Arbuscular mycorrhizal fungi (AMF) and dark septate fungi (DSE) were simultaneously colonized in the root cells of maize. Single AMF and DSE symbiosis have been proven to improve the drought tolerance of maize. However, the effects of both fungi coexisting in maize roots under drought stress are not yet known. In this study, pot experiments of maize seedlings were conducted through four inoculation treatments (single AMF inoculation of Rhizophagus irregularis, single DSE inoculation of Exophiala pisciphila, co-inoculation of AMF + DSE and non-mycorrhizal inoculation) under well-watered (WW) and drought-stressed (DS) conditions. AMF and DSE colonization status, maize physiology and aquaporin gene expression in maize roots were investigated. The objective of this paper was to evaluate whether AMF and DSE had competitive, independent or synergistic effects on regulating the drought tolerance of maize. When maize seedlings of three inoculation treatments were subjected to drought stress, single AMF inoculation had the highest shoot and root dry weight, plant height, root length, osmotic root hydraulic conductivity and hydrostatic root hydraulic conductivity in maize seedlings. However, co-inoculation of AMF + DSE induced the highest stomatal conductance in maize leaves and the lowest H₂O₂ and O₂^•− concentration, membrane electrolyte leakage, intercellular CO₂ concentration and gene expression level of ZmPIP1;1, ZmPIP1;2, ZmPIP2;1, ZmPIP2;5 and ZmPIP2;6. In addition, co-inoculation of AMF + DSE also obviously down-regulated the GintAQPF1 and GintAQPF2 expression in R. irregularis compared with single AMF inoculation treatment. Under DS stress, there were competitive relationships between AMF and DSE with regard to regulating mycorrhizal colonization, maize growth, root hydraulic conductivity and the gene expression of aquaporins in R. irregularis, but there were synergistic relationships with regard to regulating membrane electrolyte leakage, oxidative damage, photosynthesis and the aquaporin gene expression of maize seedlings. The obtained results improve our knowledge about how the mechanisms of AMF and DSE coexist, promoting the drought tolerance of host plants.

With the continuous change of climate, drought stress has emerged as the primary constraint on crop growth, posing a significant threat to the stability of global grain reserves. Arbuscular mycorrhizal fungi (AMF), as a kind of widely distributed root endophytes, enhance the drought tolerance of maize (Zea mays L.) through regulating the physiological and molecular responses. However, comprehensive transcriptome analysis to reveal the molecular mechanism of drought tolerance in the symbiotic process between AMF and maize is still limited. In the potted plant experiment, maizes inoculated with and without arbuscular mycorrhizal fungus Funneliformis mosseae were grown under well-watered (WW) or drought-stressed (DS) conditions. By using RNA-Seq and transcriptome analysis on maize roots and leaves, this work aimed to investigate the differential expressed genes (DEGs) related to the Ca²⁺ signaling pathway induced by AMF symbiosis under drought stress. Our findings indicated that F. mosseae inoculation resulted in a decrease in the net fluxes of Ca²⁺, while simultaneously elevating Ca²⁺ contents in the maize roots and leaves under well-watered or drought-stressed conditions. Notably, 189 DEGs were regulated not only by AMF symbiosis and drought stress, but also exhibited preferential expression in either leaves or roots. The annotation and enrichment of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that most of the DEGs were significantly enriched in Ca²⁺ signaling pathway genes, related to signal transduction, cellular process, and defense response. A high number of DEGs with this function (including calcineurin B-like protein (CBL), CBL-interacting protein kinase (CIPK), mitogen-activated protein kinase (MAPK), and calcium-dependent protein kinase (CDPK) receptor kinases) were upregulated-DEGs or downregulated-DEGs in F. mosseae-inoculated maizes under drought stress. Furthermore, some DEGs belong to transcription factor (TF) families, including bHLH ERF, and, MYB, were speculated to play key roles in improving the drought tolerance of maize. Based on the expression data and co-expression analysis between TF and Ca²⁺ signaling pathway genes, Whirly1 with CBL11, and BRI1-EMS-SUPPRESSOR 1 (BES1) with CBL10, CIPK24, CDPK1, CDPK14, CDPK19, and MAPK9 genes showed significant positive correlations, while B3 domain-containing transcription factors (B3 TFs) with MAPK1 and both CBL9 genes showed significant negative correlations in response to both F. mosseae inoculation and drought stress. The regulation of Ca²⁺ signaling pathways by AMF symbiosis was an important response mechanism of maize to improve their drought resistance. This study provides insightful perspectives on how AMF-induced modulation of gene expression within the Ca²⁺ signaling pathway can enhance the drought tolerance of mycorrhizal maize in the future.

Introduction: Arbuscular mycorrhizal fungi (AMF) have been demonstrated their ability to enhance the arsenic (As) tolerance of host plants, and making the utilization of mycorrhizal plants a promising and practical approach for remediating As-contaminated soils. However, comprehensive transcriptome analysis to reveal the molecular mechanism of As tolerance in the symbiotic process between AMF and host plants is still limited.Methods: In this study, transcriptomic analysis of Gossypium seedlings was conducted with four treatments: non-inoculated Gossypium under non-As stress (CK0), non-inoculated Gossypium under As stress (CK100), F. mosseae-inoculated Gossypium under non-As stress (FM0), and F. mosseae-inoculated Gossypium under As stress (FM100).Results: Our results showed that inoculation with F. mosseae led to a reduction in net fluxes of Ca²⁺, while increasing Ca²⁺ contents in the roots and leaves of Gossypium under the same As level in soil. Notably, 199 and 3129 differentially expressed genes (DEGs) were specially regulated by F. mosseae inoculation under As stress and non-As stress, respectively. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and enrichment analyses, we found that under As stress, F. mosseae inoculation up-regulated a significant number of genes related to the Ca²⁺ signaling pathway genes, involved in cellular process, membrane part, and signal transduction. This suggests a potential role in mitigating As tolerance in Gossypium seedlings. Furthermore, our analysis identified specific DEGs in transcription factor families, including ERF, MYB, NAC, and WRKY, that were upregulated by F. mosseae inoculation. Conversely, MYB and HB-other were down-regulated. The ERF and MYB families exhibited the highest number of up- and down-regulated DEGs, respectively, which were speculated to play an important role in alleviating the As toxicity of Gossypium.Discussion: Our findings provided valuable insights into the molecular theoretical basis of the Ca²⁺ signaling pathway in improving As tolerance of mycorrhizal plants in the future.

Prunella vulgaris, an essential component of traditional Chinese medicine, is suitable for growing in soil with a pH value ranging from 6.5 to 7.5. However, it is primarily cultivated in acidic soil regions of China, where its growth is frequently compromised by acidic stress. Selenium (Se) has been recognized for its potential to enhance stress tolerance in plants. However, its role in acid-stress-induced oxidative stress is not clear. In this study, the effects of varying Se concentrations on the growth and quality of P. vulgaris under acidic stress were investigated. The results showed that acid stress enhanced antioxidant enzyme activities, non-enzymatic antioxidant substances, and osmolyte content, accompanied by an increase in oxidant production and membrane damage. Furthermore, it decreased the photosynthetic capacity, inhibited root and shoot growth, and diminished the yield of P. vulgaris. In contrast, exogenous application of Se, particularly at 5 mg L⁻¹, markedly ameliorated these adverse effects. Compared to acid-stressed plants, 5 mg L⁻¹ Se treatment enhanced superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione peroxidase activities by 150.19%, 54.94%, 43.43%, and 45.55%, respectively. Additionally, soluble protein, soluble sugar, and proline contents increased by 11.75%, 23.32%, and 40.39%, respectively. Se application also improved root architecture and alleviated membrane damage by reducing hydrogen peroxide, superoxide anion, malondialdehyde, and electrolyte leakage levels. Furthermore, it significantly enhanced the photosynthetic capacity by elevating pigment levels, the performance of PSI and PSII, electron transfer, and the coordination of PSI and PSII. Consequently, plant growth and spica weight were significantly promoted, with a 12.50% increase in yield. Moreover, Se application upregulated key genes involved in flavonoid and phenolic acid metabolic pathways, leading to elevated levels of total flavonoids, caffeic acid, ferulic acid, rosmarinic acid, and hyperoside by 31.03%, 22.37%, 40.78%, 15.11%, and 20.84%, respectively, compared to acid-stressed plants. In conclusion, exogenous Se effectively alleviated the adverse effects of acid stress by improving the antioxidant system, growth, and photosynthetic capacity under acid stress, thus enhancing the yield and quality of P. vulgaris.

Cotton (Gossypium hirsutum L.) is a promising candidate for an Arsenic (As)-tolerant plant due to its low As accumulation in fibers. The individual arbuscular mycorrhizal fungi (AMF) inoculation or exogenous calcium (Ca²⁺) application is known to enhance heavy metal tolerance in higher plants; however, their synergistic mechanisms in alleviating As stress in cotton remain poorly understood. A three-factor pot experiment was conducted, including two levels of AMF (Funneliformis mosseae C.Walker & A.Schüßler) inoculation (non-inoculated/inoculated), As stress (0/100 mgAs⁵⁺·kg⁻¹soil), and exogenous Ca²⁺ (CaCl₂) application (0/20 mmol·L⁻¹ CaCl₂). AMF inoculation and Ca²⁺ application were investigated for their effects on cotton growth, root morphology, photosynthetic characteristics, osmotic regulators, antioxidant enzyme activities, and ion homeostasis under As stress. Results showed As stress significantly disrupted cotton growth (decreased plant height, shoot and root dry weight) and root morphology (reduced total root length, root area, and root fork number), photosynthetic capacity (reduced Pn, Ci, Fv/Fm, and ΦPSII), osmotic adjustment (decreased proline, soluble sugar and protein), antioxidant defense (inhibited SOD, POD, CAT activities), and K⁺/Ca²⁺ homeostasis (reduced concentration of K⁺ and Ca²⁺, and K⁺/Ca²⁺ ratio). Both AMF inoculation and Ca²⁺ application independently alleviated these adverse effects of As stress. At the same time, AMF symbiosis combined with exogenous Ca²⁺ was better than AMF inoculation or Ca²⁺ application alone in optimizing root architecture, improving stomatal function and photosynthetic efficiency, enhancing osmotic regulator accumulation and antioxidant enzyme activities, and restoring ion balance under As stress. Three-way ANOVA confirmed significant As×AMF×Ca²⁺ interactions on key parameters such as Pn and ΦPSII. In summary, both AMF inoculation and Ca²⁺ application synergistically enhanced cotton As tolerance through regulating growth, root morphology, photosynthetic characteristics, osmotic regulators, antioxidant enzyme activities, and ion homeostasis, demonstrating its potential for sustainable cotton cultivation in As-contaminated soils.

As a species of significant traditional medicinal importance, Prunella vulgaris is severely limited by drought stress, given its high sensitivity to this environmental constraint. 24-epibrassinolide (EBR) has shown promise in enhancing plant stress resilience and secondary metabolite production, yet its efficacy in mitigating drought effects on P. vulgaris requires further elucidation. In this study, foliar application of EBR (0, 0.01, 0.05, 0.1, 0.2 μmol·L⁻¹) was applied to drought-stressed P. vulgaris seedlings (maintained at 60% ± 5% field capacity, FC, for 20 days during the flowering stage; control at 75% ± 5% FC). The results showed that drought inhibited the growth and development of P. vulgaris. Compared with the control group, malondialdehyde, hydrogen peroxide, and superoxide anion increased by 77.82%, 27.47%, and 44.95%, respectively. The total chlorophyll content and the coordination between photosystem I and photosystem II decreased by 42.33% and 46.62%, respectively. Additionally, the net photosynthetic rate and biomass of P. vulgaris significantly decreased by 45.12% and 34.66%, respectively. In contrast, the 0.1 μmol·L⁻¹ EBR significantly enhanced the antioxidant and osmoregulation systems. Compared with drought stress treatment, the activities of SOD, POD, CAT, APX and GPX increased by 10.78%, 45.86%, 48.44%, 40.58% and 63.37%, respectively; soluble sugar, soluble protein and proline contents increased by 53.38%, 29.09% and 45.95%, respectively; and malondialdehyde, hydrogen peroxide and superoxide anion levels decreased by 28.37%, 15.77% and 25.73%, respectively. Total chlorophyll content, photosystem coordination and net photosynthetic rate increased by 55.68%, 43.08% and 45.88%, respectively, along with a significant 42.23% increase in total biomass. Furthermore, EBR upregulated the transcription levels of key phenylpropanoid pathway genes and elevated secondary metabolite contents. The expression of Pv4CL, PvC4H, PvPAL and PvTAT increased by 26.97%, 90.42%, 35.52% and 84.35%, respectively. Accordingly, total phenolic content, caffeic acid, ferulic acid, rosmarinic acid and hyperoside increased by 36.44%, 121.01%, 100.27%, 72.38% and 80.77%, respectively. Lower EBR concentrations (0.01 μmol·L⁻¹) had no significant effect on most indices, while 0.2 μmol L⁻¹ EBR showed weakened effects. In summary, under 60% ± 5% field capacity (FC) drought, 2,4-epibrassinolide (EBR) enhances drought adaptation, medicinal yield, and quality of P. vulgaris, with 0.1 μmol L⁻¹ EBR as the optimal concentration. This improvement is driven by enhanced antioxidant capacity, optimized photosynthesis, promoted root–shoot growth, and activated biosynthesis of medicinal compounds.