Acidity stress for the systemic elicitation of glyceollin phytoalexins in soybean plants

ABSTRACT

Glyceollins are the major pathogen- and stress-inducible natural products (phytoalexins) of soybean that possess broad-spectrum anticancer and neuroprotective properties. Yet like other phytoalexins, glyceollins are difficult to obtain because they are typically biosynthesized only transiently and in low amounts in plant tissues. We recently identified acidity stress (pH 3.0 growth medium) as an elicitor that exerted prolonged (week-long) inductive effects on glyceollin biosynthesis and identified the NAC family TF gene GmNAC42-1 that activates glyceollin biosynthesis in response to acidity stress or WGE from the soybean pathogen Phytophthora sojae. GmNAC42-1 was annotated as an SAR gene and SAR genes were statistically overrepresented in the transcriptomic response to acidity stress suggesting that acidity stress triggers the systemic elicitation of glyceollin biosynthesis. Here, we demonstrate that acidity stress acts as a systemic elicitor when provided to soybean roots. Acidity stress preferentially elicited specific glyceollins in different soybean organs with exceptionally high yields of glyceollin I in root tissues.

KEYWORDS:

• Bioproduction
• natural products
• phytoalexin
• isoflavonoid

The value of the botanical- and plant-derived drugs market worldwide was estimated to be ~$25 billion in 2016 and is expected to increase to almost $42 billion by 2023. Yet, phytoalexins represent a vast array of inducible molecules in plants that remain a mainly untapped drug resource. Phytoalexins are antimicrobial plant metabolites that are not readily accessible as drugs since they are biosynthesized only in response to pathogens or specific abiotic stresses. The chemical structures of phytoalexins vary widely among plant species and include alkaloids, terpenoids, lactones, and phenolics. With roles in plant defense, phytoalexins are naturally bioactive and have diverse activities in human cells. Mounting evidence of their value to human health and agriculture has spurred renewed interest in identifying elicitors of their biosynthesis, biosynthetic genes, and the TFs that could be used to enhance their bioproduction via bioengineering.^1–5

Glyceollins are the major phytoalexins of soybean that are biosynthesized from the isoflavonoid pathway. For decades, the study of glyceollins has served as a model for understanding the molecular aspects of elicitation and the role of inducible secondary metabolites in plant defense. Biochemical and genetic evidence suggests that glyceollins are essential for providing race-specific resistance to Phytophthora sojae, one of the most devastating pathogens in soybean agriculture.^6–8 They also exhibit anticancer, antidiabetic, and neuroprotective properties that have been the subject of recent reviews.^9–11 Glyceollin I, in particular, inhibits the proliferation and survival of a broad range of cancer types by inhibiting ER signaling and by other unclear mechanism(s).^12–19 Conversely, glyceollins promote the survival of challenged neurons by stimulating the Nrf2/HO-1 signaling pathway.²⁰

The critical barriers that limit the accessibility of glyceollins are that they cannot be synthesized economically^21–23 or obtained in sufficient amounts from soybean tissues.^24–26 Efforts to improve accessibility have included (semi)-synthesis,^21-23 fermentation of soybeans,²⁷ combined treatments of soybean seeds with pathogens and malting,²⁶ pathogens and environment stress,²⁸ and pathogens and chemicals.²⁵ A combined treatment of P. sojae WGE and the inorganic chemical silver nitrate had an additive effect, eliciting 1.5-fold more glyceollin I than the WGE treatment alone.²⁵ Yet, overexpressing the isoflavonoid biosynthesis gene encoding IFR increased glyceollin amounts more than 3-fold,²⁹ demonstrating that bioengineering can provide greater enhancements in glyceollin yields. However, overexpressing one rate-limiting biosynthetic enzyme will indefinitely uncover another. Since upregulating the expressions of all biosynthesis genes could provide greater increases in yield, we recently set-out to identify the TF gene(s) that regulate glyceollin biosynthesis.

By screening a panel of abiotic stresses, we identified acidity (pH 3.0 medium) to be a potent elicitor and dehydration to be a suppressor of glyceollin biosynthesis.³⁰ Using a comparative transcriptomics approach we found that all known glyceollin biosynthesis genes were oppositely regulated by acidity stress and dehydration, and that their expressions mirrored that of the NAC family TF gene GmNAC42-1.³⁰ Overexpressing and suppressing GmNAC42-1 in soybean hairy roots increased and decreased, respectively, the levels of glyceollin biosynthesis gene transcripts and metabolites.³⁰ GmNAC42-1 was annotated as an SAR gene and SAR genes were significantly overrepresented among those upregulated by acidity stress. SAR is a component of the plant immune system whereby tissues distant from a pathogen infection site become primed to more rapidly activate resistance during subsequent encounters with the pathogen. Yet, whether localized acidity stress could stimulate systemic phytoalexin biosynthesis remained untested.

Here, we grew seedlings in vermiculite to the first trifoliate leaf stage, then transferred them to pH 3.0 growth medium as reported previously with roots in contact with the acidic medium.³⁰ Following 9 d of treatment, seedlings were harvested and organs were dissected for analysis of glyceollins.

Acidity stress elicited greater amounts of total glyceollins compared to UV-C irradiation, which is classical abiotic elicitor of phytoalexins (Figure 1a). Transferring seedling roots to the acidic medium resulted in glyceollin accumulation in other organs with true leaves accumulating more than hypocotyls and cotyledons (Figure 1b). Yet, the roots were the major site of glyceollin accumulation (Figure 1b). Roots had an average of 1.7 mg gt⁻¹ glyceollins, which is greater than the yield observed for the combined WGE-AgNO₃ treatment of germinating seeds (837.2 µg gt⁻¹),²⁵ or that observed for soybean sprouts that were simultaneously malted and challenged with Rhizopus microsporus (1.5 mg gt⁻¹).²⁶ The organs preferentially accumulated specific glyceollins with glyceollin I being predominant in the roots and glyceollin III in the true leaves (Figure 1b). Roots exhibited a browning in color after 9 d treatment (Figure 1c). The phenotype was reminiscent of the localized browning observed during an incompatible interaction with P. sojae.^6,8 Glyceollin I represented the major isoflavonoid peak in the roots (Figure 1d). This is uncommon since the isoflavone-conjugates 6-O-malonyldaidzin and 6-O-malonylgenistin generally remain the major isoflavonoids in pathogen-treated Williams 82 roots.

Figure 1. Elicitation of glyceollins in soybean roots by pH 3.0 medium. (a) Seven-day-old seedlings were transferred to pH 5.8 or pH 3.0 medium for 9 d, or exposed to a 30 W UV-C lamp for 1 h daily. Metabolites were extracted from intact seedlings (a) or dissected organs (b) by 80% ethanol and were quantified by UPLC-PDA by comparison to purified standards. (c) Root color phenotypes following 9 days treatment with pH 5.8 or pH 3.0 medium. (d) UPLC-PDA chromatograms of isoflavonoid extracts from cotyledons, leafs, hypocotyls and roots of Harosoy 63 seedlings elicited with pH 3.0 medium for 9 d. Peaks: 1) daidzin, 2) 6-O-malonyldaidzin, 3) 6-O-malonylgenistin, 4) daidzein, 5) genistein, 6) glyceollin III, 7) glyceollin II, 8) glyceollin I and 9) βprenyl-genistein.

In conclusion, the acidic growth medium is a potent systemic elicitor of glyceollin biosynthesis. Whether acid mine drainage or other collected environmental contaminants (e.g. acid rain) could be exploited to economically elicit the biosynthesis of these anticancer and neuroprotective natural products, or other plant phytoalexins, remains to be investigated.

Methods

Plant materials and growth conditions

Harosoy 63 seeds were a kind gift from Elroy Cober (Agriculture and Agri-Food Canada, Ottawa, Ontario). Seeds (16–20 per batch) were surface sterilized with 70% ethanol 0.2% triton X (v/v) for 5 min on a mixer wheel and grown in sterile vermiculite as indicated.³⁰

Acidity stress treatments

The roots of five unblemished, uniform seedlings at their first trifoliate leaf stage (~7-day-old) were gently wrapped together with filter paper saturated with control (pH 5.8) or acidic (pH 3.0) medium that contained half-strength MS salts, MS vitamins and 1% (w/v) sucrose and were placed on the medium inside sterile beakers.³⁰ The plant organs were harvested separately and processed for metabolite analysis as indicated.³⁰

Isoflavonoid analysis

Pulverized freeze-dried tissue powder (~12 mg) from whole seedlings, or dissected roots, hypocotyls, cotyledons and leaves were extracted with 80% ethanol (10 µL mg⁻¹ dry tissue) as described.³⁰

UPLC-PDA

UPLC-PDA measurements were performed using an Accela System (Thermo Scientific, San Jose, CA, USA) containing a 1250 pump, Open AS autosampler, and photodiode array (PDA) as described.²⁵

Abbreviations

Table

ER	estrogen receptor
gt^−1	per gram dry tissue
HO	heme oxygenase-1
IFR	isoflavone reductase
MS	Murashige and Skoog
NAC	NAM/ATAF1/2/CUC2 family TF
Nrf2	nuclear factor (erythroid-derived 2)-like 2
SAR	systemic acquired resistance
TF	transcription factor
UPLC-PDA	ultrahigh performance liquid chromatography-photodiode array
WGE	wall glucan elicitor

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Additional information

Funding

This work was supported by WVU start-up funds to NK and is based upon work that is supported by the United States Department of Agriculture National Institute of Food and Agriculture Hatch project 1010200; National Institute of Food and Agriculture [1010200]; West Virginia Agricultural and Forestry Experiment Station [WVA00687]; West Virginia University [start-up].