We discuss nine in vivo studies that have used proteomics to explore the alterations in plants at the protein level, in response to metal-based NPs, including nAg, nCeO2, nAl, nAl2O3, nFe, nZnO and CdS QDs . Impact of bare or surface-functionalized nAg on plant proteomes have been studied in several plants, including arugula, wheat, rice, soybeans, and tobacco. In rice plants exposed to 30 and 60 mg/l nAg for 20 days in hydroponic growth media, 2DE-nanoLC/FTICR MS identified twenty-eight differentially-accumulated proteins, which were primarily involved in oxidative stress response, Ca2þ regulation and signaling, transcription, and protein synthesis/degradation, cell wall damage, and apoptosis . nAg exposure incremented levels of defense-related proteins including SOD, glutathione-S-transferase , L-ascorbate peroxidase, which has been shown to result from Agþ leaching. Similar defense response expressed by elevated levels of SOD and GST was reported in arugula and wheat plants, respectively, exposed to 10 mg/l polyvinyl pyrrolidone -coated nAg for 5 days . Comparative proteomic response in the roots of arugula plants exposed to nAg and AgNO3 suggested that both treatments disrupt redox regulation, biosynthesis of sulfur containing amino acids,nft hydroponic system and cellular homeostasis.However, nAg also alter endoplasmic reticulum and vacuole-associated proteins in arugula and wheat roots, not demonstrated in the Agþ treatments. Vannini et al. also reported an increased production of malate dehydrogenase in roots, which reportedly increased root exudation of organic acids such as citrate, oxalate, malate, succinate and acetate .
Zhao et al. demonstrated in a metabolomic study that uptake and translocation of nCu in cucumber plant triggers a feedback control mechanism in tandem to modulate the root exudation of amino acids and organic acids for defense response and restricting ion release, to maintain cellular homeostasis . In tobacco plants, a 7-day exposure to citrate-coated nAg in tobacco plants altered the proteins related to defense response and oxidative stress, at a similar level as AgNO3 treatments; however, although both nAg and AgNO3 affected mostly photosynthesis related proteins, in the leaves the effect was significantly higher in nAg exposed plants, highlighting a nano-specific response . In another study, proteomic analysis in soybean plants after 3-days of root exposure to three different metal nanoparticles showed significantly negative response to 5 ppm nAg treatments compared to 500 ppm nAl2O3 and 500 ppm nZnO . The drastic decrease in the proteins related to energy metabolism and a compromised defense system in the nAg exposed plants thus resulted in decreased growth of soybean plants, compared to the control, nAl2O3 and nZnO exposures. Proteomic analysis of soybean roots exposed to 200 mg/l of differentially coated-CdS-QDs in vermiculite showed over-accumulation and under-accumulation of 99 and 44 root proteins, respectively, irrespective of coating type . The response was also compared to bulk-equivalent and Cd2þ ion treatments. The affected proteins unique to QD exposures were involved in glycolysis, TCA cycle, urate oxidation, and ATP synthesis-coupled-proton transport. Stress signaling pathways were also upregulated, especially b-oxidation of fatty acids, biosynthesis of jasmonic acid and sphingosine, and lignin biosynthesis. Some proteins involved in defense response, ion binding, channel activity, and membrane organization were negatively affected. Ca2þ-transporting ATPase activity was also down regulated in all CdS-QD-treated roots. reported in nAg treatments as well.
Some altered proteins that were common to CdS bulk particles and Cd2þ ion exposure were those associated with pentose phosphate pathway, glucuronate pathway, Calvin and TCA cycle, glycolysis/gluconeogenesis, amino acid biosynthesis, catecholamine biosynthesis, GABA shunt, phenylpropanoid pathway, GSH metabolism, isoflavonoid synthesis, carbon fixation, glyoxylate/dicarboxylate metabolism, jasmonic acid biosynthesis, and terpenoid biosynthesis, and sucrose and starch catabolism.Although exploration of the plant proteome can deliver a wealth of information, the studies concerning ENMs have been primarily focused on toxicity. Tiwari et al. employed gel-based proteomics and transcriptomics to elucidate the mechanism of bio-transformation of KAuCl4 to nAu in 5-day old A. thaliana plants . A total of 10 and 15 spots from 2DE of root and shoot samples, respectively, were digested into peptides by trypsin, and analyzed using MADI-TOF-MS. nAu affected carbohydrate metabolism, electron transport chain and oxidative stress in plant tissues. The production of GSTs in A. thaliana shoots in response to increasing Au accumulation, suggested they play an important role in controlling oxidative stress during the reduction of Au ions to nAuNPs. A 14-day exposure to 250–1000 mg/l nCeO2 via foliar spray and root absorption resulted in significant effect on carbon fixation and energy production in pinto bean plants . This involved enhanced production of thylakoid proteins participating in photosynthesis, decreased production of RuBisCo and altered enzyme activities in the electron transport chain in mitochondria nCeO2 have shown to be very actively involved in the oxidative chemistry in plant cells, either acting as an antioxidant enzyme mimic or a stress elicitor .
In pinto beans, two key enzymes responsive to oxidative stress, ascorbate peroxidase and glutathione peroxidase were down-accumulated. Altered response of ascorbate peroxidase enzymes has also been reported at biochemical level in kidney beans and tomato , as well as at transcriptomics level in A. thaliana . Interestingly, transcription factors , which play a central role in protein biosynthesis and turnover were shown to be down regulated in the leaves as well as first generation seeds of bean plants exposed to 125–500 mg/kg nCeO2 . Lipoxygenase, a protein responsible for fatty acid biosynthesis, iron binding, and oxido-reductase activity was also down-accumulated in bean leaves and seeds . The differentially regulated proteins in the seeds obtained from nCeO2 treated parent plants were mostly down-accumulated compared to those harvested from untreated controls, and a dose-dependent increase in the number of candidates were noted. The candidate proteins were involved primarily in storage , carbohydrate metabolism , protein folding, and resistance mechanism .Recent advances in tools for genomics and transcriptomics in conjunction with metabolomics and proteomics have the potential to accelerate agricultural development . An area that can benefit substantially from these approaches is the plant-microbe interaction. Microbial communities play an important role in plant growth and productivity by directly controlling soil processes like stabilizing soil structure, nutrient bio-availability, degradation of organic pollutants, CO2 fixation and C degradation. They are however highly sensitive and susceptible to toxicity from stressors. Next generation sequencing technologies such as pyrosequencing and Illumina-based sequencing has resolved complexities in the microbial community with higher accuracy than conventional methods . Metagenomics allows to collectively characterize genome sequences of known and unknown members of the entire microbial community in an environmental sample, without having to isolate each into pure cultures. ENMs present in the agricultural soil from intentional use of nano-enabled agrochemicals or unintentional incorporation in the biosolids or irrigation water have the potential to impact the soil microbial community thereby affecting the agricultural productivity . Metagenomic analysis of the soil microbial community provide potential means to design sustainable ENMs with potential to bolster plant protection against pests and enhance productivity and nutritional quality . However, although the metagenomic analysis provide important information on the functional capacity of the soil microbial community, it does not reflect the metabolic activities of individual species or the community. Genomic interpretation can be complemented with gene expression analysis at the RNA level, also known as transcriptomics, which can provide critical information on sustainable ENM design for agricultural applications and impact on plants and soil microbial community. Advances in gene expression analysis using RNA-seq analysis have evolved the understanding of alteration in gene expression in complex samples, which can be further validated by RT-PCR analysis of targeted genes. The transcriptomic studies in plants exposed to ENMs have been very well reviewed in two reviews, where the authors discussed the integration of transcriptomics, micro-RNA and proteomics data in plants for discovering nano-specific biomarkers and effects . For transcriptomic studies, the knowledge of the whole genome sequence and their functional annotation is critical and hence is better addressed in smaller genomes. A. thaliana has the smallest genome that is completely annotated unlike the crop species like rice or soybeans ,hydroponic nft system and hence all global transcriptome studies focusing on ENM-plant interactions have been carried out in A. thaliana to avoid complexity .
However, complementary proteomics and metabolomics can be used to functionally annotate the genes of interest in non-model species. In theirreview, Ruotolo et al. concluded that the plants induce defense mechanisms against ENM exposure which are resolved at the transcriptome and proteome level and are primarily related to modification of root architecture, phytohormone signaling and antioxidant system activation .Integration of multiomics in plants provides a comprehensive knowledge on the regulatory mechanism at multiple subcellular organization levels in response to an external stimulus. Although the number of plant studies employing individual omic techniques to identify key biomolecules in response to ENMs is increasing, only a few studies have integrated different omics . It is critical to integrate the response at all levels, including metabolome, proteome and transcriptome to identify the molecular underpinnings that regulate the metabolic pathways in response to ENMs, which eventually is expressed in the phenotype. However, there are multiple challenges that have slowed down the use of integrative approaches, especially in crop species. The first challenge is the unavailability of transcriptomic databases and incomplete or un-annotated proteomic databases for non-model species. The second bottleneck is the difficulty in scaling very large datasets from different levels within the phenome. Being closest to the phenotype, the metabolome is easily influenced by immediate environmental conditions, which may or may not correlate with genomic and transcriptomic profile. Hence, the emergent properties at the higherlevel organization of the plant are not fully determined by the properties of the lower levels . Thirdly, metabolomic analysis is often oversimplified due to the common assumption that the sampling is performed in metabolic steady-state, characterized by constant levels of metabolites and that different metabolic pathways operate in isolation. At the whole plant level of integration, there are too many complex and dynamic processes occurring simultaneously which regulate feedback mechanisms in the cellular metabolism. In order to integrate the data from different omics in a test specimen, it is necessary to use the same sample for aliquoting into fractions for individual omic analysis. Assuming that the data acquired from each omic is of high quality and validated, they can be integrated by different approach, including postanalysis data integration; integrated data analysis; and systems modeling methods . In postanalysis data integration, the omic datasets are analyzed in isolation and the key features are networked in an overall model pathway. In integrated data analysis,specialized tools are used to merge different omics data sets prior to data analysis and interpretation. Systems modeling approaches incorporate modeling tools utilizing preexisting comprehensive omic databases . Only a handful of studies have utilized integrative approach in ENM-plant interaction studies and have relied primarily on postanalysis data integration . Integration of the plant metabolome with the proteome and transcriptome can answer several questions regarding ENM-associated mechanisms, including routes of entry, translocation, defense response and toxicity . Individual omic studies across different plant species and postanalysis integration studies over the past seven years have highlighted a few metabolic pathways of interest, depending on the mode of exposure. In root exposure studies, ENMs have shown to induce oxidative stress in the root tissues due to the immediate availability of metal ions from ENM dissolution, which is appreciated by the release of acidic exudates . As a defense response to ENMs, multiple metabolic pathways have been reported to be differentially regulated, including glutathione metabolism, GABA shunt, phenylpropanoid pathway, shikimate pathway, and flavonoid pathway. Different phenolic compounds and amino acids are evidently altered in these pathways in order to defend the plants from reactive oxygen species . Another common sign of stress in the plant roots is an increase in lignin content and alteration of membrane lipids, which protect the roots from additional stress . In several studies, nCu and CdS-QDs exposure altered levels of Ca-binding proteins, such as calmodulin or Ca2þ-ATPase . It is hypothesized that ENMs often bind to Ca2þ receptors or use the Ca-channels or Ca2þ-ATP pumps to enter the plants.The glutathione pathway and the GABA shunt play a major role in ROS defense in plants affected by exposure to ENMs. In addition to defense responses in different plant tissues, ENMs have been shown to influence carbon fixation, amino acid metabolism, and photosynthesis in the aerial tissues. In a recent study, metabolomics of leaf mesophyll protoplast showed that nFe and Mn3O4 enhanced the photosynthetic quantum yield . However nMoS2 and nAg had negative effects .