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Fungal laccase-catalyzed oxidation of naturally occurring phenols for enhanced germination and salt tolerance of Arabidopsis thaliana: a green route for synthesizing humic-like fertilizers Joon-Yung Cha, Tae-Wan Kim, Jung Hoon Choi, Kyoungsoon Jang, Laila Khaleda, Woe-Yeon Kim, and Jong-Rok Jeon J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04700 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on January 29, 2017

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Journal of Agricultural and Food Chemistry

- Title Page -

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Title: Fungal laccase-catalyzed oxidation of naturally occurring phenols for enhanced germination

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and salt tolerance of Arabidopsis thaliana: a green route for synthesizing humic-like fertilizers

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Name of Authors: Joon-Yung Chaa,c, Tae-Wan Kima,b, Jung Hoon Choie,f, Kyoung-Soon Jange, Laila

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Khaledaa,b, Woe-Yeon Kima,b,c,d* and Jong-Rok Jeonb,d*

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Addresses of Institutions:

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a

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& Technology, cPMBBRC & dIALS, Gyeongsang National University, Jinju 52727, Republic of Korea.

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e

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f

Division of Applied Life Science (BK21Plus), bDepartment of Agricultural Chemistry and Food Science

Biomedical Omics Group, Korea Basic Science Institute, Cheongju 28119, Republic of Korea

Department of Biotechnology and Bioinformatics, Korea University, Sejong 30019, Republic of Korea

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*Co-correspondence:

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Prof. Woe-Yeon Kim, Ph. D.

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E-mail [email protected]

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Phone +82-55-772-1968

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Fax +82-55-772-1969

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Prof. Jong-Rok Jeon, Ph. D.

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E-mail [email protected]

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Phone +82-55-772-1962

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Fax +82-55-772-1969 1

ACS Paragon Plus Environment


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ABSTRACT

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Fungal laccases have been highlighted as a catalytic tool for transforming phenols. Here we demonstrated that

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fungal laccase-catalyzed oxidations can transform naturally occurring phenols into plant fertilizers with properties

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very similar to those of commercial humic acids. Treatments of Arabidopsis thaliana with highly cross-linked

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polyphenolic products obtained from a mixture of catechol and vanillic acid were able to enhance the germination

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and salt tolerance of this plant. These results revealed that humic-like organic fertilizers can be produced via in vitro

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enzymatic oxidation reactions. In particular, the root elongation pattern resulting from the laccase products was

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comparable to that resulting from an auxin-like compound. A detailed structural comparison of the phenol variants

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and commercial humic acids revealed their similarities and differences. Analyses based on SEM, EFM, ERP and

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Zeta-potential measurement showed that they both formed globular granules bearing various hydrophilic/polar

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groups in aqueous and solid conditions. Solid-phase

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more nitrogen-based functional and aliphatic groups were present in the commercial humic acids. Significant

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differences were also identifiable with respect to particle size and specific surface area. High-resolution (15T) FT-

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ICR mass spectrometry-based van Krevelen diagrams showed the compositional features of the variants to be a

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subset of those of the humic acids. Overall, our study unraveled essential structural features of polyaromatics that

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affect the growth of plants, and also provided novel bottom-up eco-friendly and finely tunable pathways for

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synthesizing humic-like fertilizers.

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C NMR, FT-IR-ATR and elemental analyses showed that

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Keywords: Humic acids; fungal laccases; naturally occurring phenols; germination; salt tolerance

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INTRODUCTION

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Fungal laccases have been regarded as one of the promising green catalysts for a variety of syntheses.1,2 Unlike other

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oxidases, these enzymes use molecular oxygen, which is abundant in the atmosphere, as the final electron acceptor

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during redox cycles. In addition, they are highly reactive toward various kinds of phenols.1-3 Here, the enzymatic

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actions start with single-electron oxidations of phenolic groups, resulting in the formation of phenoxyl radicals,

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which are unstable. Depending on the reaction conditions, the radicals oxidatively couple with adjacent monomeric

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and polymeric compounds.4 When the substrates of fungal laccases are natural phenols, all agents involved in the

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reactions (e.g., catalysts, reactants and water-based media) are from natural sources. In addition, the coupling

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mimics in vivo synthetic pathways of natural polyaromatic compounds such as lignin and polyflavonoid, indicating

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that our reactions are strictly based on core principles of green chemistry.5 To date, several studies have

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demonstrated that fungal laccase-catalyzed oxidative couplings of natural phenols are useful green chemistry

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reactions for the synthesis of dyes, for food processing, and for the engineering of several solid surfaces.5-7 For more

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specific examples, the anti-oxidant capability of plant phenols can be efficiently enhanced with laccase oxidation-

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involved controls of plant phenolic molecular weights.5 Protein binding properties of plant phenolics are modulated

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with laccase-based structural diversification.5 Multifunctional coating ability to several solid surfaces is attained

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with laccases and natural phenolics.6 Lignin-derived small phenols such as ferulic and syringic acid with laccase

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action are able to transform into colorful polymers that are applicable to hair dyeing.7 The enzymes have also been

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applied on a bulk scale in the pulp bleaching and denim decolorization industries.2,8

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Humic substances (HS) play a pivotal role in soil biochemistry.9 They are heterogeneous polymeric mixtures

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containing diverse aromatic and aliphatic structures.9,10 Beyond their specific physicochemical properties, HS have

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been especially highlighted by industry due to their multifunctional effects on plant growth and physiology.11 The

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size of the global humic market used as organic fertilizers was evaluated at $326 million in 2014. In addition, HS-

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based fertilizers are increasingly being used in Europe, North America, and China.12 However, the contents and

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quality of commercial HS are highly variable because they are extracted from natural sources such as peat,

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leonardite, and brown coal.13 Also, some countries lack HS-related natural resources and must rely on imports.

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Scientific analyses of how natural HS form are still lacking, but some reports have suggested that plant lignin is the

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main source for forming HS.14 Aromatic structures of HS resemble those of lignin, and the poor biodegradability of 3



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HS is believed to derive from lignin.10,11,14 Humic-like bioactivity of plants was also confirmed by using water-

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soluble lignin from lignocellulosic biomass.15 The lignin-HS relationship strongly suggests that artificially

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synthesizing polymers that give rise to lignin-like structures should be considered as a commercially viable

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alternative route to produce HA-based organic fertilizers. We previously demonstrated that lignin-derived small

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phenols can be readily polymerized by fungal laccase to form lignin-like macromolecules.7 It was thus of great

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interest to determine whether the effects of humic-like fertilizers on plants can be re-created with such artificial

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bottom-up synthetic approaches. These synthetic approaches are easy to control, thus both assuring quality control

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of the HS-like fertilizing effects on plants and the ability to make the HS regardless of the availability of the

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otherwise necessary natural resources.

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The aim of this study was to characterize the changes in the structures of the natural phenols that occur during

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fungal laccase-catalyzed oxidation and to study the effects of the products of these reactions on the biological

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activities of plants. Commercially available catechol and vanillic acid were selected as model natural phenols.

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Laccases of Trametes versicolor were employed to transform the phenols because the fungus is regarded as one of

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the strongest laccase producers.16 Then, the effects of the oxidation products on the seed germination, root

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elongation, and salt tolerance of the Arabidopsis thaliana plant were evaluated. After confirming the organic

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fertilizer-like activities of the phenol variants, their structural and physicochemical properties were compared with

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those of commercial humic acids, and their structure-property-function relationship was in this way deciphered.

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MATERIALS AND METHODS

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Chemicals and materials

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Catechol, vanillic acid, sodium acetate, glacial acetic acid, humic acid (HA), abscisic acid (ABA), paclobutrazol

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(PAC), indoleacetic acid (IAA) and T. versicolor laccase (0.53 U mg-1 was denoted in the commercial product) were

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purchased from Sigma-Aldrich, while ethanol (HPLC grade), 5-Bromo-4-chloro-3-indolyl β-D-glucuronic acid (X-

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gluc), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were obtained from Duksan, Gold

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Biotechnology, and Fluka, respectively. The A. thaliana wild-type (Col-0 background) was used as a plant material

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to monitor the biological effects of HA or natural phenol variants. A transgenic Arabidopsis line (Col-0 background)

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harboring DR5:GUS constructs was used to monitor the auxin responses.17 Murashige and Skoog (MS) medium as a

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plant cultivation media was purchased from Duchefa Biochemie. 4


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Fungal laccase-catalyzed oxidation of natural phenols

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Laccase activity of T. versicolor (120 U g-1) was calculated as previously described.7 ABTS (1 mM, E420 = 36,000

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M-1 cm-1) was reacted with the laccase enzymes (0.1 mg mL-1) to monitor blue-color generation at 420 nm. The

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absorbance was measured at 0.0072 sec-1. To induce oxidative polymerization, either catechol (CA, 5 mg mL-1) or

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vanillic acid (VA, 5 mg mL-1) was completely dissolved in 100 mM sodium acetate buffer (pH 5.0) containing 25%

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(v/v) absolute ethanol. In case of catechol/vanillic acid reaction (CAVA), catechol (2.5 mg mL-1) was mixed with

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vanillic acid (2.5 mg mL-1). After adding T. versicolor laccases (0.1 mg mL-1), the mixtures were incubated with

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gentle shaking (25 rpm) for 24 hours at room temperature or 36℃. To exchange the reaction buffer with distilled

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water, the reaction media were centrifuged with 20,000 g for 10 min. The supernatants were then ultra-filtrated with

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5 kDa MWCO disc and distilled water (Ultracell 5 kDa, Amicon) under nitrogen pressure (Stirred ultrafiltration

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cells, Millipore). The pellets were washed with distilled water through repeated centrifugation and vigorous

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vortexing followed by mixing with the ultra-filtrated solutions for further A. thaliana treatments. Concentration was

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measured after complete drying of 0.5 mL of the final reactants under 90℃. Commercial humic acids were directly

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dissolved in distilled water with vigorous vortexing for further use.

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Seed germination assays

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Seeds of A. thaliana were surface-sterilized with a 30% bleaching solution (1.5% sodium hypochlorite and 0.02%

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Triton X-100) for 5 min, and then washed five times with sterile distilled water and incubated for 2 d at 4℃ before

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sowing. Seeds were germinated on MS media (4.3 g L-1 MS, 30 g L-1 sucrose, pH 5.8 and 0.6% (w/v) agar)

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containing HA or natural phenol variants under a 16 h/8 h light/dark cycle at 23℃ in a growth chamber.

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Germination rates were measured using radicle and cotyledon emergence after 2 d and 4 d of the incubation,

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respectively. To monitor the recovery of ABA- or PAC-induced germination inhibition by HA or natural phenol

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variants, sterilized wild-type (Col-0) seeds were grown on ABA (0.5 µM)- or PAC (3 µM)-containing MS media in

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the absence or presence of HA, CA, VA or CAVA (106 mg L-1) for 9 d. Representative images of seed germination

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were photographed under a microscope (Olympus).

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Lateral root development

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Wild-type (Col-0) seeds harboring DR5:GUS constructs were sterilized as mentioned above, and grown on MS 5



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media for 5 d. Seedlings were transferred onto MS media containing IAA, HA, CA, VA or CAVA followed by

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additional cultivation for 2 d and 7 d for GUS staining and counting a number of lateral roots, respectively.

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GUS staining

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Seedlings exposed to IAA, HA, CA, VA or CAVA for 2 d were incubated in a GUS staining buffer (50 mM sodium

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phosphate (pH 7.0), 0.2% Triton X-100, 1 mM X-gluc) overnight at 37℃ in the dark. The seedlings were

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subsequently incubated overnight in 100% EtOH at 23℃ to remove residual chlorophyll contents.

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Salt tolerance assay

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Sterilized seeds of wild-type (Col-0) plants were grown on MS media for 5 d, and transferred onto MS media

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containing HA, CA, VA or CAVA (106 mg L-1) in the absence or presence of NaCl (250 mM). Photograph was taken

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at 7 d after transplant, and plants were immediately harvested to measure chlorophyll contents for determining salt

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tolerance phenotype. The plants were soaked in 80% (v/v) acetone overnight and total chlorophyll contents were

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measured using a spectrophotometry as described previously.18

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Characterizations of commercial HA and natural phenol variants

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The liquid-phase reactants including commercial HA were lyophilized to obtain solid powders for characterizations.

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The solid particles were attached to a carbon-based adhesive tape (Tedpella) followed by a gold sputtering. The

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surface morphology was then characterized by field-emission SEM (Philips, XL30S FEG). The polymeric powders

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were pressed into flat pellets using a hydraulic press followed by EFM imaging with the tip charged by a bias of +8

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V (Nanoscope III, Veeco Instruments, Inc.). The BET (Brunauer-Emmett-Teller) specific surface area was

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monitored based on N2 adsorption methods using an ASAP 2010 system (Micromeritics Corp., USA). Elemental

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analyses (C, H, O, N, and S) were performed with 2.0 mg of each powder by Flash EA 2000series (ThermoFisher).

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Solid-state 13C NMR spectra were obtained using a 400 MHz Avance III spectrometer (Bruker BioSpin GmbH). A 4

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mm rotor spinning (10 kHz) was employed with proton pulse length of 2.4 µs, pulse repetition delay time of 3 s and

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contact time of 2.0 ms. Tetramethylsilane was used as a reference for the chemical shift. X-band continuous-wave

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EPR spectra were obtained using a Bruker EMX Plus 6/1 spectrometer equipped with a dual-mode cavity (ER

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4116DM). The experimental parameters are as the follows: microwave frequency, 9.64 GHz; microwave power, 1 6


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mW; modulation amplitude, 1 G; temperature, room temperature. Hydrodynamic size distribution and Zeta

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potentials were evaluated using an electrophoretic light scattering spectrophotometer (ELS 8000, Otsuka, Japan).

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FT-IR spectra of the polymeric powders were obtained through attenuated total reflection (ATR) mode (iS50,

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ThermoFisher).

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FT-ICR MS analysis

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Ultrahigh-resolution mass spectrometric analysis was performed on a 15 Tesla (T) FT-ICR mass spectrometer

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equipped with an electrospray ionization source (solariXTM system, Bruker Daltonics, Billerica, MA). Commercial

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humic and natural phenol variants, dissolved in methanol containing 20% NH4OH for pH adjustment to 8, were dire

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ctly infused into the 15T FT-ICR mass spectrometer by a syringe pump at a flow rate of 3 µL/min and

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analyzed in negative ion mode within the mass range of m/z 150–1200. The mass resolving power was set at

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400,000 (at m/z 400) for all spectra, and 200 scans per sample were collected with a 4 M transient. The other MS p

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arameters were as follows: an capillary voltage of 3900 V, drying gas flow rate of 4 L/min, drying gas tempera

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ture of 180°C, ion accumulation time of 0.05 s and transient length of 1.39 s. External calibration was performe

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d with quadratic regression using an arginine solution (10 µg mL-1 in methanol). Data acquisition was controlled

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by ftmsControl 2.0 software (Bruker Daltonics).

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Data processing and elemental composition assignments

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Raw data obtained from 15T FT-ICR MS were processed using DataAnalysis (ver. 4.2, Bruker Daltonics) and

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Composer (Sierra Analytics, Modesto, CA) softwares. After 15T FT-ICR MS measurements, the raw spectra were

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imported to the DataAnalysis 4.2 for peak detection and recalibration. The Composer, a formula calculator, was

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employed for assignment of elemental compositions, as described previously,19 with some modifications. Briefly, the

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empirical molecular formulae were calculated for the masses of singly charged ions in the range of m/z 150–1,000

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by allowing up to the combinations of 100

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molecular formula including up to 2

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formulas with assignment errors > 0.5 ppm were ruled out for further processing. The van Krevelen plot was used to

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visualize the assigned compositions of the samples based on their molar H/C and O/C ratios, and the Kendrick mass

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defect was used to sort homologous ion series that possess same double bond equivalents (DBE) and heteroatom

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contents but differ by increments of –CH2, respectively.21

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C, 200 1H and 50

N and 1

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O atoms, followed by additional calculations of

S atoms, as described by Koch et al.,20 and then the molecular

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RESULTS AND DISCUSSION

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Promotion of A. thaliana seed germination by commercial HA and natural phenol variants

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Of the diverse beneficial effects of HS on plant growth and physiology, their promotion of germination22-24 has been

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of particular interest for agronomists because a high germination rate contributes to lowering the costs of

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agricultural products. Therefore, we first examined whether A. thaliana seed germination rates were promoted with

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commercial HA and the natural phenol variants CA, VA and CAVA. As previously demonstrated in other plant

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species,22-24 commercial HA in our A. thaliana systems were proven to enhance seed germination rates by promoting

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the emergence of both radicles and cotyledons in a concentration-dependent manner (Figures 1A and B). However,

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the effects of either CA or VA alone were meager and in fact treating seeds with 530 mg L-1 of either CA or VA

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resulted in lower germination rates than those resulting from untreated seeds. Interestingly, as the concentration of

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CAVA was increased from 0 mg L-1 up to 106 mg L-1, the quantities of both radicles and cotyledons that emerged

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also increased. Seed germination images were consistent with the results of radicle- or cotyledon-based germination

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rates (Figure 1C). Effects of the fungal laccases alone on the germination enhancements were shown to be negligible

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compared with those of untreated seeds (data not shown), demonstrating that the observed actions of the CAVA

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reactants derived from the polymeric products of laccase-catalyzed oxidations.

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The enhancements observed as the concentration of CAVA was increased were nearly the same in extent as those of

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commercial HA (Figure 1). Some studies have shown oxidative polymerizations of phenolic compounds to give rise

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to HA-like structures,25,26 but it still remained to be determined whether such oxidative polymerizations re-create the

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fertilizing effects of natural HA and whether key monomeric structures are necessary for the plant stimulation. Our

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results suggest that bottom-up synthetic approaches can produce HA-like organic fertilizers, but monomeric

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phenolics must be carefully chosen. The dependence of the results on dose was also comparable to the results of

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previous reports showing that HA treatments facilitated the formation of seedlings and growth up to a concentration

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of 1 g of HA per Kg of soil during tomato cultivation, but with the beneficial effects decreased at a concentration of

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2 g Kg-1.24 As indicated above, the beneficial effects of our phenol variants on the plants also peaked at a finite

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concentration, in this case at 106 mg L-1.

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The natural phenols we here employed for fungal laccase-catalyzed oxidations have been previously shown to be

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strongly phytotoxic, and to thus inhibit seed germination.27 We therefore tested the toxicity of the phenols without 8


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oxidative polymerization in our A. thaliana cultivation system. CA or CAVA at a concentration of 106 mg L-1 was

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found to completely inhibit the emergence of radicles, while VA exhibited less toxicity (Figure S1 in the Supporting

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information). These effects may have been due to these natural phenols inhibiting enzymes involved in the

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glycolysis and oxidative pentose phosphate pathways as described earlier.27 Interestingly, oxidative actions of fungal

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extracellular enzymes act as a switch to modulate the conflicting biological activities of seed germination inhibition

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and enhancement with small phenols that are widespread in soils. The impact of these reactions on soil ecology

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involving plants, fungi and aromatic carbon recycling must be further assessed to fully understand their biological

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consequences.

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Phytohormone activity of natural phenol variants

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Seed germination is one of the physiological processes that initiate plant growth. Beyond external conditions such as

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temperature, water and air, phytohormones are also actively involved in controlling seed germination. Abscisic acid

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(ABA) and gibberellin (GA) have been shown to antagonistically inhibit and promote seed germination,

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respectively.28 To be more specific, ABA in seed coats was shown to inhibit embryo germination together with

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actions of the DELLA protein, which helps activate GA repressor.29 Paclobutrazol (PAC) has also been shown to

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prevent seed germination by inhibiting GA biosynthesis.30

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We next evaluated whether natural phenol variants can recover seed germination inhibited by either ABA or PAC.

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In the presence of ABA (0.5 µM) and PAC (3 µM), wild-type (Col-0) seeds of A. thaliana showed 10 ± 2% and 31 ±

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2.51% germination rates, respectively (Figure 2A). However, CAVA treatment (with ABA, 28 ± 2.82% and with

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PAC, 58 ± 2.58%) strongly countered ABA- and PAC-induced inhibition of seed germination, and hence recovered

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the germination levels to nearly the same as that from HA treatment in the presence of ABA (28 ± 3.65%) and PAC

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(57 ± 1%). In contrast, CA or VA treatments each provided only a weak recovery. Photoimages of the seed

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germinations were consistent with the germination rates based on the emergence of cotyledons (Figure 2B).

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Together with the results shown in Figure 1, these results indicated that artificial aromatic polymer structures such

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as CAVA can be used to re-create the positive effects of HA on seed germination and may exert GA-like or ABA-

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repressing activities to break seed dormancy.

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Auxin is an essential phytohormone that regulates plant development throughout the life span of the plant. The

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hormone affects cell division, vascular differentiation, embryo patterning, apical dominance, phototropism,

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gravitropism, and the architecture and growth of roots and shoots.31 HS isolated from earthworm compost has been

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previously reported to result in enhanced lateral root development in maize and A. thaliana, which is similar to the

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results of auxin hormone activity.32,33 Thus, we investigated whether auxin-mediated responses occur in the presence

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of natural phenol variants in A. thaliana plants harboring a DR5 promoter-driven GUS plasmid (DR5:GUS), which

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is widely used as a reporter of auxin responses in planta.33,34. Auxin generally accumulates in tissues undergoing cell

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division such as the apical meristem and primordial site. Thus, we examined the expression of DR5-GUS in root tips

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and lateral root development sites by applying treatments of indole-3-acetic acid (IAA, 50 nM), HA, CA, VA, or

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CAVA (106 mg L-1). Remarkably, enhanced DR5-GUS expression was observed in both the root tips and lateral

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roots of CAVA-treated A. thaliana at levels similar to the expression levels obtained from IAA or HA treatments.

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On the other hand, the expression of DR5-GUS was hardly detected in lateral roots for controls and when either a

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CA or VA treatment was applied (Figure S2A in the Supporting information).

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To monitor the actual numbers of lateral roots, we counted the roots of A. thaliana after seven days of cultivation.

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Consistent with the DR5-GUS expression patterns, the number of the roots increased with increasing concentrations

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of IAA, CAVA or HA, but was unchanged with untreated cultures or tests with either CA or VA alone (Figure S2B

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in the Supporting information). HS derived from earthworm compost has also been shown to activate the plasma

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membrane H+-ATPase, which could improve plant nutrition while maximizing the electrochemical proton

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gradient.32 In addition, enhanced lateral root formation has been found to be concomitant with the increase of both

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mitotic sites on roots and endogenous IAA content.32,33 We here demonstrated auxin-like pathways of phenolic

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polymers obtained from monomeric mixtures of CA and VA with respect to root genesis, but further

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characterization will be necessary to confirm other auxin-involved changes in plant growth and physiology such as

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H+-ATPase and mitosis-related protein expression.

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Conferring salt stress tolerance by using natural phenol variants

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Salt stress is a major abiotic stress resulting in the reduction of crop productivity. It causes osmotic stress and ion

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toxicity in plant cells by decreasing the water potential and increasing toxic ion contents, respectively. HS has been

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explored to enhance salt stress tolerance in beans and corns.35,36 We investigated whether natural phenol variants 10


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also confer salt stress tolerance. Salt tolerance conferred by the variants was examined by noting changes in leaf

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coloring and the amount of chlorophyll, which breaks down in salt stress conditions. As shown in Figure 3A, in

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conditions of salt stress and in the absence of any treatment, the cotyledon leaves were found to be dead, with the

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appearance of a white color caused by a breakdown of chlorophyll. However, this salt sensitivity was clearly

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diminished when the plant was treated with any of the phenol variants or by HA. Interestingly, CAVA and HA each

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conferred a greater salt tolerance than did either CA or VA as indicated by quantification of residual chlorophyll

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contents: the leaves of the plants treated with CAVA and salt showed a higher average chlorophyll content (38 ±

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3.17%) than those treated with CA (8.9 ± 0.38%) or VA (7.48 ± 1.36%) plus salt (Figure 3B). HA treatments

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resulted in the highest chlorophyll contents (50.1 ± 1.98%), an effect consistent with previous reports.35,36 Numerous

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studies have suggested that HS contribute to modulating the respiration, protein synthesis and enzyme activity in

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plants.37 HS are also known to enlarge the root structure by increasing root density and the number of lateral roots,

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thus enhancing nutrient uptake.35-38 Although there is no direct evidence available for how HS increases salt

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tolerance of plants, several physiological parameters affecting ion transport and nutrient uptake may be expected to

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be controlled by HS and natural phenol variants.

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SEM, EFM, elemental, surface area and Zeta-potential analysis

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SEM images of the natural phenol variants showed them to form globular and budding-like structures, similar to that

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observed for commercial HA (Figure 4A). Such repeating structures would be expected to be due to intra- or inter-

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molecular interactions of the polymeric mixtures. In fact, HA use hydrogen bonds (H-bonds) and hydrophobic

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interactions for their self-organization.39 Globular structures resulting from the self-assembly of polyaromatic

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compounds have also been reported for eumelanin, with non-covalent π–π stacking involved in the assembly. Based

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on these previous results, the globular features of the phenol variants may be attributed to the aromaticity of these

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compounds and H-bonds involving the hydrophilic groups.40 The greater smoothness of the globular structures of

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poly(vanillic acid) than of the other polymers (Figure 4A) showed that the side-chain functional groups (i.e., the

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carboxylic acid and hydroxyphenyl groups) critically affected the self-assembly pattern. This smoothness is

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consistent with the specific surface area results (Table 1); VA displayed the largest area. It is unknown whether the

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observed globular shape contributes to the biological activity of the plant. However, the self-organized structure of

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HS seems to be essential for its influence on plants. For example, changes in the supramolecular structures of HS in

11



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response to environmental stimuli have been expected to result in the HS releasing molecules beneficial for the

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plant.9,41

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EFM was employed to characterize the charge distribution in the phenol variant structures (Figure 4B). All of the

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variants exhibited significantly high unbalanced distributions (i.e., with roughness values (Rrms)) of the surface

316

charge. HA showed the highest such value, followed by CA (Table 1). The distribution was due mainly to

317

hydrophilic functional groups that are widespread in both HA and the phenol variants. This hypothesis was also

318

supported by these compounds having displayed negative Zeta potentials in aqueous media (Table 1). Our elemental

319

analyses (Table 2) showed oxygen contents from 17% to 22% in the commercial HA and phenol variants, and

320

significant amounts of nitrogen only in the commercial HA samples. The trace amounts of nitrogen in the phenol

321

variants may have been due to the presence of residual laccase enzymes among the reactants. The presence of

322

oxygen- or nitrogen-containing functional groups would be expected to contribute to the Rrms because these groups

323

readily induce dipoles.42 Together with the SEM and Zeta-potential results, the EFM results suggested that the

324

multiple polar functional groups allowed the polymers to self-aggregate with a specific 3D architecture.

325 326

It is still unclear whether 3D structures of the phenol variants are critically linked to the observed fertilizing effects

327

under our cultivation system, but it seems to be evident that quality and quantity of specific functional groups as

328

seen in unique nitrogen contents and EFM roughness variances are related with plant stimuli.

329 330

NMR, EPR, IR and size-distribution analysis

331

In solid-state

332

observed to be greater than those from the natural phenol variants. In contrast, the areas of the aromatic C peaks (90

333

– 163 ppm)43 were greater for the natural phenol variants (Figure 5A and Table 3). In the variants, the only aliphatic

334

contributor to polymerization was the methoxy group of vanillic acid (See experimental). The intense peak at 55

335

ppm from vanillic acid (Figure 5A) thus derived from the methoxy group. The strong aliphatic C peaks of

336

commercial HA were similar to those of leonardite HA that are currently used in HS-based organic fertilizers.13,44 It

337

was thus interesting that the phenol variants mainly based on aromaticity showed the fertilizer properties (Figures 1-

338

3). Indeed, both the aliphatic and aromatic portions of natural HS have been reported to be effective at stimulating

339

the growth of plants, although to different extents.11 Carboxylic acids in the HS play a critical role in plant root

13

C NMR spectra, the areas of the aliphatic C peaks (0 – 90 ppm)43 from commercial HA were

12


Page 12 of 32

Page 13 of 32


340

growth and proton pump actions.45 The effects of the phenol variants on the plant would thus derive from the

341

aromatic rings and carboxylic groups of the starting monomers (i.e., CA and VA). It was noticeable that a mixture

342

(CAVA) of CA and VA generally exhibited a stronger stimulation of the growth of the plants than did either one

343

alone (Figures 1-3). These results suggested that a balance between polyaromaticity and carboxylic/phenolic groups

344

may have simultaneously influenced the plants.

345 346

In the EPR analysis, the g-values of all polymeric powders were measured to be about 2.00, indicating that the

347

spectral signals were due to the presence of semiquinone-type free radicals. The greatest line width was from the

348

commercial HA. The width indicates the time that it takes for the excited electron to return to the ground state

349

(Figure 5B and Table 4). In general, more condensed organic compounds show smaller widths.46 Consistent with the

350

13

351

to be less than that of the phenol variants. The commercial HA having the least intense peaks was also consistent

352

with the interpretation of the line width values because of the proportional relationship between the peak intensity

353

and the concentration of semiquinone-type radicals that can be formed from dihydroxybenzene.47

C NMR spectra (Figure 5A), comparison of the line width values indicated the aromaticity of the commercial HA

354 355

Signature peaks of commercial HA at 3700 cm-1 shown in its FT-IR-ATR spectrum corresponded to the amide N-H

356

stretch (Figure S3A in the Supporting information), suggesting that the detection of N in the elemental analysis was

357

associated with the amide groups of commercial HA (Table 2). Other peaks at 910 and 1375 cm-1 observed only for

358

commercial HA corresponded to alkane C-C bending and alkene C=C bending, respectively. These results also

359

reflected the highly aliphatic nature of commercial HA. Strong absorption peaks ranging from 3500 to 3200 cm-1

360

(phenol O-H stretch) from commercial HA suggested the considerable involvement of lignin and tannin, the main

361

plant polyphenols, in the HA. The phenol peaks were also identifiable in the spectra of the phenol variants (Figure

362

S3A in the Supporting information). Phenol radicals formed by laccase-catalyzed single-electron oxidation readily

363

delocalize to the benzene rings, thus facilitating C-C bond coupling.7 Such processes can allow the phenol structures

364

to be intact during polymerization. Consistent with the

365

corresponding to the C=O stretch of the carboxylic acid was observed only for VA. All of the samples (i.e., HA, VA,

366

CA and VACA) were proven to have similar particles that are less than 103 nm hydrodynamic sizes, but the

367

distribution patterns of more than 103 nm hydrodynamic sizes were obviously different (Figure S3B in the

368

Supporting information).

13

C NMR spectra (Figure 5A), a peak at 1713 cm-1

13



369

FT-ICR-MS analysis

370

Ultra-high-resolution FT-ICR mass spectrometry has been regarded as the most powerful technique for the structure

371

determination of complex organic mixtures. Natural organic matter48,49 and products from oxidative

372

polymerization50,51 of phenolic compounds have been subjects of such mass analysis. The DBEavg values are

373

indicative of aliphatic and aromatic double bonds. Unlike the 13C NMR results (Figure 5), CAVA exhibited a lower

374

DBEavg value than did HA, but the number of ions to be analyzed in CAVA was much smaller than in other samples,

375

suggesting that many components were not integrated into the elemental calculation (Table 5). The van Krevelen

376

diagrams of all the samples (Figure 6) demonstrated that natural phenol variants can be a subset of HA. HA showed

377

a relatively even distribution of molecules, while the assigned phenol variant molecules were mainly found in two

378

regions (Figure 6). In addition, the intensity for molecules showing low O/C and high H/C ratios from CAVA was

379

noticeably less than those of either CA or VA. On the other hand, molecules assigned to the other part (i.e. high O/C

380

and low H/C ratios corresponding to condensed aromaticity) are generally overlapped with those of natural organic

381

matters21 and soil humic substances,52 suggesting that the condensed aromatic components frequently found in

382

natural matters can be derived through fungal oxidative actions on small soil phenols.

383 384

It is not unreasonable that the structural similarity of the phenol variants and commercial HA, as characterized using

385

FT-ICR mass spectrometry, gave rise to the observed fertilizing effects of the phenol variants. Together with the 13C

386

NMR, EPR, and IR results, the FT-ICR results showed that the condensed aromatic components were sufficient to

387

induce enhanced germination and salt tolerance. However, the superior fertilizing effects of commercial HA and

388

CAVA were apparently due to the aliphatic portion and specific side-chain functional groups, respectively. The

389

elemental composition may also be important since the HA we used contained significant amounts of nitrogen.

390

Further tuning of monomeric structures with, for example, long alkyl chains and different side-chain functional

391

groups will be necessary to maximize the effectiveness of the synthesized materials.

392 393

Economic feasibility analyses of our synthetic ways will be necessary to compete with currently used manufacturing

394

processes of humic acids. Laccases may be replaceable with other kinds of oxidants to economize the synthetic

395

processes. In fact, poly(phenylene oxide) resins derived from oxidative polymerization of small phenolics by

396

copper-based inorganic oxidants have been successfully commercialized.53 In addition, further studies showing a

14


Page 14 of 32

Page 15 of 32


397

comparison of fertilizing effects with more kinds of commercial humic acids will be required to assess in-depth

398

cost-effectiveness of the methods.

399 400

Conclusions

401

Overall, our study demonstrated that fungal laccase oxidative action on commercially available natural phenols can

402

lead to the synthesis of humic-like organic fertilizers. The significant enhancement of A. thaliana germination and

403

salt stress tolerance when using phenol variants such as CAVA indicates the feasibility of applying bottom-up

404

synthetic methods, including those that meet the basic requirements of green chemistry, to the production of organic

405

fertilizers for use in farming. Similarities in the structural and physicochemical properties of the artificial phenol

406

variants and commercial HA revealed the artificial macromolecules to be a compositional subset of the commercial

407

HA, while differences between them indicated the aromaticity and related hydroxyl and carboxylic groups to be

408

critical for inducing the previously reported HS-based plant bioactivity. Since bottom-up syntheses can be scaled up

409

and since the products of such syntheses can be finely tuned, these reactions should be readily amenable to quality

410

control and assurance, as has been the case for plastic engineering.

411 412

ASSOCIATED CONTENT

413

Supporting information

414

Inhibitory effects of seed germination rate with natural phenols. Recapitulation of auxin-like lateral root growth, FT-

415

IR-ATR spectra, and hydrodynamic size distribution of commercial humic acids and natural phenol variants.

416 417

AUTHOT INFORMATION

418

Corresponding Authors

419

*E-mail: [email protected] (W.-Y. Kim).

420

*E-mail: [email protected] (J.-R. Jeon).

421

Funding

422

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture,

423

Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program funded by Ministry of

424

Agriculture, Food and Rural Affairs (MAFRA) (Grant number 115085-2).

425

Notes 15



426

The authors declare no competing financial interest.

427 428

ACKNOWLEDGEMENTS

429

Solid –state

430

Institute.

13

C NMR, EFM, EPR, FT-ICR-MS, and elemental analyses were conducted at Korean Basic Science

431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 16


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573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 21



596

FIGURE CAPTION

597

Figure 1. Concentration-dependent enhancement of seed germination rate with commercial humic acids and natural

598

phenol variants. Seeds of wild-type (Col-0) were germinated on MS media containing the indicated concentrations

599

of HA, CA, VA, or CAVA. Seed germination rates were determined using radicle (A) and cotyledon (B) emergence

600

after 2 d and 4 d of incubation, respectively. Data are means ± s.e. (n=3). Significant differences are shown as

601

asterisks (*P

Fungal Laccase-Catalyzed Oxidation of Naturally ... - ACS Publications

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