Ploidy level enhances the photosynthetic capacity of a tetraploid variety of Acer buergerianum Miq.

Plant materials

The autotetraploid ‘Xingwang’ (X) originated from the colchicine-induced somatic chromosome doubling of the diploid ‘S4’ (S), and 20 clones with genetic consistency were obtained by grafting propagation. Young leaves of six-year-old ‘Xingwang’ and autodiploid ‘S4’ were used as experimental materials. All the test materials were obtained from the gardens of Shandong Agricultural University Forestry College (N36°10′ 12.44″, E117°09′ 40.33″) and were grown outdoors. The region has a temperate monsoon climate with an average annual temperature of 13 °C, average annual precipitation of 688.3 mm, and annual average relative humidity of 66%. The soil is loamy.

Polyploidy identification

The single-cell DNA content of the polyploid X was determined by flow cytometry (Handyem HPC-150, Canada) (Zhang, Guo & Deng, 2006). Normal leaves of X and S were collected; the leaves were washed, and the veins were removed. Then, 20 mg of the remaining leaf tissue was placed in a culture dish, and fresh lysis solution was added. A double-sided blade was used to chop the leaf tissue quickly in a Petri dish, and the tissue was processed for 3–5 min. A pipette was used to extract 1 ml of the chopped sample, which was filtered into a 1.5 ml centrifuge tube with a 300-mesh nylon sieve. Then, 1 mg/ml propidium iodide solution was added to the centrifuge tube to reach a final propidium iodide concentration of 0.1 mg/ml, and the solution was allowed to stand for 5 min. The processed solution was filtered through a 300-mesh nylon sieve again into a new centrifuge tube and then tested on the machine. Three replicates were employed for each test sample from the same plant, and at least 9,000 cells were collected each time. The lysate formulation was 0.1 mol/l citric acid, 0.5% TWEEN-20, 0.4 mol/l disodium hydrogen phosphate, and 1% PVP-10 (Kron & Husband, 2012).

Chromosome ploidy identification of the polyploid X was performed using the chromosome counting method (Zhang et al., 2005). The innermost 0.5 cm length of the stem tip was selected as the experimental material, which was treated at −20 °C for 18 h in a stable solution (anhydrous ethanol:glacial acetic acid:trichloromethane = 5:3:2) and then stored in 75% alcohol at 4 °C. The material was removed and washed 3 times in ultrapure water. The excess water was absorbed with clean filter paper and the sample was transferred to 0.075 mol/l KCl solution to reach hypotonicity for 30 min. Following dissociation, the material was rinsed with ultrapure water 3 times and placed in ultrapure water for 30 min to become hypotonic. The water and Carbo Fuchsin dye (Solarbio, Tongzhou, China) solution were then absorbed for 15–20 min, the material was pressed into a tablet, and 10–20 relatively clear metaphase cells were found under the microscope for identification.

Gas exchange parameters

The experimental site is in the garden of Shandong Agricultural University Forestry College (the environmental conditions are the same as those detailed above). We chose a sunny day in the middle of each month from May to September 2019 and selected tetraploid X and diploid S plants of equal size. We used an LI-6800 portable photosynthesis system (LI-COR, Lincoln, NE, USA) to measure the net photosynthetic rate (P_n), stomatal conductance (G_s), transpiration rate (T_r), and intercellular carbon dioxide concentration (C_i) of the third pair of sun-exposed, physiologically mature leaves from the top of the side branch to the trunk at the same orientation from three plants, with three leaves collected from each plant. The gas exchange parameter data were recorded every 2 h from 8:00 to 18:00, and the average value was used for the processing analysis.

Determination of chlorophyll contents and chlorophyll fluorescence parameters

Fresh leaves from diploid and tetraploid plants were cut into pieces and submerged in 80% (v/v) acetone in the dark at room temperature to extract their chlorophyll (Arnon, 1949). The chlorophyll a (Chl a), chlorophyll b (Chl b) and total chlorophyll (a+b) contents were measured using a BioMate 3S ultraviolet-visible spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 470, 649, and 665 nm according to the methods of Li (2000).

The chloroplast fluorescence parameters of the X and S leaves were measured under slightly excessive light stress. The control group was exposed to a natural light intensity (88,000 lx) while the experimental group was exposed to a 20% increased light intensity (105,600 lx). The chlorophyll fluorescence parameters were measured using a portable modulated fluorometer (PAM-2500; Heinz Walz, Pfullingen, Germany). The maximal fluorescence (F_m) and the minimal fluorescence (F_o) were recorded. The maximal photochemical efficiency of PSII (F_v/F_m) and the potential photochemical efficiency of PSII (F_v/F_o) were calculated as F_v/F_m = (F_m − F_o)/F_m and F_v/F_o = (F_m − F_o)/F_o (Kramer et al., 2004).

Nine samples from three individuals of each ploidy type were measured.

Electron microscope sample preparation of leaf chloroplasts

The mature leaves on the annual sun-exposed branches were collected at 4 °C, and the container and instruments were precooled. Fresh leaf samples (1 × 1 mm) were cut from the middle part of the leaves along the main vein, fixed in 2.5% glutaraldehyde fixative solution, and stored at 4 °C. The fixed leaves were washed 3 times with 0.1 mol/l phosphate buffer solution containing 1% osmium acid (Pelco, EM level, Fresno, CA, USA) at 4 °C for 3 h, washed 3 times with buffer solution, and then dehydrated step by step with ethanol and propylene oxide. A replacement was performed; then, the embedding material was impregnated with Spurr resin (Spi-Chem, West Chester, PA, USA). Last, the samples were polymerized in an oven at 70 °C. The embedding blocks of the different materials were placed on an ultrathin microtome (EM UC6; Leica, Wetzlar, Germany) for sectioning. The thickness of the ultrathin sections was 70 nm. The sections were stained with uranyl acetate (Spi-Chem, ACS level, West Chester, PA, USA) and lead citrate trihydrate (Spi-Chem, ACS level, West Chester, PA, USA) and observed and photographed under a transmission electron microscope (JEM1230; JEOL, Akishima, Tokyo, Japan). Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to produce statistics on the thylakoid lamella thickness. Three samples of X and S were analyzed, and for each sample, the thylakoid lamella thickness and number of plastoglobules from at least three chloroplasts were detected.

RNA extraction, library construction and RNA-seq

During the transcriptome sequencing process, for X and S, three biological replicates were taken from the same plant, and they were denoted X1, X2, and X3 and S1, S2, and S3. Tender leaves from the annual sunny branches were obtained, and an Adelaide Biologicals EASYspin Plant RNA Quick Extraction Kit was used to extract the total RNA from each X and S sample. A spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for RNA quality inspection, and the integrity of the RNA was confirmed by 1% agarose gel electrophoresis.

Oligo (dT) magnetic beads were used to enrich the mRNA with a polyA tail, and interrupting buffer was used to fragment the resulting RNA. Random N6 primers were used for reverse transcription, and two strands of cDNA were synthesized to form double-stranded DNA. The ends of synthetic double-stranded DNA were filled in, and the 5′ end was phosphorylated. The 3′ end forms a sticky end that protrudes with a base ‘A’; then, there is a complementary end with a protruding base ‘T’ on the 5′ end, and the ligated product was amplified by PCR with specific primers. The PCR products were thermally denatured into single-stranded DNA, and then the single-stranded DNA was circularized with a bridge primer to obtain a single-stranded circular DNA library. Finally, the BGISEQ-500 platform was used to obtain raw sequencing data.

D. novo assembly and functional annotation

The software SOAPnuke (Cock et al., 2010) was used to filter the raw data for sequencing, and the filtered data were called clean reads and saved in FASTQ format. Trinity software (Grabherr et al., 2011) was used to assemble then process clean reads in turn. Trinity has three independent modules: Inchworm, Chrysalis and Butterfly. The data were first assembled with Inchworm to obtain the full-length isoform transcript data and the fragment form of the isoform-containing transcript. Next, in the Chrysalis module, the contigs that may have variable splicing and parallel genes are clustered to construct a large number of individual de Bruijn maps. Last, the linear path is merged with continuous nodes in the de Bruijn maps in the Butterfly module and full-length transcript splicing subtypes are extracted.

Then, TGICL software (Pertea et al., 2003) was used to cluster the transcripts to remove redundancies and obtain the unigenes. After clustering, the unigenes were divided into two classes: clusters with the prefix CL and singletons with the prefix Unigene.

For the resulting transcript data, we used Trinity to perform open reading frame (ORF) predictions, and we annotated the transcript with complete ORFs. After assembling the unigenes, we performed bioinformatics analysis and aligned the predicted protein sequences to six functional databases, namely, NCBI nonredundant protein (Nr), NCBI nucleotide in sequences (NT), Swiss-Prot, Pfam, Kyoto Encyclopedia of Genes and Genomes (KEGG), euKaryotic Orthologous Groups (KOG), and Gene Ontology (GO), for annotation and classification. The best alignment results were used to annotate the transcriptome sequences and to determine the orientation of the sequences. The All-unigene results aligmented to the NR database were annotated to the GO database via Blast2GO software (Conesa et al., 2005), and the metabolic pathways were compared with the KEGG database (Kanehisa et al., 2008).

Differential gene analysis

The DEGseq method (Wang et al., 2010) was used to detect differentially expressed genes (DEGs), calculate the p-values (p-value, hypothesis test probability) and correct the p-values to Q-values. To improve the accuracy of the DEGs, we implemented the fold change as more than twice (fold change ≥ 2.00) and the Q-values (adjusted p-value) ≤ 0.05 to screen differential genes and detect the DEGs based on the principle of a negative binomial distribution. The DEGs related to photosynthetic characteristics were selected for analysis.

Real-time quantitative RT-PCR (qRT-PCR) verification

To confirm the reliability of the sequencing analysis, the expression of the candidate genes was measured by qRT-PCR. First, ABM’s 5X All-In-One RT MasterMix Kit (with the AccuRT Genomic DNA Removal Kit) was used to reverse transcribe the RNA into the first strand of cDNA, and the product was stored at −20 °C. Nine candidate genes were selected, the sequences of the target genes were obtained from the transcriptome results (Table S1), and primers were designed. TUB was used as the internal reference gene, and a Bio-Rad CFX96TM Real-Time PCR instrument was used to complete the qRT-PCR. The reaction procedure was as follows: 95 °C for 30 s; 39 cycles of 95 °C for 5 s and 60 °C for 30 s; and then 95 °C for 10 s. The qRT-PCR results were calculated using the 2^−ΔΔt method, and whether the expression trend of the transcriptome results was consistent with the verification results was assessed. The sequences of the primers used are shown in Table S2.

Statistical analysis

Descriptive statistical analyses were used for the measured parameters to obtain the means and standard errors (SEs). One-way analysis of variance (ANOVA) was used to analyze the significance of the differences in the gas exchange parameters, chlorophyll contents, chlorophyll fluorescence parameters and chloroplast ultrastructure between X and S, with the ploidy level as a fixed factor. The significance level was set at p < 0.05. Statistical analyses were performed with SPSS 16.0 (SPSS, Inc., Chicago, IL, USA).

Identification of the A. buergerianum tetraploid

By observing the stem tip under the microscope, we found that the chromosome number of X was significantly higher than that of S by twofold (Fig. S1).

The DNA content in a single cell was detected by flow cytometry, and the results are shown in Fig. S2. The ploidy level of the plant can be inferred from the peak figure of its relative DNA content (Doležel, Greilhuber & Suda, 2007; Paul & Husband, 2012). The main peak single-cell DNA content in the S leaves was at channel 100, while the main peak single-cell DNA content in X leaves was at channel 200. Thus, the single-cell DNA content of X was twice that of S; therefore, X was determined to be tetraploid.

Gas exchange parameters, chlorophyll contents and chlorophyll fluorescence parameters

Similar patterns of diurnal changes in T_r were observed between diploid and tetraploid A. buergerianum X and S leaves from May to September (Fig. S3). In May, July, and September, a single-peak curve was observed that first increased and then decreased, reaching a maximum at approximately 12:00. The peak T_r of X was higher than that of S, the T_r reached the maximum in July, the maximum T_r of X was 1.33 mol·m⁻²·s⁻¹, and the maximum T_r of S was 1.23 mol·m⁻²·s⁻¹. In June and August, the daily variation of the whole day showed a double-peak curve, with the first peak appearing at approximately 10:00 and the second peak appearing at 14:00. In X and S, the first peak was greater than the second peak and fell into a valley at approximately 12:00.

The daily variation trends in the P_n of X and S leaves from May to September were similar (Fig. S4). In May, July, and September, a single-peak curve first increased and then decreased. Starting at 8:00, the Pn gradually increased, reached a maximum at approximately 12:00, and then gradually decreased; the P_n reached its maximum in August. The peak P_n of X was higher than that of S. The maximum P_n of X was 11.15 µmol·m⁻²·s⁻¹, and the average P_n was 6.63 µmol·m⁻²·s⁻¹. The maximum P_n of S was 8.56 µmol·m⁻²·s⁻¹, and the average P_n was 5.72 µmol·m⁻²·s⁻¹. The diurnal changes in June and August showed a double-peaked curve. The first peak appeared at approximately 10:00, and the second peak appeared at 14:00. The first peaks of X and S were both higher than the second peak, and the values fell into a valley at approximately 12:00. Over the 5 months from May to September, the maximum P_n of X increased by 4.31%, 12.35%, 8.48%, 30.31%, and 7.76% over S.

The diurnal variation in G_s of X and S leaves from May to September was similar, and the G_s of X during different months was higher than that of S (Fig. S5). X and S showed a single-peak curve that first increased and then decreased in May, July, and September and was lower at 8:00 and 18:00. Starting from 8:00, G_s gradually increased and reached a maximum at approximately 12:00. The diurnal changes in June and August showed a bimodal curve. The peak G_s of X was higher than that of S, and the G_s of X and S reached their maximum levels in August. In June, the first peaks of X and S both appeared at approximately 10:00, and the second peak appeared at 14:00. The first peaks of X and S both appeared at approximately 10:00 in August, and the second peaks appeared at approximately 14:00 for X and at approximately 16:00 for S. The first peaks of X and S were both larger than the second peaks, and their values fell into a valley at approximately 12:00.

From May to September, the daily change curve in C_i between X and S cells presented a single valley change (Fig. S6). The diurnal variation curves of X and S both first decreased and then increased, and the C_i of X was greater than that of S during different months. Starting from 8:00, as the environmental conditions changed, the C_i gradually decreased, and the lowest value appeared at approximately 12:00 and then gradually increased.

Among the gas exchange parameters of tetraploid X and diploid S, T_r, P_n, G_s and C_i all exhibited extremely significant differences (P < 0.01). From May to September, the maximum values of all the gas exchange parameters of X were higher than those of S (Table 1).

The analysis of chlorophyll contents showed that the Chl a, Chl b and total chlorophyll contents were significantly higher in X than in S (Fig. 1A). The total chlorophyll contents of X and S were 2.47 mg·g⁻¹ and 1.52 mg·g⁻¹, respectively. The leaves of X contained 38.66% more chlorophyll than those of S. Under slightly excessive light stress, F_v/F_m was 0.77 and 0.83 and F_v/F_o was 3.45 and 4.95 in S and X, respectively, and these values were significantly different (Fig. 1B).

Ultrastructure of the chloroplast

The ultrastructures of X and S mesophyll cell chloroplasts were further compared using a transmission electron microscope (Fig. 2). The chloroplasts of X and S were well developed, with obvious thylakoids and stacked basal particles. However, differences were observed in the internal structure of the chloroplasts from the two varieties. Compared with the chloroplasts in S, the chloroplasts in X contained thylakoid lamellae that were twice as thick (Figs. 2, 3A). In addition, the thylakoid lamellae of X were arranged more closely than those of S, and more starch grains were observed in S. In the chloroplasts of the tetraploid X, the number of plastoglobules was lower than that of the diploid S (Fig. 3B).

Transcriptome sequencing and assembly

The transcriptomes of X and S produced 42,804,595 and 44,971,637 nt raw data, respectively, and the Q20 percentages were both over 97%.

The unigenes in each sample were clustered to eliminate redundancy, and 51,797 unigenes were obtained, with an average length of 1,487 nt; the average N50 was 2,034 nt.

Transcript database annotation

A total of 44,665 unigenes were annotated and accounted for 86.23% of all the unigenes. The numbers of unigenes annotated in the NR, NT, SwissProt, KEGG, KOG, Pfam, and GO functional databases were 43,076, 36,463, 32,865, 34,715, 34,085, 34,599, and 26,781, respectively (Table S3, Fig. S7).

Analysis of DEGs

A total of 24,221 DEGs (Table S4) were screened between the Acer buergerianum diploid and tetraploid transcriptomes, and they included 10,474 upregulated genes and 13,747 downregulated genes. To study the function of DEGs related to photosynthetic characteristics in diploid and tetraploid plants, GO (Table S5) and KEGG (Table S6) classifications were determined.

We analyzed the pathways of porphyrin and chlorophyll metabolism and photosynthesis. During chlorophyll synthesis in A. buergerianum, five genes encoding HemB were identified, all of which were upregulated. One gene encoded HemC, and its expression was downregulated. One gene encoded HemE, and its expression was downregulated. Three genes encoded CHLH/D/I; one was upregulated, while two were downregulated (Fig. S8, Table 2) (Matsumoto et al., 2004; Beale, 2005).

In this study, 58 DEGs were annotated as being related to the photosynthesis pathway, with 30 upregulated genes and 28 downregulated genes. Twelve were photosystem I (PSI)-related genes, of which the PsaA-, PsaB-, and PsaK-related genes were upregulated and the PsaG- and PsaH-related genes were downregulated; 28 were photosystem II (PSII)-related genes, of which the PsbE- and Psb27-related genes were upregulated and the PsbH-, PsbQ-, and PsbS-related genes were downregulated; four were cytochrome b6/f complex-related genes, of which the PetB- and PetC-related genes were downregulated; 10 were photosynthetic electron transport-related genes, of which the PetH-related genes were downregulated; eight were F-type ATPase-related genes, among which the beta-, delta-, and b-related genes were upregulated (Fig. 4) (Yamori & Shikanai, 2016).

qRT-PCR validation of the candidate DEGs

To verify the reliability of the transcriptome sequencing results, qRT-PCR validation was performed on nine randomly selected candidate genes (Table S1), including four upregulated genes (CL6641.Contig2_All, CL2285.Contig2_All, CL3396.Contig3_All, and CL335.Contig5_All) and five downregulated genes (CL7423.Contig2_All, Unigene5873_All, CL1401.Contig2_All, CL6251.Contig1_All, and CL5835.Contig1_All). The results were consistent with the expression patterns of the transcriptome sequencing results (Fig. S9), validating the transcriptome sequencing results obtained in this study.