International Journal of Molecular Sciences

Article Syringic Acid Alleviates Cesium-Induced Growth Defect in Arabidopsis

Eri Adams † , Takae Miyazaki, Ju Yeon Moon, Yuji Sawada, Muneo Sato, Kiminori Toyooka , Masami Yokota Hirai and Ryoung Shin *

RIKEN Center for Sustainable Resource Science, 1-7-22 Suehirocho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan; [email protected] (E.A.); [email protected] (T.M.); [email protected] (J.Y.M.); [email protected] (Y.S.); [email protected] (M.S.); [email protected] (K.T.); [email protected] (M.Y.H.) * Correspondence: [email protected]; Tel.: +81-045-503-9577 Current address: Galdieria, Co., Ltd., 2-4-2-709 Nihonbashi Hama-cho, Chuo-ku, Tokyo 103-0007, Japan. †  Received: 30 October 2020; Accepted: 27 November 2020; Published: 30 November 2020 

Abstract: Syringic acid, a phenolic compound, serves a variety of beneficial functions in cells. Syringic acid increases in plants in response to cesium, and exogenous application of syringic acid resulted in a significant attenuation of cesium-induced growth defects in Arabidopsis. In addition, cesium or syringic acid application to plants also resulted in increased lignin deposition in interfascicular fibers. To better understand the role of lignin and syringic acid in attenuating cesium-induced growth defects, two mutants for Arabidopsis REDUCED EPIDERMAL FLUORESCENE 4 (REF4) and fourteen laccase mutants, some of which have lower levels of lignin, were evaluated for their response to cesium. These mutants responded differently to cesium stress, compared to control plants, and the application of syringic acid alleviated cesium-induced growth defects in the laccase mutants but not in the ref4 mutants. These findings imply that lignin plays a role in cesium signaling but the attenuation of cesium stress defects by syringic acid is mediated by regulatory components of lignin biosynthesis and not lignin biosynthesis itself. In contrast, syringic acid did not alleviate any low potassium-induced growth defects. Collectively, our findings provide the first established link between lignin and cesium stress via syringic acid in plants.

Keywords: cesium; lignin; REF4; syringic acid

1. Introduction Excessive levels of cesium in soils negatively impact plant growth mainly through a restriction in the ability of plants to take up potassium due to the chemical similarity between cesium and potassium [1–8]. The regulation of cesium uptake and plant responses to cesium stress, however, have not been thoroughly elucidated. We previously reported that cesium stress in plants induces jasmonic acid biosynthesis and signaling, and that jasmonic acid functions antagonistically in the induction of the expression of HIGH AFFINITY K+ TRANSPORTER5 (HAK5)[1]. Cesium stress alters the level of amino acids in plants, including cysteine and phenylalanine, and cysteine is associated with cesium accumulation in plants [9]. Syringic acid, a natural phenolic compound, is found in most plant species and some fungal species [10,11]. Syringic acid comprises a single benzene ring with two methoxy moieties attached to the ring at positions 3 and 5, which play an important role in radical-scavenging activity, especially in conjunction with 2,2-diphenyl-1-picryl and β-carotene [12,13]. Application of syringic acid to plants increases antioxidant and peroxidation levels and can be used to alleviate oxidative stress [14]. Syringic acid protects plant, animal, and human cells against lipid and protein oxidation, inflammation,

Int. J. Mol. Sci. 2020, 21, 9116; doi:10.3390/ijms21239116 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 9116 2 of 17 cardiotoxicity, protein carbonylation, osteoporosis, neuro damage, and oxidative stress, and also plays a preventive role against diabetes and ischemia reperfusion [13,15–21]. Syringic acid is synthesized through the shikimic acid pathway [22] and is derived from phenylalanine by β-oxidation [23,24]. serves as the precursor of syringyl lignin, which is a source of lignin-derived syringic acid [13,22–24]. Lignin, a component of plant secondary cell walls, is synthesized from aromatic compounds. Lignin provides the physical strength to the secondary cell wall of xylem conduits that enables long-distance water transport in plants and creates both a physical and chemical barrier against pathogens [25]. Alterations in lignin biosynthesis and/or deposition in plants often modify defense responses and plant growth [25]. The multicopper-containing enzymes, laccases, are expressed in lignifying tissues and are involved in lignin polymerization [26]. Syringic acid contributes to the architectural integrity of lignin [13] and is a target for laccases, a monolignol catalyzing enzyme [23]. Mutation of several lignin biosynthesis enzymes, such as cinnamyl alcohol dehydrogenase, laccases, and peroxidases, reduces lignin accumulation and growth in plants [25]. The regulatory mechanisms and crosstalk that exist between lignin and plant growth, however, are thought to be complex. The plant hormone, jasmonic acid, was confirmed as an upstream component of both plant growth and lignin biosynthesis [27]. The transcriptional coregulatory mediator complex, MED5a (also called REDUCED EPIDERMAL FLUORESCENE 4, REF4, At2g48110) and MED5b (also called REF4-RELATED 1, RFR1, At3g23590), participates in the homeostatic repression of the pathway [28]. The physically interacting REF4 and RFR1 transcriptional coregulatory complex provides the bridge between the basic transcriptional machinery and the specific transcription factors, including various abiotic stress-related transcription factors and phenylpropanoid pathways [28–30]. Arabidopsis semidominant ref4 mutants exhibit partial dwarfism and reduced levels of lignin and sinapolymalate [31]. MED complexes interact with jasmonic acid-activated transcription factors and contribute to plant immunity by balancing the activity of jasmonic acid and pathways [32,33]. In the present study, an increase in syringic acid levels and a greater deposition of lignin in Arabidopsis plants were observed in response to cesium. In order to elucidate how lignin is functionally involved in cesium stress, and its association to syringic acid, the mutants of a lignin polymerization-related enzyme, laccase, and mutants related to the regulation of lignin biosynthesis, ref4, were utilized for the analysis. Results indicate that lignin mediates cesium stress in plants via syringic acid.

2. Results

2.1. Increased Levels of Syringic Acid in Cesium-Treated Arabidopsis Shoots A previous metabolic profiling analysis of Arabidopsis wild-type Col-0 revealed a large metabolic shift in plants exposed to cesium [9]. The level of syringic acid was analyzed to determine if it was affected by cesium. Arabidopsis seedlings were grown for eight days with or without exposure to 0.3 mM cesium. Afterwards, shoots and roots were separately harvested and syringic acid levels were quantified using liquid chromatography coupled with tandem quadrupole mass spectrometry (LC-QqQ-MS) (Figure1 and Figure S1). Results indicated that syringic acid levels were approximately doubled in shoots in response to the cesium treatment (Figure1). In contrast, no alterations in syringic acid levels were observed in roots (Figure S1), indicating that syringic acid-related responses to cesium occur primarily in shoots of Arabidopsis rather than roots. Int. J. Mol. Sci. 2020, 21, 9116 3 of 17

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Figure 1. Relative levels of syringic acid in Arabidopsis. The levels of syringic acid were analyzed in Figure 1. Relative levels of syringic acid in Arabidopsis. The levels of syringic acid were analyzed in shoots of cesium-treated Arabidopsis plants by liquid chromatography coupled with tandem quadrupole shoots of cesium-treated Arabidopsis plants by liquid chromatography coupled with tandem mass spectrometry (LC-QqQ-MS). Results are presented as a ratio between the level of syringic acid in quadrupole mass spectrometry (LC-QqQ-MS). Results are presented as a ratio between the level of untreated control plants versus the level in cesium-treated plants. The white bar indicates the level of syringic acid in untreated control plants versus the level in cesium-treated plants. The white bar syringic acid in shoots of control plants and the grey bar indicates the level of syringic acid in shoots of indicates the level of syringic acid in shoots of control plants and the grey bar indicates the level of cesium-treated Arabidopsis plants. Error bars represent the standard error (SE). A t-test was conducted syringic acid in shoots of cesium-treated Arabidopsis plants. Error bars represent the standard error and ** indicates a significant difference between treated and untreated plants (p < 0.01, n = 4). (SE). A t-test was conducted and ** indicates a significant difference between treated and untreated 2.2. Syringicplants (p Acid< 0.01, Treatment n = 4). Alleviates Cesium-Induced Growth Inhibition Syringic acid was applied in conjunction with cesium to Arabidopsis in order to determine how 2.2. Syringic Acid Treatment Alleviates Cesium-Induced Growth Inhibition syringic acid impacts the plant response to cesium (Figure2). Results indicated that the cesium-induced growthSyringic inhibtion acid of was plants applied was nearlyin conjunction completely with alleviated cesium byto theArabidopsis syringic acidin order application to determine (Figure how2A). syringicPotassium acid and impacts cesium the levels plant were response assessed to using cesium a flame (Figure atomic 2). absorptionResults indicated spectrometer. that the The cesium- level of inducedpotassium growth wasnot inhibtion altered of by plants syringic was acid nearly treatment completely (Figure alleviated2B), but by the the level syringic of cesium acidwas application slightly (Figureincreased 2A). by treatmentPotassium with and 100 cesiumµM syringic levels acid were (Figure assessed2C). using a flame atomic absorption spectrometer.Previous The studies level demonstrated of potassium thatwas not jasmonic altered acid by issyringic involved acid in treatm cesium-inducedent (Figure root2B), but growth the levelretardation of cesium [1, 9was]. In slightly the present increased study, by treatment the root growth with 100 retardation μM syringic by acid cesium (Figure was 2C). not found in ArabidopsisPreviousCol-0 studies plants demonstrated that were concomitantlythat jasmonic acid treated is involved with 100 inµ cesium-inducedM syringic acid root and growth cesium. retardationIn contrast, [1,9]. there wasIn the mild present retardation study, of the root root growth growth bya retardation combined treatment by cesium with was 200 notµM found syringic in Arabidopsisacid and cesium Col-0 plants (Figure that2A). were However, concomitantly a clear alleviative treated with e ff ects100 μ ofM syringic syringic acid acid was and observed cesium. In in contrast,shoots (Figure there 2was). Potassium mild retardation starvation of hasroot been growth shown by toa combined induce an increasetreatment in with ethylene 200 μ andM syringic reactive acidoxygen and species cesium (ROS) (Figure that 2A). is produced However, by a the clear activity alleviative of nicotinamide effects of adeninesyringic dinucleotideacid was observed phosphate in shoots(NADPH) (Figure oxidases 2). Potassium [34,35]. In starvation this regard, has syringic been shown acid is to also induce known an toincrease provide in a ethylene protective and role reactive against oxygenoxidative species stress [36(ROS),37]. Therefore,that is produced based on by these the previous activity findings, of nicotinamide NAPDH oxidaseadenine mutants dinucleotide [38,39] phosphatewere evaluated (NADPH) for their oxidases response [34,35]. to cesium In this and rega tord, determine syringic if acid their is response also known was alteredto provide by the a protectiveapplication role of against syringic oxidative acid (Figure stress S2). [36,37]. An ethylene Therefore, insensitive based on mutant,these previousein2 (Figure findings, S2A–C) NAPDH [38], oxidaseand an NADPHmutants oxidase [38,39] mutant, were evaluatedrespiratory burstfor their oxidase response homolog to C (atrbohcesium-c) and (Figure to S2D–F),determine were if foundtheir responseto be more was tolerant altered toby cesium the application stress. In of comparison syringic acid to (Figure control S2). plants, An ethylene most of insensitive the tested mutantsmutant, einshowed2 (Figure a similar S2A–C) level [38], of and alleviation an NADPH of cesium-induced oxidase mutant, growth respiratory defects burst in response oxidase homolog to treatment C (atrboh with- c)syringic (Figure acid S2D–F), (Figure were S2). found These to results be more indicate tolerant that to ethylene cesium andstress. ROS In producedcomparison by to NADPH control oxidases plants, mostwere of not the likely tested to mutants be involved showed in syringic a similar acid-derived level of alleviation alleviation of ofcesium-induced cesium stress. growth defects in response to treatment with syringic acid (Figure S2). These results indicate that ethylene and ROS produced by NADPH oxidases were not likely to be involved in syringic acid-derived alleviation of cesium stress.

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Figure 2. Application of syringic acid alleviates cesium-induced growth inhibition. (A) Wild-type Figure 2. Application of syringic acid alleviates cesium-induced growth inhibition. (A) Wild-type Arabidopsis Col-0 was grown on media containing 0.5 mM potassium (K) and 0.3 mM cesium (Cs) Arabidopsis Col-0 was grown on media containing 0.5 mM potassium (K) and 0.3 mM cesium (Cs) supplemented with 0 µM, 100 µM, or 200 µM syringic acid for 8 days. Potassium (B) and cesium (C) supplemented with 0 μM, 100 μM, or 200 μM syringic acid for 8 days. Potassium (B) and cesium (C) levels were quantified for each treatment. Statistically significant differences were determined by a levels were quantified for each treatment. Statistically significant differences were determined by a one-way ANOVA with Bonferroni’s multiple comparison post-test and indicated with different letters one-way ANOVA with Bonferroni’s multiple comparison post-test and indicated with different (n > 60 seedlings, p < 0.05). Error bars represent the SE. letters (n > 60 seedlings, p < 0.05). Error bars represent the SE. Since syringic acid is a phenolic compound composed of a single benzene ring, phenolic compounds Since syringic acid is a phenolic compound composed of a single benzene ring, phenolic with a similar structure were also evaluated to determine if they could alleviate cesium-induced compounds with a similar structure were also evaluated to determine if they could alleviate cesium- growth retardation (Figure3). Results indicated that the evaluated simple benzene ring compounds, induced growth retardation (Figure 3). Results indicated that the evaluated simple benzene ring benzoic acid and salicylic acid, were not able to completely alleviate growth retardation induced by compounds, benzoic acid and salicylic acid, were not able to completely alleviate growth retardation treatment with 0.3 mM cesium [1]. Additionally, negative effects on root growth were observed by induced by treatment with 0.3 mM cesium [1]. Additionally, negative effects on root growth were treatment with benzoic acid and salicylic acid, even without cesium treatment (Figure3). One of observed by treatment with benzoic acid and salicylic acid, even without cesium treatment (Figure the monolignols and a lignin precursor, sinapyl alcohol, did however provide a slight alleviation of 3). One of the monolignols and a lignin precursor, sinapyl alcohol, did however provide a slight the growth retardation induced by cesium. Treatment with 1,6-dimethoxybenzoquinone (DMBQ) alleviation of the growth retardation induced by cesium. Treatment with 1,6- resulted in a modest reduction in cesium-induced growth retardation. Specifically, growth retardation dimethoxybenzoquinone (DMBQ) resulted in a modest reduction in cesium-induced growth was 70% more alleviated in cesium-treated plants compared to controls, and approximately 30% retardation. Specifically, growth retardation was 70% more alleviated in cesium-treated plants less alleviated compared to syringic acid-treated plants (Figure3). Notably, the DMBQ treatment compared to controls, and approximately 30% less alleviated compared to syringic acid-treated improved root growth whether the plants were exposed to cesium or not. The degree of attenuation by plants (Figure 3). Notably, the DMBQ treatment improved root growth whether the plants were DMBQ, however, was much smaller than by syringic acid treatment (Figure3). Collectively, these data exposed to cesium or not. The degree of attenuation by DMBQ, however, was much smaller than by syringic acid treatment (Figure 3). Collectively, these data indicate that syringic acid is a phenolic compound that greatly alleviates cesium stress in Arabidopsis, but alleviation is not a general characteristic of phenolic compounds containing a single benzene ring.

Int. J. Mol. Sci. 2020, 21, 9116 5 of 17 indicate that syringic acid is a phenolic compound that greatly alleviates cesium stress in Arabidopsis, butInt. J. alleviation Mol. Sci. 2020 is, 21 not, x FOR a general PEER REVIEW characteristic of phenolic compounds containing a single benzene 5 ring. of 17

Figure 3.3. PhenotypePhenotype analysis analysis of of cesium-induced cesium-induced growth growth inhibition inhibition in Arabidopsis in Arabidopsisseedlings seedlings treated treated with variouswith various phenolic phenolic compounds. compounds. Wild-type Wild-typeArabidopsis ArabidopsisCol-0 was Col-0 grown was for grown 8 days for on 8 media days containingon media 0.5containing mM potassium 0.5 mM (K) potassium and 0.3 mM(K) and cesium 0.3 mM (Cs) supplementedcesium (Cs) supplemented with either 100 withµM either syringic 100 acid,μM syringic 100 µM sinapylacid, 100 alcohol, μM sinapyl 10 µM alcohol, 1,6-dimethoxybenzoquinone 10 μM 1,6-dimethoxybenzoquinone (DMBQ), 10 µ (DMBQ),M benzoic 10 acid,μM benzoic or 10 µM acid, salicylic or 10 acid.μM salicylic Statistically acid. significantStatistically di significantfferences were differences determined were bydetermined a one-way by ANOVA a one-way with ANOVA Bonferroni’s with multipleBonferroni’s comparison multiple post-test comparison and arepost-test indicated and withare indicated different letterswith different (p < 0.05). letters Error (p bars < 0.05). represent Error thebars SE. represent the SE.

2.3. Cesium and Syringic Acid Treatments ResultResult inin IncreasedIncreased LigninLignin DepositionDeposition inin ArabidopsisArabidopsis The degradation of of lignin lignin is is known known to torelease release phenolic phenolic compounds, compounds, including including syringic syringic acid, acid, and andsyringic syringic acid acidis also is alsoknown known to provide to provide structural structural integrity integrity to lignin to lignin [13,40]. [13 Therefore,,40]. Therefore, lignin lignindeposition deposition was analyzed was analyzed in Arabidopsis in Arabidopsis stemsstems to determine to determine if lignin if lignin is involved is involved in plant in plant response response to tocesium cesium stress stress through through syringic syringic acid acid (Figure (Figure 4). 4Soil-grown). Soil-grown ArabidopsisArabidopsis stemsstems were were cryosectioned cryosectioned and andstained stained for forsyringyl syringyl lignin lignin after after treatment treatment with with benzoic benzoic acid, acid, sinapyl sinapyl alcohol, alcohol, cesium cesium chloride, syringic acid, or aa combinedcombined treatmenttreatment ofof cesiumcesium andand syringicsyringic acid.acid. Benzoic acid is not known to be associated with lignin synthesis, and this was confirmedconfirmed in the present results as the levellevel ofof ligninlignin staining inin benzoicbenzoic acid-treatedacid-treated stem stem sections sections was was similar similar to to that that of of the the water-treated water-treated control control (Figure (Figure4). Treatment4). Treatment of plantsof plants with with the the syringyl syringyl lignin lignin precursor, precursor, sinapyl sinapyl alcohol, alcohol, resulted resulted in in increasedincreased ligninlignin deposition in interfascicular fibersfibers (Figure4 4).). Interestingly,Interestingly, thethe cesiumcesium andand syringic syringic acidacid treatments treatments also resultedresulted inin increasedincreased ligninlignin depositiondeposition inin interfascicularinterfascicular fibers,fibers, similarsimilar to thethe sinapylsinapyl alcoholalcohol treatment.treatment. No additional lignin deposition was obse observed,rved, however, in the sectionssections from plantsplants thatthat receivedreceived aa combinedcombined cesiumcesium andand syringicsyringic acidacid treatmenttreatment (Figure(Figure4 4).). TheseThese findingsfindings suggest suggest that that lignin lignin may play a role in regulating plant response to cesiumcesium stress. Additionally, syringicsyringic acid also appears toto bebe a a positive positive mediator mediator between between lignin lignin and cesiumand cesium stress stress resulting resulting in an increase in an increase in cesium in tolerance. cesium tolerance.

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Figure 4. Histochemical analysis of lignin deposition in Arabidopsis stems. Soil-grown Arabidopsis plants Figure 4. Histochemical analysis of lignin deposition in Arabidopsis stems. Soil-grown Arabidopsis were treated with Milli-Q water as a control, syringic acid, sinapyl alcohol, benzoic acid, CsCl, or a plants were treated with Milli-Q water as a control, syringic acid, sinapyl alcohol, benzoic acid, CsCl, combined treatment of CsCl and syringic acid. The cryosectioned samples were 20 µm thick and stained or a combined treatment of CsCl and syringic acid. The cryosectioned samples were 20 μm thick and for syringyl lignin. Images were analyzed and digitally photographed under an optical microscope. stained for syringyl lignin. Images were analyzed and digitally photographed under an optical 2.4. Absencemicroscope. of REF4 Expression Negates Syringic Acid Alleviation of Cesium-Induced Growth Retardation Two lignin defective mutants, ref4-1 and ref4-3 [28,29], and fourteen Arabidopsis laccase mutants, 2.4. Absence of REF4 Expression Negates Syringic Acid Alleviation of Cesium-Induced Growth Retardation some of which are known to be involved in monolignol polymerization [41,42], were evaluated for theirTwo response lignin to defective applications mutants, of cesium ref4-1 and and syringic ref4-3 [28,29], acid to betterand fourteen understand Arabidopsis the role laccase of lignin mutants, in plant someresponse of which to cesium are known stress (Figuresto be involved5 and6 ).in Aerial monolignol portions polymerization of cesium-treated [41,42],Arabidopsis were evaluatedseedlings for theirwere response given a score to applications of 2 when they of cesium were green and andsyringic fully expanded;acid to better a score understand of 0 when the more role than of lignin a half in of plantthe aerial response portion to ofcesium a seedling stress exhibited (Figures chlorosis; 5 and 6). and Aerial a score portions of 1 when of thecesium-treated appearance ofArabidopsis the aerial seedlingsportions waswere intermediate given a score between of 2 when a score they of were 2 and green 0 [3 ].and The fully number expanded; of wild-type a score Col-0of 0 whenArabidopsis more thanseedlings a half with of the a scoreaerial ofportion 0 was of dramatically a seedling exhibi reducedted from chlorosis; 61.3% and to 8.8% a score as a of result 1 when of the thesyringic appearance acid oftreatment. the aerial Seedlings portions withwas intermediate a score of 2 also between increased a score from of 9.0% 2 and to 0 45.6% [3]. The as anumber result of of the wild-type syringic Col- acid 0treatment Arabidopsis (Figure seedlings5B). Both withref4 a score-1 and ofref4 0 was-3 seedlings dramatically exhibited reduced slightly from improved 61.3% to tolerance8.8% as a to result cesium of thestress syringic relative acid to treatment. Col-0 wild-type Seedlings plants with when a score syringic of 2 also acid increased was not from applied 9.0% (Figure to 45.6%5). as However, a result ofthe the number syringic of acidref4-1 treatment and ref4-3 (Figure seedlings 5B). withBoth aref4 score-1 and of 0 ref4 was-3 onlyseedlings reduced exhibited from 40.2% slightly to improved 19.2% and tolerance30.1% to 23.7%,to cesium respectively, stress relative by syringic to Col-0 acid wild-type treatment. plants Furthermore, when syringic the number acidof wasref4 not-1 and appliedref4-3 (Figure 5). However, the number of ref4-1 and ref4-3 seedlings with a score of 0 was only reduced from 40.2% to 19.2% and 30.1% to 23.7%, respectively, by syringic acid treatment. Furthermore, the

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 17 number of ref4-1 and ref4-3 seedlings with a score of 2 did not exhibit a large change as a result of the syringic acid treatment (Figure 5B). Interestingly, the levels of potassium and cesium were not altered Int.in syringic J. Mol. Sci. acid-treated2020, 21, 9116 Col-0, ref4-1, and ref4-3 plants (Figure 5C,D). A slight increase in the level7 of 17of cesium was observed again in Col-0 wild-type plants treated with syringic acid (Figure 5A). Some of the laccases in Arabidopsis are known to be related to lignin synthesis through oxidative seedlingscoupling reactions with a score of radicals of 2 did and not key exhibit enzymes a large that change catalyze as athe result formation of the of syringic monolignol. acid treatment Reduced (Figureactivity5 B).of laccases Interestingly, has been the levels shown of to potassium reduce lignin and cesium content were by 20~40% not altered [41–46]. in syringic Therefore, acid-treated we also Col-0,evaluatedref4-1, Arabidopsis and ref4-3 laccase plants mutants (Figure 5(C,D).atlacs)A for slight theirincrease response in to the cesium level stress of cesium and wasthe application observed againof syringic in Col-0 acid. wild-type plants treated with syringic acid (Figure5A).

Figure 5. The absence of REF4 expression negates the effect of syringic acid on the alleviation of Figure 5. The absence of REF4 expression negates the effect of syringic acid on the alleviation of cesium-induced growth retardation. (A) Two Arabidopsis REF4 mutants (ref4-1 and ref4-3) were grown oncesium-induced media containing growth 0.5 mMretardation. potassium (A) (K)Two and Arabidopsis 0.3 mM cesiumREF4 mutants (Cs) with (ref4 or-1 without and ref4 the-3) were addition grown of 100on mediaµM syringic containing acid (syr).0.5 mM (B potassi) Percentileum (K) green and scores0.3 mM (left cesium axis and(Cs) numberswith or without inside of the bars) addition for ref4 of mutants100 μM syringic and Col-0 acid treated (syr). with(B) Percentile 0.5 mM K green and 0.3scores mM (left Cs withaxis and or without numbers the inside addition of bars) of 100 for µref4M syringicmutants acidand (syr)Col-0 (ntreated> 72). with All of0.5 the mM seedlings K and 0.3 used mM for Cs the with green or without scoring werethe addition also analyzed of 100 μ forM potassiumsyringic acid (C )(syr) and cesium(n > 72). (D All) quantification. of the seedlings Statistically used for significantthe green scoring differences were between also analyzed treatment for groups were determined by a one-way ANOVA with Bonferroni’s multiple comparison post-test and are indicated with different letters (p < 0.05). Error bars represent the SE. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 17

potassium (C) and cesium (D) quantification. Statistically significant differences between treatment Int. J. Mol. Sci.groups2020 ,were21, 9116 determined by a one-way ANOVA with Bonferroni’s multiple comparison post-test and 8 of 17 are indicated with different letters (p < 0.05). Error bars represent the SE.

FigureFigure 6. Response 6. Response of Arabidopsis of Arabidopsislaccase laccase mutants mutants ((atlacsatlacs)) to to cesium cesium treatment. treatment. (A) ( A) series A series of atlacs of atlacs were grown on media containing 0.5 mM potassium (K) and 0.3 mM cesium (Cs) with or without the were grown on media containing 0.5 mM potassium (K) and 0.3 mM cesium (Cs) with or without the addition of 100 μM syringic acid (syr). Average green score for atlac mutants and Col-0 wild-type addition of 100 µM syringic acid (syr). Average green score for atlac mutants and Col-0 wild-type plants plants after 8 days of growth on media containing 0.3 mM cesium (B) and 0.3 mM cesium and syringic B after 8acid days (C). of Statistically growth on significant media containing differences 0.3between mM cesiumthe mutant ( ) lines and compared 0.3 mM cesium to control and plants syringic (Col- acid (C). Statistically0) were determined significant by a Kruskal–Wallis differences between test with the Dunn’s mutant multiple lines comparison compared post-test to control (* for plants p < 0.05, (Col-0) were determined** for p < 0.01, by*** afor Kruskal–Wallis p < 0.001). Error testbars with represent Dunn’s the SE multiple (n > 60). comparison post-test (* for p < 0.05, ** for p < 0.01, *** for p < 0.001). Error bars represent the SE (n > 60).

Some of the laccases in Arabidopsis are known to be related to lignin synthesis through oxidative coupling reactions of radicals and key enzymes that catalyze the formation of monolignol. Reduced activity of laccases has been shown to reduce lignin content by 20~40% [41–46]. Therefore, we also evaluated Arabidopsis laccase mutants (atlacs) for their response to cesium stress and the application of syringic acid. Int. J. Mol. Sci. 2020, 21, 9116 9 of 17

Mutants for 14 out of 17 AtLAC genes in Arabidopsis were evaluated for their response to cesium Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 17 stress with or without the application of syringic acid to determine if AtLACs also play a role in syringic acid alleviationMutants of for cesium-induced 14 out of 17 AtLAC growth genes in retardation Arabidopsis were (Figure evaluated6). Results for their indicated response that to cesium all of the evaluatedstress atlacswith or, with without the exceptionthe application of atlac10 of syringicand atlac12acid to, exhibiteddetermine greaterif AtLACs cesium-induced also play a role growthin retardationsyringic relativeacid alleviation to Col-0 ofwild-type cesium-induced plants growth (Figure retardation6B). However, (Figure no 6).di Resultsfferences indicated of green that scoringall wereof observed the evaluated between atlacs Col-0, with and theatlacs exceptionmutants of atlac10 in their and response atlac12, exhibited to combined greater treatment cesium-induced with cesium and syringicgrowth retardation acid (Figure relative6C) indicating to Col-0 wild-type that the treatmentplants (Figure with 6B). syringic However, acid no was differences capable ofof alleviatinggreen scoring were observed between Col-0 and atlacs mutants in their response to combined treatment the effect of cesium in the atlacs (Figure6). Taken together, these findings suggest that lignin appears to with cesium and syringic acid (Figure 6C) indicating that the treatment with syringic acid was be functionally involved in cesium-induced growth retardation and REF4 plays a role in the alleviation capable of alleviating the effect of cesium in the atlacs (Figure 6). Taken together, these findings of cesium-inducedsuggest that lignin growth appears inhibition to be functionally by syringic in acid.volved in cesium-induced growth retardation and REF4 plays a role in the alleviation of cesium-induced growth inhibition by syringic acid. 2.5. Syringic Acid Has No Effect on Low Potassium Stress The2.5. Syringic presence Acid of Has cesium No Effect ions on is Low known Potassium to compete Stress with potassium uptake in plants and results in growthThe inhibition presence dueof cesium to low ions potassium is known levelsto compet in cellse with [1 potassium,47]. To determine uptake in plants if syringic and results acid could attenuatein growth potassium-starved inhibition due to growthlow potassium defects, levels Col-0 in cells plants, [1,47]. along To determine with ref4 if -1syringic and ref4acid-3 could mutants, wereattenuate grown under potassium-starved low potassium growth conditions defects, with Col-0 or plants, without along syringic with ref4 acid-1 and supplementation ref4-3 mutants, (Figurewere 7). Resultsgrown indicated under low that potassium the syringic conditions acid did with not or alter without the response syringic acid of any supplementation of the evaluated (FigureArabidopsis 7). linesResults to low indicated potassium that conditionsthe syringic (Figureacid did7 no).t Therefore,alter the response it appears of any that of the syringic evaluated acid Arabidopsis specifically lines to low potassium conditions (Figure 7). Therefore, it appears that syringic acid specifically functions in alleviating cesium-induced growth retardation. functions in alleviating cesium-induced growth retardation.

FigureFigure 7. Syringic 7. Syringic acid doesacid notdoes alter not thealter response the response of Arabidopsis of Arabidopsisplants plants to low to potassium. low potassium. (A) Arabidopsis (A) Col-0Arabidopsis wild-type Col-0 and wild-typeref4-1 and andref4-3 ref4-1 mutantand ref4-3 plants mutant were plants grown were grown on media on media containing containing 1750 1750µ M or 25 µMμM potassium or 25 μM (K)potassium with or (K) without with or 100 withoutµM syringic100 μM syringic acid (syr) acid (n (syr)> 60). (n Each> 60). ofEach the of treated the treated seedlings was usedseedlings to quantify was used potassium to quantify ( Bpotassium) levels. Statistically(B) levels. Statistically significant significant differences differences between between the treatment the groups were determined by a one-way ANOVA with Bonferroni’s multiple comparison post-test and are indicated with different letters (p < 0.05). Error bars represent the SE. Int. J. Mol. Sci. 2020, 21, 9116 10 of 17

3. Discussion Cesium is not considered to be beneficial to plants as it acts as a competitor for potassium uptake. The importance of understanding cesium transport and its effect in plants increased after the Fukushima nuclear power plant accident in Japan in 2011. Most studies, however, have focused on cesium-induced potassium deficiency and cesium transport in cells [7,48–50]. Potassium levels are regulated by phytohormones, such as jasmonic acid, cytokinins, auxin, ABA, and ethylene, as well as ROS [34,35,51–54]. Several studies have been conducted to determine if cesium transport or signaling is regulated by one or more of these factors. Among these factors, jasmonic acid biosynthesis and downstream signaling components were demonstrated to be induced by cesium, and the jasmonic acid-defective Arabidopsis mutants, aos and coi1-6, were shown to exhibit tolerance to cesium in roots, relative to wild-type control plants. Interestingly, the increased tolerance was not attributed to lower cesium accumulation or greater potassium accumulation [2]. A similar increase in tolerance was also observed in the present study when cesium-stressed wild-type Col-0 plants received a concurrent application of syringic acid, but this alleviation was predominantly observed in shoots (Figure2). Jasmonic acid is involved in the phenylpropanoid pathway and lignin biosynthesis [55,56]. Cesium applications resulted in increased levels of jasmonic acid [1,2], syringic acid (Figure1), and lignin deposition (Figure4). Therefore, it is plausible that a triangular interaction among syringic acid, lignin, and jasmonic acid may play a role in cesium-induced growth inhibition. Syringic acid is an abundant phenolic compound in a variety of plant species [13] and has many pharmacological properties, such as anticancer, anti