Literature DB >> 33206683

Genome-wide profiling of alternative splicing genes in hybrid poplar (P.alba×P.glandulosa cv.84K) leaves.

Ruixue Wang^1,2, Peng Yin^1,2, Yang Ruixia², Xiao Liu^1,2, Lie Luo², Jichen Xu^1,2.

Abstract

Alternative splicing (AS) is a post-transcriptional process common in plants and essential for regulation of environmental fitness of plants. In the present study, we focus on the AS events in poplar leaves to understand their effects on plant growth and development. The hybrid poplar (P.alba×P.glandulosa cv.84K) leaves were collected for RNA extraction. The extracted RNA was sequenced using on an Illumina HiSeq™ 2000 platform. Using the Populus trichocarpa genome as the reference, a total of 3810 AS genes were identified (9225 AS events), which accounted for 13.51% of all the expressed genes. Intron retention was the most common AS event, accounting for 43.86% of all the AS events, followed by alternative 3' splice sites (23.75%), alternative 5' splice sites (23.71%), and exon skipping (8.68%). Chromosomes 10 had the most condensed AS events (33.67 events/Mb) and chromosome 19 had the least (12.42 events/Mb). Association analysis showed that AS in the poplar leaves was positively correlated with intron length, exon number, exon length, and gene expression level, and was negatively correlated with GC content. AS genes in the poplar leaves were associated mainly with inositol phosphate metabolism and phosphatidylinositol signaling system pathways that would be significant on wooden plant production.

Entities: Chemical Disease Gene Species

Year: 2020 PMID： 33206683 PMCID： PMC7673502 DOI： 10.1371/journal.pone.0241914

Source DB: PubMed Journal: PLoS One ISSN： 1932-6203 Impact factor: 3.240

Introduction

Most plant genes contain introns with conserved nucleotides (GT-AG) at both ends. The initial transcription product, pre-RNA, is spliced to remove introns and the remaining exons are ligated to form mature coding mRNA sequences. Therefore, accurate splicing of gene transcripts is essential for the growth and development of plants and is helpful for their adaptation to variable environment conditions [1]. Also, the pre-mRNA sequences can undergo alternative splicing (AS) to produce different splicing isoforms. For example, the circadian clock associated gene CCA1 in Arabidopsis had two AS products, a full-size CCA1α isoform and an incomplete CCA1β isoform in which the MYB DNA-binding motif is missing [2]. Over-expression of CCA1α increased the plant’s freezing tolerance, whereas over-expression of CCA1β caused plants sensitive to freezing. β-hydroxyacyl ACP dehydratase is the key enzyme in the fatty acid synthesis pathway. In Picea mariana, the β-hydroxyacyl ACP dehydratase gene transcript retained intron 1 under normal temperature conditions and was not translated, whereas at temperatures below freezing, the normal transcript was present and was translated into the active enzyme [3]. In Arabidopsis, introns 2 and 3 of the disease resistance protein gene RPS4 were shown to be crucial for the pathogen defense response. When these two introns were deleted the gene encoded a resistance protein with the complete TIR-NBS-LRR structure, but it had no disease resistance activity [4]. In tomato, AS of a MLO gene can lead to aberrant mRNA isoforms that cause the loss-of-function [5]. Transcriptome analysis in the plant–fungus interaction showed that 31% of the reads are indicative of putative alternative transcriptional events. GO-enrichment analysis revealed them significant involving in response to stimulus, regulation of metabolic process, carboxylesterase activity, and oxigen binding [6]. In poplar, the transcripts from leaf, root, xylem, and phloem of Populus alba var. pyramidalis were analyzed. The AS events occurred in 7,536 genes, of which 4,652 genes had multiple AS events [7]. Comparably, the isochorismate synthase (ICS) gene underwent extensive AS events in poplar that was rare in Arabidopsis, suggesting alternative splicing of ICS evolved independently in Arabidopsis and Populus in accordance with their distinct defense strategies. Besides, AS was also studied in Arabidopsis [8], Eucalyptus [9], Picea abies and Pinus taeda [10]. Therefore, inaccurate AS can produce abnormal transcripts that encode varied proteins with functional diversity, which could result in an effective post-transcriptional regulatory pattern [11]. Further, AS has the evolutionary characters of economy, rapidity, and low risk. In this study, we aimed to dissect the AS events in leaves of the hybrid 84K poplar (Populus alba × Populus glandulosa), which has the advantages of easy rooting, short seedling period, and strong resistance to abiotic stress. The results will exhibit variations of AS events among poplar varieties and further contribute to a better understanding of the roles of AS in leaves on hybrid growth and development.

Materials and methods

Plant materials and growth

The seedlings of 84K poplar were grown in vermiculite mixed with nutrient soil at 1:1. The growth chamber conditions are at 25°C and in an 8 h dark/16 h light cycle. The mature leaves of well-growing plants in two months were collected and used for total RNA extraction.

Isolation of total RNA

The total RNA was isolated from the poplar leaves using Trizol (Aidlab Bio Co Ltd, China) in accordance with the manufacturer’s protocol. The residual DNA was removed with RNase-free DNase I (Takara Bio Inc, Japan) treatment for 30 min at 37°C. RNA quality was initially evaluated using an agarose gel and NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Tech, USA). Three replicates were conducted.

Construction of cDNA library and sequencing

Sequencing library was generated with 1μg RNA sample using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations. The index codes were added to attribute sequences to sample. The library was sequenced by Biomaker company (China). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS (Illumina novaseq6000, PE150). The sequencing was processed on an Illumina platform and paired-end reads were generated. The clean data was obtained from the ploy-N reads by removing adapter and low quality reads [the ratio of N >10% and the ratio of the bases (Q≤10) > 50%]. The raw sequencing data was deposited into the NCBI sequence read archive (SRA) under the BioProject ID: PRJNA647242 (accession number: SRR12280784-SRR12280783).

AS events identification and validation in the poplar leaves

Matrix clean reads were aligned to the Populus trichocarpa genome to define the gene reads locus [12], and the whole gene sequence were determined via NCBI (https://www.ncbi.nlm.nih.gov/assembly/GCF_000002775.3/) using the TopHat2 software. The assembled transcript isoforms were predicted by mapping to the corresponding gene model using Cufflinks program [13]. AS events in the poplar leaves were evaluated using ASprofile as previously described [14]. The expression level of genes or transcripts was evaluated via Fragments Per Kilobase of transcripts per Million fragments mapped (FPKM). FPKM = cDNA Fragments / [Mapped Fragments (Millions) * Transcript Length (kb)]. (cDNA Fragments means the fragments number aligned to a specific transcript. Mapped Fragments means the total fragments aligned to the transcripts). Six AS genes in the poplar leaves having two AS isoforms and in size between 200-1000bp were selected for the validation of the sequencing results. The specific primers were designed at both ends of the gene (S1 Table). Each transcript fragment was amplified using cDNA template described above with the procedure of 95°C for 10 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min. The PCR products were visualized using agarose gel electrophoresis.

Function annotation of AS genes in the poplar leaves

The AS genes in the poplar leaves were aligned with DAVID database (http://david.abcc.ncifcrf.gov/) to obtain their functions, and classified as three clusters of molecular function, biological process and cell component according to GO (Gene Ontology) [15]. The differences between the genes and the background genes were evaluated by GOseq software. The gene enrichment analysis was conducted and defined by P value < 0.05. The pathway analysis of the AS genes was performed by KEGG (Kyoto Encyclopedia of Genes and Genomes) database. BH (Benjamini-Hochberg) method was used for P value adjustment. The pathway of P value < 0.05 was defined as significantly enriched.

Statistic analysis

Based on the AS frequencies in the poplar leaves, the genes were classified into three groups: high AS genes (>5 AS events/gene), low AS genes (1–4 AS events/gene), and non-AS genes. Each group of the AS genes was then characterized according to their length, GC content, and expression levels. The data were analyzed statistically using Microsoft Excel 2013 for calculating mean and standard error. Duncan's multiple range test was used to compare the changes of the categories at the significant difference level of P <0.05.

Results

Overview of RNA-seq data

We obtained a total of 71.1 million clean data (7.11 Gb) by RNA-seq of the mature leaves of 84K poplar. The base Q30 percentage was 94.98% and the GC content was 44.93%. The average read length is 150bp. The number of reads mapped to the reference genome accounted for 68.27% of the total clean reads; 65.94% were uniquely aligned and the remaining 2.33% mapped to multiple loci. Most of the multiple mapped loci contained rRNA, repeat-associated RNA, or part of tRNA sequences. The uniquely mapped reads were mainly mRNA and some tRNA and ncRNA sequences (S2 Table). The majority of the mapped reads (93.52%) were in exonic regions, 2.84% were in intronic regions, and 3.64% were in nongene regions of the reference genome. The reads that were mapped to the exonic and intronic regions were used to analyze gene expression levels and to identify AS events for each gene.

AS genes and events in the poplar leaves transcripts

In total, 28,200 genes were assembled and annotated using StringTie. Among the annotated genes, 13.51% of them underwent 9225 AS events in an average of 2.42 AS events per gene. Gene 1718, which encodes a glycerate kinase (GLYK), underwent 28 AS events, the highest number among all the annotated genes. GLYK is a key enzyme in glycolysis which also may affect DNA replication and repair, and stimulate viral RNA synthesis. All the detected AS events in the poplar leaves were divided into four types, exon skipping (ES), intron retention (IR), alternative 5′ splicing site (A5SS), and alternative 3′ splicing site (A3SS). IR was the most common AS type, accounting for 43.86% (4046 events) of all AS events. IR was followed by A3SS and A5SS, which accounted for 23.75% and 23.71% of all AS events, respectively. ES accounted for only 8.68% of all AS events (Table 1). Some genes had more than one type of AS event; for example, gene Potri.002G124200.v3.0 (PABP1) underwent three types of AS events of IR, ES, and A5SS, and formed 6 varied AS isoforms in the poplar leaves (S1 Fig).

Table 1

Schematic diagram and distribution of the AS events in 84K poplar leaves.

Event type	Events	Proportion (%)	Genes
IR	4046	43.86	2434
A3SS	2191	23.75	869
A5SS	2187	23.71	862
ES	801	8.68	631
Total	9225		3810

*The line represents the intron, the black box represents the exon, the white box represents the exon in irregular splicing, and the gray box represents the retained intron; The straight line represents the normal splicing and the dotted line represents the irregular splicing.

Validation of AS events in the poplar leaves

We randomly selected six AS genes for validation by PCR. The results showed that all six genes each had two transcripts in the poplar leaves (Fig 1). The transcript sizes were consistent with the sizes obtained from the RNA-seq data, indicating that the sequencing data were reliable. For example, gene 6853 had two isoforms in the poplar leaves according to transcriptome data, one was from second intron retention and the other was the normal transcript. The amplified fragments were cloned and sequenced in the poplar leaves in size of 467bp and 411bp, respectively, and the results were consistent with expectation (S2 Fig).

Fig 1

Validation of AS events in the poplar leaves.

PCR results of six poplar AS genes by 1% (w/v) gel electrophoresis. 1: DL 2000 marker, 2: gene 1775, 3: gene 2340, 4: gene 14298, 5: gene 15649, 6: gene 5375, 7: gene 6835.

Validation of AS events in the poplar leaves.

PCR results of six poplar AS genes by 1% (w/v) gel electrophoresis. 1: DL 2000 marker, 2: gene 1775, 3: gene 2340, 4: gene 14298, 5: gene 15649, 6: gene 5375, 7: gene 6835.

Distribution of AS events on chromosomes

The AS events in the poplar leaves were mapped to 19 poplar chromosomes according to P. trichocarpa genome, but their distributions were uneven. Of them, chromosome 1 had the highest numbers of 1138 AS events (452 genes), and chromosome 19 had the smallest numbers of 198 AS events (90 genes) (Table 2). Moreover, chromosome 10 had the highest density of AS events (33.67 events/Mb) and AS genes (12.76%), while chromosome 11 and 19 had the smaller density of AS events (13.62 and 12.4233.67 events/Mb) and AS genes (5.99% and 6.19%). Even on a chromosome, AS events distributed unevenly such as some regions on chromosomes 8, 10, and 14 having more dense distributions of AS events (Fig 2).

Table 2

Distribution of the AS events and AS genes in the poplar leaves on poplar chromosomes.

Chromosome	AS events	Chromosome length (Mb)	AS density (AS events / Mb)	AS genes	Total gene number	AS density (AS genes / total genes)
1	1138	50.49	22.54	452	4706	9.60%
2	774	25.25	30.65	285	2589	11.01%
3	543	21.79	24.92	229	2141	10.70%
4	523	24.26	21.56	223	2334	9.55%
5	634	25.89	24.49	276	2728	10.12%
6	712	27.91	25.51	310	2771	11.19%
7	363	15.61	23.25	148	1467	10.09%
8	523	19.46	26.88	231	2267	10.19%
9	343	12.95	26.49	156	1686	9.25%
10	760	22.57	33.67	320	2507	12.76%
11	252	18.5	13.62	102	1702	5.99%
12	389	15.76	24.68	135	1452	9.30%
13	352	16.32	21.57	155	1506	10.29%
14	546	18.9	28.89	194	1929	10.06%
15	323	15.28	21.14	145	1500	9.67%
16	273	14.49	18.84	121	1432	8.45%
17	280	16.08	17.41	112	1465	7.65%
18	299	16.96	17.63	126	1385	9.10%
19	198	15.94	12.42	90	1455	6.19%

Fig 2

Genome-wide distribution of the AS events in the poplar leaves on 19 poplar chromosomes.

Characteristics of the AS Gene in the poplar leaves

Based on the AS frequencies, the genes in the poplar leaves were classified into three groups: high AS genes (>5 AS events/gene), low AS genes (1–4 AS events/gene), and non-AS genes. The RNA-seq results revealed there were 994 high AS genes, 2866 low AS genes, and 24,390 non-AS genes in the poplar transcriptome. The intron length, exon number, exon length, gene expression level, and GC content data were collected and evaluated among the three types of genes. Significantly, the AS events were positively correlated with the intron length, exon number, exon length, and gene expression level, but negatively correlated with GC content (Fig 3). Statistically (P<0.05), the average intron length of AS genes was 2.19 times that of non-AS genes, and the average intron length of high AS genes was 1.02 times that of low AS genes. The average exon number of AS genes was 2.35 times that of non-AS genes, and the average exon number of high AS genes was 1.22 times that of low AS genes. The average exon length of AS genes was 1.37 times that of non-AS genes, the average exon length of high AS genes was 1.06 times that of low AS genes. The average expression level of AS genes was 1.07 times that of non-AS genes, and the average expression level of high AS genes was 1.12 times that of low AS genes. The average GC content of AS genes was 0.96 times that of non-AS genes, and the average GC content of high AS genes was 0.99 times that of low AS genes.

Fig 3

Association between AS events and each factor in the poplar leaves.

All the transcribed genes in the poplar leaves were classified into three groups according to AS frequency in each gene as high AS genes (>5 AS events/gene), low AS genes (1–4 AS events/gene), and non-AS genes. A: Intron length, B: Exon length, C: Exon number, D: GC content, E: Expression level. The different letters indicate significant differences at the level of P < 0.05. The bars indicate SE.

Association between AS events and each factor in the poplar leaves.

Functional enrichment of AS genes in the poplar leaves

The Gene Ontology (GO) terms assigned to the AS genes in the poplar leaves were analyzed under the three main categories. Under the biological process, the 21 terms were related to metabolic processes, cellular processes, and single-organism processes (Fig 4). RNA processing and tRNA aminoacylation for protein translation etc were significantly enriched (S3 Table). Under cellular component, the 15 terms were related to cell part, cell, and organelle. Intracellular and Kinesin complex etc were more enriched. Under molecular function, the 15 terms were related to catalytic activity, binding, and transporter activity. Hydroquinone glucosyltransferase activity and ATP binding etc were significantly enriched.

Fig 4

Gene ontologies of AS genes in 84K poplar leaves.

The KEGG analysis assigned 909 AS genes in the poplar leaves to 121 pathways (Fig 5). Among the significantly enriched pathways, seventy-four genes were in the spliceosome pathway, which is related to RNA processing and splicing (S3 Fig). Twenty-six genes were in the inositol phosphate metabolism pathway (S4 Fig) and Twenty-five genes were in the phosphatidylinositol signaling system pathway (S5 Fig), both of which are related to biosensory extracellular stimuli and signal transduction. Forty-one genes were in the mRNA surveillance pathway, which is related to clearance of invalid RNA in cells (Fig 6).

Fig 5

KEGG enrichment of AS gene in 84K poplar leaves.

Fig 6

The mRNA surveillance pathway and AS genes distribution in the poplar leaves.

The purple box represents AS genes.

The mRNA surveillance pathway and AS genes distribution in the poplar leaves.

The purple box represents AS genes. In comparison with the other AS reports, mRNA surveillance pathway was no doubt more concerned in the poplar leaves. Of them, PP2A (Potri.015G079300.v3.0) gene had three transcript isoforms, isoform1 was normal while isoform 2 and 3 separately had 1 and 3 exons deletion (Fig 7). Accordingly, it resulted in 38 and 88 amino acids missing at C side for isoform 2 and 3. The predicted secondary structure of the proteins showed that the abnormal isoforms had varied composition and distribution of alpha helix, extension chain, beta turn, and random coil in comparison of the normal isoform. These three transcripts individually had the FPKM value of 1.18, 2.56, and 6.07, and supposed to function differently.

Fig 7

The splicing structure of the PP2A transcripts isoforms in the poplar leaves.

A: mRNA composition of each transcript isoform (The block represents exon, line represents intron), B: the secondary structure of each transcript isoform (The blue parts represent alpha helix, the red parts represent extension chain, the green parts represent beta turn, and the purple parts represent random coil).

The splicing structure of the PP2A transcripts isoforms in the poplar leaves.

Discussion

AS genes with species specificity

AS occurs widely in plant genomes and specifically by tissues. As the important organ, leaf contributes more for plant growth via respiration, photosynthesis, nutrient transformation, and transpiration. Thus, exploring the AS events in leaf could enhance our understanding for plant growth. In this study, we detected a total of 9225 AS events in the 84K poplar leaves transcriptome data. These AS events were from 3810 genes, which accounted for 13.51% of the genes in the transcriptome. Comparably, the AS events were detected in 8.73% of the transcripts in its parent of Populus alba var. pyramidalis [7]. Probably, the more isoforms by AS contribute more for the hybrid growth. In some other studies, the AS frequencies in kiwifruit [16], rice [17], and soybean [18] were 29%, 53.3%, and 63%, respectively, in the microorganisms Magnaporthe grisea [19], Sphaerotheca fuliginea [20], Cryptococcus [21], and Aspergillus oryzae [22] they were 1.6%, 3.6%, 4.2%, and 8.5%, respectively, and in Drosophila melanogaster [23] and human [24], there were 60% and 95%. Clearly, AS occurs more frequently in more advanced species, probably because genes in the advanced species have more introns and/or because of pleiotropism [25]. The high AS frequencies also explain why the advanced species have more complex behaviors and adapt well to environment variation [26], and why the gene numbers do not expand indefinitely as the species evolve [27]. Further, AS can be considered as a post-transcriptional regulation mechanism that acts as an effective strategy to mediate complex biological processes such as cell differentiation and organ development [28].

Gene structure has significant effects on AS types

Among the four mechanisms (ES, IR, A5SS, A3SS) [29], ES is the most common in animals, whereas IR is more frequent in plants. For example, IR and ES account for 3% and 40% of all AS events in human [30], and 43.86% and 8.68% of all AS events in 84K poplar leaves, respectively. Similar situations were also revealed in plants of Medicago truncatula [31], Populus trichocarpa [32], Arabidopsis [33], Oryza sativa [17], and 15 animal species [34]. Gene structure is related to AS events. Association analysis of the gene composition elements between AS and non-AS genes in our study here showed that the occurrence of AS events in the poplar leaves was positively correlated with intron length, exon length, exon number, and expression level, and negatively correlated with GC content, which is consistent with previous studies in maize [35] and soybean [36]. It was even more distinguishly between plants and animals. For example, plant genes generally have smaller introns and slightly larger exons than human genes. The average exon length in rice [37], Arabidopsis [37], and human [38] is 254 bp, 217 bp, and 170 bp, respectively. While, approximately 50%–70% introns in plant genes are ≤150 bp, whereas they average 5500 bp in human genes [39]. Thus, the differences in AS occurrences between plants and human described above can be understood if one considers that small introns are easier to retain and small exons are easier to skip.

AS effects on poplar growth

The KEGG analysis of AS genes in the poplar leaves showed that several pathways were enriched, including the inositol phosphate metabolism (S4 Fig) and phosphatidylinositol signaling system pathways (S5 Fig). Inositol phosphate is known to play roles in signal transmission [40]. For example, IP3 is a secondary messenger that responds to calcium currents and participates in cell signal transduction [41]. IP6 participates in the DNA repair and mRNA translocation through synergistic or binding activation with corresponding protein factors [42]. IP1 actively responds to the abiotic stresses such as low temperature, drought, and salt [43]. Therefore, the wide occurrence of AS events in inositol phosphate-related genes may provide new insights to understand the growth and development processes of poplar. Notably, the inositol related pathways have been less reported in plants such as Camellia sinensis [44] and wheat [45]. These results suggest that the AS events in inositol phosphate-related genes may be significant in poplar plant production. Another enriched pathway that involved AS genes in the poplar leaves was the mRNA surveillance system (Fig 6), which could effectively remove non-functional RNA fragments caused by AS. In 84K poplar leaves, a total of 41 genes were found to be involved in this pathway, which may help to maintain its growth and development via the nonsense-mediated mRNA decay system, the nonstop mRNA decay system, and the no-go decay system. A well-organized system of this type was proposed previously for the AS process in soybean plants [36]. More studies are required to confirm the existence of such a system, and the function variation of the AS isoforms such as three transcript isoforms of PP2A gene. Also, the AS evens in poplar here were evaluated in this paper by using the second generation sequencing technology. But the shortcomings in read length of the technique might cause the limitation and accuracy of the data although we increased the sequencing depth to minimize this impact. In future research, more validations could be conducted by PCR assay or full length isoform transcriptome analysis that could be complementary in read length and expression abundance.

Conclusions

AS events in the poplar leaves were widely involved in metabolisms that would possibly affect poplar growth and development. AS occurrences were mostly in relation to the gene features such as intron length, exon number, gene expression level, and GC content. Further dissection of the AS isoforms function in the poplar leaves would be significant to understand and improve the mechanism of wooden plants production.

Transcripts of the gene Potri.002G124200.v3.0(PABP1) in the poplar leaves.

(DOCX) Click here for additional data file.

Transcripts alignment of two Illumina sequencing isoforms and two PCR fragments of the gene 6853 in the poplar leaves.

The bases in frame indicated PCR primer. The isoform1 was normal transcript and isoform2 was included an intron. (DOCX) Click here for additional data file.

Splicesome pathway and AS genes distribution in the poplar leaves.

The purple box represents AS genes. (DOCX) Click here for additional data file.

Inositol phosphate metabolism and AS genes distribution in the poplar leaves.

The purple box represents AS genes. (DOCX) Click here for additional data file.

Phosphatidylinositol signaling system and AS genes distribution in the poplar leaves.

The purple box represents AS genes. (DOCX) Click here for additional data file.

The primer information for PCR validation.

(DOCX) Click here for additional data file.

Sequencing alignment results.

(DOCX) Click here for additional data file.

The top 10 enriched genes in each GO category in the poplar leaves.

(DOCX) Click here for additional data file. 15 Jul 2020 PONE-D-20-17398 Genome-Wide Profiling of Alternative Splicing Genes in Populus PLOS ONE Dear Dr. Xu, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. As you will see, both reviewers are quite critical of your manuscript. I have decided to give you a chance to revise your work, but please take this very seriously. A revision consisting only of explaining away the reviewers comments would not be acceptable, you need to do major changes to the work and the writing. Please submit your revised manuscript by Aug 29 2020 11:59PM. 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The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ********** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes ********** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: No ********** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors applied Illumina Next-Generation Sequencing (NGS) technology to profile the alternative splicing (AS) events in the leaf transcriptome of 2-month old Populus 84K, which has not being reported for this specific poplar hybrid. While the results could be useful for future research, there are some shortcomings in this study, making the experimental design and findings to be very preliminary. A major revision is necessary focusing on the shortcomings and comments raised appended below. Thus, I would not recommend it for publication in PloS One in its present form. Shortcomings: 1. The introduction of third-generation long-read sequencing technology (e.g. Pacific Biosciences) has overcome the traditional issues on NGS technology such as bias, false positive and misassembly. While many published research has been using long-read technology to report plant AS characterisation (including Populus species), this study presented the NGS-based findings. To improve the accuracy and impact of the results, the authors should apply long-read technology to make the ‘reference’ isoform library and use NGS data for error correction and expression counting. Alternatively, in the current manuscript, the authors should at least discuss this shortcoming and make clear to the readers about the limitation and accuracy of the data/results. 2. This study lacks a very clear and convincing problem statement and research objective. If the research is aimed to first characterise the unknown AS events in the Poplar hybrid, the samples used should cover all major plant tissues such as root, xylem, phloem etc. and not limited to the only leaf with very low transcript/isoform coverage. AS events have been known to be tissue, environment and individual specific. Also, the replication of the sample in this study seems to be only 1. With only leaf sample being sampled, the authors should justify the significance of using the leaf sample, e.g. specific scientific or economic importance of Poplar leaf, and redirect the findings and discussion toward this angle/direction. 3. There are a number of publications on the AS characterisation from Populus species, including the Populus alba, the parent of this 84K hybrid. This reviewer does not understand why there is no introduction about these scientific reports in the introduction and comparison in the Results/Discussion session. 4. Additional analysis, such as alternative polyadenylation (APA) would add more values and impact to this research and paper. 5. The Materials and Methods section is incomplete with the missing experimental procedure such as expression count, cloning and sequencing, statistical analysis. The current description on the methodology is also not sufficient, clear or detail for reproducibility. 6. The completeness of the transcriptome made in this study is unknown and should be evaluated with analysis such as BUSCO. With only one plant tissue (i.e. leaf) is presented, we have to know how comprehensive or completeness of the leaf transcriptome presented, which will provide a hint on the potential capturing of “all” AS genes. Also, the authors should consider adding GO enrichment analysis and qPCR validation of FPKM expression values. Comments: 1. Lines 1-2: the title has to be more specific (e.g. species, leaf) due to the shortcomings listed in Shortcoming No. 2 mentioned above. 2. Lines 17-20: include percentage for all types of AS events and AS density for the chromosomes discussed. 3. Line 24 and Lines 258-262: a conclusion is missing or weakly summarised, mainly because this paper has no direction as in Shortcoming No. 3 mentioned above. Perhaps the authors can elaborate on how the findings should help to shed light on plant development or wooden plant production. 4. Lines 29-49: Introduction on fundamental information or knowledge (e.g. DNA-RNA transcription, Intron/AS frequency in plants) is not adequate for general readers, including those new to this field/area. The arrangement has to be improved, e.g. the sentence from Lines 34-35 should move forward and place after sentence from lines 30-31. 5. Lines 50-51: expand the literature specific to plants (e.g. angiosperms, gymnosperms, spermatophytes, pteridophytes, lycophytes etc.) and poplar species. 6. Line 53: when “strong resistance” is mentioned, what does it mean? Abiotic? Biotic? Environment? 7. Line 56: add problem statement, objective, justification and rationale of this study by referring to Shortcoming No. 2 mentioned above. 8. Line 62: replication? 9. Line 70: data repository (raw sequencing file) to the public database such as NCBI or ENA? 10. Line 74: define clearly the company and country information 11. Line 76: define clearly which Illumina platform 12. Line 77: what is the read length? 13. Line 78: include quality assessment criteria. Q30? 14. Line 80: cite the genome paper and source (i.e. repository database where the sequences are obtained) 15. Line 80: “.. and the gene…”: the gene is referred to the Populus trichocarpa genes presented in the genome? 16. Line 83: how the transcriptome assembly being done? Software and methodology. 17. Line 85: how the FPKM value being counted and calculated? The methodology is missing. 18. Line 86: any selection criteria for the sequencing results validation? E.g. gene size, frequency of introns, AS pattern type etc. It cannot be too “random”, or else it could be bias 19. Line 90: include materials and methods on “Cloning and Sequencing” for PCR validation 20. Lines 92-95: include reference/website link for databases such as GO and DAVID 21. Lines 94-95: use the term “enrichment analysis” 22. Line 99: statistical analysis is missing (software, type of test and methodology) 23. Table 1: check the typo and alignment. Is the “clean bases” equivalent to “total GB of data obtained”? 24. Lines 128-129: when “some genes had more than one type of AS event” is mentioned, more information or details should be provided or elaborated. 25. Lines 116-129: cite the Table/Figure when describing the number/results, same for remaining sections of result description. 26. Table 2: consider to add a “Total” row to show the sum values 27. Lines 139: give the identifies of the genes when “... all five genes…” are mentioned 28. Lines 141-144: instead of showing specific example without much impact, we suggest to discuss or illustrate more solid data on the validation, e.g. comparison of sizes from NGS and size from PCR/cloning 29. Line 146: indicate the number of chromosomes 30. Lines 146-147: do you mean equal (even) or unequal (uneven) distribution? 31. Lines 151-152: how you judge on this? Visually, this reviewer see chromosomes 1, 5 and 6 have a comparable high density of AS as well 32. Lines 151-152: would it be useful to provide the average AS events/Mb (one mean value) for all chromosomes? 33. Lines 161-170: when mentioning about “XX times higher/lower than” it should be supported by statistics (e.g. P<0.05) and described in the text 34. Lines 173: did the authors verify some of the FPKM using real-time quantitative PCR? Or else, can the FPKM results be trusted? 35. Lines 171-176: please justify the rationale to show/describe this. Or else, it is better to remove it and replace with more useful information, e.g. a list of important genes exclusively expressed (with specific biochemistry importance and interest) in the leaf with unique and interesting splicing events. It will be good to verify the AS pattern and FPKM values of this set of “genes of interest” using cloning and qPCR, respectively. 36. Lines 178-183: without the GO enrichment analysis, the results presented are weak and less meaningful 37. Lines 186-289: do not start a sentence with a number 38. Line 192: why only this pathway being selected? Any justification? 39. Lines 204-209: it will be more informative to compare the AS pattern among Poplar species as several AS papers on Poplar are available 40. Line 211: “higher species” means multicellular form? Please be specific. 41. Lines 216-271: please add citation(s) 42. Lines 221: without only one example for each group (i.e. human and Populus 84k), it is less convincing to represent the entire animal and plant groups, respectively. 43. Lines 227-233: focus more on the comparison within plant groups (with more examples) 44. Line 245: “these pathways” refers to? 45. Lines 259-261: the sentences have to be rephrased and improved 46. Figure legends: the titles and descriptions are too brief. For example, revised them to “1% (w/v) gel electrophoresis of the… 1, DNA marker, ….” for Figure 1, “Genome-wide distribution of the … 19 poplar chromosomes” for Figure 2, “The splicing structure of …” for Figure 6. 47. Supplementary Materials: The authors should consider to include Fig. S1-4 in the main document (or some of them) to provide more depth in the discussion of the results. If the number of figures is too many, maybe can move Fig.1 and Fig. 4 to the supplementary materials. Reviewer #2: The study submitted by Wang et al. is focused on the raw identification of AS events in Populus 84K. The data obtained are interesting and will be usefull in future studies, but the paper need to be reviewed by a English mothertongue and the "Introduction"and "Discussion" text need to be reformulate because it is not fluent. This paper can be accepted with major revisions. See below both major and minor revisions. Please change the genus and species names in italic, in the whole manuscript. Title I suggest to add “spp.” after Populus Abstract The abstract is written very well. Please check the the word “Populus” in always in italic. Just few suggestions. Lines 10-11: Change the step in “Alternative splicing (AS) is a post-transcriptional process common in higher plants and essential for regulation of environmental fitness of plants.” Line 11: change the step in “In the present study, we dissected the poplar alternative splicing events in order to understand their effects on plant growth and development.” Keywords: Please do not use the same words you have in the title. For example, change “Populus” in “Poplar” and eliminate Alternative splicing Introduction The introduction is too short and not structured very well. You report a list of genes known to have isoforms that functionally influence plant stress response as a shopping list. Please reformulate lines 34-49. And add more informations about omic studies carried out on Populus 84k or other poplar genotypes. Lines 31-33: the AS events do not occur in plants only for adaptation to abiotic conditions, so please change this part adding even the biotic response of plants, see and add to the references “Zheng, Z., Appiano, M., Pavan, S., Bracuto, V., Ricciardi, L., Visser, R. G., ... & Bai, Y. (2016). Genome-wide study of the tomato SlMLO gene family and its functional characterization in response to the powdery mildew fungus Oidium neolycopersici. Frontiers in plant science, 7, 380.” and “De Palma, M., Salzano, M., Villano, C., Aversano, R., Lorito, M., Ruocco, M., ... & Tucci, M. (2019). Transcriptome reprogramming, epigenetic modifications and alternative splicing orchestrate the tomato root response to the beneficial fungus Trichoderma harzianum. Horticulture research, 6(1), 1-15.” Lines 34-38: first you have to start the step concerning CCA1 isoforms saying that it is an example. Second, the paper you cited is not the one about the study of CCA1 isoforms, please change the citation and use Seo et al., Plant Cell. 2012, 24: 2427-2442. 10.1105/tpc.112.098723. Third, lines 35-37 are the same of Kwon et al, it is plagiarism! And lines 37-38 are not correct, please reformulate correctly! Materials and methods Please provide information about biological and technical replicates Some details are missing. Add for each product used the company and the location (e.g. line 64 Aidlab, line 65 Takara…). Line 64: change “trizol” in “Trizol” Line 67: eliminate “Nanodrop” in brackets Line 99: change in “significantly” Results The results are very interesting but not very well written. There is an abuse of the verb “had undergone” please change the text to avoid this term. Change Table 2 in Figure 1 and modify the other figures accordingly Table 1 legend is not complete! Line 141: which gene are you talking about? In the figure 1 legend it is named 6835, in the text 6853. Line 144: add a Supplementary figure with sequencing results, for example an alignment between cloned fragments and Illumina results. Figure 1: change the legend in “Validation of AS events. PCR results of six genes randomly selected. 1: marker (ADD THE MARKER TYPE!), 2: gene 1775, 3: gene 2340, 4: gene 14298, 5: gene 15649, 6: gene 5375, 7: gene 6835. Line 146: add information about the genome used Line 196: change “isoform” in “isoform” Line 198: check the font Discussion Line 212: add a reference Line 219: move the citations at the end of the step. ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? 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Please note that Supporting Information files do not need this step. 2 Sep 2020 Journal Requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Reply: We ensure that your manuscript meets PLOS ONE's style requirements 2. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, Reply: We deposited our transcription data in NCBI GenBank and mentioned in the manuscript and cover letter. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 'Funding This work was supported by the National 267 Natural Science Foundation of China (#31672189) and Beijing Forestry University Undergraduate Training Program for Inovation and Entrepreneurship' We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. a. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 'The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.' b. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Reply: The funding information was described in cover letter 4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Reply: We ensure that the figures adhere fully to the guidelines 5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ Reply: It was done 6. Please include a copy of Table 3 which you refer to in your text on page 6. Reply: It was done 7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Reply: It was done Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Partly Reviewer #2: Partly Reply: We modified the conclusions 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes Reply: We modified the related part 3. Have the authors made all data underlying the findings in their manuscript fully available? Reviewer #1: No Reviewer #2: Yes Reply: We modified the related part 4. Is the manuscript presented in an intelligible fashion and written in standard English? Reviewer #1: No Reviewer #2: No Reply: We modified the writing 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reply: It was done Reviewer #1: 1. The introduction of third-generation long-read sequencing technology (e.g. Pacific Biosciences) has overcome the traditional issues on NGS technology such as bias, false positive and misassembly. While many published research has been using long-read technology to report plant AS characterisation (including Populus species), this study presented the NGS-based findings. To improve the accuracy and impact of the results, the authors should apply long-read technology to make the ‘reference’ isoform library and use NGS data for error correction and expression counting. Alternatively, in the current manuscript, the authors should at least discuss this shortcoming and make clear to the readers about the limitation and accuracy of the data/results. Reply: I agree with the reviewer’s points. Definitely, NGS technology has some shortcomings such as reading length in comparison with the whole length transcriptome technology but it also has the advancement in expression abundance. We did modification in discussion about the limitation and accuracy of NGS technology in AS characterisation. 2. This study lacks a very clear and convincing problem statement and research objective. If the research is aimed to first characterise the unknown AS events in the Poplar hybrid, the samples used should cover all major plant tissues such as root, xylem, phloem etc. and not limited to the only leaf with very low transcript/isoform coverage. AS events have been known to be tissue, environment and individual specific. Also, the replication of the sample in this study seems to be only 1. With only leaf sample being sampled, the authors should justify the significance of using the leaf sample, e.g. specific scientific or economic importance of Poplar leaf, and redirect the findings and discussion toward this angle/direction. Reply: AS events were much complicated and specifically by tissues. Our study here just focused on leaf and hopefully to provide some initial opinions of its effect on plant growth. Thus we modified the description of our objective. 3. There are a number of publications on the AS characterization from Populus species, including the Populus alba, the parent of this 84K hybrid. This reviewer does not understand why there is no introduction about these scientific reports in the introduction and comparison in the Results/Discussion session. Reply: We add some more publications of populus in introduction and discussion sessions. 4. Additional analysis, such as alternative polyadenylation (APA) would add more values and impact to this research and paper. Reply: We agree with the reviewer’s point. The manuscript here is an initial report. We hope to conduct the AS mechanism analysis including APA in next. 5. The Materials and Methods section is incomplete with the missing experimental procedure such as expression count, cloning and sequencing, statistical analysis. The current description on the methodology is also not sufficient, clear or detail for reproducibility. Reply: We modified the Materials and Methods section in details 6. The completeness of the transcriptome made in this study is unknown and should be evaluated with analysis such as BUSCO. With only one plant tissue (i.e. leaf) is presented, we have to know how comprehensive or completeness of the leaf transcriptome presented, which will provide a hint on the potential capturing of “all” AS genes. Also, the authors should consider adding GO enrichment analysis and qPCR validation of FPKM expression values. Reply: For the transcription data evaluation, some modifications were added in the methods and results sessions. 1. Lines 1-2: the title has to be more specific (e.g. species, leaf) due to the shortcomings listed in Shortcoming No. 2 mentioned above. Reply: It was done 2. Lines 17-20: include percentage for all types of AS events and AS density for the chromosomes discussed. Reply: It was done 3. Line 24 and Lines 258-262: a conclusion is missing or weakly summarised, mainly because this paper has no direction as in Shortcoming No. 3 mentioned above. Perhaps the authors can elaborate on how the findings should help to shed light on plant development or wooden plant production. Reply: It was done 4. Lines 29-49: Introduction on fundamental information or knowledge (e.g. DNA-RNA transcription, Intron/AS frequency in plants) is not adequate for general readers, including those new to this field/area. The arrangement has to be improved, e.g. the sentence from Lines 34-35 should move forward and place after sentence from lines 30-31. Reply: The first paragraph described the general process of the mRNA formation. And the second paragraph turned to the changed cases of AS. I do think this order might be helpful for the new to follow it. Therefore, I just changed a little. 5. Lines 50-51: expand the literature specific to plants (e.g. angiosperms, gymnosperms, spermatophytes, pteridophytes, lycophytes etc.) and poplar species. Reply: Several publications were added. 6. Line 53: when “strong resistance” is mentioned, what does it mean? Abiotic? Biotic? Environment? Reply: It was modified. 7. Line 56: add problem statement, objective, justification and rationale of this study by referring to Shortcoming No. 2 mentioned above. Reply: It was modified. 8. Line 62: replication? Reply: It was modified. 9. Line 70: data repository (raw sequencing file) to the public database such as NCBI or ENA? Reply: It was modified. 10. Line 74: define clearly the company and country information Reply: It was modified. 11. Line 76: define clearly which Illumina platform Reply: It was done. 12. Line 77: what is the read length? Reply: It was in results session 13. Line 78: include quality assessment criteria. Q30? Reply: It was in results session 14. Line 80: cite the genome paper and source (i.e. repository database where the sequences are obtained) Reply: It was modified. 15. Line 80: “.. and the gene…”: the gene is referred to the Populus trichocarpa genes presented in the genome? Reply: Both populus species of 84K and Populus trichocarpa have similar genome composition. Thus, we used the sequenced genome of Populus trichocarpa as the reference to define the gene reads locus, and further determine the whole gene sequence via NCBI blast. Some modifications were done. 16. Line 83: how the transcriptome assembly being done? Software and methodology. Reply: Matrix clean reads were aligned to the Populus trichocarpa genome to define the gene reads locus. 17. Line 85: how the FPKM value being counted and calculated? The methodology is missing. Reply: It was completed. 18. Line 86: any selection criteria for the sequencing results validation? E.g. gene size, frequency of introns, AS pattern type etc. It cannot be too “random”, or else it could be bias Reply: It was modified 19. Line 90: include materials and methods on “Cloning and Sequencing” for PCR validation Reply: It was modified 20. Lines 92-95: include reference/website link for databases such as GO and DAVID Reply: It was done 21. Lines 94-95: use the term “enrichment analysis” Reply: It was done 22. Line 99: statistical analysis is missing (software, type of test and methodology) Reply: It was done 23. Table 1: check the typo and alignment. Is the “clean bases” equivalent to “total GB of data obtained”? Reply: It was modified 24. Lines 128-129: when “some genes had more than one type of AS event” is mentioned, more information or details should be provided or elaborated. Reply: It was modified 25. Lines 116-129: cite the Table/Figure when describing the number/results, same for remaining sections of result description. Reply: It was modified 26. Table 2: consider to add a “Total” row to show the sum values Reply: It was modified 27. Lines 139: give the identifies of the genes when “... all five genes…” are mentioned Reply: It was modified 28. Lines 141-144: instead of showing specific example without much impact, we suggest to discuss or illustrate more solid data on the validation, e.g. comparison of sizes from NGS and size from PCR/cloning Reply: It was modified 29. Line 146: indicate the number of chromosomes Reply: It was modified 30. Lines 146-147: do you mean equal (even) or unequal (uneven) distribution? Reply: It was modified 31. Lines 151-152: how you judge on this? Visually, this reviewer see chromosomes 1, 5 and 6 have a comparable high density of AS as well Reply: It was modified 32. Lines 151-152: would it be useful to provide the average AS events/Mb (one mean value) for all chromosomes? Reply: It showed in table2 33. Lines 161-170: when mentioning about “XX times higher/lower than” it should be supported by statistics (e.g. P<0.05) and described in the text Reply: It was modified 34. Lines 173: did the authors verify some of the FPKM using real-time quantitative PCR? Or else, can the FPKM results be trusted? Reply: We had test 6 genes as shown in Fig1. The results showed the tendency of the isoforms FPKM was identical with the transcriptome data. Thus we verify that the description here can be trusted. 35. Lines 171-176: please justify the rationale to show/describe this. Or else, it is better to remove it and replace with more useful information, e.g. a list of important genes exclusively expressed (with specific biochemistry importance and interest) in the leaf with unique and interesting splicing events. It will be good to verify the AS pattern and FPKM values of this set of “genes of interest” using cloning and qPCR, respectively. Reply: These descriptions were removed. 36. Lines 178-183: without the GO enrichment analysis, the results presented are weak and less meaningful Reply: It was modified 37. Lines 186-289: do not start a sentence with a number Reply: It was modified 38. Line 192: why only this pathway being selected? Any justification? Reply: In the other AS studies, mRNA surveillance pathway was less reported. AS events probably caused more invalid RNAs that might influence the regular cell activity. Thus, mRNA surveillance pathway is significant for plant growth. We did some modification here and further explained in discussion session. 39. Lines 204-209: it will be more informative to compare the AS pattern among Poplar species as several AS papers on Poplar are available Reply: It was done 40. Line 211: “higher species” means multicellular form? Please be specific. Reply: It was modified 41. Lines 216-271: please add citation(s) Reply: It was modified 42. Lines 221: without only one example for each group (i.e. human and Populus 84k), it is less convincing to represent the entire animal and plant groups, respectively. Reply: It was modified 43. Lines 227-233: focus more on the comparison within plant groups (with more examples) Reply: For plants, we placed the populus, maize and soybean expamples there. We also think that the comparison between plants and human could also enance the point. Thus, some modifications were done 44. Line 245: “these pathways” refers to? Reply: It was modified 45. Lines 259-261: the sentences have to be rephrased and improved Reply: It was modified 46. Figure legends: the titles and descriptions are too brief. For example, revised them to “1% (w/v) gel electrophoresis of the… 1, DNA marker, ….” for Figure 1, “Genome-wide distribution of the … 19 poplar chromosomes” for Figure 2, “The splicing structure of …” for Figure 6. Reply: It was modified 47. Supplementary Materials: The authors should consider to include Fig. S1-4 in the main document (or some of them) to provide more depth in the discussion of the results. If the number of figures is too many, maybe can move Fig.1 and Fig. 4 to the supplementary materials. Reply: It was modified Reviewer #2: The study submitted by Wang et al. is focused on the raw identification of AS events in Populus 84K. The data obtained are interesting and will be usefull in future studies, but the paper need to be reviewed by a English mothertongue and the "Introduction"and "Discussion" text need to be reformulate because it is not fluent. Reply: The manuscript was reviewed by Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac) for editing the English text. Please change the genus and species names in italic, in the whole manuscript. Reply: It was modified Title I suggest to add “spp.” after Populus Reply: The populus here is just a hybrid variety. It was modified Abstract The abstract is written very well. Please check the the word “Populus” in always in italic. Just few suggestions. Reply: It was modified Lines 10-11: Change the step in “Alternative splicing (AS) is a post-transcriptional process common in higher plants and essential for regulation of environmental fitness of plants.” Reply: It was modified Line 11: change the step in “In the present study, we dissected the poplar alternative splicing events in order to understand their effects on plant growth and development.” Reply: It was modified Keywords: Please do not use the same words you have in the title. For example, change “Populus” in “Poplar” and eliminate Alternative splicing Reply: It was modified Introduction The introduction is too short and not structured very well. You report a list of genes known to have isoforms that functionally influence plant stress response as a shopping list. Please reformulate lines 34-49. And add more informations about omic studies carried out on Populus 84k or other poplar genotypes. Reply: It was modified Lines 31-33: the AS events do not occur in plants only for adaptation to abiotic conditions, so please change this part adding even the biotic response of plants, see and add to the references “Zheng, Z., Appiano, M., Pavan, S., Bracuto, V., Ricciardi, L., Visser, R. G., ... & Bai, Y. (2016). Genome-wide study of the tomato SlMLO gene family and its functional characterization in response to the powdery mildew fungus Oidium neolycopersici. Frontiers in plant science, 7, 380.” and “De Palma, M., Salzano, M., Villano, C., Aversano, R., Lorito, M., Ruocco, M., ... & Tucci, M. (2019). Transcriptome reprogramming, epigenetic modifications and alternative splicing orchestrate the tomato root response to the beneficial fungus Trichoderma harzianum. Horticulture research, 6(1), 1-15.” Reply: It was modified Lines 34-38: first you have to start the step concerning CCA1 isoforms saying that it is an example. Second, the paper you cited is not the one about the study of CCA1 isoforms, please change the citation and use Seo et al., Plant Cell. 2012, 24: 2427-2442. 10.1105/tpc.112.098723. Third, lines 35-37 are the same of Kwon et al, it is plagiarism! And lines 37-38 are not correct, please reformulate correctly! Reply: All were modified Materials and methods Please provide information about biological and technical replicates Reply: It was modified Some details are missing. Add for each product used the company and the location (e.g. line 64 Aidlab, line 65 Takara…). Reply: It was modified Line 64: change “trizol” in “Trizol” Reply: It was modified Line 67: eliminate “Nanodrop” in brackets Reply: It was modified Line 99: change in “significantly” Reply: It was modified Results The results are very interesting but not very well written. There is an abuse of the verb “had undergone” please change the text to avoid this term. Reply: It was modified Change Table 2 in Figure 1 and modify the other figures accordingly Reply: It was modified Table 1 legend is not complete! Reply: It was modified Line 141: which gene are you talking about? In the figure 1 legend it is named 6835, in the text 6853. Reply: It was modified Line 144: add a Supplementary figure with sequencing results, for example an alignment between cloned fragments and Illumina results. Reply: It was modified Figure 1: change the legend in “Validation of AS events. PCR results of six genes randomly selected. 1: marker (ADD THE MARKER TYPE!), 2: gene 1775, 3: gene 2340, 4: gene 14298, 5: gene 15649, 6: gene 5375, 7: gene 6835. Reply: It was modified Line 146: add information about the genome used Reply: It was modified Line 196: change “isoform” in “isoform” Reply: It was done Line 198: check the font Reply: It was done Discussion Line 212: add a reference Reply: It was done Line 219: move the citations at the end of the step. Reply: It was done Submitted filename: Response to Reviewers.doc Click here for additional data file. 6 Oct 2020 PONE-D-20-17398R1 Genome-Wide Profiling of Alternative Splicing Genes in Hybrid Poplar (P.alba×P.glandulosa cv.84K) Leaf PLOS ONE Dear Dr. Xu, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please address the minor comments of Reviewer 1. In addition, please check the writing carefully. A rapid reading finds many small mistakes, such as "Chromosomes 10" (should be "Chromosome 10") in the Abstract, or "Of thm" (should be "Of them") on line 182. Please submit your revised manuscript by Nov 20 2020 11:59PM. 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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Marc Robinson-Rechavi Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The revised manuscript have shown great improvement and authors have addressed most of the reviewers' comments. The manuscript is ready for publication if authors can address some minor comments as below: 1. Please be careful with typo and some of terms used as they are not accurate. Please make changes to these issues in your revised documents: Line 141: " stand error" to "standard error" Line 360: "...evaluated initially" to "...evaluated in this paper..." Line 361: "...reading length..." to "..read length.." Line 363: "...sequencing amounts.." to "...sequencing depth..." Line 363: "...In next..." to "In future research, ,,," Line 364: "...whole length..." to "...full length isoform..." Line 154: change to "...showed it is comprehensive..." Line 378: sequencing alignment results 2. Line 141: which multiple comparison test is used? 3. "...mRNA fragment randomization test, insert length test, and transcriptome sequencing data saturation test...". Any supporting table or figure for this? 4. The authors mentioned that the description of the objective has been modified to tailor to this study that focus on leaf as the major organ and to generate some initial data. However, I could not find the revised sentence where I expect to have both revision to the objective and justification of using leaf to be in both abstract and last paragraph of introduction. Also, please include the section, paragraph or line information in the authors' response to help the reviewer to identify the revision (at least for the major revision). 5. The conclusion in the abstract and main text body is not specifically linked to the objective of using leaf as sample for the AS study. Reviewer #2: All my comments have been addressed, only publication number 6 is not correctly named. Must be De Palma M et al. ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. 21 Oct 2020 Reviewer #1: The revised manuscript have shown great improvement and authors have addressed most of the reviewers' comments. The manuscript is ready for publication if authors can address some minor comments as below: 1. Please be careful with typo and some of terms used as they are not accurate. Please make changes to these issues in your revised documents: Line 141: " stand error" to "standard error" Line 360: "...evaluated initially" to "...evaluated in this paper..." Line 361: "...reading length..." to "..read length.." Line 363: "...sequencing amounts.." to "...sequencing depth..." Line 363: "...In next..." to "In future research, ,,," Line 364: "...whole length..." to "...full length isoform..." Line 154: change to "...showed it is comprehensive..." Line 378: sequencing alignment results Reply: All were corrected 2. Line 141: which multiple comparison test is used? Reply: Duncan's multiple range test was used in our experiment. The description was modified in the method section (Statistic analysis). 3. "...mRNA fragment randomization test, insert length test, and transcriptome sequencing data saturation test...". Any supporting table or figure for this? Reply: The description was from the sequencing company but they did not provide the relative data. Thus, the sentence was removed. 4. The authors mentioned that the description of the objective has been modified to tailor to this study that focus on leaf as the major organ and to generate some initial data. However, I could not find the revised sentence where I expect to have both revision to the objective and justification of using leaf to be in both abstract and last paragraph of introduction. Also, please include the section, paragraph or line information in the authors' response to help the reviewer to identify the revision (at least for the major revision). Reply: It was modified in line 13-14 in abstract, and last paragraph of introduction. 5. The conclusion in the abstract and main text body is not specifically linked to the objective of using leaf as sample for the AS study. Reply: It was modified in line 23-25 in abstract, and main text body Reviewer #2: All my comments have been addressed, only publication number 6 is not correctly named. Must be De Palma M et al. Reply: It was corrected. Submitted filename: Response to reviewers1.doc Click here for additional data file. 23 Oct 2020 Genome-Wide Profiling of Alternative Splicing Genes in Hybrid Poplar (P.alba×P.glandulosa cv.84K) Leaves PONE-D-20-17398R2 Dear Dr. Xu, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Marc Robinson-Rechavi Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: 3 Nov 2020 PONE-D-20-17398R2 Genome-Wide Profiling of Alternative Splicing Genes in Hybrid Poplar (P.alba×P.glandulosa cv.84K) Leaves Dear Dr. Xu: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Marc Robinson-Rechavi Academic Editor PLOS ONE

43 in total

1. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.

Authors: M Ashburner; C A Ball; J A Blake; D Botstein; H Butler; J M Cherry; A P Davis; K Dolinski; S S Dwight; J T Eppig; M A Harris; D P Hill; L Issel-Tarver; A Kasarskis; S Lewis; J C Matese; J E Richardson; M Ringwald; G M Rubin; G Sherlock
Journal: Nat Genet Date: 2000-05 Impact factor: 38.330

2. Genomewide comparative analysis of alternative splicing in plants.

Authors: Bing-Bing Wang; Volker Brendel
Journal: Proc Natl Acad Sci U S A Date: 2006-04-21 Impact factor: 11.205

3. Marker assisted selection of low phytic acid trait in maize (Zea mays L.).

Authors: S Sureshkumar; P Tamilkumar; N Senthil; P Nagarajan; A U Thangavelu; M Raveendran; S Vellaikumar; K N Ganesan; R Balagopal; G Vijayalakshmi; V Shobana
Journal: Hereditas Date: 2014-03-13 Impact factor: 3.271

Review 4. Complexity of the alternative splicing landscape in plants.

Authors: Anireddy S N Reddy; Yamile Marquez; Maria Kalyna; Andrea Barta
Journal: Plant Cell Date: 2013-10-31 Impact factor: 11.277

5. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.

Authors: Cole Trapnell; Brian A Williams; Geo Pertea; Ali Mortazavi; Gordon Kwan; Marijke J van Baren; Steven L Salzberg; Barbara J Wold; Lior Pachter
Journal: Nat Biotechnol Date: 2010-05-02 Impact factor: 54.908

6. Genome-wide analysis of alternative splicing in Volvox carteri.

Authors: Arash Kianianmomeni; Cheng Soon Ong; Gunnar Rätsch; Armin Hallmann
Journal: BMC Genomics Date: 2014-12-16 Impact factor: 3.969

7. Deep RNA sequencing reveals a high frequency of alternative splicing events in the fungus Trichoderma longibrachiatum.

Authors: Bin-Bin Xie; Dan Li; Wei-Ling Shi; Qi-Long Qin; Xiao-Wei Wang; Jin-Cheng Rong; Cai-Yun Sun; Feng Huang; Xi-Ying Zhang; Xiao-Wei Dong; Xiu-Lan Chen; Bai-Cheng Zhou; Yu-Zhong Zhang; Xiao-Yan Song
Journal: BMC Genomics Date: 2015-02-06 Impact factor: 3.969

8. Gene Expression Profiling of Development and Anthocyanin Accumulation in Kiwifruit (Actinidia chinensis) Based on Transcriptome Sequencing.

Authors: Wenbin Li; Yifei Liu; Shaohua Zeng; Gong Xiao; Gan Wang; Ying Wang; Ming Peng; Hongwen Huang
Journal: PLoS One Date: 2015-08-24 Impact factor: 3.240

9. Genome-Wide Study of the Tomato SlMLO Gene Family and Its Functional Characterization in Response to the Powdery Mildew Fungus Oidium neolycopersici.

Authors: Zheng Zheng; Michela Appiano; Stefano Pavan; Valentina Bracuto; Luigi Ricciardi; Richard G F Visser; Anne-Marie A Wolters; Yuling Bai
Journal: Front Plant Sci Date: 2016-04-06 Impact factor: 5.753

10. Analysis of Alternative Splicing and Alternative Polyadenylation in Populus alba var. pyramidalis by Single-Molecular Long-Read Sequencing.

Authors: Hongyin Hu; Wenlu Yang; Zeyu Zheng; Zhimin Niu; Yongzhi Yang; Dongshi Wan; Jianquan Liu; Tao Ma
Journal: Front Genet Date: 2020-02-07 Impact factor: 4.599

1 in total

1. Functional analysis of PagNAC045 transcription factor that improves salt and ABA tolerance in transgenic tobacco.

Authors: Xuemei Zhang; Zihan Cheng; Gaofeng Fan; Wenjing Yao; Wei Li; Sixue Chen; Tingbo Jiang
Journal: BMC Plant Biol Date: 2022-05-25 Impact factor: 5.260

1 in total