American elderberry (Sambucus nigra
) is a specialty crop grown in many parts of North America. Overall elderberry cultivation is expanding in the U.S. with the potential for further growth [1
]. The cultivation of elder plants requires the use of fertilizers; most commonly nitrogen (N)–based. Therefore, improving and understanding the role of N in elder production is needed [2
]. Fertilizers typically contain N in the form of nitrate and ammonium compounds, which rapidly dissolve in water, resulting in loss to the soil, and low nitrogen use efficiency (NUE) [3
]. A better understanding of the molecular interactions and mechanisms that underlie elder response to different N-treatments may aid in more efficient utilization of N-fertilizer as well as furthering the understanding of elderberry growth.
Several studies have examined gene expression changes in plants in response to N treatment. Bi and co-workers found a set of N-responsive genes in three genotypes of maize under N-limiting conditions by high throughput RNA sequencing [4
]. Transcriptome analyses have identified differentially expressed genes under N-deprivation conditions and yielded a complex pathway in the model plant Arabidopsis thaliana
]. However, the exact function of these genes remains unknown. Proteomic approaches can aid in the understanding of changes and functions of the plant proteome to N [6
]. Proteomics can complement many functional genomics approaches [10
] and provide useful information about post translational modifications (PTM) with annotation of the corresponding gene [11
]. While several studies have found a proportional relationship between transcript levels and the abundance of proteins [12
], it is still difficult to predict the relationship between cellular protein concentrations and the abundance of corresponding mRNAs [15
]. Part of the difficulty is likely due to PTMs and the effects of micro RNAs on expression of proteins [16
]. A comparative proteomics approach could provide a better assessment of differentially abundant proteins and metabolic processes.
Here, we present a comparative proteomics investigation of the impact of N-fertilizer treatment on American elderberry (Sambucus nigra
). Three genotypes are used in the investigation; including an heirloom genotype (Adams II) and the more recently developed genotypes (Bob Gordon [17
] and Wyldewood [18
]). The selection of suitable N-fertilizer usage is important to ensure agriculture productivity and sustainability [2
]. Elder leaves were chosen for the study due to their sensitivity to varying environmental conditions [19
]. Two-dimensional electrophoresis (2-DE), followed by in-gel digestion, and mass spectrometry (MS) allowed us to identify 165 proteins that showed altered abundance upon exposure to different levels of N-fertilizer treatment. Protein functional studies helped to further our understanding of protein profile changes in response to treatment. These results provide novel insight into cellular changes which could lead to new N-treatment management strategies to improve NUE and plant production in the future.
2. Materials and Methods
Acrylamide, phenol (Tris-equilibrated), CHAPS, 3-(4-heptyl) phenyl-3-hydroxypropyl) dimethylammoniopropanesulfonate (C7BzO), dithiothreitol (DTT), iodoacetamide (IAA), EDTA and 2-hydroxyethyl disulfide (2-HED) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ammonium persulfate, bis-acrylamide, urea (electrophoresis grade), thiourea (electrophoresis grade), ammonium acetate, glycerol, Coomassie Brilliant Blue G-250, agarose (low EEO) and 2-mercaptoethanol were obtained from Thermo Fisher Scientific (Houston, TX, USA). N,N,N′,N′-tetramethylethylenediamine (TEMED) was obtained from BioRad Laboratories, Inc. (Hercules, CA, USA). EZQ protein quantification kits were obtained from Molecular Probes (Invitrogen, Carlsbad, CA, USA). IPG strips and IPG buffer 3–10 were obtained from GE Healthcare (Piscataway, NJ, USA). Modified porcine trypsin was acquired from Promega (Madison, WI, USA).
Study materials (leaves) were harvested from an active research trial in southwest Missouri that incorporated three elderberry genotypes and four N-fertilization treatments (applied 9 May 2014 and the two previous springs), as previously described [20
]. Fresh, fully mature elder leaves from the middle portions of branches were randomly collected from 36 of the 48 plots in August 2014, promptly refrigerated, and transported under refrigeration to the laboratory for analysis. About 1000 cm3
of leaves were collected from each plot. The leaves were collected after fruit had been harvested. The middle portions of the compound elder leaves were then washed, ground under liquid nitrogen, and 100 mg of each replicate was aliquoted into 2 mL microcentrifuge tubes and stored at −80 °C prior to protein extraction.
The proteins were extracted according to a method described by Hurkman and Tanaka with some modifications [21
]. Briefly, proteins were isolated by phenol and precipitated overnight by five volumes of pre-chilled 0.1 M ammonium acetate in 100% methanol. Protein concentration was determined by EZQ quantitation kits (Life Technologies, Grand Island, NY, USA) using the manufacturer’s instructions. Proteins were separated by 2-DE as previously described [22
]. All 2-DE stained gels were scanned using a UMAX PowerLook 2100 XL scanner (UMAX Systems GmbH, Willich, Germany). At least triplicate gels were performed for each treatment, and a total of 36 gel images were analyzed by ImageMaster 2D Platinum software version 7.0 (GE Healthcare). To compensate for gel-to-gel variation, the volume of each spot was normalized as a relative volume as previously described [23
Three replicates were performed for each genotype at each N-fertilizer treatment. One-way analysis of variance (ANOVA), part of ImageMaster 2D Platinum (Version 7.0; GE Healthcare), was initially performed for each group to identify statistical significance by using the relative volume (vol %). A significance level of 95% was chosen. To evaluate the quality of spots, several parameters were set manually. The 2-fold difference calculated as the ratio of the average spot volume of each treatment to that of the control was used; spots were observed in at least 80% of spot maps and repeated on triplicates. Once the spots were considered to be statistically significant, the average values of these spots were analyzed by Tukey’s honest significant difference test, and then filtered by two-way ANOVA by genotype, two-way ANOVA by N-treatment, and/or two-way ANOVA interaction (p < 0.05) using the SAS software package (Version 9.4, SAS Institute, Cary, NC, USA). Only spots of interest were selected for further MALDI-TOF/TOF or LC-MS/MS analysis. Additionally, principal component analysis (PCA) was performed using the SAS software.
Six pooled gels were generated by pooling equal amounts (200 µg) of three biological replicates onto one gel following the same steps as described above. Each protein spot of interest was excised from the pooled gel, trypsin digested and identified by MALDI-TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems, Foster City, CA, USA) in the positive ion reflector mode as previously described [22
]. For peptide and protein identification of MALDI-TOF/TOF data, the resulting peptide peak lists were submitted to the MASCOT database search engine against the National Center for Biotechnology Information non-redundant (NCBInr) database. A homology search was performed due to the limited sequences of Sambucus nigra
genome (164 sequences as of May 2018). The following parameters were selected: Viridiplantae
(Green Plant) as taxonomy, trypsin as digesting enzyme, one missed cleavage, fixed modification of carbamidomethyl (Cys), variable modification of oxidation (Met), precursor ion mass error tolerance of 100 ppm, MS/MS fragment ion mass error tolerance of 0.1 Da, peptide charge of 1+, monoisotopic and MALDI-TOF/TOF as instrument. Confident protein identifications were defined as: (1) the highest protein score on the database searching report, (2) a minimum of two matched peptides, (3) less than 15% deviation between theoretical and experimental Mr and pI values (gel-based). An in-house BlastP search at NCBI was performed to verify all matches and update annotations and identification of all hypothetical or unknown proteins (sequence similarity > 80%).
In cases where the MASCOT search did not reveal confident identification, we performed LC-MS/MS experiments as previously described [24
]. For peptide and protein identification by LC-MS/MS, raw files were analyzed and quantified using the SEQUEST algorithm implemented with a Scorcerer2 integrated Data Appliance (SageN Research, Milpitas, CA, USA) against a local copy of the Viridiplantae
database downloaded in FASTA format via file transfer protocol from NCBInr (released in November, 2013; 2,355,794 proteins). Homology searches included full trypsin specificity (KR/P), two missed cleavage sites, peptide mass tolerance of 50 ppm, fragment ion mass tolerance of 1 Da, carbamidomethylation of Cys, as a static modification and Met oxidation as variable modification. Search results were verified by Scaffold viewer V4.0.5 (Proteome Software Inc., Portland, OR, USA). For each identification, a minimum of two matched peptides; peptide threshold (p
< 0.05) and protein threshold (p
< 0.001); cross-correlation factor (Xcorr) 2.0, 3.0 and 3.5 for the charge states and +2, +3 and +4 respectively, and minimum Delta CN (Delta correlation) of 0.1. Results were then filtered using the Protein- and PeptideProphet [25
] implemented in the Scaffold software to achieve a peptide and protein global false discovery rate of <5 and 0.1%, respectively. An extra BlastP search was also performed to update the best matches and align all hypothetical or unknown proteins (sequence similarity > 80%).
Hierarchical clustering was constructed using the software PermutMatrix [27
]. The average abundance of identified proteins, which were classified in the same orthologous group and displayed similar trends of abundance profiles across all genotypes, were taken and the fold change calculated between each group and control, followed by log2 transformation for heat map representation. The dissimilarities were calculated based on Euclidean distances and hierarchical clustering was carried out according to Ward’s method. The trees were generated using the multiple-fragment heuristic algorithm (MF) as a seriation rule.
The function of the identified proteins was sought by transferring original sequences to the Arabidopsis
genome and orthologous genes using the Mercator web-based pipeline (http://mapman.gabipd.org/app/mercator
), which divides protein into 35 hierarchical, nonredundant functional classes using MapMan bin codes [28
]. The cellular locations of identified proteins were determined by searching the best matched Arabidopsis
orthologous proteins in the SUBA database (Version 3) [29
It is essential to develop a more comprehensive understanding of how different plants respond to different levels of N-fertilizer in the field. In this study, we exploited a comparative proteomics approach to identify specificity and dynamic changes in leaf proteomes of American elderberry affected by N-treatment (Table 1
). Protein functional classification with their Arabidopsis
orthologues was adopted to enable us to evaluate the expression level of the same proteins in different genotypes. Understanding how American elderberry responds to different levels of fertilizer not only permits us to better manage its utilization, but also to understand the growth and development of elder, possibly improving the yields of different genotypes with optimized choice of N-fertilizer usage.
2-DE coupled with MALDI-TOF/TOF and LC-MS/MS and advanced statistical methods were used in this study as an efficient proteomics approach, allowing us to observe spatial difference in protein expression. 165 protein spots (approximately 5% of all detectable protein spots) showed significant changes in response to N-treatment compared to control. PCA analysis revealed that the protein expression profile of Bob Gordon differs from that of the other two genotypes, indicating that Bob Gordon is a distinct phenotype and sensitive to N-treatment. This is consistent with the observation that Bob Gordon has the highest yield among the three genotypes under same growing conditions [31
]. Our study suggests that Bob Gordon may possess widespread usefulness as a commercial elder cultivar and is worth further study.
According to the results of the two-way ANOVA analysis (Table S1
), some of the differentially abundant proteins were assigned to N-treatment as the main effect. Phenotypically, three genotypes behave differently when cultivated side-by-side, suggesting that the different inherent genetic backgrounds play a role [31
]. However, some differentially abundant proteins still had the same pattern of regulation across different genotypes with the same N-treatment indicating common pathways among genotypes.
Proteins tied to photosynthesis were differentially abundant in the study. These proteins have been previously highlighted in different species upon varied N-treatment [9
]. Most of these photosynthetic proteins showed a lower expression level with increasing N-fertilizer concentrations, regardless of genotype (Figure 4
). Our findings are consistent with earlier work which showed that the Rubisco activation state decreased at high N-concentrations and that Rubisco may serve as a storage protein in apple leaves [38
]. A previous study on maize found lower intercellular CO2
concentrations in response to high N supply also consistent with our results [39
]. A recent genetic study showed that the OsPIPs gene may be responsible for mediating insufficient CO2
]. We speculate one of the outcomes of decreased level of RCA in elderberry leaves with a high N-treatment could lower the CO2
concentration in the chloroplasts, that may be an indication of over-fertilization.
In addition to photosynthetic proteins, we could observe a considerable distribution of proteins involved in protein metabolism (synthesis, PTM, degradation, folding, assembly and cofactor ligation). Several components of the protein synthesis machinery (translation elongation and initiation factors and ribosomal proteins) except EF, were only expressed at a treatment of 112 kg N/ha. Elongation factors are known for regulating the translocation step in polypeptide chain elongation. The VAJ/GFA1/CLO gene encodes a translational elongation factor-2 family protein, which controls directional floral organ growth in Arabidopsis thaliana
possibly through pre-mRNA splicing [41
]. Similarly, eukaryotic initiation factors (EIF) are phosphorylated to modulate the rate of mRNA binding [42
]. In tobacco, the expression of elongation factor EF-1 alpha increased in meristems, rapidly growing tissues, and developing gametophytes [43
]. In our study, EF2 and EIF4A8 were found to be enriched in Bob Gordon and Wyldewood, suggesting enhanced protein synthesis to support the development and growth of plant. In Arabidopsis thaliana
, mutations in genes encoding ribosomal proteins affect leaf developmental processes [44
]. We suspect deficiency of the EF protein may play a role in perturbing the development of leaf under high N-treatment. Chaperone proteins (CPN21 and 60) are essential for facilitating protein folding, unfolding and preventing protein aggregation [45
]. Others suggest it may regulate increased oxidative stress [46
]. Consequently, the increased amounts of CPN21 may help to lower oxidative stress at a treatment of 112 kg N/ha. A comparative genetics study of mutants in Arabidopsis thaliana
has implicated an inverse relationship between 26S proteasome (26SP) and 20S proteasome (20SP) abundance, with defectiveness 26SP level invariably causing the elevated abundances of free 20SP lead to increased tolerance to oxidative stress [47
]. Our results agree well with the established correlation between 26SP and 20SP. We constantly observed enhanced expression of 20SP concurrently with the down-regulation of 26SP at different N levels. The enriched abundance of serine/threonine-protein phosphatase PP2A-4 catalytic subunit (PP2A-4) was presented for Adams II and Wyldewood at a treatment of 169 kg N/ha. Many functional roles of PP2A in stress signal pathways were discussed under biotic and abiotic conditions [48
]. Its role in N stress is yet to be determined.
Proteins involved in amino acid metabolism, such as ketol-acid reductoisomerase (KARI), s-adenosylmethionine synthase 1, aspartate aminotransferase (AST), and GDH, were differently abundant in the three N groups. Amino acid biosynthesis and degradation tightly correlate with N-availability [49
]. These proteins are involved in the biosynthesis of the branched-chain amino acids [50
], glycine, serine, aspartate [51
] and cysteine, respectively. KARI was also identified in maize ear with 2-DE gels under different N-conditions, which correlates with ear growth and grain yield [52
]. In the case of AST, it is involved the signal transduction pathway to regulate asparagine to glutamine ratio in cobs and that ratio shows the plant N status [53
]. Additionally, the ascending amount of AST likely coordinates with increasing maize productivity [54
]. Taken together, the increased amount of these two proteins in Bob Gordon at a treatment of 169 kg N/ha may help explain the higher yield that is often observed under field conditions [31
]. GDH and AMT, which are involved in amino acid degradation were found down-regulated for all three genotypes at a treatment of 112 kg N/ha.
Several proteins involved in redox homeostasis were found to exhibit altered expressions, including CAT, GDP-mannose 3,5-epimerase isoform X2 (GME), MDAR, 2-cys peroxiredoxin BAS1 (BAS1) and APX. Redox changes show well-organized interactions to prevent tissue damage and death under environmental disturbance [55
]. Ascorbate-glutathione cycle plays a key role in H2
metabolism in plant cells [56
is reduced to water by APX using ascorbate as the electron donor and the reducing agent ascorbate is regenerated by MDAR and dehydroascorbate reductase using reduced glutathione [57
]. The increased levels of APX and MDAR for Adams II were found at a treatment of 169 kg N/ha. Similarly, GME is involved in the regulation of ascorbate biosynthesis [58
] and was found enriched for Bob Gordon at a treatment of 112 kg N/ha. APX was increased in the same genotype. Both BAS1 and CAT participate in the reduction and degradation of H2
into water [59
]. There was an enhanced abundance of BAS1 and CAT in Wyldewood at a treatment of 112 kg N/ha. In summary, these results suggest a possible enhanced defense in the plant against oxidative stress at higher N-conditions.
One notable feature of this study was the identification of multiple protein isoforms associated with N-treatment [61
]. About 62% of 101 non-redundant proteins were recognized as isoforms. These isoforms show different Mr and/or pI changes on the 2-DE gels in response to N-treatment. There are several reasons for this phenomena, including genetic variation, alternative splicing of RNA transcripts, and PTMs (ubiquitination, acetylation, glycosylation, and/or phosphorylation) [62
]. Many of the protein isoforms reported in the present study would be omitted using the label free mass spectrometry method (i.e. geLC-MS), which provides overall changes in protein abundance [63
]. Different protein isoforms have been found to play an important role in mediating N. In maize, three GS2 isoforms were identified in the root and the accumulation trend of phosphorylated isoform is consistent with plastidial GS activity. Moreover, nitrate and ammonium have been found to induce the accumulation of different isoforms [64
]. Similarly, the physiological role of a new isoform of GS has been reported in the leaf of wheat, suggesting a complex and flexible regulation for GS isoforms underlying the wheat that is associated with N utilization and plant growth [65
]. In the present study, two isoforms of GS were consistently found to have opposite expression levels for Adams II and Bob Gordon among the three N-treatments, while the same down-regulated changes were observed for Wyldewood. The modification of multiple protein isoforms may give us novel perspectives to understand plant response to N-treatment.
As described above, the comparison among different levels of N in all genotypes reveled 23 unique proteins representing 58 isoforms. Among these, 18 unique proteins illustrating 23 isoforms showed a significant genotype × N level interaction (p
< 0.05). These conserved proteins were then classified into protein orthologous groups and summarized in the cellular critical pathways. As protein modification may play an important role in this study, the detection of PTMs by three-dimensional gel electrophoresis may warrant further investigation [66
]. However, we note the stringent conditions used in our study for the identification of differential abundant proteins by 2DE should help to diminish issues with co-migration of proteins.
It is well known that under N starvation, accumulation of lipid degradation and toxic compounds in plants can induce ROS [67
]. To protect the cell from oxidative damage, ROS-scavenging pathways are activated [68
]. Our proteomics results revealed that a number of enzymes involved in protein metabolism, redox homeostasis and transportation (indicated as red squares in Figure 4
) were up-regulated for the three genotypes at a treatment of 169 kg N/ha, suggesting that oxidative stress could be more efficiently reduced at high N-concentrations. As part of the plant defense system, detoxification of non-enzymatic components (i.e. GSH) is also activated to destroy ROS [69
]. LGL, which is found up-regulated with N-treatment in our study, is involved in the GSH-based detoxification of methylglyoxal [70
Changes in protein expression profiles in photosynthesis, the OPP pathway and glycolysis suggest that plants may require a certain amount of N to enable these proteins to produce energy for plant growth in its most efficient way. Similar results were found in maize leaf [37
]. GS is a key enzyme in ammonium assimilation [71
] and an overall decrease in abundance of GS among the three genotypes in response to increasing levels of N lower its utilization. SNAlm plays a role in carbohydrate recognition [30
]. The abundance of SNAlm decreased at the lower treatment level and increased at the higher levels. Other proteins associated with minor CHO metabolism and development were down-regulated at highest fertilizer levels.