Insights into the Response of Soybean Mitochondrial Proteins to

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Insights into the Response of Soybean Mitochondrial Proteins to Various Sizes of Aluminum Oxide Nanoparticles under Flooding Stress Ghazala Mustafa, and Setsuko Komatsu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00572 • Publication Date (Web): 26 Oct 2016 Downloaded from http://pubs.acs.org on October 28, 2016

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Journal of Proteome Research

Insights into the Response of Soybean Mitochondrial Proteins to Various Sizes of Aluminum Oxide Nanoparticles under Flooding Stress

Ghazala Mustafa 1, 2, and Setsuko Komatsu1, 2 *

1

Graduate School of Life and Environmental Science, University of Tsukuba, Tsukuba

305-8572, Japan. 2

National Institute of Crop Science, National Agriculture and Food Research Organization,

Tsukuba 305-8518, Japan.

* Corresponding authors: Setsuko Komatsu, National Institute of Crop Science, National Agriculture and Food Research Organization, Kannondai 2-1-18, Tsukuba 305-8518, Japan. Tel.: +81-29-838-8693, Fax: +81-29-838-8694, Email: [email protected]

Running title: Al2O3 NPs mediated soybean mitochondrial proteomics

Abbreviations: Al2O3, aluminum oxide; LC, liquid chromatography; MS, mass spectrometry; ROS, reactive oxygen species; NPs, nanoparticles.

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ABSTRACT: Rapid developments in nanotechnology have led to the increasing use of nanoparticles (NPs) in the agricultural sector. To investigate the possible interactions between NPs and crops under flooding stress, the molecular mechanisms in soybean affected by exposure to various sizes of Al2O3 NPs were analyzed using a proteomic technique. In plants exposed to 30-60 nm Al2O3 NPs, the length of the root including hypocotyl was increased and proteins related to glycolysis were suppressed. Exposure to 30-60 nm Al2O3 NPs mediated the scavenging activity of cells by regulating the ascorbate/glutathione pathway. Hierarchical clustering analysis indicated that ribosomal proteins were also increased on exposure to flooding-stressed plants with 30-60 nm Al2O3 NPs. Mitochondrion was the target organelle of Al2O3 NPs under flooding stress conditions. Mitochondrial proteomic analysis revealed that the abundance of voltage-dependent anion channel protein was increased on exposure to flooding-stressed soybean with 135 nm Al2O3 NPs, indicating the permeability of the mitochondrial membrane was increased. Furthermore, isocitrate dehydrogenase was increased on exposure of plants to 5 nm Al2O3 NPs under flooding conditions. These results suggest that Al2O3 NPs of various sizes affect mitochondrial proteins under flooding stress by regulating membrane permeability and tricarboxylic acid cycle activity.

KEYWORDS: soybean, flooding stress, root, aluminum oxide nanoparticles, mitochondria, proteomics.

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INTRODUCTION Nanoparticles (NPs) are increasingly being used in commercial applications in the fields of electronics, biotechnology, pharmaceuticals, biosensors, and cosmetics (1). In the last decade, numerous studies have examined the properties and synthesis of NPs (2). In addition, due to overwhelming production and NPs’s release into the environment, the impacts of NPs, both positive and negative, on the environment and biological systems, including plants, animals, and humans, have received considerable attention (3). The elevated production and use of NPs in agricultural products has recently been reviewed (4). To examine the toxicity of NPs, it is essentail to determine the interactions between NPs and plants, which are the primary producers in the ecosystem (5); however, the interaction mechanisms of NPs with plants remains poorly understood. The uptake, transport, and accumulation of NPs is influenced by the plant species, and the size, functionalization, chemical composition, and stability of NPs. Rico et al. (6) reviewed the interactions between NPs and edible plants and the implications of NPs entering the food chain. NPs are reported to cause variable growth effects on plants. For example, gold NPs reportedly accumulate in barley roots, which displayed reduced growth (7). In wheat, aluminum oxide (Al2O3) NPs affected growth at different concentrations (8). In soybean, zinc oxide NPs increased the germination rate and length/fresh weight of radicals under drought stress (9), whereas titanium oxide and silicon oxide NPs activated the antioxidant defense system (10). In flooding-stressed soybean, silver NPs enhanced growth (11), and Al2O3 NPs ameliorated the flooding stress-induced impairment of growth (12). Despite these findings, the molecular mechanisms underlying these NP-mediated effects on plant growth need to be explored. NPs affect several biological activities, including reactive oxygen species (ROS) generation (13). Buzea et al. (14) reported that deposition and accumulation of metal NPs 3



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on the cellular surface and also within organelles caused oxidative stress. NP-mediated oxidative stress depends on the NPs surface area, size, and composition, including the nature of metals, and is also influenced by cellular factors, like mitochondrial respiration, NP-cell interactions, and activation of immune cell (13). In tobacco, exposure of cell suspensions to Al2O3 NPs triggered programmed cell death and altered the permeability of the plasma membrane (15). Verano-Braga et al. (16) reported that 20 nm silver NPs were able to enter cells and cause cellular stress, including the ROS generation and protein carbonylation; however, larger NPs (100 nm) only indirectly induced oxidative stress. Based on these studies, it appears that NPs damage different plant organelles and that these effects are dependent on particle size. Changing climatic conditions alter the physiological state of plants and stimulate different biological pathways in order to combat unfavorable stress conditions (17). Among the different abiotic stresses, flooding has shattering effects on growth and production of crop (18). Under flooding stress conditions, exposure of soybean to Al2O3 NPs improved growth by regulating energy metabolism (12). In addition, 50 ppm Al2O3 NPs were found to facilitate growth under flooding conditions, whereas at a higher concentration (500 ppm), growth was inhibited. In soybean, flooding response mechanisms that are induced by silver and Al2O3 NPs have been identified (11,12); however, the specific modes of action of NPs of variable sizes on plants and subcellular organelles have yet to be explored. Because of the growing use of NPs in agricultural products (19), the molecular-level effects of NPs on plants warrant investigation. To investigate the size-dependent effects of Al2O3 NPs on early-stage of soybean under flooding stress, morphological and proteomic analyses were performed. Soybeans were treated with Al2O3 NPs of varying sizes at 50 ppm, as this concentration was previously reported to promote soybean growth under flooding stress (12). Based on the 4


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proteomic analysis of total proteins, the mitochondrion was identified as the target organelle affected by Al2O3 NPs under flooding stress. Therefore, mitochondrial proteins were isolated and analyzed by proteomic technique along with the bioinformatic and transcriptional techniques.

EXPERIMENTAL PROCEDURES Plant Material and Treatments Seeds of soybean (Glycine max L. cv. Enrei) were first sterilized with sodium hypochlorite solution, twice rinsed in water, and sown on 500 mL silica sand with 150 mL water in a plastic case (180 mm x 140 mm x 45 mm). Soybeans were kept to grow in growth chamber illuminated with white fluorescent light (16 h light period/day) at 25ºC. For treatment, 2-day-old soybeans were transferred to glass tubes (38 mm ID x 130 mm) with 120 mL of reverse osmosis water (20). After covering glass tube with plastic cap that allow air flow, the tubes were kept at 25°C in the dark. Untreated plant served as control. The Al2O3 NPs of 5, 135 (US Research Nanomaterials, Houston, TX, USA), and 30-60 nm (Sigma-Aldrich, St Louis, MO, USA) particle sizes, were used at 50 ppm. For morphological analysis, length of the root including hypocotyl was measured at 0, 1, 2, 3, and 4 days after treatments. For crude protein extraction, proteins were extracted from root including the hypocotyl at 0, 1, 2, and 3 days after treatments. For mitochondrial protein isolation, mitochondrial proteins were extracted from root tip at 0 and 2 days after treatments. For mRNA expression experiment, RNA was extracted from root tip at 0, 1, and 2 days after treatments. Three independent experiments were carried out as biological replicates for all the experiments (Figure 1).

Total Protein Extraction and Purification 5



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A portion (300 mg) of each collected sample was used for protein isolation. Three independent biological replicates were used for each sample. Proteins were isolated from the 300mg sample using the previously described method (21). Protein concentration was determined using the Bradford method (22) with bovine serum albumin as the standard. Furthermore, proteins (150 µg) were purified by phase separation using standard procedures (21). The purified samples were obtained in dried pellets.

Enrichment of Mitochondrial Fraction For the preparation of mitochondrial fractions, root tip was used and mitochondria were isolated using a previously described method (23). Protein concentration was determined using the Bradford method [22] with bovine serum albumin as the standard. The proteins were desalted with Monospin C18 column (GL Science, Tokyo, Japan) and dried using Speed-Vac concentrator.

Enzyme Activity Assay In order to assess the mitochondrial purity, marker enzymes were used. For enzyme analysis, root tip for crude protein extract was homogenized in extraction buffer. The enzyme extract and mitochondrial fractions were used for enzyme activity analyses. The enzyme assays for fumarase [24], glucose-6-phosphate dehydrogenase [25], NADH cytochrome c reductase [26], and catalase [27] were performed as previously described. The enzyme activities were calculated with the formula: units/mL = (∆A x total volume x dilution factor)/ (Extinction coefficient x sample volume).

Immunoblot Analysis Proteins were separated by 17% sodium dodecyl sulfate-polyacrylamide gel 6


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electrophoresis by a previously described protocol [28]. After separation, the proteins were blotted and analyzed by a method previously described [23].

Protein Digestion and Mass Spectrometry Analysis The dried pellets from crude and mitochondrial protein fractions were digested and alkylated. The resulting tryptic peptides were acidified and analyzed by liquid chromatography (LC) mass spectrometry (MS) as methods described previously [29,30].

Protein Identification of Acquired Mass Spectrometry Data Identification of proteins was performed using the Mascot search engine (version 2.5.1; Matrix Science, London, UK) and Proteome Discoverer software (version 1.4.0.288; Thermo Fisher Scientific, San Jose, CA, USA) against a soybean peptide database (54,175 sequences; Phytozome, version 9.1; http://www.phytozome.net/soybean) [31,32].

Differential Analysis of Identified Proteins The relative abundances of peptides and proteins were compared using the commercial label-free quantification package SIEVE software (version 2.1.377; Thermo Fisher Scientific).

Bioinformatic Analysis To determine the functional role of proteins identified in the MS analysis, functional categorization was performed using MapMan bin codes and MapMan software [33,34]. The MitoPred [35], MitoProt [36], TargetP (ver 1.1) [37], and SUBA3 [38] were used for subcellular localization of identified mitochondrial proteins.

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Cluster Analysis using Protein Abundance Protein abundance ratios at different time points of flooding-stress with Al2O3-NPs treatments were analyzed with Genesis software (version 17.6, http://genome.tugraz.at) (39).

RNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction Analysis Total RNA extraction and qRT-PCR were performed as previously described [20]. The primers were designed using the Primer3 web interface (http://frodo.wi.mit.edu). Speciﬁcity of designed primers (Supplemental Table 1) and separation of qRT-PCR products were performed by a previously described method [20].

Statistical Analysis Significant changes in the abundance of proteins were analyzed by two-way ANOVA using GraphPad Prism 7.01 software (GraphPad Prism Software, La Jolla, CA, USA). Morphological and mRNA expression alterations were analyzed for significance using Tukey’s Multiple Range test. A p value of

Insights into the Response of Soybean Mitochondrial Proteins to

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