Protein profile changes during priming explants to embryogenic response in Coffea canephora: identification of the RPN12 proteasome subunit involved in the protein degradation

Plant material and somatic embryos quantification

Pretreatment of individual in vitro 9-months old plantlets with six pairs of leaves was followed by induction cut-leaf explants to promote the somatic embryogenesis at the cut-leaf edges of C. canephora, as previously described with the following modifications (Quiroz-Figueroa et al., 2006). In vitro plants were maintained under a long-day photoperiod in Murashige and Skoog (MS) media until they were 9-months old, then transferred to supplemented MS media with 0.54 µM 1-naphthaleneacetic acid (NAA) and 2.32 µM kinetin (KIN) for 14 days, which are referred to as +NAA–KIN plantlets, or retained in MS without any PGR, which are referred to as −NAA–KIN plantlets. Then 0.8-cm circular explants from the third to the fourth mature leaf of five plants from each treatment, either +NAA–KIN or −NAA–KIN, were cultivated in liquid Yasuda medium supplemented with 5 µM 6-benzyladenine (BA) in darkness for up to 100 days. Somatic embryos derived from +NAA–KIN and −NAA–KIN explants were examined and quantified under a stereomicroscope (SMZ745T; Nikon, Tokyo, Japan) with an adapted camera (EOS Rebel T3i; Canon, Melville, NY, USA) at magnification of 10× for up to 100 days after induction of cut-leaf explants in the Yasuda-based media (Yasuda, Fujii & Yamaguchi, 1985). The Student paired-samples t-test in GraphPad prism 9 version 9.0.1 static analysis software was used to determine significant differences between treatments. Differences were considered statistically significant with values of P ≤ 0.0001.

Protein sample preparation for two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Nine-month-old plantlets were transferred to MS medium either with or without NAA-KIN, kept for 14 days, and then the leaf explants were dissected and frozen for protein analysis. The frozen leaf powder was homogenized in an extraction buffer that contained 0.7 M sucrose, 100 mM KCl, 500 mM Tris-Cl (pH 7.5), 50 mM EDTA, 50 mM dithiothreitol (DTT), 1 mM phenyl methyl sulfonyl fluoride, and a cocktail of protease inhibitors under icy conditions (Hurkman & Tanaka, 1986). All steps were performed on ice and with ice-cold solutions. After centrifuging the homogenate at 15,000 g for 10 min, the supernatant was made 10% trichloroacetic acid/50% acetone and 5 mM DTT and incubated on ice for 5 min (Niu et al., 2018). The protein precipitate obtained by centrifugation at 15,000 g for 3 min was further washed twice with 80% acetone supplemented with 5 mM DTT. When the residual acetone in samples was removed by air drying, the protein precipitate was dissolved in rehydration buffer consisting of 8 M urea, 1.5 M thiourea, 1.5% CHAPS, and 50 mM DTT. The protein amount in the samples was determined by the Peterson method (Peterson, 1977).

Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel image analysis

Approximately 900 μg of protein was loaded on a 24-cm IPG-strip with a linear pH range between 3 and 10 (ReadyStrip IPG; Bio-Rad, Hercules, CA, USA) by 12-h passive in-gel rehydration. Isoelectric focusing was performed at 20 °C and initiated with a linear gradient to 100 V over 4 h, followed by 250 V for 4 h, 1,000 V for 1-h, linear gradient to 10,000 over 2 h, 10,000 V for 80,000 V-h, and 200 V for 1-h. The gel strips were then equilibrated in 5 mL of the equilibrated solution I (6 M urea, 375 mM Tris-HCl, pH 8.8, 2% sodium dodecyl sulfate (SDS), 20% glycerol, and 2% DTT), followed by an equilibration in 5 mL of a solution supplemented with 2.5% iodoacetamide without DTT. For SDS-PAGE, equilibrated gel strips were placed on top of 12% gel and overlaid with 1% (wt/vol) agarose in equilibration solution I. Next, the electrophoresis was performed at 12 °C with 200 V until the dye reached the bottom of the gel. The gels (25 × 20.5 × 1.5 mm) were run in the Protean plus Dodeca Cell System (Bio-Rad, Hercules, California, USA). After electrophoresis, the gels were rinsed three times with Milli-Q water, stained for 1-h with G-250 staining (PageBlue; Thermo Fisher Scientific, Waltham, MA, USA), and rinsed with Milli-Q water to remove the background. Three biological replicates were made. Digital images of gels were acquired at 300 dpi using the ChemiDoc MP System (Bio-Rad, Hercules, California, USA). Comparison of gel images and processing, and quantitative comparison of protein spots based on their percent volume were performed with Melanie version 8 (GE Healthcare, Chicago, IL, USA). Significant changes in protein abundance between +NAA–KIN and −NAA–KIN samples were found with one-way analysis-of-variance test at P < 0.05 and at least 1.4-fold change using Melanie software.

Protein in-gel digestion and identification by liquid chromatography tandem mass spectrometry

Spots with statistical significance were manually excised and subjected to in-gel digestion. Tryptic peptides were desalted with ziptip tips, as described in Huerta-Ocampo et al. (2012). The peptides were resuspended in 0.1% formic acid and analyzed using the Easy-nLC-1000 liquid chromatography system (Thermo Fisher Scientific, Waltham, MA, USA) with the analytical nanoscale liquid chromatography column PepMAP (Thermo Fisher Scientific, Waltham, MA, USA). Peptide elution was made with a binary gradient of water as mobile phase A and acetonitrile as mobile phase B supplemented with 0.1% formic acid and delivered at 250 μL min⁻¹ using the following program: 5% of B to 55% B in 35 min, 55% B to 100% B in 5 min, 100% B for 5 min, 100% B to 5% B in 5 min followed by column equilibration with 5% B for 7 min. Mass spectrometry was performed using the LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) operated in positive ion mode and with an easy-nanospray potential of 1.9 kV. Data-dependent acquisition was performed at Orbitrap for the top-10 method with 120,000 resolution for full MS and an AGC target of 1,000,000 and 15,000 resolution and AGC target of 50,000 for tandem mass spectrometry (MS/MS). The ions were fragmented in the higher energy dissociation cell (HCD). The ion threshold for triggering MS/MS events was 5,000, and dynamic exclusion was 90 s. Proteome Discovery 2.2 (Thermo Fisher Scientific, Waltham, MA, USA) was used for data processing using MASCOT (Matrix Science Inc., Boston, MA, USA) and SEQUEST as search engines and the Coffea canephora protein sequence database (GCA_900059795.1) from the National Center for Biotechnology Information. The search engine parameters were as follows: carbamidomethylating of cysteine as a fixed modification, oxidation of methionine as a variable modification, trypsin enzyme, maximum missed cleavage 2, and Coffea taxonomy. We used a decoy database and only recovered protein hits based on two successful peptide identifications. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2022) partner repository with the dataset identifier PXD055039.

Western blot analysis

Nine-month-old plantlets were transferred to MS medium either with or without NAA–KIN and kept for 14 days. Equivalent to 100 mg of leaf explantes from +NAA–KIN and −NAA–KIN plantlets were frozen in liquid nitrogen, ground with a pestle and mortar, homogenized in Laemmli buffer (Laemmli, 1970). Proteins were resolved onto 12% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Merck Millipore). The membrane was blocked with 5% bovine serum albumin (BSA) in TBS or 5% fat-free milk in PBS for 1 h at room temperature and then probed with anti-Ub (dilution 1:5000; ab19169; Abcam, Cambridge, MA, USA) or with anti-HIS3 (dilution 1:10,000) as loading control (ab1791; Abcam, Cambridge, MA, USA), respectively (Aguilar-Hernández et al., 2017). AP conjugated antibody was utilized to detect immuno complexes by using 1-step NBT/BCIP regents (Thermo Fisher Scientific, Rockford, IL, USA).

Sequence analysis and primer design for C. canephora

Given that two RPN12 genes were reported in Arabidopsis thaliana AtRPN12A (AT1G64520.1) and AtRPN12B (AT5G42040.1), and that AtACT2 (AT3G18780.2) is a reference gene for quantitative real-time PCR (RT-qPCR) (Book et al., 2010; Zhou et al., 2019), orthologs of those genes in C. canephora were retrieved in BLAST searches against C. canephora implemented in Coffee Genome Hub resource (http://www.coffee-genome.org). C. canephora encodes only one CcRPN12 gene (Cc08t16370.1), with 75.13% and 75.84% of identity to A. thaliana AtRPN12A and AtRPN12B, respectively. The homolog of A. thaliana AtACT2 in C. canephora was identified as Cc07t17400.1 (CcACT2). Specific primers for RT-qPCR analysis of CcRNP12 and CcACT2 in C. canephora were designed from the coding sequences (CDS) using the software Primer 3 (https://primer3.ut.ee). The primers were synthesized by Adn-Artifitial (Guanajuato, Mexico) on a scale of 25 nmol, with the option of desalted purification, without modifications in the 5′ and 3′ ends. The amplicon of the CcRPN12 gene spans exons one to three and has a length of 249 bp. The CcACT2 gene amplicon was 233 bp in length.

RNA extraction, cDNA synthesis and quantitative real-time PCR (RT-qPCR) analysis

Nine-month-old plantlets were transferred to MS medium either with or without NAA–KIN and kept for 14 days. An equivalent of 100 mg of leaf explants was collected, frozen with liquid nitrogen, and preserved in the Thermo Scientific™ Revco ExF Series Ultra Freezer for 30 days. The total RNA was extracted using TRIzol reagent (Invitrogen, Waltham, Massachusetts, U.S.), in accordance with previous reports by Chomczynski (1993). RNA integrity was assessed by examining samples in a 1.2% agarose gel, and by measuring both A260/A280 and A260/A230 ratios using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The samples used had measurements of A260/A280 ratio between 1.89 and 1.99. The RNA concentrations of the processed samples ranged from 240.4 to 353.4 ng/μL. One μg of RNA was used to synthesize cDNA with 200 units of the ImProm-II™ reverse transcriptase (Cat # A3802; Promega, Madison, WI, USA) and 0.5 μg oligo dT₁₈ primers (Cat # SO131; Thermo Fisher Scientific, Waltham, MA, USA), as indicated by the manufacturer’s instructions, in a volume of 20 μL.

The C. canephora ACT2-specific primers CcACT2Fwd (5′-AGCAACTGGGATGACATGGA) and CcACTRev (5′-TCCAGCACAATACCAGTCGT), and the RPN12-specific primers CcRPN12Fwd (5′-ATTCAAAGCTGCCTTCGTCC) and CcRPN12Rev (5′-CCTCGGGCATCAGTGTAGTA) were utilized in the RT-PCR reaction setup. For each individual PCR reaction, 100 ng of cDNA was utilized as a template for PCR amplification in a 20 μL reaction volume using 10 μL of QuantiNova Probe PCR Master Mix (Cat # 208054; QIAGEN, Hilden, Germany), 1 μL each of 8 μM forward and reverse primers, and Rotor Gene Q MDx instrument (QIAGEN, Venlo, The Netherlands). Three technical replicates were made for each reaction with Rep. Ct (95% CI). The reaction parameters were 95 °C for 2 min, 40 cycles of 95 °C for 15 s and 61 °C for 30 s. Fluorescence data were collected during each annealing-extension step at 61 °C. Five dilution points were used for the standard curve 1, 1:10, 1:100, 1:1,000, and 1:10,000. For the ACT2 gene pair of primers, the efficiency was 141.18%, R2 was 0.8922, and the slope was −2.6155. For the RPN12 gene primer pair, the efficiency was 85.78%, the R2 value was 0.9914, and the slope was −3.7175. The RT-qPCR data reactions that were used to calibrate curves, melt, identify outliers, reproducibility (Rep. Ct (95% CI)), and variation were first evaluated using the Rotor-Gene Q 2.1.0.9 Windows software version, and then data were extracted for use in Excel templates. Subsequently, the relative transcript abundance of the RPN12 gene was determined using the Delta-Delta Ct (2^−ΔΔCt) method (Livak & Schmittgen, 2001), with the ACT2 reference gene as internal control (Gutierrez et al., 2008). The significant changes in RPN12 gene expression in the +NAA–KIN and −NAA–KIN explants were determined using a Student’s t-test. GraphPad Prism 9 version 9.0.1 statistical analysis software was employed to analyze gene expression data with statistically significant values of P ≤ 0.05.

Histology

To carry out histological and histochemical analysis on +NAA–KIN and −NAA–KIN explants, the samples were immersed in FAA solution (containing 10% formaldehyde, 5% acetic acid, and 50% ethanol) for 48 h before washing five times with distilled water. Next, the samples were dehydrated in a graded ethanol series starting with 10%, followed by 30%, 50%, 70%, 85%, 96%, and 100%. The steps were repeated, and the samples were vacuum infiltrated for 20 min, followed by an hour of incubation at 4 °C. After that, the samples were coated with JB-4 resin (JB-4 Embedding kit; Polysciences, Warrington, PA, USA). The blocks were cut into 3 µm slices using a MICROM HM 325 microtome and were double-stained with a solution of toluidine blue to characterize their structural features and with Xylidine Ponceau to detect their total protein (XP, Vidal 1970). Images were acquired using a Leica MZFL III stereomicroscope.

Conditioning explants with NAA and KIN

We previously defined the auxin plus cytokinin priming plantlet step to promote forming somatic embryos along cut-leaf edges of C. canephora explants. The in vitro plantlets were given 0.54 µM auxin 1-naphtaleneacetic acid (NAA) plus 2.32 µM cytokinin kinetin (KIN) in semisolid culture media for 14 days, which is referred to as +NAA–KIN. The next step was to give 5 µM cytokinin 6-benzyladenine (BA) to cut-leaf explants in liquid media for up to 100 days (Quiroz-Figueroa et al., 2006). The aim of our analysis was to discover how NAA plus KIN affect the embryogenic response of this SE setting. Plantlets were cultivated for 14 days either with or without NAA–KIN which are referred to as +NAA–KIN and −NAA–KIN, respectively, and then used as cut-leaf explants to induce SE through BA. NAA–KIN treatment did not affect the morpho-anatomical patterns of C. canephora plant leaves during a 14-day period (Fig. S1). Compared with +NAA–KIN explants, −NAA–KIN explants showed less growth of meristematic-like cells at the edge, and proembryonary masses were observed at 28 days of explant cultivation, as shown in Fig. S2. Next, embryos produced along the edge of the explant were recorded for up to 100 days (Fig. 1A). NAA–KIN further increased the globular stage embryo yield by two-fold (128/64) after 75 days of explant cultivation (Fig. 1B). After another 25 days of explant cultivation, the yield of embryos at the cotyledonary stage increased to 31 times (31/1). When comparing the number of embryos produced after 75 and 100 days of induction, it was found that the number of embryos at the globular stage in +NAA–KIN explants decreased dramatically while it remained constant in −NAA–KIN explants. The significant increase in the number of cotyledonary embryos in +NAA–KIN explants over −NAA–KIN explants suggests that NAA–KIN is involved in explant conditioning to improve SE.

Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE) analysis

Next, we followed to compare the protein patterns of +NAA–KIN and −NAA–KIN explants using two-dimensional polyacrylamide gel electrophoresis (2D SDS-PAGE). Multiple 2D SDS-PAGE gels were obtained to represent three technical and biological repeats. The reference map contained 230 protein spots and was populated between pI from 4 to 7 and from 50- to 100 kDa MW. Forty-two differential spots were identified with at least a 1.4-fold change and a P-value of 0.05 in the one-way analysis of variance test. Forty-one spots were differentially up-accumulated, and one spot was down-accumulated (Fig. 2, Fig. S3). The top five protein spots with the most abundance ratios were 1, 3, 4, 2 and 7. Twelve out 42 2D-PAGE differential spots were identified with liquid chromatography MS/MS using the C. canephora genome dataset deposited at the National Center for Biotechnology Information dataset. The ratio changes for spots 1 (CDP17851), spot 4 (CDP17853), and spot 2 (CDP17852), all of which are chitinases A class III, and spot 3 (CDP12187), which is a subunit RPN12 of the 26S proteasome, were more than five-fold (Table 1).

Proteins with a role in the protein metabolic process include a ribosomal protein CDP14047 (spot 37), leucine aminopeptidase CDP04332 (spot 17), papain-like protease CDP01294 (spot 7), and the subunit of the ATP-dependent proteolytic complex 26S proteasome CDP12187 (spot 3). Proteins participating in folding and refolding proteins include a chloroplast chaperonin CPN60 CDP18583 (spot 53) and a mitochondrial heat shock protein 60 CDO99385 (spot 53). Phosphatase protein is implicated in the reversible phosphorylation of proteins CDP17727 (spot 7) and in chromatin organization by recognition of methylated dinucleotides CpG as well CDP05220 (spot 46). Proteins associated with oxidation–reduction were NADPH-dependent thioredoxin reductase CDP01751 (spot 59) and isoflavone reductase CDP07496 (spot 59), glycine catabolic process CDP08258 (spot 17), photosynthesis CDP20139 and CDP17859 (spots 50 and 7), and carbohydrate metabolic process CDP17851, CDP17853, and CDP17852 (spots 1, 4, and 2).

RPN12 gene expression analysis and western blot analysis of extracts from −NAA–KIN or +NAA–KIN explants with ubiquitin antibody

The embryogenic response and altering proteome constituents through activating protein biosynthesis, post-translational modifications of proteins or removal of unnecessary proteins have been correlated by biochemical and morphological analysis (Aguilar-Hernández & Loyola-Vargas, 2018; Gomez-Garay et al., 2013; Kumaravel et al., 2017). The multicatalytic ATP-dependent complex 26S proteasome is responsible for specific protein degradation after proteins are marked with ubiquitin (Vierstra, 2009). In this study, the protein subunit CcRPN12 of the ATP-dependent proteolytic complex 26S proteasome was found to be up-regulated. Then, we opted to investigate if the rise in CcRPN12 protein abundance in +NAA–KIN explants was determined by the transcript-level using qPCR with the CcACT2 gene as a reference. There was no difference in the amount of CcRPN12 in +NAA–KIN or −NAA–KIN explants (Fig. 3A), suggesting a post-transcriptional mechanism leading the up-regulation of CcRNP12 in +NAA–KIN explants. The RPN12 protein is essential for the proteasome assembly (Boussardon et al., 2022; Smalle et al., 2002), thus altering CcRPN12 abundance might impact the pool of ubiquitinated proteins. One of the overall ways to visualize ubiquitination can be achieved by western blotting against ubiquitin (Aguilar-Hernández et al., 2017). Given that within identified proteins in this study are implicated in the proteasome 26S dependent protein catabolic process, we determined the ubiquitination smear in −NAA–KIN and +NAA–KIN explants by western blotting with anti-ubiquitin and using anti-histone 3 as loading control. Both −NAA–KIN and +NAA–KIN explants displayed a similar ubiquitination smear at the high MW (Fig. 3B), suggesting that the 26S proteasome is active and that probably a small pool of ubiquitin conjugates engages in the totipotency of explants to produce efficiently somatic embryos.