**3. Results and Discussion**

#### *3.1. A. Thaliana Proteome after CdS QDs Treatment*

2D-PAGE profiling (technical triplicates) of the plant proteomes of A. thaliana generated overall about 600 visible protein features for each of the wt and two mutants, exposed or not exposed to CdS QDs. After minor spots were eliminated to allow consistent MALDI-TOF/TOF analysis, about 250 reproducible spots remained (Figure S3). For the control samples, we had a total of 103 reproducible spots, of which 79 were present in all three plants; 2 were found only in wt, and 1 only in *atnp*01, with no unique spots for atnp02 (Figure S4A). For the treated samples, we had 105 reproducible spots, of which 81 were common to all the plant types. A further 4, 0 and 3 were unique to wt, *atnp*01, and *atnp*02 respectively (Figure S4B). The proteins in common between wt and mutants are shown in Figure S4 and explained in the Figure caption. Of these spots, 98 were designated as 'differentially abundant', meaning that they varied in intensity with p-value ≤ 0.05 between the treated (trt) and untreated (ctr) plant pairs. Of these, 61 were from the wt pair, 31 from the *atnp*01 pair and 31 from the *atnp*02 pair (Figure 1A). The numbers of commonalities and up- and downregulated proteins between wt and mutants and between mutants are listed in the caption to Figure 1.

**Figure 1.** Venn diagrams showing proteins common and non-common to wt and *atnp01*, to wt and *atnp02*, to *atnp01* and *atnp02* and to all treatment conditions. (**a**) There were 15 proteins common between wt and *atnp01*, 8 between wt and *atnp02*, and 3 between the two mutants; (**b**) The proteins upregulated in wt, *atnp01* and *atnp02* lines between control and treatment conditions. There were four upregulated proteins in common between wt and *atnp01*, two between wt and *atnp02*, and none between the two mutants; (**c**) The proteins downregulated in wt, *atnp01* and *atnp02* mutant lines between control and treatment conditions. There were four downregulated proteins in common between wt and *atnp01*, two between wt and *atnp02*, and two between the two mutants.

The identities of the differentially abundant proteins are reported in Table S1, and the associated heat map is shown in Figure 2. The heat map was obtained after the computation of the protein abundances in the control and treated samples for all the plant types, and by dividing the treated by the control to obtain the final differential heat map. Figure S4 shows the heat maps for all the treatments and controls of wt, *atnp*01, and *atnp*02. For all 98 proteins, the MapMan ontology BIN assignations are listed in Table S2. Most of the proteins in wt were downregulated, annotating in processes such as biotic and abiotic stress responses, protein folding, and protein degradation (Figure 2). Conversely, in both mutants, there is a balance between the numbers of up- and downregulated proteins (Figure 2). Comparing the heat map of treated vs. control, it can be observed that, whereas the treatment for the wt led to a general down-regulation of proteins, for the two mutants antnp01 and atnp02 there was also a significant number of proteins upregulated (Figure 2). The proteome of each mutant differed from that of the wt, both when the plants were grown under control conditions and when they were exposed to CdS QDs. Only one of the variable features was in common among all comparisons, namely putative protein phosphatase 2C 58 (At4g28400), downregulated in both mutants and upregulated in wt (Figures 1A and 2). This is a protein phosphatase known to mediate abiotic stress pathways and it is a member of major phosphatase class PP2C [34,35]. The phytohormone abscisic acid (ABA) is a major player in the regulation of responses to abiotic stresses, in particular drought and salinity. In Arabidopsis, ABA signaling, triggered particularly under abiotic stresses, involve various PP2Cs members as key regulators [36]. Two proteins were common to both mutants in the treatment condition, namely bifunctional enolase 2/transcriptional activator (Eno2) and putative pectinesterase/pectinesterase inhibitor VGDH2 (Vgdh2) (Figure 1A). Eno2, downregulated in the mutants (Figure 2), is the key glycolytic enzyme and encoded by LOS2 (Low expression of osmotically responsive genes 2). The ENO2 locus is highly expressed throughout plant development and is on average 10-fold more abundant than ENO1 and ENO3 in all tissues and organs [37]. Interestingly, Eno2 is a key protein in glycolysis and might be involved in the response to CdS QDs which can include impaired glycolysis. Vgdh2, downregulated in *atnp*01 and upregulated in *atnp*02 (Figure 2), acts in the modification of cell walls via demethylesterification of cell wall pectin [38].

Separately considering the up- and downregulated proteins, we constructed two new Venn diagrams (Figure 1B,C). The diagram for the upregulated proteins indicates that there are five proteins in common between treated wt and treated atnp01: pathogenesis-related protein 5 (At1g75040), F-box only protein 7 (Fbx7), oxygen-evolving enhancer protein 1-1, chloroplastic (Psbo1), probable fatty acyl-CoA reductase 4 (Far4), and gamma carbonic anhydrase 2 (Gammaca2). Pathogenesis-related protein 5 is partially responsible for acquired pathogen resistance [39]. Fbx7 is required for protein synthesis during temperature stress. [40]. Psbo1 stabilizes the manganese cluster which is the primary site of water splitting [41]. Far4 provides the fatty alcohols required for the synthesis of suberin in roots, seed coat, and wound-induced leaf tissue [42]. Gammaca2 mediates complex I assembly in mitochondria and respiration [43]. There are two proteins in common between treated wt and treated *atnp*02: vacuolar protein sorting-associated protein 24 homolog 1 (Vps24-1) and 30S ribosomal protein S5, chloroplastic (At2g33800). Vps24-1 is required for multivesicular body (MVB) formation and sorting of endosomal cargo proteins into MVBs. [44]. At2g33800 binds directly to 16s ribosomal RNA [45]. In the groups considered, there are no proteins in common between the two treated mutants. Among the downregulated proteins we found four proteins in common between treated wt and treated *atnp*01: heat shock 70 kDa protein 3 (Hsp70-3), histone H2B.7 (At3g46030), L-ascorbate peroxidase 1 (Apx1), and filament-like plant protein 6 (Fpp6). Hsp70-3, in collaboration with other chaperone proteins, assists translocation of precursor proteins into organelles, facilitates folding of de novo synthesized proteins, and is responsible for the degradation of damaged proteins undergoing stress conditions such as from Cd [46]; At3g46030 is a core component of the nucleosome [39]; Apx1 is a key component of the reactive oxygen species gene network, moreover, its synthesis can be induced by Cd exposure [46]. The function of Fpp6 is unknown. We found two proteins in common between treated wt and treated *atnp*02: glycine-rich RNA-binding protein 8 (Rbg8) and Ras-related protein RABF1(Rabf1). Rbg8 plays a role in RNA transcription or processing during stress [47]. Rabf1 is an endosomal protein probably involved in endocytosis [48]. There are two proteins in common between the two treated mutant lines: probable protein phosphatase 2C 58 and enolase 2. The first of these proteins was found in common between the wt and two mutants (Figure 1A), but it was upregulated in the wt while being downregulated in the two mutants (Figure 2). The second protein was found in common between the two mutants and was downregulated.

From the lower part of the heat map (Figure 2), it seems that the majority of the downregulated proteins are specific to the mutant *atnp*01, with the exception of Sap1, a zinc-finger protein and Scl28, a serine-arginine-like splicing factor, both upregulated. The most strongly downregulated proteins are: Pcmp-E25, a pentatricopeptide; Hsp70-3, a heat shock protein; Sam1, a S-adenosylmethionine synthetase; Xi-J, myosione 16; Far4, fatty-acyl-CoA reductase, At3g46030, histone H2B.7, VHA-B1, V-type proton ATPase, SEN1, t-RNA splicing endonuclease, and FPP6, filament like plant protein. Instead, ENO2 and PP2C were downregulated in both mutants. For the proteins Vgdh, Gapa1, Aba3, Pux10, and At4g05080, we found that they are strongly downregulated in the mutant *atnp*01, but strongly upregulated in *atnp*02 (Figure 2). This behavior indicates that this group of proteins is involved in an opposite manner within the response pathways that the two different mutants display towards the treatment. In the upper part of the heatmap in Figure 2, it is possible to observe several strongly upregulated proteins only in the mutant *atnp*02. These proteins are: Hsp70-10, a mitochondrial heat shock protein involved in the response to stress, Cat2, catalase 2, involved in the detoxification of hydrogen peroxide, Clpc2, a chloroplast chaperon protein with ATPase activity, Gldp2, a glycine dehydrogenase involved in the degradation of glycine in the mitochondrion, Cul3-B, cullin 3B, active in protein ubiquitination, and Atk5, kinesin 5, with microtubule-binding activity in the cytoskeletron. These proteins reflect the activity of the plant to control the oxidative stress and damage to mitochondria brought about by the CdS QDs.

In wt, the set of reprogrammed proteins was associated with the following major MapMan bins: protein synthesis, protein degradation and protein post-transcriptional modification; RNA regulation of transcription; DNA synthesis and chromatin structure; amino acid metabolism; hormone metabolism; redox ascorbate and glutathione biosynthesis; photosynthesis and photorespiration and abiotic and biotic stresses (Figure 3). In *atnp*01, the set of reprogrammed proteins was associated with protein degradation, RNA regulation of transcription, abiotic and biotic stress, and mitochondrial electron transport and ATP synthesis (Figure 3). In *atnp*02, the set of reprogrammed proteins was associated with protein folding, protein degradation, RNA regulation of transcription, photosynthesis (PS), secondary metabolism, mitochondrial electron transport/ATP synthesis, hormone metabolism, abiotic and biotic stress (Figure 3).

**Figure 2.** Heat map representing the effect on the A. thaliana proteome of CdS QDs (80 mg L−1) for wt and for the mutant lines *atnp*01 and *atnp*02. This heat map was obtained, similarly to Figure S5, by dividing the abundances of treated samples by the control samples. The proteins underlined in red are those in common among the wt and the two mutants *atnp*01 and *atnp*02, those that were found in the networks related to the mutated genes in the two mutants and those in common with the proteomic study with PF2D and the transciptome.

**Figure 3.** The distribution of differentially abundant proteins in wt, atnp01 and atnp02 according to MapMan ontology classification (BINs). The horizontal axis shows the different MapMan bin types, with the number of proteins for each bin on the vertical axis.
