Two Poplar Hybrid Clones Differ in Phenolic Antioxidant Levels and

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Agricultural and Environmental Chemistry

Two poplar hybrid clones differ in phenolic antioxidant levels and polyphenol oxidase activity in response to high salt and boron irrigation Khanh Nguyen, Carlos Cuellar, Prabhjot Sandy Mavi, Danika LeDuc, Gary Bañuelos, and Monika Sommerhalter J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01106 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 22, 2018

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Journal of Agricultural and Food Chemistry

Two poplar hybrid clones differ in phenolic antioxidant levels and polyphenol oxidase activity in response to high salt and boron irrigation Khanh K. Nguyena, Carlos Cuellara, Prabhjot (Sandy) Mavia, Danika LeDuca, Gary Bañuelosb, Monika Sommerhaltera a

Department of Chemistry and Biochemistry, CSU East Bay, 25800 Carlos Bee Blvd., Hayward,

CA 94542, USA b

USDA, Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, 9611

S. Riverbend Ave., Parlier, CA 93648, USA Corresponding author: Monika Sommerhalter e-mail: [email protected]; phone: 001-510-885-3427

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Abstract

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Poplar hybrids can be used for selenium (Se) and boron (B) phytoremediation under

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saline conditions. The phenolic antioxidant stress response of two salt and B tolerant poplar

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hybrids of parentage Populus trichocarpa x nigra x deltoides was studied using high

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performance liquid chromatography (HPLC) and absorption based assays to determine the

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antioxidant capacity, total phenolic content, hydroxycinnamic acid levels, and the enzyme

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activity of L-phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), phenol peroxidase

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(POD), and laccase. Most remarkable was the contrasting response of the two poplar clones for

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PPO activity and phenolic levels to irrigation with high salt/B water. To cope with stressful

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growing conditions, only one clone increased its phenolic antioxidant level and each clone

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displayed different PPO isoform patterns. Our study shows that poplar hybrids of the same

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parentage can differ in their salt/B stress coping mechanism.

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Keywords: Populus trichocarpa x nigra x deltoides; poplar hybrids; combined salt and boron

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stress; stress response; antioxidants; phenolic compounds; polyphenol oxidase; L-phenylalanine

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ammonia lyase


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Introduction

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The sustainability of forestry or agriculture in arid or semi-arid regions is highly desirable

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and also challenging due to high salinity in the soil and a limited supply of high quality irrigation

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water.1 Furthermore, saline soils in the Western US can also accumulate large amounts of

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natural-occurring boron (B).2 Although B is an essential plant micronutrient, high B levels are

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toxic to plants. The tolerance level depends on the plant species and form of B.3 In some

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regions, such as the San Joaquin Valley (SJV) of Central California, irrigation with B,

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molybdenum, and selenium (Se) contaminated water has led to further ecological problems.4 In

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Fall 1998, as part of a general Se and B phytoremediation strategy, a screening program was

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initiated at the USDA-ARS San Joaquin Valley Research Center (Parlier, CA, USA) to test

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hybrid poplars for their ability to tolerate typical high salt/B irrigation water present in the west

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side of the SJV.5 Hybrid poplars are fast growing trees with economic relevance for the veneer,

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lumber, and paper industry. Ideally, a poplar plantation could be used to recycle salt and B laden

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waters, e.g., drainage or ground water, while the harvested tree products could provide an

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economical resource.6 Several poplar species have already been identified with the ability to

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grow under high salt irrigation,7,8 while others have demonstrated that poplar clones can tolerate

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B contaminated soils and accumulate B.9

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The combined stress of high salinity and B exposure to plants can lead to a wide range of

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effects, including elevating phenolic levels11 and affecting (synergestic or antagonistic) the

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uptake of other ions.10,11 Typically, excessive salinity results in an increase of plant phenolic

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levels.12–15 With some exceptions,16 treatments with excess B decrease phenolic levels.17 Very

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few studies have considered the effect of combined salt and B stress on plant phenolic levels.



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More studies are needed to predict synergistic or antagonistic trends in the plant phenolic

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response to excessive salt/B irrigation when trying to identify salt- and B-tolerant plant species.

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Many phenolic compounds involved in stress response are generated in the

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phenylpropanoid pathway.18 These phenolic compounds effectively quench radicals, chelate

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metal ions, or act as UV light scavengers.19 The increase in phenolic acids of the

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phenylpropanoid pathway can often be linked to raised biochemical activity or elevated mRNA

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levels of the enzyme L-phenylalanine ammonia lyase (PAL).19–21 PAL (EC 4.3.1.5) catalyzes

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the first step in the phenylpropanoid pathway and generates trans-cinnamic acid from L-

47

phenylalanine. Hydroxylations and methyl transfer reactions then generate the hydroxycinnamic

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acids p-coumaric acid, caffeic acid, and ferulic acid followed by further derivatives.

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Enzymes that use phenolic compounds as substrates are polyphenol oxidase (PPO),

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laccase, and phenol peroxidase (POD). PPO is best known for the initiation of the browning

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reaction in cut or bruised plant tissue.22 Some PPO enzymes catalyze the oxidation of

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monophenols to diphenols (monophenolase activity; EC 1.14.18.1), and all PPO enzymes

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catalyze the oxidation of o-diphenols into highly reactive o-quinones (diphenolase activity; EC

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1.10.3.1). Quinones form dark-colored pigments via polymerization or bind to proteins or other

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biomolecules. The oxidation of p-diphenols to p-quinones is catalyzed by laccase enzymes (EC

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1.10.3.2). Laccases might be involved in lignification.23 Phenol peroxidases (EC 1.11.1.7)

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require hydrogen peroxide to oxidize phenolic compounds and are associated with numerous

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processes including lignification and stress response.24,25

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Changes in PPO activity were most prominently associated with wounding and herbivore

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attack.14 However, they have also been linked to salinity,26 heavy metal exposure,27 drought28

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and thermal stress.21 Some plants, including poplar trees, contain numerous PPO genes, whereas 4 ACS Paragon Plus Environment

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other plants, such as walnut or thale cress, contain only one or no PPO gene at all.29–31 Boeckx

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and coworkers discussed the possible involvement of PPO in photosynthesis and abiotic stress

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response.32 Sullivan summarized several examples of special PPO isoforms with biosynthetic

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roles.33 The full range of possible physiological roles for PPO gene products has yet to be

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determined.

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In this study, we investigate the phenolic antioxidant stress-response of two poplar

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hybrids, clone 345-1 and 347-14, exposed to excessive salinity and B. These two clones were

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identified to be the most salt and B tolerant poplar hybrids among 100 hybrid poplar clones

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subjected to a range of high salt and B irrigation waters (10-30 dS/m salinity and 10-15 mg/L B)

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for up to 150 days under micro-field conditions in a multi-year screening program at the USDA-

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ARS San Joaquin Valley Research Center, Parlier, CA, USA.5 The tested poor quality waters

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closely mimicked ground and drainage waters associated with excessive salt and B-laden soils

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present in over 400,000 acres within the westside of San Joaquin Valley in California. The

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clones 345-1 and 347-14 originated from a trichocarpa x deltoides x nigra parentage and have

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been considered to be useful for Se and B phytoremediation strategies under saline conditions.5

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Identifying phenolic antioxidant stress responses for these two clones may help us to identify

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other potential salt/B tolerant plant species that can be used for Se and B phytoremediation

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strategies.

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Materials and Methods

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Materials

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Leaf material was collected from two hybrid poplar clones (345-1 and 347-14) with

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trichocarpa x deltoides x nigra parentage, irrigated with two types of water quality for 150 days:

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low salinity at < 1 dS/m and low B at < 1 mg B/L and high salinity at 10-30 dS/m and high B at 5 ACS Paragon Plus Environment


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10-15 mg B/L, respectively. A detailed description of the field-growing conditions and irrigation

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scheduling with both good and poor quality waters can be found in Ref.5 Generally, each sample

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consisted of 5-6 leaves taken from the northwestern top (120-180 cm), middle (60-120 cm), and

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bottom (0-60 cm) section of the poplar trees. Each sampling was replicated 4 times, and each

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replicate consisted of three trees per water quality treatment corresponding to a total of 12 trees

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per each water treatment for each clone. Leaf samples were flash frozen in liquid nitrogen and

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stored at -80 oC. For the analyses described here, leaf samples from top, middle and bottom tree

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positions were pooled, and three to four sample preparations were performed per clone for each

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water treatment. All chemicals used in this study were procured from Sigma-Aldrich or Fisher

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Scientific, and water from a Millipore purification system was used to prepare all buffer

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solutions.

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DPPH assay

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The leaves were ground with mortar and pestle and placed for one week into a drying

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oven set at 60oC. Each sample was prepared in quadruplet by grinding 5 mg dried leaf material

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in 0.5 mL of 95% ice-cold methanol. The extracts were centrifuged at 15,000 rpm for 5 minutes.

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A volume of 20 µL methanol (blank) or 10-fold diluted sample was mixed with 200 µL of 0.95

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mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) prepared in methanol. After 30 minutes of

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incubation, the absorbance at 510 nm was recorded using a Synergy H1 plate reader from

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BioTek. The quenching effect was calculated according to the following formula: 100 –

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(Abssample/Absmethanol) x 100.

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Folin Ciocalteu assay

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Each sample was prepared in triplicate by grinding 10 mg dried leaf material in 1 mL of

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95% ice-cold methanol. Accoring to Ainsworth and Gillespie, the extracts were incubated in the 6 ACS Paragon Plus Environment

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dark at room temperature for 2 days.34 All samples were centrifuged at 15,000 rpm for 5

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minutes. A 100 µL portion of supernatant or 10-fold diluted supernatant was combined with 200

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µL of 1:10 diluted Folin-Ciocalteu reagent and 700 µL of 0.70 M Na2CO3. After 2 hours of

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incubation, the samples were centrifuged once more at 15,000 rpm for 10 minutes. A volume of

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200 µL centrifuged solution was pipetted into a micro-plate for absorbance measurements at 765

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nm. A standard curve was prepared in parallel based on a dilution series of 8.5 to 170 µg/mL

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gallic acid monohydrate in methanol.

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HPLC quantification of selected phenolic compounds

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Approximately 0.05 g leaf material, previously stored at -80°C, was ground in a pre-

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chilled mortar with 1.5 mL ice-cold methanol and centrifuged at 15,000 rpm for 15 minutes. The

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supernatant was briefly bubbled with argon gas. A 6.0 M sodium hydroxide solution was added

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to reach a final concentration of 2.0 M sodium hydroxide. The sample was incubated for 2 hours

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at room temperature followed by acid hydrolysis. A 6.0 M hydrochloric acid solution was added

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to reach a final concentration of 2.0 M, and the sample was placed for 30 minutes in a boiling

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water bath. Finally, the pH of the extracts was adjusted to 3.0 using 1.0 M sodium hydroxide

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and 1.0 M hydrochloric acid solutions. Prior to injection, all samples were filtered with a 0.22

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µm PVDF (polyvinylidene fluoride) syringe filter from Fisherbrand. Standard solutions in a

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concentration range of 0.5 µg/mL to 50 µg/mL containing either caffeic acid, ferulic acid, p-

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coumaric acid, sinapic acid, protocatechuic acid, catechin, gallic acid, or catechol were prepared

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in methanol.

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Separation of phenolic compounds was performed with an Agilent HP 1100 series HPLC

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system using a Luna C18 250 mm x 4.6 mm column from Phenomenex. The flow rate of the

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mobile phase was kept at 0.5 mL/min. Mobile phase A consisted of HPLC-grade water with 7 ACS Paragon Plus Environment


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0.1% v/v TFA (trifluoroacetic acid), and mobile phase B was acetonitrile. The gradient

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conditions were as follows: 0-5 min: 15% B; 5-33 min: 15%-22% B; 33-50 min: 22%-25% B;

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50-52 min: 25%-80% B; 52-57 min: 80% B, 57-58 min: 80%-15% B, 58-70 min: 15% B. The

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temperature of the column was controlled at 25oC. The injection volume was 10 µL. One

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chromatogram is shown as example in Supplemental Fig. 1. The detection wavelengths of the

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DAD were set to 320 nm to detect caffeic and p-coumaric acid and to 300 nm to detect ferulic

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acid. Only the dominant E-isomers were detected. We were not able to identify peaks for

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sinapic acid, catechol, catechin, gallic acid, and protocatechuic acid. Consequently, these

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compounds are not mentioned further in the text. The retention times for caffeic, p-coumaric,

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and ferulic acid were 22.62 ± 0.28, 34.93 ± 0.40, and 39.52 ± 0.10 min, respectively. All three

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calibration curves of the form Y = slope ∙ X with Y as peak area in mAU sec and X as

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concentration in mg/mL had R2 values above 0.996. The slope values were 91,900, 103,500, and

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81,650 for caffeic, p-coumaric, and ferulic acid, respectively.

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Enzyme activity assays

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A total amount of 0.15 g frozen leaves were ground with 0.750 mL extraction buffer in a

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pre-cooled mortar with a pestle for several minutes. The extraction buffer was composed of 0.10

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M sodium phosphate buffer, pH 7.0, 1% w/v polyvinylpyrrolidone, 1 % v/v Triton-X100, and the

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protease inhibitor cocktail “HALT” supplied by Thermofisher. Each extract was transferred into

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a microcentrifuge tube and centrifuged at 15,000 rpm for 10 minutes.

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To determine the activity of the enzyme PAL, a 75 mM stock solution of L-phenylalanine

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was prepared in 0.1 M sodium borate buffer, pH 8.8. The assay mixtures contained 0.15 mL of

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75 mM L-phenylalanine and 0.10 mL poplar extract and were incubated in a water bath set to 30

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°C. Control reactions with 0.15 mL 0.1 M sodium borate buffer, pH 8.8 and 0.10 mL poplar 8 ACS Paragon Plus Environment

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extract were incubated in the same water bath. After 15, 30, and 45 minutes, 70 µL aliquots

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were removed and mixed with 35 µL 6 M hydrochloric acid to stop the reaction. After

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centrifugation for 20 minutes at 15,000 rpm, the supernatants were placed in HPLC vials. An

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Agilent HP 1100 series HPLC system with a Luna C18 250 mm x 4.6 mm column from

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Phenomenex was used to quantify the formation of trans-cinnamic acid. HPLC-grade water with

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0.1% v/v TFA (mobile phase A) and acetonitrile (mobile phase B) was applied in a gradient with

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the following conditions: 0-25 min: 40% B-80%B; 25-28 min: 80% B; 28-29 min: 80%-40% B;

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29-35 min: 40% B. The flow rate of the mobile phase was 0.5 mL/min. The temperature of the

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column was controlled at 25oC. The injection volume was set to 10 µL. The detection

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wavelength of the DAD was 290 nm. The retention time for trans-cinnamic acid was 14.31 ±

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0.25 min for the standards with a calibration curve of Y = 122.83 X with X in µg/mL and an R2-

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value of 0.9947. Some sample assays exhibited a shift to higher retention times of

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approximately 15.2 min. PAL activity is given in units of mIU corresponding to the formation of

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one nanomol trans-cinnamic acid per minute at pH 8.8 and 30 °C.

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Laccase and PPO activity were determined with a coupled assay developed by Esterbauer

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and coworkers.35 The decline in absorbance of 5-thio-2-nitrobenzoate, which readily reacts with

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the p-quinone or o-quinone products, was monitored at 412 nm for two to five minutes in five-

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second intervals using the kinetics option of a nanodrop 2000c spectrophotometer from

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Thermofisher. A typical PPO assay contained 10 mM substrate (catechol or caffeic acid), 50

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mM sodium phosphate buffer, pH 7.0, 1% w/v sodium dodecyl sulfate (SDS), 0.040 mM 5-thio-

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2-nitrobenzoate, and 10 µL 5-fold diluted crude extract in a total volume of 2 mL. To test PPO

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activity with monophenolic substrates (p-coumaric and ferulic acid), the crude extract was not

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diluted, and the time was increased to one hour with measurements taken every minute. Laccase



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assays were conducted with hydroquinone as substrate, but without SDS. Control reactions

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without crude extract and without substrate were performed. PPO and laccase activity are

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reported in IU corresponding to the oxidation of one micromol phenolic substrate in one minute

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at room temperature and pH 7.0. Molar absorptivity values of 11.4 mM-1 cm-1 for PPO assays

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and 13.1 mM-1 cm-1 for laccase assays with hydroquinone were obtained from Ref. 35 To test the

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substrate preference of laccase, assays were also conducted with syringaladzine36 and ABTS.37

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Phenol peroxidase activity was measured according to Chance and Maehly.38 A typical

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reaction mixture contained 50 mM sodium phosphate buffer, pH 7.0, 10 mM guaiacol, 5 mM

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H2O2, and 10 µL 5-fold diluted crude extract in a total reaction volume of 2 mL. Absorbance

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increase was followed at 470 nm (epsilon = 26,600 M-1 cm-1).

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The total protein content was determined via the Bradford assay with six bovine gamma

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globulin standards in the range of 125 µg/mL to 1,500 µg/mL. The protein standards and the

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Bradford reagent were procured from Thermofisher. The crude leaf extracts were diluted five-

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fold with 0.10 M sodium phosphate buffer, pH 7.0, to yield protein concentrations within the

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range of the standard curve. Each sample and protein standard was measured in triplicate.

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SDS-PAGE analysis

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SDS-PAGE was performed with 10% Criterion TGX midi gels (13.3 cm x 8.7 cm x 0.1

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cm) from Bio-Rad. The running buffer contained 3.0 g TRIS-base, 14.4 g glycine, and 1.0 g

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SDS per one liter. Crude leaf extracts were prepared as already described for the enzyme

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activity assays. These extracts were mixed in a one-to-one ratio with loading buffer composed of

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100 mM TRIS-HCl, pH 6.8, 4% w/v SDS, 0.2% w/v bromophenol blue, and 20% v/v glycerol.

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Notably, the loading buffer did not contain mercaptoethanol, and none of the samples was

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heated. A pre-stained See Blue standard was procured from Life Technologies. To investigate 10 ACS Paragon Plus Environment

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the effect of SDS on band migration, Novex 10% TRIS-glycine mini gels were used. SDS was

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omitted from the sample loading buffer described above. Running buffers were prepared at

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different SDS concentrations of 0 mM, 1.2 mM, 2.4 mM, and 3.6 mM. Most gels were run for

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90 to 120 minutes at a constant voltage of 125 V. The gel staining method included a color

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enhancement step based on the coupling of quinones with the reagent anilinediethylamine sulfate

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(ADA).39 The gels or cut gel pieces were first immersed for 10 minutes in 40 mL of a sodium

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phosphate buffer set to a pH value of either 6.0 or 7.9. Next, 10 mL of 100 mM catechol were

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added. After 10 min incubation, the gels were washed three times with MilliQ water. The

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washed gels were then immersed for 5 to 15 min in a mixture of 25 mL 0.2 M TRIS-HCl, pH 8.0

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and 5 mL 25 mM ADA prepared in 10 mM hydrochloric acid. Gel images were taken and

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analyzed with a ChemiDoc station and the program Image Lab from BioRad.

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Isoelectric focusing (IEF)

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Novex IEF mini gels with a pH range of 3-10, IEF sample loading buffer, as well as

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cathode and anode buffers were procured from Thermo Fisher Scientific and used according to

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the manufacturer's instructions. The gel was run at 100 V, 200 V, and finally 500 V for time

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periods of 1 hr, 1hr, and 30 min, respectively. The lane loaded with the standard (IEF markers 3-

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10, SERVA liquid mix) was separated from the lanes loaded with poplar leaf extracts with a

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razor blade and stained with Coomassie staining solution. The remaining part of the gel was

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immersed in 40 mL of a 0.1 M sodium phosphate buffer of either pH 6.0 or pH 7.9 supplemented

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with 5 mM SDS, stained with catechol, and later enhanced by ADA addition as described above.

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The pI values of the activity-stained bands were estimated based on a linear fit performed for the

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IEF marker. The trendline equation for the retention factor versus the pI value of the standard

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proteins in the range of pI = 7.4 to 4.5 was y = -0.1363 x + 1.4165 (R2 = 0.9996). 11 ACS Paragon Plus Environment


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Statistical analysis Statistical analysis was performed with the program Minitab (edition 17, Minitab Inc.,

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US). The alpha factor was set to 0.05 for ANOVA analysis. The sample size was n=3, except for

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the DPPH assay with n=4. All data were expressed as mean values plus/minus one standard

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deviation. Letters (a-d) indicate the grouping of samples according to the Tukey method.

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Results

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Changes in phenolic levels upon high salt and B irrigation

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The antioxidant capacity and the total phenolic content of poplar leaves from clones 345-

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1 and 347-14 exposed to excessive salinity and B were measured using the DPPH quenching

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assay and the Folin-Ciocalteu assay (Fig. 1A,B). Antioxidant capacity notably increased in

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leaves of clone 345-1 exposed to high salt/B irrigation. In contrast, clone 347-14 showed no

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change in leaf antioxidant capacity for the same salt/B treatment. Under normal irrigation

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conditions, clone 345-1 had higher antioxidant capacity than clone 347-14. The data on total

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phenolic content followed the same pattern as the antioxidant capacity. However, total phenolic

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content significantly decreased in leaves of clone 347-14 exposed to high salt/B irrigation.

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Specific phenolic compounds within the phenylpropanoid pathway were quantified via

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HPLC. Phenolic compounds are often conjugated with sugar molecules or organic acids (e.g.,

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glucose or quinic acid). Base and acid hydrolysis of methanolic plant extracts releases free

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phenolic acids and facilitates the detection of phenolic acids in HPLC chromatograms.40 We

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were able to quantify p-coumaric and ferulic acid in all leaf samples and caffeic acid, which was

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lowest in abundance, in most leaf samples (Fig. 1C). Overall, clone 345-1 exhibited significantly

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higher amounts of p-coumaric, ferulic, and caffeic acid than clone 347-14 when exposed to


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excessive salinity and B. Comparisons between control and high salt/B irrigation revealed a

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significant rise in phenolic acids for clone 345-1 for high salt/B irrigation, while constant levels

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were measured for clone 347-14 for control and high salt/B irrigation.

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Changes in activity of enzymes involved in phenolic metabolism

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PAL converts L-phenylalanine into trans-cinnamic acid, which is a key precursor for

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phenolic acids of the phenylpropanoid pathway. Overall, clone 345-1 displayed lower PAL

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activity than clone 347-14 (Fig. 2A). Both clones show large variability for their biological

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replicates, and no statistically significant changes were observed with respect to PAL activity

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and irrigation treatment. The highest concentration of trans-cinnamic acid was detected in crude

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extracts prepared from leaves of clone 345-1 grown under high salt/B irrigation (Fig. 2B). Clone

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345-1 also had the highest levels of phenolic acids for high salt/B irrigation. The accumulation

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of trans-cinnamic acid and the subsequent metabolites of the phenylpropanoid pathway might be

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due to a decrease in the activity of enzymes that consume phenolic compounds rather than an

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increase in PAL activity. Consequently, POD, laccase, and PPO activity was measured.

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Figures 3A and 3B summarize the activity data for POD and laccase, respectively. POD

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activity decreased upon high salt/B irrigation for both clones. Laccase activity determined with

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the substrate hydroquinone was low with large relative deviations among biological replicates.

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Hence, no dependence on irrigation treatment was observed. In agreement with observations

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made by Harvey,41 we were not able to detect laccase activity with the substrates syringaldazine

264

or 2,2′-azino-di-(3-ethylbenzthiazoline) sulfonic acid (ABTS).

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PPO activity was determined with the substrate catechol with and without addition of

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sodium dodecyl sulfate (SDS); PPO enzymes are activated by the anionic detergent SDS.42 The

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presence of 5 mM SDS caused an increase in PPO activity by a factor of 1.7 to 5.1 (Fig. 3C). 13 ACS Paragon Plus Environment


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Wang and Constabel had previously demonstrated that two PPO isoforms from poplar hybrids of

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parentage trichocarpa x deltoides x nigra reached maximum activity at 5 mM SDS.43 As

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expected, addition of SDS did not result in an activation of laccase or POD activity (data not

271

shown). PPO activity was dependent on irrigation treatment, but the direction of the change in

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PPO activity was different for the two clones. The highest PPO activity was found for clone

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347-14 after high salt/B irrigation. In contrast, clone 345-1 either showed similar or lower PPO

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activity values after high salt/B irrigation. The same trend was observed for experiments

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conducted with caffeic acid. The activity values for caffeic acid determined in the presence of

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SDS were approximately 2-fold lower compared to the other diphenolic substrate, catechol. In

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contrast, p-coumaric and ferulic acid, both monophenolic, were very poor substrates. Activity

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values for clone 345-1 only reached 2.58 ± 0.54 and 3.60 ± 0.24 mIU/(mg protein) for p-

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coumaric and ferulic acid, respectively. Similar values were obtained for clone 347-14 with 1.38

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± 0.78 and 3.06 ± 0.42 mIU/(mg protein) for p-coumaric and ferulic acid, respectively.

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Monophenolase activity was only determined for samples obtained under normal irrigation

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conditions.

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Clone dependent PPO isoform patterns detected via protein electrophoresis

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The unique SDS activation of PPO was exploited to visualize different PPO isoforms.

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Figure 4 shows SDS-PAGE gels of poplar leaf extracts stained for PPO activity with the

286

substrate catechol. The positions of the bands are reported as apparent but not actual molecular

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weights. The apparent molecular weights are determined by comparing the positions of activity-

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stained bands to a pre-stained standard protein ladder. PPO isoforms differ in their pH

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optima.22,43 In the gel stained at pH 6 (Fig. 4A), one band, located at approximately 52 kDa for

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clone 345-1 and 53 kDa for clone 347-14, is clearly visible. More bands were detected for both 14 ACS Paragon Plus Environment

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clones with the alkaline staining condition at pH 7.9 (Fig. 4B). Clone 345-1 showed two weak

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bands at 57 and 52 kDa for high salt/B irrigation and four bands at 57, 54, 51, and 47 kDa for the

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control condition (normal irrigation). Both samples of clone 347-14 showed three bands at

294

approximately 62, 53, and 51 kDa under the alkaline staining condition. The relative staining

295

intensity of all visible bands in Fig. 1 displayed the same sample-dependent trend as the solution

296

activity assays (Fig. 3C). The bands for clone 345-1 with high salt/B irrigation were the faintest,

297

whereas clone 347-14 with high salt/B irrigation showed the most intense bands.

298

Isoelectric focusing (IEF) separates proteins by their native charge. The IEF gel shown

299

in Figure 5 was cut into three pieces after the run. The standard IEF marker loaded in Lane 1

300

required staining with Coomassie (Fig. 5A). The lanes loaded with poplar leaf extract were

301

stained with catechol at either pH 6 (Fig. 5B) or pH 7.9 (Fig. 5C). The alkaline staining

302

condition revealed more bands than the acidic condition. No bands were detected for the sample

303

from clone 345-1 under high salt/B irrigation. Two bands at approximate pI values of 5.2 and

304

5.4 were visible after staining at pH 6.0. More bands appeared after staining at pH 7.9; the most

305

prominent new bands were located at approximate pI values of 6.2 and 6.4. These new bands

306

most likely originated from the same PPO isoform as the SDS-PAGE bands at higher apparent

307

molecular weight (62kDa, Fig. 4B), as both appeared under more alkaline staining conditions.

308

Supplemental Table S1 summarizes the predicted molecular weights and pI values of 23

309

currently available PPO protein sequences from diverse poplar species. The molecular weights

310

for almost all processed chloroplastic PPO isoforms fall between 56.0 and 57.6 kDa, and their

311

predicted pI values are between 5.2 and 6.15. Only the vacuolar isoform PPO13 is heavier at

312

64.0 to 65.8 kDa. The predicted pI values for PPO13 range from 5.9 to 6.3. It is tempting to

313

speculate that the high molecular weight band (62 kDa, Fig. 4B) and the pI band above 6.2 (Fig. 15 ACS Paragon Plus Environment


Page 16 of 37

314

5C) are both associated with a vacuolar PPO13-like isoform. However, no amino acid sequences

315

specific to the PPO isoforms in the clones 345-1 or 347-14 are currently available, and the SDS-

316

PAGE method reveals only apparent molecular weights. The presence of SDS can increase the

317

electrophoretic mobility of PPO isoforms (Supplemental Fig. 2). This limitation was also

318

encountered in other SDS-PAGE based characterizations of PPO isoforms.42–44

319

In summary, the electrophoretic experiments clearly revealed different PPO staining

320

patterns for the two clones 345-1 and 347-14. Both clones have the same parentage, but each

321

clone ended up with a different set of PPO genes in the hybridization process.

322

Discussion

323

The growth of various poplar hybrids under high salt/B irrigation had been evaluated

324

previously in a multi-year screening program at the USDA-ARS San Joaquin Valley Research

325

Center, Parlier, CA, USA.5 In this study, we focused on clones 345 -1 and 347-14, which both

326

originate from a trichocarpa x deltoides x nigra parentage and predominantly received “good”

327

evaluation scores. Good performance was defined as retaining more than 50% of the leaves and

328

exhibiting only slight leaf toxicity in which necrosis was limited to the leaf margins after

329

excessive irrigation with salinity B-laden waters.5 The phenotype response for all poplar hybrids

330

irrigated with high salt/B water was eventually leaf abscission and leaf necrosis. Although

331

phenotypically similar, the two salt/B tolerant poplar clones accumulated B and chloride in a

332

different manner.5 Clone 345-1 accumulated a considerable amount of chloride but very little B

333

in its leaves. In fact, B levels in the lower leaves of clone 345-1 did not significantly increase

334

with salt/B irrigation. In contrast, clone 347-14 accumulated more B and some chloride in its

335

lower leaves upon salt/B irrigation. These were the first observations that indicated a diverging

336

coping mechanism for the two clones. 5 Since B levels influence phenolic metabolism,17 the 16 ACS Paragon Plus Environment

Page 17 of 37


337

present study focuses on the phenolic antioxidant response of the two salt and B tolerant clones

338

exposed to excessive salinity and B.

339

Plants often raise antioxidant levels to cope with stressful growing conditions, and clone

340

345-1 exemplifies this coping mechanism with potentially toxic levels of Cl and B. As shown in

341

Figure 1, the antioxidant response and the total phenolic content significantly increased upon

342

high salt/B irrigation. Furthermore, an increase in concentration of specific phenolic acids (Fig.

343

1C) and of their precursor, i.e., trans-cinnamic acid (Fig. 2B), was observed. The activity of the

344

enzyme PAL, which generates trans-cinnamic acid, however, remained unchanged in our study.

345

Notably, clone 345-1 exhibited a decline in PPO and POD activity upon high salt/B irrigation

346

(Fig. 3). Low PPO and POD activity might enable plants to maintain high levels of phenolic

347

compounds as part of a strong antioxidant stress response.21,26,45

348

In contrast, clone 347-14 showed significantly increased PPO activity levels with salt/B

349

irrigation. Clone 347-14 also showed low total soluble phenolic content and low levels of

350

specific phenolic acids. Despite overall high PAL activity and a slight decrease in POD activity,

351

clone 347-14 was unable to accumulate phenolic antioxidants. The stress response of clone 347-

352

14 hence diverged from the stress response of clone 345-1. In another study, clone specific

353

changes in PPO mRNA levels were detected using cDNA microarrays: a salt-induced increase in

354

PPO expression was found for the salt tolerant Populus alba clone 14P11, but no significant

355

changes were detected for the salt sensitive clone 6K3.7 A high salinity shock, as well as

356

dehydration, resulted in increased expression of a PPO gene identified as PPO3 in Populus

357

euphratica.46 The salt-tolerant P. alba clone 14P11, P. euphratica, and clone 347-14 share an

358

unexpected stress response with respect to a stress-triggered increase of either PPO expression or

359

PPO activity. 17 ACS Paragon Plus Environment


360

Page 18 of 37

More examples of contradictory experimental results with respect to PPO activity in

361

response to abiotic stress were highlighted in a recent review.32 The authors pointed out that

362

PPO can function as both a pro- and anti-oxidant; the PPO-catalyzed formation of o-quinones is

363

linked to the reduction of oxygen to water, but the presence of highly reactive quinones can

364

result in the generation of reactive oxygen species (ROS). There is a great variety of possible

365

physiological roles for PPO enzymes.33 As such, possible benefits of increased PPO activity

366

could include an antioxidant role, sealing of physical injuries, or other undiscovered biosynthetic

367

roles. Some PPO genes, notably PPO1 from P. trichocarpa x deltoides, are wound inducible.14,29

368

The polymerization or the attachment of quinones to other biomolecules could seal physical

369

injuries, similar to the wounding response after an herbivore attack.25

370

Previously, others suggested that lower PPO activity can preserve phenolic antioxidant

371

levels.26,45 Our data for both clones support the notion that the level of phenolic antioxidants is

372

influenced by PPO activity, albeit in a different direction. But how might an increase in PPO

373

activity lower the pool of available phenolic antioxidants in clone 347-14? In a first scenario,

374

high PPO activity would directly deplete phenolic antioxidants. In vitro, the diphenolic

375

compound caffeic acid and its quinic acid conjugate, chlorogenic acid, are good PPO

376

substrates.29 Wang and Constabel detected diphenolase but no monophenolase activity for two

377

poplar PPO isoforms.43 Our study confirmed that monophenolic compounds are poor substrates

378

for poplar leaf PPO. However, if caffeic acid is removed from the phenylpropanoid pathway by

379

PPO catalyzed oxidation, the concentration levels of the other two phenolic acids might decrease

380

as an indirect consequence of sharing the same pathway. A P450-like hydroxylase can convert

381

p-coumaric acid into caffeic acid, and caffeic acid can be turned into ferulic acid via caffeic acid

382

O-methyltransferase.47-49


Page 19 of 37

383


In a second scenario, the cellular location of PPO and potential PPO substrates must also

384

be considered. Phenolic compounds are predominantly found in vacuoles. For example, only

385

minor amounts of ferulic, p-coumaric, and caffeic acid were found in the cytosol of soybean

386

leaves, while over 88% was found in vacuoles.50 In contrast, most PPO enzymes are localized in

387

plastids, particularly the chloroplast.22,29,32 The leaves from the tested poplar trees grown with

388

high salt/B irrigation waters eventually showed some necrosis on the edges of the leaves.

389

Booker and Miller suggested that cellular compartmentalization might be lost under stressful

390

conditions,20 which would greatly facilitate reactive encounters between plastid localized PPO

391

enzymes and numerous phenolic substrates. It is also conceivable that chloroplastic

392

concentrations of the potential PPO substrates adjust to the growth conditions. For example,

393

Boeckx and coworkers discovered an increase of caffeoyl malate in red clover leaves upon light

394

exposure.51

395

The third possible scenario is based on the discovery of new PPO isoforms localized to

396

other cellular compartments, including the Golgi apparatus and vacuoles.52 For example, a

397

vacuolar PPO was shown to catalyze aurone formation in snapdragon flowers.53 Aurone

398

synthases belong to a new group of plant PPOs with unusual structural and functional

399

properties.54 Tran and Constabel demonstrated that one poplar PPO isoform, called PPO13, is

400

located in vacuoles of poplar leaves.29 The functional role of the vacuolar isoform PPO13 in

401

poplar is still unknown. Tran and Constabel suggested that PPO13 carries out a specialized,

402

possibly biosynthetic role in poplar.29 The corresponding gene in P. trichocarpa contains an

403

intron, and two splicing variants of PPO13 can be expressed (Supplemental Table S1). Provided

404

that PPO13 is indeed expressed in our leaf samples, a direct PPO-catalyzed depletion of phenolic

405

compounds would be possible even in leaves with intact cellular structure. We speculated that



Page 20 of 37

406

clone 347-14 contains a PPO13-like isoform visualized at an apparent molecular weight of 62

407

kDa in activity stained SDS-PAGE gels (Fig. 4B) and a pI value above 6 in activity-stained IEF

408

gels (Fig. 5B), but the corresponding bands did not increase in staining intensity under high

409

salt/B stress. Other SDS-PAGE bands at lower apparent molecular weight, in contrast, increased

410

in staining intensity.

411

The fourth scenario is based on studies suggesting an indirect influence of PPO on

412

phenolic content via metabolic flux control 52 or altered gene expression.31 Downregulation of

413

PPO in potato tubers increased the concentration of chlorogenic acid, which in turn resulted in

414

higher blight resistance.55 Silencing of the single PPO gene in walnut resulted in leaf necrosis,

415

altered the concentration levels of diverse phenolic metabolites and changed expression levels of

416

several genes in the phenylpropanoid pathway. For example, mRNA levels of coumaric acid 4-

417

hydroxylase (C4H) increased significantly.31 Considering the chloroplastic localization of most

418

PPO isoforms, such an indirect mechanism would provide the most likely link between PPO

419

activity levels and phenolic antioxidants in poplar leaf extracts of clones 345-1 and 347-14.

420

In general, poplar trees contain many different PPO genes with expression levels that

421

depend on the tissue, the developmental stage of the tissue, and external triggers.29 PPO most

422

likely performs multiple different roles in plants with large PPO gene families.30,32 As

423

demonstrated via activity stained electrophoresis experiments, the two poplar clones 345-1 and

424

347-14 express a different array of PPO isoforms. We propose that the presence of different

425

PPO isoforms contrastingly affected the phenolic antioxidant response of the two poplar hybrids

426

upon high salt/B irrigation. High or low PPO activity therefore cannot be used to predict salt/B

427

tolerance in plants with larger PPO protein families, such as poplar hybrids.


Page 21 of 37

428


The two salt and B tolerant poplar clones 345-1 and 347-14 were previously identified

429

from a multiyear microfield study as promising candidates for Se and B phytoremediation under

430

saline conditions. In this study, biochemical assays were performed on their leaves to evaluate

431

their responses to cope with adverse growing conditions, exposure to excessive B and salinity.

432

Although both clones originated from the same parentage, they diverged in their antioxidant

433

stress response. This observation prompts the question of whether further contrasting molecular

434

responses might be observed as part of other coping mechanisms, such as expression of salt

435

transporters or biosynthesis of osmolytes. Our future efforts with these two clones will include

436

comparing the levels of soluble sugars and amino acids, such as proline and glycine-betaine, in

437

leaf extracts of the two poplar hybrids after high salt and B exposure. In addition, we plan to

438

determine the PPO isoform sequences in the clones 345-1 and 347-14. These two clones provide

439

a unique opportunity to unravel the diverse roles that PPO enzymes can play in abiotic and biotic

440

stress response. Such information can aid in developing more robust biomarker screens for

441

identifying salt and B tolerance in more diverse plant species. With reoccurring droughts and

442

limited supplies of good-quality water, we need efficient and reliable means to identify tolerant

443

plants that are crucial to sustain agriculture in arid or semi-arid regions.

444

Conflict of interest

445

The authors declare no competing financial interest.

446

Abbreviations: ADA, anilinediethylamine sulfate; ANOVA, analysis of variance; B, boron;

447

DAD, diode array detector; DPPH, 1,1-diphenyl-2-picrylhydrazyl; HPLC, high performance

448

liquid chromatography; IEF, isoelectric focusing; PAGE, polyacrylamide gel electrophoresis;

449

PAL L-phenylalanine ammonia lyase, POD, phenol peroxidase, PPO, polyphenol oxidase;



Page 22 of 37

450

PVDF, polyvinylidene fluoride; SDS, sodium dodecyl sulfate; TFA, trifluoroacetic acid; TRIS-

451

HCL, trisaminomethane hydrochloride

452

Funding Sources Funding for this work was obtained via a Faculty Support Grant from CSU East Bay

453 454

awarded to MS. KKN, CC, and PM received research fellowships from the Center of Student

455

Research at CSU East Bay. Financial support is also acknowledged by CSU Fresno Agricultural

456

Research Initiative (CC#350034 - ARI Poplar Genomics ARI# 10-1-008-23).

457

Supporting Information

458

HPLC chromatogram of a poplar leaf extract; table with information on PPO sequences from

459

diverse poplar species; graph of electrophoretic mobility of SDS-PAGE bands stained for PPO

460

activity as a function of SDS concentration. This material is available free of charge via the

461

Internet at http://pubs.acs.org.

462

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AcPPO from cherimoya (Annona cherimola Mill.) is localized to the Golgi apparatus.

603

Plant Sci. 2018, 266, 46-54.


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Ono, E.; Hatayama, M.; Isono, Y.; Sato, T.; Watanabe, R.; Yonekura-Sakakibara, K.;

605

Fukuchi-Mizutani, M.; Tanaka, Y.; Kusumi, T.; Nishino, T.; et al. Localization of a

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flavonoid biosynthetic polyphenol oxidase in vacuoles. Plant J. 2006, 45, 133–143.

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(54)

Molitor, C.; Mauracher, S. G.; Pargan, S.; Mayer, R. L.; Halbwirth, H.; Rompel, A.

608

Latent and active aurone synthase from petals of C. grandiflora: a polyphenol oxidase

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with unique characteristics. Planta 2015, 242, 519–537.

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(55)

Llorente, B.; López, M. G.; Carrari, F.; Asís, R.; Di Paola Naranjo, R. D.; Flawiá, M. M.;

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Alonso, G. D.; Bravo-Almonacid, F. Downregulation of polyphenol oxidase in potato

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tubers redirects phenylpropanoid metabolism enhancing chlorogenate content and late

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blight resistance. Mol. Breed. 2014, 34, 2049–2063.



614

Page 30 of 37

Figure captions

615

Fig. 1: Antioxidant capacity of poplar leaf extracts determined with the DPPH quenching

616

assay (n=4) (A) and total phenolic content (n=3) (B). The levels of specific phenolic acids were

617

determined via HPLC (C). Columns (white for control and grey for high salt/B irrigation)

618

represent the mean ±1 SD. The letters (a-d) indicate statistically significant differences as

619

determined with the Tukey test.

620

Fig. 2: The activity of the enzyme PAL (A) and the concentration of the PAL substrate

621

trans-cinnamic acid (B). The columns (white for control and grey for high salt/B irrigation)

622

represent the mean ±1 SD. Same letters above the columns indicate that the values are not

623

significantly different from each other.

624

Fig. 3: Enzyme activity of poplar leaf extracts for phenol peroxidase (POD) (A), laccase

625

(B), and polyphenol oxidase (PPO) (C). White and grey columns represent the mean ±1 SD

626

(n=3) for leaf extracts from control and high salt/B irrigated conditions. The letters (a-d) indicate

627

statistically significant differences as determined with the Tukey test.

628

Fig. 4: SDS-PAGE gel stained for catecholase activity at pH 6.0 (A) and pH 7.9 (B).

629

Lane 1 contained the SeeBlue pre-stained standard. Lanes 2-5 were loaded with crude extracts

630

from clone 345-1 high salt/B, clone 345-1 control, clone 347-14 high salt/B, and clone 347-14

631

control (2.5 µg total protein per lane), respectively.

632

Fig. 5: IEF gel pieces after applying Coomassie staining solution (A) or staining for

633

catecholase activity in the presence of SDS at pH 6.0 (B) and 7.9 (C). The IEF marker “Serva

634

liquid mix” was run in the first lane and then cut from the main gel. Lanes 1-4 in gel pieces (B)


Page 31 of 37


635

and (C) were loaded with crude extracts from clone 345-1 high salt/B, clone 345-1 control, clone

636

347-14 high salt/B, and clone 347-14 control (2.5 µg total protein per lane), respectively.

637



Page 32 of 37

Figure 1

30

A

25

B

a

a

20

Total phenolic content (mg GAE/ g DW)

DPPH quenching response (%)

25

b

15

c c

10 5 0

phenolic acid (mg/g FW)

15

c d

10 5 0

345-1

C

20

b

347-14

345-1

347-14

0.3 0.25

a a

0.2 0.15

a b

0.1

b

c c

0.05

a b

b

not detected

0 345-1

347-14

p-coumaric acid

345-1

347-14

ferulic acid

345-1

347-14

caffeic acid


Page 33 of 37


Figure 2:

B

0.06

b 0.04

ab

0.02

12

a

10

a a trans-cinnamic acid (µg/g FW))

PAL activity (mIU/mg protein)

A 0.08

8 6 4

b

b b

2 0

0.00

345-1

347-14

345-1

347-14



Page 34 of 37

Figure 3:

B 0.14 a

laccase activity (IU/mg protein)

POD activity (IU/mg protein)

A 1.2 1.0

ab

0.8

b

b

0.6 0.4 0.2 0.0

345-1

PPO activity (IU/mg protein)

C

a

0.12 0.10

a a a

0.08 0.06 0.04 0.02 0.00

347-14

345-1

347-14

8

a

7 6 5 4 3

c

a

2 1

a

b b b

b

345-1

347-14

d

b c

b

0

catechol no SDS

345-1

347-14

catechol and SDS

345-1

347-14

caffeic acid and SDS


Page 35 of 37


Figure 4



Page 36 of 37

Figure 5


Page 37 of 37


TOC Graphic

Clone 345-1 Phenolic levels Salt/B PPO PPO activity activity

Clone 347-14 PPO activity Salt/B

Phenolic levels


Two Poplar Hybrid Clones Differ in Phenolic Antioxidant Levels and

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