nanomaterials Article Growth-Promoting Gold Nanoparticles Decrease Stress Responses in Arabidopsis Seedlings Eleonora Ferrari 1 , Francesco Barbero 2,3 , Marti Busquets-Fité 4 , Mirita Franz-Wachtel 5 , Heinz-R. Köhler 6 , Victor Puntes 2,7,8 and Birgit Kemmerling 1, * 1 ZMBP, University of Tübingen, 72076 Tübingen, Germany; eleonora.ferrari@uni-tuebingen.de 2 Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Bellaterra, 08193 Barcelona, Spain; fra.barbero@gmail.com (F.B.); victor.puntes@icn.cat (V.P.) 3 Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193 Barcelona, Spain 4 Applied Nanoparticles, S.L., 08018 Barcelona, Spain; marti.busquets@appliednanoparticles.eu 5 Proteome Center, University of Tübingen, 72076 Tübingen, Germany; mirita.franz-wachtel@uni-tuebingen.de 6 Animal Physiological Ecology, University of Tübingen, 72076 Tübingen, Germany; heinz-r.koehler@uni-tuebingen.de 7 Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain 8 Vall d’Hebron Institut de Recerca (VHIR), 08032 Barcelona, Spain * Correspondence: birgit.kemmerling@zmbp.uni-tuebingen.de Abstract: The global economic success of man-made nanoscale materials has led to a higher pro- duction rate and diversification of emission sources in the environment. For these reasons, novel nanosafety approaches to assess the environmental impact of engineered nanomaterials are required. While studying the potential toxicity of metal nanoparticles (NPs), we realized that gold nanoparticles   (AuNPs) have a growth-promoting rather than a stress-inducing effect. In this study we established stable short- and long-term exposition systems for testing plant responses to NPs. Exposure of Citation: Ferrari, E.; Barbero, F.; plants to moderate concentrations of AuNPs resulted in enhanced growth of the plants with longer Busquets-Fité, M.; Franz-Wachtel, M.; Köhler, H.-R.; Puntes, V.; primary roots, more and longer lateral roots and increased rosette diameter, and reduced oxidative Kemmerling, B. Growth-Promoting stress responses elicited by the immune-stimulatory PAMP flg22. Our data did not reveal any detri- Gold Nanoparticles Decrease Stress mental effects of AuNPs on plants but clearly showed positive effects on growth, presumably by Responses in Arabidopsis Seedlings. their protective influence on oxidative stress responses. Differential transcriptomics and proteomics Nanomaterials 2021, 11, 3161. https:// analyses revealed that oxidative stress responses are downregulated whereas growth-promoting doi.org/10.3390/nano11123161 genes/proteins are upregulated. These omics datasets after AuNP exposure can now be exploited to study the underlying molecular mechanisms of AuNP-induced growth-promotion. Academic Editor: Anne Kahru Keywords: engineered nanomaterial (ENM); nanoparticle (NP); gold nanoparticle (AuNP); plant; Received: 19 October 2021 Arabidopsis thaliana; plant growth; stress response; transcriptomics; proteomics Accepted: 18 November 2021 Published: 23 November 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- Engineered nanomaterials (ENMs) are distributed into the environment in drastically iations. increasing amounts, yet knowledge on the resulting effects of ENMs on the environment is limited [1,2]. Although naturally occurring nanomaterials have always existed, in the last decade the emission rate of anthropogenic nanoparticles (NPs), intentionally or unintentionally released, has been continuously rising [3,4]. For these reasons, novel Copyright: © 2021 by the authors. nanosafety approaches to assess the environmental impact of ENMs are required [5]. Licensee MDPI, Basel, Switzerland. Gold ENMs are used worldwide in various fields, including medicine, biology, chem- This article is an open access article istry, physics, electronics, and cosmetics [6–8]. The unique optical and electrochemical distributed under the terms and properties of AuNPs [9], as well as their accessibility for various surface functionaliza- conditions of the Creative Commons tions [10], have been exploited in many applications ranging from diagnostics and cancer Attribution (CC BY) license (https:// therapy [11] to industrial catalysis [12] and water purification [13]. Furthermore, the latest creativecommons.org/licenses/by/ developments in nanotechnology have opened up new opportunities in the food safety in- 4.0/). dustry [14,15] and agronomy [16]. Several studies have shown that biosynthesized AuNPs Nanomaterials 2021, 11, 3161. https://doi.org/10.3390/nano11123161 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2021, 11, 3161 2 of 24 have larvicidal and nematicidal effects in crop cultivation without adversely affecting their growth and development [17,18], implicating the application of more agro-ecological practices in the future. The increasing production, use, and disposal of ENMs has translated into a higher and uncontrolled release of such materials in the environment [19]. As a result of the unique size-dependent physicochemical properties of NMs, such as higher reactivity than their respective bulk materials, concerns about potential adverse effects have arisen [20,21]. In order to assess the risk of ENMs on biosystems, a comprehensive characterization of the material is required, as their physicochemical properties and behavior after release into the environment depend on their chemical composition, size, shape, and surface [22,23]. Stable colloidal ENMs can be obtained by steric or electrostatic surface functionalization with coating materials [24,25]. Despite the stabilizing function of such surface modifications, it must be taken into account that the chemistry of coatings can change under natural conditions, affecting the properties of ENMs and their biocompatibility [26,27]. To study the risk of ENMs, ecological effects should be studied under natural condi- tions, but model systems also need to be optimized for stable, non-toxic assay conditions and adequate characterization of ENMs. As ENMs can change their properties in differ- ent environments, characterization is necessary for the starting material as well as under experimental conditions. Positive, negative, and no effects of ENMs on human health, animals, and plants have been described. In vitro toxicity studies of AuNPs did not find detectable changes in the concentration of inflammatory markers in either humans or animals [28–31]. On the other hand, AuNPs were associated with dose-dependent imbalances of oxidative stress levels in vitro [32,33], with higher doses of particles responsible for initial oxidative cell damage [34,35]. Many studies have reported that the effects of AuNPs on living organisms are strictly related not only to their concentration, but especially to their size and the physicochemical properties of the coatings [36,37]. Environmental AuNP concentration predicted by screening models are 0.14 µg L−1 in natural waters and 5.99 µg kg−1 in soils [38]. Under lab conditions, for these concentrations no measurable physiological effect on plants have been reported [39]. To evaluate whether AuNPs have any effect on plants, higher concentrations are tested to assess the maximum potential risk that could be caused by AuNP accumulation. The effects of AuNPs on plants were reviewed by Siddiqi and Husen [40]. Overall, despite a few contradictory studies, AuNPs have been found to have detrimental effects at high concentrations (≥100 mg/L) [41], with particles with a diameter below 5 nm showing increasing toxicity [42–44]. By contrast, lower concentrations of AuNPs can enhance seed germination and chlorophyll content, and improve growth and productivity in several crops and model plants under laboratory conditions [45–47]. Enhanced and reduced toxic effects and stress responses, such as reactive oxygen species (ROS) production and activation of immune responses, have both been reported in plants after AuNP treatment [40,47–50]. Furthermore, growth-promoting effects are common for nanofertilizers such as ZnNPs, for example, but were also observed for inert AuNPs [18,47]. AuNPs are widely inert and the special physicochemical properties of NPs can be studied without physiological side effects of the bulk material [51]. The mechanism underlying AuNP-induced growth-promotion is not yet understood. Understanding how NPs influence plant growth could be useful for improving crop yield in the future [52]. AuNP uptake is controversially discussed in the community, with studies showing uptake and even transport within the plant and others ruling it out [53–56]. The pore size of cell walls has been determined to be approximately 3–6 nm, a size that precludes the uptake of larger molecules, but some properties of NPs, such as surface charge, may induce morphological changes in the cell wall, thereby affecting pore size and uptake rate [57,58]. Though many plants have been tested for their ability to take up various kinds of ENMs, our knowledge is still limited and many aspects remain elusive. The mechanisms underlying the physiological effects caused by AuNPs in or outside the plant cells will be interesting to elucidate in the future. Nanomaterials 2021, 11, 3161 3 of 24 Transcriptomics and proteomics studies after exposure to multi-walled carbon nan- otubes (MWCNT), titanium dioxide (TiO2 ), cerium dioxide (CeO2 ), and silver (Ag) NPs have been reported in Arabidopsis plants [59–62]. Furthermore, Simon et al. [63] performed a transcriptome sequencing study on the eukaryotic green alga Chlamydomonas reinhardtii after treatment with Ag, TiO2 , zinc oxide (ZnO) NPs, and quantum dots (QDs). Conversely, the transcriptome and proteome changes in plants exposed to AuNPs have not yet been adequately studied. Here, we show that well-characterized AuNPs stabilized with sodium citrate and tannic acid (SCTA) are stable, sterilizable, and functional in promoting the growth of Ara- bidopsis seedlings and do not show any negative effects at moderate concentration levels. Stress responses are downregulated after AuNP exposure at the ROS burst level and also on the transcriptome and proteome level. We studied transcriptome and proteome changes after AuNP-SCTA treatment, and those omics data revealed candidate genes/proteins that could explain the growth-promoting effect of AuNPs on a molecular level. 2. Materials and Methods 2.1. AuNP Synthesis Aqueous dispersions of citrate-stabilized AuNPs were synthesized following two kinetically controlled seeded growth approaches as reported by Bastús et al. [64] and Piella et al. [65]. Tetrachloroauric (III) acid trihydrate (99.9% purity), sodium citrate tribasic dihydrate (99%), and tannic acid were purchased from Sigma-Aldrich (Madrid, Spain). Briefly, 150 mL of sodium citrate (SC) aqueous solution (2.2 mM) were brought to a boil in a three-neck flask under reflux; subsequently, 1 mL of 25 mM chloroauric acid (HAuCl4 ) was injected into the citrate solution. After few minutes the solution became reddish, indicative of AuNP formation (~10 nm, seeds). Afterwards, different sequential steps of growth, consisting of sample dilution plus further addition of gold precursor, led to the desired AuNP size. In the second method, the main difference was the addition of traces of tannic acid (TA) (200 µM) as a co-reducer and an increase in the starting pH to produce highly monodispersed and stable AuNPs. Several batches of the AuNPs were synthesized with and without the addition of TA. All batches presented very similar features and produced similar results in the assays described. The AuNPs were synthesized by the Catalan Institute of Nanoscience and Nanotechnology and purchased from Applied Nanoparticles SL. The AuNP dispersions were concentrated by Amicon® Ultra Centrifugal Filters (100 kDa) (Merck, Darmstadt, Germany). 2.2. AuNP Characterization 2.2.1. Size Determination by Electron Microscopy The diameter of the synthesized AuNPs was measured by analysis of images obtained by scanning electron microscopy (SEM) with FEI Magellan XHR SEM (FEI, Hillsboro, OR, USA) in transmission mode (STEM) operated at 20 kV. Samples were prepared by drop-casting 3 µL of the NP dispersion onto a carbon-coated copper TEM grid and left to dry under mild vacuum. To prevent aggregation of the NPs during the drying procedure, they were previously conjugated with 55 kDa polyvinylpyrrolidone (PVP) (Sigma-Aldrich, Madrid, Spain). More than 500 particles from different regions of the grid were measured. 2.2.2. UV-Vis Spectroscopy The UV-Vis absorption spectra of AuNPs are due to the collective oscillation of their metallic surface electrons, called localized surface plasmon resonance (LSPR). The LSPR profile and maximum position strictly depend on the material, shape, and size of the NPs, as well as the refractive index of the solvent and the vicinity of the NP surfaces. The LSPR profile is highly sensitive to NP aggregation. At inter-particle distances that are less than their diameter, the NP near-field electromagnetic coupling applies, leading to significant UV-Vis spectra changes that translate to a LSPR red-shift and/or to the occurrence of a Nanomaterials 2021, 11, 3161 4 of 24 second peak at a higher wavelength. For large aggregates, an increase in the baseline can be observed [66–68]. UV-Vis spectra were acquired with a Shimadzu UV-2400 spectrophotometer (SSI, Kyoto, Japan). One mL of sample was placed in a plastic cuvette and analyses were performed at time zero or over time in the 300–800 nm range at room temperature. In the case of solidified media, samples were poured into the cuvette prior to jellification. MilliQ water or 12 Murashige and Skoog (MS) agar (Duchefa, Haarlem, The Netherlands) were taken as reference for the different samples. 2.2.3. Size and Zeta Potential Measurements Laser doppler velocimetry and dynamic light scattering were used to determine the Z potential and the hydrodynamic diameter of the AuNPs, respectively, employing a Malvern Zetasizer Nano ZS instrument (light source wavelength at 638.2 nm; detector at a fixed scattering angle of 173◦ ) (Malvern Panalytical Ltd., Malvern, UK). Measurements were performed at 25 ◦ C. Diameters were reported as Z-average and polydispersity index (PDI) calculated by cumulative analysis. 2.2.4. Au Quantification Samples were digested with aqua regia (1:3 HNO3 (70%):HCl (37%)) for 24 h and then diluted with MilliQ water to be further analyzed by induced coupled plasma-mass spectroscopy (ICP-MS) using an ICP-MS NexION 300 from Perkin Elmer (Shelton, CT, USA). 2.3. AuNP Sterilization AuNP suspensions were sterilized by filter sterilization with cellulose mixed ester (CME) and polyethersulfone (PES) filters (Carl Roth, Karlsruhe, Germany), both with a pore size of 0.2 µm, according to the manufacturer’s protocol. UV-Vis spectra of AuNPs before and after filtration were acquired as previously mentioned. 2.4. Plant Growth Conditions and Plant NP Exposition 2.4.1. Seed Sterilization Arabidopsis thaliana ecotype Columbia 0 (Col-0) seeds were surface sterilized by chlo- rine gas treatment in a desiccator containing 50 mL of 12% sodium hypochlorite and 2 mL of 37% HCl for 4 h. The chemicals were purchased from Merck (Darmstadt, Germany). The seeds were dried in a laminar air flow sterile bench for 30 min. The seeds were grown on agar-solidified medium or in hydroponic cultures. 2.4.2. Plant Growth Conditions All plants were grown in long day conditions with 16 h light, 8 h dark, 22 ◦ C, 110 µmol m−2 s−1 light, and 60% relative humidity. 2.4.3. NP Exposure in Agar-Solidified Medium Sterilized seeds were sown on agar plates containing 12 MS plant medium and stratified at 4 ◦ C for 2 days in the dark. Afterwards, plates were incubated for 7 days under controlled long day conditions. Plates were placed vertically to allow root growth along the agar surface. The reducing and stabilizing agents sodium citrate and tannic acid (SCTA) or AuNP-SCTA were mixed with the medium at the indicated concentrations before jellification. Physicochemical characterization of AuNP-SCTA dispersed in 12 MS agar showed high colloidal stability up to 3 weeks, allowing for 1 week exposure experiments. 2.4.4. NP Exposure in Hydroponic Culture Sterilized seeds were sown on a thin layer of 12 MS agar medium and stratified at 4 ◦C for 2 days in the dark. Subsequently, the seeds were germinated and grown for 2 weeks under controlled long day conditions. The seedling roots grew through the agar Nanomaterials 2021, 11, 3161 5 of 24 into liquid 12 MS plant medium. After 2 weeks, SCTA or AuNP-SCTA were mixed in the indicated concentration with the 12 MS medium and were incubated for 6 h. Since UV-Vis spectroscopic analyses of AuNP-SCTA dispersed in 12 MS revealed no changes in the shape of the spectrum at 6 h, whereas a typical aggregation profile was shown after 9 h, this interval was chosen for short-term experiments. 2.5. Physiological Effects Arabidopsis thaliana seedlings were grown in agar-solidified medium, as described previously, with SCTA or AuNP-SCTA to a final concentration of 10 mg/L. Photographs of 7 day-old seedlings were taken. Growth parameters, i.e., rosette diameter, primary root length, and lateral root length, were measured using the software ImageJ. The lateral root number was determined by counting the number of lateral roots per seedling with 20 seedlings being analyzed for each individual parameter (n = 20). 2.6. Statistical Analysis Statistical significance between groups was evaluated using one-way ANOVA com- bined with Tukey’s honest significant difference (HSD) test. FOX assay data were tested with a two-way nested ANOVA followed by Dunnett’s post-hoc test; data were normally distributed (Shapiro–Wilk test) and showed homogeneity of variances (Levene’s test). Significant differences are indicated with different letters (p < 0.01). Statistical evaluations were performed using JMP (version 15.0.0, Heidelberg, Germany) software. 2.7. Detection of Immune-Related Responses 2.7.1. Oxidative Burst Production of reactive oxygen species (ROS) was measured in a luminol-based assay using a microplate luminometer (CentroPRO LB 962; Berthold Technologies, Bad Wildbad, Germany) as described by Albert et al. [69]. The elicitor flg22 (final concentration 100 nM) was used in the assay as positive control. The horseradish peroxidase, in the presence of ROS, catalyzed the oxidation of luminol to 3-aminophthalate with emission of light at 428 nm. The monitored oxidative burst was measured as emitted light and recorded as relative light units (RLU). The ROS burst was monitored for 30 min for 3 plants per treatment and three leaf pieces per plant (n = 9). 2.7.2. FOX Assay The level of lipid hydroperoxides (LOOHs) was assessed with the modified colorimet- ric ferrous oxidation xylenol orange (FOX) assay as described by Hermes-Lima et al. [70] and adjusted by Schmieg et al. [71]. Leaves of 5 week-old A. thaliana plants were cut into square pieces (about 2 mm2 ) and left to equilibrate overnight in milliQ water. Then the leaf pieces were elicited for 30 min with flg22 (100 nM) or the tested compounds and immedi- ately stored at −80 ◦ C. Three plants and three leaf pieces per plant (n = 9) were used for each sample. Samples were homogenized in ice-cold HPLC-grade methanol in a 1:15 ratio, and 30 µL of the supernatant was used in the reaction mixture. Cumene hydroperoxide equivalents (CHPequiv./mg wet weight) were calculated using the following equation: ABS570 Total Volume CHPE = ABS570+CHP × Volume CHP × Sample Volume × Dilution Factor ABS570 = ABS570+CHP × 1 × 200 30 × 15 2.8. Transciptomics 2.8.1. RNA Extraction RNA was extracted from 100 mg of Arabidopsis seedling roots treated for 6 h and 7 d with 10 mg/L Au-SCTA or SCTA (SC 2.2 mM; TA 200 µM) in triplicate using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) followed by on-column DNA digestion with the RNase-Free DNase Set (QIAGEN, Hilden, Germany). The total RNA concentration, Nanomaterials 2021, 11, 3161 6 of 24 RNA Integrity Number (RIN) value, and rRNA ratio (28S/18S) were evaluated using Agilent2100 Bionalyzer (RNA 6000 Nano Kit; Agilent, Waldbronn, Germany). 2.8.2. Transcriptome Sequencing Analysis The BGI Group (Shenzhen, China) performed the total transcriptome sequencing (RNA-Seq) analysis. Samples were sequenced on the Illumina HiSeq platform. The internal software SOAPnuke v1.5.2 was used to filter low-quality reads, reads with adaptors, or those containing more than 5% of unknown bases (N). Genome mapping of clean reads was performed using HISAT v2.0.4 (Hierarchical Indexing for Spliced Alignment of Transcripts) software [72]. The assembler of RNA-Seq alignments into potential transcripts StringTie v1.0.4 was used to reconstruct transcripts [73]. Cuffcompare, a tool of Cufflinks [74], was used to identify novel transcripts by comparing reconstructed transcripts with genome reference annotation information. The coding ability of those new transcripts was predicted using CPC v0.9-r2 [75]. After novel transcript detection, novel coding transcripts were merged with reference transcripts to get a complete reference and clean reads were mapped to it using Bowtie2 v2.2.5 [76]. For each sample, the gene expression level was calculated with RSEM, a software package for estimating gene and isoform expression levels from RNA-Seq data [77]. Differentially expressed genes (DEGs) were detected with the nonpara- metric approach NOIseq method (parameters: fold change ≥ 2.00 and probability ≥ 0.8) as described by Tarazona et al. [78]. 2.9. Mass Spectrometry Analysis 2.9.1. Total Protein Extraction For total protein extraction from seedlings, 100 mg of material were ground in liquid nitrogen and mixed in a ratio of 1:3 with ice-cold extraction buffer (10% glycerol, 150 mM Tris/HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 10 mM DTT, 0.2% Nonidet P-40, 2% PVPP, 1 tablet of proteinase inhibitor cocktail (Roche, Mannheim, Germany) per 10 mL solution). Protein extraction was performed on a rotor at 4 ◦ C for 1 h and the extract was purified by a centrifuging at 4 ◦ C, 5000× g for 20 min. The supernatant was then transferred through a one-layer Miracloth (Merck, Darmstadt, Germany) in a fresh pre-chilled 1.5 mL tube on ice. 2.9.2. NanoLC-MS/MS Analysis The Proteome Center Tübingen performed the nanoscale liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on total protein extracts as described. Proteins were purified in a 12% NUPAGE Novex Bis-Tris Gel (Invitrogen, Karlsruhe, Germany) for 10 min at 200 V and stained with Colloidal Blue Staining Kit (Invitrogen, Karl- sruhe, Germany). In-gel digestion of proteins was performed as previously described [79]. Extracted peptides were first desalted and then labeled using C18 StageTips [80] as de- scribed elsewhere [81]. Samples were labeled with dimethyl “light” ((CH3 )2 ) and dimethyl “intermediate” ((CH1 D2 )2 ). Complete incorporation levels of the dimethyl labels were achieved in all cases. Eluted peptides were mixed in a 1:1 ratio according to the measured protein amounts. The analysis of the peptide mixture was performed on an Easy-nLC 1200 system coupled to an LTQ Orbitrap Elite or a QExactive HF mass spectrometer (all Thermo Fisher Scientific) as described elsewhere [82] with slight modifications: Peptides were injected onto the column in HPLC solvent A (0.1% formic acid) at a flow rate of 500 nL/min and subsequently eluted with a 227 min (Orbitrap Elite) or 127 min (QExactive HF) gradient of 10–33–50–90% HPLC solvent B (80% ACN in 0.1% formic acid). During peptide elution the flow rate was kept constant at 200 nL/min. In each scan cycle, the 15 (Orbitrap Elite) or 12 (Q Exactive HF) most intense precursor ions were sequentially fragmented using collision-induced dissociation (CID) and higher energy collisional dissociation (HCD) fragmentation, respectively. In all measurements, sequenced precursor masses were excluded from further selection for 60 (Orbitrap Elite) Nanomaterials 2021, 11, 3161 7 of 24 or 30 s (Q Exactive HF). The target values for MS/MS fragmentation were 5000 and 105 charges, and for the MS scan 106 and 3 × 106 charges. 2.9.3. MS Data Processing The MS data were processed with MaxQuant software suite v1.5.2.8 and v1.6.3.4 (Cox and Mann 2008), respectively. A database search was performed using the Andromeda search engine [83], which is a module of the MaxQuant. MS/MS spectra were searched against an Arabidopsis thaliana database obtained from Uniprot, and a database consisting of 285 commonly observed contaminants. In the database search, full tryptic specificity was required and up to two missed cleavages were allowed. Protein N-terminal acetylation and oxidation of methionine were set as variable modifications. Initial precursor mass tolerance was set to 4.5 ppm and to 0.5 Da at the MS/MS level (CID fragmentation), or 20 ppm (HCD fragmentation). Peptide, protein, and modification site identifications were filtered using a target-decoy approach at a false discovery rate (FDR) set to 0.01 [84]. For protein group quantitation a minimum of two quantified peptides were required. Perseus software (v1.6.1.3), a module from the MaxQuant suite [85], was used for calculation of the significance B (psigB ) for each protein ratio with respect to the distance of the median of the distribution of all protein ratios as well as the intensities. All proteins with a fold change ≥2.00- and psigB <0.01 in a pairwise comparison were considered to be differentially expressed. 3. Results 3.1. Physicochemical Characterization of AuNPs Dispersed in Plant Growth Media Two different types of gold nanoparticles (AuNPs) with an average diameter of about 12 nm were synthesized with the two seeded-growth methods reported by Bastús et al. [64] and Piella et al. [65], with the only difference being the addition of tannic acid (TA), which can interact with the NP surface, inferring higher colloidal stability. The physicochemical characterization of one of the used batches of AuNPs prepared in the presence of TA (AuNP- SCTA) is shown in Figure 1a,b, whereas the characterization of AuNP-SC is reported in Supplemental Figure S1a,b. It is important to characterize the evolution of the AuNPs once dispersed in the working media to correctly correlate the pristine and the evolving NP features with the observed biological effects [86]. Thus, over time, physicochemical characterization of AuNP-SC and AuNP-SCTA in the used working media, i.e., 12 MS and agar-solidified 12 MS media, was performed. Once dispersed in 12 MS, AuNP-SC underwent fast aggregation, pointed out by an immediate emergence of a second localized surface plasmon resonance (LSPR) peak at around 650 nm in the UV-Vis spectra (Supplemental Figure S1c) [87]. This aggregation was probably due to the increase in the ionic strength by mono- and divalent inorganic ions in the media ( 12 MS has a salinity of 23 mM). The ions in the media can screen the negative charges provided by the SC present on the surface of the AuNPs, responsible for the electrostatic repulsion between particles [88,89]. By contrast, the UV-Vis spectra of AuNP-SCTA dispersed in 12 MS showed no changes until 6 h of exposure. After 9 h, changes in the spectrum shape were observed, showing the start of a typical aggregation profile that led to complete aggregation after 15 h, de- tectable by a drastic change in the UV-Vis spectrum (Figure 1c) [87]. This result indicates that AuNP-SCTA had a good colloidal stability up to 6 h of exposure to the hydroponic medium, whereas after 9 h the NPs started to slowly aggregate. Therefore, in this study all experiments carried out in hydroponic cultures were short time exposures, in a time range of 6 h. The presence of TA was the only difference between the two types of AuNPs. Thus, we hypothesize that this organic molecule functions as a NP stabilizer, increas- ing the particle stability against salt-driven aggregation. TA confers an effective higher surface charge or partial steric stabilization, preventing NP aggregation. Regarding the observed aggregation of the AuNP-SCTA in 12 MS after 9 h, it could be speculated that organic molecules (e.g., sucrose), present in the medium in excess compared to the NP Nanomaterials 2021, 11, 3161 8 of 24 stabilizers, could progressively replace the NP stabilizers on the NP surface, conferring a negative effect on stabilization and supporting aggregation. However, further studies Nanomaterials 2021, 11, x FOR PEER REVIEW 8 of 25 will be necessary to precisely understand the role and nature of TA in the stabilization of AuNP-SCTA. Figure 1. 1 and ½ MS 1agar. 1.Physicochemical Physicochemical characterization characterizationof AuNP-SCTA of AuNP-SCTA dispersed dispersedin Hin 2O,H½ MS, 2 O, 2 MS, and 2 MS (a) agar. (a) Bright field—scanning transmission electron microscopy (STEM) of AuNP-SCTA.(b) Bright field—scanning transmission electron microscopy (STEM) of AuNP-SCTA. NP (b) NP average diameter measured by STEM; hydrodynamic diameters (in H 2O) measured by dynamic average diameter measured by STEM; hydrodynamic diameters (in H2 O) measured by dynamic light light scattering, reported as Z average and poly dispersity index (PDI); Z potential of the AuNP- scattering, reported as Z average and poly dispersity index (PDI); Z potential of the AuNP-SCTA SCTA dispersed in H2O (pH 6.5, conductivity 0.85 mS/cm). (c) UV-Vis spectra of AuNP-SCTA dispersed in H O (pH 6.5, conductivity 0.85 mS/cm). (c) UV-Vis spectra of AuNP-SCTA dispersed in dispersed in H22O (red) and over time (from time 10 to 15 h) in ½ MS (black). (d) UV-Vis spectra of H2 O (red) and AuNP-SCTA over timein(from dispersed ½ MStimeagar0 at totime 15 h)0in 2 MS (red (black). dashed) and(d)after UV-Vis spectra 3 weeks of AuNP-SCTA of exposure (blue); 1 dispersed in MS agar at time 0 (red dashed) and after 3 weeks of exposure representative2 photograph of Arabidopsis seedlings germinated and grown for 1 week (blue); representative on an agar photograph plate of Arabidopsis containing ½ MS andseedlings AuNPs, germinated and grown showing a typical for 1color reddish weekof onnon-aggregated an agar plate containing AuNPs- 1 MS SCTA. 2 and Absorbance A in arbitrary units (a.u.). All experiments were repeated twiceAbsorbance AuNPs, showing a typical reddish color of non-aggregated AuNPs-SCTA. A with similar results. in arbitrary units (a.u.). All experiments were repeated twice with similar results. UV-Vis It spectroscopy is important of AuNP-SCTA to characterize exposed of the evolution to 12the MSAuNPs agar showed high stability once dispersed at in the least up to 3 weeks (Figure 1d), allowing for long-term exposure experiments. working media to correctly correlate the pristine and the evolving NP features with the Conversely, 1 AuNP-SC biological observed dispersed in 2 MS[86]. effects agar Thus, showed an initial over time, aggregation physicochemical that, unlike in 12 MS, did characterization of not evolveand AuNP-SC over time (Supplemental AuNP-SCTA in the used Figure workingS1d),media, probably due i.e., ½ MStoand the agar-solidified interaction with ½ agar media, MS molecules wasandperformed. the fast viscosity Once increase dispersed dueinto medium jellification underwent ½ MS, AuNP-SC as well as to fast the reduced number aggregation, of particles pointed out by(aggregation an immediate is directly proportional emergence to concentration). of a second Note localized surface that below 10 10 NP mL−1 the collision probability decreases to almost zero, so even if their plasmon resonance (LSPR) peak at around 650 nm in the UV-Vis spectra (Supplemental surfaceS1c) Figure is not passivated, [87]. NPs do not This aggregation wasaggregate. probably due to the increase in the ionic strength In light of these observations, by mono- and divalent inorganic ions the AuNP-SCTA in the media were(½ MS chosen has afor the following salinity of 23 mM).physio- The logical ions in and the molecular media canstudies, screen permitting the negative allcharges the experiments providedtoby bethe conducted SC presentwithonstable the AuNPs, thereby allowing for the correct NP size to be correlated with the surface of the AuNPs, responsible for the electrostatic repulsion between particles [88,89]. observed effects on Arabidopsis. By contrast, the UV-Vis spectra of AuNP-SCTA dispersed in ½ MS showed no Several changes untilAuNP-SCTA batches 6 h of exposure. with After 9 h,very similar changes physicochemical in the spectrum shape features were pro- were observed, duced and tested. The results of the physiological studies after AuNP-SCTA showing the start of a typical aggregation profile that led to complete aggregation after 15 treatment were h, fully reproducible detectable between by a drastic change theindifferent the UV-Visbatches, showing spectrum that the (Figure 1c)synthesis [87]. Thisprotocol result is very robust and produced reliable results. indicates that AuNP-SCTA had a good colloidal stability up to 6 h of exposure to the hydroponic medium, whereas after 9 h the NPs started to slowly aggregate. Therefore, in this study all experiments carried out in hydroponic cultures were short time exposures, in a time range of 6 h. The presence of TA was the only difference between the two types of AuNPs. Thus, we hypothesize that this organic molecule functions as a NP stabilizer, Nanomaterials 2021, 11, 3161 9 of 24 3.2. AuNP-SCTA Sterilization In order to grow seedlings under sterile conditions and to discriminate the AuNP ef- fects from the possible physiological and molecular changes induced in plants by microbial contaminants such as, e.g., (pathogenic) bacteria and fungi, the sterility of the colloidal solution is a fundamental requirement. To sterilize AuNP-SCTA solutions, physical fil- tration methods were chosen. Two different filter materials, i.e., cellulose mixed ester (CME) and polyethersulfone (PES), both with a pore size of 0.2 µm, were tested. To analyze possible changes in the particle concentration or their aggregation state, UV-Vis spectra before and after filtration were acquired (Figure 2). Both filtering procedures were effec- tive in removing all contaminating microorganisms and allowed plant cultivation under sterile conditions. The CME filter, a standard hydrophilic membrane commonly used for a broad range of applications, was shown to significantly affect the amplitude of the spectra, revealing a drastically reduced AuNP-SCTA concentration. By contrast, the PES filter, a hydrophilic and low protein-binding membrane, did not change the spectra and therefore did not affect the concentration of AuNP-SCTA. No changes in the overall shape of the UV-Vis spectrum were observed, indicating that no alterations of the physicochemical properties of the AuNPs occurred. Therefore, PES membranes were used10in Nanomaterials 2021, 11, x FOR PEER REVIEW all subsequent of 25 experiments for NP sterilization. Figure 2.2.UV-Vis Figure UV-Vis spectra of AuNP-SCTA spectra before before of AuNP-SCTA and afterand sterilization with different after sterilization filterdifferent with materials.filter materials. UV-Vis spectra of AuNP-SCTA before (red dashed) and after sterilization with PES (blue) and CME UV-Vis spectra (black) filters of AuNP-SCTA were acquired with before (red dashed) a Shimadzu UV-2400 and after sterilization spectrophotometer. with PES Absorbance A (blue) in and CME (black) arbitraryfilters units were (a.u.). acquired with was The experiment a Shimadzu UV-2400 repeated twice with spectrophotometer. similar results. Absorbance A in arbitrary units (a.u.). The experiment was repeated twice with similar results. 3.3. Physiological Effects of AuNPs 3.3. Physiological Although goldEffects of AuNPs (Au) can be present in the environment from natural sources, in the last decade Although gold (Au)and the increased use candisposal of AuNPs be present in has theaffected the level of environment this chemical from natural sources, in element in soil and water [1]. Although many studies on the accumulation and the last decade the increased use and disposal of AuNPs has affected the level of this physiological effects of Au in various plant species have been conducted [90], a chemical element in soil and water [1]. Although many studies on the accumulation comprehensive investigation on AuNP fate and action after their release into plant growth and media physiological and their effectseffects ofatAu on plants the in various plant physiological, species and transcriptomic, have been conducted proteomic level [90], a comprehensive is missing. In theinvestigation present study, onArabidopsis AuNP fate and action thaliana afterastheir was used release a model intotoplant growth plant media andthetheir investigate effects effects on on of AuNPs plants growth atandthedevelopment. physiological, transcriptomic, As shown in Supplementaland proteomic FigureisS2b, level AuNP-SCTA missing. In theinpresent a range from study, 0 toArabidopsis 20 mg/L affected Arabidopsis thaliana was used rootas growth a model plant to in a dose-dependent investigate manner the effects with a maximal of AuNPs on growth effectand at 10development. mg/L, whereas theAs NP stabilizer shown in Supplemental SCTA (SC 2.2 mM; TA 200 μM) did not affect the primary root length at any of the tested Figure S2b, AuNP-SCTA in a range from 0 to 20 mg/L affected Arabidopsis root growth in concentrations (Supplemental Figure S2a). For this reason, 10 mg/L AuNP-SCTA was achosen dose-dependent manner with as the final concentration in allasubsequent maximal experiments. effect at 10 mg/L, whereas the NP stabilizer SCTA Physiological analyses were performed in orderthe (SC 2.2 mM; TA 200 µM) did not affect primaryArabidopsis to evaluate root length at any of the tested responses concentrations (Supplemental to abiotic stress caused by AuNP-SCTA Figure S2a).Seedlings, exposure. For thisgrown reason, 10controlled under mg/L AuNP-SCTA long was day conditions, chosen as the finalwereconcentration harvested after in 7alldays, and representative subsequent experiments. parameters were recorded, i.e., primary Physiological root length, analyses were rosette diameter, performed innumber order toof evaluate lateral roots, and lateral responses to Arabidopsis root length. For each parameter, another set of plants was grown in the presence of SCTA abiotic stress caused by AuNP-SCTA exposure. Seedlings, grown under controlled long (SC 2.2 mM; TA 200 μM) as a control. Although SCTA did not affect plant growth and development, AuNP-SCTA had a positive influence on all parameters tested. Seedlings germinated and grown on AuNP- SCTA-containing medium developed a longer primary root, with an enhancement of 1.2 folds compared to control seedlings (Figure 3a). Furthermore, the lateral root number and length were positively affected upon AuNP-SCTA treatment, displaying, compared to the Nanomaterials 2021, 11, 3161 10 of 24 day conditions, were harvested after 7 days, and representative parameters were recorded, i.e., primary root length, rosette diameter, number of lateral roots, and lateral root length. For each parameter, another set of plants was grown in the presence of SCTA (SC 2.2 mM; TA 200 µM) as a control. Although SCTA did not affect plant growth and development, AuNP-SCTA had a positive influence on all parameters tested. Seedlings germinated and grown on AuNP- SCTA-containing medium developed a longer primary root, with an enhancement of 1.2 folds compared to control seedlings (Figure 3a). Furthermore, the lateral root number and length were positively affected upon AuNP-SCTA treatment, displaying, compared to the controls, an increase of 1.7- and 1.5-fold, respectively (Figure 3e,f). Shoot develop- ment was also influenced by AuNP-SCTA exposure in the same way as the root system (Figure 3c). The size of the rosette diameters was enhanced by 1.3-fold in comparison to Nanomaterials 2021,the 11, xcontrol FOR PEERseedlings. REVIEW These data show that AuNP-SCTA is not acutely toxic to plants, but 11 of 25 rather have a positive effect on plant growth. Figure 3. AuNP-SCTA enhances growth Figure 3. AuNP-SCTA of Arabidopsis enhances growth seedlings. Wild-type seedlings. of Arabidopsis ArabidopsisWild-type seedlings were grown for 7seedlings Arabidopsis d on agar-solidified ½ MS medium containing 10 mg/L of 1AuNP-SCTA or SCTA (SC 2.2 mM; TA 200 μM) (control) or on were grown for 7 d on agar-solidified 2 MS un-supplemented medium (untreated). Growth parameters medium containing 10 mg/L of AuNP-SCTA or SCTA were scored: (a) primary root length and (b) representative (SC(c)2.2 picture of (a). mM;diameter Rosette TA 200and µM) (d)(control) or onphotograph representative un-supplemented medium of (c). (e) Lateral (untreated). root number. Growth (f) Lateral parameters root length. Results shown wereare means (a) scored: ± SEprimary with n = root 20. Different length labels and (b)a and b indicate statistically representative picturedifferent groups of (a). (c) according Rosette to diameter and multiple comparisons following one-way ANOVA analysis at a probability level of p < 0.01. All experiments were repeated (d) representative twice with similar results. photograph of (c). (e) Lateral root number. (f) Lateral root length. Results shown are means ± SE with n = 20. Different labels a and b indicate statistically different groups according 3.4. Immune Responses to multiple comparisons followingupon AuNP ANOVA one-way Treatment analysis at a probability level of p < 0.01. All experiments were NPs have twice repeated been reported to affect with similar the innate immune system in animals [5]. To assess results. whether they have an influence on plant immune responses, different innate immune defensive reactions in plants were evaluated. The production of reactive oxygen species (ROS) in the apoplast and lipid peroxidation are typical cellular events triggered by the plant surveillance system that detects highly conserved microbe- or pathogen-associated molecular patterns (M/PAMPs) via cell surface-located pattern-recognition receptors The cellular response of Arabidopsis thaliana to abiotic stress resulting from NP exposure was initially measured as reactive oxygen species (ROS) production or oxidative burst, using a luminol-based chemiluminescence assay. As shown in Figure 4a, no ROS production was detected after exposure to milliQ water (untreated control), AuNP-SCTA Nanomaterials 2021, 11, 3161 11 of 24 (100 mg/L), or coating solution SCTA (SC 2.2 mM; TA 200 μM) (control). This also confirms that the NP suspensions were free of endotoxins such as LPS that would induce ROS in plants [93]. As positive control, the PAMP flg22 was added at a final concentration of 3.4.100 nM. Responses Immune The same concentration upon AuNP Treatmentof the elicitor was used as treatment also in combination with AuNP-SCTA (10 or 100 mg/L) or SCTA as control. Although the coating NPs have been reported to affect the innate immune system in animals [5]. To assess solution did not affect the level of ROS production caused by flg22 treatment, AuNP- whether they have an influence on plant immune responses, different innate immune de- SCTA influenced the level of recorded ROS. In particular, in the presence of 10 mg/L fensive reactions in plants were evaluated. The production of reactive oxygen species (ROS) AuNP-SCTA the PAMP (flg22) signal decreased. Furthermore, a 10x higher NP in the apoplast and lipid peroxidation are typical cellular events triggered by the plant concentration (100 mg/L) surveillance system was highly that detects tested,conserved and a further decrease microbe- in the level of ROS or pathogen-associated was molecu- detected. lar patterns (M/PAMPs) via cell surface-located pattern-recognition receptors (PRRs) in a In order process calledto discriminate between pattern-triggered a real immunity decrease (PTI) [91,92].in the ROS production and a mere technical interference The cellular with the response light detection, of Arabidopsis a lipid thaliana peroxidation to abiotic assay wasfrom stress resulting performed. NP ex- Cellular and organelle membranes, due to their high polyunsaturated posure was initially measured as reactive oxygen species (ROS) production or oxidative fatty acid (PUFA) content, are aparticularly burst, using luminol-based susceptible to ROS-induced chemiluminescence assay. peroxidation [94]. The As shown in Figure applied 4a, no ROS colorimetric ferrous oxidation xylenol orange (FOX) assay was modified production was detected after exposure to milliQ water (untreated control), AuNP-SCTA to quantify lipid hydroperoxides (100 (LOOHs) mg/L), or coating in plant solution SCTAextracts. (SC 2.2Upon mM; treatment TA 200 µM) with 10 andThis (control). 100 also mg/Lconfirms AuNP- SCTA that theplus NPflg22, the levelwere suspensions of lipid free peroxidation of endotoxinsdecreased such as LPSsignificantly that would in induce comparison ROS in to flg22 alone (Figure 4b). Treatment with 100 mg/L AuNP-SCTA resulted plants [93]. As positive control, the PAMP flg22 was added at a final concentration of 100 in a more pronounced nM. The same decrease in the lipid concentration of thehydroperoxide elicitor was usedlevelascompared treatment to 10in also mg/L AuNP-SCTA. combination with The FOX assay confirmed the oxidative burst assay results, clearly AuNP-SCTA (10 or 100 mg/L) or SCTA as control. Although the coating solution pointing out that co- did not exposure to PAMPs affect the level of ROS and AuNP-SCTA production caused byreduced the PAMP-induced flg22 treatment, AuNP-SCTAROS burst and influenced the subsequent lipid peroxidation. level of recorded The underlying ROS. In particular, mechanism in the presence of thisAuNP-SCTA of 10 mg/L effect is still elusive, the PAMP but the datasignal (flg22) suggest that AuNP-SCTA decreased. Furthermore,might a 10be×able higherto detoxify ROS and shift NP concentration the balance (100 mg/L) was between growth tested, and a furtheranddecrease immunity intrade-off the level to of the ROSgrowth side. was detected. Figure 4.4. AuNP-SCTA decreases ROS production and and lipid lipid peroxidation peroxidation levels. levels. (a) ROS production measured with a luminol-based assay in leaf squares of Arabidopsis Col-0. ROS production is represented luminol-based assay in leaf squares of Arabidopsis Col-0. ROS production is represented asas relative relative light light units units (RLU) (RLU) after after elicitation with milliQ water (untreated control), flg22 (100 nM) (positive control), AuNPs-SCTA (100 elicitation with milliQ water (untreated control), flg22 (100 nM) (positive control), AuNPs-SCTA (100 mg/L), SCTA (control), mg/L), SCTA (control), flg22 + SCTA, or flg22 + AuNPs-SCTA 10 or 100 mg/L. Results are mean ± SE (n = 9). The experiment was repeated flg22 + SCTA, or flg22 + AuNPs-SCTA 10 or 100 mg/L. Results are mean ± SE (n = 9). The experiment was repeated two two times with similar results. (b) Lipid peroxides level, expressed as CHP equiv./ mg ww, was measured in Arabidopsis times with similar results. (b) Lipid peroxides level, expressed as CHP equiv./mg ww, was measured in Arabidopsis leaves leaves with the FOX assay. The results after treatment with milliQ water (untreated), flg22 (100 nM) (positive control), or with the FOX assay. The flg22+AuNPs-SCTA results (10 or after treatment 100 mg/L) with milliQ are presented as mean water ± SE(untreated), flg22 (100 nM) of three independent (positive Based experiments. control), on or flg22 + two-way AuNPs-SCTA (10 or 100 mg/L) are presented as mean ± SE of three independent experiments. nested ANOVA followed by Dunnett’s post-hoc test, data were normally distributed (Shapiro–Wilk test) and showed Based on two-way nested ANOVA followed homogeneity by Dunnett’s of variances post-hoc (Levene’s test).test, data were Different normally letters distributed indicate (Shapiro–Wilk statistically test) and showed significant differences homogeneity at p < 0.01. Different labels of a–c indicate variances statistically (Levene’s different test). Different groups letters according indicate to multiple statistically comparisons significant following differences at p