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Protoplast isolation and transcriptome analysis of developing xylem in Pinus massoniana (Pinaceae)

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Abstract

Background

With active physiological and biochemical activities, tissue-specific protoplasts from cambial derivatives, could serve as a specific source for information on xylogenesis for softwood species resistant to stable genetic transformation and lacking available mutants.

Methods and results

In this study, protoplasts were isolated from developing xylem of the Chinese red pine, Pinus massoniana, by enzymolysis. High-quality RNAs were extracted from developing xylem and their protoplasts for constructing transcriptome libraries. Using Illumina HiSeq 2500 PE150 platform, a total of 362,328,426 clean paired-end reads (54.35G) were generated from multiple cDNA libraries and assembled into 146,422 unigenes. The transcriptome data were further analysed to identify 1567 differentially expressed genes (DEGs) between the isolated protoplasts and developing xylem of P. massoniana (Masson pine), 1126 DEGs were upregulated in protoplasts relative to developing xylem cells and 441 were downregulated. Most of the differentially expressed genes in biological process terms are related to plant response, which may be due to the response to cell wall removal. Further, the expression pattern of 71 unigenes involved in lignin biosynthesis was verified by RNA-seq.

Conclusions

This study is the first to report the transcriptome profiles of the developing xylem and its protoplasts of coniferous trees, which provide a new perspective and valuable resource for tracking transcriptional regulatory events in wood formation of Masson pine.

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Data availability

The raw data has been submitted to SRA database with accession numbers SRR12596930 (PM_XP2), SRR12596931 (PM_XP1), SRR12596932 (PM_X2) and SRR12596933 (PM_X1).

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Funding

This work was supported by Grants from the Open Research Fund of Guangxi Key Laboratory of Superior Timber Trees Resource Cultivation (17-B-03-01), the Science and Technology Major Program of Guangxi (AA17204087-1) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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  1. Tengfei Shen and Mengxuan Xu contributed equally to this work.

Authors and Affiliations

  1. Guangxi Key Laboratory of Superior Timber Trees Resource Cultivation, Guangxi Forestry Research Institute, Nanning, 530002, China

    Tengfei Shen, Yuanheng Feng & Zhangqi Yang

  2. Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics and Biotechnology Ministry of Education, Nanjing Forestry University, Nanjing, 210037, China

    Tengfei Shen, Mengxuan Xu, Haoran Qi & Meng Xu

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  1. Tengfei Shen
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Contributions

MX participated in the conception and design of the experiments and reviewed a draft of the manuscript. TS and MXX participated in conducting the experiments and analyzing the data and drafted the manuscript. HQ participated in conducting the experiments and analyzing the data. FY and ZY participated in the conception and design of the experiments. All authors read and approved the final version of the manuscript.

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Correspondence to Meng Xu.

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Supplementary Information

Below is the link to the electronic supplementary material.

11033_2021_6995_MOESM1_ESM.jpg

Supplementary file1 (JPG 44 kb). Supplementary Figure S1 Active protoplasts under light microscope of big (red) and small cell (green

11033_2021_6995_MOESM2_ESM.jpg

Supplementary file2 (JPG 36 kb). Supplementary Figure S2 Agilent 2100 Bioanalyzer analysis of total RNA extracted from developing xylem and its protoplasts of masson pine, PM1/2_X-developing xylem, PM1/2_XP- protoplasts isolated from developing xylem

11033_2021_6995_MOESM3_ESM.jpg

Supplementary file3 (JPG 303 kb). Supplementary Figure S3 Annotation information of assembled unigenes in masson pine. (A) Number and percentage of unigenes annotated to the seven databases. (B) A seven-way Venn diagram, which indicating 9,994 unigenes matched in seven different databases

11033_2021_6995_MOESM4_ESM.jpg

Supplementary file4 (JPG 738 kb). Supplementary Figure S4 Unigenes annotation of GO, KOG, and KEGG databases. (A) GO categories of all unigenes. (B) KOG function classification. (C) KEGG annotation of putative proteins. The y-axis indicates the name of the KEGG metabolic pathway. The x-axis indicates the percentage of the number of unigenes annotated to the pathway out of the total number of unigenes annotated

11033_2021_6995_MOESM5_ESM.jpg

Supplementary file5 (JPG 265 kb). Supplementary Figure S5 The FPKM of unigenes in PM_XP and PM_X. (A) The FPKM boxplots of all unigenes in PM_X and PM_XP. (B) The distribution of FPKM values of all unigenes in PM_X and PM_XP

Supplementary file6 (JPG 76 kb). Supplementary Figure S6 The PCA analysis results of PM_XP and PM_X

Supplementary file7 (JPG 1,382 kb). Supplementary Figure S7 GO enrichment analysis of DEGs

Supplementary file8 (JPG 200 kb). Supplementary Figure S8 GO enrichment of PM_X-specific unigenes

Supplementary file9 (JPG 192 kb). Supplementary Figure S9 KEGG enrichment of PM_X -specific unigenes

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Supplementary file11 (XLSX 223 kb)

Supplementary file12 (XLSX 1197 kb)

Supplementary file13 (XLSX 12 kb)

Supplementary file14 (XLSX 10 kb)

Supplementary file15 (XLSX 12 kb)

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Shen, T., Xu, M., Qi, H. et al. Protoplast isolation and transcriptome analysis of developing xylem in Pinus massoniana (Pinaceae). Mol Biol Rep 49, 1857–1869 (2022). https://doi.org/10.1007/s11033-021-06995-6

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