The heterologous expression of a chrysanthemum TCP-P transcription factor CmTCP14 suppresses organ size and delays senescence in Arabidopsis thaliana

Accepted Manuscript The heterologous expression of a chrysanthemum TCP-P transcription factor CmTCP14 suppresses organ size and delays senescence in Arabidopsis thaliana Ting Zhang, Yixin Qu, Haibin Wang, Jingjing Wang, Aiping Song, Yueheng Hu, Sumei Chen, Jiafu Jiang, Fadi Chen PII:

S0981-9428(17)30120-1

DOI:

10.1016/j.plaphy.2017.03.026

Reference:

PLAPHY 4846

To appear in:

Plant Physiology and Biochemistry

Received Date: 18 January 2017 Revised Date:

23 March 2017

Accepted Date: 31 March 2017

Please cite this article as: T. Zhang, Y. Qu, H. Wang, J. Wang, A. Song, Y. Hu, S. Chen, J. Jiang, F. Chen, The heterologous expression of a chrysanthemum TCP-P transcription factor CmTCP14 suppresses organ size and delays senescence in Arabidopsis thaliana, Plant Physiology et Biochemistry (2017), doi: 10.1016/j.plaphy.2017.03.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title: The heterologous expression of a chrysanthemum TCP-P transcription

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factor CmTCP14 suppresses organ size and delays senescence in Arabidopsis

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thaliana

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Authors: Ting Zhang1, Yixin Qu1, Haibin Wang1, Jingjing Wang1, Aiping Song1,

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Yueheng Hu1, Sumei Chen1, Jiafu Jiang1, Fadi Chen1, *

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Affiliation:

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College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

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* Corresponding author: Fadi Chen

Address: College of Horticulture, Nanjing Agricultural University, Nanjing 210095,

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China

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Tel: +86-25-84395592

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Fax: +86-25-84395266

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E-mail: [email protected]

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Ting Zhang, [email protected]

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Yixin Qu, [email protected]

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Haibin Wang, [email protected]

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Jingjing Wang, [email protected]

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Aiping Song, [email protected]

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Yueheng Hu, [email protected]

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Sumei Chen, [email protected]

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Jiafu Jiang, [email protected]

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Fadi Chen, [email protected]

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ACCEPTED MANUSCRIPT Abstract

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TCP transcription factors are important for plant growth and development, but their

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activity in chrysanthemum (Chrysanthemum morifolium) has not been thoroughly

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explored. Here, a chrysanthemum TCP-P sequence, which encodes a protein

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harboring the conserved basic helix-loop-helix (bHLH) motif, was shown to be

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related phylogenetically to the Arabidopsis thaliana gene AtTCP14. A yeast-one

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hybrid assay showed that the encoding protein had no transcriptional activation ability,

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and a localization experiment indicated that it was localized in the nucleus.

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Transcription profiling established that the gene was most active in the stem and leaf.

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Its heterologous expression in A. thaliana down-regulated certain cell cycle-related

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genes, reduced the size of various organs and increased the chlorophyll and

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carotenoid contents of the leaf which led to delayed senescence and a

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prolonged flowering period. Moreover, by screening the cDNA library of

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chrysanthemum, we found that the CmTCP14 can interact with CmFTL2 and some

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CmDELLAs.

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Keywords: Chrysanthemum morifolium; TCP transcription factor; organ size; leaf

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senescence

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1. Introduction

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Organ size is determined by a combination of cell number and cell size. The

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mechanisms controlling organ size are rather complex in plants because of their needs

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to adapt to unstable external environments (Mizukami, 2001). Studying mutants

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defective for cell proliferation and/or expansion has exposed a number of regulatory

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factors (Hu and Chua, 2003; Xia et al., 2013; Johnson et al., 2015), among which is

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the family of plant-specific TCP transcription factors (Martin-Trillo and Cubas, 2010).

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The TCPs harbor a 59 residue basic helix-loop-helix (bHLH) motif (the so-called TCP

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domain) which specifies their DNA binding behavior and the protein-protein

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interactions in which they are involved (Cubas et al., 1999). The Arabidopsis thaliana

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genome harbors 24 TCP genes, and the rice genome at least 20 (Yao et al., 2007).

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These genes have been classified into class I (also known as PCF or TCP-P) and class

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II (TCP-C) types (Cubas, 2002; Navaud et al., 2007). These two groups have been

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suggested to antagonistically modulate plant cell division and growth via their

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competitive binding to similar cis-regulatory modules: the TCP-Ps are thought to

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promote growth and the TCP-Cs to inhibit it (Hervé et al., 2009). However, the

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classification is not always clear, for example, the action of two TCP-P members

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AtTCP14 and AtTCP15 promotes cell proliferation in young internodes, but restricts it

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in the leaf (Kieffer et al., 2011). In addition to their participation in cell proliferation and expansion, the TCPs also

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contribute to the control of leaf shape, axillary meristem development, floral organ

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asymmetry and hormone synthesis (Martin-Trillo and Cubas, 2010). Loss-of-function

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mutants for AtTCP2, AtTCP4 and AtTCP10 produce somewhat enlarged leaves

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(Koyama et al., 2007). In rice, the TB1 product interacts with MADS57 to control the

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outgrowth of axillary buds by regulating the expression of D14 (Guo et al., 2013).

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Over-expression of CYC2 induces petal fusion and the formation of tube-shaped disk

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florets in Gerbera hybrida (Broholm et al., 2008). In A. thaliana, TCP1 regulates the

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transcription of DWARF4, a key brassinosteroid synthesis-related gene (Guo et al.,

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2010).

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Chrysanthemum is one of the most popular ornamental plants in the world. So far,

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the documented TCP genes are mainly class II types in chrysanthemum. For example,

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DgBRC was reported to inhibit the formation of shoot branching and CmCYC genes

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were involved in the regulation of ray floret development (Chen et al., 2013; Huang et

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al., 2016). To further elucidate the function of TCP gene family in chrysanthemum,

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we isolated and characterized a TCP-P gene in our study, which was homologous to

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AtTCP14 and designated as CmTCP14. Its heterologous expression in A. thaliana

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resulted in a major effect on whole plant phenotype and the development of various

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organs, such as root, leaf, flower and silique, was significantly inhibited. Moreover,

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the chlorophyll content was increased and the onset of flowering and senescence was

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greatly delayed.

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2. Materials and methods

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2.1. Plant materials and growing conditions

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The chrysanthemum cultivar ‘Jinba’ was obtained from the Chrysanthemum

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Germplasm Resource Preserving Center (Nanjing Agricultural University, China).

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Uniform rooted cuttings were potted into a 1:1 mixture of soil and vermiculite and

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grown in a greenhouse under a 14 h photoperiod (day/night temperature 25℃/18℃, 3

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relative humidity 70%). A. thaliana (ecotype Col-0) plants were grown in a 1:1:1

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mixture of perlite, vermiculite and soilrite under a 16 h photoperiod (80-100 µ m−2·s−1

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illumination), with a day/night temperature of 23°C/18°C. Plants were kept well and

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watered throughout.

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2.2. Cloning and sequencing of full length cDNA

Chrysanthemum leaves were snap-frozen in liquid nitrogen, and the total RNA was

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isolated from the frozen tissue using the RNAiso reagent (TaKaRa, Tokyo, Japan)

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according to the manufacturer’s instructions. The first cDNA strand was synthesized

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from a 1 µg aliquot of total RNA using SuperScript III reverse transcriptase

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(Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. A CmTCP14

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specific primer pair (TCP14-M-F and -R, Table S1) was designed to amplify a

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fragment of the chrysanthemum EST (Unigene5654) (Chen et al., 2009), and then

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5’RACE and 3’RACE were conducted to obtain the full length. The 5’ RACE reaction

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used a 5' RACE System kit (Invitrogen, Carlsbad, CA, USA) along with the

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gene-specific primers GSP5’-1, -2 and -3 (Table S1), while the 3’ RACE reaction

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required the cDNA first strand (synthesized using an oligo (dT) primer) to be

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amplified using the adaptor primer Adaptor-R and the gene-specific primers GSP3’-1

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and -2 (Table S1). The PCR products were purified and inserted into pMD19-T

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(TaKaRa, Tokyo, Japan) for sequencing. Finally, a pair of gene-specific primers

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(TCP14-Full-F and -R, Table S1) was designed from the putative 5’ and 3’-UTR

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sequences, and these were used to amplify the complete ORF.

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2.3. Phylogenetic analysis The amino acid sequences of TCP14 homologs were obtained from GenBank, and

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used to conduct a phylogenetic analysis based on the neighbor-joining method

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implemented in MEGA 5.0 software (Tamura et al., 2011). Bootstrap values were

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estimated from 1,000 replicates.

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2.4. Subcellular localization of CmTCP14 4

ACCEPTED MANUSCRIPT CmTCP14 cDNA was amplified with the primer pair TCP14-GFP-F/R (Table S1)

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using a Phusion High-Fidelity PCR kit (New England Biolabs, Ipswich, MA, USA).

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The amplicon and the pENTRTM 1A vector (Invitrogen, Carlsbad, CA, USA) were

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both restricted with KpnI and EcoRV, the fragments were ligated with T4 DNA ligase

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(TaKaRa, Tokyo, Japan) and the ligated sequence was validated by sequencing. The

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LR ClonaseTM II enzyme mix (Invitrogen, Carlsbad, CA, USA) was used to obtain a

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35S::GFP-CmTCP14 fusion gene (Earley et al., 2006). The construct was transiently

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expressed in onion epidermal cells via particle bombardment with a PDS-1000 device

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(Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions. The

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35S::D53-RFP construct was co-transformed to act as a nuclear marker (Zhou et al.,

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2013). After incubation in the dark at 28℃ for 16 h, the GFP signal was detected by a

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confocal

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A. tumefaciens (strain EHA105) cells harboring the 35S::GFP-CmTCP14 construct

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were infiltrated into five-week-old N. benthamiana leaves. After two days, leaf

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epidermal cells were monitored by the confocal laser scanning microscopy (Leica

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TCS SP5).

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2.5. Transactivation activity assay

The CmTCP14 ORF was introduced into the EcoR1/Pst1 cloning site of the

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pGBKT7 vector (Clontech, Mountain View, CA, USA) and transformed into

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Saccharomyces cerevisiae strain MAV203 to test the transactivation activity. A

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transactivation activity assay was performed by growing the cells on a solidified

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medium lacking tryptophan and uracil. The assay was based on β-gal / LacZ and used

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CPRG as the substrate. VP16 and an empty pGBKT7 were used as the positive and

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negative controls, respectively. All protocols were performed according to the

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manufacturer’s recommendations (Clontech, Mountain View, CA, USA).

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2.6. A. thaliana transformation

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Agrobacterium tumefaciens (strain EHA105) carrying the construct 35S::CmTCP14

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was transformed into A. thaliana Col-0 using the floral dip method (Clough and Bent, 5

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1998). Transformed progeny were selected by culturing on 1/2MS medium agar plates

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containing 20 µg/mL hygromycin, and these were advanced by self-pollination to

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obtain the T3 generation. Transgene zygosity was checked using RT-PCR based on the

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primer pair TCP14-test-F/R (Table S1).

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2.7. Chlorophyll and carotenoid quantification

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Chlorophyll and carotenoid contents were determined using available methods with

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only minor modifications (Lichtenthaler, 1987). Briefly, approximately 20 mg (fresh

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weight) of the fifth leaf was incubated in 5 mL 95% ethanol for 48 h in the dark, after

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which the absorbance of the supernatant was analyzed using a DU 800 UV/Vis

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spectrophotometer (Beckman Coulter, California, CA, USA), scanning at 665, 649

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and 470 nm.

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2.8. Scanning electron microscopy (SEM)

The fifth leaves of WT and 35S::CmTCP14 transgenic plants were harvested, fixed

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in FAA solution (50% v/v ethanol, 5% v/v glacial acetic acid, 10% v/v formaldehyde)

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and held overnight at 4℃. The samples were then dehydrated by passing through an

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ethanol series, coated with gold using an E-100 ion sputter and imaged using SU8010

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scanning electron microscope (Hitachi, Japan) at acceleration voltage of 10 kV.

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2.9. Transcription profiling by qRT-PCR Total RNA was extracted from various tissues of chrysanthemum and the leaves of

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4-week-old Arabidopsis with an RNAprep pure Plant Kit (TIANGEN, Beijing), and

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treated with DNase I to remove genomic DNA contamination. First-strand cDNA was

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synthesized with oligo (dT18) based on a PrimeScript Reverse Transcriptase Kit

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(TaKaRa, Tokyo, Japan). ABI7500 real-time PCR system was applied using SYBR

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Premix Ex Taq (TaKaRa, Tokyo, Japan) with EF1α (KF305681) as an endogenous

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control in chrysanthemum and AtActin2 (At3g18780) in Arabidopsis. The relative

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expression levels of related genes were calculated using the 2−∆∆Ct method (Livak and

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Schmittgen, 2001). All primer pairs for expression analysis are listed in Table S1. 6

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2.10. Leaf senescence assay An established detached leaf senescence assay was used to test the fifth and sixth

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leaves of 30-day-old A. thaliana plants (Li et al., 2016). The leaves were harvested

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and incubated in 30mL 3mM MES (pH 5.7), then kept in the dark at 22°C for four

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days. Images were taken after 0 and 4 days, and the transcription levels of some leaf

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senescence related genes were tested.

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2.11. Y2H assay

The pGBKT7-CmTCP14 construct was transformed into the yeast strain AH109 for

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screening the chrysanthemum cDNA libraries. Full lengths of positive clones were

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ligased into the pGADT7 vector (Clontech, Mountain View, CA, USA) at the EcoRI

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and XhoI cloning sites. The recombined constructs were co-transformed with

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pGBKT7-CmTCP14 to AH109 yeast cells for further validation. All protocols were

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performed following the manufacturer’s instructions (Clontech, Mountain View, CA,

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USA).

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3. Results

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3.1. Sequence of the chrysanthemum TCP14 homolog

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Based on the conserved TCP domain, a corresponding chrysanthemum EST was

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isolated from our cDNA library and the full length was further obtained by RACE

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PCR. The targeted chrysanthemum sequence encoded a class I TCP protein with a

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conserved bHLH-type DNA-binding domain in its N-terminal region and was most

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closely related to AtTCP14 (Fig. 1), therefore, this gene was designated as CmTCP14.

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The full length of CmTCP14 cDNA was 1447 nt, of which 1,125 nt constituted the

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gene's open reading frame (ORF), predicted to encode a 374 residue product of

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molecular mass 40.60 kDa and pI 7.02.

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3.2. Subcellular localization, transcription profiling and transcriptional activation of

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CmTCP14

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35S::GFP-CmTCP14 construct was generated and transiently transformed into onion 7

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with the sites of deposition of the RFP signal produced by the co-transformed

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35S::D53-RFP construct (Fig. 2A). The result was further confirmed by the behavior

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of the 35S::GFP-CmTCP14 construct expressed in Nicotiana benthamiana epidermal

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cells (Fig. 2B). A quantitative real time RT-PCR (qRT-PCR) experiment revealed that

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CmTCP14 was constitutively expressed in various tissues, with high expression levels

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in stem and leaf (Fig. 3). When the transcriptional activation of CmTCP14 was tested

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in yeast, neither cells carrying the pGBKT7-CmTCP14 construct nor those carrying an

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empty vector were able to grow on the SD/-Trp-Ura plate, while the positive control

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(VP16) cells grew well (Fig. 4), indicating that CmTCP14 exhibited no transcriptional

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activation activity in yeast cells and we speculated that CmTCP14 exerted the

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transcriptional function by interacting with other factors.

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3.3. The phenotype of A. thaliana plants heterologously expressing CmTCP14 To study the function of CmTCP14 in A. thaliana, two independent overexpression

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lines from T3 generation were selected (Fig. 5). When grown on half strength

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Murashige and Skoog (1962) medium (1/2MS) for a week, roots of both lines were

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significantly shorter than the wild type (WT) (Fig. 6). At the vegetative stage, the leaf

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development of transgenic plants was greatly restricted with shorter petiole length and

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elevated chlorophyll contents (Fig.7 A-I). SEM imaging of the fifth leaf revealed that

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the epidermal cells in both lines were smaller than those of WT (Fig. 7J-K). At the

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reproductive developmental stage, the petal size, silique length and plant height were

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all dramatically reduced in the transgenic plants (Fig. 8). Taken together, these

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phenotypic observations indicated that overexpression of CmTCP14 can significantly

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suppress the organ size of Arabidopsis.

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3.4. The effect of CmTCP14 heterologous expression on gene transcription

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The transcriptomic effect of expressing CmTCP14 heterologously in four-week-old

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A. thaliana plants was monitored for 25 genes associated with cell division; the

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chosen genes all harbor at least one class I TCP binding site in their upstream 8

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targets of AtTCP14 (Daviere et al., 2014). Transcription levels of most genes were

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down-regulated in the transgenic lines, especially E2F3, CDKB1;2 and CDKB2;2 (Fig.

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9A), consistent with the restricted cell size. Since both chlorophyll and carotenoid

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contents were enhanced in the transformants (Fig. 7E), the qRT-PCR platform was

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also used to track the transcription of a set of chlorophyll synthesis-related genes

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(Nagata et al., 2005) and PSY; the latter encodes phytoene synthase, the main

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rate-determining enzyme of carotenoid synthesis (Toledoortiz et al., 2010). Most of

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the chlorophyll synthesis-related genes were up-regulated by the presence of the

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transgene; in particular, the abundance of PORB transcript was almost three fold

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higher in the transgenic plants (Fig. 9B). The abundance of PSY transcript was also

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increased (Fig. 9C). The transcriptomic analysis revealed that CmTCP14 is involved

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in the cell division and chlorophyll and carotenoid biosynthesis pathways.

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3.5. The heterologous expression of CmTCP14 delayed leaf senescence Besides the influence on organ size, overexpression of CmTCP14 also led to the

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delayed leaf senescence phenotype. The dark-induced leaf senescence assay applied to

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the leaves of 30-day-old plants revealed that WT leaves became strongly chlorotic,

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while those of the CmTCP14 overexpressors still remained green (Fig. 10A).

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Transcription levels of SGR, SAG12, SAG20 and SAG29, which are known to be

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up-regulated during leaf senescence (Miller et al., 1999), were significantly lower in

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the transgenic lines than the WT (Fig. 10B). At the late stage of development, the

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overexpression lines displayed delayed senescence of the whole plant and still

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produced inflorescences while WT plants started to die (Fig. S1). In addition to the

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effect on leaf senescence, the transgenic lines also showed delayed flowering time

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(Fig. S2).

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3.6. CmTCP14 interacted with CmFT2 and CmDELLAs

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To further explore the function of CmTCP14, a Y2H experiment was performed.

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Among the positive clones, two clones encoding CmFTL2 and CmDELLA2 were 9

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obtained. The homologs of FTL2 and DELLA2 were also isolated in our

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chrysanthemum cDNA library to test the interactions. We found that CmTCP14 can

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interact with FTL2 and some DELLA proteins (Fig. 11).

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4. Discussion Members of the TCP transcription factor family are typically associated with cell

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proliferation and expansion (Martin-Trillo and Cubas, 2010). The product of A.

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thaliana AtTCP14 promotes cell proliferation in young internodes by binding to the

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promoter of certain cell cycle-related genes (Daviere et al., 2014), although it, along

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with AtTCP15, also acts to repress cell proliferation in the developing leaf (Kieffer et

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al., 2011). In our study, the heterologous expression of CmTCP14 in A. thaliana was

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found to suppress the growth of root, leaf, stem and flower. Expression levels of a

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number of cell cycle-related genes were down-regulated in the transgenic plants,

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which may have contributed to the inhibition of cell proliferation. To confirm that

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whether the phenotype of CmTCP14 in Arabidophsis was influenced by the native

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AtTCP14 and AtTCP15, qPCR assay was performed. We found the relative expression

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levels of the two genes did not change significantly (Fig. S3). Perhaps CmTCP14 can

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compete with AtTCP14 and AtTCP15 in target DNA-binding and this need to be

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further explored.

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During organ development, the timing of onset of cell proliferation and expansion

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often correlates with a switch from the mitotic cell cycle to endoreduplication (Breuer

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et al., 2014). Changes in the level of endoreduplication influence cell division and cell

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expansion, thereby modulating organ size (Sugimoto-Shirasu and Roberts, 2003). The

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up-regulation of RBR and CYCA2;3, which act downstream of AtTCP14 and

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AtTCP15, can strongly repress endoreduplication (Peng et al., 2015). However, here,

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both genes were down-regulated in the transgenic lines, leading to the suggestion that

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the control of organ size in transgenic lines probably did not reflect any reduction in

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endoreduplication or besides of RBR and CYCA2;3 functioning, another pathway

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down-regulating endoreduplication exists. DA1, DAR1 and DRA2 were previously

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reported to act redundantly to regulate endoreduplication during leaf development

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(Peng et al., 2015). In our study, the relative expression level of DA1 was significantly

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decreased but not for DAR1 and DAR2 (Fig. S4). This raised the possibility that

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CmTCP14 affected the organ size of Arabidophsis partially by regulating the

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transcriptional level of DA1. Besides of regulating the expression level of some cell-cycle related genes,

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CmTCP14 may also influence the distribution and homeostasis of auxin to affect

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organ size like GhTCP14 (Wang et al., 2013). From the result of qRT-PCR assay, we

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can find the expression of IAA3, which encodes an auxin-responsive protein and is the

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negative regulator of root growth, increased significantly (Fig. S5). By contrast,

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expression of PIN1 and PIN5, which encode auxin efflux transporters were

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down-regulated (Fig S5). However, the expression of AUX1 and PIN2 were not

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significantly changed and we did not observe the loss of gravitropism phenotype like

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in GhTCP14-ovrexpressing plants, which implying functional divergence of TCP14

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among different species.

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In addition to suppressing organ size, the constitutive expression of CmTCP14 also

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enhanced the synthesis of chlorophyll and carotenoid in the leaf. Higher contents of

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chlorophyll and carotenoid are associated with increased photosynthetic activity,

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delayed leaf senescence, and greater resistance against disease (Eckhardt et al., 2004;

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Mcquinn et al., 2015). Here, the constitutive expression of CmTCP14 in A. thaliana

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indeed delayed leaf senescence and the transgenic plants continued to produce

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inflorescences when the WT plants had completed their life cycle (Fig. S1). There is

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therefore the possibility that the over-expression of CmTCP14 may have the same

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effect of prolonging the flowering period in Chrysanthemum.

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The reduction in stature and the intensification of leaf color induced by the

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heterologous expression was reported to associate with suppressed GA responses

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(Steiner and Weiss, 2012). DELLA proteins are known as the repressor of GA

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signaling pathway and proved to interact with TCP14 and other class I TCPs in A.

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thaliana (Daviere et al., 2014; Resentini et al., 2015). Besides, AtTCP14 has also been

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shown to interact with the product of SPY to suppress GA signaling (Steiner et al.,

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2012). In our Y2H assay, we hooked a DELLA protein, further experiment indicated 11

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that CmTCP14 can interact with some other CmDELLAs which implied that the

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CmTCP14 may function in the GA signaling pathway. Besides, the CmTCP14 transgenic plants also experienced a delay to their flowering

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time (Fig. S2). The possibility that the transgene product interacted with

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flowering-related effectors was suggested by the known interaction between TCPs

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and FT in A. thaliana (Ho and Weigel, 2014). However, yeast two hybrid screens

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involving CmTCP14 with one of AtFT, AtTSF, AtFD, AtFLC and AtCO all failed to

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show any evidence for interaction in vitro. We also examined the interactions between

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CmTCP14 and the three FT-like genes in chrysanthemum (Oda et al., 2012), and

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found that CmTCP14 only interacted with CmFTL2 (Fig. 11A), which may reflect the

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functional divergence among FT-like genes.

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The determination of flowering time in A. thaliana involves several pathways, and

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is influenced by both endogenous developmental signals and external environmental

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cues (Fornara et al., 2010). How CmTCP14 contributes to flowering time still remains

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unclear: it is possible that its effect may differ between A. thaliana and

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chrysanthemum, given that CmTCP14 does interact with CmFTL2 but not with AtFT

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in vitro. The delayed flowering phenotype induced by CmTCP14 heterologous

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expression may also be a pleiotropic consequence of the reduced size of the root

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system, which can be expected to compromise nutrient uptake.

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Chrysanthemum is an important ornamental plant and studying genes controlling

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organ size, leaf senescence and flowering time has potential application values.

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Different size of the products meets the diversified demands of customers. Besides,

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during the transport, chrysanthemum are often exposed to prolonged dark storage, so

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extended greenness is a highly desirable characteristic for chrysanthemum. The

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function of CmTCP14 revealed in our study makes it a candidate gene for molecular

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breeding in the future.

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Contributions

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Jiafu Jiang and Fadi Chen conceived and designed the project; Fadi Chen and

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Sumei Chen provided the materials. Ting Zhang, Yixin Qu and Jingjing Wang 12

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performed the experiments. Ting Zhang, Aiping Song and Yueheng Hu analyzed the

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data. Ting Zhang and Haibin Wang wrote the manuscript.

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Acknowledgements This work is supported by the National Science Fund for Distinguished Young

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Scholars (31425022), the National Natural Science Foundation of China (31372092,

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31500570), Project of "333 project" in Jiangsu Province (BRA2015315), the Natural

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Science Fund of Jiangsu Province (BK20150661), the Fundamental Research Funds

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for the Central Universities (KJQN201652), and Fund for Independent Innovation of

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Agricultural Sciences in Jiangsu Province [CX（16）1025]. We thank Prof. Jianmin

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Wan (National Key Facility for Crop Gene Resources and Genetic Improvement,

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Institute of Crop Science, Chinese Academy of Agricultural Sciences) for the

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experimental help and suggestions.

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Conflicts of interest

The authors declare that they have no conflicts of interest.

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Breuer, C., Braidwood, L., Sugimoto, K., 2014. Endocycling in the path of plant development. Curr. Opin. Plant Biol. 17C (1), 78-85. Broholm, S.K., Elomaa, P., 2008. A TCP domain transcription factor controls flower type specification along the radial axis of the Gerbera (Asteraceae) inflorescence. P. Natl. Acad. Sci. USA. 105 (26), 9117-9122. Chen, S., Miao, H., Chen, F., Jiang, B., Lu, J., Fang, W., 2009. Analysis of expressed sequence tags (ESTs)

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collected from the inflorescence of chrysanthemum. Plant Mol. Biol. Rep. 27 (4), 503-510. Chen, X., Zhou, X., Xi, L., Li, J., Zhao, R., Ma, N., Zhao, L., 2013. Roles of DgBRC1 in regulation of lateral

branching in chrysanthemum (Dendranthema× grandiflora cv. Jinba). PloS one 8(4), e61717-e61717. Clough, S.J., Bent, A.F., 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16(6), 735-43.

Developmental genetics and Plant Evolution, 247-266.

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Cubas, P., 2002. Role of TCP genes in the evolution of key morphological characters in Angiosperms.

Cubas, P., Lauter, N., Doebley, J., 1999. The TCP domain: a motif found in proteins regulating plant growth and development. Plant J. 18(2), 215-222.

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Daviere, J.M., Wild, M., Regnault, T., Baumberger, N., Eisler, H., Genschik, P., Achard, P., 2014. Class I TCP-DELLA interactions in inflorescence shoot apex determine plant height. Curr. Biol. 24 (16), 1923-1928.

Earley, K.W., Haag, J.R., Pontes, O., Opper, K., Juehne, T., Song, K., Pikaard, C.S., 2006. Gateway-compatible vectors for plant functional genomics and proteomics. Plant J. 45 (45), 616-629. Eckhardt, U., Grimm, B., Hörtensteiner, S., 2004. Recent advances in chlorophyll biosynthesis and breakdown in higher plants. Plant Mol. Biol. 56 (1), 1-14.

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Guo, S., Xu, Y., Liu, H., Mao, Z., Zhang, C., Ma, Y., Zhang, Q., Meng, Z., Chong, K., 2013. The interaction between OsMADS57 and OsTB1 modulates rice tillering via DWARF14. Nat. Commun. 4, 1566. Guo, Z., Fujioka, S., Blancaflor, E.B., Miao, S., Gou, X., Li, J., 2010. TCP1 modulates brassinosteroid biosynthesis by regulating the expression of the key biosynthetic gene DWARF4 in Arabidopsis thaliana. Plant Cell 22 (4), 1161-1173.

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interacts with the E3 ubiquitin ligase DA2 to regulate seed and organ size in Arabidopsis. Plant Cell 25

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Fig. 1. bHLH domain comparison and phylogenetic analysis of TCP proteins. (A)

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Neighbor-joining phylogenetic tree of CmTCP14 and related TCP proteins, (B)

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Alignment of the amino acid sequences of the bHLH domains of the TCP proteins.

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The accession numbers of the proteins are: AcCYC2(AFP66931), AmCYC

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(CAA76176), AtTCP1 (At1G67260), AtTCP2 (At4G18390), AtTCP3 (At1G53230),

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AtTCP6 (At5G41030), AtTCP7 (At5G23280), AtTCP12 (At1G68800), AtTCP14

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(At3G47620),

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(At1G30210), CmCYC2a (KU595430), CmCYC2b (KU595431), CmCYC2c

(At1G69690),

AtTCP18

(At3G18550),

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AtTCP15

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AtTCP24

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(KU595428),

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(KU595429), DgBRC1-1 (JX870411), GbTCP (ABL86669), GhCYC3(ACC54348),

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GhTCP14 (AAD48836), HaCYC2c (CCE25961), PCF1 (BAA23142), PCF2

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(BAA23143), PsCYC1 (EU574913), PsLST1 (EU574914), SbTB1 (AAK37494),

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ZmTB1 (AAL66761). The bar (0.05) indicates branch length.

(KU595426),

CmCYC2e

(KU595427),

CmCYC2f

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Fig. 2. Subcellular localization of CmTCP14 in (A) onion and (B) N. benthamiana

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epidermal cells. 35S::D53-RFP was used as a nuclear marker. Bars: 100 µm in (A),

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30 µm in (B).

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Fig. 3. The transcription of CmTCP14 in various chrysanthemum organs. Values were

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given in the form (mean ± SD, n=3).

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Fig. 4. Transactivation activity analysis of CmTCP14 in yeast cells. VP16 and an

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empty pGBKT7 were used as, respectively, the positive and negative controls. The

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β-gal values were given in the form (mean ± SD, n=3).

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Fig. 5. Transcription levels of CmTCP14 in WT and transgenic A. thaliana plants

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constitutively expressing CmTCP14, as determined by RT-PCR. Actin2 was used as

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the reference gene.

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Fig. 6. Root growth of 7-day-old A. thaliana seedlings constitutively expressing

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CmTCP14. (A) Root development. Bar: 1 cm, (B) Quantitative analysis of root length.

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determined by Student's t-test (**P<0.01).

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Fig. 7. Phenotypes of WT and transgenic A. thaliana seedlings (lines #1 and #2)

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expressing CmTCP14 driven by the CaMV 35S promoter. (A-C) Four week old WT,

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#1 and #2 plants, (D) The fifth leaf of WT, #1 and #2, (E) The chlorophyll and

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carotenoid contents of WT, #1 and #2. Values were given in the form (mean ± SD,

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n=3), (F-I) Petiole length, leaf length, leaf width and the ratio of leaf length/width of

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the fifth leaf. Values were given in the form (mean ± SD, n=10), (J) SEM images of

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the fifth leaf of WT, #1 and #2, (K) Cell size in WT, #1 and #2. Values were given in

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the form (mean ± SD, n=3). Bars: 1 cm in A-C, 3 mm in D, 50µm in J, and statistical

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differences were determined by Student's t-test (**P<0.01).

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Fig. 8. Phenotypes of 5-week-old WT and transgenic A. thaliana seedlings (lines #1

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and #2) expressing CmTCP14 driven by the CaMV 35S promoter. (A) The whole

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plant, (B-D) Flower, (E) Silique, (F-H) Petal and (I-K) Calyx. Scale bars: 2 cm in (A),

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5 mm in (B-D), 3 mm in (E), 2.5 mm in (F-K). (L-O) Plant height, petal length, calyx

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length and silique length. Values were given in the form (mean ± SD, n=10) and

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statistical differences were determined by Student's t-test (**P<0.01).

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Fig. 9. The abundance in WT and in transgenic A. thaliana seedlings expressing

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CmTCP14 driven by the CaMV 35S promoter of transcripts associated with the Cell

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cycle regulation (A) and the synthesis of Chlorophyll (B), Carotenoids (C). Values

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were given in the form (mean ± SD, n=3).

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Fig. 10. The effect of constitutively expressing CmTCP14 on the senescence of the

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fifth and sixth leaves of 30-day-old A. thaliana plants. (A) Images were taken before

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and after holding in the dark for four days, (B) Transcriptional abundance of

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senescence-related genes in WT and two transgenic lines. Values were given in the form (mean ± SD, n=3).

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Fig. 11. Yeast two-hybrid experiment verifying the interactions between CmTCP14

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and CmFTLs (A) and CmDELLAs (B). -WL: SD –Trp/-Leu, -WLHA: SD –

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Trp/-Leu/-His/-Ade. The accession numbers of the proteins are: CmFTL1

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(AB679270),

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(ALF46585), CmDELLA2 (ALF46588), CmDELLA3 (ALF46586).

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(AB679271),

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CmFTL2

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CmFTL3

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CmDELLA1

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Supplementary data

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Table S1. The PCR Primers used in this study.

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Fig. S1. The constitutive expression of CmTCP14 in A. thaliana delays whole plant

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senescence. (A) Two-month-old WT, #1 and #2 plants, (B) Flowering days of the

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main stem of WT, #1 and #2 plants. Bar: 3 cm. Values were given in the form (mean ±

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SD, n=20) and statistical differences were determined by Student's t-test (**P<0.01).

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Fig. S2. Flowering phenotype of WT and transgenic A. thaliana seedlings (lines #1

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and #2) expressing CmTCP14 driven by the CaMV 35S promoter. (A) 30-day-old

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plants, (B) The total leaves of WT, #1 and #2 plants at the time of bolting. Bar: 1 cm.

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Values were given in the form (mean ± SD, n=20) and statistical differences were

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Fig. S3. The relative expression level of native AtTCP14 and AtTCP15 in WT, #1 and

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#2. Values were given in the form (mean ± SD, n=3).

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Fig. S4. The relative expression level of DA1, DAR1 and DRA2 in WT and transgenic

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plants. Values were given in the form (mean ± SD, n=3).

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Fig. S5. Transcriptional abundance of some auxin related genes in WT, #1 and #2.

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Values were given in the form (mean ± SD, n=3).

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1.This study describes the isolation of a TCP-P transcription factor CmTCP14 from Chrysanthemum. 2.Heterologous expression studies revealed that CmTCP14 suppresses organ size, delays leaf senescence and prolong flowering time. 3.qRT-PCR analysis revealed overexpression of CmTCP14 in Arabidophsis down regulated some cell-cycle related and up regulated chlorophyll synthesis-related genes. 4. Y2H assay indicated CmTCP14 can interact with CmFTL2 and some CmDELLAs.

ACCEPTED MANUSCRIPT Contributions Jiafu Jiang and Fadi Chen conceived and designed the project; Fadi Chen and Sumei Chen provided the materials. Ting Zhang, Yixin Qu and Jingjing Wang performed the experiments. Ting Zhang, Aiping Song and Yueheng Hu analyzed the

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data. Ting Zhang and Haibin Wang wrote the manuscript.