**1. Introduction**

One result of the expansion of nanotechnology has been the large-scale production of engineered nanomaterials (ENMs), which are becoming widely diffused as industrial products for everyday use. However, ENMs can be released into the environment through the recycling of waste, manufacturing, and deliberate or accidental environmental release [1]. Soil is becoming more frequently contaminated with released ENMs, some types of which, due to their small size and high surface reactivity, may enter into plant cells, where both detrimental and positive effects have been noted [2]. Among the smallest ENMs are colloidal cadmium sulfide quantum dots (CdS QDs). The market for colloidal quantum dots (QDs) alone is projected to increase from the current \$3 billion to \$8.5 billion by 2023, an almost three-fold increase in production and marketing (https://www.marketsandmarkets.com/Market (accessed on 2 February 2021)). QDs are semiconductor nanocrystals measuring around 2–10 nm [3], and their chemical and physical properties are closely size-dependent. Based on their excellent optical properties, QDs have important applications, with development of platforms for simultaneous

**Citation:** Gallo, V.; Zappettini, A.; Villani, M.; Marmiroli, N.; Marmiroli, M. Comparative Analysis of Proteins Regulated during Cadmium Sulfide Quantum Dots Response in *Arabidopsis thaliana* Wild Type and Tolerant Mutants. *Nanomaterials* **2021**, *11*, 615. https://doi.org/10.3390/ nano11030615

Academic Editor: Thierry Rabilloud

Received: 7 February 2021 Accepted: 25 February 2021 Published: 1 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

imaging, sensing, and therapy [4,5]. Indeed, QDs are pivotal constituents of innovative nanotechnology-enabled tools for medical diagnostic and ex vivo imaging [6,7]. QDs have also been used to improve resolution in TVs, digital cameras, computers, and smartphone displays and to increase energy conversion efficiency in quantum dot light-emitting diodes (QLEDs) and in quantum solar cells (QDSSCs) [8,9]. Consequently, the increasing use of QDs-enabled products is expected to result in larger releases of these materials into the environment [10,11]. CdS QDs are very stable and have high reactivity and high surface charge [12]. However, different properties have been connected with CdS QDs toxicity; some studies have implicated Cd release as the main toxicity factor, but under other circumstances, particle properties like shape and size seem to be more relevant [13,14]. Our previous work showed that only about 2% of CdS QDs dissolved in MS medium in 21 days [14]. The main toxicity mechanisms of CdS QDs nanoparticles within a cell or an organism are the generation of reactive oxygen species (ROS), which damage cell membranes, proteins and DNA; the production of protein coronas, removing proteins important to normal cell metabolism [15]; inhibition of the functionality of chloroplasts and mitochondria [16,17]; and the induction of apoptosis through the activation of post-translational regulatory mechanisms such as miRNA [18].

Generally, the influence of exposure duration on Cd-based QDs toxicity is insufficiently understood [19,20]. Only a limited number of studies have analyzed and compared the impacts of CdS QDs and Cd ions on whole organism development, as well as on organ, tissue or cellular structure [21–24]. Marmiroli et al. [14,25] characterized the transcriptomic and changes associated with exposure to CdS QDs in *A. thaliana*; two mutants (*atnp01* and *atnp02*) of Landsberg *erecta*, collection Soll-Jonson, with Ac/Ds maize transposon insertion, were identified as tolerant to CdS QDs but not to Cd2+. Genotypic characterization of *atnp01* and *atnp02* was performed; Southern hybridization profiles based on either EcoRI or BamHI digestion identified two hybridizing fragments in *atnp01,* and a single different one in *atnp02*, demonstrating that the two mutants harbored three different Ds elements. The number of Ds elements inserted in each mutant and their genomic locations were determined by genome walking. Upon sequencing the amplicons produced in the genome walking, a BLAST-based alignment with the full genome sequence identified that the candidate genes potentially affected by the Ds transposition events in *atnp01* were *At3g46880*, coding an unknown chloroplast-localized protein, *At1g13870* (*DRL1*, deformed roots and leaves) coding a cytoplasm-localized calmodulin-binding protein possibly involved in leaf development and meristem structural organization, and *At1g13880* (*ELM2*), a member of the MYB transcription factors category. The *atnp02* event could have affected either *At3g24330*, which encodes an O-glycosyl hydrolase localizing to the endomembrane, or *At3g24430* (*HCF101*), coding for a chloroplast-localizing ATP-binding protein. The Ds element lay within the *At3g24400* pseudogene (*AtPERK2*), which may code for a proline-rich extensin-like receptor kinase.

These mutants were characterized at physiological, genetic, and transcriptomic levels, and a comparison with the response to Cd2+ demonstrated the nano-specific nature of the molecular mechanisms and the pathways involved in the tolerance. It was proved by Marmiroli et al. [26] that CdS QDs once inside the plant cell undergo biotransformation rendering them less active and diminishing their power to hamper photosynthesis, respiration, and DNA transcription; this occurs through the inactivation of specific enzymes and by the chaperone action of hard corona proteins, including some heat shock proteins (Hsp70) [15]. The correspondence between transcriptomics and proteomics results for the same toxicant is typically in the range 20–80% [25,27], but proteomics furnishes important information about the cellular response to toxicants, protein–protein interactions, and post-translational modifications that cannot be detected by transcriptomics. Proteins differentially expressed in the presence and absence of CdS QDs were identified by 2D-PAGE. Image analysis was used to compare spots between gels of treated and untreated wt and mutants (*atnp01*, *atnp02*), and those with different intensities were excised and fingerprinted using MALDI-TOF/TOF mass spectrometry. Results obtained with 2D-PAGE were then compared with

those obtained in previous work [25], which used the same conditions of treatment but a different separation technique: two-dimensional protein fractionation (ProteomeLab PF2D), employing a gradient isoelectric focusing (IEF) separation in the first dimension, and high-performance liquid chromatography (HPLC) in the second dimension. These two techniques use a different pH range (5–8 vs. 4–8.5), a different solubilization for proteins (SDS vs. urea), and a different quantity of proteins (4 mg vs. 400 μg); therefore, they are often seen as complementary [28]. However, other proteomic techniques, like iTRAQ, have been shown to give a greater number of identifications [29].
