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Multispectral imaging by unoccupied aerial vehicles provides a nondestructive, high-throughput approach to measure biomass accumulation over successive alfalfa (Medicago sativa L. subsp. sativa) harvests. Information from estimated growth curves can be used to infer harvest biomass and to gain insights into the relationship between growth dynamics and forage biomass stability across cuttings and years. In this study, multispectral imaging and several common vegetation indices were used to estimate genetic parameters and model growth of alfalfa cultivars to determine the longitudinal relationship between vegetation indices and forage biomass. Results showed moderate heritability for vegetation indices, with median plot level heritability ranging from 0.11 to 0.64, across multiple cuttings in three trials planted in Ithaca, NY, and Las Cruces, NM. Genetic correlations between the normalized difference vegetation index and forage biomass were moderate to high across trials, cuttings, and the timing of multispectral image capture. To evaluate the relationship between growth parameters and forage biomass stability across cuttings and environmental conditions, random regression modeling approaches were used to estimate the growth parameters of cultivars for each cutting and the variance in growth was compared to the variance in genetic estimates of forage biomass yield across cuttings. These analyses revealed high correspondence between stability in growth parameters and stability of forage yield. The results of this study indicate that vegetation indices are effective at modeling genetic components of biomass accumulation, presenting opportunities for more efficient screening of cultivars and new longitudinal modeling approaches that can provide insights into temporal factors influencing cultivar stability., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Plant Growth-Promoting Rhizobacteria (PGPR) are gaining increasing attention, but their interactions with indigenous rhizosphere microbiomes remain unclear. To address this issue, we isolated a strain of Priestia aryabhattai with a growth-promoting effect. Under greenhouse conditions, its growth-promoting effect on alfalfa was evaluated, and amplicon sequencing was used to analyze changes in the rhizosphere microbial community to explore the growth promotion mechanism. Our study shows that inoculation with Priestia aryabhattai increases the α-diversity index of the alfalfa rhizosphere microbiome and enhances the abundance of beneficial bacterial genera. This is likely because inoculation with Priestia aryabhattai increased the abundance of carbon-sequestering genera, particularly Gemmatimonas, thereby improving the soil environment. The increased abundance of beneficial bacteria stimulates root development in alfalfa and enhances nutrient uptake, particularly phosphorus, which in turn boosts photosynthesis and promotes alfalfa growth. In summary, Priestia aryabhattai improves soil environment and promotes alfalfa growth by enhancing the carbon sequestration capacity of the rhizosphere microbial community. This work provides theoretical support and insight for the development of PGPR inoculants and for further research on their mechanisms., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

To explore the feasibility of using cold plasma as a mutagenesis breeding technology for forage crops, in this study we used the Medicago sativa L. cultivar, Zhongcao No. 3, as the experimental material. The effects of plasma treatments on Medicago sativa L. were analyzed through the use of plasma and activated water. Treatments with plasma and activated water inhibited plant height but promoted root growth. By creating a closed environment, adding a dielectric barrier plate, and combining these two treatment methods, the greatest impact can be had on the growth of Medicago sativa L. seeds. After treatment, the plant heights were approximately half those of the control group, and the root lengths were approximately 1.6 times those of the control group. Through emission spectroscopy, it was found that active particles such as O, NO 2 , and N 2 * were present and could be considered to have produced plasma-activated water through contact with the water surface, thus affecting the survival and growth of the seeds. Whole-genome resequencing (WGRS) was performed on the wild-type and selected mutants after treatment, with an average sequencing depth of 115.93×, an average genome alignment rate of 91.72 %, and an average genome coverage rate of 91.85 %. Various types of mutations were detected and annotated. After filtering, 7,822,324 SNP (single nucleotide polymorphisms) sites, 2,161,917 indel sites, 200,544 SV sites, 238 CNV (copy number variation) sites. The SNPs, indels (insertions/deletions), and SVs (structural variations) were mainly heterozygous, with heterozygosity rates of 87.13 %, 92.16 %, and 83.49 %, respectively. The CNVs were dominated by low copy numbers, accounting for 53.77 %. These results indicate that plasma treatment has significant effects on plant growth and genome of Medicago sativa L., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: Timely and accurate monitoring of soil salinity content (SSC) is essential for precise irrigation management of large-scale farmland. Uncrewed aerial vehicle (UAV) low-altitude remote sensing with high spatial and temporal resolution provides a scientific and effective technical means for SSC monitoring. Many existing soil salinity inversion models have only been tested by a single variable selection method or machine learning algorithm, and the influence of variable selection method combined with machine learning algorithm on the accuracy of soil salinity inversion remain further studied., Methods: Firstly, based on UAV multispectral remote sensing data, by extracting the spectral reflectance of each sampling point to construct 30 spectral indexes, and using the pearson correlation coefficient (PCC), gray relational analysis (GRA), variable projection importance (VIP), and support vector machine-recursive feature elimination (SVM-RFE) to screen spectral index and realize the selection of sensitive variables. Subsequently, screened and unscreened variables as model input independent variables, constructed 20 soil salinity inversion models based on the support vector machine regression (SVM), back propagation neural network (BPNN), extreme learning machine (ELM), and random forest (RF) machine learning algorithms, the aim is to explore the feasibility of different variable selection methods combined with machine learning algorithms in SSC inversion of crop-covered farmland. To evaluate the performance of the soil salinity inversion model, the determination coefficient (R 2 ), root mean square error (RMSE) and performance deviation ratio (RPD) were used to evaluate the model performance, and determined the best variable selection method and soil salinity inversion model by taking alfalfa covered farmland in arid oasis irrigation areas of China as the research object., Results: The variable selection combined with machine learning algorithm can significantly improve the accuracy of remote sensing inversion of soil salinity. The performance of the models has been improved markedly using the four variable selection methods, and the applicability varied among the four methods, the GRA variable selection method is suitable for SVM, BPNN, and ELM modeling, while the PCC method is suitable for RF modeling. The GRA-SVM is the best soil salinity inversion model in alfalfa cover farmland, with R v 2 of 0.8888, RMSE v of 0.1780, and RPD of 1.8115 based on the model verification dataset, and the spatial distribution map of soil salinity can truly reflect the degree of soil salinization in the study area., Conclusion: Based on our findings, the variable selection combined with machine learning algorithm is an effective method to improve the accuracy of soil salinity remote sensing inversion, which provides a new approach for timely and accurate acquisition of crops covered farmland soil salinity information., Competing Interests: The authors declare that they have no competing interests., (© 2024 Ma et al.)

Understanding the effects of different land use modes on the spatial and temporal variations of soil moisture and temperature in the deep profile and revealing the regulatory effects of various vegetation covers on regional water and heat resources can provide a theoretical basis for the optimization of land management and vegetation restoration. Taking the advantage of different land use patterns in the Liudaogou watershed in the northern part of Loess Plateau, we monitored soil moisture content as well as temperature in the 0-1000 cm soil layer in 2022 to analyze the temporal variation and vertical profile distribution characteristics of soil moisture and temperature under four land use modes (woodland, grassland, farmland, and wild grassland). The results showed that soil moisture and temperature distributions varied significantly across different land use types. In the growing season (April-October), total soil water storage in the 0-1000 cm soil layer of the four land use types, in a descending order, was as follows: soybean farmland (1393 mm), wild grassland (1374 mm), Caragana korshinkii forest (1218 mm), and alfalfa grassland (557 mm). Soil moisture of C. korshinkii forest and soybean farmland changed obviously in the 0-300 cm soil layer, and that of wild grassland and alfalfa grassland was in 0-500 and 0-200 cm soil layers, respectively, while soil moisture of deep soil layers fluctuated little. The impact of land use modes on soil temperature was primarily manifested in the 0-200 cm soil layer, and the depth was up to 300 cm. The depth of precipitation infiltration replenishment of the four land use modes was approximately 200 cm. The depth of soil moisture depletion was 200 cm in both C. korshinkii forest and alfalfa grassland, and was 100 cm in soybean farmland and wild grassland. Soil hydrothermal processes in the deep profile varied across vegetation types.

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.

Plant-beneficial bacteria (PBB) have emerged as a promising approach for assisting phytoremediation of heavy metal (HM)-contaminated soils. However, their colonization efficiency is often challenged by complex soil environments. In this study, we screened one rhizobacterium (Klebsiella variicola Y38) and one endophytic bacterium (Serratia surfactantfaciens Y15) isolated from HM-contaminated soils and plants for their high resistance to Cd and strong growth-promoting abilities. These strains were encapsulated individually or in combination with alginate and applied with Medicago sativa in Cd-contaminated soil pot experiments. The effectiveness of different bacterial formulations in promoting plant growth and enhancing Cd bioconcentration in M. sativa was evaluated. Results showed that PBB application enhanced plant growth and antioxidant capacity while reducing oxidative damage. Encapsulated formulations outperformed unencapsulated ones, with combined formulations yielding superior results to individual applications. Quantitative PCR indicated enhanced PBB colonization in Cd-contaminated soils with alginate encapsulation, potentially explaining the higher efficacy of alginate-encapsulated PBB. Additionally, the bacterial agents modified Cd speciation in soils, resulting in increased Cd bioaccumulation in M. sativa by 217-337 %. The alginate-encapsulated mixed bacterial agent demonstrated optimal effectiveness, increasing the Cd transfer coefficient by 3.2-fold. Structural equation modeling and correlation analysis elucidated that K. variicola Y38 promoted Cd bioaccumulation in M. sativa roots by reducing oxidative damage and enhancing root growth, while S. surfactantfaciens Y15 facilitated Cd translocation to shoots, promoting shoot growth. The combined application of these bacteria leveraged the benefits of both strains. These findings contribute to diversifying strategies for effectively and sustainably remediating Cd-contaminated soils, while laying a foundation for future investigations into bacteria-assisted phytoremediation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Co-evolution of plant beneficial microbes in contaminated environments enhances plant growth and mitigates abiotic stress. However, few studies on heavy metal (HM) tolerant plant growth-promoting bacteria (PGPB) promoting crop growth in Morocco's farming areas affected by drought and salinity are available. Plant associated bacteria tolerant to HM and able to produce indole acetic acid and siderophores, display ACC-deaminase activity and solubilize phosphate, were isolated from long-term metal exposed environments. Tolerance to HM and biofilms formation in the absence or presence of HM were assessed. A consortium including two Ensifer meliloti strains (RhOL6 and RhOL8), one Pseudomonas sp. strain (DSP17), and one Proteus sp. strain (DSP1), was used to inoculate alfalfa (Medicago sativa) seedlings under various conditions, namely, salt stress (85 mM) and water stress (30% water holding capacity). Shoot and root dry weights of alfalfa were measured 60 days after sowing. In the presence of HM, DSP17 showed the greatest auxin production, whereas RhOL8 had the highest ACC-deaminase activity and DSP17 formed the densest biofilm. Root dry weight increased 138% and 195% in salt and water stressed plants, respectively, regarding non-inoculated controls. Our results confirm the improvement of alfalfa growth and mitigation of salt and drought stress upon inoculation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Sowing date and soil fertility are very important factors in the overwintering and production performance of alfalfa (Medicago sativa L.), yet there's a knowledge gap in knowledge on how late-seeded alfalfa responds to phosphorus (P) fertilization. A field study was conducted in Inner Mongolia from 2020 to 2022 using a split-plot design. The main plots consisted of five sowing dates (31 July, 8, 16, and 24 August, and 1 September), while the subplots involved five P application rates (0, 40, 70, 100, and 130 kg P 2 O 5 ha -1 ). Throughout the growing seasons, the overwintering rate, root traits, forage yield, and yield components were measured. The results revealed a consistent decrease in overwintering ability and productivity with the delayed sowing. This reduction in overwintering rate was mainly due to diminished root traits, while the decrease in forage yield was largely associated with a reduction in plants per square meter. However, P fertilizer application to late-seeded alfalfa demonstrated potential in enhancing the diameter of both the crown and taproot, thus strengthening the root system and improving the overwintering rate, the rate of increase ranges from 11.6 to 49%. This adjustment could also improve the shoots per square meter and mass per shoot, increasing by 9.4-31.3% and 15.0-27.1% respectively in 2 years, which can offset the decline in forage yield caused by late sowing and might even increase the forage yield. Regression and path analysis indicated that alfalfa forage yield is primarily affected by mass per shoot rather than shoots per square meter. This study recommended that the sowing of alfalfa in similar regions of Inner Mongolia should not be later than mid-August. Moreover, applying P fertilizer (P 2 O 5 ) at 70.6-85.9 kg ha -1 can enhance the forage yield and persistence of late-seeded alfalfa. Therefore, appropriate late sowing combined with the application of P fertilizer can be used as an efficient cultivation strategy for alfalfa cultivation after a short-season crop harvest in arid and cold regions., (© 2024. The Author(s).)

The chloroplast biogenesis occurs in cotyledon during alfalfa seed germination before true leaf formation, and is extremely important for the followed plant development and growth. In this study, we conducted a simulation of alfalfa seed germination in the soil by using tin foil and focused on 10 pivotal time points of chloroplast biogenesis in cotyledons before and after light exposure, which showed significant differences in multispectral images, and covered the whole process of chloroplast biogenesis from proplastid, etioplast to mature chloroplast. We revealed three phases that referred to the programmed involvements of photosynthesis promotion, ultrastructure maturity, transcriptomic expression, and protein complex construction, and observed distinct transcriptional expressions of genes from nuclear and chloroplast genomes. In phase I at dark germination before light exposure, chloroplast-encoded genes showed up-regulated expressions together with the importation of chloroplast proteins. In phase II for the first day after light exposure, nuclear-encoded genes' expressions were initiated at 2 h after light exposure (E2h), followed by swift assembly of chloroplast thylakoid membrane protein complexes, and roaring F v /F m and contents of chlorophyll a, chlorophyll b and carotenoid. The initiation at E2h was pronounced by the observation of gradual accumulation of single lamella, and facilitated the formation of granum stacks (thylakoid) at E8h in phase II. In phase III from the second day after light exposure, chloroplast became gradually complete with the fully established photosynthetic capacity. Altogether, our results layed a theoretical foundation for enhancing potential photosynthetic efficiency in alfalfa and related species., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shangang Jia, Peisheng Mao reports financial support was provided by the Key R & D project of Sichuan Science and Technology Program (2023YFSY0012), earmarked fund for CARS (CARS-34), and National Key R&D Program of China (2022YFD1300804). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The phosphoenolpyruvate carboxylase kinase of Medicago sativa L. (MsPPCK1) modulates the phosphorylation status and activity of the C4 pathway phosphoenolpyruvate carboxylase enzyme, which is pivotal for photosynthetic carbon assimilation in plants. This study investigated the role of MsPPCK1 in alfalfa by creating transgenic plants overexpressing MsPPCK1 under the control of the CaMV35S promoter. The enhanced alkali tolerance of transgenic plants indicated an important role of MsPPCK1 gene in regulating plant alkali tolerance. Transgenic plants exhibited heightened antioxidant activity (SOD, POD, and CAT), reduced MDA, H 2 O 2 , OFR and REC% content, increased activity of key photosynthetic enzymes (PEPC, PPDK, NADP-ME, and NADP-MDH), and enhanced photosynthetic parameters (Pn, E, Gs, and Ci). Moreover, MsPPCK1 overexpression increased the content of organic acids (oxaloacetic, malic, citric, and succinic acids) in the plants. The upregulation of MsPPCK1 under rhizobial inoculation showcased its other role in nodule development. In transgenic plants, MsDMI2, MsEnod12, and MsNODL4 expression increased, facilitating root nodule development and augmenting plant nodulation. Accelerated root nodule growth positively influences plant growth and yield and enhances alfalfa resistance to alkali stress. This study highlights the pivotal role of MsPPCK1 in fortifying plant alkali stress tolerance and improving yield, underscoring its potential as a key genetic target for developing alkali-tolerant and high-yielding alfalfa varieties., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hua Cai reports financial support was provided by National Natural Science Foundation of China with grant number (32071866). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

As emerging persistent pollutants, microplastic (MPs) pollution attracted increasing attention worldwide since it is posing several environmental concerns. MPs interact with heavy metals in soil and may provoke damages on soil properties and ultimately impaired plants and human health. The present study aims to evaluate alfalfa plants (Medicago sativa) response after exposure to heavy metal polluted soils from mine site in the North of Tunisia in presence of environmental microplastic. For that, soils were sampled from two sites of Jebel Ressass mine in addition to a control soil. Plants were exposed to the three soils in presence of two increasing rates of microplastics D1 (1 mg/kg of soil) and D2 (100 mg/kg of soil) for 60 days. After harvest, agronomic parameters, chlorophyll content as well as heavy metal accumulation in plants were analyzed. Furthermore, oxidative status was evaluated in terms of malondialdehyde accumulation (MDA), catalase (CAT) activities and glutathion-S-transferase (GST). Overall, our finding highlights that MPs disrupted agronomic parameters and the photosynthetic activities of alfalfa plants. Additionally, our results revealed that the presence of MPs in polluted soils cause an increase on heavy metal accumulation in alfalfa shoots. Biochemical analyses demonstrated that the combined exposure to MPs and heavy metal induced oxidative stress in alfalfa plants by increasing CAT activity and MDA accumulation. The present investigation highlights the ecological risks of microplastics in terrestrial environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Soil salinization poses a serious threat to crop growth. The selection of appropriate soil modifiers and water-soluble fertilizers for saline soils represents a crucial method for enhancing crop yields. The modifiers and medium-element water-soluble fertilizers were prepared using hydrochar derived from rice straw. Two distinct experiments were designed to study the effect of modifiers and water-soluble fertilizers on saline soils. The first experiment, designated as the "Soil Cultivation Experiment" , sought to investigate the impact of various modifiers on soil quality. The second experiment, designated as the "Method of Field Micro-Area Experiment", aimed to assess the influence of water-soluble fertilizers on saline soils. The results showed that the application of modifiers and water-soluble fertilizers significantly enhanced comprehensive soil physical and chemical properties, crop growth, soil enzyme activity, and other key indicators in saline and alkaline soils. The optimal dosage of the modifier was 20 g/kg, which reduced the pH value from 8.62 to 8.21 and the decreased alkalinity by 8.26%. Furthermore, their application effectively boosted nutrient levels, including organic matter, and increased soil enzyme activity. The biomass of alfalfa showed enhancements of 63.01% and 20.87% and the biomass of leymus chinensis increased by 29.39% and 9.02% for the two batches, respectively. Notably, the application of water-soluble fertilizer yielded achieved superior results. This study also provided a theoretical basis for their future application in soda saline-alkali soil., (© 2024. The Author(s).)

As a perennial forage crop, alfalfa (Medicago sativa L.) has been extensively utilized for the vegetation restoration of degraded soil and provides feedstock for forage. Its high usage can be attributed to its high yield potential and the increasing soil organic carbon (SOC) sequestration of alfalfa cultivation. However, the impact of land conversion to alfalfa on SOC content and its underlying drivers remain unclear. We performed a meta-analysis at the global scale to explore the quantified effects of alfalfa cultivation on SOC content and identify its controlling factors. We employed 1699 pairwise data points from 90 publications based on cropland/abandoned land conversion to alfalfa. Globally, cropland (cropland-alfalfa) and abandoned land (abandoned land-alfalfa) conversion to alfalfa enhanced SOC content by 12.1 % and 13.7 %, respectively. Alfalfa exhibited greater SOC content benefits in the surface soils (0-20 cm) with a lower level of initial SOC (<16 g kg -1 ), regardless of the land conversion type. Cropland-alfalfa was observed to increase SOC content with fertilization, irrigation, and conventional tillage in the long term (>5 years). Furthermore, abandoned land-alfalfa enhanced SOC content in the absence of alfalfa biomass removal and for longer cultivation durations (>5 years). Boosted regression tree analyses indicated variations in soil properties (75 % for cropland-alfalfa and 65 % for abandoned land-alfalfa) as the primary factors driving changes in SOC content. The dominant drivers were determined as the soil layer (51.6 %), cultivation duration (13.1 %), and initial SOC (12.9 %) for cropland-alfalfa, and initial SOC (43.7 %), soil layer (24.6 %) and cultivation duration (17.1 %) for abandoned land-alfalfa. Land conversion to alfalfa has great potential for SOC sequestration, particularly in low-fertility soils. Therefore, alfalfa cultivation is highly recommended for degraded lands due to its SOC sequestration benefits in vegetation restoration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Alfalfa ( Medicago sativa subsp. sativa ), the "queen of forage," is the most important perennial legume, with high productivity and an excellent nutritional profile. Medicago sativa subsp. falcata is a subspecies of the alfalfa complex and exhibits better drought tolerance. However, drought stress significantly hampers their development and yield. The molecular mechanisms underlying the aboveground and underground tissues of sativa and falcata responding to drought stress remain obscure. Here, we performed a comprehensive comparative analysis of the physiological and transcriptomic responses of sativa and falcata under drought stress. The results showed that photosynthesis was inhibited, and antioxidant enzymes were activated under drought stress. MsC3H29 , a CCCH-type zinc finger protein, was identified as a hub gene through weighted gene coexpression network analysis (WGCNA) and was significantly induced by drought in underground tissue. The MsC3H29 protein was localized in the nucleus. Overexpression (OE) of MsC3H29 can increase the primary root length and fresh weight of transgenic alfalfa hairy roots, while RNA interference (RNAi) decreases them under drought stress. The 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA) staining revealed that MsC3H29 promoted drought tolerance of alfalfa hairy roots through decreasing ROS accumulation. The targeted metabolome analysis showed that the overexpression of MsC3H29 resulted in higher levels of accumulation for flavonoid monomers, including vicenin, daidzein, apigenin, isorhamnetin, quercetin, and tricin, in transgenic alfalfa hairy roots before and after drought stress, while RNAi led to a reduction. Our study provided a key candidate gene for molecular breeding to improve drought resistance in alfalfa.

The morphological architecture of inflorescence influences seed production. The regulatory mechanisms underlying alfalfa ( Medicago sativa ) inflorescence elongation remain unclear. Therefore, in this study, we conducted a comparative analysis of the transcriptome, proteome, and metabolome of two extreme materials at three developmental stages to explore the mechanisms underlying inflorescence elongation in alfalfa. We observed the developmental processes of long and short inflorescences and found that the elongation capacity of alfalfa with long inflorescence was stronger than that of alfalfa with short inflorescences. Furthermore, integrative analysis of the transcriptome and proteome indicated that the phenylpropanoid biosynthesis pathway was closely correlated with the structural formation of the inflorescence. Additionally, we identified key genes and proteins associated with lignin biosynthesis based on the differential expressed genes and proteins (DEGs and DEPs) involved in phenylpropanoid biosynthesis. Moreover, targeted hormone metabolome analysis revealed that IAA, GA, and CK play an important role in the peduncle elongation of alfalfa inflorescences. Based on omics analysis, we detected key genes and proteins related to plant hormone biosynthesis and signal transduction. From the WGCNA and WPCNA results, we furthermore screened 28 candidate genes and six key proteins that were correlated with lignin biosynthesis, plant hormone biosynthesis, and signaling pathways. In addition, 19 crucial transcription factors were discovered using correlation analysis that might play a role in regulating candidate genes. This study provides insight into the molecular mechanism of inflorescence elongation in alfalfa and establishes a theoretical foundation for improving alfalfa seed production.

Key Message: MsTFL1A is an important gene involved in flowering repression in alfalfa (Medicago sativa) which conditions not only above-ground plant shoot architecture but also root development and growth. Delayed flowering is an important trait for forage species, as it allows harvesting of high-quality forage for a longer time before nutritional values decline due to plant architecture changes related to flowering onset. Despite the relevance of delayed flowering, this trait has not yet been thoroughly exploited in alfalfa. This is mainly due to its complex genetics, sensitivity to inbreeding and to the fact that delayed flowering would be only advantageous if it allowed increased forage quality without compromising seed production. To develop new delayed-flowering varieties, we have characterized the three TERMINAL FLOWERING 1 (TFL1) family of genes in alfalfa: MsTFL1A, MsTFL1B and MsTFL1C. Constitutive expression of MsTFL1A in Arabidopsis caused late flowering and changes in inflorescence architecture, indicating that MsTFL1A is the ortholog of Arabidopsis TFL1. Overexpression of MsTFL1A in alfalfa consistently led to delayed flowering in both controlled and natural field conditions, coupled to an increase in leaf/stem ratio, a common indicator of forage quality. Additionally, overexpression of MsTFL1A reduced root development, reinforcing the role of MsTFL1A not only as a flowering repressor but also as a regulator of root development.We conclude that the precise manipulation of MsTFL1A gene expression may represent a powerful tool to improve alfalfa forage quality., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Saline and alkaline stresses limit plant growth and reduce crop yield. Soil salinization and alkalization seriously threaten the sustainable development of agriculture and the virtuous cycle of ecology. Biofertilizers made from plant growth-promoting rhizobacteria (PGPR) not only enhance plant growth and stress tolerance, but also are environmentally friendly and cost-effective. There have been many studies on the mechanisms underlying PGPRs enhancing plant salt resistance. However, there is limited knowledge about the interaction between PGPR and plants under alkaline-sodic stress. To clarify the mechanisms underlying PGPR's improvement of plants' tolerance to alkaline-sodic stress, we screened PGPR from the rhizosphere microorganisms of local plants growing in alkaline-sodic land and selected an efficient strain, Bacillus altitudinis AD13-4, as the research object. Our results indicate that the strain AD13-4 can produce various growth-promoting substances to regulate plant endogenous hormone levels, cell division and differentiation, photosynthesis, antioxidant capacity, etc. Transcriptome analysis revealed that the strain AD13-4 significantly affected metabolism and secondary metabolism, signal transduction, photosynthesis, redox processes, and plant-pathogen interactions. Under alkaline-sodic conditions, inoculation of the strain AD13-4 significantly improved plant biomass and the contents of metabolites (e.g., soluble proteins and sugars) as well as secondary metabolites (e.g., phenols, flavonoids, and terpenoids). The 16S rRNA gene sequencing results indicated that the strain AD13-4 significantly affected the abundance and composition of the rhizospheric microbiota and improved soil activities and physiochemical properties. Our study provides theoretical support for the optimization of saline-alkali-tolerant PGPR and valuable information for elucidating the mechanism of plant alkaline-sodic tolerance.

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

Shoot branching is an important biological trait affecting alfalfa (Medicago sativa L.) production, but its development is complicated and the mechanism is not fully clear. In the present study, pectin acetylesterase 12 (MsPAE12) and NAM/ATAF/CUC-domain transcription factor gene (MsNAC73) were isolated from alfalfa. MsPAE12 was highly expressed in shoot apexes, and MsNAC73 was found to be a key transcriptional repressor of MsPAE12 by directly binding to salicylic acid (SA) and jasmonic acid (JA) elements in the MsPAE12 promoter. The biological functions of MsPAE12 and MsNAC73 were studied through overexpression (OE) and down-expression (RNAi) of the 2 genes in alfalfa. The numbers of shoot branches increased in MsPAE12-OE lines but decreased in MsPAE12-RNAi and MsNAC73-OE plants, which was negatively related to their indole-3-acetic acid (IAA) accumulation in shoot apexes. Furthermore, the contents of acetic acid (AA) in shoot apexes decreased in MsPAE12-OE plants but increased in MsPAE12-RNAi and MsNAC73-OE plants. The changes of AA contents were positively related to the expression of TRYPTOPHAN AMINOTRANSFERASE 1 (MsTAA1), TRYPTOPHAN AMINOTRANSFERASE-RELATED 2 (MsTAR2), and YUCCA flavin monooxygenase (MsYUCC4) and the contents of tryptophan (Trp), indole-3-pyruvic acid (IPA), and IAA in shoot apexes of MsPAE12-OE, MsPAE12-RNAi, and MsNAC73-OE plants. Exogenous application of AA to wild type (WT) and MsPAE12-OE plants increased Trp, IPA, and IAA contents and decreased branch number. Exogenous IAA suppressed shoot branching in MsPAE12-OE plants, but exogenous IAA inhibitors increased shoot branching in MsPAE12-RNAi plants. These results indicate that the MsNAC73-MsPAE12 module regulates auxin-modulated shoot branching via affecting AA accumulation in shoot apexes of alfalfa., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Biochar is a promising solution to alleviate the negative impacts of salinity stress on agricultural production. Biochar derived from food waste effect was investigated on three plant species, Medicago sativa, Amaranthus caudatus, and Zea mays, under saline environments. The results showed that biochar improved significantly the height by 30%, fresh weight of shoot by 35% and root by 45% of all three species compared to control (saline soil without biochar adding), as well as enhanced their photosynthetic pigments and enzyme activities in soil. This positive effect varied significantly between the 3 plants highlighting the importance of the plant-biochar interactions. Thus, the application of biochar is a promising solution to enhance the growth, root morphology, and physiological characteristics of plants under salt-induced stress., (© 2024. The Author(s).)