Skip to main content

Advertisement

A coumarin analogue NFA from endophytic Aspergillus fumigatus improves drought resistance in rice as an antioxidant

Abstract

Background

Drought and its resulting oxidative damage are the major yield limiting factors for crops in arid and semi-arid regions. Recent studies have found that endophytic fungi coexisting in plants can alleviate biotic or abiotic damage to plant growth and development. In order to screen for the endophytes associated with drought stress, 12 strains of endophytic fungi with high antioxidant activity isolated from riparian plants Myricaria laxiflora were evaluated for their effects in rice by the crude extracts.

Results

Of the 12 endophytic fungi, Aspergillus fumigatus SG-17 functioned most effectively, with the crude extract exhibiting relatively higher antioxidant capacity both in vivo and in vitro. The subsequent MS and NMR analysis showed that the primary substance responsible for the antioxidant activity in the extract was (Z)-N-(4-hydroxystyryl) formamide (NFA), an analogue of coumarin. Enzyme activity assay in nerve cells SH-SY5Y showed that NFA could maintain the membrane integrity and regulate the antioxidase activity under oxidative stress. In rice suffering drought stress, NFA effectively alleviated the harm by regulating the contents of NADPH oxidases, antioxidants and heat shock proteins, all of which are closely related with the reactive oxygen species pathway.

Conclusion

These findings indicated that some endophytes from plants often subjected to flooding and oxidative stress could enhance drought resistance by producing compounds such as NFA to regulate the oxidative pathway.

Background

Endophytic fungi can be beneficial to the host plant through evolutionary adaptation [13, 26]. They have a rich biodiversity [9, 32], and actively regulate the growth and development of the host or other plants [12, 40]. The metabolites present in endophytic fungi exhibit extensive physiological activity, such as anti-bacterial, anti-tumor, pesticidal, immunosuppressive and antioxidative effects [18, 32, 42]. Over the past few decades, endophytic fungi have become a potential resource for development of new drugs [10, 23]. In particular, they can improve the ability of plants to resist biotic or abiotic stresses [31, 38].

Drought is a yield limiting factor to rice [11, 15]. Physiological indexes such as Proline (Pro) content, malondialdehyde (MDA) content, membrane relative permeability (MRP), relative water content (RWC) and antioxidant enzyme activity are closely involved in drought response [45]. Under drought stress, the Pro and MDA content and MRP increase, while RWC declines. There is also a strong link between the damage by drought and the metabolic balance of reactive oxygen species (ROS) [21], such as hydrogen peroxide (H2O2), superoxide anion (O2), singlet oxygen (1O2) and hydroxyl radical (•OH) [21]. Therefore, to some extent, drought resistance is dependent on the antioxidative ability [21]. However, ROS has a dual function in plants [24]. On one hand, they can trigger oxidative bursts that improve resistance to stress [5]; on the other hand, they can induce membrane lipid peroxidation and cause a detrimental effect [33]. Thus, the dynamic equilibrium of ROS is crucial to plants, and the stronger the antioxidant capacity is, the higher the stress tolerance would be [33]. ROS is mainly produced by NADPH oxidase [39], and eliminated by both non-enzymatic antioxidants, such as polysaccharides, flavonoids, polyphenols, alkaloids, saponins and vitamins [18, 21, 41], and antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD; [1, 33]).

Heat shock proteins (HSPs) are a class of proteins which are activated when plants are subjected to adversity [22]. As “molecular chaperones”, HSPs are often involved in folding, refolding, processing and transporting proteins [22]. Under drought stress, HSPs are actively involved in ROS metabolism in two ways: they affect the synthesis of ROS by inhibiting the activity of NADPH oxidase [14], and promote the expression of antioxidant enzymes related to ROS clearance and subsequent apoptotic metabolism [6, 14]. Thus, they have important applied value, and can provide a theoretical basis for selecting strongly resistant plant varieties [19]. Among them, HSP70 plays an important role in plant drought resistance [4, 29].

Recent reports have shown that the metabolites present in endophytic fungi are closely related to the habitat of the host plant [10]. Myricaria laxiflora is a shrub distributed specifically in the water draw-down zone around the Three Gorges Reservoir [34]. The plants can survive in nearly 6 months of seasonal flooding, indicating a strong adaptive ability to cope with deficient oxygen and the resulting oxidative stress [34]. As expected, in a previous study many endophytic fungi with high antioxidant activity were isolated from M. laxiflora [43].

Aspergillus fumigatus is commonly found in moist environments, negatively impacting yields and quality of crops [20]. Here, we found that the crude extract of endophytic A. fumigatus strain SG-17 had strong antioxidant activity, reaching 31.86% of that of vitamin C (Vc), and could help rice resist drought stress [8]. The main substance involved was an analogue of coumarin, named (Z)-N-(4-hydroxystyryl) formamide (NFA), which had been isolated from Streptomyces amakusaensis as a selective antibiotic treatment of Mycobacterium involved in tuberculosis [3]. NFA was also found in A. finnigatus as an inhibitor against rabbit platelet aggregation induced by arachidonic acid and collagen [35]. In the salt-tolerant fungus Penicillium chrysogenum from the Yellow River Delta [30] and in the marine-derived endophytic fungi [2], NFA was discovered to exhibit antibacterial activity [30], as well as moderate cytotoxicity against Du145, A-549 and Hela cell lines [2]. So far, there are few reports on the antioxidant activity of NFA. Here, we found that NFA from A. fumigatus was able to effectively alleviate drought stress in rice. This role may be mediated by regulation of oxidative pathway, involving antioxidant enzymes, HSP70 and NADPH oxidase. These findings indicate that the endophytic fungi from plants adapted to oxidative stress have a positive effect on plant resistance to drought stress.

Methods

Strain resource and medium

Twelve endophytic fungi from various tissues of M. laxiflora pre- and post- flooding [34] were used in a preliminarily screening (Table 1). With the permission of the local forestry administration, the plant samples were collected from the island of Yanzhiba, located in 109°32′E to 110°52′E and 30° 53′N to 31 °3′N. Dr. Wang YB of Biotechnology Center in China Three Gorges University carried out the formal identification, and the specimens (LSP201404220) was deposited in the Herbarium of Three Gorges University. All endophytic strains were stored at 4 °C in paraffin. The storage and activation medium was potato dextrose agar (PDA), comprising 200 g potato, 20 g glucose and 15–20 g agar, made up with distilled water to 1000 mL. The pH was adjusted to 6.0 and the medium was sterilized at 121 °C for 20 min. Sabouraud’s dextrose (SD) medium was used for liquid fermentation, which consisted of 10 g peptone and 40 g of glucose, made up with distilled water to 1000 mL. D-MEM/F-12 complete medium, used to culture nerve cells SH-SY5Y for the in vivo antioxidant activity assay, was sterilized by filtration through 0.45 μm microporous membranes [16].

Table 1 Characteristics of twelve endophytes isolated from M. laxiflora

Preparation of the crude extract

Hyphae of the 12 endophytic fungi were inoculated individually in 150 mL of SDA liquid medium, and then grown for 14 d at 28 °C. Fungi in the fermented liquid were harvested by suction and extracted 3 times with an equal volume of ethyl acetate. The organic phases were combined and distilled under vacuum at 40 °C to harvest the extraction of ethyl acetate.

Antioxidant activity measurement

Both in vitro and in vivo methods were employed to evaluate the antioxidative ability. For in vitro assessment, total antioxidant capacity (T-AOC) kit [28, 43] and 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) radical scavenging kit [36] were used to screen the fermented broth and the crude extract, respectively, by following the manufacturer’s instructions. Both kits were from Nanjing Jiancheng Bioengineering Institute, Nanjing, China (http://www.njjcbio.com/). The in vivo antioxidant activity was determined by nerve cells SH-SY5Y according to the manufacturer’s instructions (Beyotime Biotechnology Company, Shanghai, China, http://beyotime.bioon.com.cn/) [16]. In order to characterize the possible mechanism of antioxidant protective effect on nerve cells, we determined cysteine-dependent aspartate-directed protease (Caspase) 3, Caspase 9, SOD and lactate dehydrogenase (LDH) activity [16]. LDH leakage rate was regarded as a reliable indicator of cellular membrane integrity, and calculated as [16]: OD value of the supernatant in the medium/OD value of the total cells × 100%, and data were normalized from control (100%). All the results were replicated 3 times.

Drought resistance analysis in rice

Seeds of rice cultivar Nipponbare were placed first in 1% nitric acid for 12 h and then in tap water for 2 d to break dormancy. The rooting seeds were planted into the plastic cups or pots containing about 100 g of local soil, and then grew in a light incubator (30 °C, 30,000 lx light intensity and approximately 60% relative humidity) for 20 d. The volume of the cup was 245 cm3 with a height of 9.5 cm. Before transferred to the cups, the soil had been air-dried and well-mixed. After 20 d, rice seedlings with similar size were selected and grown for another 20 d under drought stress. One day before drought treatment, 50 mL of 0.1 mg/mL treatment solution (crude extract or NFA) or the living hyphae were applied to the seedlings. Proline solution at 50 μM was used as a positive control. In order to compare the effects of other antioxidants, 0.1 mg/mL Vc solution was applied to rice seedlings. In the drought period, seedlings were deprived of water. Each treatment was replicated 3 times.

Physiological indexes investigation

After 20-d drought stress at room temperature, the survival rate of rice seedlings and physiological indexes were investigated. Pro and MDA contents were assayed using kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Analysis of SOD, POD, HSP70 and NADPH oxidase was preformed using kits from Jiangsu Baolai Biotechnology Co., Ltd. (Jiangsu, China). RWC was calculated based on the equation: RWC (%) = [(Fresh weight − dry weight)/(turgid weight − dry weight)] × 100 [7]. MRP (%) was measured according to published methods [45], and calculated as: Conductivity of leakage before death/ Conductivity of leakage of after death× 100.

Isolation and identification of the primary substance from SG-17

The SG-17 crude extract was firstly separated by preparative thin layer chromatography (Pr-TLC) with the expansion condition of petroleum ether: acetone = 1: 1 (V/V). Four bands were visible when the Pr-TLC plate was held under a UV 254 nm lamp. These bands were eluted by methanol to obtain four fractions (Fraction1~4, respectively). After in vitro antioxidant activity analysis using T-AOC and DPPH method, fraction 2 with the strongest activity was separated and purified by semi-preparative high performance liquid chromatography (HPLC) (250 × 10 mm id, Cosmosil MS-II). The conditions were as follows: mobile phase, acetonitrile: water = 60:40 (V/V) with a flow rate of 3.0 mL/min and a UV detection wavelength of 254 nm. The isolated and purified compounds by HPLC were freeze-dried, then dissolved in 0.5 mL deuterium DMSO. 1H-NMR and 13C-NMR were determined by Bruker AVANCE 400 MHz nuclear magnetic resonance (NMR) spectroscopy. All reagents were analytical grade, except that acetonitrile and methanol used for high HPLC were chromatography-grade. The water used was triple-distilled.

Data processing

The data were expressed as mean ± standard deviation. Significant differences among groups were calculated using One-Way ANOVA, followed by multiple comparisons using Ducan’s test, provided by the statistical software SPSS 20.0 (For windows version, SPSS Inc., Chicago, IL, USA).

Results

Preliminary screening of fungi assisting rice against drought stress

In the previous study, 163 endophytic fungi from various tissues of M. laxiflora pre- and post- flooding were isolated [34]. Here, 12 strains were selected for their relatively high antioxidant activity (43, Table 1). To evaluate the contribution to drought tolerance in rice, the living hyphae or the crude extracts of 12 fungi were applied to the seedlings. After 20-d drought stress at room temperature, none of the living hyphae had any effect on drought response (Fig. 1). However, crude extracts of QY-1, SG-4, SG-17, and SY-15 increased the survival rate of rice under drought stress (Table 1). The most effective strain was SG17, identified as an A. fumigatus [9], by which the effect was nearly equivalent to the positive control of Proline (Fig. 1). Besides, its crude extract did not affect the normal growth of rice (Fig. 1).

Fig. 1
figure1

Effect of SG-17 crude extract on rice against drought stress. a Rice seedlings after 20 days of drought. b Drought + SG-17 crude extract. c Drought + Proline. d Well watered + SG-17 crude extract. e Drought + SG-17 living hyphae. f Well watered seedlings. g Dry weight (DW) of the seedlings, * means significant difference with P < 0.05 and ** for P < 0.01 compared with drought group, n = 3

Effects of SG-17 crude extract on rice susceptibility to drought

To analyze the effects of SG-17 crude extract, we investigated some physiological indexes of rice seedlings after 20 days drought treatment. Under drought conditions, Proline content of rice seedlings treated with the crude extract was significantly lower than that of the untreated control, and was even lower than that of seedlings treated with Proline (Fig. 2a). Similar results were found on the indexes of MDA and MRP (Fig. 2b and c), indicating that SG-17 crude extract could effectively enhance drought tolerance of rice. This effect was even better than the positive control of Proline. The conclusion was further supported by the result of RWC, which was significantly higher in the crude extract treated samples than in the untreated or positive (+Pro) control (Fig. 2d). It was also noted that RWC in the seedlings of Dr. + SG17 was even higher than in those well watered, implying that SG-17 extracts could trigger a certain water metabolism to keep rice away from drought.

Fig. 2
figure2

Physiological analysis of rice against drought by SG-17 crude extract. a Proline content. Dr.: seedlings subjected to drought for 20 days. Dr. + SG-17: Drought + SG-17 crude extract. Pro: Drought + Proline. Normal: well watered. Ducan’s multiple range test, p < 0.05 and n = 3. b MDA content. c Membrane relative permeability. d Relative water content

Antioxidant activity of SG-17 crude extract and the constituents in vitro

The antioxidant activity of SG-17 crude extract was measured by the T-AOC kit and the DPPH radical scavenging kit in vitro. The extract showed a certain degree of antioxidant activity. Although the T-AOC value of the extract was lower than Vc, the DPPH free radical scavenging rate reached 90.57%, almost equivalent to that of Vc (Table 2). After separation by thin layer chromatography and HPLC, four fractions were purified from the extract. Through tracing the antioxidant activity, we found that all four fractions exhibited a certain degree of antioxidant ability. Among them, fraction 2 had the highest activity, with the T-AOC value over 16 times more than that of Vc (Table 2).

Table 2 Antioxidant activity of SG-17 crude extract assayed by two methods

Antioxidant activity of fraction 2 in vivo

We determined the antioxidant protective effect of fraction 2 on nerve cells SH-SY5Y subjected to oxidative stress induced by 800 μM H2O2. Although, under normal conditions without H2O2, fraction 2 at 12.50 μg/mL showed some toxicity to cell growth (Fig. 3a), it increased the relative survival rate of SH-SY5Y by 56.49% in the presence of oxidative stress (Fig. 3a), exhibiting an antioxidant protective effect on nerve cells. This result suggested that the underlying substance in fraction 2 may function through alleviating the oxidative damage.

Fig. 3
figure3

Effect of fraction 2 in protecting SH-SY5Y cells from oxidative stress. a Various concentration of fraction 2 and the resultant relative survival rate with or without simulated oxidant, t-test, * means P < 0.05, and ** for 0.01. b Leakage rate of lactate dehydrogenase under oxidant stress. Enzyme activity of SOD (c), Caspase 3 (d) and Caspase 9 (e) response to simulated oxidant and fraction 2

In order to identify the potential antioxidant mechanism, we measured the LDH leakage rate and enzyme activity of SOD, apoptotic protein caspase 3 and caspase 9 in SH-SY5Y cells upon treatment of 12.50 μg/mL fraction 2. We found that fraction 2 decreased the LDH leakage rate by 81.5% under oxidative stress (Fig. 3b), suggesting that fraction 2 could maintain membrane integrity (Fig. 3b). The activity of SOD was also enhanced by fraction 2 treatment under oxidative stress (Fig. 3c), while that of caspase 3 or caspase 9 was not affected (Fig. 3d and e). These results suggested that fraction 2 may be involved in antioxidant metabolic pathways mainly through maintaining membrane integrity and regulating the activity of antioxidant enzymes.

Structure determination of NFA

Based on the above results, we speculated that fraction 2 isolated from SG-17 crude extract by thin layer chromatography (TLC) might contain the main constituent responsible for the antioxidant activity. Chromatographic analysis showed that the purity of this fraction had got to 98%. After MS and NMR analysis, the structure of fraction 2 was confirmed as a coumarin analogue named (Z)-N-(4-hydroxystyryl) formamide (NFA, Fig. 4a) according to the spectral characteristics (Fig. 4f), which was consistent with previous data [30, 35]. Moreover, it was reported that NFA can gradually transform to an isomer (E)-N-(4-hydroxystyryl) formamide (ENFA) ([30, 35], Fig. 4b). Here, similar results were found. In TLC seperation, ENFA appeared below NFA after 1 day at room temperature (Fig. 4c and d). In semi-preparative HPLC analysis, both compounds were eluted almost together (Fig. 4e), with the retention time of ENFA being about 9.58–10.00 min and that of NFA about 10.78–11.30 min (Fig. 4e).

Fig. 4
figure4

Identification of fraction 2 as NFA. a Structure of NFA. b Structure of ENFA. Separation of NFA (c) and ENFA (d) by TLC. e Separation of fraction 2 by semi-preparative HPLC. f 1H-NMR and 13C-NMR data of compound fraction 2

Effect of NFA on rice resistance to drought stress

In order to prove that NFA is responsible for the effects against drought stress, we applied the purified NFA solution directly to rice seedlings. After drought for 20 days, most seedlings even treated by Vc died, whereas some in the NFA treatment survived and grew better than that of the positive control Proline (Fig. 5), indicating that NFA could effectively protect rice from drought damage. To an extent, the effect of NFA was superior to that of the osmotic modulator, Proline. After applying NFA, the contents of Pro and MDA were significantly lower than those of other treatments under drought stress (Fig. 2a and b). Besides, NFA decreased the membrane relative permeability (Fig. 2c), indicating a protective effect on membrane integrity under drought. Moreover, during drought stress, NFA maintained relatively high water content in rice leaves (Fig. 2d). These results proved that NFA was able to effectively assist rice in drought resistance.

Fig. 5
figure5

Effects of NFA on rice resistance to drought stress. a Well watered rice seedlings. b Seedlings under drought. c Drought + NFA. d Drought + Proline. e Drought + Vc

NFA regulated oxidative pathway of rice under drought stress

It has been known that drought is closely associated with oxidative stress [15, 39]. To interrogate the relationship between NFA and the oxidative pathway under drought adversity, we measured the dynamic activity of SOD, POD, HSP70 and NADPH oxidase in rice seedlings stressed by drought. After 15 days of drought, the enzyme activity of SOD was significantly higher for NFA-treated group (P < 0.01) compared with other groups (Fig. 6a). The variation of POD enzyme activity had a similar tendency after 10 days (Fig. 6b). Both SOD and POD are important scavengers of ROS induced by drought [33], therefore, NFA could help rice resist drought possibly by regulating antioxidant enzymes involved in the oxidative pathway.

Fig. 6
figure6

Oxidative metabolism affected by NFA in rice under drought adversity. Dynamic data were acquired after drought for 5, 10, 15 and 20 days. a SOD enzyme activity. b POD enzyme activity. c NADPH oxidase content. d HSP70 content

Once plants suffer from drought, NADPH oxidase will be induced to produce a certain range of ROS [39]. Here, the NADPH oxidase content after treatment with NFA was significantly increased (P < 0.01), especially after 5 days of drought (Fig. 6c). This result indicated that in early drought, NFA could upregulate NADPH oxidase to produce ROS and subsequently participate in the anti-stress metabolism. In early drought not longer than 5 days, NFA also significantly induced the expression (P < 0.01) of HSP70 (Fig. 6d), which is known to be extensively involved in the metabolism of ROS [4, 19]. This result further suggested that NFA enhanced drought resistance in rice through regulation of oxidative pathway.

Discussion

Recent studies have shown that endophytic fungi in plants are species-rich, and show great utilization potential in medicine, food and agriculture [37, 44]. In this study, we showed that the endophytic fungus A. fumigatus isolated from M. laxiflora, a plant tolerant to hypoxia stress, exhibited strong in vivo and in vitro antioxidant activity. By producing the active compound NFA, the fungus could effectively assist rice to resist drought.

To our knowledge, by far no one has reported the antioxidant activity of NFA, nor its effect on drought resistance in plants. In this study, we identified an antioxidant NFA from the endophytic fungus A. fumigatus and found that it could maintain membrane integrity, and regulate the contents of NADPH oxidase, antioxidases and HSPs. As NADPH oxidase directly controlls the ROS production, whereas antioxidase and HSPs affect the degradations of ROS, NFA alleviating drought stress in rice may be mediated by regulation of oxidative pathway. Our physiological data indicated that the drought resistance of rice conferred by NFA might be attributed to NFA’s dual effects: it induced NADPH oxidase after a short period of drought, while activated antioxidant enzyme system to eliminate ROS after a long period of drought (Fig. 6c and d). These findings are consistent with the dual role of ROS, implying that NFA may regulate the homeostasis of ROS in a drought stage-dependent manner.

In addition to A. fumigatus SG-17, other endophytic fungi, such as SG-4, SY-15 and QY-1, could help rice resist drought stress as well. In rice anti-flooding test, we obtained similar results (data not shown). Thus, endophytic fungi, especially those in the special habitat plant M. laxiflora, are of potential application value in oxidative stress. It is worth to mention that although SG-17 did not show the highest antioxidant activity, it generated the best effect of enhancing the drought resistance of rice among the 12 endophytic fungi tested.

In identifying the structure of fraction 2, the antioxidant activity determined by two in vitro methods of T-AOC and DPPH differed. Although NFA exhibited relatively high T-AOC value, its free radical scavenging rate was lower. The possible reason was that when measured by DPPH method, some substances might be generated in the process, which deepened the solution color (Additional file 1: Figure S1), resulting in less photometric count.

In this study, both HSP70 and NADPH oxidase in rice were induced by NFA at the early stages of drought stress. Hence, HSP70 is unlikely to inhibit the synthesis of NADPH oxidase. The roles of HSP70 in the antioxidative pathway need further research. Meanwhile, there are a series of enzymes associated with oxidative stress, such as mitogen-activated protein kinase (MAPK) [25], rho-related GTPase from plants (ROP) [27], apoptosis proteins [17], and so on. To further clarify the mechanism of drought resistance mediated by NFA, activities of these downstream enzyme families need to be studied in the future.

Conclusions

To maintain stable crop yields and guarantee global food security, it is of great significance to improve drought resistance via clarifying the underlying mechanisms. Here, a new antioxidant, NFA, could alleviate drought stress in rice by regulation of oxidative pathway. The antioxidant activity and the physiological effects on plants of NFA were analyzed in detail, potentially providing a new clue for antioxidant development by chemiecology and for our understanding of the symbiosis between endophytic fungi and host plants subjected to oxidative stress.

Abbreviations

A. fumigatus :

Aspergillus fumigatus

Caspase:

Cysteinyl aspartate specific proteinase

DPPH:

1, 1-diphenyl-2-picrylhydrazyl

ENFA:

(E)-N-(4-hydroxystyryl) formamide

HPLC:

High performance liquid chromatography

HSP:

Heat shock proteins

LDH:

Lactate dehydrogenase

M. laxiflora :

Myricaria laxiflora

MDA:

Malondialdehyde

MRP:

Membrane relative permeability

NFA:

(Z)-N-(4-hydroxystyryl) formamide

PDA:

Potato dextrose agar

POD:

Peroxidase

Pro:

Proline

ROS:

Reactive oxygen species

RWC:

Relative water content

SD:

Sabouraud’s dextrose

SOD:

Superoxide dismutase

T-AOC:

Total antioxidant capacity

Vc:

Vitamin C

References

  1. 1.

    Alscher RG, Erturk N, Heath LS. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot. 2002;53:1331–41.

  2. 2.

    An CY, Li XM, Li CS, Gao SS, Shang Z, Wang BG. Triazoles and other N-containing metabolites from the marine-derived endophytic fungus Penicillium chrysogenum EN-118. Helvetica Chimica Acta. 2013;96:682–7.

  3. 3.

    Anzai K, Okuma K, Nagatsu J, Suzuki S. Chemical structure of tuberin. J. Antibiot. 1962;15:110–1.

  4. 4.

    Augustine SM, Cherian AV, Syamaladevi DP, Subramonian N. Erianthus arundinaceus HSP70 (EaHSP70) acts as a key regulator in the formation of anisotropic interdigitation in sugarcane (Saccharum spp. hybrid) in response to drought stress. Plant Cell Physiol. 2015;56:2368–80.

  5. 5.

    Brosché M, Kangasjärvi J. Low antioxidant concentrations impact on multiple signaling pathways in Arabidopsis thaliana partly through NPR1. J Exp Bot. 2012;63:1849–61.

  6. 6.

    Chen F, Yu Y, Qian J, Wang Y, Cheng B, Dimitropoulou C, Patel V, Chadli A, Rudic RD, Stepp DW, Catravas JD, Fulton DJ. Opposing actions of heat shock protein 90 and 70 regulate nicotinamide adenine dinucleotide phosphate oxidase stability and reactive oxygen species production. Arterioscler Thromb Vasc Biol. 2012;32:2989–99.

  7. 7.

    Gao JF. 2006. Plant physiology experiment guide, Higher Education Press, Beijing, China.

  8. 8.

    Gao Y, Lei Q, Jiang W, Kong YS, Xue YH, Liu SP. Molecular characterization and phenolic acids analysis of an endophytic fungus with high antioxidant activity. Microbiol China. 2016;43:1235–43.

  9. 9.

    García A, Rhoden SA, Rubin CJ, Nakamura CV, Pamphile JA. Diversity of foliar endophytic fungi from the medicinal plant Sapindus saponaria L. and their localization by scanning electron microscopy. Biol Res. 2012;45:139–48.

  10. 10.

    Gouda S, Das G, Sen SK, Shin HS, Patra JK. Endophytes: a treasure house of bioactive compounds of medicinal importance. Front Microbiol. 2016;7:1538.

  11. 11.

    Guan YS, Serraj R, Liu SH, Xu JL, Ali J, Wang WS, Venus E, Zhu LH, Li ZK. Simultaneously improving yield under drought stress and non-stress conditions: a case study of rice (Oryza sativa L.). J Exp Bot. 2010;61:4145–56.

  12. 12.

    Hanada RE, Pomella AW, Costa HS, Bezerra JL, Loguercio LL, Pereira JO. Endophytic fungal diversity in Theobroma cacao (cacao) and T. grandiflorum (cupuacu) trees and their potential for growth promotion and biocontrol of black-pod disease. Fungal Biol. 2010;114:901–10.

  13. 13.

    Herre EA, Mejia LC, Kyllo DA, Rojas E, Maynard Z, Butler A, Van Bael SA. Ecological implications of anti-pathogen effects of tropical fungal endophytes and mycorrhizae. Ecology. 2007;88:550–8.

  14. 14.

    Hsieh LT, Frey H, Nastase MV, Tredup C, Hoffmann A, Poluzzi C, Zeng-Brouwers J, Manon-Jensen T, Schröder K, Brandes RP, Iozzo RV, Schaefer L. Bimodal role of NADPH oxidases in the regulation of biglycan-triggered IL-1β synthesis. Matrix Biol. 2016;49:61–81.

  15. 15.

    Hu Y, Wu Q, Peng Z, Sprague SA, Wang W, Park J, Akhunov E, Jagadish KSV, Nakata PA, Cheng N, Hirschi KD, White FF, Park S. Silencing of OsGRXS17 in rice improves drought stress tolerance by modulating ROS accumulation and stomatal closure. Sci Rep. 2017;7:15950.

  16. 16.

    Huang SL, He HB, Zou K, Bai CH, Xue YH, Wang JZ, Chen JF. Protective effect of tomatine against hydrogen peroxide-induced neurotoxicity in neuroblastoma (SH-SY5Y) cells. J Pharm Pharmacol. 2014;66:844–54.

  17. 17.

    Huang WR, Zhang Y, Tang X. Shikonin inhibits the proliferation of human lens epithelial cells by inducing apoptosis through ROS and caspase-dependent pathway. Molecules. 2014;19:7785–97.

  18. 18.

    Huang WY, Cai YZ, Xing J, Corke H, Sun M. A potential antioxidant resource: endophytic fungi from medicinal plants. Econ Bot. 2007;61:14–30.

  19. 19.

    Jungkunz I, Link K, Vogel F, Voll LM, Sonnewald S, Sonnewald U. AtHsp70-15-deficient Arabidopsis plants are characterized by reduced growth, a constitutive cytosolic protein response and enhanced resistance to TuMV. Plant J. 2011;66:983–95.

  20. 20.

    Liang Z, Zhang T, Zhang X, Zhang J, Zhao C. An alkaloid and a steroid from the endophytic fungus Aspergillus fumigatus. Molecules. 2015;20:1424–33.

  21. 21.

    Liu Y, He C. Regulation of plant reactive oxygen species (ROS) in stress responses: learning from AtRBOHD. Plant Cell Rep. 2016;35:995–1007.

  22. 22.

    McLellan CA, Turbyville TJ, Wijeratne EM, Kerschen A, Vierling E, Queitsch C, Whitesell L, Gunatilaka AA. A rhizosphere fungus enhances Arabidopsis thermotolerance through production of an HSP90 inhibitor. Plant Physiol. 2007;145:174–82.

  23. 23.

    Mei C, Flinn BS. The use of beneficial microbial endophytes for plant biomass and stress tolerance improvement. Recent Pat Biotechnol. 2010;4:81–95.

  24. 24.

    Mittler R, Blumwald E. The roles of ROS and ABA in systemic acquired acclimation. Plant Cell. 2015;27:64–70.

  25. 25.

    Nagano M, Ishikawa T, Fujiwara M, Fukao Y, Kawano Y, Kawai-Yamada M, Shimamoto K. Plasma membrane microdomains are essential for Rac1-RbohB/H-mediated immunity in rice. Plant Cell. 2016;28:1966–83.

  26. 26.

    Nisa H, Kamili AN, Nawchoo IA, Shafi S, Shameem N, Bandh SA. Fungal endophytes as prolific source of phytochemicals and other bioactive natural products: a review. Microb Pathog. 2015;82:50–9.

  27. 27.

    Oda Y, Fukuda H. Rho of plant GTPase signaling regulates the behavior of Arabidopsis kinesin-13A to establish secondary cell wall patterns. Plant Cell. 2013;25:4439–50.

  28. 28.

    Prior RL, Wu XL, Schaich K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agr Food Chem. 2005;53:4290–302.

  29. 29.

    Qi Y, Wang H, Zou Y, Liu C, Liu Y, Wang Y, Zhang W. Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice. FEBS Lett. 2011;585:231–9.

  30. 30.

    Qu P, Liu PP, Fu P, Wang Y, Zhu W. Secondary metabolites of halotolerant fungus Penicillium chrysogenum HK14-01 from the Yellow River Delta area. Acta Microb Sinica. 2012;52:1103–12.

  31. 31.

    Redman RS, Sheehan KB, Stout RG, Rodriguez RJ, Henson JM. Thermo-tolerance generated by plant fungal symbiosis. Science. 2002;298:1581.

  32. 32.

    Rodriguez RJ, White JF Jr, Arnold AE, Redman RS. Fungal endophytes: diversity and functional roles. New Phytol. 2009;182:314–30.

  33. 33.

    Shigeoka S, Maruta T. Cellular redox regulation, signaling, and stress response in plants. Biosci Biotechnol Biochem. 2014;78:1457–70.

  34. 34.

    Tian W, Bi YH, Zeng W, Jiang W, Xue YH, Wang GX, Liu SP. Diversity of endophytic fungi of Myricaria laxiflora grown under pre- and post-flooding conditions. Genet Mol Res. 2015;14:10849–62.

  35. 35.

    Umehara K, Yoshida K, Okamoto M, Iwami M, Tanaka H, Kohsaka M, Imanaka H. Studies on WF-5239, a new potent platelet aggregation inhibitor. J Antibiot. 1984;37:469–74.

  36. 36.

    Valavanidis A, Nisiotou C, Papageorgiou Y, Kremli I, Satravelas N, Zinieris N, Zygalaki H. Comparison of the radical scavenging potential of polar and lipidic fractions of olive oil and other vegetable oils under normal conditions and after thermal treatment. J Agr Food Chem. 2004;52:2358–65.

  37. 37.

    Venugopalan A, Srivastava S. Endophytes as in vitro production platforms of high value plant secondary metabolites. Biotechnol Adv. 2015;33:873–7.

  38. 38.

    Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, Fischer M, Heier T, Hückelhoven R, Neumann C, von Wettstein D, Franken P, Kogel KH. The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. PNAS. 2005;102:13386–91.

  39. 39.

    Wang X, Zhang MM, Wang YJ, Gao YT, Li R, Wang GF, Li WQ, Liu WT, Chen KM. The plasma membrane NADPH oxidase OsRbohA plays a crucial role in developmental regulation and drought-stress response in rice. Physiol Plant. 2016;156:421–43.

  40. 40.

    Waqas M, Khan AL, Kamran M, Hamayun M, Kang SM, Kim YH, Lee IJ. Endophytic fungi produce gibberellins and indoleacetic acid and promotes host-plant growth during stress. Molecules. 2012;17:10754–73.

  41. 41.

    Ye Y, Xiao Y, Ma L, Li H, Xie Z, Wang M, Ma H, Tang H, Liu J. Flavipin in Chaetomium globosum CDW7, an endophytic fungus from Ginkgo biloba, contributes to antioxidant activity. Appl Microbiol Biotechnol. 2013;97:7131–9.

  42. 42.

    Yu H, Zhang L, Li L, Zheng C, Guo L, Li W, Sun P, Qin L. Recent development and future prospects of antimicrobial metabolites produced by endophytes. Microbiol Res. 2010;165:437–9.

  43. 43.

    Zeng W, Qin W, Tian W, Xue Y, Wang G, Liu S. Antioxidant activity in vitro of endophytic fungi from Myricaria laxiflora, a riparian plant with strong tolerance ability of flooding. J Pure Appl Microbiol. 2015;9:87–95.

  44. 44.

    Zhang HW, Song YC, Tan RX. Biology and chemistry of endophytes. Nat Prod Rep. 2006;3:753–71.

  45. 45.

    Zhang Z, Zhang Q, Wu J Zheng X, Zheng S, Sun X, Qiu Q, Lu T. 2013. Gene knockout study reveals that cytosolic ascorbate peroxidase 2 (OsAPX2) plays a critical role in growth and reproduction in rice under drought, salt and cold stresses PLoS One, 8:e57472.

Download references

Acknowledgements

Thanks Dr. Ma Q for offering suggestions in statistical methods and providing professional editing services.

Funding

This work was financially supported by National Natural Science Foundation of China (No. 31270389 and No. 31540065) in the design of the study and collection, analysis, and interpretation of data.

Availability of data and materials

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Author information

QW isolated and analyzed the fungi. LCX identified the structure of NFA. JW preformed the antioxidant activity. XYH carried out the physiological experiments and completed the manuscript. WGX drafted the work and analyzed the data. LSP designed and co-supervised the study. All authors have read and approved the final manuscript.

Correspondence to Shiping Liu.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Additional file

Additional file 1:

Figure S1. Antioxidant activity of fraction 2 by DPPH method. (A) DPPH alcohol solution. (B) fraction 2 solution. (C) Vc solution. (PPT 6256 kb)

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Keywords

  • Endophytic fungus
  • Aspergillus fumigatus
  • NFA
  • Antioxidant
  • Drought stress