Elsevier

Gene

Volume 626, 30 August 2017, Pages 215-226
Gene

Research paper
Molecular cloning and functional characterization of CoFT1, a homolog of FLOWERING LOCUS T (FT) from Camellia oleifera

https://doi.org/10.1016/j.gene.2017.05.044Get rights and content

Highlights

  • CoFT1 expression increased during the floral induction stage and showed a photoperiod-dependent manner.

  • Overexpression of CoFT1 in Arabidopsis caused early flowering and elevated the expression of flowering time genes.

  • CoFT1 interacted with both Arabidopsis FD and Camellia oleifera CoFD proteins.

  • CoFT1 gene promoter contained light-responsive elements and flowering related transcriptional factor binding sites.

Abstract

FLOWERING LOCUS T (FT) is an important integrator of flowering genetic pathways and plays crucial roles in flowering transition. The tea-oil tree (Camellia oleifera Abel.) is a valuable woody oil crop, but the molecular mechanisms controlling flowering are still unclear. In this study, a FT-like gene, CoFT1, was isolated and characterized from C. oleifera. The CoFT1 protein was localized in the nucleus and cytoplasm of Arabidopsis protoplasts, and exhibited no transactivation activity in yeast cells. CoFT1 was highly expressed in mature leaves and reproductive organs, such as flower buds, flowers, flower organs, and young fruits. Its expression showed diurnal rhythms under both long-day and short-day conditions, and was photoperiod-dependent. Seasonal expression analysis revealed that the CoFT1 transcript in leaves increased during the floral induction period. Overexpression of CoFT1 in wild-type Arabidopsis resulted in precocious flowering and elevated the transcription levels of flowering related genes, such as SOC1, AP1, and LFY. Furthermore, the yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that CoFT1 was able to interact with both Arabidopsis FD and C. oleifera CoFD proteins. The sequence analysis revealed that the CoFT1 promoter contained a number of light-responsive elements, several hormonal- and stress-responsive motifs, and flowering related transcriptional factor binding sites, including CORE, CCAATBOX1, and CArG motifs. Our results suggested that CoFT1 might function as a flowering promoter in C. oleifera.

Introduction

Flowering, the switch from the vegetative to the reproductive development phase, is a critical step for plants and ensures reproductive success. The timing of flowering is precisely regulated by complex networks in response to both environmental signals and endogenous cues (Amasino and Michaels, 2010, Niu et al., 2016). In the annual model plant Arabidopsis thaliana, six distinct genetic pathways controlling flowering have been defined: the autonomous, photoperiod, vernalization, gibberellin, thermosensory, and age pathways (Fornara et al., 2010). FLOWERING LOCUS T (FT) is a crucial integrator of these flowering pathways and encodes a small mobile protein that induces flowering (Wigge et al., 2005). FT belongs to a protein family that contains the phosphatidylethanolamine-binding (PEBP) domain (Pin and Nilsson, 2012, Putterill and Varkonyi-Gasic, 2016). In Arabidopsis, another five members of this gene family have also been elucidated: MOTHER OF FT AND TFL1 (MFT), TWIN SISTER OF FT (TSF), TERMINAL FLOWER1 (TFL1), BROTHER OF FT AND TFL1 (BFT), and ARABIDOPSIS THALIANA CENTRORADIALIS HOMOLOGUES (ATC). TSF and MFT are functionally redundant with FT in during flowering activation (Yoo et al., 2004, Yamaguchi et al., 2005), while ATC, BFT, and TFL1 act as flowering repressors (Mimida et al., 2001, Yoo et al., 2010).

In Arabidopsis, FT mRNA is induced in the leaf vascular areas by COSTANS (CO) during inductive photoperiods (Samach et al., 2000, An et al., 2004). Subsequently, FT protein moves from the leaf to the shoot apical meristem (SAM) via long-distance transport in phloem companion cells (Corbesier et al., 2007, Jaeger and Wigge, 2007, Mathieu et al., 2007). In SAM, FT associates with the bZIP transcription factor FD to directly or indirectly activate the expression of APETALA1 (AP1), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and LEAFY (LFY) (Abe et al., 2005, Wigge et al., 2005, Yoo et al., 2005, Corbesier et al., 2007). TSF, the paralog of FT in Arabidopsis, probably acts through a similar regulatory mechanism to activate flowering (Yamaguchi et al., 2005). Additionally, evidence of mobility from other annual species, including cucurbit (Lin et al., 2007), rice (Tamaki et al., 2007, Komiya et al., 2009), and tomato (Lifschitz et al., 2006) suggests that the FT-like proteins serve as major florigenic signals that promote flowering.

Recently, FT-like genes have been extensively characterized in herbaceous plants, including rice (Kojima et al., 2002), wheat (Yan et al., 2006), tomato (Lifschitz et al., 2006), cucurbit (Lin et al., 2007), maize (Danilevskaya et al., 2008), sugar beet (Pin et al., 2010), potato (Navarro et al., 2011), sorghum (Wolabu et al., 2016), and switchgrass (Niu et al., 2016), as well as in perennial woody plants, such as poplar (Hsu et al., 2006), apple (Kotoda et al., 2010), blueberry (Song et al., 2013), kiwifruit (Varkonyi-Gasic et al., 2013), Jatropha curcas (Li et al., 2014, Ye et al., 2014), avocado (Ziv et al., 2014), and loquat (Zhang et al., 2016). Although most FT-like genes function as flowering promoters, some of them exhibit distinct expression patterns or divergent functions, even antagonistic roles in flowering. For example, Apple MdFT1 and MdFT2 have the potential to act as floral promoters, but show distinct tissue-specific and seasonal expression patterns (Kotoda et al., 2010). In potato, the floral and tuberization transitions are regulated by StSP3D and StSP6A, respectively (Navarro et al., 2011). Poplar FT1 and FT2 control floral induction and vegetative growth, respectively (Hsu et al., 2011), and in sugar beet, BvFT1 inhibits flowering, whereas BvFT2 is a flowering activator (Pin et al., 2010). Therefore, the actual FT-like genes that activate flowering need to be identified, particularly in economically significant crops, so that flowering time can be altered to improve seed yields or fruit loads.

The tea-oil tree (Camellia oleifera Abel.) is a perennial evergreen plant that belongs to the Theaceae family. It originated and is widely cultivated in Southern China (Ma et al., 2011, Wang et al., 2011). The tea oil extracted from C. oleifera seeds contains high levels of monounsaturated fatty acids (FA, e.g. oleic acid) and polyunsaturated FA (Ma et al., 2011), which resembles the olive oil in FA composition. These features make it a health food for humans. C. oleifera is recognized worldwide as an important woody oil crop, along with oil palm, coconut, and olive trees (Xia et al., 2014). The floral induction of the tea-oil tree generally occurs between late April and early May, and then flowering occurs from late autumn to early winter (Wang et al., 2011, Yuan et al., 2011). After pollination, the ovaries stop developing during the cold winter and growth resumes in spring next year with the fruit repining in late autumn (Wang et al., 2011). These flowering traits result in poor pollination and fertilization at low temperature (Lin and Xu, 1981), long-term overlap and nutrient competition between fruit development and the flowering process (Wang et al., 2011), and a prolonged fruit growth stage (Cao et al., 2015). Therefore, the timing of flowering is crucial for tea oil production and has also become an important target for C. oleifera breeding. However, the mechanisms underlying flowering time control in C. oleifera are still largely unknown.

In this study, an FT homolog gene, named CoFT1, was isolated and characterized from the C. oleifera cultivar ‘Xiang lin 27’. The sequence bioinformatics, subcellular targeting, transactivation activity, and expression patterns of CoFT1 were analyzed. Alteration to flowering times and the expression levels of flowering related genes were also investigated in Arabidopsis plants overexpressing CoFT1. Furthermore, the interactions between the CoFT1 and CoFD/FD proteins and the putative cis-acting elements of the CoFT1 promoter were also analyzed. Our data suggest a potential role for CoFT1 in promoting flowering in C. oleifera.

Section snippets

Plant materials, growth conditions, and samples collection

The ten-year-old adult plants of C. oleifera (cv. ‘Xiang lin 27’) used in this study were grown in an experimental field at the National Oil-tea Camellia Engineering & Technology Research Center (Hunan Academy of Forestry, Changsha, China). The root, stem, leaf, and leaf bud samples for tissue-specific expression analysis of CoFT1 were collected on July 1, 2014; the flower and flower organ (bract, sepal, petal, stamen, and pistil) samples were harvested on November 1; the floral bud was sampled

Isolation and characterization of CoFT1 from C. oleifera

The ORF of the CoFT1 gene was 540 bp long and encoded a putative protein of 179 amino acids, which had a calculated molecular mass of 20.2 kD and a theoretical isoelectric point of 7.74. A BLAST homology search of GenBank database indicated that CoFT1 protein shared 90, 88, 88, and 87% identity with AcFT, MdFT2, CsFT, and JcFT from Actinidia chinensis, Malus × domestica, Cucumis sativus, and Jatropha curcas, respectively. Therefore, we named this gene CoFT1 (GenBank: KX346229). Multiple sequence

Discussion

In this study, we isolated and characterized a FT-like gene, named CoFT1, from C. oleifera. Phylogenetic analysis of the PEBP protein family indicated that CoFT1 was classified into the FT-like clade, whose proteins mainly act to promote flowering (Karlgren et al., 2011). Sequence alignment revealed that CoFT1 possessed the FT-specific critical residues Tyr85 and Gln140, and the conserved segment B and C (Fig. 1A), which suggested a potential role for CoFT1 in flowering activation. Recent

Conclusions

The results obtained from the molecular identification and functional characterization of the CoFT1 gene will improve our understanding of the mechanism underlying flowering time control in C. oleifera. The increased CoFT1 transcript levels in C. oleifera leaves during the floral induction stage, the induced early flowering, and the upregulated expression levels of flowering time genes by CoFT1 in transgenic Arabidopsis strongly suggested that CoFT1 functioned as a flowering promoter in C.

Conflict of interest

The authors declare that they have no competing interests.

Authors' contributions

S. Su and L. Ma conceived and designed the research, H. Lei, Y. Wen and X. Wang performed the experiment, H. Lei and S. Su analyzed the data and wrote the manuscript. All authors have read and approved the final manuscript.

Acknowledgements

This work was supported by the Special Fund for Forest Scientific Research in the Public Welfare (201404702).

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