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Whole transcriptome sequencing reveals genes involved in plastid/chloroplast division and development are regulated by the HP1/DDB1 at an early stage of tomato fruit development

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Abstract

The phenotype of tomato high pigment-1 (hp1) mutant is characterized by overproduction of pigments including chlorophyll and carotenoids during fruit development and ripening. Although the increased plastid compartment size has been thought to largely attribute to the enhanced pigmentation, the molecular aspects of how the HP1/DDB1 gene manipulates plastid biogenesis and development are largely unknown. In the present study, we compared transcriptome profiles of immature fruit pericarp tissue between tomato cv. Ailsa Craig (WT) and its isogenic hp1 mutant. Over 20 million sequence reads, representing > 1.6 Gb sequence data per sample, were generated and assembled into 21,972 and 22,167 gene models in WT and hp1, respectively, accounting for over 60 % official gene models in both samples. Subsequent analyses revealed that 8,322 and 7,989 alternative splicing events, 8833 or 8510 extended 5′-UTRs, 8,263 or 8,939 extended 3′-UTRs, and 1,136 and 1,133 novel transcripts, exist in WT and hp1, respectively. Significant differences in expression level of 880 genes were detected between the WT and hp1, many of which are involved in signaling transduction, transcription regulation and biotic and abiotic stresses response. Distinctly, RNA-seq datasets, quantitative RT-PCR analyses demonstrate that, in hp1 mutant pericarp tissue at early developmental stage, an apparent expression alteration was found in several regulators directly involved in plastid division and development. These results provide a useful reference for a more accurate and more detailed characterization of the molecular process in the development and pigmentation of tomato fruits.

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Abbreviations

A5SS:

Alternative 5′ splice sites

A3SS:

Alternative 3′ splice sites

ABA:

Abscisic acid

AFE:

Alternative first exons

ALA:

Aminolevulinate

ALE:

Alternative last exons

DAP:

Days after pollination

DDB1 :

UV-DAMAGED DNA BINDING PROTEIN-1

DET1:

DE-ETIOLATED-1

FDR:

False discovery rate

FPKM:

Fragments Per Kilobase of exon per million fragments mapped reads

GLK:

Golden 2-like

GO:

Gene Ontology

HP :

High-Pigment

MXE:

Mutually exclusive exons

nr databases:

Non-redundant databases

RI:

Retained intron

RIN:

RNA Integrity Number

SE:

Skipped exons

SGN:

SOL Genomics Network

SNP:

Single nucleotide polymorphism

SRA:

Sequence read archive

TSS:

Transcription start site

U :

Uniform ripening

UTRs:

Untranslated regions

WT:

Wild type

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Acknowledgments

This work was supported by the National Science Fund for Distinguished Young Scholars (No. 30825030), the National Science and Technology Key Project of China (No. 2011CB100401), the National Natural Science Foundation of China (No. 31171179), and Advanced Program of Doctoral Fund of Ministry of Education of China (20110181130009) for Y. Liu, and the Fundamental Research Funds for the Central Universities for S. Huang.

Author information

Correspondence to Xiao Li or Yongsheng Liu.

Additional information

Xiaofeng Tang, Zizhi Tang, Shengxiong Huang, and Jikai Liu contributed equally.

Electronic supplementary material

Below is the link to the electronic supplementary material.

425_2013_1942_MOESM1_ESM.doc

Supplemental Table S1 The sequences of specific primers used in RT-PCR for validation of novel untranslated region, novel transcripts, and alternative splicing isoforms. “Locus” shows the exact location of each gene relative to S. lycopersicum genome (the SL2.40 version). “Gene model” displays the corresponding official gene ID in SGN (http://solgenomics.net). Primer A (upper) and primer B (lower) represent the positive primer for detected gene sequence in official gene models and determined by RNA-Seq, respectively. Primer C represents the negative primer sequence (DOC 49 kb)

425_2013_1942_MOESM2_ESM.doc

Supplemental Table S2 Primer sequences used in RT-PCR validation of differentially expressed genes involved in chlorophyll metabolism, plastid division and other biological processes in immature fruits of WT and hp1 mutant. “Gene” and “Gene model” represent the gene symbol and corresponding official gene ID in SGN (http://solgenomics.net), respectively(DOC 69 kb)

425_2013_1942_MOESM3_ESM.xls

Supplemental Table S3 Annotation results of transcriptome assembly. The “Gene model” stands for the corresponding official gene ID in SGN (http://solgenomics.net). “Direction” represents the positive/negative strand of DNA strand. “FPKM of WT” and “FPKM of hp1” means FPKM value of the transcripts in WT and hp1 mutant, respectively. The “log (fold_change)” is the value of log (FPKM of hp1/FPKM of WT). The “p_value” is calculated by the Fisher’s exact test. “Seq_Description”, “Seq_Length”, “GOs”, “Enzyme_Codes” and “sequence” are the annotation for putative gene function, length, GO annotation, enzyme annotation and the exact nucleotide sequence, respectively. “--NA--” indicates the transcripts in WT or hp1 mutant without significant hit(XLS 31165 kb)

425_2013_1942_MOESM4_ESM.xls

Supplemental Table S4 Statistical results of indentified and extended untranslated regions (UTRs). The sheet “WT_5” and “WT_3” displays statistical results of indentified and extended 5′- and 3′- UTRs of transcripts in WT, respectively. The sheet “hp1_5” and “hp1_3” displays statistical results of indentified and extended 5′- and 3′- UTRs of transcripts in hp1 mutant, respectively. In each sheet, the following contents are included. The “Gene model” represents official gene ID in SGN (http://solgenomics.net). “identified UTRs” shows the length of identified 5′ or 3′ end boundaries, and “extended UTRs” shows the length of extended 5′ or 3′ end boundaries which are extended relative to the end of official gene models in WT, respectively(XLS 3390 kb)

425_2013_1942_MOESM5_ESM.xls

Supplemental Table S5 Statistical results of five types of alternative splicing events. “WT only”, “hp1 only” and “WT&hp1” stand for the alternative splicing events in AC, hp1 mutant and both genotypes, respectively. “Gene model” displays the corresponding official gene ID in SGN (http://solgenomics.net). “Alternative splicing events” indicates specific alternative splicing events for each gene(XLS 1712 kb)

425_2013_1942_MOESM6_ESM.xls

Supplemental Table S6 Statistical results of novel transcripts. “WT”, “hp1” and “WT&hp1″ stand for the novel transcripts in WT, hp1 mutant and both genotypes, respectively. “Gene model” displays the corresponding official gene ID in SGN (http://solgenomics.net). “Direction” represents the positive or negative direction of the DNA strand. “Locus” shows the exact location of each gene relative to S. lycopersicum genome (the SL2.40 version). “Seq_Description”, “Seq_Length” and “sequence” represent the annotation for putative gene function, length, and the exact nucleotide sequence, respectively. “--NA--” indicates the transcripts in WT or hp1 mutant without significant hit, comparing to the official annotation (nr database) (XLS 1274 kb)

425_2013_1942_MOESM7_ESM.xls

Supplemental Table S7 Genes differentially expressed in the WT and hp1 mutant. The sheets “up diff” and “down diff” indicate genes were significantly up-regulated or down-regulated in the hp1 mutant compared to those in the WT, respectively (p < 0.05). “Gene model” represents the corresponding official gene ID in SGN (http://solgenomics.net). “Locus” shows the exact location of each gene relative to S. lycopersicum genome (the SL2.40 version). “FPKM of WT” and “FPKM of hp1” stand for the FPKM value of the transcripts in WT and hp1 mutant, respectively. The “ln (fold_change)” is the value of ln (FPKM of hp1/FPKM of WT). The “p_value” is determined by the Student’s t-test(XLS 548 kb)

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Tang, X., Tang, Z., Huang, S. et al. Whole transcriptome sequencing reveals genes involved in plastid/chloroplast division and development are regulated by the HP1/DDB1 at an early stage of tomato fruit development. Planta 238, 923–936 (2013). https://doi.org/10.1007/s00425-013-1942-9

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Keywords

  • Alternative splicing
  • DDB1
  • Pigmentation
  • Plastid/Chloroplast
  • Tomato
  • Transcriptome