A peach gene, PpEBB1 has been transformed into poplar as described in a previous study (Zhao et al., 2020). The WT and transgenic poplars were grown in an aseptic Murashige and Skoog (MS) medium containing 0.1 mg·l⁻¹ naphthyl acetic acid (NAA) under a 14:10 h day and (200 μmol·m⁻²·s⁻¹) night light regimes at temperatures of 25 and 18°C, respectively. The stems were cut into several segments (about 1 cm) for subculture, each segment containing at least two axillary buds. The 6-week seedlings were used in this study. The axillary shoots of transgenic poplar and WT poplar were used for RNA sequencing (RNA-seq) analysis and protein extraction. The basal portions of the shoots were used for sugar content determination, brassinosteroid (BR) content determination, and individual amino acid determination.

Total RNA was isolated using the RNA prep Pure Plant Kit (polysaccharide- and polyphenolic-rich; TIANGEN, Beijing, China) with three biological replicates. A NanoPhotometer P360 (Implen, Munich, Germany) was used to assess the quality and quantity of RNA. The PrimeScript RT reagent kit and gDNA Eraser (Perfect Real Time; Takara Biotechnology, Dalian, China) were used for reverse transcription. SYBR Premix Ex Taq (Takara Biotechnology, Dalian, China) was used for qRT-PCR. Primers are listed in Supplementary Table S1.

RNA was isolated using the RNA prep Pure Plant Kit (polysaccharide- and polyphenolic-rich; Tiangen, Beijing, China). The concentration and purity were assessed by NanoPhotometer® spectrophotometer (Implen, CA, United States), Qubit®3.0 Fluorometer (Life Technologies, CA, United States), and Agilent 2100 RNA Nano 6,000 Assay Kit (Agilent Technologies, CA, United States). Oligo (dT) magnetic beads were used for mRNA enrichment and purification. The messenger RNA (mRNA) was fragmented by adding the fragmentation buffer. Then, the first strand of complementary DNA (cDNA) was synthesized using 6-base random primers with mRNA segments as the template. The second strand of cDNA was synthesized by adding buffer, dNTPs, RNaseH, and DNA Polymerase I. QIAQuick PCR kit was used for purification using VAHTS® Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme, Nanjing, China). Then the double-stranded cDNA was treated with terminal repair, base A, and a sequencing connector. Then the 450–500 bp fragment was recovered by agar-gel electrophoresis and amplified by PCR, to complete the preparation of the library. The prepared library was sequenced using the PE150 method in the Illumina platform. The clean data was obtained by removing low-quality sequences and joint contamination. Reads Count for each gene in each sample was counted by HTSeq v0.6.0, and fragments per kilobase million mapped reads (FPKM) were then calculated to estimate the expression level of genes in each sample. P. trichocarpa genome annotations were downloaded from the JGI database.¹ DESeq2 v1.6.3 was used to identify DEGs between WT and transgenic poplar. Genes with q≤ 0.05 and |log2_ratio| ≥ 1 were identified as DEGs. The Gene Ontology (GO; http://geneontology.org/) enrichment of DEGs was determined by the hypergeometric test, in which the p-value was calculated and adjusted as the q-value, and the background data was genes in the whole genome. GO terms with q < 0.05 were considered to be significantly enriched. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of DEGs was determined by the hypergeometric test, in which the p-value was adjusted by multiple comparisons as the q-value. KEGG terms with q < 0.05 were considered to be significantly enriched.

Total protein was extracted with three biological replicates. Samples were collected and frozen in liquid nitrogen immediately. Then, the powder was transferred into a 5 ml centrifuge tube with lysis buffer (containing 1% Triton X-100, 10 mm dithiothreitol, 1% protease inhibitor cocktail, 50 μm PR-619, 3 μm TSA, 50 mm NAM, and 2 mm EDTA; v:v = 1:4) and sonicated three times on ice using a high-intensity ultrasonic processor (Scientz Biotechnology, Ningbo, China). An equal volume of Tris-saturated phenol (pH 8.0) was added, and the mixture was vortexed for 5 min. The upper phenol phase was transferred to a new centrifuge tube after centrifugation at 5,000 g at 4°C for 10 min, and five volumes of ammonium sulfate-saturated methanol were added and incubated at −20°C for at least 6 h to precipitate proteins. The supernatant was removed after centrifugation at 4°C for 10 min, and the precipitate was washed once with ice-cold methanol, followed by ice-cold acetone three times. Finally, 8 m urea was used to redissolve the protein, and the protein concentration was determined using a BCA protein assay kit (Beyotime, Nanjing, China) according to the instructions of the manufacturer.

For digestion, the protein solution was reduced with 5 mm dithiothreitol for 30 min at 56°C and alkylated with 11 mm iodoacetamide for 15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mm triethylammonium bicarbonate (TEAB) to urea concentrations less than 2 min. Finally, trypsin was added at a 1:50 trypsin-to-protein mass ratio for the first digestion overnight and a 1:100 trypsin-to-protein mass ratio for a second 4 h digestion.

After trypsin digestion, the peptide was desalted by a Strata X C18 SPE column (Phenomenex, Los Angeles, United States) and vacuum-dried. The peptide was reconstituted in 0.5 M TEAB and processed according to the protocol of the manufacturer for the tandem mass tag (TMT) kit. Briefly, one unit of TMT reagent was thawed and reconstituted in acetonitrile. The peptide mixtures were then incubated for 2 h at room temperature and pooled, desalted, and dried by vacuum centrifugation.

The tryptic peptides were fractionated by high pH reverse-phase HPLC using an Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). Briefly, peptides were first separated with a gradient of 8–32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into 18 fractions and dried by vacuum centrifugation.

The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient was comprised of an increase from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, from 23 to 35% in 8 min and increasing to 80% in 3 min, and then holding at 80% for the last 3 min, all at a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system.

The peptides were subjected to nanospray ionization (NSI) source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo Fisher Scientific, MA, United States) coupled online to the ultra-performance liquid chromatography (UPLC). The electrospray voltage applied was 2.0 kV. The m/z scan range was from 350 to 1,800 for a full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using the NCE setting of 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan followed by 20 MS/MS scans with 15 s dynamic exclusions. Automatic gain control (AGC) was set at 5E4. The fixed first mass was set as 100 m/z.

Samples (1 g) of lyophilized liquid nitrogen were ground into a powder and extracted for 2 h at 4°C in 10 ml of 80% methanol. The extracts were centrifuged at 10,000 r/min for 5 min at 4°C. The supernatant was added to a Bond Elut column and eluted with 3 ml methanol. Then, the eluant was added into a Strata-X column and eluted with 3 ml methanol. Nitrogen flow was used to dry eluant and 200 μl methanol was added to redissolve. The sample was filtered through a 0.22 μm syringe for HPLC-MS/MS detection.

The extracted samples were quantified using a ZORBAX SB-C18 reversed-phase column (150 mm × 2.1 mm, 3.5 μm). Five microlitres of the sample were injected at a flow rate of 0.35 ml·min⁻¹ and a column temperature of 35°C.

Individual amino acids were measured according to the method used in the previous study (Gayen et al., 2016). Sample power (0.5 g) was placed in 10 ml of 6 M HCl containing 0.1% phenol at 110°C for approximately 20 h. The digested sample was filtered through a 0.22 μm syringe filter. Two hundred microlitres of the clear extract were taken in a 2 ml microcentrifuge tube along with 20 μl of n-leucine internal standard solution; then, 100 μl of triethylamine acetonitrile and 100 μl of phenyl isothiocyanate acetonitrile were added into the tube in turn. The sample was mixed well and allowed to stand at 25°C for 1 h. A sample of 10 μl was injected into an Amethyst C18-H column (250 mm × 4.6 mm, 5 μm) using a RIGOL L3000 HPLC at a flow rate of 0.4 ml·min⁻¹, 40°C column temperature, and detected by a UV detector at a wavelength of 254 nm.

GraphPad Prism software was used to construct charts. Statistical analysis was performed using IBM SPSS Statistics 19 software to analyze the significance of differences among data, with a significance level of p < 0.05 under Duncan’s test.

PpEBB1-oe poplars have been obtained from a previous study (Zhao et al., 2020). WT and PpEBB1-oe plantlets were cultured in an aseptic MS medium for long-days photoperiod for about 6 weeks. In PpEBB1-oe poplar, some axillary buds in the axils of leaves were released to grow out while the axillary buds of WT were inhibited (Figure 1A). As a result, overexpressing EBB1 led to more syllectic branches compared with WT (Figure 1B; Supplementary Figure S1). Meanwhile, overexpression of PpEBB1 led to smaller and curly leaves, which was also observed by Yordanov et al. (2014).

To identify the molecular mechanisms by which PpEBB1 promotes branching, line 29, which has the highest PpEBB1 expression level among all of the transgenic lines (Zhao et al., 2020), was used for transcriptome and tandem mass tag (TMT) proteome analysis. Significantly differentially expressed genes (DEGs) were identified based on their |log2Ratio| ≥ 1 and q < 0.05 threshold between the PpEBB1-oe line 29 and WT poplar. DEGs were used for GO term analysis and KEGG enrichment analysis. Results showed that a large number of DEGs were involved in light response, sugar metabolism, BR metabolism, plant hormone signal transduction, and photosynthesis (Datasets S1 and S2). The RNA-seq data were reliably validated by qRT-PCR and principal component analysis (PCA; Supplementary Figures S2, S3). Proteins with significant (p < 0.05) > 1.3 or <1/1.3-fold changes in relative abundance between PpEBB1-oe line 29 and WT were identified as differentially expressed proteins (DEPs). The result of PCA verified the repeatability and reliability of the proteomics data (Supplementary Figure S4). These DEPs were categorized into numerous metabolic processes by GO and KEGG analysis. Results showed that a large number of DEPs were associated with sugar metabolism, nitrogen metabolism, and amino acid metabolism (Datasets S3 and S4).

Light is a key environmental factor for bud outgrowth. Light quality and light intensity have a great influence on regulating branching. In this study, a large number of DEGs (Dataset S5) were enriched in light-associated GO terms, including “response to light stimulus” (GO:0009416), the response to light qualities (blue light, GO:0009637; red light, GO:0010114; and far-red light, GO:0010218), and light intensity (GO:0009642; GO:0009644; Dataset S1). Hence, these results indicate that EBB1 may influence the response of a plant to light, which may be one of the factors by which EBB1 promotes branching.

The positive function of sugars on bud outgrowth has been well studied (Mason et al., 2014; Barbier et al., 2015a). Notably, from the transcriptomic data in the study, some GO terms related to sugar metabolism were significantly enriched (Dataset S1), including starch catabolic (GO:0005983) and metabolic (GO:0005982) processes, “response to sucrose” (GO:0009744), “hexose metabolic process” (GO:0019318), “fructose metabolic process” (GO:0006000), and “sucrose synthase activity” (GO:0016157). In addition, a KEGG pathway “starch and sucrose metabolism” (map00500) was identified (Dataset S2). DEGs involved in these sugar metabolism processes were selected and searched for the orthologs in Arabidopsis for detailed annotation (Dataset S6). A heatmap was formed using these DEGs (Figure 2A), and from Figure 2A, we can see that most of the DEGs involved in sugar metabolism are downregulated.

In addition, different sugar metabolism processes, including “fructose 1,6-bisphosphate 1-phosphatase activity” (GO:0042132), “sugar-phosphatase activity” (GO:0050308), “hexose biosynthetic process” (GO:0019319), “glucose metabolic process” (GO:0006006), “fructose metabolic process” (GO:0006000), “fructose and mannose metabolism” (pop00051), and “starch and sucrose metabolism” (pop00500), were significantly influenced, as verified by both the GO (Dataset S3) and KEGG (Dataset S4) enrichment analyses of the DEPs performed in this study. Most of these DEPs were downregulated in the PpEBB1-oe poplar (Figure 2B; Dataset S7), which was consistent with the transcriptomic results. These results indicate that PpEBB1 could have a great influence on sugar metabolism so that the levels of various types of sugar were measured to exactly understand the function of PpEBB1 on sugars. As shown in Figure 2C, the contents of different types of sugars detected in this study, including fructose, glucose, sucrose, trehalose, and starch, were all significantly increased in the PpEBB1-oe poplar. Due to a decrease in the expression of metabolism genes and proteins, the rates of catabolism or utilization may be affected resulting in an increase in PpEBB1-oe poplars.

GO (Dataset S1) and KEGG terms (Dataset S2) related to BR were identified, and they included “brassinosteroid biosynthetic process” (GO:0016132), “brassinosteroid metabolic process” (GO:0016131), “brassinosteroid homeostasis” (GO:0010268), “regulation of brassinosteroid biosynthetic process” (GO:0010422), and “brassinosteroid biosynthesis” (map00905). The expression levels of genes involved in the biosynthesis of BR (Supplementary Figure S5) including DWARF (DWF) 4, DWF6/de-etiolated 2 (DET2), rotundifolia 3 (ROT3)/CYP90C1, constitutive photomorphogenesis and dwarfism (CPD)/CTY90A1, and BR6ox1 were changed (Figure 3A; Dataset S8). BAS1 is a BR-inactivating gene that converts castasterone (CS) and brassinolide (BL) to their C26 hydroxylated derivatives. Two differentially expressed BAS1 genes in the PpEBB1-oe poplar were both downregulated (Figure 3A; Dataset S8). BKI1, interacting with brassinosteroid insensitive1 (BRI1) and negatively regulating BRI1 signaling (Wang and Chory, 2006; Wang et al., 2014), were downregulated, whereas BSK and BES1/BZR1 genes were upregulated in PpEBB1-oe poplar.

The contents of BL, CS, and 6-deoxocathasterone (6-deoxoCS) were measured (Figure 3B). The 6-deoxoCS level was significantly decreased in PpEBB1-oe poplar, which could be transformed into CS under the action of BR6ox1/2 in the campestanol (CN)-independent pathway. CS, the immediate precursor of BL, was significantly increased in PpEBB1-oe poplar, while BL was significantly decreased. The total contents of CS and BL were compared between PpEBB1-oe poplar and WT because only CS and BL possess detectable biological activity (Yokota et al., 1982; Zhao and Li, 2012), and the results showed that the content of active BRs in PpEBB1-oe poplar was slightly higher than that in WT.

Nitrogen is one of the most important mineral nutrients for plant growth and development, including regulating shoot branching (Drummond et al., 2015). Twelve DEPs were mapped in the nitrogen metabolism pathway (pop00910; Dataset S4) by KEGG analysis. Among these DEPs (Figure 4; Dataset S9), one nitrite reductase and two glutamate synthase proteins were downregulated, while two glutamate dehydrogenase proteins were upregulated, and two of the glutamine synthetase proteins were downregulated; the other was upregulated in PpEBB1-oe poplars. Nitrogen is partially converted to amino acids after assimilation by plants, and studies (Fichtner et al., 2017; Lu et al., 2018) found that there is a relationship between amino acids and shoot branching. GO and KEGG analysis identified some terms (Datasets S3 and S4) related to amino acids, including “aspartate family amino acid biosynthetic process” (GO:0009067), “cysteine and methionine metabolism” (pop00270), “histidine metabolism” (pop00340), “glycine, serine, and threonine metabolism” (pop00260), “tryptophan metabolism” (pop00380), “phenylalanine, tyrosine, and tryptophan biosynthesis” (pop00400), “arginine biosynthesis” (map00220), “alanine, aspartate, and glutamate metabolism” (map00250), and “biosynthesis of amino acids” (map01230). Hence, the content of 20 amino acids in PpEBB1-oe and WT poplars was determined. Compared with WT, PpEBB1-oe poplar only had higher levels of aspartic acid (Asp), arginine (Arg), cysteine (Cys), and tryptophan (Trp), whereas other amino acids were all significantly decreased (Figure 5).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.