Introduction

Arbuscular mycorrhizal fungi (AMF) are among the most widely distributed soil microorganisms, forming symbiotic relationships with approximately 70% of terrestrial vascular plants (Brundrett and Tedersoo, 2018). In this symbiosis, referred to as arbuscular mycorrhiza (AM), AMF develop an extensive extraradical hyphal network in the soil, which facilitates nutrient uptake for host plants—particularly phosphorus (P)—and enhances plant resilience to both biotic and abiotic stressors (Smith and Read, 2008). In return, host plants allocate 4−20% of their photosynthetically fixed carbon (C) to their fungal partners in the form of sugars and fatty acids (Helber et al., 2011; Jiang et al., 2017). This symbiotic C-P exchange, the cornerstone of AM symbiosis (Smith and Read, 2008), not only plays a pivotal role in the biogeochemical cycling of C and P elements but also acts as a key mechanism through which AMF influence plant community structure and productivity (Johnson, 2010; Wipf et al., 2019).

As one of the most ancient symbiotic relationships, AM has undergone extensive evolutionary processes (Foster and Kokko, 2006; Rich et al., 2021), with its establishment and development intricately regulated by a set of symbiotic genes and signaling molecules influenced by both biotic and abiotic factors (Maclean et al., 2017; Shi et al., 2021). For instance, in AM plants, the two pathways for P uptake—the direct P pathway via root hairs and epidermis and the mycorrhizal P pathway via AMF hyphae—are coordinately regulated by the same phosphate-sensing pathway (Shi et al., 2021; Das et al., 2022). Under P-deficiency conditions, host plants synthesize and release strigolactones into the soil, activating the mycorrhizal P pathway by stimulating the elongation and branching of AMF hyphae (Mori et al., 2016; Yu et al., 2022). Additionally, the extent of AMF colonization and the benefits conferred by AMF largely depend on the host plant species (genotypes), with some being “responsive plants” that exhibit positive growth responses (e.g., alfalfa, maize) and others being “nonresponsive plants” that show minimal or no growth enhancement while exhibiting increased AM-activated defense responses (e.g., rice, wheat; Hata et al., 2010; Li et al., 2024).

AMF are considered obligate symbionts that rely on living host plants to complete their life cycle by producing daughter spores (Smith and Read, 2008), which presents challenges for basic research on AM and their applications in agriculture and other fields. To date, achieving a pure (plant-independent) culture of asymbiotic AMF has not been successful, partially due to their dependence on plant-derived symbiotic C (Bravo et al., 2017; Luginbuehl et al., 2017; Kameoka et al., 2019). Genomic and transcriptomic analyses have shown that AMF lack the genes required for the biosynthesis of long-chain fatty acids, and subsequent studies have demonstrated that fatty acids are transferred from host plants to AMF as their primary C source (Bravo et al., 2017; Jiang et al., 2017; Keymer et al., 2017; Luginbuehl et al., 2017). However, the mechanism underlying the allocation of plant-derived fatty acids to fungi in AM symbiosis remains unclear. In host plants, AMF colonization triggers the expression of lipid biosynthesis and transport genes (Gutjahr et al., 2012; Bravo et al., 2017; Keymer et al., 2017; Luginbuehl et al., 2017), with lipid transport to the AM symbiotic interface relying on the periarbuscular membrane-localized transporters stunted arbuscule (STR) and STR2 (Zhang et al., 2010; Gutjahr et al., 2012), and fungal uptake of plant-derived lipids mediated by fatty acid transporter 1 (RiFAT1) and RiFAT2 in the model AMF species Rhizophagus irregularis (Brands and Dörmann, 2022). This symbiotic C allocation is functionally linked to fungal P delivery within a framework of mutual nutritional rewards (Kiers et al., 2011), which have been disrupted during AMF-nonhost interactions due to the strong AM-activated defense responses in non-host roots (Wang et al., 2023).

Fatty acids and their derivatives, essential structural components and energy sources for plant, animal, and microbial cells, are commonly found in soil, particularly in the rhizosphere, where plant-derived fatty acids are made available through root exudates and rhizodeposition (Swenson et al., 2018; Dai et al., 2022; Liu et al., 2024). Following the discovery that AMF acquire fatty acids from their hosts during symbiosis, the use of fatty acids in cultivating asymbiotic AMF has attracted considerable attention. Kameoka et al. (2019) demonstrated that under asymbiotic conditions, exogenous palmitoleic acid and several other fatty acids stimulated the hyphal elongation and branching in Rhisophagus irregularis and R. clarus, but not in Gigaspora margarita, resulting in the formation of infection-competent but morphologically and functionally incomplete spores with reduced size, lower germination rates, and decreased infection capacity compared to symbiotic spores. Additionally, Sugiura et al. (2020) showed that exogenous myristate (tetradecanoic acid)—a common organic acid in various plant root exudates (Li et al., 2017; Liu et al., 2024) and soil substrates (Swenson et al., 2018; Dai et al., 2022)—can serve as a C source for the asymbiotic growth of R. irregularis and R. clarus, promoting hyphal growth, the formation of branched absorbing structures, and the production of morphologically incomplete, infection-competent spores. Subsequent studies revealed that combining plant hormones with myristate enhanced the efficiency of myristate as a C source in asymbiotic R. clarus, leading to the generation of numerous infection-competent spores that were smaller than those generated under symbiotic conditions (Sugiura et al., 2020; Tanaka et al., 2022). These discoveries have significantly advanced the pure culture of AMF and, importantly, identified myristate as the first external C source accessible to AMF under asymbiotic conditions, providing exceptional opportunities for understanding and utilizing the ecological functions of AM symbiosis in plant community regulation and global C cycling (Rillig et al., 2020).

However, the effects of external myristate on the growth and development of symbiotic AMF—the typical state of functioning AMF in nature—remain unclear. Regarding sugars, another type of host-derived C source, symbiotic AMF may be unable to absorb them additionally through extraradical hyphae (Bücking et al., 2008; Helber et al., 2011; Sugiura et al., 2020). A recent study reported that while exogenous myristate does not affect the extraradical biomass of symbiotic R. irregularis and R. intraradices, it enhances their colonization rates by stimulating the growth and development of extraradical hyphae. Based on this, the authors inferred that symbiotic AMF may acquire exogenous myristate (Liu et al., 2023). To date, the effects of exogenous myristate on AM symbiosis and their underlying mechanisms remain limited. Specifically, whether symbiotic AMF can uptake exogenous myristate via extraradical hyphae in the presence of plant-derived symbiotic C is yet to be determined. This question is crucial for understanding the symbiotic mechanisms and ecological roles of AM symbiosis (Rillig et al., 2020).

To address this significant knowledge gap, this study investigated the ability of symbiotic AMF to absorb external non-symbiotic myristate and examined the effect of exogenous myristate on symbiotic C-P exchanges in AM. Specifically, we aimed to determine: (i) whether symbiotic AMF can take up exogenous non-symbiotic myristate, (ii) how this additional non-symbiotic C source influences the symbiotic C allocation from plants to AMF, and (iii) if and how fungal P transfer to the host plant is affected by exogenous myristate. This work provides, to our knowledge, the strongest evidence to date that symbiotic AMF can absorb non-symbiotic C sources like myristate in the presence of plant-derived symbiotic carbohydrates. Furthermore, our findings reveal that exogenous myristate may, unexpectedly, disrupt the C-P exchange in AM symbiosis.

Results

Tracing of ¹³C in AMF extraradical hyphae (Exp. 1)

In the two-compartment AMF-carrot hairy root co-cultivation system (Exp. 1, Fig. 1a), exogenous myristate specifically induced the formation of branched absorbing structures in R. irregularis, R. intraradices, and R. diaphanus in the HC approximately two weeks after Myr⁺ treatment (Fig. 1b). For R. irregularis, the ¹³C/¹²C ratio of extraradical hyphae in HC (trial 1: 1.1504 ± 0.0575%, trial 2: 1.0919 ± 0.0001%) and RC (trial 1: 1.1026 ± 0.0055%, trial 2: 1.0918 ± 0.0002%) under ¹³C-Myr treatment was slightly but significantly higher than that in the corresponding HC (trial 1: 1.0922 ± 0.0003%, trial 2: 1.0908 ± 0.0003%) and RC (trial 1: 1.0923 ± 0.0002%, trial 2: 1.0908 ± 0.0002%) from the non-labeled control (¹²C-Myr), respectively (P < 0.05, Fig. 1c). Similarly, for R. intraradices and R. diaphanus, the ¹³C/¹²C ratio of extraradical hyphae in HC (R. intraradices: 1.0933 ± 0.0002%, R. diaphanus: 1.0922 ± 0.0005%) and RC (R. intraradices: 1.0927 ± 0.0005%, R. diaphanus: 1.0930 ± 0.0004%) under ¹³C-Myr treatment was slightly but significantly higher than those in HC (R. intraradices: 1.0917 ± 0.0001%, R. diaphanus: 1.0908 ± 0.00003%) and RC (R. intraradices: 1.0918 ± 0.00004%, R. diaphanus: 1.0908 ± 0.00003%) from the non-labeled control (P < 0.01, Fig. 1c). Additionally, the calculated ¹³C enrichment in extraradical hyphae between HC and RC showed no significant difference for R. irregularis and R. diaphanus (Fig. 1d, e), although it was higher in HC than in RC for R. intraradices (P < 0.05), excluding the possibility that the ¹³C enrichment in AMF extraradical hyphae originated from ¹³C-myristate retention on the hyphal surface from the HC. Collectively, these results suggest that these three AMF species can take up a small amount of exogenous myristate through their extraradical hyphae when associated with host roots.

Fig. 1

In HC, the calculated ¹³C enrichments in extraradical hyphae of R. irregularis (trial 1: 0.5823 ± 0.4111‰, trial 2: 0.0107 ± 0.0014‰), R. intraradices (0.0163 ± 0.0031‰), and R. diaphanus (0.0136 ± 0.0067‰) showed no significant differences in the ¹³C-Myr treatment (Fig. 1d). In RC, the ¹³C enrichment in extraradical hyphae also showed no significant differences (Fig. 1e), except for a slightly but significantly higher ¹³C enrichment in R. diaphanus (0.0217 ± 0.0031‰) compared to R. intraradices (0.0082 ± 0.0037‰) and R. irregularis in trial 2 (0.0093 ± 0.0017‰, P < 0.05). These results suggest a generally similar myristate absorption capacity among these AMF species under the tested conditions.

Effects of myristate on AMF colonization and expression of fatty acid uptake and metabolism marker genes (Exp. 1)

The expression of fatty acid uptake and metabolism marker genes in R. irregularis extraradical hyphae were analyzed to investigate their uptake of myristate from HC at the transcriptional level. The results showed that exogenous myristate significantly increased the expression levels of the fungal fatty acid transporter gene RiFAT2 (P < 0.05), while had no significant effect on RiFAT1 (Fig. 2a). Additionally, exogenous myristate significantly or marginally significantly increased the expression levels of the fungal genes involved in fatty acid β-oxidation (RiFAD1, P < 0.05), palmitvaccenic acid synthesis (RiOLE1, P = 0.057), and TCA cycle activity (RiCIT1, P < 0.05) (Fig. 2b). These results suggest that exogenous myristate activates the expression of fungal genes related to fatty acid uptake and metabolism in the extraradical hyphae of R. irregularis. For AMF colonization intensity, exogenous myristate significantly increased the hyphal and arbuscular colonization rates of R. irregularis, R. intraradice, and R. diaphanus in carrot hairy roots (P < 0.05, Fig. 2c−e) but had no effect on vesicle colonization.

Fig. 2

Effects of exogenous myristate on AMF spore germination, root colonization, and extraradical structure development (Exp. 2)

In the single-compartment R. irregularis-carrot hairy root co-cultivation system (Exp. 2, Fig. 3a), exogenous myristate significantly promoted spore germination (P < 0.001, Fig. 3b), and enhanced hyphal branching and length (P < 0.001, Fig. 3c, d) before visible physical contact between hyphae and hairy roots at four days after AMF inoculation. Morphological examinations showed that exogenous myristate significantly enhanced the colonization intensities of hyphae and arbuscules, but not vesicles, in carrot hairy roots across various application levels (Fig. 3e). Exogenous myristate also significantly increased the extraradical biomass of AMF (hyphae and spore) at different application concentrations (P < 0.05, Fig. 3f).

Fig. 3

Meanwhile, exogenous myristate significantly increased the density of AMF spores from eight weeks post-AMF inoculation (P < 0.05, Fig. 3g), and the diameter of newly produced AMF spores in media with exogenous myristate was identical to those in media without myristate (Fig. 3h).

Environmental myristate levels and the effects of exogenous myristate on AMF intraradical and extraradical biomass during symbiosis with alfalfa and rice (Exp. 3)

In Exp. 3 (Fig. 4a), our field survey revealed that myristate was extensively detected in soil and plant samples from the paddy field, grassland, and woodland at a 2.87‒14.68 mg kg^-1 level (Fig. 4b). On average, the myristate level in plant (fresh leaf or root) is mostly higher than that in soil (leaf litter, rhizosphere soil, or bulk soil) (P < 0.05, Fig. 4b). Additionally, the myristate levels in leaf litter and rhizosphere soil were usually greater than in bulk soil (P < 0.05), suggesting that plants are significant source of myristate in the soil. The myristate contents from the same sample type typically varied across different habitats (P < 0.05).

Fig. 4

In the pot experiment (Exp. 3), all typical AMF intraradical structures (hyphae, arbuscules, and vesicles) were observed in the roots of both alfalfa and rice in the R. irregularis-inoculated groups (AM), while no AMF colonization was detected in the non-inoculated (NM) groups. Under low-P conditions, exogenous myristate significantly increased arbuscule abundance in alfalfa roots (P < 0.05, Fig. 4c) but did not significantly affect hyphae or vesicle abundance (Supporting Information Fig. S1a, c). Exogenous myristate significantly enhanced arbuscule, hyphae, and vesicle abundance in rice roots (P < 0.05, Fig. 4d, Supporting Information Fig. S1b, d). However, under high-P conditions, exogenous myristate had no effect on AMF abundance in either alfalfa or rice roots (Fig. 4, Supporting Information Fig. S1).

For intraradical AMF biomass, exogenous myristate significantly increased the levels of the AMF signature fatty acid (NLFA 16:1ω5) in both alfalfa and rice roots under low-P conditions (P < 0.05, Fig. 4e, f); however, it had no significant impact under high-P conditions. In rice roots, NLFA 16:1ω5 levels were higher under high-P than low-P conditions (P < 0.05). For extraradical AMF biomass, exogenous myristate increased NLFA 16:1ω5 levels in the rhizosphere soils of alfalfa (P < 0.05) but not in rice (P = 0.14) under low-P conditions (Fig. 4e, f). Under high-P conditions, myristate had no effect on NLFA 16:1ω5 levels in the rhizosphere soil of either plant.

Effects of exogenous myristate on the transfer of symbiotic C to AMF (Exp. 3)

The analysis of ¹³C-NLFA-isotope ratio mass spectrometry was performed to investigate the impact of exogenous myristate on the allocation of symbiotic C from plants to AMF. In AM plants, there was a notable enrichment of ¹³C (δ¹³C = 1.75 ± 0.6‰) in the AMF signature fatty acid (NLFA 16:1ω5) compared to NM plants (δ¹³C = −24.8 ± 2.3‰), demonstrating the transfer of newly fixed ¹³C from plants to AMF. The calculated ¹³C flow from alfalfa to root-colonizing AMF (0.3400‒1.2642 μg) was over ten times higher than that from rice (0.0261‒0.0596 μg), highlighting a substantially greater efficiency of plant-derived symbiotic C delivery to AMF in AM-responsive alfalfa compared to non-responsive rice. In alfalfa, exogenous myristate reduced the symbiotic C transfer to AMF (Myr effect: P < 0.001, Fig. 5a), although this effect was not significant under high-P conditions. In rice, myristate also significantly influenced the symbiotic C flow to AMF, with its effect significantly interacting with soil P level (Myr × P: P = 0.004, Fig. 5b): myristate significantly increased the allocation of ¹³C to AMF under low-P conditions (P < 0.05) but decreased it under high-P conditions (P < 0.01).

Fig. 5

At the transcriptional level, the qRT-PCR analysis revealed that the expressions of the symbiotic lipid transfer marker genes STR2 (in alfalfa and rice) and the fungal genes responsible for symbiotic fatty acid uptake (RiFAT1 and RiFAT2) or monosaccharide uptake (Monosaccharide Transporter 2 (RiMST2)) were exclusively detected in AM roots, except for a low expression level of alfalfa MsSTR2 occasionally observed in NM roots (Fig. 5c–h). In AMF-inoculated alfalfa roots, exogenous myristate significantly downregulated the expressions of the symbiotic C transfer genes MsSTR2 in the plant and RiMST2 in AMF (P < 0.05), while having no effect on RiFAT1 and RiFAT2 in AMF (Fig. 5c, g, e). In AMF-inoculated rice roots, the effects of myristate on the tested symbiotic C transfer genes were largely influenced by soil P levels (P < 0.05). Under low-P conditions, myristate significantly increased the expressions of plant OsSTR2 and fungal RiFAT1 and RiFAT2, while decreasing the expression of fungal RiMST2 (P < 0.05, Fig. 5d, f, h). Under high-P conditions, myristate significantly increased the expression of RiMST2 (P < 0.05) but had no effects on OsSTR2, RiFAT1, and RiFAT2. In both plant species with AMF inoculation, soil P level showed significant effects on STR, RiFAT1, and MST2 genes (P < 0.05), with generally lower expression levels of these genes under high-P compared to low-P conditions.

Effects of exogenous myristate on the mycorrhizal P pathway and mycorrhizal growth response (Exp. 3)

In the pot experiments (Exp. 3), AMF inoculation significantly increased P concentrations in both alfalfa and rice plants (P < 0.001, Fig. 6a, b). Under low-P conditions, exogenous myristate significantly decreased the mycorrhizal P pathway in both alfalfa and rice (P < 0.05, Fig. 6c, d). Under high-P conditions, while exogenous myristate significantly decreased the mycorrhizal P response in alfalfa (P < 0.05), it had no significant effect on rice.

Fig. 6

At the transcriptional level, the expression of mycorrhizal P pathway marker genes in plants (Phosphate Transporter, MsPT8 or OsPT11) and AMF (RiPT) was detected exclusively in the AM roots of rice and alfalfa, except for a low expression level of rice OsPT11 observed in the NM root under low-P and Myr⁻ conditions. This finding confirmed that AM symbiosis activated the mycorrhizal P pathway at the transcriptional level in both plant species (Fig. 6e, f). Exogenous myristate significantly influenced the transcriptional responses of the mycorrhizal P pathway in both alfalfa and rice (P < 0.01). In alfalfa, exogenous myristate downregulated the expressions of MsPT8 and RiPT under both low- and high-P conditions (P < 0.05, Fig. 6e). In rice, exogenous myristate decreased the expression levels of OsPT11 and RiPT under low-P conditions (P < 0.05), but not under high-P conditions (Fig. 6f).

In alfalfa, AMF inoculation significantly increased plant biomass under both low- and high-P conditions (P < 0.05, Supporting Information Fig. S2a), with mycorrhizal growth responses significantly higher under high-P than low-P conditions (P < 0.05, Supporting Information Fig. S2c). In rice, AMF inoculation had no effect on plant biomass under low-P conditions, but reduced rice biomass under high-P conditions (P < 0.05); mycorrhizal growth responses were significantly lower under high-P than low-P conditions (P < 0.05, Supporting Information Fig. S2b, d). Exogenous myristate had no effect on plant biomass or mycorrhizal growth response in either alfalfa or rice, while soil P levels significantly influenced mycorrhizal growth response: high-P treatment increased mycorrhizal growth response in alfalfa but decreased it in rice compared to low-P conditions (P < 0.01, Supporting Information Fig. S2c, d).

Effects of exogenous myristate on the transcriptome of AM roots (Exp. 3)

Transcriptome analysis was performed to preliminarily explore the effects of exogenous myristate on mycorrhizal roots at the transcriptional level. The RNA-seq data identified 39,944 and 33,056 genes (transcripts) in alfalfa and rice, respectively. Principal component analysis of gene expression profiles in mycorrhizal roots of alfalfa and rice showed that the first and second principal components together explained 32.4% and 44.1% of the variation, respectively (Supporting Information Fig. S3a, b). In both alfalfa and rice roots, gene expression profiles with or without myristate application could be partially separated under low-P conditions, but not under high-P conditions, suggesting the importance of P status in myristate’s effect.

Exogenous myristate induced a variety of differentially expressed genes (DEGs, Myr⁺ vs. Myr⁻) in mycorrhizal alfalfa (LP: 308, HP: 137) and rice (LP: 590, HP: 855) roots (Supporting Information Fig. S3c, d). Gene Ontology (GO) analysis of these DEGs enriched several GO terms related to plant stress or defense, most of which were downregulated by exogenous myristate (Fig. 7a, b). These included “defense response”, “response to salicylic acid”, “peroxidase activity”, etc., in rice, and “glycosyltransferase activity” and “alternative oxidase activity” in alfalfa.

Fig. 7

We subsequently analyzed the expression profiles of the component genes associated with enriched GO terms that are related to defense or stress, using the RNA-seq dataset (Fig. S4a, b). In alfalfa, exogenous myristate significantly downregulated several key genes involved in reactive oxygen species balance (alternative oxidase 2 (AOX2), AOX3) and stress tolerance (UDP-glycose flavonoid glycosyltransferase (UFGT), UDP-glycosyltransferase (UGT) 73C3, UGT73C6) under low-P conditions, as well as typical stress-responsive genes (peroxidase (PRX) P7, cytochromes P450 (CYP) 83B1), and gene families such as geraniol 8-hydroxylase-like (G8HL), CYP71D10, and CYP736A12 under high-P conditions (Supplementary Fig. S4a). In rice, exogenous myristate downregulated several typical defense-associated transcriptional factors (TGA2, TGA4, WRKY45, WRKY62, WRKY76, and bZIP79) and marker genes (pathogenesis-related 5-like (PR5L), protein phosphatase 2C 35 (OsPP2C35), 1-aminocyclopropane-1-carboxylate synthase 2 (ACS2), DWARF4, pyricularia oryzae resistance-ta (PITA), mildew resistance locus O2 (MLO2), NRR repressor homologue 1 (RH1), and chitinase 1 (CHT1)) under low-P conditions (Supplementary Fig. S4b). However, most of these genes were upregulated following myristate application under high-P conditions.

qRT-PCR verification of the effects of exogenous myristate on AM-activated defense genes in mycorrhizal roots (Exp. 3)

To verify the impact of exogenous myristate on the AM-activated defense response, qRT-PCR was performed to analyze the expression of defense marker genes in both plant species. In alfalfa roots, exogenous myristate significantly decreased the expression of alternative oxidase 3 (MsAOX3) and UDP-glycosyltransferase 85A (MsUGT85A) (P < 0.05), while its effect on peroxidase P7 (MsPRXP7) depended on soil P level (Myr × P: P < 0.05): exogenous myristate significantly decreased MsPRXP7 expression under high-P conditions (P < 0.05) but had no effect under low-P conditions (Fig. 7c). In rice roots, the effect of exogenous myristate on chitinase 1 (OsCHT1) was also dependent on soil P level (Myr × P: P < 0.05): it marginally decreased OsCHT1 expression under low-P conditions (P = 0.053) but increased it under high-P conditions (P = 0.050). Exogenous myristate significantly suppressed the expression of defense-related transcriptional factor OsTGA2 and peroxidase 39 (OsPRX39) under low-P and high-P conditions, respectively (P < 0.05, Fig. 7d). Overall, the qRT-PCR results largely confirmed the inhibitory effects of myristate on AM-activated defense responses in alfalfa and low-P rice.

Discussion

AMF are obligate biotrophs that rely on host-derived symbiotic carbohydrates for reproduction (Smith and Read, 2008). However, whether symbiotic AMF can uptake external non-symbiotic organic C remains poorly understood (Rillig et al., 2020; Liu et al., 2023). Under asymbiotic conditions, Sugiura et al. (2020) found that among eight saturated or unsaturated fatty acids (C12 to C18) and two β-monoacylglycerols supplied, only myristate led to an increase in the biomass of R. irregularis. In our study, we observed slight yet statistically significant ¹³C enrichments in the extraradical hyphae of three AMF species from both hyphal and root compartments during the ¹³C₁-myristate tracing experiment (Fig. 1c), indicating that the extraradical hyphae of the tested AMF species can absorb external non-symbiotic myristate in the presence of plant-derived symbiotic carbohydrates. Several additional observations further supported this finding. First, exogenous myristate specifically induced the formation of branched absorbing structures in AMF extraradical hyphae (Fig. 1b), which may serve as preferential sites for myristate uptake (Bago et al., 1998; Sugiura et al., 2020). Second, exogenous myristate activated the transcription of marker genes associated with fatty acid uptake and metabolism in extraradical hyphae (Fig. 2). Third, myristate application frequently enhanced both intraradical and extraradical AMF biomass (Figs. 2, 3, and 4), even when the symbiotic C supply from the plant was reduced (Figs. 4 and 5), accompanied by decreased mycorrhizal P responses (Fig. 6). In a priority study, myristate application also increased colonization intensity, but not the extraradical biomass of AMF in tomato roots (Liu et al., 2023). Overall, our findings indicate that the extraradical hyphae of symbiotic AMF can uptake external non-symbiotic organic C sources like myristate beyond those directly delivered by the host, at least under the conditions studied.

Notably, myristate has been frequently detected in root exudates and soil substrates from non-targeted metabolome analyses (Li et al., 2017; Swenson et al., 2018; Dai et al., 2022; Liu et al., 2024), although its specific levels in various soil environments remain largely unknown. Our field survey demonstrates that myristate is widely present in soil, litter, and plant (Fig. 4b), with its environmental level (2.87−14.68 mg kg^-1) encompassing the myristate treatments applied in our greenhouse experiment (Exp. 3, totaling 1.83 mg per 1000 ml or 300 ml soil substrate in rice and alfalfa, respectively). Therefore, it is likely that the widespread AMF extraradical hyphae network may absorb myristate from soil environments such as root exudates, decaying plants, or litter in realistic environments. Further research is still needed to validate this hypothesis; however, it effectively explains a long-observed but poorly understood phenomenon— AMF often colonize and thrive in leaf litter and undecomposed plant material despite their limitations in assimilating organic forms of nutrients (Went and Stark, 1968; Zhang et al., 2018; Bunn et al., 2019)—as the myristate concentration in plant and leaf litter is higher than that in soil (Fig. 4b).

During AM establishment, plant roots typically activate low-level defense responses to limit excessive C consumption caused by over-colonization (Blilou et al., 2000; Feng et al., 2019). In our study, myristate increased both intraradical and extraradical biomass of the symbiotic AMF and promoted the formation of normal-sized secondary spores (Figs. 2 and 3), unlike the small spores induced by myristate under asymbiotic conditions (Sugiura et al., 2020). This suggests that plant roots supply additional substances to AMF beyond myristate, the identification of which is essential for the pure culture of AMF. Meanwhile, exogenous myristate transcriptionally downregulated plant genes associated with AM-activated defenses under low-P conditions (Fig. 7), suggesting that myristate may interfere with normal defense mechanisms in host roots to facilitate AMF colonization. This effect contrast with AM signals like strigolactones and quercetin, which enhance plant defenses (Sudheeran et al., 2020; Kusajima et al., 2022). Further research is required to clarify myristate’s role in regulating AMF colonization and development.

Despite increased arbuscule abundance (Fig. 4)—where symbiotic nutritional exchange occurs (Smith and Read, 2008)—exogenous myristate unexpectedly reduced the mycorrhizal P response and downregulated plant and fungal mycorrhizal P transporter gene expression in both alfalfa and rice roots (Fig. 6), indicating a diminished mycorrhizal P benefit through inactivation of mycorrhizal P pathway transcription. Meanwhile, exogenous myristate also reduced C supply to AMF from both alfalfa and rice, except in low-P rice (Fig. 5). Together, these results suggest that exogenous myristate disrupts the symbiotic C-P exchange, which is functionally coupled in AM symbiosis (Kiers et al., 2011). This disruption could be attributed to several factors. First, it may result from AMF partially shifting from utilizing symbiotic C to absorbing external C (myristate, Fig. 1), thus reducing their competition for symbiotic C at the AM interaction interface (Bitterlich et al., 2014). Correspondingly, this decline in symbiotic C supply to AMF may decrease mycorrhizal P benefit, since AMF can modulate P transport based on the rate at which plants supply C (Kiers et al., 2011; Argüello et al., 2016). Second, exogenous myristate may interfere with AM signaling, as implied by the interference in normal AM-activated defenses (Fig. 7), thereby limiting nutrient exchange in the symbiosis. Third, AMF P transport proteins expressed in arbuscular cells can re-uptake P from the symbiotic interface, creating P competition between the plant and AMF (Xie et al., 2022). In our study, the increased fungal biomass with exogenous myristate suggests a higher P demand by AMF, reducing the feasibility of nutritional trade.

In contrast to its effect under high-P treatment, myristate increased, rather than decreased, the allocation of rice-derived C to AMF under low-P conditions, accompanied by enhanced expression of marker genes for fatty acid (but not sugar) allocation from rice to AMF (Fig. 5b, f, g). These results highlight that myristate’s impact on AM symbiosis is influenced by soil P status, a significant regulatory factor of the symbiosis by coordinating the direct P pathway and mycorrhizal P pathway in AM plants (Shi et al., 2021; Das et al., 2022). The notable effects of soil P status on myristate’s impact on C-P exchange were also observed in alfalfa (Figs. 5 and 6), where the unexpectedly higher AMF intraradical biomass (Fig. 4) and greater mycorrhizal growth response (Fig. S2) in the high-P compared to the low-P treatment are likely due to the inhibitory effects of extremely low-P environments on AMF colonization and functioning (Janos, 2007; Wang et al., 2020). Our findings regarding the disruptive effects of myristate on symbiotic C-P exchange, along with the broad yet varied presence of myristate in different soil components (Fig. 4), imply that myristate may be a critical influencing factor in AM symbiosis. Interestingly, myristate also exhibited inhibitory effects on rhizospheric Devosia, which is responsible for promoting growth and suppressing disease in Arabidopsis (Liu et al., 2024). Further research is required to clarify the mechanisms underlying the disruptive effect of exogenous myristate on symbiotic C-P exchange and its interaction with soil P.

Despite these encouraging findings, our work has several limitations. First, the absorption of myristate by symbiotic AMF was observed only after exogenous application under artificial conditions, which may not accurately reflect natural environments. Second, our study focused exclusively on myristate and AMF species from the genus Rhizophagus, although different AMF groups may respond to myristate or other fatty acids in various ways, at least under asymbiotic conditions (Kameoka et al., 2019; Sugiura et al., 2020). Third, our investigation into the mechanism by which myristate disrupts C-P exchange in AM symbiosis remains preliminary, and the limited P supply along with varied growth conditions between alfalfa and rice further complicate the mechanistic analysis. Therefore, further research is needed to verify and quantitatively assess the absorption of external myristate and other C sources by symbiotic AMF under natural conditions, as well as to elucidate their regulatory mechanisms and ecological significance in AM symbiosis.

Conclusions

In summary (Fig. 8), our study shows that symbiotic AMF can uptake external non-symbiotic C sources such as myristate, which is commonly present in soil components. Exogenous myristate enhanced both intraradical and extraradical biomass of symbiotic AMF, possibly linked to the suppression of AM-activated defense responses in host roots. Unexpectedly, exogenous myristate disrupted the C-P exchange in AM symbiosis, resulting in reduced mycorrhizal P benefits for plants and decreased allocation of symbiotic C to AMF, although these effects varied depending on soil P conditions. These findings provide new insights into the nutritional interactions in AM symbiosis, which are crucial for understanding and managing this ancient relationship in a changing world.

Fig. 8

Materials and methods

Biological materials

In vitro cultures of AMF species Rhizophagus irregularis (strain: MUCL 41833), R. intraradices (strain: MUCL 49410), and R. diaphanus (strain: MUCL 49416) associated with carrot (Daucus carota) hairy roots were generously supplied by the Glomeromycota In vitro Collection (GINCO). Our study consisted of three interconnected and complementary experiments.

Exp. 1: ¹³C₁-labeled myristate (¹³C₁-Myr) tracing in two-compartment AMF-hairy root co-culture systems

In Exp. 1, ¹³C₁-Myr tracing experiments were performed to determine whether exogenous myristate can be directly absorbed by the extraradical hyphae of Rhizophagus irregularis (two independent trials), R. intraradices, and R. diaphanus associated with carrot hairy root cultivated in solid modified Strullu–Romand (MSR) medium, utilizing a two-compartment Petri dish system (90 × 15 mm, see Fig. 1a) as described by St-Arnaud et al. (1996), with minor modifications (see Supporting Information Methods S1).

When dense AMF extraradical hyphae were observed in the hyphae compartment (HC), 12–14 weeks later, 200 μL of 0.1 mM myristate with 98% ¹³C₁ labeling (¹³C₁-Myr) or unlabeled (¹²C-Myr, control) myristate was added weekly to the HC until harvest, two or three weeks later. At harvest, AMF extraradical hyphae from the HC and root compartment (RC) were collected separately by dissolving the medium in 10 mM sodium citrate, filtering through a 37 µm nylon mesh, and thoroughly rinsing five times with sodium citrate followed by ultrapure water. To check for possible contamination of ¹³C-myristate from the HC to the RC, a small portion of the MSR medium from the RC was also collected randomly from three ¹³C₁-Myr and three ¹²C-Myr (control) treatments, with roots and AMF hyphae removed using tweezers under a stereomicroscope, and then used for determination of ¹³C enrichment. During the experiment, the MSR medium in the RC was not contaminated by ¹³C-myristate from the HC, as evidenced by the identical ¹³C/¹²C ratios of the MSR medium collected in the RC in ¹³C₁-Myr (1.0966 ± 0.0012) and ¹²C-Myr (1.0967 ± 0.0032) treatments.

The determination of ¹³C enrichment in AMF extraradical hyphae and solid MSR medium was performed as previously described (Bao et al., 2019), with the details provided in the Supporting Information Methods S2.

Quantitative real-time PCR (qRT-PCR) targeting fungal marker genes and quantification of AMF colonization intensity (Exp. 1)

In Exp. 1, the expression of fungal marker genes related to fatty acid uptake (RiFAT1 and RiFAT2, Brands and Dörmann, 2022) and metabolism—β-oxidation (fatty acid degradation 1 (RiFAD1)), palmitvaccenic acid synthesis (oleate desaturase-like enzyme 1 (RiOLE1), Brands et al., 2020), and the TCA cycle (citrate synthase 1 (RiCIT1))—in extraradical hyphae of R. irregularis from the HC, with or without myristate addition, was assessed, using the elongation factor-1 beta gene (RiEF-1β) as the reference gene (Das et al., 2022). Relative gene expression levels were calculated using the comparative 2^−ΔΔCt method (Livak and Schmittgen, 2001), with four independent biological replicates and three technical replicates per biological replicate. The collection of extraradical hyphae from the HC and qRT-PCR procedures is detailed in the Supporting Information (Method S3, Table S1).

Carrot hairy roots in the RC from the two-compartment AMF-hairy root co-culture system (Exp. 1) were also harvested using sterile tweezers and used to assess AMF colonization intensity (see Supporting Information Methods S4).

Exp. 2: Effects of exogenous myristate on AMF development in single-compartment AMF-hairy root co-culture systems

In Exp. 2, we assessed the effects of exogenous myristate at varying concentrations on spore germination and the development of R. irregularis intraradical and extraradical structures associated with carrot hairy roots in a single-compartment co-culture system. Briefly, three 4-cm-long sterile carrot hairy root segments were cultivated in MSR medium with or without R. irregularis inoculation (200 sterilized spores per Petri dish). Potassium myristate solution (0, 0.1, 0.5, 1, 5, or 10 mM, 200 μL) was applied to the plates weekly until harvest, with five biological replicates (plates) per treatment. The germination rate of AMF spores, as well as extraradical hyphal branching and length, were monitored under a stereomicroscope (M250FA, Leica, Germany) and calculated using ImageJ at 4 days post-inoculation (Schneider et al., 2012). Spore density was monitored every 2 weeks starting 8 weeks after AMF inoculation.

Carrot hairy roots and MSR medium were harvested at 14 weeks post-AMF inoculation. The roots and solid culture medium were carefully separated using sterile tweezers. The medium was weighed and dissolved in 10 mM sodium citrate, and the AMF collection was preliminarily separated into hyphae and spores using a blender, followed by filtration through a 250 µm and 37 µm nylon meshes, respectively, and manual selection with tweezers under a stereomicroscope. Biomasses of the extraradical hyphae and spores were oven-dried at 80°C for 48 hours and quantified separately. To evaluate the development of AMF intraradical structures in the carrot hairy roots, hyphal, vesicle, and arbuscular colonization intensities were assessed (Supporting Information Methods S4).

Exp. 3: Environmental distribution of myristate and the effect of exogenous myristate on AM symbiosis

In Exp. 3, a field survey and a pot experiment were conducted. The field survey aimed to evaluate the environmental distribution of myristate in soil and plants. We analyzed myristate concentrations in bulk soil (0-10 cm) and in plant leaves, roots, leaf litter, and rhizosphere soil from the dominant species O. sativa, Axonopus compressus, and Morus alba in a paddy field, grassland, and woodland, respectively. The procedures regarding sample collection, pretreatment, and analysis of myristate were detailed in Supporting Information Methods S5.

The pot experiment in Exp. 3 was conducted to assess the effect of exogenous myristate on C-P nutritional exchanges in AM symbiosis between R. irregularis and two phylogenetically distant plant species—the AM-responsive plant Medicago sativa L. cv. Sanditi (alfalfa, dicot) and the AM-nonresponsive plant Oryza sativa L. cv. Zhonghua 11 (rice, monocot) (Hata et al., 2010). This was achieved using ¹³C-neutral lipid fatty acid (NLFA)-isotope ratio mass spectrometry technology, along with physiological and transcriptome analyses. The pot experiment employed a full factorial design with four treatment factors: myristate addition (with or without 0.1 mM myristate), AMF inoculation (with or without R. irregularis), host species (alfalfa or rice), and soil P level (relatively low or high). Ten biological replicates were included per treatment, totaling 160 pots. The experimental procedures for plant cultivation, P or myristate treatment, plant harvest, and measurement of plant P concentration are detailed in Supporting Information Methods S6.

RNA sequencing (RNA-seq) on alfalfa and rice roots and bioinformatics (Exp. 3)

In Exp. 3, alfalfa and rice roots inoculated with AMF, with or without myristate application at two P levels, were subjected to RNA sequencing (RNA-seq). Three biological replicates (roots from a single pot per replicate) were randomly selected from ten replicates per treatment, resulting in a total of 24 samples. The procedures for RNA-seq and bioinformatics followed our previous study (Wang et al., 2023), with details provided in Supporting Information Methods S7.

Quantitative real-time PCR (qRT-PCR, Exp. 3)

In Exp. 3, RNA extracted from alfalfa or rice roots was reverse-transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA). qRT-PCR was performed, with three independent biological replicates (randomly selected from ten replicates) for each treatment and three technical replicates per biological replicate. The relative expression levels of plant and fungal genes involved in C delivery from plants to AMF (MsSTR2, OsSTR2, RiFAT1, RiFAT2, and RiMST2) (Brands and Dörmann, 2022; Das et al., 2022), the mycorrhizal P pathway (MsPT8, OsPT11, and RiPT) (Yang et al., 2012; Fiorilli et al., 2013), and AM-activated plant defense— specifically, reactive oxygen species balance (MsAOX3), stress responses (MsUGT85A, MsPRXP7, and OsPRX39), and typical defense marker (OsCHT1 and OsTGA2) (Rehman et al., 2018; Suleman et al., 2021; Zhang et al., 2021)—were analyzed to characterize the impact of exogenous myristate on C-P nutritional exchange in AM symbiosis and the underlying mechanisms at the transcriptional level. The alfalfa actin 2 (MsACT2), rice cyclin 2 (OsCyc2), or R. irregularis elongation factor-1 beta (RiEF-1β) genes were used as reference genes (Yang et al., 2012; Das et al., 2022), with the procedures and primers provided in the Supporting Information (Method S3, Table S1).

13C labeling of plants, lipid extraction, measurement of AMF signature fatty acids, and quantification of ¹³C enrichment and ¹³C flow (Exp. 3)

In Exp. 3, ¹³C labeling of plants, lipid extraction, measurement of AMF signature fatty acids (NLFA 16:1ω5, Olsson et al., 2005), and quantification of ¹³C enrichment and ¹³C flow to NLFA 16:1ω5 were performed as described in previous studies (Olsson et al., 2005; Bao et al., 2019), with details provided in Supporting Information Methods S8.

Statistical analysis

Mycorrhizal response was calculated based on plant biomass (mycorrhizal growth response) and plant P concentration (mycorrhizal P response), respectively, following Corrêa et al. (2014): Mycorrhizal response (%) = (AM − NM) / NM × 100%, where AM denotes the observed value for the target parameter (biomass or P concentration) in mycorrhizal plants, and NM represents the mean value of nonmycorrhizal plants. Multiple comparisons among different treatments were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (equal variances) or Games-Howell’s post hoc (unequal variances) test. A univariate two-way ANOVA, followed by a post hoc Tukey test, was employed to assess the effects of myristate, P level, and their interactions on AMF colonization intensity and plant P concentration. The significant differences between AM and NM groups were determined using Student’s t test (for normally distributed data) or Mann-Whitney U-test (for non-normally distributed data).

Data availability

The RNA-seq data generated in this study have been deposited in the Sequence Read Archive at the National Center for Biotechnology Information under BioProject ID PRJNA1083641. The raw data are provided as a Source Data File.

Acknowledgements

We thank the financial support from the National Key R&D Program of China (Key special Project for Marine Environmental Security and Sustainable Development of Coral Reefs 2022-702), the National Natural Science Foundation of China (nos. 32272203 and 31772397), the Fujian Province Science and Technology Plan Project (no. 2024I1011), the Key-Area Research and Development Program of Guangdong Province (2022B0202110001), the Agricultural and Social Development Project of Guangzhou Municipal Science and Technology Bureau (no. 202206010058), and the Guangdong Provincial Key Laboratory of Plant Resources (no. 2023PlantKFP03).

Additional information

Author Contributions

• Hanwen Chen: Investigation, Formal analysis, Methodology, Visualization.

• Tian Xiong: Investigation, Formal analysis, Data curation, Methodology.

• Baoxing Guan: Writing – original draft, Investigation, Formal analysis, Data curation, Visualization.

• Jiaqi Huang: Investigation, Methodology, Resources.

• Danrui Zhao: Investigation, Resources.

• Yao Chen: Investigation, Resources.

• Haoran Liang: Investigation, Visualization.

• Yingwei Li: Investigation, Resources.

• Jingwen Wu: Investigation, Resources.

• Shaoping Ye: Resources.

• Ting Li: Resources.

• Wensheng Shu: Supervision.

• Jin-tian Li: Supervision.

• Yutao Wang: Conceptualization, Data curation, Writing – original draft, Writing – review & editing, Funding acquisition, Supervision, Project administration.

Funding

National Key R&D Program of China (Key special Project for Marine Environmental Security and Sustainable Development of Coral Reefs) (2022-702)

• Yutao Wang

MOST | National Natural Science Foundation of China (NSFC) (32272203)

• Yutao Wang

MOST | National Natural Science Foundation of China (NSFC) (31772397)

• Yutao Wang

Fujian Province Science and Technology Plan of Guangdong Province (2022B0202110001)

• Yutao Wang

Agricultural and Social Development Project of Guangzhou Municipal Science and Technology Bureau (202206010058)

• Yutao Wang

Guangdong Provincial Key Laboratory of Plant Resources (2023PlantKFP03)

• Yutao Wang

Additional files

Supporting Information