Research article
Transcriptome-wide identification and expression analysis of chrysanthemum SBP-like transcription factors

https://doi.org/10.1016/j.plaphy.2016.02.009Get rights and content

Highlights

  • 12 SPL genes were isolated and characterized in chrysanthemum.

  • Conserved motifs in the SBP-box proteins were shared by Arabidopsis and chrysanthemum.

  • 6 CmSPLs contained a miR156 target site, while 5 CmSPLs were targeted by miR157.

  • The hormone and stress treatments arouse the expression of 12 CmSPLs variously.

Abstract

SQUAMOSA promoter-binding protein (SBP) transcription factors are known to function in a number of processes in plants. Here, we have characterized twelve SBP-like (SPL) genes in the important ornamental species chrysanthemum (Chrysanthemum morifolium). A total of twelve distinct sequences were isolated and amplified based on transcriptomic sequences. Phylogenetic analysis identified two pairs of orthologous proteins for Arabidopsis and chrysanthemum and two pairs of paralogous proteins in chrysanthemum. Conserved motifs in the SPL proteins shared by Arabidopsis and chrysanthemum were scanned using MEME. A bioinformatics analysis revealed that six of these genes contained a miR156 target site, while five CmSPLs were targeted by miR157. Moreover, we used 5′ RLM-RACE to map the cleavage sites in CmSPL2 and CmSPL3. The expression of these twelve genes in response to a variety of phytohormone treatments and abiotic stresses was characterized. This work improves our understanding of the various functions of SPL gene family members in the stress response.

Introduction

Transcriptional control relies on transcription factors, a class of proteins also known as trans-acting factors, which are located within the nucleus. They are capable of activating and/or repressing transcription. Plant transcription factors can modulate the expression of plant genes in response to cold, drought, hormones, high salt and pathogen signals, thereby playing essential roles in the regulation of gene networks for many important developmental processes and defense responses in plants.

The SQUAMOSA promoter-binding protein (SBP) genes are a transcription factor family found only in plants (Cardon et al., 1999). The genes in this family code for a SBP-box domain, which includes 79 highly conserved amino acid residues that comprise DNA-binding and nuclear localization domains and two zinc binding sites (Cardon et al., 1999).

SBP-like (SPL) proteins were first identified in Antirrhinum majus (AmSBP1 and AmSBP2), based on their interaction with the promoter region of the floral meristem identity gene SQUAMOSA (Klein et al., 1996). Since then, SPL genes have been identified, isolated, and characterized in many plant species, most notably in Arabidopsis thaliana. To date, sixteen SPL genes have been identified in the Arabidopsis genome (Cardon et al., 1999). Wang et al. (2005) reported that the maize SPL family gene TGA1 (teosinte glumearchitectue 1) affects the shape of the corn apical meristem. Xie et al. (2006) identified 19 OsSPL genes in the rice genome, and Manning et al. (2006) conducted a study of the tomato family members of LeSPL epigenetic mutants.

Several physiological and biochemical studies have reported that the SPL transcription factor family exhibits many important biological functions. In Arabidopsis, the SPL gene family plays an important role in sporogenesis (Unte et al., 2003), shoot development (Wu and Poethig, 2006), flower development (Gandikota et al., 2007), leaf primordia interval formation (Wang et al., 2008), nutrition and reproductive stage changes (Jung et al., 2011), leaf development (Usami et al., 2009), fertility (Xing et al., 2010), plant hormone signal transduction (Jung et al., 2012) and copper homeostasis (Yamasaki et al., 2009).

As a gene family encoding transcription factors, more than half of the SPL genes identified to date are targeted by miR156/157. These microRNAs mainly affect the expression of SPL genes, thereby influencing various processes in plant development (Salinas et al., 2012). miR156/157 share a 14–16 nt-long homologous stretch in their mature sequences and exhibit overlapping binding sites in the same target genes (Zhang and Ling, 2014). In A. thaliana, the miR156-targeted SPL3 gene can be regulated by mRNA cleavage (Wu and Poethig, 2006) and through translational repression (Gandikota et al., 2007). Sequence and experimental analyses suggested that 11 out of 19 OsSPL genes were putative targets of OsmiR156. Furthermore, overexpression of two OsmiR156 genes (OsmiR156b and OsmiR156h) in rice resulted in severe dwarfism, greatly reduced panicle size, and delayed flowering, indicating that OsmiR156 and OsSPL target genes are involved in various developmental processes, and especially flower development in rice (Xie et al., 2006).

With the application of high-throughput sequencing technology, many researchers have used phylogenetic trees of conserved SBP domains to study the structural and evolutionary relationships between various species of the SPL family. Bioinformatic tools can be employed to study the SPL gene family through the comprehensive analysis of the whole genome in the increasing numbers of available plant genomes (Cardon et al., 1999, Salinas et al., 2012, Zhang and Ling, 2014, Xu et al., 2015).

Chrysanthemum (Chrysanthemum morifolium Ramat.) is an important ornamental species, second only to the rose in terms of its market value. With the rapid development of molecular biology, molecular genetic improvement of chrysanthemum is increasingly becoming a hot topic. However, the various activities of SPL proteins in chrysanthemum have not been explored. Here, we report the isolation of 12 chrysanthemum SPL putative transcription factors based on a set of transcriptome data, and we have analyzed their expression levels in response to various stressors and phytohormone treatments.

Section snippets

Plant materials and growth conditions

Cuttings of the cut flower chrysanthemum cultivar ‘Jinba’, maintained by the Chrysanthemum Germplasm Resource Preservation Center (Nanjing Agricultural University, Nanjing, China), were rooted in vermiculite in the absence of fertilizer in a greenhouse. After 14 days, they were transplanted to the growth substrate in order to prepare for exposure to a range of stress and phytohormone treatments.

Database searches and sequencing of full-length CmSPL cDNAs

All of the putative SPL proteins were retrieved from C. morifolium transcriptome data (Zhang et al.,

The SPL gene content of chrysanthemum

The 12 SPL sequences that were isolated were designated CmSPL1 through CmSPL12 (GenBank: KT253086–KT253097). The full-length cDNAs varied in length from 693 to 3191 bp, and their predicted protein products comprised between 142 and 954 residues. The full details of the CmSPL gene and derived protein sequences are given in Table 1. Ten CmSPL proteins were predicted to show nuclear localization, excluding CmSPL3 and CmSPL6. However, both of these proteins exhibit the conserved bipartite nuclear

Discussion

In previous studies, the SPL transcription factor family has been identified in Arabidopsis (Cardon et al., 1999), tomato (Salinas et al., 2012), A. majus (Klein et al., 1996), Ricinus communis (Zhang and Ling, 2014), Prunus mume (Xu et al., 2015) and other plants. The SPL gene family codes for a type of plant-specific zinc finger transcription factors. The proteins encoded by the SPL genes can specifically bind to the promoters of the floral meristem identity gene SQUAMOSA and its orthologous

Conclusions

This study is the first transcriptome-wide analysis of the SPL gene family in chrysanthemum. The present study characterized the expression of 12 CmSPLs in response to a range of phytohormones and stress treatments. These findings lay the foundation for future research on the function of SPL genes in the plant stress response. Future studies on the CmSPL genes should shed light on the fundamental functions of these genes and promote their application in chrysanthemum breeding.

Contributions

Conceived and designed the experiments: SAP CSM CFD. Performed the experiments: SAP GTW XJJ JLL. Analyzed the data: SAP CSM CFD. Contributed reagents/materials/analysis tools: GZY WHB CFD. Wrote the paper: SAP WD.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

This study was funded by the National Natural Science Foundation of China (31501792, 31500570), the Fundamental Research Funds for the Central Universities (KJQN201658, KYTZ201401, KJQN201652), the Natural Science Fund of Jiangsu Province (BK20150657, BK20150661), the National Science Fund for Distinguished Young Scholars (31425022), Special Fund for Agro-scientific Research in the Public Interest (201403039), and the China Postdoctoral Science Foundation (2014M561673, 2015T80564).

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