Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2019, 67, 6911−6920
Enhanced Properties of Chitosan Microparticles over Bulk Chitosan on the Modulation of the Auxin Signaling Pathway with Beneficial Impacts on Root Architecture in Plants María José Iglesias,† Silvana Lorena Colman,† María Cecilia Terrile,† Ramiro París,† Sergio Martín-Saldaña,‡ Alberto Antonio Chevalier,‡ Vera Alejandra Á lvarez,§ and Claudia Anahí Casalongué*,† Downloaded via BUFFALO STATE on July 18, 2019 at 07:04:57 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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UNMdP, CONICET, Instituto de Investigaciones Biológicas, UE-CONICET-UNMdP, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3250, B7600 Mar del Plata, Argentina ‡ Gihon Laboratorios Químicos SRL, Calle 4 y 5 Parque Industrial General Salvio, B7600 Mar del Plata, Argentina § UNMdP, CONICET, Instituto Investigación de Ciencia & Tecnología de Materiales INTEMA, UE-CONICET-UNMDP, Grupo Materiales Compuestos Termoplásticos, Facultad de Ingeniería, Universidad Nacional de Mar del Plata, Avenida Colón 10850, B7600 Mar del Plata, Argentina S Supporting Information *
ABSTRACT: Improving the root system architecture (RSA) under adverse environmental conditions by using biostimulants is emerging as a new way to boost crop productivity. Recently, we have reported the characterization of novel chitosan-based microparticles (CS-MPs) with promising biological properties as rooting agents in lettuce. In this work, we demonstrated that in contrast to bulk chitosan (CS), which exerts root growth inhibition, CS-MPs promoted root growth and development from 1 to 10 μg mL−1 without cytotoxicity effects at higher doses in Arabidopsis and lettuce seedlings. In addition, we studied the mechanistic mode of action of CS-MPs in the development of early RSA in the Arabidopsis model. CS-MPs unchained accurate and sustained spatio-temporal activation of the nuclear auxin signaling pathway. Our findings validated a promising scenario for the application of CS-MPs in the modulation of RSA to respond to changing soil environments and improve crop performance. KEYWORDS: Arabidopsis, auxin, chitosan microparticles, lettuce, root system architecture
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INTRODUCTION
The patterns of plant development are consolidated by the action of hormonal regulation mechanisms. The dynamic and versatile modulation of auxin biosynthesis, transport, and signaling has been found to be required for RSA development under changing environmental conditions.13,14 Auxin regulates root development mainly through the nuclear signaling pathway mediated by TIR1/AFB receptors.15 Auxin binding to TIR1/AFB receptors triggers the degradation of Aux/IAA repressors, with the consequent activation of auxin response genes.16 The early response genes involved the Aux/IAA, SAUR, and GH3 gene families.17 The inhibition of auxininduced growth and the repression of auxin gene expression required for root development has been evidenced in CS treated wheat coleoptiles and sweet orange plants, respectively.18,19 Recently, Lopez-Moya et al. reported the inhibition of PR and LR development in barley and tomato plants.20 The same authors demonstrated that CS modulates RSA in Arabidopsis plants through repression of the transcription factor Wuschel-related homeobox 5 (WOX5), which is a major regulator of root stem cell activity. WOX5 repression was associated with alterations in auxin biosynthesis and transport,
The root system architecture (RSA) involves the coordinated growth and development of primary roots (PRs), lateral roots (LRs), and adventitious roots in order to improve soil exploration and resource acquisition, which are pivotal for plant fitness and crop productivity.1 Because the development of RSA is a crucial factor in determining plant survival, particularly under adverse environmental conditions, its modulation is emerging as a strategy to generate improvement in crop yields.2,3 In this context, the development of new bioactive materials with emerging properties fits with the actual challenge of augmenting crop productivity with reduced environment impact.4,5 Chitosan (CS) is composed of β-1,4linked glucosamine and N-acetyl glucosamine residues and is generated by the partial deacetylation of chitin polymer. Because of its unique properties, such as biodegradability, biocompatibility, ubiquity, and low cost, CS has multiple applications in several fields, including agriculture.6,7 CS action in the protection of plants against biotic stress by inhibiting the growth of microorganisms and eliciting plant innate immunity has been extensively studied in multiple species.8−10 Although CS has been suggested as a biostimulant to promote plant growth in several horticultural plants, even a small imbalance from optimal concentrations leads to growth inhibition with a high impact on root development.11,12 © 2019 American Chemical Society
Received: Revised: Accepted: Published: 6911
February 11, 2019 May 27, 2019 June 3, 2019 June 3, 2019 DOI: 10.1021/acs.jafc.9b00907 J. Agric. Food Chem. 2019, 67, 6911−6920
Article
Journal of Agricultural and Food Chemistry Scheme 1. Synthesis of CS-MPs
Chitosan-Based Materials and Treatments. The CS-MPs and CS used in this study were described and characterized by Martı ́nSaldaña et al.24 The CS exhibited a mass-average molecular weight (Mw) of 1531 ± 372 kDa, a number-average molecular weight (Mn) of 559 ± 95 kDa, a polydispersity index (PI = Mw/Mn) of 1.95 ± 0.32 as determined by gel permeation chromatography, and a deacetylation degree (DD) higher than 87% as determined by Fourier-transform infrared (FTIR) spectroscopy.30 CS-MPs were prepared by the gelation method with modifications, using sodium tripolyphosphate (TPP) as the cross-linker, and they had a mean diameter of 2.10 ± 0.78 μm and a PDI of 0.14 as determined by scanning electron microscopy (SEM, JEOL JSM-6100 with 15 kV). Samples were previously coated with metallic gold for 30 s with an Auto Sputter Coater 108 (Cressington Scientific Instruments). Micrographs were analyzed with ImageJ software (U.S. National Institutes of Health, http://rsb.info.nih.gov/ij/).24,31 CS-MPs also present a zeta potential value (ζ) of 27.65 ± 1.22 mV at pH 6.8 as determined by a laser particle sizer (Z-sizer 3000 HS, Malvern). The materials were developed and characterized by Gihon Laboratorios Quı ́micos SRL, Argentina. Figure S1a,b shows the morphology of the CS-MPs determined by SEM as well as their FTIR spectra and the relevant peaks assigned to CS-MPs and bulk CS. FTIR was performed on an IRAffinity-1S FTIR spectrophotometer (Shimadzu) in attenuated total reflection mode (ATR-FTIR). To analyze the efficacy of CSMPs on root growth parameters, dry CS-MPs were resuspended in water from 0.1 to 100 μg mL−1. Bulk CS was diluted in 0.1% acetic acid. The pH of each assayed dilution of both bulk CS and CS-MPs was in the range of 6.0−6.5. Fresh Weight, Primary Root, and Lateral Root Measurements. Five days post-germination (dpg),Arabidopsis and lettuce seedlings were transferred to 1/2 MS medium supplemented with CSMPs or CS and grown vertically in a growth chamber at 25 °C under 250 μmol m−2 s−1 photons with 16:8 h light−dark cycles until analysis. Root and aerial fresh weights (FWs) were weighed on a laboratory scale (Sartorius). Seedlings were photographed after 3 days to determine PR length and after 5 days for LR number and LR length. The PR and LR lengths were quantified using the ImageJ image-analysis software (U.S. National Institutes of Health, http:// rsb.info.nih.gov/ij/). Measurements of Root Hair Length and Density. Seedlings (5 dpg) were transferred to liquid 1/2 MS medium supplemented with CS-MPs or CS for 48 h. Bright-field images from Arabidopsis roots were taken using a Zeiss Axioplan Imaging 2 microscope with an Axiocam HRC CCD camera (Zeiss) using the Axiovision program
leading to overaccumulation of auxin in the root tip with detrimental impacts on root development, suggesting that the dose, frequency, and formulation of CS should be adjusted to prevent negative effects on plant development. Another point to take into account for the application of CS in the field is its limited solubility in water. The complex behavior of bulk CS on plant physiology as a consequence of chemically heterogeneous copolymer preparations has slowed down its promising potential use in agriculture.21,22 Therefore, CS particulated systems are emerging as an alternative to bulk CS and all of its complex problems. They are easy to obtain and also to modify in terms of their water solubility and interactive biological abilities.23 Nevertheless, it is necessary to demonstrate how the type, concentration, and particle size impact on plant tissues. We previously reported the characterization of CS-MPs developed with high molecular weight CS obtained from Pleoticus mulleri fishing industry waste from the Argentine Sea (Scheme 1). Preliminary assays have shown that CS-MPs stimulate PR elongation in lettuce seedlings, suggesting a novel potential use as rooting agents.24 In this work, we studied the properties of CS-MPs as biostimulants of root development compared with that of bulk CS and the hormonal mechanism by which CS-MPs impact the modulation of RSA in Arabidopsis. Arabidopsis is suggested as an adequate model for dicot plant root research because its root system fits with the typical eudicot root topography.25
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MATERIALS AND METHODS
Plant Materials and Growth Conditions. Wild-type (WT) and transgenic pMSG2/IAA19:GUS, DR5:GUS, BA3:GUS, and DIIVENUS Arabidopsis thaliana (Arabidopsis) lines are in the Columbia (Col-0) ecotype.26−29 Butterhead lettuce (Lactuca sativa L.) cv. Reina de Mayo seeds were purchased from El Colono local seed market, Mar del Plata, Argentina. Arabidopsis and lettuce seeds were surfacesterilized in 30% sodium hypochlorite and 0.2% Tween-20 solution for 10 min, followed by three washing steps in sterilized distilled water. Then, the seeds were stratified at 4 °C for 2−3 days in the dark. The seeds were placed on half-strength Murashige and Skoog medium (1/2 MS, Sigma-Aldrich) with 0.8% agar in Petri plates and grown vertically at 23 °C under 250 μmol m−2 s−1 photons with 16:8 h light−dark cycles until analysis. 6912
DOI: 10.1021/acs.jafc.9b00907 J. Agric. Food Chem. 2019, 67, 6911−6920
Article
Journal of Agricultural and Food Chemistry
Figure 1. Promotion of root development in Arabidopsis by CS-MPs: (a) PR elongation 3 days post-treatment, (b) LR number 5 days posttreatment, (c) LR length 5 days post-treatment, (d) seedling FW 9 days post-treatment, and (e) representative images of seedlings 9 days posttreatment. Col-0 Arabidopsis seedlings (5 dpg) grown in 1/2 MS medium were treated with increasing concentrations of CS-MPs or CS as a control. Data are mean values of five independent experiments (n = 60; ANOVA, Dunnet’s post hoc test against the control; *p < 0.05, **p < 0.01, ***p < 0.001). the addition of 10 μg mL−1 CS-MPs. A liquid solution of CS-MPs (200 μL) was poured at the surface of each root to ensure homogeneous absorption. Seedlings were grown for 24 h. Fluorescence from VENUS protein was detected in root cells using a 20× objective, a 0.5 numerical aperture, and 470/40−525/50 nm excitation and detection wavelengths in a Zeiss Axioplan Imaging 2 microscope with an Axiocam HRC CCD camera (Zeiss). Images were analyzed by the FIJI software bundle.33 Glucuronidase (GUS) Staining. Transgenic BA3:GUS, DR5:GUS, and pMSG2/IAA19:GUS seedlings (5 dpg) were transferred into liquid 1/2 MS medium containing 1, 10, or 100 μg mL−1 CS-MPs and then incubated with mild shaking for 24, 48, 72, or 96 h at 23 °C. For the BA3:GUS line, CS-MPs particles were applied together with 100 nM indole acetic acid (IAA) and incubated for 6 h. After treatment, the BA3:GUS, DR5:GUS, and pMSG2/IAA19:GUS
(version 4.2). Root hair (RH) density and length were analyzed in a 5 mm section from the beginning of the PR differentiation zone.32 RH length was analyzed using ImageJ software. Root Gravitropic Assay. Seedlings (3 dpg) were transferred to fresh 1/2 MS medium supplemented with 10 μg mL−1 CS-MPs. To ensure homogeneous absorption and action, liquid medium was also poured at the surface of each root. The plates were mounted vertically on a scanner (Epson Perfection V600) and let sit for 60 min. After root gravistimulation, images were taken every 15 min for 8 h. Root growth and tip angle were measured by using the FIJI software bundle.33 Treatment of DII-VENUS Transgenic Sensor Plants with CSMPs. DII-VENUS Arabidopsis transgenic sensor seedlings were designed to map auxin signaling response at a high resolution in plant cells.29 Seedlings (5 dpg) were transferred to fresh plates with 6913
DOI: 10.1021/acs.jafc.9b00907 J. Agric. Food Chem. 2019, 67, 6911−6920
Article
Journal of Agricultural and Food Chemistry seedlings were fixed in 90% acetone for 1 h at -20 °C, washed twice in 50 mM sodium phosphate buffer (pH 7.0) and incubated in staining buffer containing 50 mM sodium phosphate (pH 7.0), 5 mM EDTA, 0.1% Triton X-100, 5 mM K4Fe(CN)6, 0.5 mM K3Fe(CN)6, and 1 mg mL−1 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid cyclohexylammonium salt (X-Gluc, Gold Biotechnology) from 2 h to overnight at 37 °C. Bright-field images were taken using a Zeiss Axioplan Imaging 2 microscope (Zeiss). Measurement of Nitric Oxide (NO) Production. Arabidopsis seedlings (5 dpg) were loaded in the dark with 5 mM of the specific NO dye 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM-DA, Calbiochem) in 20 mM HEPES−NaOH buffer at pH 7.5 for 30 min. After three washes, seedlings were examined by epifluorescence with a Nikon DS-Fi 1 digital camera coupled to a Nikon Eclipse Ti epifluorescence microscope (excitation, 495 nm; emission, 515−555 nm). RNA Extraction and Quantitative Real-Time PCR (RT-qPCR). Seedlings (5 dpg) were transferred to liquid 1/2 MS medium supplemented with increasing concentrations of CS-MPs or 10 μg mL−1 CS and H2O as controls. After 24 h, total RNA from Arabidopsis seedlings was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s recommendations. Samples were treated with RQ1 RNase-free DNase (Promega) for DNA contamination removal. For cDNA synthesis, 1 μg of total RNA was reverse-transcribed by IMPROM II (Thermo Fisher Scientific) using random primers (Biodynamics). The expression of a subset of early auxin response genes was analyzed by real-time PCR (qPCR), using the following primers: IAA5F, 5′-CCGGAGAAAGAACAGTCTCG-3′; IAA5R, 5′AGCATCCGAACAGAATTTGC-3′; IAA14F, 5′-GAAGCAGAGGAGGCAATGAG-3′; IAA14R, 5′-CCCATGGTAAAGGAGCTGAA3′; GH3.5F, 5′-CCATCTCTGAGTTCCTCACAAGC-3′; GH3.5R, 5′-TCCTCTTCGATTGTTGGCATTAGC-3′; GH3.17F, 5′ACGCAGACACGTCATCAATCCC-3′; GH3.17R, 5′TGCTGTGACGTGGCTTTAGCTC-3′; ACTINF, 5′GC C A T C CA A GC T G TT C TC T C - 3 ′ ; and AC T INR, 5 ′ GAAACCCTCGTAGATTGGCA-3′. qPCR reactions were conducted in triplicate (95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s) in a Step One realtime PCR system (Applied Biosystems) using FastStart Universal SYBR Green Master Rox (Roche) following the manufacturer’s instructions. The results were normalized to the expression level of the gene for actin and expressed as fold-change over the levels of the controls using the comparative cycle threshold (CT) method.34 PCR products were analyzed by melting curve analysis to confirm the presence of a single product. Statistical Analysis. The values shown in the figures are mean values ± standard errors (SE) of at least three experiments. The data were subjected to analysis by t-tests or ANOVA with Dunnet’s post hoc comparisons against the controls in GraphPad Prism version 5.01 software (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 2. Promotion of RHs upon CS-MPs exposure: (a) RH density, (b) RH length, and (c) representative images of root segments. Col-0 Arabidopsis seedlings (5 dpg) were transferred to liquid 1/2 MS medium supplemented with increasing concentrations of CS-MPs for 48 h. RH density and length were analyzed in a 5 mm section from the beginning of the differentiation zone. Data are mean values of four independent experiments (n = 30; ANOVA, Dunnet’s post hoc test against the control; *p < 0.05, **p
