Proteomics of Micronutrient Deficiency and Toxicity

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Sabine Lüthje*, Claudia N. Meisrimler†, David Hopff* University of Hamburg, Hamburg, Germany* Utrecht University, Utrecht, The Netherlands†

1 ­INTRODUCTION Micronutrients (iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), and boron (B)) are essential for a multitude of physiological functions in plant growth, development, and in oxidative stress response. They are required at low concentrations in electron transport and antioxidative systems, but also for the stabilization of proteins and cellular structures (O'Neil et al., 2004). They are cofactors of metalloproteins that are involved in storage and transport, enzymatic reactions, signal transduction, and other functions. Micronutrients permeate biomembranes either by passive diffusion or by unspecific ion transporters, metal effluxer, or heavy metal transporting ATPases. Decreased levels of one micronutrient can affect bioavailability of others (Patterson et al., 2007). Homeostasis of micronutrients has to be tightly regulated to maintain cellular processes and to prevent oxidative stress. Bioavailability of micronutrients depends on several factors. Hydrous Fe and Mn oxides affect the solubility of Zn and Cu by precipitation and specific adsorption reactions (Rieuwerts et  al., 1998). Common farming practices, such as intensive farming, monocultures, and liming of acid soils, deplete micronutrients from the soil or decrease their bioavailability. As a consequence micronutrient deficiency is widespread in agricultural soils. Furthermore, extensive use of glyphosate has been shown to interfere with Fe, Zn, and Mn uptake (Tsui et al., 2005; Eker et al., 2006). An additional factor affecting bioavailability of micronutrients is the climate. Drought, strong rain falls, or waterlogging cause either deficiency or toxicity of micronutrients (Steffens et al., 2005; da Silva et al., 2011; Waraich et al., 2011). Due to global warming and climate change those events become more frequent and affect crop production. In the last century studies have been focused mostly on morphological symptoms, physiological functions, and transport of micronutrients (for reviews see, Belvins & Lukaszewski, 1998; Brown et al., 2002; Kobayashi & Nishizawa, 2012). Nowadays, the fast development of omics technologies and the decoding of crop genomes promote a more systems biology view on the molecular mechanisms in micronutrient deficiency and toxicity (Yan et al., 2006; Ahsan et al., 2009). Bottom-up and top-down strategies with gel-free and gel-based proteomics have been used to study aspects of nutrient availability on plants. A major focus was set on Fe homeostasis, while only few studies have been published for other micronutrients such as Cu, Zn, Mn, and B. In this chapter general and specific mechanisms of micronutrient deficiency and toxicity will be discussed. Plant Micronutrient Use Efficiency. https://doi.org/10.1016/B978-0-12-812104-7.00010-1 Copyright © 2018 Elsevier Inc. All rights reserved.

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2 ­IRON Iron is the sixth most abundant element in the universe, the fourth most abundant element in the earth's crust (Morgan & Anders, 1980), and the most abundant transition metal in plants (Merchant, 2010), which launch Fe into the nutrition chain. It is essential for virtually all forms of life. With increasing oxygenation of the atmosphere the availability of Fe decreased dramatically because its oxides have a very low solubility. Because Fe shortage is a growth limiting factor, plants developed two strategies to face this challenge. With the exception of grasses plants enhance the solubility of Fe by a concerted action of increased ATP-dependent acidification of the rhizosphere, increased allocation of NADH for the reduction of Fe(III) to Fe(II) (Römheld & Marschner, 1983; Bienfait et al., 1983) and an induction of a transporter for Fe(II) (Eide et al., 1996). This system is also referred to as “Strategy I”. In Arabidopsis thaliana, it has been shown that the release of certain coumarins is essential for Fe acquisition (Clemens & Weber, 2016), whereat a proteomic study (Lan et al., 2011) contributed to this discovery. Strategy II has been evolved by grasses, which synthesize high-affinity Fe chelatores, so-called phytosiderophores. These are released into the rhizosphere and are capable of solving Fe(III) directly from the soil. Those Fe-phytosiderophore complexes are taken up by proton coupled oligopeptide transporters such as yellow stripes 1 (Curie et al., 2009). In opposition to other graminaceous species, rice (Oryza sativa) has a transporter, which can take up Fe(II) and thereby combines the two strategies (Ishimaru et al., 2006). Research on Fe homeostasis in plants has focused on the key players involved in uptake, distribution, and storage of Fe (Curie et  al., 2009; Jeong & Guerinot, 2009). But as Fe is involved in fundamental physiological processes such as energy metabolism, nitrogen fixation, or defense against oxidative stress, it is obvious that a reprogramming of several metabolic and developmental pathways have to occur. These changes are partly reflected in the alterations of protein abundances and therefore proteomics is a valuable tool to achieve a better understanding of the mechanisms plants evolved to manage Fe shortage. The information derived from a holistic point of view might ultimately be helpful in developing strategies to counteract the decrease of crop yield and food quality caused by Fe deficiency (López-Millán et al., 2013). The importance of proteomics in this context is underlined by the weak correlation between transcriptome and proteome, indicating that most responsive proteins are post-transcriptionally regulated (Pan et al., 2015). Furthermore, posttranslational modifications such as phosphorylation, which has been investigated in only one single study so far (Lan et al., 2012) constitute an additional level of complexity.

2.1 ­IRON DEFICIENCY Most proteomic studies about Fe homeostasis in plants are dealing with Fe deficiency in Strategy I plants. The knowledge on changes of the proteome profiles in Strategy II plants is strongly limited as there are only three studies of which one has been carried out with Fe deficient shoots and roots of rice (Chen et al., 2015b), one with root hairs of Fe deficient maize (Zea mays) (Li et al., 2015) and one using plasma membrane preparations of Fe deficient maize roots (Hopff et al., 2013). The proteomic view on Fe deficiency has been reviewed thoroughly (López-Millán et al., 2013). The majority of proteomic studies about Fe starvation in plants have been carried out using protein extracts of whole roots because this is the entry site of nutrients where the major adaption leading to an improved Fe acquisition have to occur (Donnini et al., 2010; Lan et al., 2011; Lan et al., 2012;

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Chen et  al., 2015b (+leaves); Rodríguez-Celma et  al., 2013; Rodríguez-Celma et  al., 2011; Pan et al., 2015 (+leaves)). Attempts to analyze subproteomes of Fe deficient plants including root plasma membrane (Gutierrez-Carbonell et  al., 2016; Hopff et  al., 2013; Meisrimler et  al., 2011), detergent resistant membranes (Gutierrez-Carbonell et al., 2016), thylakoid membranes (Andaluz et al., 2006; Timperio et al., 2007), microsomal fractions of shoots (Zargar et al., 2015) and roots (Meisrimler et al., 2016), phloem saps (Gutierrez-Carbonell et al., 2015), root hairs (Li et al., 2015) and apoplastic fluids (Ceballos-Laita et al., 2015) are getting more popular. The methodological strategy most frequently applied to study changes of protein profiles upon Fe starvation is the classical two dimensional polyacrylamide gel elelectrophoresis (2D-PAGE) with isoelectric focusing (IEF) in the first dimension and separation according to the molecular weight in the second dimension by sodium dodecylsulfate (SDS) PAGE (Chen et  al., 2015b; RodríguezCelma et al., 2011, 2013, 2016; Ceballos-Laita et al., 2015; Donnini et al., 2010; Gutierrez-Carbonell et al., 2015; Lattanzio et al., 2013; Rellán-Álvarez et al., 2010; Sudre et al., 2013). Advantages of this gel-based approach have a higher robustness and reproducibility compared to MS-based approaches (Magdeldin et al., 2012). Shotgun proteomic approaches, no matter if the strategy used is labeling with an isobaric tag for relative and absolute quantification (iTRAQ) (Zargar et al., 2015; Lan et al., 2011, 2012; Pan et al., 2015) or unlabeled (Hsieh et al., 2013; Hopff et al., 2013; Gutierrez-Carbonell et al., 2016; Meisrimler et al., 2016) usually provide more comprehensive datasets with more proteins changed in abundance and offer the chance to identify proteins, which are absent in 2D-PAGE based studies such as hydrophobic membrane proteins, very alkaline or acidic proteins or isoforms, which co-migrate in gels. The deepest proteome analysis with 13.706 identified proteins in roots of which 9.110 could be quantified and 12.124 in leaves of which 8.303 could be quantified has been carried out in Arabidopsis using iTRAQ (Pan et al., 2015). Proteins changed in abundance were 481 and 445 in roots and leaves, respectively. A combination of blue native PAGE (BN-PAGE) with IEF-SDS-PAGE has been used to analyze the response of thylakoids from Beta vulgaris to Fe deficiency (Andaluz et al., 2006). While classical 2D-PAGE could separate more distinct protein spots than BN-PAGE, BN-PAGE could provide additional information about protein-protein interactions and a separation of hydrophobic proteins. Due to technical variations, different growth conditions and other variations at any stage of a proteomic experiment the comparison of proteomic studies is not straightforward (Li & Lan, 2017; LópezMillán et al., 2013). Therefore it is difficult to determine, if differences between proteomic studies are species-related or due to experimental conditions. Furthermore, changes in abundance of a peptide or an altered spot intensity can also be caused by posttranslational modifications.

2.1.1 ­Energy metabolism

Fe shortage induces a strong energy demand for its acquisition and simultaneously impairs electron transfer chains, which are required for energy production. Respiration is dependent on several Fe-S cluster and heme-containing proteins and therefore strongly affected by Fe shortage (Vigani et  al., 2009). A decrease of subunits belonging to the respiratory chain complexes has been found in Medicago truncatula (Rodríguez-Celma et  al., 2011), cucumber (Cucumis sativus) (Donnini et  al., 2010), and Chlamoydomonas reinhardtii under autotrophic conditions (Höhner et  al., 2013). Contrarily, in Arabidopsis, several proteins that participate in the respiratory chain were increased (Lan et al., 2011). The authors discussed this finding as transient because the plants were exposed to Fe deficiency for only three days after they were grown under Fe sufficient conditions. Proteins involved in glycolysis are often increased with Fe starvation (Donnini et al., 2010; Rodríguez-Celma et al., 2011; Höhner et al., 2013;

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Rellán-Álvarez et al., 2010). This mechanism is thought to be a first aid to compensate the reduced ATP production (Vigani & Zocchi, 2009). Similar to that, proteins of the tricarboxylic acid (TCA) cycle often are increased as well (Rodríguez-Celma et al., 2011; Donnini et al., 2010), which is according to the increased demand for reducing power. In maize root plasma membranes (Hopff et al., 2013) the abundance of glycolytic enzymes decreased as well as in root hairs (Li et al., 2015), where also proteins of the TCA cycle decreased. It is difficult to conclude if this finding is related to Strategy II, because Fe acquisition requires less energy than in Strategy I plants or restricted to the analyzed subproteomes. In rice, however, proteins involved in glycolysis and TCA cycle showed an increase (Chen et al., 2015b). Photosynthesis is strongly affected by Fe deprivation. Proteomic studies with thylakoid preparations (Andaluz et al., 2006; Timperio et al., 2007) indicate a decrease of electron transporting protein complexes including photosystem (PS) I, PS II and cytochome b6/f. The abundances of proteins involved in carbon fixation including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were increased in sugar beet (B. vulgaris) (Andaluz et al., 2006) and rice (Chen et al., 2015b), but decreased in spinach (Spinacia oleracea) (Timperio et al., 2007).

2.1.2 ­Nitrogen-metabolism

Nitrogen (N) is a limiting factor in plant growth because it is a major constituent of proteins, nucleotides, as well as chlorophyll and other metabolites and cellular components (Marschner, 1995). Fe starvation impairs nitrogen assimilation because it is an essential cofactor of nitrate reductase, nitrite reductase, and glutamate synthase (Borlotti et al., 2012). A decrease of nitrite reductase has been found in M. truncatula (Rodríguez-Celma et al., 2011, 2013), rice (Chen et al., 2015b), and tomato (Solanum lycopersicum) (Li et al., 2008). Other proteins involved in N metabolism as glutamine synthase (Chen et al., 2015b), glutamate synthase (Li et al., 2008), and others increased, which indicates an increased N recycling to countervail the reduced N assimilation (López-Millán et al., 2013). The abundance of nitrate transporters (Lan et  al., 2011; Gutierrez-Carbonell et  al., 2016) and ammonium transporters (Gutierrez-Carbonell et al., 2016) decreased with Fe deprivation. Nitrate assimilation requires ATP and reducing equivalents and therefore the decrease of related proteins could result from a competition for resources in favor of Fe acquisition. Nitrate uptake is proton-coupled and thus counteracts rhizosphere acidification, which is crucial for the response of Strategy I plants to Fe shortage. The decrease of ribosomal subunits in several proteomic studies (Lan et al., 2011; Hopff et al., 2013; Gutierrez-Carbonell et al., 2016) could exemplify a reduced protein production as a consequence of Fe deficiency impaired N assimilation. On the other hand, there is an increased demand for N for methionine and S-adenosyl methionine (SAM) production. SAM appears to be a very important point of intersection in the response to Fe starvation as SAM synthases are frequently increased under Fe limiting conditions (Li et al., 2008; Donnini et al., 2010; Lan et al., 2011; Rodríguez-Celma et al., 2011; Gutierrez-Carbonell et al., 2015; Pan et al., 2015). SAM is a precursor of nicotianamine, which is necessary for Fe chelation and trafficking and which is the precursor for phytosiderophores in Strategy II plants (Curie et al., 2009). SAM is also the precursor for ethylene, which has an important role in the regulation of genes for Fe acquisition (Li & Lan, 2017). The phenylpropanoid pathway, which leads to the synthesis of coumarins and lignin, requires SAM as well (Lan et al., 2011).

2.1.3 ­Cell wall

An increase of proteins involved in cell wall enhancement and a decrease of proteins involved in degradation of cell wall components has been found in roots of Arabidopsis (Lan et al., 2011), plasma

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membrane of maize roots (Hopff et  al., 2013), and rootstocks of Prunus dulcis x Prunus persica (Rodríguez-Celma et al., 2013). This is in line with histochemical observations (Forner-Giner et al., 2010) and the detection of changes in the lignin composition in stems of M. truncatula (RodríguezCelma et al., 2016). An increase of cell wall rigidity might decrease its permeability and thereby act as a protective barrier to reduce lateral movement of Fe, uptake of toxic cadmium (Cd), or entry of pathogens due to an increased vulnerability under Fe limitation.

2.1.4 ­Redox homeostasis

Iron is an integral part of many redox proteins as peroxidases, catalases (CAT), and Fe containing superoxide dismutase (SOD) that detoxify reactive oxygen species (ROS). When Fe is scarce detoxification of ROS is impaired, which leads to oxidative stress. A decrease of CAT and an increase of manganese SOD and copper/zinc SOD (Rodríguez-Celma et al., 2013; Lan et al., 2011) has been observed while peroxidases changed differentially (Rodríguez-Celma et al., 2013). Plants possess a high number of peroxidases, which indicates a very complex Fe dependent redox network that is far from being understood (Lüthje et al., 2014). PM bound peroxidases decreased in pea (Pisum sativum) roots (Meisrimler et al., 2011) while those detected in PM of maize roots increased (Lüthje et al., 2011). Due to its Fe independence glutathionylation is possibly a strategy to counteract oxidative stress (LópezMillán et al., 2013).

2.2 ­IRON TOXICITY The same properties that make iron a favorable element for redox processes can be deleterious when its concentration exceeds the organism's capacity to chelate this metal. The toxicity of Fe is based on its direct involvement in ROS production and on its ability to bind to thiol-, carboxyl- and imidazole residues, which leads to alterations in protein structures and thereby protein functions. Further, an oversupply of Fe may lead to a competition concerning uptake between Fe and other essential metals, which finally results in metal imbalance (Singh et al., 2016). Fe oversupply is sometimes used as a tool to investigate the role of certain proteins in Fe homeostasis (Zhu et al., 2016; Divol et al., 2013; Chungopast et al., 2016) but to our knowledge there is only one proteomic study, which shows changes of a protein profile in response to Fe toxicity (Hopff et al., 2013). In that study, several ribosomal subunits, the plasma membrane bound ascorbate reducible cytochrome AIR 12 (auxin induced in root cell cultures) and plasma membrane associated glycolytic enzymes were increased with Fe toxicity in opposite to Fe deficiency. Some proteins related to cell wall restructuring, stress signaling and growth regulator perception were differentially changed in abundance with Fe toxicity and Fe starvation as well.

3 ­COPPER Copper has a natural abundance of 60 mg/kg in the Earth's crust (Printz et al., 2016). Average ambient background Cu concentrations in European soils vary between 11.4 and 17 mg Cu/kg (Alloway, 2013). Cu is essential to all living organisms as a trace mineral. It plays key roles in photosynthetic and respiratory electron transport chains, in ethylene sensing, cell wall metabolism, oxidative stress protection and biogenesis of molybdenum cofactor. Cu homeostasis in plant cells is a tightly controlled process that ensures a sufficient delivery but also avoids the harmful effect of Cu excess through

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the generation of ROS by the Fenton reaction (Drazkiewicz et al., 2004). The two common states in plants are Cu(I) and Cu(II). In higher plants, the main function of Cu is the transport of electrons in the electron transport chains in mitochondria and chloroplasts. Based on this fact, most Cu deficient plants show impairment in the photosynthetic electron transport chain and a reduction in nonphotochemical quenching, which is consistent with an impaired plastocyanin function. Cu acts also as a cofactor for enzymes involved in the cellular redox state and the modeling of the cell wall (Cohu & Pilon, 2010). Under Cu deficiency genes involved in Cu acquisition and redistribution are activated. SQUAMOSA promoter-binding protein-like7 (SPL7) a transcription factor is a central regulator of Cu homeostasis (Garcia-Molina et  al., 2014). Cu homeostasis is mainly maintained by two Cu transporter families: (1) P1B-type ATPases and (2) high affinity Cu transport proteins from the copper transporter (Ctr/ COPT) family with a variety of functions (Abdel-Ghany & Pilon, 2008; Garcia-Molina et al., 2014). Intracellular Cu transport involves a Cu chaperone (antioxidant protein 1, ATX1) and one P1B-ATPase (Flores & Unger, 2013; Printz et al., 2016). Generally, mechanisms of Cu uptake in plants have not been completely elucidated; however, a strong overlap between Fe-uptake and Cu uptake mechanisms has been suggested (Printz et al., 2016). Furthermore, Cu plays an important role in cell wall composition and lignin production, which is regularly reflected in proteome analysis.

3.1 ­COPPER DEFICIENCY In old leaves of rape (Brassica napus) Cu content decreased under Cu deficiency, demonstrating a remobilization process between leaves (Billard et al., 2015). Cu deficiency also triggered an increase in Cu transporter expression in roots (copper transporter 2, COPT2) and leaves (heavy metal ATPase1, HMA1), and more surprisingly, the induction of the MOT1 gene encoding a molybdenum (Mo) transporter associated with a strong increase in Mo uptake (Billard et al., 2015). The proteomic analysis of leaves by 2D-PAGE revealed 33 proteins differentially regulated by Cu deficiency, among which more than half were located in chloroplasts. Eleven differentially expressed proteins are known to require Cu for their synthesis and/or activity. Enzymes that were located directly upstream or downstream of Cu-dependent enzymes were also differentially expressed. Similar to rape leaves, analysis of alfalfa (Medicago sativa) stems showed that the concentrations of Mo were increased in comparison with the optimum Cu level (Billard et al., 2015; Printz et al., 2016). The same was observed for Zn levels in the stem of alfalfa plants. Furthermore, 2D-PAGE analysis of alfalfa stems revealed accumulation of specific Cu chaperones. The parallel conducted expression analysis showed a decrease of genes involved in stem development (Printz et al., 2016). Additionally, proteins involved in the cytoskeleton, lignin and methionine metabolism were significantly decreased. In the green algae Chlamydomonas the main responses to Cu deficiency are controlled by the transcription factor CRR1 (Sommer et al., 2010). Transcriptome studies have revealed a Cu deficiency induced increase in the abundance of different hydrogenases (HYD1, HYDEF, HYDG), putative hybrid cluster protein (HCP2, HCP3), and pyruvate ferredoxin oxidoreductase (PFR1) transcripts, which are related to anaerobic responses in Chlamydomonas (Mus et al., 2007; Castruita et al., 2011). It was hypothesized that the corresponding proteins to these transcripts cannot function in aerobic conditions because their active site clusters are O2 labile, leading to miss-expression because Cu deficiency results in faulty O2 sensing. In addition, proteins of the tetrapyrrole biosynthetic pathway, the isoprenoid pathway and the SAM biosynthesis pathway changed. Lastly, a change in two selenocysteine tRNA synthases and a selenium (Se) binding-proteins were decreased in Cu deficiency. In the

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future, it will be of interest to see results in context with the protective function of Se under heavy metal exposure in plants, published recently (Sun et al., 2016).

3.2 ­COPPER TOXICITY In comparison to Cu deficiency, Cu toxicity has been rather extensively studied in different plants and algae using the proteomic approach. In most cases phenolic extraction of total proteins was performed, which was analyzed by 2D-PAGE coupled with different forms of MS. In the brown algae Sargassum fusiforme, also known as hijiki, Cu stress at low concentration was tolerable, but it had a negative impact at high concentration. These high concentrations lead to decreased photosynthetic pigments, impairment of the photosynthesis apparatus, photosynthesis activity, and induction of lipid oxidation. Furthermore SOD, CAT and nitrate reductase activity are modified (Zhu et  al., 2016). In S. fusiforme, energy ­metabolism-related proteins were significantly induced by chronic Cu stress, whereas acute Cu toxicity reduction of proteins involved in energy metabolism and photosynthesis were observed. Proteomic results suggest that energy production and conversion are inhibited under oxidative toxicity caused by high Cu concentrations. Photosynthesis inhibition is then a consequence of the interference of Cu with the biosynthesis of the photosynthetic machinery, leading to pigment changes (Zou et al., 2015). The positive effects by heterotrophic-Cu(II) stressed HCuS coupling cultivation on Chlorella protothecoides in lipid accumulation has been observed in another mixed omics approach (Li et al., 2013a). The results showed that the optimized biomass and lipid yield were achieved with this strategy, resulting in an ideal lipid composition for the preparation of high quality biodiesel. Further, 30 differentially expressed proteins involved in carbohydrate metabolism, carbon fixation, TCA cycle, lipid metabolism, protein biosynthesis, transportation and regulation, ATP and RNA biosynthesis, nucleotide metabolism, and ROS scavenging have been identified. The proteomic data suggested that the glycolysis pathway might be the important contributor for lipid accumulation (Li et al., 2013a). In addition to algae, various proteomic studies were published for land plants, including monocots and dicots. Proteomic studies on Cu toxicity were mainly performed for crop plants. In the only available study for a tree, crack willow (Salix fragilis) was compared to eared willow (Salix aurita) clones on a proteomic and physiological level (Evlarda et al., 2014). Data elucidated that growth reduction and the proteomic changes in crack willow indicate that this clone adjusts its metabolism to maintain cellular homeostasis, whereas eared willow maintains its growth. Physiological and proteomic data suggested that this can only be done at the cost of cellular deregulation. Therefore it became clear that high biomass is not linked with a good tolerance strategy. In grasses, multiple studies are available for rice (seedlings, germinated rice seeds, embryos) and common wheat (Triticum aestivum) seedlings (Li et al., 2013b; Chen et al., 2015a,b; Ahsan et al., 2007; Zhang et al., 2009). For wheat, the growth of shoots and roots was markedly inhibited by Cu toxicity and lipid peroxidation was greatly increased. It has also been shown that Cu was readily absorbed by wheat seedlings (Li et al., 2013a,b). Proteins with a function in signal transduction, stress defense, and energy production were significantly enhanced, while many protein species involved in carbohydrate metabolism, protein metabolism, and photosynthesis were severely reduced. Network analysis revealed multiple phytohormones, such as abscisic acid (ABA), ethylene, and jasmonic acid (JA) as key regulators. Results of germinated rice seedlings, Cu sensitive rice varieties, as well as winter cherry (Withania somnifera), show similar ­tendencies than observed for wheat (Ahsan et  al., 2007; Song et  al., 2014; Chen et  al., 2015a,b; Rout et  al.;, 2013), leading to the general observation that excess Cu generates oxidative stress that disrupts other

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important metabolic processes. In addition, data on rice embryos (Zhang et  al.;, 2009) included a metallothionein-like substance, which is thought to be a potential marker for heavy metal contaminated soil. Metallothioneins have a high content of cysteine residues that bind various heavy metals, bestowing some resistance against heavy metal toxicity.

4 ­ZINC Zinc (Zn) is an essential element in all organisms. In its oxidized Zn(II) form, which is the form of Zn found throughout biology, it acts as a catalytic or structural co-factor in a large number of enzymes and regulatory proteins (Maret, 2009). Well-known examples in plants include the enzymes carbonic anhydrase and alcohol dehydrogenase, and the structural Zn-finger domains mediating DNA-binding of transcription factors and protein-protein interactions (Sriram & Lonchyna, 2009). Similar to Fe uptake in Strategy I plants, acidification via H+-extrusion by proton pumps in the plasma membrane is important for Zn acquisition from the soil. In case of Zn deficiency, plant responses include increased uptake into the root symplast (Sinclair & Krämer, 2012). It was postulated that re-mobilization of Zn from the vacuoles and increased xylem loading is enhanced under Zn deficiency. The ZIP (Zn IRTlike Proteins) family of metal transporters plays an important role in Zn acquisition. According to the current model, the active bZIP19/bZIP23 complex enhances transcription of ZIP1, ZIP3, ZIP4, ZIP5, ZIP9, ZIP10, ZIP12, IRT3, NAS2, and NAS4 (Sinclair & Krämer, 2012). HMA2 (heavy metal associated) transcript levels are also known to increase under Zn deficiency. On a proteomic level, Zn has been less studied than Fe but more than Cu and other micronutrients. Zn was often studied in context with Fe availability and compared to Fe, because of high similarities and co-regulations for both metals (Fukao et al., 2011).

4.1 ­ZINC DEFICIENCY Zn deficiency has been studied less than Zn toxicity using proteomic approaches. Zn deficiency was either analysed by 2D-gels or by label-free quantitative proteomics. Previous high-scale studies on Zn deficiency have mainly used transcriptomic approaches, showing a high impact of Zn deficiency on the regulation of transcription factors. A high amount of transcription factors represent Zn finger proteins. Even though Zn finger proteins are numerous they represent only 0.7% of the total amount of proteins (Billard et al., 2015). It was shown that Zn is not remobilized in rape and from the ionomic points of view, Mn and Mo were increased under Zn deficiency. In rape five proteins were decreased by Zn deficiency with nine cis-epoxycarotenoid dehydrogenase as the most repressed. Among the 12 increased proteins, chitinase showed the highest increase. Most of the identified proteins were Zn proteins. In general, proteins of energy metabolism, protein synthesis, and defense or stress related (HSC70, myrosinase) were identified. Regulation of chitinase and HSC70 were also found in a previous transcriptomic study on roots of Arabidopsis (van de Mortel et al., 2006). Billard et al. (2015) suggests after comparing results with data from Fukao et al. (2011) about Zn toxicity that the protein disulphide isomerase and triosephosphate isomerase are under constant repression by Zn. In Arabidopsis roots, Zn deficiency also increased two defensin-like (DEFL) family proteins, but the function of this regulation is unclear to date (Fukao et al., 2011).

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In the single cell green algae Chlamydomonas, a large accumulation of two COG0523 domain containing proteins and a decrease of the zinc-containing carbonic anhydrase (CAH) isoform1 was observed (Hsieh et al., 2013). CAH constitute a major group of proteins that bind zinc. It interconverts CO2 and bicarbonate to facilitate CO2 fixation for carbon assimilation and plays a role in photosynthetic growth under limiting CO2 conditions. The cah1 null mutants in Chlamydomonas do not exhibit severe growth phenotypes, even under low CO2 growth conditions; therefore, CAH1 might be a dispensable enzyme and would make a good target for degradation if the cell was in need of intracellular Zn for recycling and redistribution (Hsieh et al., 2013). Furthermore, redox proteins for example, thioredoxin 5 has been increased in Zn deficient cells, but was undetectable in Zn-repleted cells—showing the importance of the cellular redox-regulation under Zn deficiency. This was reinforced by the observation that multiple thioredoxin 5 interacting proteins of the energy metabolism were also regulated.

4.2 ­ZINC TOXICITY Environmental pollution by high Zn concentration occurs frequently and was studied quite regularly in the past using different proteomic approaches. General mechanisms and proteins changed in abundance with Zn toxicity. These were related to ROS scavenging and carbohydrate/energy metabolism, which is similar to other proteomic studies on heavy metal toxicity. Effects on the root proteome showed that proteins of the antioxidant system increased, proteins of carbohydrate/energy metabolism decreased and amino acid metabolisms were changed by Zn excess in Populus × euramericana (Romeo et al., 2014). Mitochondria and vacuoles were the cellular organelles predominately affected by Zn stress in poplar. Similar results for the mitochondrial proteome were shown for sugar beet roots (GutierrezCarbonell et al., 2013), confirming the strong linkage of Zn toxicity with ROS production in the mitochondria. Gutierrez-Carbonell et al. (2013) observed a general metabolism shutdown at excessive Zn supply, which was denoted by decreases in the aerobic respiration and by an impairment of the defense systems against oxidative stress. Accordingly, an increased lipid peroxidation was observed at high Zn supply, which is comparable to the Cu toxicity discussed above. Both studies suggest that metabolic changes at high Zn supply reflect cell death. Additionally, Zn excess affects the polar root secondary metabolism in poplar throughout the down-regulation of anthocyanidin synthase, which is a key enzyme of the phenylpropanoid pathway (Romeo et al., 2014). The anthocyanidin synthase is usually lower in expression when the phenylpropanoid pathway shifts toward lignin production (Peng et al., 2008). Furthermore, Zn-responsive proteins in microsomal fractions from leaves of Arabidopsis plants were found to be involved in the one-carbon metabolism pathway (Barkla et al., 2014). The results included glycine decarboxylase P protein, serine hydroxymethyltransferase (SHMT), and methionine synthase, all of which showed reduced abundance in the Zn-treated samples. Additionally, plants showed increased petiole length, a phenotype that may reflect the reduced levels of methionine, a key product of the one-carbon metabolism pathway. Plants with high heavy metal tolerance and accumulation have been analyzed for proteomic changes, showing another view of molecular regulation and application (Farinati et al., 2009; Schneider et al., 2013). Arabidopsis halleri accumulates Zn in the leaves. A. halleri plants showed a general upregulation of photosynthesis-related proteins exposed to metals and to metals plus microorganisms, suggesting that metal accumulation in shoots is an energy-demanding process (Farinati et al., 2009). In contrast, proteins involved in plant defense mechanisms were down-regulated, indicating that heavy metal accumulation in leaves supplies a protection system and highlights a cross-talk between heavy

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metal signaling and defense signaling. Schneider et al. (2013) showed that epidermal cell of alpine pennycress (Noccaea caerulescens), which can accumulate high Zn concentrations in the leaf epidermis without showing any toxicity symptoms, differed from mesophyll cell proteome. Epidermal cells had an increased capability to cope with the oxidative stress, as indicated by a higher abundance of glutathione S-transferase proteins. A Zn importer of the ZIP family was more abundant in the epidermal tissue than in the mesophyll tissue. The vacuolar Zn transporter MTP1 was equally distributed between the two cell types. Almost all of the Zn located in the mesophyll was stored as Zn-nicotianamine complexes. In contrast, a much lower proportion of the Zn was found as Zn-nicotianamine complexes in the epidermis. However, these cells have higher concentrations of malate and citrate. Both organic acids are probably responsible for complexation of most epidermal Zn in the vacuole after import by MTP1 (Schneider et al., 2013). Finally, cross-talk between excess Zn and Fe deficiency was studied in Arabidopsis roots, using iTRAQ for microsomal proteins (Fukao et al., 2011; Zargar et al., 2015), because plant symptoms under excess Zn resemble symptoms of Fe deficient plants. Protein expression changes between excess Zn and Fe deficiency showed similar pattern. Changes due to excess Zn were restored by the addition of Fe. Several membrane proteins were identified. Among them, IRT1, an iron and zinc transporter, and FRO2 increased greatly in response to excess zinc. The expression of these two genes has been previously reported to increase under Fe deficient conditions. Indeed, the concentration of Fe was significantly decreased in roots and shoots under excess Zn (Fukao et al., 2011). Also, V-ATPases were identified, and three of them decreased significantly in response to excess zinc. Excess Zn in the wild type decreased V-ATPase activity and the length of roots and cells to levels comparable to those of the untreated de-etiolated 3-1 mutant, which bears a mutation in V-ATPase subunit C. Results pointed out that growth defects caused by excess Zn in which cross-talk between Fe and Zn homeostasis happens, the V-ATPase activity might play a central role.

5 ­MANGANESE In acidic soils, Mn becomes a major limiting factor in plant production systems (Wu et  al., 2010). Manganese is essential for photosynthesis and respiration; it takes part in the activation of numerous enzymes (Ref. Graham et  al., 1988). Gene families have been identified that are involved in Mn2+ uptake and transport. Among these are natural resistance-associated macrophage protein (Nramp) transporters, ZIP transporters, the cation diffusion facilitator (CDF) transporter family, cation/H+ antiporters, and P-type ATPases (Pittmann, 2005; Vatansever et al., 2016).

5.1 ­MANGANESE DEFICIENCY Mn deficiency often occurs as a latent disorder without any growth restrictions or clear leaf symptoms, despite significant disintegration of PS II under these conditions (Schmidt et al., 2016). A study on two barley genotypes showed that the amount of PS II supercomplexes was drastically reduced in response to Mn deficiency (Schmidt et al., 2015). Similar results were found for Chlamydomonas (Hsieh et al., 2013). The latter study used a label-free, quantitative proteomics strategy. At least 270 proteins altered in abundance in the soluble proteome of the algae under conditions of Mn deficiency. A reduced abundance of MnSOD1 was observed. Proteins involved in photosynthetic function remained unchanged,

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which allows a fast recovery upon the addition of supplemental manganese to the growth medium (Hsieh et al., 2013). The increase of oxygen evolving enhancer proteins (Oee1, Oee2, Oee3) in the soluble protein fraction supported the hypothesis that these proteins become less associated with thylakoid membranes under Mn deficiency. The overall increase in the amount of proteins involved in proteolysis could be explained by an increase of oxidized proteins, which are a target for the proteasome.

5.2 ­MANGANESE TOXICITY Excess of Mn causes various phytotoxic effects (Foy et  al., 1978; Führs et  al., 2008). It has been suggested that the leaf apoplast is the most important compartment in plants for defense against Mn stress (Horst et  al., 1999). The apoplastic proteome of cowpea (Vigna uniculata) grown under high Mn supply has been studied by 2D-PAGE and BN-PAGE (Fecht-Christoffers et  al., 2003). Besides pathogen-related proteins (PR-proteins), acidic peroxidases altered in abundance under those conditions. BN-PAGE analysis combined with a guaiacol peroxidase staining revealed an increase of several apoplastic peroxidases (~30 kDa). After 2 days of Mn treatment, additional peroxidase bands appeared. Identification of the guaiacol spots revealed sequence similarity to thaumatin-like proteins, chitinases, glucanases, fascilin-like arabinogalactan-protein, hevein-like and wound-induced proteins, and ­pathogenesis-related (PR) proteins. The increase of peroxidase correlated with the formation of Mn toxicity symptoms (i.e., brown spots). Apoplastic peroxidase activity increased in an Mn sensitive cultivar but not in an Mn tolerant cultivar of cowpea (Fecht-Christoffers et al., 2006). Similar results have been observed in barley and rice, suggesting that acidic peroxidases may have an important function in the first level response to Mn induced oxidative stress (Führs et al., 2010). Furthermore it has been proposed that browning of leaf tissues in cowpea under Mn stress may be due to the oxidation of phenolic compounds by acidic peroxidases with a high affinity for monophenols in the apoplast (Fecht-Christoffers et al., 2003; Führs et al., 2010). The effects of Mn (3  days) have been further studied in sensitive and tolerant cowpea cultivars (Führs et  al., 2008). Total soluble proteins have been analyzed for samples grown under low and high Mn supply by 2D-PAGE. More than 25 proteins altered in abundance in dependence on the Mn treatment. Significant changes were observed for eight proteins with increased Mn supply. In the Mn ­sensitive cultivar, five proteins involved in primary carbon fixation decreased and two PR-proteins increased in abundance. Induction of PR-related proteins appears to be a more general stress mechanism, which can be observed also for other heavy metals and abiotic stresses (Hossain & Skomatsu, 2013). The oxygen-evolving enhancer protein 1 (OEC33) was reduced in the sensitive cultivar, whereas the oxygen-evolving enhancer protein 2 (OEC23) was increased in abundance in the Mn tolerant cultivar. Since Mn is involved in the water splitting complex of PS II, the subunit composition of PS I and II was investigated by BN-PAGE (Führs et al., 2008). Mn toxicity increased a slightly enlarged form of PS I (~650 kDa). This increase in abundance was observed for both cultivars, but was higher in the Mn sensitive cultivar. The higher molecular mass arose by attachment of trimeric LHCII during transition from state 1 to state 2 photosynthesis (Haldrup et al., 2001). The authors suggested that a coordinated interplay of apoplastic and symplastic reactions may be important during the Mn stress response in cowpea (Führs et al., 2008). Long-term effects (4  weeks) of Mn toxicity on poplar (Populus cathayana) leaf proteome have been studied by 2D-PAGE (Chen et al., 2013). The treatment induced 10 proteins that were related

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to photosynthesis, ROS scavenging, and cell signaling related to ROS, plant cell death, heat shock, cell defense and rescue, and gene expression and regulation. Four proteins increased (peroxiredoxin BAS1, trypsin/chemotrypsin inhibitor, HSP, actin-1) and one protein decreased (zinc finger protein) in abundance in male leaves. In females, two proteins increased (2-cys thioredoxin BAS1, RuBisCo) and one protein (actin-1) decreased in abundance. Actin-1 increased in males, but decreased in females. In total six proteins were observed at higher abundance in stressed males, whereas seven proteins showed higher abundance in control males compared to females. In addition increased Mn absorption leads to greater morphological injuries in females. Differences in antioxidant systems of males and females were concluded by this study (Chen et al., 2013).

6 ­BORON Boron has a function in the cross-linking of the pectin rhamnogalacturonan II in the cell wall (e.g., O'Neil et  al., 2004). In addition, B appears to play a role in plant membranes by the cross-linking of glycoproteins, and may be involved in their recruitment to membrane microdomains. At least 26 B-binding proteins have been identified in microsomal preparations of Arabidopsis and nine in maize (Wimmer et al. (2009). Boron is transported by nodulin-like intrinsic proteins (NIPs), boric acid channels, and B exporters (BORs) (Miwa & Fujiwara, 2010). NIP5;1 and BOR1 expressions are regulated by B conditions, which could maintain B homeostasis. At-BOR1 exports B into the xylem under B deficiency and is down-regulated under high B supply. In contrast Hv-BOR2/Bot1, Ta-BOR2, and AtBOR4 export B from plant cells and thereby may increase B tolerance. An iTRAQ analyses of B tolerant and sensitive barley (Hordeum vulgare L.) revealed the identifica­ tion of three enzymes involved in siderophore production (Fe deficiency sensitive 2 and 3, methylthioribose kinase) that were more abundant in B-tolerant cultivars (Patterson et al., 2007). It was also shown that Fe-deficient plants of the B tolerant cultivar Sahara accumulated more B compared to Fe-replete plants. In contrast, the B sensitive cultivar Clipper, did not show such effect. A similar accumulation of B was observed for Zn deficiency in barley (Graham et al., 1987). Thus Fe-dependent phytosiderophore production may need further elucidation.

6.1 ­BORON DEFICIENCY Boron deficiency is the most extensive deficiency of any plant micro-nutrient worldwide (Shorrocks, 1997; Belvins & Lukaszewski, 1998). Besides the iTRAQ study by Patterson et  al. (2007), several proteomic approaches have been published on long-term B deficiency. Alves et al. (2011) studied total protein extracts of roots from white lupin (Lupinus albus) by 2D-PAGE. In total, 128 proteins could be identified by mass spectrometry. These proteins were related to cell wall metabolism, cell structure, defense, energy pathways, and protein metabolism. Since multiple isoforms with different isoelectric points and molecular masses were identified for several proteins, a prominent role of posttranslational modifications was suggested in B deficiency. Observations made for cytoskeletal associated proteins suggested an important function of B in cytoskeletal biosynthesis. Roots of Orange (Citrus sinensis) seedlings were used for iTRAQ analysis of whole cell extracts. In this experiment the abundance of 164 proteins increased and that of 225 proteins decreased by B deficiency (Yang et al., 2013). Most of these proteins were related to protein metabolism, nucleic

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acid metabolism, stress responses, carbohydrate and energy metabolism, cell transport, cell wall and cytoskeleton metabolism, biological regulation and signal transduction, and lipid metabolism. Several new B responsive candidates were identified that are involved in cell transport (i.e., PIPs and VDACs), biological regulation and signal transduction (i.e., BTF3, GRFs, AIP3 and MBF1), stress responses (i.e., Nudix hydrolase, ALDH, allene oxide cyclase) and other metabolic processes (i.e., RNA-binding K-homology (KH) domain-containing proteins, small ubiquitin-like modifier 2 and RmlC-like cupins superfamily proteins). Besides iTRAQ analysis, Yang et al. (2013) measured expression and activity of acid-metabolizing enzymes and antioxidant enzymes in roots. The final conclusion of these experiments was that adaptation to B deficiency occurred by a decrease in root respiration, an increase of antioxidants, and ROS scavenging proteins and enhancement of cell transport.

6.2 ­BORON TOXICITY Alkaline and saline soils are characteristic for B toxicity. It is often associated with low rainfall or it can be a consequence of over-fertilization and/or watering with high B levels (Nable et al., 1997). Boron toxicity-responsive proteins of pomelo (Citrus grandis) (B-sensitive) and orange (B-tolerant) have been compared by 2D-PAGE (Sang et al., 2015). The analyses revealed several differences in the protein profiles of the two species by B toxicity. In phenolic extracts, 50 (B-tolerant) and 45 (B-sensitive) proteins altered in abundance. The higher B-tolerance of orange could be explained by a better adaptation of proteins with a function in photosynthesis and energy metabolism. For example, abundance of Chl a/b-binding protein (CAB) decreased. CAB binds to Chlorphyll and forms light harvesting complexes (Li et al., 2000). The adaptation of photosynthesis and energy metabolism in orange may cause a higher CO2 assimilation and a better maintenance of energy homeostasis. A lowered photosynthesis may reduce the requirement of antioxidants and ROS scavenging systems as well. However, in barley leaves the abundance of PSI type III CAB increased by B toxicity (Atik et al., 2011). Finally, B-toxicity-responsive proteins with a function in coenzyme biosynthesis (i.e., Pyridoxal biosynthesis protein and S-adenosylmethionine synthetase 4) showed higher abundance in orange.

7 ­CONCLUSIONS AND FUTURE PERSPECTIVE Micronutrients play important roles in enzymes of photosynthesis, respiration, and detoxification systems. It was shown that deficiency or toxicity cause the regulation of these processes and other systems. Some micronutrients revealed partially quite similar stress responses, for example, Fe and B (upregulation of phytosiderophores) others cross‐talk (Zn excess/Fe deficiency). Thus future studies should be focused on combination of different stress treatments. Fe deficiency is best characterized from a proteomic point of view. Beside whole cell extracts of soluble proteins, subproteomes, and membrane proteins have been characterized by gel-based and gel-free approaches. However, most publications investigated the effects of iron deficiency on Fe uptake Strategy I plants, whereas only a few studies were published on Strategy II plants or Fe toxicity. Future research should concentrate more on plasma membranes and microdomains that are important targets for nutrient transport, stress signaling, and adaptation. Furthermore, multiple proteomic studies showed the importance of Cu, Fe, and Zn on cell wall-related proteins. It would be of interest to see more detailed studies on cell wall and apoplastic proteins. Studies on micronutrients other than Fe

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are at the very beginning of understanding molecular mechanisms in stress response. The next steps in proteomics for micronutrient acquisition and adaptation process will be to include more available mutant-lines on master-regulators, such as specific transcription factors, as well as analysis of posttranslational modifications and protein-protein interactions to better understand signaling networks and regulation mechanisms. A combination of proteomics with genomics, transcriptomics, and metabolomics (so-called plant nutriomics) will help to understand the complex molecular mechanisms involved in plant nutrition.

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