DNA AND CELL BIOLOGY Volume 36, Number 11, 2017 ª Mary Ann Liebert, Inc. Pp. 1–9 DOI: 10.1089/dna.2017.3850
ORIGINAL ARTICLE
Downloaded by Kings College London-journal Section from online.liebertpub.com at 09/26/17. For personal use only.
Transcriptome Profiling of Clematis apiifolia: Insights into Heat-Stress Responses Lulu Gao, Yuzhu Ma, Peng Wang, Shu’an Wang, Rutong Yang, Qing Wang, Linfang Li, and Ya Li
Clematis apiifolia, belonging to the Clematis L., is a woody vine native to China. It is characterized as heat resistant and fast growing. To better understand potential mechanisms involved in heat-stress responses in Clematis, we characterized the digital gene expression signatures of C. apiifolia under heat-stress conditions. Using RNA sequencing technology, we sequenced six libraries, three biological replicates of control samples and three of heat-stressed samples. In total, 61,708 unigenes were obtained, 36,447 (59.06%) of which were annotated. There were 1941 differentially expressed genes (DEGs) under heat stress, including 867 upregulated and 1074 downregulated genes. Gene ontology enrichment of DEGs revealed that ‘‘metabolic process,’’ ‘‘cellular process,’’ and ‘‘single organism’’ were the top three functional terms under heat stress. A Kyoto Encyclopedia of Genes and Genomes analysis led to the identification of ‘‘protein processing in metabolic pathways,’’ ‘‘phenylpropanoid biosynthesis,’’ and ‘‘biosynthesis of secondary metabolites’’ as significantly enriched pathways. Among the upregulated genes, heat-shock factors and heat-shock proteins, especially small heat-shock proteins, were particularly abundant under heat stress. The data will aid in elucidating the molecular events underlying heat-stress responses in Clematis L. Keywords: gene expression, heat stress, queen of vines, qRT-PCR, RNA-Seq
2007). Transcriptome analysis is an effective and widely used technique for exploring genes associated with heat resistance. In switch grass, a transcriptome analysis identified 2000 upregulated and 2809 downregulated unigenes under long-term heat stress (Li et al., 2013). In lotus, a transcriptome analysis found that small heat-shock proteins (sHsps) and genes related to cell morphogenesis were upregulated under heat-shock stress (Liu et al., 2016). Hightemperature stress inhibits the synthesis of normal proteins in plants and increases the synthesis of heat-shock proteins (Hsps) (Mittler et al., 2012). Hsps function as molecular chaperones under normal conditions. They help in nascent peptide folding, assembly, transport, and membrane transport under stress conditions. They are also involved in the degradation of misfolded proteins and Hsps repair protein aggregation to restore normal functions under the stress of denatured proteins (Kotak et al., 2007). Based on their molecular weights, Hsps were divided into five categories: Hsp100, Hsp90, Hsp70, Hsp60, and sHsps (Narberhaus, 2002; Ijaz, 2016). Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts, and they are the most evolutionarily conserved Hsps (Mayer and Bukau, 2005). In plants, sHsps are the most widely distributed. Presently, there are 16 kinds of plant sHsps that have been identified in arabidopsis, rice,
Introduction
C
lematis, the ‘‘Queen of vines,’’ is a popular climbing plant worldwide. It has a high ornamental value and is used in landscapes and floriculture as a garden or potted plant (Xie et al., 2011; Sheng et al., 2014). Most Clematis varieties are bred in high latitudes or cold regions and are cold resistant but heat sensitive. Temperatures of 30–40C in the summer lead to constrained growth, twig wilting, leaf wilting, shorter flowering periods, and abnormal flowers (Ma, 2016). Understanding the heat-resistant mechanism of Clematis is becoming more important. Clematis apiifolia is an excellent heat-resistant species (Ma, 2016) that provides important material for investigating the molecular mechanisms of heat stress in Clematis L. Presently, the molecular mechanism of heat resistance in C. apiifolia is poorly understood. Heat stress is often defined as the rise in temperature beyond a threshold level for a period of time that is sufficient to cause irreversible damage to plant growth and development. In general, a transient elevation in temperature, usually 10–15C above the ambient temperature, is considered heat shock or heat stress. Plants require physiological and biochemical adaptations to avoid or reduce the damage caused by high-temperature stress and to adapt to changes in the surrounding environment (Wahid et al.,
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, China.
1
Downloaded by Kings College London-journal Section from online.liebertpub.com at 09/26/17. For personal use only.
2
poplar, soybean, corn, lily, pea, and plum trees (Sarkar et al., 2009). High-temperature stress can induce the synthesis of sHsps. The heterologous expression of sHsps in plants to improve plant stress resistance is presently achieved through transgenic technology (Wahid et al., 2007). However, it is still important to determine the key sHsps in different species because of their level of diversity. Heat transcription factors (Hsfs) regulate the expression levels of Hsps genes at the transcriptional level under heat shock and heat stress. The DNA-binding domain and oligomerization domain (HR-A/B) of Hsfs in plants can be divided into three categories: A, B, and C (Takii and Fujimoto, 2016). A wide range of cognitive plant Hsfs was first cloned in tomato (Scharf et al., 1990). HsfA1, which is the main factor in heat-shock responses, can regulate the expression of Hsps and other Hsfs (Mishra et al., 2002). Under heat stress, Hsfs can regulate the expression levels of Hsp101s, Hsp70s, and sHsps in Arabidopsis thaliana (At). Hsf mutants have increased heat sensitivity levels, and plants overexpressing Hsfs have strong heat resistance levels (Li et al., 2005; Charng et al., 2007; Ogawa et al., 2007). In this study, we used RNA sequencing (RNA-Seq) and quantitative real-time-PCR (qRT-PCR) to identify genes in C. apiifolia seedlings under a 40C heat treatment. In total, six transcriptomes were used in the analysis and numerous differentially, specifically expressed transcripts of heatregulated genes were identified. These data provide a valuable foundation for genetic studies on heat stress in Clematis L. Materials and Methods Plant materials and treatments
Seedlings of C. apiifolia were collected from the Clematis germplasm nursery at the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, China. Each seedling was placed in a light incubator for 7 days under 25C conditions for pre-incubation. Then, they were placed at 40C (heat stress) and 25C (control) for 6 h. Three independent biological replicates were included for both heat-stress and control treatments. The seedlings were taken, immediately frozen in liquid nitrogen, and stored at -80C for subsequent analysis. RNA extraction and library preparation for transcriptome analysis
Total RNAs were extracted by using Trizol reagent according to manufacturer’s instructions (Invitrogen, Carlsbad, CA). The total RNA concentration was quantified by using UV spectrophotometry, and the quality was checked by electrophoresis in a 1% agarose gel. Equal volumes of RNA from each of the three replications of the heat-stress treatment and control were pooled. Six independent paired-end libraries were subjected to RNA-Seq analysis. Paired-end libraries with average insert lengths of *200 bp were synthesized by using a Genomic Sample Prep Kit (Illumina, San Diego, CA) according to the manufacturer’s instructions. Before cluster generation, library concentration and size were assayed by using an Agilent DNA 1000 Kit (Agilent, Palo Alto, CA) on a 2100 Bioanalyzer. Libraries were sequenced on an Illumina HiSeq 2500 instrument by a customer sequencing service (Novogene, Beijing, China). The raw data are available in the
GAO ET AL.
National Center for Biotechnology Information Sequence Read Archive (www.ncbi.nlm.nih.gov/Trace/sra) under accession number SRP108804. Sequencing, de novo assembly, and functional annotation
The raw reads were filtered and cleaned by removing lowquality raw reads and adaptor sequences. Clean reads were assembled into non-redundant transcripts by using Trinity, which has been developed specifically for the de novo assembly of transcriptomes using short reads (Grabherr et al., 2011). Short sequences (
ORIGINAL ARTICLE
Downloaded by Kings College London-journal Section from online.liebertpub.com at 09/26/17. For personal use only.
Transcriptome Profiling of Clematis apiifolia: Insights into Heat-Stress Responses Lulu Gao, Yuzhu Ma, Peng Wang, Shu’an Wang, Rutong Yang, Qing Wang, Linfang Li, and Ya Li
Clematis apiifolia, belonging to the Clematis L., is a woody vine native to China. It is characterized as heat resistant and fast growing. To better understand potential mechanisms involved in heat-stress responses in Clematis, we characterized the digital gene expression signatures of C. apiifolia under heat-stress conditions. Using RNA sequencing technology, we sequenced six libraries, three biological replicates of control samples and three of heat-stressed samples. In total, 61,708 unigenes were obtained, 36,447 (59.06%) of which were annotated. There were 1941 differentially expressed genes (DEGs) under heat stress, including 867 upregulated and 1074 downregulated genes. Gene ontology enrichment of DEGs revealed that ‘‘metabolic process,’’ ‘‘cellular process,’’ and ‘‘single organism’’ were the top three functional terms under heat stress. A Kyoto Encyclopedia of Genes and Genomes analysis led to the identification of ‘‘protein processing in metabolic pathways,’’ ‘‘phenylpropanoid biosynthesis,’’ and ‘‘biosynthesis of secondary metabolites’’ as significantly enriched pathways. Among the upregulated genes, heat-shock factors and heat-shock proteins, especially small heat-shock proteins, were particularly abundant under heat stress. The data will aid in elucidating the molecular events underlying heat-stress responses in Clematis L. Keywords: gene expression, heat stress, queen of vines, qRT-PCR, RNA-Seq
2007). Transcriptome analysis is an effective and widely used technique for exploring genes associated with heat resistance. In switch grass, a transcriptome analysis identified 2000 upregulated and 2809 downregulated unigenes under long-term heat stress (Li et al., 2013). In lotus, a transcriptome analysis found that small heat-shock proteins (sHsps) and genes related to cell morphogenesis were upregulated under heat-shock stress (Liu et al., 2016). Hightemperature stress inhibits the synthesis of normal proteins in plants and increases the synthesis of heat-shock proteins (Hsps) (Mittler et al., 2012). Hsps function as molecular chaperones under normal conditions. They help in nascent peptide folding, assembly, transport, and membrane transport under stress conditions. They are also involved in the degradation of misfolded proteins and Hsps repair protein aggregation to restore normal functions under the stress of denatured proteins (Kotak et al., 2007). Based on their molecular weights, Hsps were divided into five categories: Hsp100, Hsp90, Hsp70, Hsp60, and sHsps (Narberhaus, 2002; Ijaz, 2016). Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts, and they are the most evolutionarily conserved Hsps (Mayer and Bukau, 2005). In plants, sHsps are the most widely distributed. Presently, there are 16 kinds of plant sHsps that have been identified in arabidopsis, rice,
Introduction
C
lematis, the ‘‘Queen of vines,’’ is a popular climbing plant worldwide. It has a high ornamental value and is used in landscapes and floriculture as a garden or potted plant (Xie et al., 2011; Sheng et al., 2014). Most Clematis varieties are bred in high latitudes or cold regions and are cold resistant but heat sensitive. Temperatures of 30–40C in the summer lead to constrained growth, twig wilting, leaf wilting, shorter flowering periods, and abnormal flowers (Ma, 2016). Understanding the heat-resistant mechanism of Clematis is becoming more important. Clematis apiifolia is an excellent heat-resistant species (Ma, 2016) that provides important material for investigating the molecular mechanisms of heat stress in Clematis L. Presently, the molecular mechanism of heat resistance in C. apiifolia is poorly understood. Heat stress is often defined as the rise in temperature beyond a threshold level for a period of time that is sufficient to cause irreversible damage to plant growth and development. In general, a transient elevation in temperature, usually 10–15C above the ambient temperature, is considered heat shock or heat stress. Plants require physiological and biochemical adaptations to avoid or reduce the damage caused by high-temperature stress and to adapt to changes in the surrounding environment (Wahid et al.,
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, China.
1
Downloaded by Kings College London-journal Section from online.liebertpub.com at 09/26/17. For personal use only.
2
poplar, soybean, corn, lily, pea, and plum trees (Sarkar et al., 2009). High-temperature stress can induce the synthesis of sHsps. The heterologous expression of sHsps in plants to improve plant stress resistance is presently achieved through transgenic technology (Wahid et al., 2007). However, it is still important to determine the key sHsps in different species because of their level of diversity. Heat transcription factors (Hsfs) regulate the expression levels of Hsps genes at the transcriptional level under heat shock and heat stress. The DNA-binding domain and oligomerization domain (HR-A/B) of Hsfs in plants can be divided into three categories: A, B, and C (Takii and Fujimoto, 2016). A wide range of cognitive plant Hsfs was first cloned in tomato (Scharf et al., 1990). HsfA1, which is the main factor in heat-shock responses, can regulate the expression of Hsps and other Hsfs (Mishra et al., 2002). Under heat stress, Hsfs can regulate the expression levels of Hsp101s, Hsp70s, and sHsps in Arabidopsis thaliana (At). Hsf mutants have increased heat sensitivity levels, and plants overexpressing Hsfs have strong heat resistance levels (Li et al., 2005; Charng et al., 2007; Ogawa et al., 2007). In this study, we used RNA sequencing (RNA-Seq) and quantitative real-time-PCR (qRT-PCR) to identify genes in C. apiifolia seedlings under a 40C heat treatment. In total, six transcriptomes were used in the analysis and numerous differentially, specifically expressed transcripts of heatregulated genes were identified. These data provide a valuable foundation for genetic studies on heat stress in Clematis L. Materials and Methods Plant materials and treatments
Seedlings of C. apiifolia were collected from the Clematis germplasm nursery at the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, China. Each seedling was placed in a light incubator for 7 days under 25C conditions for pre-incubation. Then, they were placed at 40C (heat stress) and 25C (control) for 6 h. Three independent biological replicates were included for both heat-stress and control treatments. The seedlings were taken, immediately frozen in liquid nitrogen, and stored at -80C for subsequent analysis. RNA extraction and library preparation for transcriptome analysis
Total RNAs were extracted by using Trizol reagent according to manufacturer’s instructions (Invitrogen, Carlsbad, CA). The total RNA concentration was quantified by using UV spectrophotometry, and the quality was checked by electrophoresis in a 1% agarose gel. Equal volumes of RNA from each of the three replications of the heat-stress treatment and control were pooled. Six independent paired-end libraries were subjected to RNA-Seq analysis. Paired-end libraries with average insert lengths of *200 bp were synthesized by using a Genomic Sample Prep Kit (Illumina, San Diego, CA) according to the manufacturer’s instructions. Before cluster generation, library concentration and size were assayed by using an Agilent DNA 1000 Kit (Agilent, Palo Alto, CA) on a 2100 Bioanalyzer. Libraries were sequenced on an Illumina HiSeq 2500 instrument by a customer sequencing service (Novogene, Beijing, China). The raw data are available in the
GAO ET AL.
National Center for Biotechnology Information Sequence Read Archive (www.ncbi.nlm.nih.gov/Trace/sra) under accession number SRP108804. Sequencing, de novo assembly, and functional annotation
The raw reads were filtered and cleaned by removing lowquality raw reads and adaptor sequences. Clean reads were assembled into non-redundant transcripts by using Trinity, which has been developed specifically for the de novo assembly of transcriptomes using short reads (Grabherr et al., 2011). Short sequences (
