Metabolomics-Based Analysis on the Effect and Metabolic Response of Mycelia by Sawdust Addition from Hypsizygus marmoreus.

The composition of culture substrate is an important environmental factor that affects the growth and metabolism of Hypsizygus marmoreus, and sawdust is commonly used as the substrate for cultivating mushrooms. However, the influences of sawdust on metabolic level of H. marmoreus in mycelial growth is little reported. In this study, the effect of sawdust addition on mycelial growth rate, morphological characteristics and nutrient content of H. marmoreus was explored, and the metabolic response was analyzed based on LC-MS/MS. The results showed the mycelial growth rates and the number of mycelial clamp connections in sawdust medium A and sawdust medium B were significantly higher than that of the basic medium (Control). The mycelial morphology in sawdust medium A was denser, with higher edge trimness and stronger aerial mycelia. The contents of crude fiber, crude protein and polysaccharide of the mycelia from sawdust medium A increased by 85.15%, 90.65% and 92.61%, respectively, compared to that in the basic medium. A total of 551 metabolites were identified and obtained. The differential accumulated metabolites (DAMs) were mainly amino acids, lipids compounds and carbohydrates. It was speculated that the addition of sawdust played a vital role in promoting the cell division and, thus, the formation of clamp connections in H. marmoreus mycelia. Regarding amino acids, the metabolism of glycine, serine and ABC transporters was active with the increase in sawdust, thereby increasing the protein content. And some valuable bioactive molecules were found, such as docosahexaenoic acid (DHA). This study will lay the foundation for further research on the substance transformation and quality improvement of cultivation substrate for mushrooms.

The Promoting Mechanism of the Sterile Fermentation Filtrate of Serratia odorifera on Hypsizygus marmoreus by Means of Metabolomics Analysis.

Hypsizygus marmoreus has become one of the most popular edible mushrooms due to its high nutritional and economic value. Previous researchers found that Serratia odorifera could promote the growth of H. marmoreus by producing and secreting some of its inducers. However, the specific mechanism of action was still unclear. In this study, we found that the exogenous addition of sterile fermentation filtrate (HZSO-1), quorum sensing (QS) signaling molecules, 3-oxo-C6-HSL, cyclo(Pro-Leu), and cyclo(Tyr-Leu) could significantly promote the growth of H. marmoreus, increase the number of clamp junctions, and the diameter of mycelium (p < 0.05). In addition, non-targeted metabolomic analysis revealed that 706 metabolites were detected in the treated group. Of these, 307 metabolites were significantly different (p < 0.05). Compared with the control, 54 and 86 metabolites were significantly increased and decreased in the HZSO-1 group, respectively (p < 0.05). We speculate that the sterile fermentation filtrate of S. odorifera could mediate the carbohydrate and amino acid metabolism of H. marmoreus by influencing the pentose phosphate pathway (PPP) to increase the energy supply for the growth and development of the mycelium. The above results will further reveal the growth-promoting mechanism of S. odorifera on H. marmoreus.

Analysis and modification of central carbon metabolism in Hypsizygus marmoreus for improving mycelial growth performance and fruiting body yield.

Hypsizygus marmoreus is one of the main industrially cultivated varieties of edible fungi, with a delicious taste and high nutritional value. However, the long harvest period of 130-150 days greatly limits its large-scale expansion. This study aimed to investigate the effects of central carbon metabolism (CCM) on the mycelial growth performance and fruiting body formation of H. marmoreus. Nine edible fungi with different harvest periods were collected and used to evaluate their intracellular carbon metabolic differences in the CCM, which revealed that the imbalanced distribution of intracellular carbon metabolic levels in the CCM of H. marmoreus might be one of the key factors resulting in a slow mycelial growth rate and a long harvest period. Further analysis by three strategies, including metabolomics, adaptation of different carbon sources, and chemical interference, confirmed that low carbon flux into the pentose phosphate pathway (PPP) limited the supply of raw materials, reduced power, and thus influenced the mycelial growth of H. marmoreus. Furthermore, four transformants with increased expression levels of glucose-6-phosphate dehydrogenase (G6PDH), a key rate-limiting enzyme in the PPP of H. marmoreus, were developed and showed more extracellular soluble protein secretion and higher sugar assimilation rates, as well as improved mycelial growth rates in bottle substrate mixtures. Finally, cultivation experiments indicated that the maturation periods of the fruiting body with ~4-5 days in advance and the maximum fruiting body yield of 574.8 g per bag with an increase of 7.4% were achieved by improving the G6PDH expression level of the PPP in H. marmoreus. This study showed that CCM played an important role in the mycelial growth and development of H. marmoreus, which provided new insights for future advancements in cultivating and breeding edible fungi.

A chromosome-scale genome and proteome draft of Tremella fuciformis.

In this study, we first reported a high-quality chromosome-scale genome of Tremella fuciformis using Pacbio HiFi sequencing combining Hi-C technology. According to 21.6 Gb PacBio HiFi reads and 18.1 Gb Hi-C valid reads, we drafted a T. fuciformis genome of 27.38 Mb assigned to 10 chromosomes, with the contig N50 of 2.28 Mb, GC content of 56.51 %, BUSCOs completeness of 93.1 % and consensus quality value of 33.7. The following annotation of genomic components predicted 5,171 repeat sequences, 283 RNAs, and 10,150 protein-coding genes. Next, the intracellular proteins at three differential life stages of T. fuciformis (conidium, hyphal and fruiting body) were identified by the shot-gun proteomics. 6,823 canonical proteins (68.1 % of predicted proteome) have been identified with protein FDR cut-off of 0.01, establishing the first proteome draft of predicted protein-coding genes of T. fuciformis. Finally, 24 T. fuciformis polysaccharides (TPS) biosynthesis-related genes in mycelia were identified by comparative transcriptomics and proteomics, which may be more active than in conidium and revealed the TPS biosynthesis process in mycelia. This present study elucidated T. fuciformis genome composition and organization, drafted its associated proteome, and provided a genome-view of TPS biosynthesis, which will be a powerful platform for biological and genetic studies in T. fuciformis.

Transcriptome Analysis Reveals the Function of a G-Protein α Subunit Gene in the Growth and Development of Pleurotus eryngii.

Pleurotus eryngii is a commercially important edible fungus with high nutritional and economic value. However, few functional studies have examined key genes affecting the growth and development of P. eryngii. In this study, transformed strains, including over-expression (PeGNAI-OE) and RNA interference (PeGNAI-RNAi) lines, were constructed to elucidate the role of GNAI in P. eryngii growth. GNAI expression was found to affect the mycelial growth and the number of clamp connections. Moreover, the transformed strains were shown to have higher endogenous cAMP levels, thus affecting amylase and laccase activity. Fruiting experiments showed that GNAI expression revealed the formation of P. eryngii primordia and the number of buttons, while transcription analysis identified GNAI gene involvement in the growth and development of P. eryngii. Seven downstream genes regulated by GNAI were differentially expressed in PeGNAI-OE and PeGNAI-RNAi compared to wild type (WT). These genes may be related to mycelial growth and enzyme activity. They were involved in the MAPK signaling pathway, inositol phosphate metabolism, ascorbate, aldarate metabolism, and starch and sucrose metabolism. In summary, GNAI performs different physiological functions in regulating the growth and development of P. eryngii. Importantly, the molecular mechanisms of GNAI regulatory function are relatively complex and need further study.

Deaminative-cleaved S. monotuberculatus fucosylated glycosaminoglycan: Structural elucidation and anticoagulant activity.

Stichopus monotuberculatus is a tropical sea cucumber species and used as a folk medicine and tonic food. In this study, a fucosylated glycosaminoglycan (SmFG), the depolymerized SmFG (dSmFG) and its oligosaccharide fractions were prepared. The SmFG and its depolymerized products were comprised of a chondroitin-sulfate-E backbone, and various sulfated fucose side chains, including an unusual disaccharide side chain connected to the C-3 position of D-glucuronic acid (GlcA) or GlcA-ol. A peeling reaction occurred during the deaminative depolymerization process. The dSmFG and its fractions showed strong anticoagulant activity by selectively inhibiting intrinsic tenase complex, and had no anti-factor IIa, Xa and VIIa activity. The anticoagulant activity reduced with the decrease of molecular weight, and the unusual branch and novel reducing end may enhance the anticoagulant activity. These findings can provide significant information for development and utilization of depolymerized products from SmFG in food and pharmaceutical industries.

Simulated digestion, dynamic changes during fecal fermentation and effects on gut microbiota of Avicennia marina (Forssk.) Vierh. fruit non-starch polysaccharides.

Grey mangrove (Avicennia marina (Forssk.) Vierh.) fruit is a traditional folk medicine and health food consumed in many countries. In this study, its polysaccharides (AMFPs) were obtained and analyzed by chemical and instrumental methods, with the results indicating that AMFPs consisted of galactose, galacturonic acid, arabinose, and rhamnose in a molar ratio of 4.99:3.15:5.38:1.15. The dynamic changes in AMFPs during the digestion and fecal fermentation processes were then investigated. The results confirmed that AMFPs were not depolymerized by gastric acid and various digestive enzymes. During fermentation, 56.05 % of the AMFPs were utilized by gut microbiota. Galacturonic acid, galactose, and arabinose from AMFPs, were mostly consumed by gut microbiota. AMFPs obviously decreased harmful bacteria and increased some beneficial microbiota, including Megasphaera, Mistuokella, Prevotella, and Megamonas. Furthermore, AMFPs obviously increased the levels of various short-chain fatty acids. These findings suggest that AMFPs have potential prebiotic applications for improving gut health.

Variation in community structure and network characteristics of spent mushroom substrate (SMS) compost microbiota driven by time and environmental conditions.

Global mushroom production is growing rapidly, raising concerns about polluting effects of spent mushroom substrate (SMS) and interest in uses in composts. In this study, SMS composting trials and high-throughput sequencing were carried out to investigate to better understand how the structure, co-occurrence patterns, and functioning of bacterial and fungal communities vary through compost time and across environmental conditions. The results suggested that both bacterial and fungal microbiota displayed significant variation in community composition across different composting stages. Enzyme activity levels showed both directional and fluctuating changes during composting, and the activity dynamics of carboxymethyl cellulase, polyphenol oxidase, laccase, and catalase correlated significantly with the succession of microbial community composition. The co-occurrence networks are "small-world" and modularized and the topological properties of each subnetwork were significantly influenced by the environmental factors. Finally, seed germination and seedling experiments were performed to verify the biosafety and effectiveness of the final composting products.

A fungal cellulose nanocrystals-based approach to improve the stability of triterpenes loaded Pickering emulsion.

Depolysaccharide residues of edible fungus Pleurotus eryngii (dePSR-Pe), a mushroom industry waste, have abundant cellulose. In this study, the cellulose nanocrystals of P. eryngii (PeCNs) were extracted by hydrochloric acid. Results showed that the length of PeCNs is 469 ± 76.41 nm with a high aspect ratio of 40-100 and needle morphology. The structural characterization revealed that PeCNs had good thermal stability (approach 300 °C) and high crystallinity (84.2 %). An O/W Pickering emulsion stabilized with PeCNs was prepared to inhibit lipid oxidation and improve the loading capacity of triterpenes of P. coco. Unimodal size distribution of emulsion droplets was obtained under an optimized aqueous-phase condition to form a metastable emulsion, regardless of varying oil-water volume ratio <50/50. In vitro digestion study suggested that triterpenes-loaded Pickering emulsion had 1-3 times higher drug stability than bulk oil. These metastable Pickering emulsions call for fewer nanoparticles and provide a new strategy for the industry application of cellulose nanocrystals at less cost.

Study of Dimorphism Transition Mechanism of Tremella fuciformis Based on Comparative Proteomics.

Tremella fuciformis is a dimorphic fungus that can undertake a reversible transition between yeast-like conidia and hyphal forms. The transformation mechanism and proteomic differences between these two forms have not been reported. Therefore, in this study, we attempted to explore the differential protein profiles of dikaryotic yeast-like conidia from fruiting bodies and mycelia (FBMds) and dikaryotic mycelia (DM) by synthetically applying high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) full proteomics and parallel reaction monitoring (PRM) targeted proteomics. The results showed that a total of 5687 proteins were quantified, and 2220 of them (39.01%) showed more than a two-fold change in expression. The functional analysis of the differentially expressed proteins (DEPs) confirmed that the DEPs were mainly located in the membrane and nucleus. The FBMds tended to express proteins involved in biosynthesis, metabolism, DNA replication and transcription, and DNA damage repair. At the same time, DM exhibited an increased expression of proteins involved in signal transduction mechanisms such as the mitogen-activated protein kinase (MAPK) signaling pathway and the Ras signaling pathway. Further, phosphorylation analysis confirmed the importance of the MAPK signaling pathway in T. fuciformis dimorphism, and comparative metabolism analysis demonstrated the metabolic difference between FBMds and DM. The information obtained in the present study will provide new insights into the difference between FBMds and DM and lay a foundation for further research on the dimorphism formation mechanism of T. fuciformis.

Physicochemical properties and in vitro digestion behavior of a new bioactive Tremella fuciformis gum.

A new easy-dissolved Tremella fuciformis gum (TFG) from fruiting body was investigated in detail from three aspects: physicochemical characteristics, rheological behavior and in vitro digestion behavior. The results showed that TFG consisted of 73.9% polysaccharides, exhibiting easy solubility in water and good colloidal characteristics and stability. The physical and chemical treatments could decrease the apparent viscosity of TFG solution. The antioxidation activity of TFG remained constant at each static in vitro digestion phase, revealing that this gum could be used as a potential food thickener and antioxidant. The digestion behavior of TFG was also determined using a dynamic in vitro digestive system, DIVRS-II. The results demonstrated that the digestion behavior of TFG should be attributed to the morphology of digestive tracts, continuous secreting and continuous emptying. The antitussive effect of TFG was related to the increase in serum IL-10 content.

Bacterial Community Composition in the Growth Process of Pleurotus eryngii and Growth-Promoting Abilities of Isolated Bacteria.

The effects of biological factors on the vegetative growth process of mushrooms remain largely unexplored. We investigated the bacterial community in different growth stages of Pleurotus eryngii by high-throughput sequencing technology to explore the relationship between interacting bacteria and the growth and development of P. eryngii. We found significant variances in mushroom interacting association bacteria (MIAB) compositions among the samples from different growth stages, and 410 genera were identified. The bacteria in the full-bag and post-ripe stages were shifted to the biocontrol and growth-promotion ones. The mushroom growth-promoting bacteria (MGPB) were also isolated successfully and identified as B. cereus Bac1. The growth speed and density of mycelial pellets of P. eryngii, and activities of two exoenzymes (laccase and amylase), were analyzed by adding the different volumes of cell-free fermentation broth of B. cereus Bac1 to fungal culture media. The results showed that when a 5 mL cell-free fermentation broth was used, the growth speed of P. eryngii hyphae was enhanced by 1.15-fold over the control and reached 0.46 mm/h. The relative activity of laccase and amylase was increased by 26.9 and 43.83%. Our study revealed that the abundant interacting bacteria coexist with P. eryngii hyphae. Moreover, the abundance of some bacteria exhibiting a positive correlation with the growth periods of their host fungi can effectively promote the growth of the host, which will provide technical supports on the high-efficiency production of P. eryngii in factory cultivation.

Effects of Drying Methods on the Physicochemical Aspects and Volatile Compounds of Lyophyllum decastes.

In this study, fresh Lyophyllum decastes was dried using hot air drying (HAD), hot air combined with vacuum drying (HAVD), and vacuum freeze drying (VFD). Additionally, the quality and volatile compounds were analyzed. VFD achieved the best color retention, the highest rehydration capacity, and the slightest damaged tissue structure; however, it recorded the longest drying time and the highest energy consumption. HAD was the most energy-efficient of the three methods. Furthermore, the products with more hardness and elasticity were obtained by HAD and HAVD-this finding was convenient for transportation. In addition, GC-IMS demonstrated that the flavor components had significantly changed after drying. A total of 57 volatile flavor compounds was identified, and the aldehyde, alcohol, and ketone compounds were the primary ingredient of the L. decastes flavor component, whereby the relative content of the HAD sample was apparently higher than HAVD and VFD. Taken together, VFD was better at preserving the color and shape of fresh L. decastes, but HAD was more appropriate for drying L. decastes because of the lower energy consumption, and was more economical. Meanwhile, HAD could be used to produce a more intense aroma.

Nitrite removal by Acinetobacter sp.TX: a candidate of curbing N2O emission.

The nitrite removal pathway in Acinetobacter sp. TX5 was explored through the key gene identification and the corresponding enzyme purification, after which the capability to reduce nitrite by immobilized beads was investigated in a fixed-bed reactor. Results revealed that a nosZ gene encoding nitrous oxide reductase (N2OR) exists in TX5 cells, and a N2OR responsible for the reduction of N2O to N2 was purified successfully with a molecular weight of 70.05 kDa, a purification fold of 16.30 and a recovery rate of 5.17%. For TX5 immobilization, the optimal values of polyvinyl alcohol (PVA), spent mushroom substrate (SMS) and Aci (TX5) obtained by response surface methodology (RSM) were 6.32%, 2.92% and 4.57%, respectively. In a fixed-bed reactor packed with immobilized TX5, the removal efficiency (RE) achieved 90% (at 50 h) for NO2--N and 85% (at 96 h) for total nitrogen (TN). On the basis of these results, a nitrite removal pathway in TX5 was proposed. Overall, Acinetobacter sp. TX5 might be a promising candidate for nitrite removal with an ability to suppress N2O accumulation.

Screening for Arabidopsis mutants with altered Ca2+ signal response using aequorin-based Ca2+ reporter system.

Environmental stimuli evoke transient increases of the cytosolic Ca2+ level. To identify upstream components of Ca2+ signaling, we have optimized two forward genetic screening systems based on Ca2+ reporter aequorin. AEQsig6 and AEQub plants were used for generating ethyl methanesulfonate (EMS)-mutagenized libraries. The AEQsig6 EMS-mutagenized library was preferably used to screen the mutants with reduced Ca2+ signal response due to its high effectiveness, while the AEQub EMS-mutagenized library was used for screening of the mutants with altered Ca2+ signal response. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020) and Zhu et al. (2013).

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Some fungi engage symbiosis with bacteria, which can effectively promote the metabolism and growth of fungi. The diversity and community structure of bacteria can reflect the growth and substrate utilization of fungi. In this study, we analyzed the effects of different sawdust dosages on the community structure and diversity of bacteria associated with the hyphae of Pleurotus eryngii using high-throughput sequencing technology (HST) based on PCR-amplified 16S rRNA V3-V4 fragments. The results showed that the high-quality sequences from five groups of mycelia samples were clustered into 25 phyla, 52 classes, 114 orders, 199 families, and 406 genera. Proteobacteria (35.0%-85.9%) and Firmicutes (6.5%-38.4%) were the most abundant bacterial phyla, while Acinetobacter (14.8%-71.6%) and Pseudomonas (1.7%-22.3%) were the dominant symbiotic genera. Compared with the mycelia grown on the complete culture medium, sawdust addition could increase the diversity of bacteria coexisting with P. eryngii mycelia, and change the community structure of 10 dominant phyla and 9 dominant genera. P. eryngii cultivated in substrate containing 5 g sawdust had the fastest mycelium growth rate, thick mycelia, and neatly edges. Furthermore, judging from the abundance and diversity, Pseudomonas and Lactobacillus became the dominant genera, which were positively correlated with the mycelia growth vigour. Sawdust, as an important carbon source, could affect the growth and development of P. eryngii and the community structure and diversity of bacteria coexisting with mycelia. This study would lay a theoretical foundation for exploring the molecular mechanism of sawdust and mycelium symbiosis affecting the growth and development of P. eryngii.

BONZAI Proteins Control Global Osmotic Stress Responses in Plants.

Hyperosmotic stress caused by drought and salinity is a significant environmental threat that limits plant growth and agricultural productivity. Osmotic stress induces diverse responses in plants including Ca2+ signaling, accumulation of the stress hormone abscisic acid (ABA), reprogramming of gene expression, and altering of growth. Despite intensive investigation, no global regulators of all of these responses have been identified. Here, we show that the Ca2+-responsive phospholipid-binding BONZAI (BON) proteins are critical for all of these osmotic stress responses. A Ca2+-imaging-based forward genetic screen identified a loss-of-function bon1 mutant with a reduced cytosolic Ca2+ signal in response to hyperosmotic stress. The loss-of-function mutants of the BON1 gene family, bon1bon2bon3, are impaired in the induction of gene expression and ABA accumulation in response to osmotic stress. In addition, the bon mutants are hypersensitive to osmotic stress in growth inhibition. BON genes have been shown to negatively regulate plant immune responses mediated by intracellular immune receptor NLR genes including SNC1. We found that the defects of the bon mutants in osmotic stress responses were suppressed by mutations in the NLR gene SNC1 or the immunity regulator PAD4. Our findings indicate that NLR signaling represses osmotic stress responses and that BON proteins suppress NLR signaling to enable global osmotic stress responses in plants.

Study on the community structure and function of symbiotic bacteria from different growth and developmental stages of Hypsizygus marmoreus.

BACKGROUND: The symbiotic bacteria associated with edible fungi are valuable microbial resources worthy of in-depth exploration. It is important to analyze the community structure and succession of symbiotic bacteria in mushrooms. This can assist in the isolation of growth-promoting strains that have an essential relationship with the cultivation cycle as well as the agronomic traits and yields of fruiting bodies. RESULTS: In all of the samples from cultivation bags of Hypsizygus marmoreus, 34 bacterial phyla were detected. Firmicutes was the most abundant bacterial phylum (78.85%). The genus Serratia showed an exponential increase in abundance in samples collected from the cultivation bags in the mature period, reaching a peak abundance of 55.74% and the dominant symbiotic flora. The most predominant strain was Serratia odorifera HZSO-1, and its abundance increased with the amount of hyphae of H. marmoreus. Serratia odorifera HZSO-1 could reside in the hyphae of H. marmoreus, promote growth and development, shorten the fruiting cycle by 3-4 days, and further increase the fruiting body yield by 12%. CONCLUSIONS: This study is a pioneering demonstration of the community structure of the symbiotic microbiota and bacteria-mushroom interaction in the growth and development of edible fungi. This work lays a theoretical foundation to improve the industrial production of mushrooms with symbiotic bacteria as assisting agents.

Cyclic Dipeptides Mediating Quorum Sensing and Their Biological Effects in Hypsizygus Marmoreus.

A novel quorum sensing (QS) system was discovered in Serratia odorifera, the symbiotic bacterium of Hypsizygus marmoreus. This system uses cyclo(Pro-Phe), cyclo(Pro-Tyr), cyclo(Pro-Val), cyclo(Pro-Leu), cyclo(Tyr-Leu), and cyclo(Tyr-Ile) as autoinducers. This discovery is the first attempt to characterize cyclic dipeptides as QS signaling molecules in S. odorifera and improves the classical QS theory. Significantly, except for cyclo(Tyr-Leu), these QS autoinducers can increase the transcription level of lignin-degrading enzyme genes of H.marmoreus. The cyclo(Pro-Phe) can increase the activity of extracellular laccase (1.32-fold) and manganese peroxidase (20%), which may explain why QS potentially regulates the hyphal growth, primordium formation, and fruit body development of H. marmoreus. Furthermore, it was demonstrated that the cyclo(Tyr-Ile) biosynthesis in S. odorifera was catalyzed by the nonribosomal peptide synthetase (NRPS). This study supports exploring the growth and development of H.marmoreus promoted by its symbiotic bacteria at QS signal transduction level.

Three CNGC Family Members, CNGC5, CNGC6, and CNGC9, Are Required for Constitutive Growth of Arabidopsis Root Hairs as Ca2+-Permeable Channels.

The genetic identities of Ca2+ channels in root hair (RH) tips essential for constitutive RH growth have remained elusive for decades. Here, we report the identification and characterization of three cyclic nucleotide-gated channel (CNGC) family members, CNGC5, CNGC6, and CNGC9, as Ca2+ channels essential for constitutive RH growth in Arabidopsis. We found that the cngc5-1cngc6-2cngc9-1 triple mutant (designated shrh1) showed significantly shorter and branching RH phenotypes as compared with the wild type. The defective RH growth phenotype of shrh1 could be rescued by either the expression of CNGC5, CNGC6, or CNGC9 single gene or by the supply of high external Ca2+, but could not be rescued by external K+ supply. Cytosolic Ca2+ imaging and patch-clamp data in HEK293T cells showed that these three CNGCs all function as Ca2+-permeable channels. Cytosolic Ca2+ imaging in growing RHs further showed that the Ca2+ gradients and their oscillation in RH tips were dramatically attenuated in shrh1 compared with those in the wild type. Phenotypic analysis revealed that these three CNGCs are Ca2+ channels essential for constitutive RH growth, with different roles in RHs from the conditional player CNGC14. Moreover, we found that these three CNGCs are involved in auxin signaling in RHs. Taken together, our study identified CNGC5, CNGC6, and CNGC9 as three key Ca2+ channels essential for constitutive RH growth and auxin signaling in Arabidopsis.