Piriformospora indica alleviates soda saline-alkaline stress in Glycine max by modulating plant metabolism.

Soil salinization is one of the major factors limiting agricultural production. Utilizing beneficial microorganisms like Piriformospora indica (P. indica) to enhance plant tolerance to abiotic stresses is a highly effective method, but the influence of P. indica on the growth of soybean in natural saline-alkaline soil remains unclear. Therefore, we investigated the effects of non-inoculation, P. indica inoculation, and fertilization on the growth, antioxidant defense, osmotic adjustment, and photosynthetic gas exchange parameters of soybean under two different levels of saline-alkaline stress in non-sterilized natural saline-alkaline soil. The study found that: 1) P. indica inoculation significantly promoted soybean growth, increasing plant height, root length, and biomass. Under mildly saline-alkaline stress, the increases were 11.5%, 16.0%, and 14.8%, respectively, compared to non-inoculated treatment. Under higher stress, P. indica inoculation achieved the same level of biomass increase as fertilization, while fertilization only significantly improved stem diameter. 2) Under saline-alkaline stress, P. indica inoculation significantly increased antioxidant enzyme activities and reduced malondialdehyde (MDA) content. Under mildly stress, MDA content was reduced by 47.1% and 43.3% compared to non-inoculated and fertilized treatments, respectively. Under moderate stress, the MDA content in the inoculated group was reduced by 29.9% and 36.6% compared to non-inoculated and fertilized treatments, respectively. Fertilization only had a positive effect on peroxidase (POD) activity. 3) P. indica inoculation induced plants to produce more osmotic adjustment substances. Under mildly stress, proline, soluble sugars, and soluble proteins were increased by 345.7%, 104.4%, and 6.9%, respectively, compared to non-inoculated treatment. Under higher stress, the increases were 75.4%, 179.7%, and 12.6%, respectively. Fertilization had no significant positive effect on proline content. 4) With increasing stress, soybean photosynthetic capacity in the P. indica-inoculated treatment was significantly higher than in the non-inoculated treatment, with net photosynthetic rate increased by 14.8% and 37.0% under different stress levels. These results indicate that P. indica can enhance soybean's adaptive ability to saline-alkaline stress by regulating ROS scavenging capacity, osmotic adjustment substance content, and photosynthetic capacity, thereby promoting plant growth. This suggests that P. indica has great potential in improving soybean productivity in natural saline-alkaline soils.

Insights into the ameliorative effect of ZnONPs on arsenic toxicity in soybean mediated by hormonal regulation, transporter modulation, and stress responsive genes.

Arsenic (As) contamination of agricultural soils poses a serious threat to crop productivity and food safety. Zinc oxide nanoparticles (ZnONPs) have emerged as a potential amendment for mitigating the adverse effects of As stress in plants. Soybean crop is mostly grown on marginalized land and is known for high accumulation of As in roots than others tissue. Therefore, this study aimed to elucidate the underlying mechanisms of ZnONPs in ameliorating arsenic toxicity in soybean. Our results demonstrated that ZnOB significantly improved the growth performance of soybean plants exposed to arsenic. This improvement was accompanied by a decrease (55%) in As accumulation and an increase in photosynthetic efficiency. ZnOB also modulated hormonal balance, with a significant increase in auxin (149%), abscisic acid (118%), gibberellin (160%) and jasmonic acid content (92%) under As(V) stress assuring that ZnONPs may enhance root growth and development by regulating hormonal signaling. We then conducted a transcriptomic analysis to understand further the molecular mechanisms underlying the NPs-induced As(V) tolerance. This analysis identified genes differentially expressed in response to ZnONPs supplementation, including those involved in auxin, abscisic acid, gibberellin, and jasmonic acid biosynthesis and signaling pathways. Weighted gene co-expression network analysis identified 37 potential hub genes encoding stress responders, transporters, and signal transducers across six modules potentially facilitated the efflux of arsenic from cells, reducing its toxicity. Our study provides valuable insights into the molecular mechanisms associated with metalloid tolerance in soybean and offers new avenues for improving As tolerance in contaminated soils.

How a Long-Term Cover Crop Cultivation Impacts Soil Phosphorus Availability in a No-Tillage System?

The growth of cover crops can contribute to the increase in phosphorus content at depth by root decomposition. The aim of this work was to verify the effect of cover crops on soil phosphorus availability and use by successive plants, and the accumulation of soil P in a no-tillage system conducted for 14 years. This research was carried out during the 2016/2017 and 2017/2018 crop seasons, whose treatments have been installed and maintained since 2003. The experimental design was a randomized block design, and the plots consisted of spring crops: pearl millet, forage sorghum, sunn hemp, and additionally, a fallow/chiseling area. The evaluation of available P was determined by P fractionation. In general, in the two years of evaluation, the accumulation of P in the shoot dry matter was higher in sunn hemp growth, on average 25% higher than pearl millet in 2016 and 40% higher than sorghum in 2017. The highest contents of labile inorganic P were in the sorghum-soybean and fallow/chiseling-soybean successions, with values higher than 50 mg kg-1 of P in the 0-0.1 m soil layer. However, in the other layers analyzed, the cover crops obtained higher availability of labile inorganic P. The systems using cover crops recovered 100% of the P fertilized in soybean.

GmIRT1.1 from soybean (Glycine max L.) is involved in transporting Fe, Mn and Cd.

Soybean is one of the most important crops for producing high quality oil and protein. Mineral nutrient deficiencies are frequently observed in soybeans. However, there are few studies to understand the absorption process of mineral nutrients in soybeans. Here, we investigated the functions of soybean (Glycine max L.) IRT1.1 (IRON-REGULATED TRANSPORTER 1.1) in the transportation of mineral elements. Heterologous expression of GmIRT1.1 in yeast mutants revealed that GmIRT1.1 compensated for the growth defects of Δfet3fet4 and Δsmf1 mutants under iron (Fe) and manganese (Mn) deficiency conditions, respectively, and enhanced the sensitivity of the Δycf1 mutant to cadmium (Cd) toxicity. Expression analysis revealed that GmIRT1.1 was only significantly induced by Fe deficiency and was primarily expressed in roots. Furthermore, the GmIRT1.1 overexpression lines enhanced Arabidopsis tolerance to Fe deficiency, leading to increased accumulation of Fe in the roots and shoots. Additionally, the transgenic lines increased the sensitivity to Mn and Cd toxicity. Subcellular localization analysis revealed that GmIRT1.1 was localized on the plasma membrane. Moreover, the results obtained from the soybean hairy roots system indicated that the localization of GmIRT1.1 was dependent on the regulation of Fe homeostasis in plant. Consequently, these results suggested that GmIRT1.1 was responsible for the transportation of Fe, Mn and Cd.

Artificial neural networks as a tool for seasonal forecast of attack intensity of Spodoptera spp. in Bt soybean.

Soybean (Glycine max) is the world's most cultivated legume; currently, most of its varieties are Bt. Spodoptera spp. (Lepidoptera: Noctuidae) are important pests of soybean. An artificial neural network (ANN) is an artificial intelligence tool that can be used in the study of spatiotemporal dynamics of pest populations. Thus, this work aims to determine ANN to identify population regulation factors of Spodoptera spp. and predict its density in Bt soybean. For two years, the density of Spodoptera spp. caterpillars, predators, and parasitoids, climate data, and plant age was evaluated in commercial soybean fields. The selected ANN was the one with the weather data from 25 days before the pest's density evaluation. ANN forecasting and pest densities in soybean fields presented a correlation of 0.863. It was found that higher densities of the pest occurred in dry seasons, with less wind, higher atmospheric pressure and with increasing plant age. Pest density increased with the increase in temperature until this curve reached its maximum value. ANN forecasting and pest densities in soybean fields in different years, seasons, and stages of plant development were similar. Therefore, this ANN is promising to be implemented into integrated pest management programs in soybean fields.

Occurrence and distribution of phytic acid and its degradation products in soybeans in China: Analytical challenges.

Phytic acid (IP6) and its degradation products lower myo-inositol phosphates exert different impacts on nutrient bioavailability and product quality characteristics. However, information regarding the occurrence of IP6 and its degradation products is scarce. In this work, simultaneous determination of IP6 and its degradation products in soybeans was developed, with emphasis on analysis by UPLC-MS/MS and a BEH Amide column both with hybrid surface technology. The retention and analyte/metal surface interactions issues were effectively addressed without ion-pairing reagents addition or derivatization. This method was applied to analyze soybeans from China. Total contents were 0.44-13.2 mg/g, and IP6 and its degradation product myo-inositol pentakisphosphate (IP5) were the predominant analytes, accounting for over 99%. Accession type significantly affected IP5 content, and landraces had significantly higher IP5 than cultivars. Geographically, the lowest IP6 was concentrated in the Huanghuaihai region. Significant correlations existed between IP6 and longitude, altitude, and annual cumulative sunshine hours. This study provides comprehensive insights into the IP6 and its degradation product profile in soybeans, which will benefit breeding soybeans based on specific requirements.

Non-CG DNA hypomethylation promotes photosynthesis and nitrogen fixation in soybean.

Non-CG DNA methylation, a plant-specific epigenetic mark mainly regulated by chromomethylase (CMT), is known to play important roles in Arabidopsis thaliana. However, whether and to what extent non-CG DNA methylation modulates agronomic traits in crops remain to be explored. Here, we describe the consequences of non-CG DNA hypomethylation on development, seed composition, and yield in soybean (Glycine max). We created a Gmcmt mutant line lacking function of all four CMT genes. This line exhibited substantial hypomethylation of non-CG (CHG and CHH) sites. Non-CG hypomethylation enhanced chromatin accessibility and promoted or repressed the expression of hundreds of functionally relevant genes, including upregulation of GOLDEN-LIKE 10 (GmGLK10), which led to enhanced photosynthesis and, unexpectedly, improved nitrogen fixation efficiency. The Gmcmt line produced larger seeds with increased protein content. This study provides insights into the mechanisms of non-CG methylation-based epigenetic regulation of soybean development and suggests viable epigenetic strategies for improving soybean yield and nutritional value.

Long-Term Effect of Crop Succession Systems on Soil Chemical and Physical Attributes and Soybean Yield.

Most soybean producers in the Cerrado biome use the direct seeding system, making it essential to cultivate cash or cover crops in the off-season, to promote soil protection, as well as increase organic matter, which is directly related to improvements in the chemical and physical characteristics of these soils. In this sense, this work was conducted in Jataí, state of Goias, Brazil, to evaluate the physical-chemical attributes of the soil and the performance of soybeans cultivated in different crop succession systems cultivated for 6 years in the region of Jataí, GO. The experimental design was randomized blocks with four plots and four replications; the crops that followed soybeans were arranged as follows: T1-corn (Zea mays); T2-pearl millet (Pennisetum glaucum); T3-Urochloa ruziziensis; and T4-corn + Urochloa ruziziensis. Soybean yield components and grain yield were evaluated in two harvests (2020/2021 and 2021/2022). Deformed and undisturbed soil samples were collected in 2022 to assess soil fertility and for physical analysis. The data were subjected to analysis of variance (F test) and the means were compared using the Tukey test at 5% probability. The soybean-millet succession system stood out for the chemical and physical attributes of the soil: calcium, magnesium, base saturation, hydrogen + aluminum, and total porosity. The crop succession system did not affect yield for the two years analyzed, but the accumulated grain yields were higher in the crop succession soybean/corn intercropped. The results highlight the importance of using cover crops in improving the physical and chemical qualities of the soil in the long term. However, in the Cerrado, there is a predominance of the soybean/corn succession system motivated by financial issues to the detriment of the qualitative aspects of the soil, in which the introduction of Urochloa ruziziensis in intercropping with corn would improve the chemical attributes of the soil and have a long-term impact on the accumulated grain production.

Comparative quantitative phosphoproteomic and parallel reaction monitoring analysis of soybean roots under aluminum stress identify candidate phosphoproteins involved in aluminum resistance capacity.

Aluminum (Al) toxicity adversely impacts soybean (Glycine max) growth in acidic soil. Reversible protein phosphorylation plays an important role in adapting to adverse environmental conditions by regulating multiple physiological processes including signal transduction, energy coupling and metabolism adjustment in higher plant. This study aimed to reveal the Al-responsive phosphoproteins to understand their putative function and involvement in the regulation of Al resistance in soybean root. We used immobilized metal affinity chromatography to enrich the key phosphoproteins from soybean root apices at 0, 4, or 24 h Al exposure. These phosphoproteins were detected using liquid chromatography-tandem mass spectrometry measurement, verified by parallel reaction monitoring (PRM), and functionally characterized via overexpression in soybean hairy roots. A total of 638 and 686 phosphoproteins were identified as differentially enriched between the 4-h and 0-h, and the 24-h and 0-h Al treatment comparison groups, respectively. Typically, the phosphoproteins involved in biological processes including cell wall modification, and RNA and protein metabolic regulation displayed patterns of decreasing enrichment (clusters 3, 5 and 6), however, the phosphoproteins involved in the transport and metabolic processes of various substrates, and signal transduction pathways showed increased enrichment after 24 h of Al treatment. The enrichment of phosphoproteins in organelle organization bottomed after 4 h of Al treatment (cluster 1). Next, we selected 26 phosphoproteins from the phosphoproteomic profiles, assessed their enrichment status using PRM, and detected enrichment patterns similar to those observed via phosphoproteomic analysis. Among them, 15 phosphoproteins were found to reduce the accumulation of Al and callose in Al-stressed soybean root apices when their corresponding genes were individually overexpressed in soybean hairy roots. In summary, the findings of this study facilitated a comprehensive understanding of the protein phosphorylation events involved in Al resistance responses and revealed some critical phosphoproteins that enhance Al resistance in soybean roots.

CRISPR/Cas genome editing in soybean: challenges and new insights to overcome existing bottlenecks.

BACKGROUND: Soybean is a worldwide-cultivated crop due to its applications in the food, feed, and biodiesel industries. Genome editing in soybean began with ZFN and TALEN technologies; however, CRISPR/Cas has emerged and shortly became the preferable approach for soybean genome manipulation since it is more precise, easy to handle, and cost-effective. Recent reports have focused on the conventional Cas9 nuclease, Cas9 nickase (nCas9) derived base editors, and Cas12a (formally Cpf1) as the most commonly used genome editors in soybean. Nonetheless, several challenges in the complex plant genetic engineering pipeline need to be overcome to effectively edit the genome of an elite soybean cultivar. These challenges include (1) optimizing CRISPR cassette design (i.e., gRNA and Cas promoters, gRNA design and testing, number of gRNAs, and binary vector), (2) improving transformation frequency, (3) increasing the editing efficiency ratio of targeted plant cells, and (4) improving soybean crop production. AIM OF REVIEW: This review provides an overview of soybean genome editing using CRISPR/Cas technology, discusses current challenges, and highlights theoretical (insights) and practical suggestions to overcome the existing bottlenecks. KEY SCIENTIFIC CONCEPTS OF REVIEW: The CRISPR/Cas system was discovered as part of the bacterial innate immune system. It has been used as a biotechnological tool for genome editing and efficiently applied in soybean to unveil gene function, improve agronomic traits such as yield and nutritional grain quality, and enhance biotic and abiotic stress tolerance. To date, the efficiency of gRNAs has been validated using protoplasts and hairy root assays, while stable plant transformation relies on Agrobacterium-mediated and particle bombardment methods. Nevertheless, most steps of the CRISPR/Cas workflow require optimizations to achieve a more effective genome editing in soybean plants.

Rhizobia cystathionine γ-lyase-derived H2S delays nodule senescence in soybean.

Hydrogen sulphide (H2S) is required for optimal establishment of soybean (Glycine max)-Sinorhizobium fredii symbiotic interaction, yet its role in regulating the nitrogen fixation-senescence transition remains poorly understood. A S. fredii cystathionine γ-lyase (CSE) mutant deficient in H2S synthesis showed early nodule senescence characterized by reduced nitrogenase activity, structural changes in nodule cells, and accelerated bacteroid death. In parallel, the CSE mutant facilitated the generation of reactive oxygen species (ROS) and elicited antioxidant responses. We observed that H2S-mediated persulfidation of cysteine C31/C80 in ascorbate peroxidase (APX) and C32 in APX2 modulated enzyme activity, thereby participating in hydrogen peroxide (H2O2) detoxification and delaying nodule senescence. Comparative transcriptomic analysis revealed a significant up-regulation of GmMYB128, an MYB transcription factor (TF), in the CSE mutant nodules. Functional analysis through overexpression and RNAi lines of GmMYB128 demonstrated its role as a positive regulator in nodule senescence. MYB128-OE inoculated with the CSE mutant strain exhibited a reduction in nitrogenase activity and a significant increase in DD15 expression, both of which were mitigated by NaHS addition. Changes at the protein level encompassed the activation of plant defenses alongside turnover in carbohydrates and amino acids. Our results suggest that H2S plays an important role in maintaining efficient symbiosis and preventing premature senescence of soybean nodules.

First report of Colletotrichum chlorophyti causing soybean anthracnose in Brazil.

Soybean [Glycine max (L.) Merr.] is one of the world's five major food crops, and Brazil produces the highest share at around 42%. Anthracnose caused by Colletotrichum is an important limiting factor to soybean production. In November 2013, anthracnose symptoms, characterized by brown irregular-shaped lesions on petioles, stems, and pods were observed in soybean fields (1% of incidence) in Vera, Mato Grosso State, Brazil. From the five plants gathered in the field, three leaves along with their corresponding petioles were meticulously chosen for the removal of symptomatic tissues. Sampling of these tissues involved carefully cutting a 0.5 × 0.5 cm fragment in the lesion area. The fragments were disinfected with 70% ethanol for 1 min, followed by 1% sodium hypochlorite for 2 min. Then the fragments were rinsed three times in sterile distilled water, placed on water-agar, and incubated at 25 °C for four days, in a 12/12 h photoperiod. Hyphal tips were transferred to potato dextrose agar (PDA) plates and incubated as previously described for seven days. A Colletotrichum sp. single-spore isolate (LFN0461) was selected, grown, preserved in filter paper, and stored at -80 °C. In 2023, it was reactivated for molecular characterization. On PDA, colony showed a rough-like mycelial growth, violaceous-black (front/reverse), with curved-shaped conidia 14.7 - 28.2 × 2.1 - 8.96 µm (average 18.4 × 4.7 µm). The DNA was extracted from 10-day-old mycelium using the cetyltrimethylammonium bromide (CTAB) method. The rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone (HIS3), and ß-tubulin 2 (TUB2) regions were amplified by polymerase chain reaction (PCR), using the primer pairs ITS-1F + ITS-4 (Gardes and Bruns 1993; White et al. 1990), GDF1 + GDR1 (Guerber et al. 2003), CYLH3F + CYLH3R (Crous et al. 2006), and Bt2A + Bt2B (Glass and Donaldson 1995), respectively. The sequences were deposited in the GenBank database (accession numbers: PP209207 - ITS; PP213392 - GAPDH; PP213393 - HIS3; MN688797 - TUB2). The reconstruction of the multilocus phylogenetic tree revealed that the LFN0461 isolate clustered with C. cholorophyti reference strain (IMI 103806) with 99.9% of Bayesian probability. Given the seed-borne nature of soybean anthracnose (Boufleur et al. 2021; Yang et al. 2013), pathogenicity tests were carried out by soybean seeds inoculation. Fifty seeds of NS6220 IPRO (Nidera) cultivar were inoculated by water restriction method, with LFN0461 colonies grown on PDA amended with mannitol (Machado et al. 2004), while 50 seeds were placed on PDA amended with mannitol as negative control. Soybean seeds remained in contact with the inoculum for 48 hours. Subsequently, seeds were sown in 2 L pots (n = 10) containing sterilized substrate, which were placed in a greenhouse at 25 ± 5 ºC. After 10 days, inoculated soybean seedlings exhibited characteristic necrotic lesions on cotyledons and hypocotyls, while negative control plants remained asymptomatic. Colletotrichum chlorophyti was successfully reisolated from the symptomatic tissues. Currently, C. chlorophyti has been reported to cause soybean anthracnose and infect seeds in the United States (Yang et al. 2013, 2012). Although this pathogen has not been reported since our first observation in 2013 in Brazil, many Colletotrichum isolates are misidentified due to reliance on morphology (Boufleur et al. 2021). To our knowledge, this study is the first report of C. chlorophyti causing soybean anthracnose in Brazil, joining a new group of emergent Colletotrichum spp. associated with this disease.

Morphological and Molecular Analysis of Two Mycophagous Nematodes, Aphelenchoides bicaudatus and A. rutgersi (Nematoda: Aphelenchoididae) from Florida Strawberry.

From 2016 to 2021, nematode surveys in Florida strawberry fields revealed several species of foliar nematodes (Aphelenchoides spp.). Aphelenchoides besseyi sensu stricto was detected only in 2016 and 2017 on photosynthetic strawberry leaves/buds, but other not well characterized populations of Aphelenchoides sp. were found on declining/dessicated leaves. Morphological analyses showed that these samples of Aphelenchoides sp. consisted of A. bicaudatus, a species detected in Florida for the first time, and A. rutgersi, a species previously reported in Florida from the citrus rhizosphere. These two species differed from A. besseyi in the shape of their tail terminus: bifurcate in A. bicaudatus; mucronate with a ventral thin mucro in A. rutgersi; and stellate in A. besseyi. One population each of these species was used for morphological and molecular analyses after being reared on Monilinia fructicola. Body and tail length differences were observed among Florida A. bicaudatus and other populations from the Far East and South Africa. Phylogenetic analyses of the rRNA gene sequences showed that Florida A. bicaudatus grouped with those of species from South Korea, Taiwan, and the Netherlands and several other populations listed as Aphelenchoides sp. from Brazil, Costa Rica, and Japan, which were considered as representatives of A. bicaudatus in this study. Similarly, sequences of Florida A. rutgersi grouped with those from environmental samples in Japan and North Carolina, which were listed as Aphelenchoides sp. and were considered as representatives of A. rutgersi in this study. Photosynthetic strawberry leaf samples were free from both A. bicaudatus and A. rutgersi, indicating that these two species did not damage strawberry. They were associated with desiccated leaves and/or propagative stolons, usually infected by fungi, confirming that they are mycetophagous under field conditions in this study. Results of soybean leaf inoculation on moist filter paper containing A. bicaudatus specimens showed that this species could become phytophagous under artificial conditions. Nematodes penetrated the leaf epidermis and migrated into the mesophyll causing leaf tissue discoloration/necrosis, which remained localized within the infested area. Soybean leaf damage was almost negligible, and no nematode reproduction was observed in the inoculated soybean areas.

Analysis of ion transport properties of Glycine max HKT transporters and identifying a regulation of GmHKT1;1 by the non-functional GmHKT1;4.

The HKT transporter plays an important role for plants in response to salt stress, but the transport property of the soybean HKT transporters at the molecular level is still unclear. Here, using Xenopus oocyte as a heterologous expression system and two-electrode voltage-clamp technique, we identified four HKT transporters, GmHKT1;1, GmHKT1;2, GmHKT1;3, and GmHKT1;4, which all belong to type I subfamily, but having distinct ion transport properties. While GmHKT1;1, GmHKT1;2 and GmHKT1;3 function as Na+ transporters, GmHKT1;1 is less selective against K+ than the two other transporters. Astonishingly, GmHKT1;4, which lacks transmembrane segments and has no ion permeability, is significantly expressed, and its gene expression pattern is different from the other three GmHKTs under salt stress. Interestingly, GmHKT1;4 reduced the Na+/K+ currents mediated by GmHKT1;1. Further study showed that the transport ability of GmHKT1;1 regulated by GmHKT1;4 was related to the structural differences in the first intracellular domain and the fourth repeat domain. Overall, we have identified one unique GmHKT member, GmHKT1;4, which modulates the Na+ and K+ transport ability of GmHKT1;1 via direct interaction. Thus, we have revealed a new type of HKTs interaction model for altering their ion transport properties.

Comparative proteomics analysis of root and nodule mitochondria of soybean.

Legumes perform symbiotic nitrogen fixation through rhizobial bacteroids housed in specialised root nodules. The biochemical process is energy-intensive and consumes a huge carbon source to generate sufficient reducing power. To maintain the symbiosis, malate is supplied by legume nodules to bacteroids as their major carbon and energy source in return for ammonium ions and nitrogenous compounds. To sustain the carbon supply to bacteroids, nodule cells undergo drastic reorganisation of carbon metabolism. Here, a comprehensive quantitative comparison of the mitochondrial proteomes between root nodules and uninoculated roots was performed using data-independent acquisition proteomics, revealing the modulations in nodule mitochondrial proteins and pathways in response to carbon reallocation. Corroborated our findings with that from the literature, we believe nodules preferably allocate cytosolic phosphoenolpyruvates towards malate synthesis in lieu of pyruvate synthesis, and nodule mitochondria prefer malate over pyruvate as the primary source of NADH for ATP production. Moreover, the differential regulation of respiratory chain-associated proteins suggests that nodule mitochondria could enhance the efficiencies of complexes I and IV for ATP synthesis. This study highlighted a quantitative proteomic view of the mitochondrial adaptation in soybean nodules.

GmANKTM21 Positively Regulates Drought Tolerance and Enhanced Stomatal Response through the MAPK Signaling Pathway in Soybean.

Drought stress is one of the significant abiotic stresses that limit soybean (Glycine max [L.] Merr.) growth and production. Ankyrin repeat (ANK) proteins, being highly conserved, occupy a pivotal role in diverse biological processes. ANK genes were classified into nine subfamilies according to conserved domains in the soybean genome. However, the function of ANK-TM subfamily proteins (Ankyrin repeat proteins with a transmembrane domain) in the abiotic-stress response to soybean remains poorly understood. In this study, we first demonstrated the subcellular localization of GmANKTM21 in the cell membrane and nucleus. Drought stress-induced mRNA levels of GmANKTM21, which encodes proteins belonging to the ANK-TM subfamily, Transgenic 35S:GmANKTM21 soybean improved drought tolerance at the germination and seedling stages, with higher stomatal closure in soybean, lower water loss, lower malondialdehyde (MDA) content, and less reactive oxygen species (ROS) production compared with the wild-type soybean (Dongnong50). RNA-sequencing (RNA-seq) and RT-qPCR analysis of differentially expressed transcripts in overexpression of GmANKTM21 further identified potential downstream genes, including GmSPK2, GmSPK4, and GmCYP707A1, which showed higher expression in transgenic soybean, than those in wild-type soybean and KEGG enrichment analysis showed that MAPK signaling pathways were mostly enriched in GmANKTM21 overexpressing soybean plants under drought stress conditions. Therefore, we demonstrate that GmANKTM21 plays an important role in tolerance to drought stress in soybeans.

Predict the characteristics of the DI engine with various injection timings by Glycine max oil biofuel using artificial neural networks.

Glycine max oil biofuel (GMOB) is a product of the transesterification of soybean oil. It contains a substantial amount of thermal energy. In this study, the result of varying fuel injection timings on the performance, ignition, and exhaust parameters of a research engine with single-cylinder, four-stroke with direct injection (DI) diesel was experimentally investigated and optimised using artificial neural networks (ANN). The results demonstrated that a 20% fuel blend with 24.5° before top dead centre (b TDC) decreased brake thermal efficiency (BTE), NOx emissions, and exhaust cylinder temperature but improved fuel consumption, carbon dioxide emissions (CDE), and smoke emissions. With 26.5° b TDC, the BTE was found to be approximately 5.0% higher while the fuel consumption was approximately 2.0% lower than with the original injection timing of 24.5° b TDC. At 26.5° b TDC, the NOx emission was approximately 8.6% higher, and the smoke emission was approximately 4.07% lower than at the original injection timing (24.5° b TDC).

Colonization compatibility with Bacillus altitudinis confers soybean seed rot resistance.

The plant microbiome and plant-associated bacteria are known to support plant health, but there are limited studies on seed and seedling microbiome to reveal how seed-associated bacteria may confer disease resistance. In this study, the application of antibiotics on soybean seedlings indicated that seed-associated bacteria were involved in the seed rot resistance against a soil-borne pathogen Calonectria ilicicola, but this resistance cannot be carried to withstand root rot. Using PacBio 16S rRNA gene full-length sequencing and microbiome analyses, 14 amplicon sequence variants (ASVs) including 2 ASVs matching to Bacillus altitudinis were found to be more abundant in the 4 most resistant varieties versus the 4 most susceptible varieties. Culture-dependent isolation obtained two B. altitudinis isolates that both exhibit antagonistic capability against 6 fungal pathogens. Application of B. altitudinis on the most resistant and susceptible soybean varieties revealed different colonization compatibility, and the seed rot resistance was restored in the 5 varieties showing higher bacterial colonization. Moreover, qPCR confirmed the persistence of B. altitudinis on apical shoots till 21 days post-inoculation (dpi), but 9 dpi on roots of the resistant variety TN5. As for the susceptible variety HC, the persistence of B. altitudinis was only detected before 6 dpi on both shoots and roots. The short-term colonization of B. altitudinis on roots may explain the absence of root rot resistance. Collectively, this study advances the insight of B. altitudinis conferring soybean seed rot resistance and highlights the importance of considering bacterial compatibility with plant varieties and colonization persistence on plant tissues.

Growth and Yield Dynamics in Three Japanese Soybean Cultivars with Plant Growth-Promoting Pseudomonas spp. and Bradyrhizobium ottawaense Co-Inoculation.

Co-inoculation of soybeans with Bradyrhizobium and plant growth-promoting bacteria has displayed promise for enhancing plant growth, but concrete evidence of its impact on soybean yields is limited. Therefore, this study assessed the comparative efficacy of two 1-aminocyclopropane-1-carboxylate deaminase-producing Pseudomonas species (OFT2 and OFT5) co-inoculated with Bradyrhizobium ottawaense (SG09) on the growth, physiology, nodulation efficiency, and grain yield of three major Japanese soybean cultivars: Enrei, Fukuyutaka, and Satonohohoemi. The experiments were conducted in a warehouse under natural light conditions. The treatments included the inoculation of SG09, SG09 + OFT2, and SG09 + OFT5. Compared with Bradyrhizobium inoculation alone, co-inoculation led to significant improvements in nodulation efficiency, growth, and physiological performance in the Enrei and Fukuyutaka cultivars, but not in the Satonohohoemi cultivar. Furthermore, co-inoculation significantly boosted the total nitrogen content and ion uptake in the shoots, ultimately leading to a remarkable improvement in the grain yield in the Enrei and Fukuyutaka cultivars. These findings contribute to clarifying the interplay among Bradyrhizobium, Pseudomonas, and the plant host cultivar. Notably, Bradyrhizobium-Pseudomonas co-inoculation represents a potentially effective biofertilization strategy for soybean production, highlighting promising avenues for sustainable agricultural practices.

Diverse bacterial consortia: key drivers of rhizosoil fertility modulating microbiome functions, plant physiology, nutrition, and soybean grain yield.

Soybean cultivation in tropical regions relies on symbioses with nitrogen-fixing Bradyrhizobium and plant growth-promoting bacteria (PGPBs), reducing environmental impacts of N fertilizers and pesticides. We evaluate the effects of soybean inoculation with different bacterial consortia combined with PGPBs or microbial secondary metabolites (MSMs) on rhizosoil chemistry, plant physiology, plant nutrition, grain yield, and rhizosphere microbial functions under field conditions over three growing seasons with four treatments: standard inoculation of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens consortium (SI); SI plus foliar spraying with Bacillus subtilis (SI + Bs); SI plus foliar spraying with Azospirillum brasilense (SI + Az); and SI plus seed application of MSMs enriched in lipo-chitooligosaccharides extracted from B. diazoefficiens and Rhizobium tropici (SI + MSM). Rhizosphere microbial composition, diversity, and function was assessed by metagenomics. The relationships between rhizosoil chemistry, plant nutrition, grain yield, and the abundance of microbial taxa and functions were determined by generalized joint attribute modeling. The bacterial consortia had the most significant impact on rhizosphere soil fertility, which in turn affected the bacterial community, plant physiology, nutrient availability, and production. Cluster analysis identified microbial groups and functions correlated with shifts in rhizosoil chemistry and plant nutrition. Bacterial consortia positively modulated specific genera and functional pathways involved in biosynthesis of plant secondary metabolites, amino acids, lipopolysaccharides, photosynthesis, bacterial secretion systems, and sulfur metabolism. The effects of the bacterial consortia on the soybean holobiont, particularly the rhizomicrobiome and rhizosoil fertility, highlight the importance of selecting appropriate consortia for desired outcomes. These findings have implications for microbial-based agricultural practices that enhance crop productivity, quality, and sustainability.