Aquaporin-0-protein interactions elucidated by crosslinking mass spectrometry.

Aquaporin-0 (AQP0) constitutes 50 % of the lens membrane proteome and plays important roles in lens fiber cell adhesion, water permeability, and lens transparency. Previous work has shown that specific proteins, such as calmodulin (CaM), interact with AQP0 to modulate its water permeability; however, these studies often used AQP0 peptides, rather than full-length protein, to probe these interactions. Furthermore, the specific regions of interaction of several known AQP0 interacting partners, i.e. αA and αB-crystallins, and phakinin (CP49) remain unknown. The purpose of this study was to use crosslinking mass spectrometry (XL-MS) to identify interacting proteins with full-length AQP0 in crude lens cortical membrane fractions and to determine the specific protein regions of interaction. Our results demonstrate, for the first time, that the AQP0 N-terminus can engage in protein interactions. Specific regions of interaction are elucidated for several AQP0 interacting partners including phakinin, α-crystallin, connexin-46, and connexin-50. In addition, two new interacting partners, vimentin and connexin-46, were identified.

Inhibition of aquaporin-9 ameliorates severe acute pancreatitis and associated lung injury by NLRP3 and Nrf2/HO-1 pathways.

Inflammation, apoptosis and oxidative stress play crucial roles in the deterioration of severe acute pancreatitis-associated acute respiratory distress syndrome (SAP-ARDS). Unfortunately, despite a high mortality rate of 45 %[1], there are limited treatment options available for ARDS outside of last resort options such as mechanical ventilation and extracorporeal support strategies[2]. This study investigated the potential therapeutic role and mechanisms of AQP9 inhibitor RG100204 in two animal models of severe acute pancreatitis, inducing acute respiratory distress syndrome: 1) a sodium-taurocholate induced rat model, and 2) and Cerulein and lipopolysaccharide induced mouse model. RG100204 treatment led to a profound reduction in inflammatory cytokine expression in pancreatic, and lung tissue, in both models. In addition, infiltration of CD68 + and CD11b + cells into these tissues were reduced in RG100204 treated SAP animals, and edema and SAP associated tissue damage were improved. Moreover, we demonstrate that RG100204 reduced apoptosis in the lungs of rat SAP animals, and reduces NF-κB signaling, NLRP3, expression, while profoundly increasing the Nrf2-dependent anti oxidative stress response. We conclude that AQP9 inhibition is a promising strategy for the treatment of pancreatitis and its systemic complications, such as ARDS.

Aquaporin 2 in Cerebral Edema: Potential Prognostic Marker in Craniocerebral Injuries.

Despite continuous medical advancements, traumatic brain injury (TBI) remains a leading cause of death and disability worldwide. Consequently, there is a pursuit for biomarkers that allow non-invasive monitoring of patients after cranial trauma, potentially improving clinical management and reducing complications and mortality. Aquaporins (AQPs), which are crucial for transmembrane water transport, may be significant in this context. This study included 48 patients, with 27 having acute (aSDH) and 21 having chronic subdural hematoma (cSDH). Blood plasma samples were collected from the participants at three intervals: the first sample before surgery, the second at 15 h, and the third at 30 h post-surgery. Plasma concentrations of AQP1, AQP2, AQP4, and AQP9 were determined using the sandwich ELISA technique. CT scans were performed on all patients pre- and post-surgery. Correlations between variables were examined using Spearman's nonparametric rank correlation coefficient. A strong correlation was found between aquaporin 2 levels and the volume of chronic subdural hematoma and midline shift. However, no significant link was found between aquaporin levels (AQP1, AQP2, AQP4, and AQP9) before and after surgery for acute subdural hematoma, nor for AQP1, AQP4, and AQP9 after surgery for chronic subdural hematoma. In the chronic SDH group, AQP2 plasma concentration negatively correlated with the midline shift measured before surgery (Spearman's ρ -0.54; p = 0.017) and positively with hematoma volume change between baseline and 30 h post-surgery (Spearman's ρ 0.627; p = 0.007). No statistically significant correlation was found between aquaporin plasma levels and hematoma volume for AQP1, AQP2, AQP4, and AQP9 in patients with acute SDH. There is a correlation between chronic subdural hematoma volume, measured radiologically, and serum AQP2 concentration, highlighting aquaporins' potential as clinical biomarkers.

Aquaporins: Gatekeepers of Fluid Dynamics in Traumatic Brain Injury.

Aquaporins (AQPs), particularly AQP4, play a crucial role in regulating fluid dynamics in the brain, impacting the development and resolution of edema following traumatic brain injury (TBI). This review examines the alterations in AQP expression and localization post-injury, exploring their effects on brain edema and overall injury outcomes. We discuss the underlying molecular mechanisms regulating AQP expression, highlighting potential therapeutic strategies to modulate AQP function. These insights provide a comprehensive understanding of AQPs in TBI and suggest novel approaches for improving clinical outcomes through targeted interventions.

Peroxiporins in Triple-Negative Breast Cancer: Biomarker Potential and Therapeutic Perspectives.

Triple-negative breast cancer (TNBC) remains one of the most challenging subtypes since it is initially characterized by the absence of specific biomarkers and corresponding targeted therapies. Advances in methodology, translational informatics, genomics, and proteomics have significantly contributed to the identification of therapeutic targets. The development of innovative treatments, such as antibody-drug conjugates and immune checkpoint inhibitors, alongside chemotherapy, has now become the standard of care. However, the quest for biomarkers defining therapy outcomes is still ongoing. Peroxiporins, which comprise a subgroup of aquaporins, which are membrane pores facilitating the transport of water, glycerol, and hydrogen peroxide, have emerged as potential biomarkers for therapy response. Research on peroxiporins reveals their involvement beyond traditional channeling activities, which is also reflected in their cellular localization and roles in cellular signaling pathways. This research on peroxiporins provides fresh insights into the mechanisms of therapy resistance in tumors, offering potential avenues for predicting treatment outcomes and tailoring successful TNBC therapies.

Simultaneous Native Mass Spectrometry Analysis of Single and Double Mutants To Probe Lipid Binding to Membrane Proteins.

Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions because lipids can simultaneously bind membrane proteins at different sites with different specificities. Here, we developed a native mass spectrometry (MS) approach using single and double mutants to measure the relative energetic contributions of specific residues on Aquaporin Z (AqpZ) toward cardiolipin (CL) binding. We first mutated potential lipid-binding residues on AqpZ, and mixed mutant and wild-type proteins together with CL. By using native MS to simultaneously resolve lipid binding to the mutant and wild-type proteins in a single spectrum, we directly determined the relative affinities of CL binding, thereby revealing the relative Gibbs free energy change for lipid binding caused by the mutation. Comparing different mutants revealed that W14 contributes to the tightest CL binding site, with R224 contributing to a lower affinity site. Using double mutant cycling, we investigated the synergy between W14 and R224 sites on CL binding. Overall, this novel native MS approach provides unique insights into the binding of lipids to specific sites on membrane proteins.

Modulation of physio-biochemical and photosynthesis parameters by overexpressing SbPIP2 gene improved abiotic stress tolerance of transgenic tobacco.

The present study aims to explore the potential of a plasma-membrane localized PIP2-type aquaporin protein sourced from the halophyte Salicornia brachiata to alleviate salinity and water deficit stress tolerance in a model plant through transgenic intervention. Transgenic plants overexpressing SbPIP2 gene showed improved physio-biochemical parameters like increased osmolytes (proline, total sugar, and amino acids), antioxidants (polyphenols), pigments and membrane stability under salinity and drought stresses compared to control plants [wild type (WT) and vector control (VC) plants]. Multivariate statistical analysis showed that, under water and salinity stresses, osmolytes, antioxidants and pigments were correlated with SbPIP2-overexpressing (SbPIP2-OE) plants treated with salinity and water deficit stress, suggesting their involvement in stress tolerance. As aquaporins are also involved in CO2 transport, SbPIP2-OE plants showed enhanced photosynthesis performance than wild type upon salinity and drought stresses. Photosynthetic gas exchange (net CO2 assimilation rate, PSII efficiency, ETR, and non-photochemical quenching) were significantly higher in SbPIP2-OE plants compared to control plants (wild type and vector control plants) under both unstressed and stressed conditions. The higher quantum yield for reduction of end electron acceptors at the PSI acceptor side [Φ( R0 )] in SbPIP2-OE plants compared to control plants under abiotic stresses indicates a continued PSI functioning, leading to retained electron transport rate, higher carbon assimilation, and less ROS-mediated injuries. In conclusion, the SbPIP2 gene functionally validated in the present study could be a potential candidate for engineering abiotic stress resilience in important crops.

A bamboo 'PeSAPK4-PeMYB99-PeTIP4-3' regulatory model involved in water transport.

Water plays crucial roles in expeditious growth and osmotic stress of bamboo. Nevertheless, the molecular mechanism of water transport remains unclear. In this study, an aquaporin gene, PeTIP4-3, was identified through a joint analysis of root pressure and transcriptomic data in moso bamboo (Phyllostachys edulis). PeTIP4-3 was highly expressed in shoots, especially in the vascular bundle sheath cells. Overexpression of PeTIP4-3 could increase drought and salt tolerance in transgenic yeast and rice. A co-expression pattern of PeSAPK4, PeMYB99 and PeTIP4-3 was revealed by WGCNA. PeMYB99 exhibited an ability to independently bind to and activate PeTIP4-3, which augmented tolerance to drought and salt stress. PeSAPK4 could interact with and phosphorylate PeMYB99 in vivo and in vitro, wherein they synergistically accelerated PeTIP4-3 transcription. Overexpression of PeMYB99 and PeSAPK4 also conferred drought and salt tolerance in transgenic rice. Further ABA treatment analysis indicated that PeSAPK4 enhanced water transport in response to stress via ABA signaling. Collectively, an ABA-mediated cascade of PeSAPK4-PeMYB99-PeTIP4-3 is proposed, which governs water transport in moso bamboo.

Evaluation of the aquaporin molecules characterization in the sperm cells of men from different aged.

Background/aim: Male infertility rises for many reasons, along with age; therefore, we aimed to research the characterization of aquaporin-3, 7, and 8 in human sperm belonging to different age groups. Material and methods: This study was conducted on sperm samples of men aged over 18 years. A total of 60 men were included in the study and divided into three age groups: group 1, age 18-25 years (n = 20); group 2, age 26-35 years (n = 20); and group 3, age ≥35 years (n = 20). Sperm ejaculates obtained from each participant were used for spermiogram tests, Kruger strict morphology analysis, and immunohistochemistry. Results: We observed no statistically significant differences in terms of macroscopic and microscopic sperm testing. The immunostaining score of aquaporin-3 was the lowest in group 1 and increased in group 3 and group 2, respectively (p < 0.05). Aquaporin-8 immunostaining only increased in group 2 (p < 0.05). Aquaporin-7 immunostaining scores were not different between the groups (p > 0.05). When the immunostaining scores of aquaporin molecules were compared with each other, aquaporin-7 was significantly increased compared with the others (p < 0.05). Conclusion: According to the results, it can be stated that aquaporin-3 and aquaporin-8 molecules were more expressed at age 26 to 35 years, and aquaporin-7 was densely expressed from age 18 to 25 years. If the characterization of these molecules is adversely affected, male infertility may eventually emerge. We recommend further advanced-level studies on this subject.

Aquaporins in colorectal cancer: exploring their role in tumorigenesis, metastasis, and drug response.

Aquaporins (AQPs) are small, integral proteins facilitating water transport across plasma cell membranes in response to osmotic gradients. This family has 13 unique members (AQP0-12), which can also transport glycerol, urea, gases, and other salute small molecules. AQPs play a crucial role in the regulation of different cellular processes, including metabolism, migration, immunity, barrier function, and angiogenesis. These proteins are found to aberrantly overexpress in various cancers, including colorectal cancer (CRC). Growing evidence has explored AQPs as a potential diagnostic biomarker and therapeutic target in different cancers. However, there is no comprehensive review compiling the available information on the crucial role of AQPs in the context of colorectal cancer. This review highlights the significance of AQPs as the biomarker and regulator of tumor cells metabolism. In addition, the proliferation, angiogenesis, and metastasis of tumor cells related to AQPs expression as well as function are discussed. Understanding the AQPs prominent role in chemotherapy resistance is of great importance clinically.

Fast collective motions of backbone in transmembrane α helices are critical to water transfer of aquaporin.

Fast collective motions are widely present in biomolecules, but their functional relevance remains unclear. Herein, we reveal that fast collective motions of backbone are critical to the water transfer of aquaporin Z (AqpZ) by using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. A total of 212 residue site-specific dipolar order parameters and 158 15N spin relaxation rates of the backbone are measured by combining the 13C- and 1H-detected multidimensional ssNMR spectra. Analysis of these experimental data by theoretic models suggests that the small-amplitude (~10°) collective motions of the transmembrane α helices on the nanosecond-to-microsecond timescales are dominant for the dynamics of AqpZ. The MD simulations demonstrate that these collective motions are critical to the water transfer efficiency of AqpZ by facilitating the opening of the channel and accelerating the water-residue hydrogen bonds renewing in the selectivity filter region.

Aquaporin expression in cryopreserved human sperm: exploring the capabilities of natural deep eutectic solvents (NADEs).

BACKGROUND: Aquaporins (AQPs) are essential proteins that facilitate the rapid movement of water and cryoprotective agents (CPAs) during the cryopreservation process, and ensure the cryo-tolerance of sperm cells. OBJECTIVE: This study evaluated the preservation of aquaporin levels in human sperm after undergoing freezing using natural deep eutectic solvents (NADES) as CPAs for cryoprotection. MATERIALS AND METHODS: From June 2021 to October 2022, 35 semen samples with normal sperm parameters were acquired from the Mehr Infertility Treatment Institute in Rasht, Iran. The samples were divided into several groups for analysis: control group (not frozen), group frozen with SpermFreeze Solution, and groups frozen with different NADESs, including ChS, ChX, ChU, ChG, GlyP, and EtP. After thawing, various aspects for each group were assessed, including the integrity and condensation of sperm chromatin, viability, motility, integrity of acrosome, and the expression of AQP1, AQP3, AQP7, AQP8, and AQP9 genes. RESULTS: The analysis of gene expression revealed that freezing with ChS and GlyP preserved the expression of the AQP1 and AQP3 genes compared to the control group. Regarding AQP7 and AQP8, significant differences were not observed in expression levels between certain NADES groups (e.g., ChS, ChU, and GlyP) and the control group. Additionally, samples frozen with specific NADESs, such as ChS, ChG, EtP, and GlyP, exhibited preserved levels of AQP9 expression when compared to the control group. CONCLUSION: These findings emphasize the importance of NADES in preserving the expression of aquaporins in cryopreserved human sperm and their important fertility parameters. Doi.org/10.54680/fr24310110512.

AQP8 Modulates Mitochondrial H2O2 Transport to Influence Glioma Proliferation.

BACKGROUND: Aquaporin-8 (AQP8) is involved in impacting glioma proliferation and can effect tumour growth by regulating Intracellular reactive oxygen species (ROS) signalling levels. In addition to transporting H2O2, AQP8 has been shown to affect ROS signaling, but evidence is lacking in gliomas. In this study, we aimed to investigate how AQP8 affects ROS signaling in gliomas. MATERIALS AND METHODS: We constructed A172 and U251 cell lines with AQP8 knockdown and AQP8 rescue by CRISPR/Cas9 technology and overexpression of lentiviral vectors. We used CCK-8 and flow cytometry to test cell proliferation and cycle, immunofluorescence and Mito-Tracker CMXRos to observe the distribution of AQP8 expression in glioma cells, Amplex and DHE to study mitochondria release of H2O2, mitochondrial membrane potential (MMP) and NAD+/NADH ratio to assess mitochondrial function and protein blotting to detect p53 and p21 expression. RESULT: We found that AQP8 co-localised with mitochondria and that knockdown of AQP8 inhibited the release of H2O2 from mitochondria and led to increased levels of ROS in mitochondria, thereby impairing mitochondrial function. We also discovered that AQP8 knockdown resulted in suppression of cell proliferation and was blocked at the G0/G1 phase with increased expression of mitochondrial ROS signalling-related p53/p21. CONCLUSIONS: This finding provides further evidence for mechanistic studies of AQP8 as a prospective target for the treatment of gliomas.

Multi-hormonal analysis and aquaporins regulation reveal new insights on drought tolerance in grapevine.

Disentangling the factors that foster the tolerance to water stress in plants could provide great benefits to crop productions. In a two-year experiment, two new PIWI (fungus resistant) grapevine varieties, namely Merlot Kanthus and Sauvignon Kretos (Vitis hybrids), grown in the field, were subjected to two different water regimes: weekly irrigated (IR) or not irrigated (NIR) for two months during the summer. The two varieties exhibited large differences in terms of performance under water-limiting conditions. In particular, Merlot Kanthus strongly decreased stem water potential (Ψs) under water shortage and Sauvignon Kretos maintained higher Ψs values accompanied by generally high stomatal conductance and net carbon assimilation, regardless of the treatment. We hypothesized differences in the hormonal profile that mediate most of the plant responses to stresses or in the regulation of the aquaporins that control the water transport in the leaves. In general, substantial differences were found in the abundance of different hormonal classes, with Merlot Kanthus reporting higher concentrations of cytokinins while Sauvignon Kretos higher concentrations of auxins, jasmonate and salicylic acid. Interestingly, under water stress conditions ABA modulation appeared similar between the two cultivars, while other hormones were differently modulated between the two varieties. Regarding the expression of aquaporin encoding genes, Merlot Kanthus showed a significant downregulation of VvPIP2;1 and VvTIP2;1 in leaves exposed to water stress. Both genes have probably a role in influencing leaf conductance, and VvTIP2;1 has been correlated with stomatal conductance values. This evidence suggests that the two PIWI varieties are characterized by different behaviour in response to drought. Furthermore, the findings of the study may be generalized, suggesting the involvement of a complex hormonal cross-talk and aquaporins in effectively influencing plant performance under water shortage.

White matter hyperintensities and the surrounding normal appearing white matter are associated with water channel disruption in the oldest old.

INTRODUCTION: Age-related magnetic resonance imaging (MRI) T2 white matter hyperintensities (WMHs) are common and associated with neurological decline. We investigated the histopathological underpinnings of MRI WMH and surrounding normal appearing white matter (NAWM), with a focus on astroglial phenotypes. METHODS: Brain samples from 51 oldest old Oregon Alzheimer's Disease Research Center participants who came to autopsy underwent post mortem (PM) 7 tesla MRI with targeted histopathological sampling of WMHs and NAWM. Stained slides were digitized and quantified. Mixed-effects models determined differences in molecular characteristics between WMHs and the NAWM and across NAWM. RESULTS: PM MRI-targeted WMHs are characterized by demyelination, microglial activation, and prominent astrocytic alterations, including disrupted aquaporin (AQP) expression. Similar changes occur within the surrounding NAWM in a pattern of decreasing severity with increased distance from WMHs. DISCUSSION: Decreased AQP expression within WMH and proximal NAWM suggest an overwhelmed system wherein water homeostasis is no longer maintained, contributing to WM damage in older individuals. HIGHLIGHTS: Post mortem magnetic resonance imaging (MRI) was used to characterize the pathology of white matter hyperintensities (WMHs) and surrounding normal appearing white matter (NAWM). Stained immunohistochemical (IHC) slides from targeted WMH and NAWM samples were digitized and quantified. WMHs and NAWM were associated with inflammation, demyelination, and gliosis. WMHs and NAWM astrocytic changes included decreased AQP1 and AQP4 expression. Abnormal NAWM pathology diminished in severity with increasing distance from WMH.

Targeting OsNIP3;1 via CRISPR/Cas9: A strategy for minimizing arsenic accumulation and boosting rice resilience.

Arsenic (As) contamination in rice poses a significant threat to human health due to its toxicity and widespread consumption. Identifying and manipulating key genes governing As accumulation in rice is crucial for reducing this threat. The large NIP gene family of aquaporins in rice presents a promising target due to functional redundancy, potentially allowing for gene manipulation without compromising plant growth. This study aimed to utilize genome editing to generate knock-out (KO) lines of genes of NIP family (OsLsi1, OsNIP3;1) and an anion transporter family (OsLsi2), in order to assess their impact on As accumulation and stress tolerance in rice. KO lines were created using CRISPR/Cas9 technology, and the As accumulation patterns, physiological performance, and grain yield were compared against wild-type (WT) under As-treated conditions. KO lines exhibited significantly reduced As accumulation in grain compared to WT. Notably, Osnip3;1 KO line displayed reduced As in xylem sap (71-74%) and grain (32-46%) upon treatment. Additionally, these lines demonstrated improved silicon (23%) uptake, photosynthetic pigment concentrations (Chl a: 77%; Chl b: 79%, Total Chl: 79% & Carotenoid: 49%) overall physiological and agronomical performance under As stress compared to WT. This study successfully utilized genome editing for the first time to identify OsNIP3;1 as a potential target for manipulating As accumulation in rice without compromising grain yield or plant vigor.

Tobacco aquaporin NtAQP1 and human aquaporin hAQP1 contribute to single cell photosynthesis in Synechococcus.

BACKGROUND INFORMATION: Aquaporins are H2O-permeable membrane protein pores. However, some aquaporins are also permeable to other substances such as CO2. In higher plants, overexpression of such aquaporins has already led to an enhanced photosynthetic performance due to improved CO2 mesophyll conductance. In this work, we investigated the effects of such aquaporins on unicellular photosynthetically active organisms, specifically cyanobacteria. RESULTS: Overexpression of aquaporins NtAQP1 or hAQP1 that might have a function to improve CO2 membrane permeability lead to increased photosynthesis rates in the cyanobacterium Synechococcus sp. PCC7002 as concluded by the rate of evolved O2. A shift in the Plastoquinone pool state of the cells supports our findings. Water permeable aquaporins without CO2 permeability, such as NtPIP2;1, do not have this effect. CONCLUSIONS AND SIGNIFICANCE: We conclude that also in single cell organisms like cyanobacteria, membrane CO2 conductivity could be rate limiting and CO2-porins reduce the respective membrane resistance. We could show that besides the tobacco aquaporin NtAQP1 also the human hAQP1 most likely functions as CO2 diffusion facilitator in the photosynthesis assay.

Cartilage protective and anti-edema effects of JTF in osteoarthritis via inhibiting NCOA4-HMGB1-driven ferroptosis and aquaporin dysregulation.

BACKGROUND: Preventing joint edema is crucial in halting osteoarthritis (OA) progression. Growing clinical evidence indicate that Jianpi-Tongluo Formula (JTF) may have a promising anti-edema effect. However, the therapeutic properties of JTF and the underlying mechanisms remains unclear. MATERIALS AND METHODS: An OA rat model was established and employed to evaluate pharmacological effects of JTF in vivo based on dynamic histopathologic assessments and micro-CT observations. Then, OA-related genes and potential targets of JTF were identified through clinical transcriptomic data analysis and "disease gene-drug target" network analysis, which were verified by a series of in vivo experiments. RESULTS: JTF administration effectively reduced pain and joint edema, inhibited matrix degradation, chondrocyte apoptosis, and aquaporin expression in OA rats. Notably, JTF dose-dependently reversed damage-associated molecular patterns and inflammatory factor upregulation. Mechanically, our "disease gene-drug target" network analysis indicated that the NCOA4-HMGB1-GSK3B-AQPs axis, implicated in ferroptosis and aquaporin dysregulation, may be potentially served as a target of JTF against OA. Accordingly, JTF mitigated NCOA4, HMGB1, and GSK3B expression, oxidative stress, and iron metabolism aberrations in OA rats. Furthermore, JTF treatment significantly attenuated the aberrant upregulation of AQP1, AQP3, and AQP4 proteins observed in cartilage tissues of OA rats. CONCLUSION: Our data reveal for the first time that JTF may exert cartilage protective and anti-edema effects in osteoarthritis therapy by inhibiting NCOA4-HMGB1-driven ferroptosis and aquaporin dysregulation.

Aquaporin ZmTIP2;3 Promotes Drought Resistance of Maize through Symbiosis with Arbuscular Mycorrhizal Fungi.

Arbuscular mycorrhizal fungi symbiosis plays important roles in enhancing plant tolerance to biotic and abiotic stresses. Aquaporins have also been linked to improved drought tolerance in plants and the regulation of water transport. However, the mechanisms that underlie this association remain to be further explored. In this study, we found that arbuscular mycorrhiza fungi symbiosis could induce the gene expression of the aquaporin ZmTIP2;3 in maize roots. Moreover, compared with the wild-type plants, the maize zmtip2;3 mutant also showed a lower total biomass, colonization rate, relative water content, and POD and SOD activities after arbuscular mycorrhiza fungi symbiosis under drought stress. qRT-PCR assays revealed reduced expression levels of stress genes including LEA3, P5CS4, and NECD1 in the maize zmtip2;3 mutant. Taken together, these data suggest that ZmTIP2;3 plays an important role in promoting maize tolerance to drought stress during arbuscular mycorrhiza fungi symbiosis.