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.

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.