materials at different fertility periods, with internode length identified as the main trait determining the differences in alfalfa plant height. Leaf characteristics, which are closely related to photosynthetic capacity, are crucial energy sources supporting the expression of plant height traits, and we found that an increase in the number of leaves contributed to a proportional increase in plant height. Additionally, a significant positive correlation was observed between plant height and leaf dry weight per plant during the branching and early flowering stages of alfalfa. The leaves of alfalfa affect plant height through photosynthesis, with the budding stage identified as the key period for efficient light energy utilization. Plant height at the budding stage showed a significant positive correlation with soluble sugar (SS) content and a significant negative correlation with intercellular CO2 concentration. Moreover, we found that alfalfa plant height was significantly correlated with the contents of indole-3-acetic acid in stem tips (SIAA), gibberellin A3 in leaves (LGA3), zeatin in stem tips (SZT), and abscisic acid in leaves (LABA). Further investigation revealed that SS, SIAA, and LGA3 contents were important physiological indicators affecting alfalfa plant height. This study provides a theoretical basis for understanding the formation of alfalfa plant height traits and for genetic improvement studies.

The antennal transcriptome analysis and characterizations of odorant-binding proteins in Megachile saussurei (Hymenoptera, Megachilidae).

BACKGROUND: Odorant-binding proteins (OBPs) are essential in insect's daily behaviors mediated by olfactory perception. Megachile saussurei Radoszkowski (Hymenoptera, Megachilidae) is a principal insect pollinating alfalfa (Medicago sativa) in Northwestern China. The olfactory function have been less conducted, which provides a lot of possibilities for our research. RESULTS: Our results showed that 20 OBPs were identified in total. Multiple sequence alignment analysis indicated MsauOBPs were highly conserved with a 6-cysteine motif pattern and all belonged to the classic subfamily, coding 113-196 amino acids and sharing 41.32%-99.12% amino acid identity with known OBPs of other bees. Phylogenetic analysis indicated there were certain homologies existed among MsauOBPs and most sequences were clustered with that of Osmia cornuta (Hymenoptera, Megachilidae). Expression analysis showed the identified OBPs were mostly enriched in antennae instead of other four body parts, especially the MsauOBP2, MsauOBP3, MsauOBP4, MsauOBP8, MsauOBP11 and MsauOBP17, in which the MsauOBP2, MsauOBP4 and MsauOBP8 presented obvious tissue-biased expression pattern. Molecular docking results indicated MsauOBP4 might be the most significant protein in recognizing alfalfa flower volatile 3-Octanone, while MsauOBP13 might be the most crucial protein identifying (Z)-3-hexenyl acetate. It was also found the lysine was a momentous hydrophilic amino acid in docking simulations. CONCLUSION: In this study, we identified and analyzed 20 OBPs of M. saussurei. The certain homology existed among these OBPs, while some degree of divergence could also be noticed, indicating the complex functions that different MsauOBPs performed. Besides, the M. saussurei and Osmia cornuta were very likely to share similar physiological functions as most of their OBPs were clustered together. MsauOBP4 might be the key protein in recognizing 3-Octanone, while MsauOBP13 might be the key protein in binding (Z)-3-hexenyl acetate. These two proteins might contribute to the alfalfa-locating during the pollination process. The relevant results may help determine the highly specific and effective attractants for M. saussurei in alfalfa pollination and reveal the molecular mechanism of odor-evoked pollinating behavior between these two species.

Enriched endogenous free Spd and Spm in alfalfa (Medicago sativa L.) under drought stress enhance drought tolerance by inhibiting H2O2 production to increase antioxidant enzyme activity.

Drought stress is a major factor limiting agricultural development, and exogenous polyamines (PAs) can increase plant drought resistance by enhancing antioxidant activity, but few studies have examined whether endogenous PAs enhance the plant antioxidant system. Here, to investigate the effects of endogenous PAs on the antioxidant system of alfalfa under drought stress and the underlying mechanisms, two alfalfa cultivars, Longzhong (drought resistant) and Gannong No. 3 (drought sensitive), were used as test materials, and their seedlings were treated with polyethylene glycol (PEG-6000) for 8 days at -1.2 MPa to simulate drought stress. The levels of free PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)], hydrogen peroxide (H2O2), malondialdehyde (MDA), key PA metabolism enzyme [arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), polyamine oxidase (PAO), and diamine oxidase (DAO)] activities, and antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD)] activities were measured. These physiological indicators were used for correlation analysis to investigate the relationship between PA metabolism and the antioxidant enzyme system. The results showed that PA synthesis in alfalfa under drought stress was dominated by the ADC pathway. Spd and Spm played an important role in improving drought tolerance. The high levels of ADC and SAMDC activities were facilitated by the conversion of Put to Spd and Spm. H2O2 generation by oxidative decomposition of PAs was mainly dependent on the oxidative decomposition of DAO but not PAO. Low DAO activity favored low H2O2 production. Spd, Spm, ADC, ODC and SAMDC were positively correlated with the antioxidant enzymes SOD, CAT and POD in both cultivars under drought. Therefore, we concluded that high ADC and SAMDC activities in alfalfa promoted the conversion of Put to Spd and Spm, leading to high accumulation of Spd and Spm and low Put accumulation. Low Put levels led to low H2O2 production through low DAO activity, and low H2O2 levels induced the expression of antioxidant enzyme-encoding genes to improve antioxidant enzyme activity and reduce MDA accumulation and thereby enhanced drought resistance in alfalfa.

Integrative analysis of multi-omics data for discovery of ferroptosis-related gene signature predicting immune activity in neuroblastoma.

Immunotherapy for neuroblastoma remains unsatisfactory due to heterogeneity and weak immunogenicity. Exploring powerful signatures for the evaluation of immunotherapy outcomes remain the primary purpose. We constructed a ferroptosis-related gene (FRG) signature by least absolute shrinkage and selection operator and Cox regression, identified 10 independent prognostic FRGs in a training cohort (GSE62564), and then verified them in an external validation cohort (TCGA). Associated with clinical factors, the signature accurately predicts overall survival of 3, 5, and 10 years. An independent prognostic nomogram, which included FRG risk, age, stage of the International Neuroblastoma Staging System, and an MYCN status, was constructed. The area under the curves showed satisfactory prognostic predicting performance. Through bulk RNA-seq and proteomics data, we revealed the relationship between hub genes and the key onco-promoter MYCN gene and then validated the results in MYCN-amplified and MYCN-non-amplified cell lines with qRT-PCR. The FRG signature significantly divided patients into high- and low-risk groups, and the differentially expressed genes between the two groups were enriched in immune actions, autophagy, and carcinogenesis behaviors. The low-risk group embodied higher positive immune component infiltration and a higher expression of immune checkpoints with a more favorable immune cytolytic activity (CYT). We verified the predictive power of this signature with data from melanoma patients undergoing immunotherapy, and the predictive power was satisfactory. Gene mutations were closely related to the signature and prognosis. AURKA and PRKAA2 were revealed to be nodal hub FRGs in the signature, and both were shown to have significantly different expressions between the INSS stage IV and other stages after immunohistochemical validation. With single-cell RNA-seq analysis, we found that genes related to T cells were enriched in TNFA signaling and interferon-γ hallmark. In conclusion, we constructed a ferroptosis-related gene signature that can predict the outcomes and work in evaluating the effects of immunotherapy.

Corrigendum: Neutrophil extracellular traps in autoimmune diseases: Analysis of the knowledge map.

[This corrects the article DOI: 10.3389/fimmu.2023.1095421.].

Exogenous application of salicylic acid improves freezing stress tolerance in alfalfa.

Freezing stress is one of the most detrimental environmental factors that can seriously impact the growth, development, and distribution of alfalfa (Medicago sativa L.). Exogenous salicylic acid (SA) has been revealed as a cost-effective method of improving defense against freezing stress due to its predominant role in biotic and abiotic stress resistance. However, how the molecular mechanisms of SA improve freezing stress resistance in alfalfa is still unclear. Therefore, in this study, we used leaf samples of alfalfa seedlings pretreatment with 200 µM and 0 µM SA, which were exposed to freezing stress (-10°C) for 0, 0.5, 1, and 2h and allowed to recover at normal temperature in a growth chamber for 2 days, after which we detect the changes in the phenotypical, physiological, hormone content, and performed a transcriptome analysis to explain SA influence alfalfa in freezing stress. The results demonstrated that exogenous SA could improve the accumulation of free SA in alfalfa leaves primarily through the phenylalanine ammonia-lyase pathway. Moreover, the results of transcriptome analysis revealed that the mitogen-activated protein kinase (MAPK) signaling pathway-plant play a critical role in SA alleviating freezing stress. In addition, the weighted gene co-expression network analysis (WGCNA) found that MPK3, MPK9, WRKY22 (downstream target gene of MPK3), and TGACG-binding factor 1 (TGA1) are candidate hub genes involved in freezing stress defense, all of which are involved in the SA signaling pathway. Therefore, we conclude that SA could possibly induce MPK3 to regulate WRKY22 to participate in freezing stress to induced gene expression related to SA signaling pathway (NPR1-dependent pathway and NPR1-independent pathway), including the genes of non-expresser of pathogenesis-related gene 1 (NPR1), TGA1, pathogenesis-related 1 (PR1), superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), glutathione-S-transferase (GST), and heat shock protein (HSP). This enhanced the production of antioxidant enzymes such as SOD, POD, and APX, which increases the freezing stress tolerance of alfalfa plants.

Potential Effect of DIMBOA (2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one) on Alleviating the Autotoxic Coumarin Stress in Alfalfa (Medicago sativa) Seedlings.

The allelopathic theory has garnered considerable attention in the field of agricultural production for its efficient plant protection, rapid crop yield increase, and scientific establishment of the crop rotation system. To study the effects of the main maize allelochemical DIMBOA (2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one) on the growth and development of alfalfa under autotoxic coumarin stress, we treated alfalfa seedlings with DIMBOA under coumarin stress and non-stress conditions in this study. Results show that 0.0342 mM coumarin significantly inhibited alfalfa seed germination percentage(Gp), germination potential(GP), radicle length, germ length, seeding height, and simple viability index (SVI), with decreases of 37.29%, 59.91%, 7.60%, 30.90%, 13.27%, and 45.70%, respectively. An amount of 0.6 mM DIMBOA could promote alfalfa seed Gp, GP, radicle length, germ length, seeding height, dry fresh ratio, and SVI, with increases of 12.38%, 23.91%, 48.69%, 48.65%, 48.68%, 295.12%, and 67.17%, respectively. However, the addition of DIMBOA under conditions of coumarin stress could effectively alleviate coumarin effects on alfalfa seedlings. Coumarin + DIMBOA treatment for 24 h mainly decreased reactive oxygen species (ROSs) and malondialdehyde (MDA) as well as soluble protein and soluble sugar, increasing some antioxidant enzyme activities and antioxidant content to alleviate the oxidative damage of alfalfa caused by coumarin stress. Administration of treatment for 72 h significantly promoted the morphological development of alfalfa seeding roots. Administration of treatment for 96 h significantly enhanced the photosynthetic capacity of alfalfa seedlings. The results of principal component analysis demonstrated that chlorophyll b(Chl b)and net photosynthetic rate(Pn) were the key indicators for coumarin + DIMBOA treatment to promote photosynthesis in alfalfa seedlings. Additionally, root length, mean root diameter, and root volume were the key indicators of root growth and development. Coumarin + DIMBOA treatment primarily increased catalase(CAT), peroxidase (POD), and ascorbate peroxidase (APX) activity and antioxidants(ASA) while reducing MDA and superoxide anion radical(O2•-). This study strongly suggested that DIMBOA can effectively improve the tolerance of alfalfa seedlings to coumarin stress through a combination of effects on root morphology, photosynthesis, and physiological indicators.

Comparative Transcriptome Analysis Reveals New Insight of Alfalfa (Medicago sativa L.) Cultivars in Response to Abrupt Freezing Stress.

Freezing stress is a major limiting environmental factor that affects the productivity and distribution of alfalfa (Medicago sativa L.). There is growing evidence that enhancing freezing tolerance through resistance-related genes is one of the most efficient methods for solving this problem, whereas little is known about the complex regulatory mechanism of freezing stress. Herein, we performed transcriptome profiling of the leaves from two genotypes of alfalfa, freezing tolerance "Gannong NO.3" and freezing-sensitive "WL326GZ" exposure to -10°C to investigate which resistance-related genes could improve the freezing tolerance. Our results showed that a total of 121,366 genes were identified, and there were 7,245 differentially expressed genes (DEGs) between the control and treated leaves. In particular, the DEGs in "Gannong NO.3" were mainly enriched in the metabolic pathways and biosynthesis of secondary metabolites, and most of the DEGs in "WL326GZ" were enriched in the metabolic pathways, the biosynthesis of secondary metabolites, and plant-pathogen interactions. Moreover, the weighted gene co-expression network analysis (WGCNA) showed that ATP-binding cassette (ABC) C subfamily genes were strongly impacted by freezing stress, indicating that ABCC8 and ABCC3 are critical to develop the freezing tolerance. Moreover, our data revealed that numerous Ca2+ signal transduction and CBF/DREB1 pathway-related genes were severely impacted by the freezing resistance, which is believed to alleviate the damage caused by freezing stress. Altogether, these findings contribute the comprehensive information to understand the molecular mechanism of alfalfa adaptation to freezing stress and further provide functional candidate genes that can adapt to abiotic stress.

Effect of estradiol as a continuous variable on breast cancer survival by menopausal status: a cohort study in China.

High levels of circulating estradiol (E2) are associated with increased risk of breast cancer, whereas its relationship with breast cancer prognosis is still unclear. We evaluated the effect of E2 concentration on survival endpoints among 8766 breast cancer cases diagnosed between 2005 and 2017 from the Tianjin Breast Cancer Cases Cohort. Levels of serum E2 were measured in pre-menopausal and post-menopausal women. Multivariable-adjusted Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) between quartile of E2 levels and overall survival (OS) and progression-free survival (PFS) of breast cancer. The penalized spline was then used to test for non-linear relationships between E2 (continuous variable) and survival endpoints. 612 deaths and 982 progressions occurred over follow-up through 2017. Compared to women in the quartile 3, the highest quartile of E2 was associated with reduced risk of both PFS in pre-menopausal women (HR 1.79, 95% CI 1.17-2.75, P = 0.008) and OS in post-menopausal women (HR 1.35, 95% CI 1.04-1.74, P = 0.023). OS and PFS in pre-menopausal women exhibited a nonlinear relation ("L-shaped" and "U-shaped", respectively) with E2 levels. However, there was a linear relationship in post-menopausal women. Moreover, patients with estrogen receptor-negative (ER-negative) breast cancer showed a "U-shaped" relationship with OS and PFS in pre-menopausal women. Pre-menopausal breast cancer patients have a plateau stage of prognosis at the intermediate concentrations of E2, whereas post-menopausal patients have no apparent threshold, and ER status may have an impact on this relationship.

Identification of an Autophagy-Related Risk Signature Correlates With Immunophenotype and Predicts Immune Checkpoint Blockade Efficacy of Neuroblastoma.

Neuroblastoma is one of the malignant solid tumors with the highest mortality in childhood. Targeted immunotherapy still cannot achieve satisfactory results due to heterogeneity and tolerance. Exploring markers related to prognosis and evaluating the immune microenvironment remain the major obstacles. Herein, we constructed an autophagy-related gene (ATG) risk model by multivariate Cox regression and least absolute shrinkage and selection operator regression, and identified four prognostic ATGs (BIRC5, GRID2, HK2, and RNASEL) in the training cohort, then verified the signature in the internal and external validation cohorts. BIRC5 and HK2 showed higher expression in MYCN amplified cell lines and tumor tissues consistently, whereas GRID2 and RNASEL showed the opposite trends. The correlation between the signature and clinicopathological parameters was further analyzed and showing consistency. A prognostic nomogram using risk score, International Neuroblastoma Staging System stage, age, and MYCN status was built subsequently, and the area under curves, net reclassification improvement, and integrated discrimination improvement showed more satisfactory prognostic predicting performance. The ATG prognostic signature itself can significantly divide patients with neuroblastoma into high- and low-risk groups; differentially expressed genes between the two groups were enriched in autophagy-related behaviors and immune cell reactions in gene set enrichment analysis (false discovery rate q -value < 0.05). Furthermore, we evaluated the relationship of the signature risk score with immune cell infiltration and the cancer-immunity cycle. The low-risk group was characterized by more abundant expression of chemokines and higher immune checkpoints (PDL1, PD1, CTLA-4, and IDO1). The risk score was significantly correlated with the proportions of CD8+ T cells, CD4+ memory resting T cells, follicular helper T cells, memory B cells, plasma cells, and M2 macrophages in tumor tissues. In conclusion, we developed and validated an autophagy-related signature that can accurately predict the prognosis, which might be meaningful to understand the immune microenvironment and guide immune checkpoint blockade.

Association between low density lipoprotein cholesterol and all-cause mortality: results from the NHANES 1999-2014.

The association between low density lipoprotein cholesterol (LDL-C) and all-cause mortality has been examined in many studies. However, inconsistent results and limitations still exist. We used the 1999-2014 National Health and Nutrition Examination Survey (NHANES) data with 19,034 people to assess the association between LDL-C level and all-cause mortality. All participants were followed up until 2015 except those younger than 18 years old, after excluding those who died within three years of follow-up, a total of 1619 deaths among 19,034 people were included in the analysis. In the age-adjusted model (model 1), it was found that the lowest LDL-C group had a higher risk of all-cause mortality (HR 1.708 [1.432-2.037]) than LDL-C 100-129 mg/dL as a reference group. The crude-adjusted model (model 2) suggests that people with the lowest level of LDL-C had 1.600 (95% CI [1.325-1.932]) times the odds compared with the reference group, after adjusting for age, sex, race, marital status, education level, smoking status, body mass index (BMI). In the fully-adjusted model (model 3), people with the lowest level of LDL-C had 1.373 (95% CI [1.130-1.668]) times the odds compared with the reference group, after additionally adjusting for hypertension, diabetes, cardiovascular disease, cancer based on model 2. The results from restricted cubic spine (RCS) curve showed that when the LDL-C concentration (130 mg/dL) was used as the reference, there is a U-shaped relationship between LDL-C level and all-cause mortality. In conclusion, we found that low level of LDL-C is associated with higher risk of all-cause mortality. The observed association persisted after adjusting for potential confounders. Further studies are warranted to determine the causal relationship between LDL-C level and all-cause mortality.

The complete chloroplast genome sequence of Potentilla glabra Lodd.

Potentilla glabra Lodd is an important traditional Chinese medicine, which is widely distributed in many areas of China. It has a variety of pharmacological activities and has been drunk as a tea for a long time. Therefore, it has great economic, research, and social value. However, no plastid genome has been reported to date. Here, we report species of this genus complete chloroplast genome of Potentilla glabra Lodd. The chloroplast genome of Potentilla glabra Lodd is found to be 152,900 bp in length with 37.24% GC contents. The cp genome sequences contained 130 genes, including 84 mRNA genes, 37 tRNA, eight rRNA genes, and one pseudogenes, respectively. A phylogenetic tree reconstructed by 42 chloroplast genomes reveals that Potentilla glabra Lodd is most related with Potentilla parvifolia. The complete chloroplast genome of these plants will be benefit for studies on the general characteristics and evolution of the Potentilla family genome.

Emergence of Invasive Serotype Ib Sequence Type 10 Group B Streptococcus Disease in Chinese Infants Is Driven by a Tetracycline-Sensitive Clone.

Background: Group B streptococcus (GBS) is a leading cause of serious infections in infants. The extensive use of tetracycline has led to the selection of specific resistant and infectious GBS clones. The sequence type (ST) 10 GBS strain, causing invasive infections in infants, is becoming prevalent in China. We aimed to understand the clinical and microbiological characteristics of this GBS strain. Methods: We conducted a retrospective study on infants with invasive GBS disease from the largest women's and children's medical center in Shanxi and collected data between January 2017 and October 2020. GBS isolates were analyzed by capsule serotyping, genotyping, antibiotic resistance, and surface protein genes. Results: All ST10 isolates belonged to serotype Ib; type Ib/ST10 strains were responsible for 66.7% (14/21, P < 0.05) of infant invasive GBS infections during the period and all resulted in late-onset (LOD) and late LOD disease (14/14). Infants with type Ib/ST10 GBS disease had significantly higher rates of meningitis (9/14, 64.3%, p < 0.05) and clinical complications (5/14, 35.7%, p < 0.05). The Ib/ST10 GBS isolates had limited genetic diversity, clustered in the CC10/bca/PI-1 + PI-2a genetic lineage, showed resistance to erythromycin, lincomycin, and fluoroquinolones and sensitivity to tetracycline, and possessed genes ermT, ermB, and amino acid changes in gyrA and parC. Conclusions: The probable clonal expansion can result in severe infections in infants and ongoing emergence of multi-drug resistant isolates. Continued monitoring for type Ib/ST10 GBS infections is warranted.

The complete chloroplast genome sequence of Rubus sachalinensis Lévl.

Rubus is a medicinal plant distributed in northern China and has high economic and social value. In this study, we reported the complete chloroplast genome sequence of the plant. We determined that the length of the chloroplast genome of Rubus sachalinensis Lévl is found to be 155,787 bp and the GC content is 37.24%. The cp genome sequences contains 132 genes, including 37 tRNA genes, eight rRNA genes, and 87 mRNA genes, respectively. The genomic data can help the classification and evolution of Rubus plants, and provide a theoretical basis for the study of Rubus sachalinensis Lévl.

Physiological and biochemical response of different resistant alfalfa cultivars against thrips damage.

To investigate physiological and biochemical changes of thrips-resistant alfalfa (Medicago sativa L. cv. Gan-nong No. 9), we aimed at clarifying the response mechanisms of alfalfa against thrips. Medicago sativa L. cv. including thrips-resistant Gan-nong No.9 (G9), thrips-susceptible Gan-nong No.3 (G3) and highly thrips-susceptible WL363HQ (363) were infested with different thrips densities (3, 5, 7 and 9-thrips per branch). The quantitative change in specific nutrients, secondary metabolites, defensive and antioxidant enzymes were measured at seedling stage of the three alfalfa cultivars. The results showed that with the increase of thrips densities, the damage indices, SS, Pro, flavonoids, tannin and H2O2 in G9, G3 and 363 were significantly increased, but PPO and SOD significantly reduced, compared with CK. Furthermore, the tannin and lignin contents of G9 were significantly higher compared to 363, but SP content was significantly lower than G3 and H2O2 content which was further significantly less compared to 363. Correlation analysis observed that the damage index of the three alfalfa cultivars showed a significant positive association with SS, Pro, flavone, tannin, and H2O2 (P < 0.01), while damage index and DW, Chl (a, b, a + b), PPO and SOD showed a significant negative correlation (P < 0.01). Based on principal component comprehensive evaluation, the 5-thrips adults per branch were the critical inoculation threshold for G9 against thrips injury because the score was - 0.048. These results revealed that thrips damage significantly increased the contents of SS, Pro, flavonoids, tannins and H2O2, as well as significantly declined the activities of PPO and SOD in the three cultivars (P < 0.05), moreover, thrips-resistant G9 markedly accumulated lignin content, POD and CAT activity, inhibited Chl (a + b, b) and SP biosynthesis to resist thrips damage.

Plant transcriptome analysis reveals specific molecular interactions between alfalfa and its rhizobial symbionts below the species level.

BACKGROUND: Leguminous plants alter patterns of gene expression in response to symbiotic colonization and infection by their cognate rhizobial bacteria, but the extent of the transcriptomic response has rarely been examined below the species level. Here we describe the identification of 12 rhizobial biotypes of Ensifer meliloti, which form nitrogen-fixing nodules in the roots of alfalfa (Medicago sativa L.), followed by a comparative RNA-seq analysis of four alfalfa cultivars each inoculated with two E. meliloti strains varying in symbiotic performance and phylogenetic relatedness. RESULTS: Rhizobial biotypes were identified on the basis of their symbiotic performance, particularly shoot dry weight. Differentially expressed genes (DEGs) and metabolic pathways were determined by comparing the RNA-seq data with that of the uninoculated control plant. Significant differences were found between DEGs generated in each cultivar with the inoculation of two rhizobial strains in comparison (P < 0.01). A total of 8111 genes was differentially expressed, representing ~ 17.1% of the M. sativa genome. The proportion of DEGs ranges from 0.5 to 12.2% for each alfalfa cultivar. Interestingly, genes with predicted roles in flavonoid biosynthesis and plant-pathogen interaction (NBS-LRR) were identified as the most significant DEGs. Other DEGs include Medsa002106 and genes encoding nodulins and NCR peptides whose expression is specifically induced during the development of nitrogen-fixing nodules. More importantly, strong significant positive correlations were observed between plant transcriptomes (DEGs and KEGG pathways) and phylogenetic distances between the two rhizobial inoculants. CONCLUSIONS: Alfalfa expresses significantly distinct sets of genes in response to infection by different rhizobial strains at the below-species levels (i.e. biotype or strain). Candidate genes underlying the specific interactions include Medsa002106 and those encoding nodulins and NCR peptides and proteins in the NBS-LRR family.

Genetic diversity and symbiotic efficiency difference of endophytic rhizobia of Medicago sativa.

Research on rhizobium diversity has paved the way for diversification of rhizobial germplasm resources. Seventy-three endophytic bacterial isolates were collected from seven tissues of five alfalfa cultivars in three geographic locations in Gansu, China. Restriction fragment length polymorphism (RFLP) fingerprinting of 16S rRNA and analysis of concatenated sequence of three housekeeping genes (atpD, glnII, and recA) and two symbiotic genes (nodC and nifH) were used for strain identification. Results showed that the endophytic strains were genetically diverse at different taxonomic levels, and Ensifer meliloti (31) and Agrobacterium radiobacter (12) are common Medicago sativa endophytic bacteria in Gansu, China. The nifH genes (97%-98% sequence identity) of E. meliloti strains were more diverse than the nodC genes (99%-100% sequence identity), even though the strains evolved from a common ancestor. The degree of dispersion of symbiotic phenotypes of E. meliloti strains on M. sativa 'Gannong No. 3', 'Gannong No. 9', and 'Qingshui' was much less than that on M. sativa 'Longzhong' and 'WL168HQ'. This suggested that the symbiotic efficiency of E. meliloti strains on the former three alfalfa cultivars was similar but on the latter two was discrepant. Their symbiotic efficiency differed primarily according to alfalfa cultivars and, to a lesser extent, to the tested strains, indicating the difference in the sensitivity of different alfalfa cultivars to rhizobial strains.