Leaf Proteomic Analysis in Two Maize Landraces With Different Tolerance to Boron Toxicity

1. Introduction

Boron (B) is an essential element for plants being well known its structural role in both cell walls and membranes [1,2,3,4,5]. Actually, B establishes diester bonds between apiose residues of two rhamnogalacturonan-II (RGII) molecules forming RGII-B complexes that stabilize the pectin network of the cell wall [6,7,8]. Furthermore, B contributes to the preservation of plasmalemma integrity and function [9], likely through the formation of B complexes with membrane components that contain cis-diol groups [10,11]. Thereby, B forms complexes with major constituents of membrane lipid rafts, such a as glycosyl inositol phosphoryl ceramides (GIPCs) [12]. Moreover, B participates in the formation of GIPCs-B-RGII complexes, which connect the plasmalemma to the cell wall [13]. Besides these structural roles, B is also involved in plant development participating in root and shoot elongation, pollen-tube growth, flowering, and fruiting [14,15,16]. In addition, B has been reported to participate in several physiological processes, such as photosynthesis, nucleic acid synthesis, phenolic, nitrogen and polyamines metabolisms, proteins stabilization and biosynthesis, and gene expression, among others [16,17,18,19,20,21,22].

Since B is a micronutrient, the range between its deficient, optimal, and toxic concentrations for plants is very narrow [23]. Therefore, it is common to find soils with inadequate B content for optimal plant development. Soils with high B contents predominantly occurs in arid and semi-arid countries, where this micronutrient accumulates in the topsoil mainly owing to high evapotranspiration and tiny leaching caused by low rainfall, a situation that is often aggravated by irrigation with B-enriched water [22,23]. Additionally, excess B is also found in lands close to coastal areas due to the hydraulic connection between their coastal aquifers and seawater [24] or in regions with recurrent geothermal activities [2]. Climate change is another factor that is contributing to the B increase in soils. Increasing temperatures and decreasing rainfall are predicted in the coming years, which will lead to an increase in agricultural areas with excessive B levels [3,25].

Excessive B contents in soils cause adverse effects such as chlorosis and necrosis in leaves, damages to stems and buds, and misshapen fruits [17,22]. Furthermore, an excess of B provokes DNA damages, inhibition of protein folding, impairment of protein functions and activities, and alterations in photosynthesis and nitrogen and carbon metabolisms, among other processes [2,22,26]. In fact, several photosynthetic parameters, such as CO₂ assimilation (P_N), photosynthetic electron transport rate (ETR), maximum quantum yield of chlorophyll fluorescence (Fv/Fm), and CO₂ use efficiency decreased under B toxicity conditions [22,27]. Because of the aforementioned effects of B toxicity in plant physiology, elevated B contents in agricultural lands reduce crop growth, yield, and quality [22,28]. In fact, a noteworthy decrease in the yield of several main crops subjected to B toxicity has been reported [28]. Despite the large number of effects caused by B toxicity in plants, it is not well known how B produces these alterations. However, it has been suggested that the ability of B to form bonds with molecules containing mono-, di- and poly-hydroxyl groups could be the chemical basis by which B toxicity could trigger morphophysiological alterations [29].

Maize is an important crop that provides approximately half of the calories consumed worldwide being, in addition, one of the principal genetic model plants for crop improvement and food security [30,31,32]. However, maize production is seriously constrained by abiotic and biotic stresses [33]. In particular, B toxicity causes a decrease in maize production as well as in other cereals [28,34,35]. Therefore, the search and molecular characterization of new maize varieties with improved tolerance to B toxicity has become an interesting research topic. In a recent work, two Peruvian maize landraces (Pachía and Sama) were tested for tolerance to high B. The Sama landrace had greater tolerance to B excess than Pachía [27]. In this work, a comparative proteomic characterization of these two maize landraces with different tolerance to B toxicity was performed to improve our molecular knowledge about which proteins are involved in B-toxicity tolerance.

2. Results

A total of 2793 proteins were identified in at least one of the biological replicates of a landrace (Sama or Pachía) and a B treatment analyzed (Table S1a). In addition, the number of proteins detected in both Pachía and Sama in each of the B treatments studied was similar being close to 1100 proteins (Table 1).

Table S1a shows the dataset of the identified proteins indicating their gene ontology (GO) biological processes (GOBP), GO molecular functions (GOMF), and GO cellular compartments (GOCC), and Table S1b summarizes the statistical analysis and fold changes of the proteins. To study the differentially accumulated proteins in Pachía and Sama in both B treatments, four comparison groups were established: 1) Sama and Pachía seedlings subjected to the control B condition (S0.05/P0.05), 2) Sama and Pachía treated with 10 mM B (S10/P10), 3) Sama subjected to 10 mM and 0.05 mM B (S10/S0.05), and 4) Pachía treated with 10 mM and 0.05 mM B (P10/P0.05). A total of 303 proteins had statistically significant differential expression (P ≤ 0.05) in the above groups (Table S2). The S0.05/P0.05 and S10/P10 groups contain those proteins that were differentially expressed between Sama and Pachía in 0.05 mM or 10 mM B, respectively. In media with 0.05 mM B, more proteins were up- and down-accumulated between Sama and Pachía than in 10 mM B (Figure 1 and Table 1). In addition, the S10/S0.05 and P10/P0.05 comparison groups included proteins that were differentially expressed in response to B toxicity in Sama or Pachía, respectively. Pachía had a higher number of proteins induced and repressed by B toxicity than Sama, thus 98 proteins were up-expressed in Pachía in 10 mM B while only 38 in Sama and 51 proteins were down-expressed in Pachía under B toxicity versus 28 in Sama (Figure 1).

2.1. Classification into several functional categories of differentially accumulated proteins in both maize landraces and B treatments

All significant differentially expressed proteins in the four comparison groups described above were functionally classified into 26 categories using several databases (Table S2). The functional categories that included the largest number of differentially accumulated proteins were transcription and translation processes (57), photosynthesis (25), amino acid metabolism (24), protein degradation (23), carbohydrate metabolism (20), and protein stabilization and folding (18) (Figure 2 and Table S2). These main categories together contained more than 50% of the total differentially expressed proteins.

2.2. Differentially expressed proteins in Sama and Pachía in response to B toxicity

Considering that the major aim of this work was to analyze the changes provoked by B toxicity on protein expressions in Pachía and Sama, we will now focus on the proteins that were differentially expressed by B toxicity in these landraces. Thus, 66 and 149 proteins were differentially expressed in response to B toxicity in Sama and Pachía, respectively (Table 1). The main functional categories containing the highest number of differentially expressed proteins under B toxicity in both Sama and Pachía were transcription and translation, photosynthesis, amino acid metabolism, protein degradation, protein stabilization and folding, and reactive oxygen species (ROS) (Figure 3 and Figure 4). Interestingly, most of the proteins belonging to the transcription and translation category were induced in response to B toxicity in both Sama and Pachía, the number of differentially induced proteins being remarkably higher in Pachía (Figure 3 and Figure 4). However, almost all proteins included in the photosynthesis category were repressed in 10 mM B, the number of down-accumulated proteins being also higher in Pachía than in Sama (Figure 4 and Table S2). Regarding protein degradation, and protein stabilization and folding, most of the differentially expressed proteins in 10 mM B were found in Pachía, suggesting that B toxicity would alter the structure and folding of proteins in this landrace. In addition, many of the proteins in the ROS category were induced by B toxicity in both landraces (Figure 4 and Table S2). Although the groups of carbon assimilation and metabolism, lipid metabolism, and respiration included a smaller number of proteins that those mentioned above, nevertheless, a larger number of differentially expressed proteins were found in Pachía under B toxicity (Figure 4 and Table S2). Other interesting categories were cell death, cell division, cell wall, ribosome biogenesis, and RNA binding and processing which, despite having a very small number of proteins regulated by B toxicity, had an interesting distribution in both landraces and B treatments. In fact, in the cell death and cell wall categories, only proteins whose expressions were induced by B toxicity were found in Pachía, however, the cell division, ribosome biogenesis, and RNA binding and processing categories also contained proteins with higher accumulation in 10 mM B but in both landraces (Figure 4 and Table S2).

A total of 18 proteins were commonly expressed (repressed or induced) in both landraces in response to B toxicity, with the amino acid metabolism and photosynthesis categories having the highest number of proteins (Table 2). All proteins of the amino acid metabolism group were up-accumulated under B toxicity conditions, with these inductions being slightly greater in Pachía than in Sama. Interestingly, however, all commonly expressed proteins from the photosynthesis category were repressed by B toxicity, these repressions being remarkably higher in Pachía than in Sama (Table 2).

Table 3 and Table 4 list the most strongly differentially expressed proteins that were up- or down-regulated more than twofold by B toxicity in Pachía and Sama, respectively. In Pachía, 105 proteins had strong differential expression under B toxicity, while only 27 were found in Sama. Photosynthesis was the functional category containing the highest number of proteins whose expressions were strongly down-accumulated in response to B toxicity in both Pachía and Sama, however, interestingly, both minor number of repressed and very strongly repressed (FC <0.33) proteins were observed in Sama (Tables 3,4, and S2). Different subunits of the NDH complex (NDHS, B1, B2, J, and H) were strongly repressed by B toxicity in Pachía but not in Sama (Tables 3, 4, and S2). In addition, only in Pachía were detected proteins related to protein degradation processes whose expressions were mainly induced by B toxicity suggesting that enhanced damage would be provoked by 10 mM B in Pachía proteins (Table 3). Furthermore, B toxicity markedly induced a larger number (15) of proteins in Pachía belonging to the transcription and translation category (Table 3).

Table 5 shows the proteins that were strongly up- or down-accumulated when protein expressions of Sama were compared to those of Pachía in media with 10 mM B. Sama had a remarkable up-accumulation of four proteins involved in photosynthesis (ZmPIFI and OEE2-1), chlorophyll biosynthesis (ChlH1), and secondary metabolism (PAO1) being, in addition, this last protein strongly induced in response to B toxicity (Table 4 and Table 5). However, in Pachía several proteins were detected with a strong accumulation in 10 mM B when compared with Sama (shown in Table 5 as strongly down-accumulated proteins in Sama) highlighting, among them, histone H1 and ribosomal protein S7 which, besides, were strongly induced by B toxicity (Table 3 and Table 5).

Finally, in both Pachía and Sama, proteins exclusively detected in one of these landraces were found, among them, Nfc103a and eIF3a, which were only identified in Pachía in 10 mM B (Table 6).

3. Discussion

Although 2793 proteins were detected in this proteomic analysis, only 303 proteins were differentially accumulated (Tables S1a and S2), which were classified into 26 functional categories. Functional analysis indicated that pathways involved in transcription and translation processes, amino acids metabolism, photosynthesis, carbohydrate metabolism, protein degradation, and protein stabilization and folding were highly enriched categories in both landraces (Figure 2). Remarkably, the expression levels of proteins related to these enriched processes were significantly different between Pachía and Sama.

3.1. Several proteases and translation-related proteins would allow Pachía to survive in media with B excess

Pachía is a B-sensitive maize cultivar described by Mamani-Huarcaya et al. [27]. Interestingly, the highest number of differentially accumulated proteins (DAPs) was found in the comparison group P10/P0.05 (Figure 1) suggesting that the B toxicity damage caused in Pachía could be partially relieved by these proteins. A remarkable number of these DAPs included in the categories of protein degradation (11), and transcription and translation (15) were strongly overexpressed in Pachía (Table S2 and 3). However, only four proteins of the transcription and translation group were markedly induced by 10 mM B in Sama (Table 4). The B-sensitive Citrus grandis had a higher number of proteins involved in protein degradation that was also overexpressed under B toxicity conditions in comparison with B-tolerant Citrus sinensis [36]. These authors concluded that B toxicity caused greater protein damage and proteolysis in C. grandis. Therefore, the high number of protein degradation-related proteins that were overexpressed in Pachía in 10 mM B would suggest that B toxicity would cause greater damage in Pachía proteins than in those of Sama leading to increased proteolysis in B-sensitive Pachía. Proteins related to protein degradation strongly overexpressed in Pachía included, among others, cysteine protease14 and four serine proteases (Table 3). Proteases have been implicated in plant acclimation to abiotic stress, playing a major role in the degradation of damaged and misfolded proteins, thus contributing to cell survival. In fact, cysteine and serine proteases are involved in degradation of misfolded proteins and protection against abiotic stresses [37,38,39,40]. Hence, these five proteases could have a main role in the degradation of damaged and misfolded proteins in Pachía under excess B, contributing to maintaining the correct conformation of Pachía proteins and, therefore, to the survival of this landrace under this stressful condition. In addition, a noteworthy number of proteins involved in transcription and translation processes were overexpressed at 10 mM B in Pachía, namely, 30 in contrast to only nine of Sama (Table S2). Proteomic analysis performed with dehydration, salt, and temperature stresses in cereals also displayed alterations in the levels of translation-related proteins, such as initiation factors and the ribosome constituent proteins ([41] and references therein). Furthermore, it has been suggested that a B excess provokes inhibition of RNA-dependent processes, such as transcription and translation, owing to the ability of B to form complexes with ribose molecules [42]. In this regard, Tanaka et al. [43] have suggested that B or boric acid acts on the translation machinery likely forming complexes with cis-diol groups of rRNA and tRNA. In addition, it has currently been proposed that high-B stress enhances ribosome frequency on stop codons leading to a global ribosome stalling [44]. Consequently, the high contents of leaf-soluble B in Pachía seedlings subjected to 10 mM B reported by Mamani-Huarcaya et al. [27] would generate an increased formation of B complexes with cis-diol groups of RNA that would damage ribosomes leading to a drop in protein synthesis likely through a global ribosome stalling. The strong overexpression of several ribosomal proteins would maintain the Pachía ribosome stability in B toxicity (Tables S2 and 3). These results are consistent with those reported for rice, where several ribosomal protein large subunit genes were upregulated under temperature stress, suggesting that their encoded proteins might be involved in stress amelioration, likely maintaining the proper functioning of ribosomes [41]. Interestingly, the eukaryotic translation initiation factor 3 subunit A (eIF3a) was exclusively detected in B toxicity in Pachía (Table 6). These factors are one of the most significant components involved in plant protein synthesis and, specifically, rice eIF3A has been proposed to play an important role in different stresses [45]. Therefore, eIF3a would also help to alleviate the drop in protein synthesis in Pachía. Thereby, Pachía would partly ameliorate injuries caused by B toxicity on protein synthesis and ribosome by overexpressing a high number of transcription- and translation-related proteins, abolishing a non-viable reduction of transcription and translation processes.

3.2. Proteins that would confer Sama more B toxicity tolerance

Polyamine oxidase 1 (PAO1) is an interesting protein that was clearly up-accumulated in Sama when compared to Pachía at 10 mM B and was also strongly induced in Sama by B toxicity (Table 4 and Table 5). This enzyme catalyzes the back conversion of spermine (Spm) to spermidine (Spd), and Spd to putrescine (Put) [46]. Maize polyamines play a crucial role in abiotic stress response [33]. In fact, it has been reported that Put protects the plant photosynthetic apparatus against several abiotic stresses [47]. Moreover, the conjugation of Put to PSII proteins may lead to the structural and functional stability of PSII [46,48]. Therefore, the over-accumulation of PAO1 in Sama plants subjected to B toxicity would generate an increase in Put levels that would protect their photosynthetic apparatus resulting in the higher P_N observed in Sama under this stress, as described by Mamani-Huarcaya et al. [27]. This finding is consistent with results reported for Karoon, a drought-tolerant maize cultivar. Pakdel et al. [46] proposed that higher expression of PAO genes and enzymatic polyamine oxidation activity would protect the photosynthetic apparatus of Karoon under water stress.

3.2.1. Lower repression of photosynthesis-related proteins would enhance the B-toxicity tolerance of Sama

Photosynthesis is one of the essential physiological processes affected by B toxicity [2,22]. Photosynthetic efficiency could be achieved in Sama under B toxicity conditions increasing the synthesis of photosynthetic pigments, since chlorophyll content is a major limiting component of the photosynthetic efficiency [49]. Interestingly, Sama had a strong over-accumulation of magnesium-chelatase subunit H1 chloroplastic (ChlH1) at 10 mM B in comparison with those from Pachía (Table 5). ChlH binds to porphyrin and catalyzes the insertion of Mg²⁺ into protoporphyrin IX [50]. Accordingly, the over-accumulation of ChlH1 in Sama would explain its higher contents of chlorophyll a in B toxicity and the higher P_N described by Mamani-Huarcaya et al. [27].

In this study, 25 proteins related to photosynthetic light reactions were differentially accumulated, most of them involved in electron transport, light harvesting, and oxygen evolving processes (Table S2). Pachía and Sama presented several photosynthesis-related proteins that were repressed by B toxicity when their expressions were compared with those of Pachía and Sama, respectively, in media with 0.05 mM B (Table S2). However, the number of these DAPs was lower in Sama than in Pachía (11 versus 16, respectively; Table S2) and, besides, those proteins commonly down-accumulated in both landraces had a weaker decrease in Sama (Table 2). In addition, only two photosynthetic proteins were strongly down-expressed 3-fold or more (corresponding to FC≤ 0.33) by B toxicity in Sama in contrast to ten proteins found in Pachía (Table 3 and Table 4). This decreased accumulation of photosynthesis related-proteins may cause lower photosynthetic performance in B-toxicity-treated Pachía plants than in Sama plants, as described by Mamani-Huarcaya et al. [27]. Therefore, Sama would retain sufficient levels of photosynthesis-related proteins in 10 mM B, which would allow it to maintain photosynthetic parameters at similar levels to those of the control conditions, as reported by Mamani-Huarcaya et al. [27]. Furthermore, three photosynthesis-related proteins were up-accumulated in Sama when their expressions were compared with those of Pachía in 10 mM B, namely, oxygen-evolving enhancer protein 2-1 chloroplastic (OEE2-1), post-illumination chlorophyll fluorescence increase (ZmPIFI), and NAD(P)H-quinone oxidoreductase subunit S chloroplastic (NDHS) (Tables 5 and S2). OEE2-1 is likely an extrinsic protein of the oxygen-evolving complex (OEC) (UniProt; https://www.uniprot.org/). The OEC is stabilized and protected by extrinsic polypeptides [51]. The strong OEE2-1 over-accumulation in 10 mM B in Sama could facilitate the stability and protection of the OEC leading to the higher photosynthetic electron transporter rate (ETR) observed in this landrace [27]. Regarding ZmPIFI, it is homologous to the PIFI protein of Arabidopsis thaliana (AtPIFI), an essential component of the NAD(P)H dehydrogenase (NDH) complex involved in chlororespiratory electron transport around PSI [52]. The Atpifi mutant had a lower nonphotochemical quenching (NPQ) than wild type under high light irradiances, suggesting that AtPIFI would protect plants from photooxidative stress triggered by excessive light [52]. Consequently, both ZmPIFI over-accumulation and the higher NPQ values that Sama showed in 10 mM B, unlike those from Pachía (Table 5; [27]), suggest that ZmPIFI would also be a component of the maize NDH complex playing a role in oxidative photoprotection of this landrace under B-toxicity conditions. Furthermore, unlike Sama, several subunits of the NDH complex were markedly repressed in Pachía by B toxicity (Tables S2, 3, and 4). The NDH complex mediates cyclic electron transport around PSI playing a crucial role in C₄ photosynthesis [53,54]. NDH-mediated cycle electron transport (NDH-CET) performs two functions: 1) maintaining photosynthetic redox balance in the electron transfer avoiding stromal overreduction and functioning as a safety valve for excess electrons under stress, and 2) supplying ATP for efficient carbon assimilation, especially under stressful conditions [53,54,55,56]. The finding that none of the above components of the NDH complex was significantly repressed by B-toxicity in Sama suggests that its NDH-CET would prevent stromal overreduction and would protect against photooxidation. This fact would explain the high values of net photosynthetic CO₂ assimilation (P_N), maximum photochemical efficiency (Fv’/Fm’), and quantum yield efficiency of PSII electron transport (Φ_PSII) reported in Sama at 10 mM B, which were similar to those of control conditions [27]. Consistent with our data, Zhu et al. [56] have suggested that an increased abundance of NDH subunits in salt-stressed wheat would enhance NDH-CET alleviating the accumulation of excess electrons and maintaining energy homeostasis. Moreover, the subunit S of the NDH complex was over-accumulated in Sama under B toxicity when compared to those from Pachía, leading to a likely higher amount of NDH-complex that would provide extra ATP to achieve better P_N and growth at this landrace in media with 10 mM B as, in fact, was observed by Mamani-Huarcaya et al. [27]. In addition, a higher supply of ATP could be obtained in Sama in comparison to Pachía under B toxicity from a weaker decrease of the α- and β-chloroplastic subunits of ATP synthase in Sama (Table 2). Although B excess causes photosynthetic damage [2,22], plants have evolved mechanisms to repair these injuries that require a high amount of ATP from chloroplastic ATP synthase [57,58]. In Sama, B toxicity barely affected photosynthetic parameters [27]. This finding points out that this landrace would own mechanisms to repair its photosynthetic machinery. Likely, one of these mechanisms would be to provide greater ATP availability, which would be achieved by maintaining sufficient levels of NDH and ATP synthase complexes that would synthesize the amounts of ATP needed to repair its photosynthetic machinery and, therefore, to maintain its photosynthetic values at levels similar to those of control conditions.

4. Materials and Methods

4.1. Plant materials and growth conditions

Sama and Pachía, two Peruvian maize landraces from the Sama valley and the Pachía district (to the east of Tacna), were used in this study. Seeds were surface-sterilized as described by Mamani-Huarcaya et al. [27]. Afterwards, the seeds were placed in seedbeds filled with a perlite/vermiculite mixture (1/1, v/v) and watered with deionized H₂O. After seven days, seedlings were transplanted to 30-L plastic containers with a nutrient solution (NS) that was identical to the one used by Mamani-Huarcaya et al. [27]. After two days of acclimation to hydroponic medium, the seedlings were divided into groups and transferred to fresh NS supplemented with 10 mM H₃BO₃ (B toxicity conditions) or 0.05 mM H₃BO₃ (control conditions). This medium was aerated by air pumps and renewed twice a week. The seedlings were germinated and grown hydroponically in a growth chamber under a 12 h light/12 h dark regime (215 µmol m^–2 s^–1 of photosynthetically active radiation at plant height), at 22ºC and 50% relative humidity. The plants were randomly harvested 10 days after the onset of the B treatments and their leaves were quickly separated with a scalpel, frozen in liquid nitrogen and stored at –80°C until further analysis.

4.2. Protein extraction and digestion

Maize leaves (200-250 mg fresh weight) from four separate seedlings per condition (B treatment and maize landrace) were ground to a fine powder in a mortar precooled with liquid nitrogen. Proteins were extracted with trichloroacetic acid (TCA)/acetone-phenol [59], solubilized in a solution containing 7 M urea, 2 M thiourea and 2% (w/v) CHAPS (3 [(3-cholamidopropyl) dimethylammonium]-1-propanesulfonate), and quantified by the Bradford method using bovine serum albumin (BSA) as a standard [60].

The cleaning of maize protein extract, protein digestion, and mass spectrometry determinations were carried out at the Proteomics Facility for Research Support Central Service (SCAI) of the University of Córdoba (Spain) as follows.

Biological quadruplicate samples were separated and cleaned as described. Leaf protein extracts (50 µg of BSA protein equivalents per sample) were electrophoretically pre-concentrated in a centimeter band of 10% (w/v) SDS-PAGE gel. Protein bands were excised from the gels and, afterwards, the gel pieces were distained in 200 mM ammonium bicarbonate/50% acetonitrile for 15 min, followed by 5 min in 100% acetonitrile. Proteins were reduced by addition of 20 mM dithiothreitol in 25 mM ammonium bicarbonate and incubated for 20 min at 55 °C. The mixture was cooled to room temperature and then free thiols were alkylated by adding 40 mM iodoacetamide in 25 mM ammonium bicarbonate for 20 min in the dark. Finally, the gel pieces were washed twice in 25 mM ammonium bicarbonate.

Proteolytic digestion was performed by addition of trypsin to a final concentration of 12.5 ng/µL in 25 mM ammonium bicarbonate at 37 ºC overnight. Protein digestion was stopped by adding trifluoroacetic acid at a final concentration of 1% (v/v). Finally, the digested samples were vacuum-dried and dissolved in a mixture of 2% (v/v) acetonitrile and 0.05% (v/v) trifluoroacetic acid.

4.3. Shotgun-DDA-LC-MS/MS analysis

Peptide separations were performed on a nano-LC using Dionex Ultimate 3000 nano UPLC (Thermo Scientific, San Jose, CA, USA), equipped with a C18 75 μm × 50 cm Acclaim Pepmap column (Thermo Scientific, San Jose, CA, USA), at 40 °C at a flow rate of 300 nL/min. Peptide mixtures were previously concentrated and cleaned up on a 300 µm x 5 mm Acclaim Pepmap precolumn (Thermo Scientific, San Jose, CA, USA) using 2% acetonitrile/0.05% trifluoroacetic acid at 5 µL/min for 5 min. Peptides were eluted with a gradient of 60 min ranging from 96% solvent A (0.1% formic acid) to 90% solvent B (80% acetonitrile and 0.1% formic acid), followed by an 8 min wash at 90% solvent B and a 12 min re-equilibration at 4% solvent B. Eluted peptides were converted to gas-phase ions by nanoelectrospray ionization and analyzed on a Thermo Orbitrap Fusion mass spectrometer (Thermo Scientific, San Jose, CA, USA) operated in the positive mode. Survey scans of peptide precursors were acquired over the m/z range 400−1500 at 120K resolution (at 200 m/z) with a 4 × 10⁵ ion count target. Tandem MS was performed by isolation at 1.2 Da with the quadrupole. Monoisotopic precursor ions were fragmented by CID (Chemically Induced Dimerization) in the ion trap, which was set up as follows: automatic gain control, 2 × 10³; maximum injection time, 50 ms; and normalized collision energy of 35%. Only those precursors with charge state 2–5 were sampled for MS2. A dynamic exclusion time of 15 s and a tolerance of 10 ppm around the selected precursor and its isotopes were used to avoid redundant fragmentations. The instrument was run in top 30 mode with 3-s cycles, meaning the instrument would continuously perform MS2 events until a maximum of top 30 non-excluded precursors or 3 s, whichever was shorter.

4.4. Protein quantification

Charge state deconvolution and deisotoping were not performed. MS2 spectra were searched using MaxQuant software v. 1.5.7.4 [61]. MS2 spectra were searched with Andromeda engines against a database of Uniprot Zea mays_Jun19. Peptides generated from tryptic digestion were searched employing the following parameters: up to one missed cleavage, carbamidomethylation of cysteines as fixed modifications, and oxidation of methionine as variable modifications. The precursor mass tolerance was 10 ppm and product ions were searched at 0.6 Da tolerances. A target-decoy search strategy was applied, which integrates multiple peptide parameters such as length, charge, number of modifications, and identification score into a single quality that acts as statistical evidence on the quality of each single peptide spectrum match. The identified peptides were grouped into proteins according to the law of parsimony and filtered to 1% false discovery rate (FDR). Peptide quantification was carried out using MaxQuant software, in a MaxLFQ label-free quantification method [62]. In the MaxLFQ label-free quantification method, a retention time alignment and identification transfer protocol (“match-between runs” feature inMaxQuant) was applied. Proteins identified from only one peptide were not taken into account in this analysis. Peak intensities across the whole set of quantitative data for all peptides in the samples were imported from the LFQ intensities of proteins from the MaxQuant analysis and normalized according to Cox et al. [62]. LFQ normalized intensity values were transformed to a logarithmic scale with base two. Protein quantification and calculation of statistical significance were carried out using Student-t test and error correction (P-value ≤ 0.05). The criteria used to consider a protein as differentially expressed were as follows: (a) the protein was consistently present in at least three biological replicates per condition; (b) it had statistically significant differences (Student-t test, P ≤ 0.05) between genotypes or B treatments; and (c) a fold change ≥ 1.5 or ≤ 0.66667. The differentially accumulated proteins were manually categorized by function using different databases (Uniprot, https://www.uniprot.org/; Maize Genetics and Genomics, https://www.maizegdb.org/; ExplorEnz, https://www.enzyme-database.org/; BRENDA, https://www.brenda-enzymes.org/; KEGG: Kyoto Encyclopedia of Genes and Genomes, https://www.genome.jp/kegg/; and PANTHER: Protein ANalysis THrough Evolutionary Relationships, http://pantherdb.org/).

5. Conclusions

Overexpression of several proteases and transcription- and translation-related proteins would allow Pachía to degrade and replace partially the proteins damaged by B toxicity achieving survival under this stress condition.

In Sama, PAO1 over-accumulation and weaker knockdown of several subunits of NDH and ATP synthase complexes under B excess would confer a greater B toxicity tolerance to this landrace by: 1) acting as an electron safety valve that would avoid stromal overreduction, and thus decrease photosynthetic damage and, 2) providing an additional supply of ATP that would contribute to repair the photosynthetic system of Sama.