Identification and expression profiling of the auxin response factors (ARFs) in the tea plant (Camellia sinensis (L.) O. Kuntze) under various abiotic stresses

Plant Physiology and Biochemistry 98 (2016) 46e56

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Research article

Identification and expression profiling of the auxin response factors (ARFs) in the tea plant (Camellia sinensis (L.) O. Kuntze) under various abiotic stresses Yan-Xia Xu, Juan Mao, Wei Chen, Ting-Ting Qian, Sheng-Chuan Liu, Wan-Jun Hao, Chun-Fang Li, Liang Chen* National Center for Tea Improvement, Tea Research Institute of the Chinese Academy of Agricultural Science/ Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, 9 South Meiling Road, Hangzhou 310008, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 September 2015 Received in revised form 18 November 2015 Accepted 18 November 2015 Available online 22 November 2015

Auxin response factor (ARF) proteins are a multigene family of regulators involved in various physiological and developmental processes in plants. However, their modes of action in the tea plant (Camellia sinensis) remain largely unknown. In this study, we identified 15 members of the tea ARF gene family, using the public information about C. sinensis, both in our laboratory, as well as in other laboratories, and analyzed their phylogenetic relationships, conserved domains and the compositions of the amino acids in the middle region. A comprehensive expression analysis in different tissues and organs revealed that many ARF genes were expressed in a tissue-specific manner, suggesting they have different functions in the growth and development processes of the tea plant. The expression analysis under three forms of auxin (indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, naphthylacetic acid) treatment showed that the majority of the ARF genes were down-regulated in the shoots and up-regulated in the roots, suggesting opposite action mechanisms of the ARF genes in the shoots and roots. The expression levels of most ARF genes were changed under various phytohormone and abiotic stresses, indicating the ARF gene family plays important roles in various phytohormone and abiotic stress signals and may mediate the crosstalk between phytohormones and abiotic stresses. The current study provides basic information for the ARF genes of the tea plant and will pave the way for deciphering the precise role of ARFs in tea developmental processes and breeding stress-tolerant tea varieties. © 2015 Published by Elsevier Masson SAS.

Keywords: ARF genes family Camellia sinensis Expression Phytohormone Stress Transcriptional regulator

1. Introduction The phytohormone auxin is vital for plant growth and development, including root and shoot architecture, organ patterning, vascular development and tropic responses to light and gravity (Woodward and Bartel, 2005; Xu et al., 2014a). Auxin regulates many genes, and the auxin response factors (ARFs) are genes that regulate the expression of their down-stream target genes (Guilfoyle and Hagen, 2007). Most ARF proteins consist of three characteristic domains: the B3 DNA-binding domain (DBD), which is located in the N-terminal region; the C-terminal dimerization domain (CTD), which is located in the C-terminal region, which is a proteineprotein interaction domain related in its amino acid

* Corresponding author. E-mail address: [email protected] (L. Chen). http://dx.doi.org/10.1016/j.plaphy.2015.11.014 0981-9428/© 2015 Published by Elsevier Masson SAS.

sequence to domains III and IV found in the C-terminal of Aux/IAA proteins and which allows the dimerization of ARFs or ARF and Aux/IAA proteins; and the variable middle region (MR) that confers activator or repressor activity (Guilfoyle and Hagen, 2007). The ARF gene family has been demonstrated to be involved in various developmental processes. Previously, the molecular functions of many ARFs were best characterized in Arabidopsis. The Arabidopsis genome encodes 23 ARF members: AtARF1-AtARF23. AtARF1, 2, 3, 4, 5, 7 and 19 were suggested to be involved in leaf development, including leaf longevity and leaf shape (AtARF1 and 2) (Ellis et al., 2005), leaf expansion timing and patterning (AtARF3, 7 and 19) (Fahlgren et al., 2006; Wilmoth et al., 2005), vascular development (AtARF3 and 4) (Zhou et al., 2007), leaf initiation and vein pattern formation (AtARF5) (Garrett et al., 2012). AtARF2, 3, 4, 5, 7, 8, 10, 16 and 19 were indicated to regulate root development, such as lateral root growth (AtARF2, 3 and 4) (Marin et al., 2010), embryonic root initiation (AtARF5) (Schlereth et al., 2010), lateral root

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formation (AtARF7, 8 and 19) (Okushima et al., 2007; Tian et al., 2004; Wilmoth et al., 2005) and root-stem cell differentiation (AtARF10 and 16) (Ding and Friml, 2010). AtARF1, 2, 6, 8 and 17 were demonstrated to be involved in floral organ development. Among them, AtARF1 and 2 regulated floral organ abscission (Ellis et al., 2005); AtARF6 and 8 regulated female and male reproduction (Tabata et al., 2010; Wu et al., 2006) and AtARF17 regulated pollen wall pattern formation (Yang et al., 2013). In addition, ARF also influenced seed development. For example, AtARF2 regulated seed size, as its knockout conferred bigger seeds in mutants than in wild types (Schruff et al., 2006). AtARF8 was also associated to fruit morphology (Goetz et al., 2006). Because ARF is the key transcription factor for auxin in plants, considerable progress has been achieved in cloning and characterizing its functions in other species. Our previous studies demonstrated that OsARF12, 16 and 19 were involved in auxinmediated root elongation, the phosphate starvation response and leaf angle change, respectively (Qi et al., 2012; Shen et al., 2013; Zhang et al., 2015). The reports from other laboratories also showed that SlARF4 and SlARF7 were negative regulators of fruit development, and SlARF9 regulated cell division activity during early fruit development in the tomato (de Jong et al., 2015; Sagar et al., 2013). The capsicum gene CaARF8 and eggplant gene SmARF8 were involved in parthenocarpy (Du et al., 2015; Tiwari et al., 2011). However, ARFs remain largely uncharacterized in woody plants. Thirty-nine poplar ARFs, 19 grapevine ARFs and 19 sweet orange ARFs, were identified recently (Kalluri et al., 2007; Li et al., 2015b; Wan et al., 2014). Although the tea plant (Camellia sinensis (L.) O. Kuntze) is an economically important perennial, an evergreen woody crop that is used to produce an alcohol-free beverage, there were few reports on its ARFs. Here, we summarized the public information about C. sinensis from our laboratory and the other laboratories and identified 15 CsARF genes and analyzed their phylogenetic relationships, conserved domains and amino acid compositions of the MR domain. We investigated the expression pattern of these CsARFs in different tissues and organs of tea plant and in response to various abiotic stresses. These distinctive spatio-temporal expression patterns and different responses to abiotic stresses will provide important insights for researching gene functions and breeding stress-tolerant varieties of the tea plant.

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2. Materials and methods 2.1. Plant materials and treatments The tea plant (C. sinensis (L.) O. Kuntze) cultivar ‘Longjing 43’ was used in the current study. For various phytohormone treatments, three-year-old plants grown in pots at the greenhouse of the Tea Research Institute of the Chinese Academy of Agricultural Sciences (TRICAAS), Hangzhou, China, were selected and their roots were washed carefully with distilled water. Then, they were grown for 6 h and 24 h in water with or without 50 mm indole-3acetic acid (IAA), 5 mm 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mm naphthylacetic acid (NAA), 50 mm naphthoxyacetic acid (NOA), 2.5 mm salicylic acid (SA), 50 mm gibberellic acid (GA), or 5 mm 6-benzyladenine (6-BA) until shoots and roots were sampled. The concentration of stock solution was 1 M, 0.5 M, 0.5 M, 1 M, 2.5 M, 1 M and 0.5 M for IAA, 2,4-D, NAA, NOA, SA, GA and 6-BA, respectively. All of them were first dissolved with 1 M NaOH and then diluted with water to suitable volume. For salinity treatment, tea plants in the same growth environment mentioned above were treated for 1 d and 3 d with 300 mM NaCl. For drought treatment, irrigation was withheld for up to 6 d, and the shoots were sampled after 1 d and 6 d of treatment. In addition, we simulated field drought with a 20% polyethylene glycol (PEG) treatment and sampled at 6 h and 24 h. For tissuespecific expression analysis, we examined six-year-old tea plants grown in the experimental field of the TRICAAS and sampled their roots, stems, young leaves, mature leaves, buds, flowers, fruit peels and seeds. Total RNA from the above tissues was analyzed with qRT-PCR. All experiments included three separate biological replicates. 2.2. Sequences dataset and ARF genes identification in tea plants The annotated unigenes from several transcriptomes of the tea cultivar ‘Longjing 43’ in our laboratory (SRP034436, Wang et al., 2015) and in previous reports from the other laboratories (SRP002436, SRP016951, SRP039585) were used for identification of ARF genes. A total of 64 tea plant proteins identified in this initial search were analyzed using the hidden Markov model (HMM) profiles of the ARF protein family Pfam 02362: B3 DNA binding domain (B3); Pfam 02309: AUX/IAA family; Pfam 06507: Auxin

Table 1 ARF gene family members in C. sinensis. Gene namea

CsARF1-1 CsARF1-2 CsARF2-1 CsARF2-2 CsARF3-1 CsARF3-2 CsARF4 CsARF6 CsARF8-1 CsARF8-2 CsARF9 CsARF11 CsARF16-1 CsARF16-2 CsARF19 a b c d

ORF length (bp)b

2028 2031 2598 2511 2130 2112 2382 2727 2463 2496 2076 2082 2004 2052 3225

Deduced polypeptidec Length (aa)

MW (kDa)

PI

675 676 865 836 709 703 793 908 820 831 691 693 667 683 1074

75.53 75.85 96.19 93.29 77.41 77.40 87.89 100.25 91.59 92.46 77.22 77.28 73.76 75.45 120.18

6.08 6.17 6.62 6.67 6.69 6.84 6.18 6.36 6.06 6.01 6.13 6.20 6.51 6.88 6.52

Domaind

Localization

DBD, ARF, CTD DBD, ARF, CTD DBD, ARF, CTD DBD, ARF, CTD DBD, ARF DBD, ARF DBD, ARF, CTD DBD,ARF,CTD DBD, ARF, CTD DBD, ARF, CTD DBD, ARF, CTD DBD, ARF, CTD DBD, ARF DBD, ARF DBD, ARF, CTD

Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear

Names referred to the identified CsARF genes in the tea plant in this work. Length of open reading frame in base pairs. The number of amino acids, molecular weight (kilodaltons) and isoelectric point of deduced polypeptide calculated by DNASTAR. DBD: B3 DNA-binding domain; ARF: auxin response factor domain; CTD: C-terminal dimerization domain.

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response factor (AUX_RESP), thus, we eliminated 49 redundant and invalid proteins and retained 15 proteins. Subsequently, the retained proteins were sorted as unique sequences for a further protein domain search using InterProScan Sequence Search (http:// www.ebi.ac.uk/Tools/pfa/iprscan/). All of the proteins obtained as a result of this search were used as queries in BLASTP searches to find their potential orthologs in Arabidopsis and were named according to their potential Arabidopsis orthologs. 2.3. Multiple sequence alignment, phylogenetic analysis, motif analysis and subcellular localization Multiple sequence alignment of all of the full-length protein sequences was performed using the software ClustalX1.83. A phylogenetic tree was constructed with the MEGA4 software program by neighbor-joining methods with 1000 bootstrap replicates. The conserved motif was analyzed using the NCBI Conserved Domain Database (CDD) and the Multiple Em for Motif Elicitation (MEME) (http://meme.nbcr.net/meme/cgi-bin/meme. cgi). The subcellular localizations were predicted by the subcellular localization predictor (CELLO v.2.5; http://cello.life.nctu.edu. tw). 2.4. qRT-PCR Total RNA was extracted from 100 mg of frozen tea tissue using the RNAprep Pure Plant Kit (TIANGEN, China) according to the manufacturer's protocol. The methods of reverse transcription and qRT-PCR were as described in a previous report (Qi et al., 2012). The primer sequences for qRT-PCR are listed in Table S1. All of the qRT-PCR experiments were analyzed using three independent biological replicates. The CsACTIN gene was used as an internal control. 3. Results 3.1. Identification of 15 auxin response factors (ARFs) in tea plant To identify putative ARFs in the tea plant, whose genome has not been released, we employed Pfam 02362 (B3 DNA binding domain), Pfam 02309 (AUX/IAA family) and Pfam 06507 (Auxin response factor: AUX_RESP) to search the CsARF genes from several reported transcriptomes of the tea cultivar ‘Longjing 43’ in our laboratory and the other laboratories (SRP002436, SRP016951, SRP034436, SRP039585, Wang et al., 2015). Additional searches were performed in NCBI using the obtained proteins as queries in BLASTP searches. After eliminating the redundant and invalid gene sequences, we identified 15 CsARF genes. To further confirm whether the identified sequences are full lengths or not, we employed multiple alignments of three species, Arabidopsis, Populus and tea plant to check the start and stop codons and the amino acid sequences of identified tea ARF sequences. The result was shown in Fig. S1. All of the 15 CsARFs were well aligned with their homologous genes in Arabidopsis and Populus. We named these identified genes according to their homologous genes in Arabidopsis. The information of the 15 CsARF genes, including the gene name, open reading frame (ORF) length, the basic parameters of the deduced polypeptide, the typical domain and subcellular localization are shown in Table 1. As shown in Table 1, the 15 CsARF genes encode proteins ranging from 667 to 1074 amino acids in length with molecular weights (MW) that vary from 73.76 to 120.18 kDa. Their predicted isoelectric point (PI) varies from 6.01 to 6.88. These data suggested that different ARF proteins could work under different conditions. In addition, all of the CsARF proteins were predicted to locate at the nucleus,

Fig. 1. Phylogenetic relationships of Arabidopsis, Populus and tea ARF proteins. Amino acid sequences of full-length predicted ARF protein were aligned using the ClustalX 1.83 program. A phylogenetic tree was generated using MEGA4 software using neighbor-joining methods. Bootstrap values from 1000 replicates are indicated at each branch.

indicating their function of transcriptional regulation. However, because the genome of the tea plant has not been released, the number of CsARF family members of C. sinensis in this study may be not full. Considering the high heterozygosity and large genome size of the tea plant, we have reason to believe that there are more than 15 ARF proteins in the tea plant.

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Fig. 2. Twenty conserved motifs in CsARFs analyzed by MEME search tool. The width of the motifs ranged from 6 to 50, zero or one repeat per sequence. (A) The motifs were mapped on CsARF proteins by different colors. (B) The height of each box represents the specific amino acid conservation in each motif.

3.2. Phylogenetic relationship and protein structure analysis To explore the phylogenetic relationship of the ARF proteins among different species, the ARF proteins in the tea plant, Arabidopsis and Populus were used for constructing a phylogenetic tree (Fig. 1). The phylogenetic analysis showed that all of these ARFs could be divided into six classes: class I (AtARF1/2-like), class II (AtARF3/4-like), class III (AtARF5-like), class IV (AtARF7/19-like), class V (AtARF6/8-like) and class VI (AtARF10/16/17-like), with each class containing 6, 3, 0, 1, 3 and 2 CsARFs, respectively (Fig. 1). A similar scenario was reported in several other studies (Shen et al., 2015; Xing et al., 2011). The locus IDs of the Arabidopsis and Populus ARFs for the phylogenetic tree construction in this work are listed in Table S2 and Table S3. Multiple alignments of the CsARF proteins were obtained with the ClustalX program, and the results showed that most of the CsARF proteins contained three conserved domains: DBD, ARF and CTD (Fig. S1). The domains were further analyzed using NCBI CDD and the MEME program. Twenty conserved motifs were identified and their distribution in the proteins of the respective classes has been shown in Fig. 2. Motifs 1 and 2 correspond to DBD; motifs 5, 8 and 11 correspond to ARF, while motifs 4 and 9 correspond to CTD. Of the 15 identified CsARFs, only CsARF 3-1, 3-2 (both belong to class II) and 16-1, 16-2 (both belong to class VI) lack the CTD. The result was consistent with multiple alignments (Fig. S1). Much evidence showed that the amino acid composition of the

MR of each ARF determines whether it acts as an activator or repressor. These enriched in glutamine (Q), serine (S) and leucine (L) in MR are often activators, while those enriched in S, L, proline (P) and glycine (G) are often repressors (Guilfoyle and Hagen, 2007). As shown in Fig. 3, these 15 CsARFs could be divided into three groups according to the amino acid composition and the presence or absence of the CTD. CsARF6, 8-1, 8-2 and 19 were QSLrich in the MR, suggesting they are probable transcriptional activators, while CsARF1-1, 1-2, 2-1, 2-2, 4, 9 and 11 were SP-rich in the MR, suggesting they are probable transcriptional repressors. The rest of the proteins, CsARF3-1, 3-2, 16-1 and 16-2, lack the CTD and may be repressors due to SG/SP-rich MR. 3.3. Expression of CsARFs in various tissues and organs of tea plant To some extent, gene expression patterns can provide useful insights into the physiological functions of the related proteins. To estimate the physiological roles of 15 CsARF genes, the expression patterns of these genes in the roots, stems, young leaves, mature leaves, buds, flowers, fruit peels and seeds were determined using real-time PCR (qRT-PCR) (Fig. 4). As shown in Fig. 4, the heat map visualized the relative expression of 15 CsARFs in various tissues and organs and showed that the genes could be clustered into three main groups. Group I was composed of CsARF1-1, 1-2, 16-1 and 16-2 and showed a generally lower expression in all of the detected tissues compared to the other genes. However, CsARF1-1, 1-2 and

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Fig. 3. Analysis of amino acid compositions and classification of CsARFs. (A) Amino acid compositions in the MR domains of CsARF proteins. The horizontal axis indicates CsARF proteins and the vertical axis indicates the corresponding amino acid compositions. The types of amino acids are distinguished by different colors. (B) The classification of CsARF proteins based on their structures. DBD: B3 DNA-binding domain; MR: middle region; CTD: C-terminal dimerization domain; Q: glutamine; S: serine; L: leucine; G: glycine; P: proline; AD: activation domain; RD: repression domain.

16-1 displayed preference for leaves and CsARF16-2 displayed preference for fruit peels. Group II was the largest group with 8 members (CsARF2-1, 2-2, 3-1, 3-2, 8-1, 8-2, 9 and 11) and mainly expressed in the roots and flowers. Group III was composed of 3 genes (CsARF4, 6 and 19), with the highest expression in the buds. There was a high variability in the transcript abundance of the CsARF genes in the different tissues and organs, suggesting they have different functions in the growth and development processes of the tea plant. 3.4. Expression analysis of CsARFs in response to auxin treatment The hormone auxin is a central regulator of plant growth and development. It influences plant shoot and root system architecture through inducing or suppressing the expression of a series of genes. To examine the response of CsARFs to exogenous auxin stimuli, the expression patterns of the 15 identified CsARFs in the shoots and roots at 6 h and 24 h after three forms of auxin, indole-

3-acetic acid (IAA), 2,4edichlorophenoxyacetic acid (2,4eD) and naphthylacetic acid (NAA) treatment were investigated using qRTPCR. The results revealed that most of these genes were downregulated in the shoots and up-regulated in the roots by the exogenous auxin treatment (Fig. 5). For IAA treatment, 11 genes (CsARF1-2, 2-1, 2-2, 3-2, 4, 8-2, 9, 11, 16-1, 16-2 and 19) were downregulated across all time points in the shoots. In addition, 3 genes (CsARF1-1, 3-1 and 8-1) were down-regulated after the 6 h treatment, but there was no significant change after the 24 h treatment and 1 gene (CsARF6) was up-regulated after the 6 h treatment and down-regulated later (Fig. 5A). In the roots, 11 genes (CsARF1-1, 2-1, 3-1, 3-2, 4, 6, 8-1, 8-2, 11, 16-1 and 19) were up-regulated after the 6 h treatment and fell later. CsARF1-2, 2-2 and 16-2 were downregulated and CsARF9 had no significant change across all time points (Fig. 5B). For the 2,4-D treatment, with the exception of CsARF6 and 9, all of the other genes were suppressed across all time points in the shoots (Fig. 5C). In the roots, with the exception of CsARF11, all of the other genes that were up-regulated after 6 h

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in the roots under the SA treatment. CsARF8-2 was down-regulated in the shoots and up-regulated in the roots under all three treatments. However, nearly all of these genes shared similar expression patterns in the shoots and roots later, as the factor that nearly all of these genes were observed to be down-regulated after the 24 h treatment under all three signaling molecule treatments (Fig. 6). 3.6. Expression analysis of CsARFs in response to salt and drought treatment

Fig. 4. Heat map showing the spatio-temporal expression patterns of CsARF genes. Red and green represent relatively higher and lower expression (log2 ratios) than the control, respectively. The values used in the heat map were determined by qRT-PCR and normalized with a control sample. R: root; ST: stem; YL: young leaf; ML: mature leaf; B: bud; F: flower; P: peel; SE: seed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

To determine the potential roles of the CsARFs in response to environmental stresses, we analyzed the transcription levels of 15 CsARF genes under salinity (NaCl) and drought (PEG simulation and irrigation) treatments. The salt stress causes different responses among the CsARF genes in the shoots and roots. For instance, CsARF1-1, 1-2, 9 and 16-2 were significantly up-regulated in the shoots and down-regulated in the roots. CsARF2-1, 4, 8-1, 8-2, 11, 161 and 19 were down-regulated in both the shoots and roots. The greatest change in expression (nearly 14-folds) was found in CsARF4 in the 1 d salt treatment. However, CsARF3-1 and 3-2 presented no significant difference under the salt treatment in both the shoots and roots (Fig. 7A, B). The detected CsARF genes can be classified into four groups according to the expression patterns under drought treatment (Fig. 7C, D): A) sustained up-regulated: CsARF6, 9; B) sustained down-regulated: CsARF4, 8-1, 8-2, 11, 19; C) no significant difference: CsARF1-1, 1-2, 3-1, 16-1; D) irregular: the remainder of the CsARF genes. 4. Discussion 4.1. Characterization of 15 ARFs in the tea plant

under IAA treatment were also up-regulated (Fig. 5B, D). For the NAA treatment, in the shoots, 11 genes (CsARF1-2, 2-1, 2-2, 3-1, 3-2, 4, 8-1, 8-2, 11, 16-2 and 19) and 1 gene (CsARF9) were downregulated and up-regulated across the two time points. Additionally, CsARF16-1 was down-regulated after the 6 h treatment but upregulated significantly after the 24 h treatment, CsARF6 was upregulated after the 6 h treatment but down-regulated significantly after the 24 h treatment and CsARF1-1 had no significant change across the two time points (Fig. 5E). In the roots, with the exception of CsARF11, all of the other genes were up-regulated after the 6 h treatment (Fig. 5F). Taken together, most of the CsARF genes followed a similar expression pattern after treatment by the different forms of auxin. We also analyzed the expression of these genes in response to naphthoxyacetic acid (NOA), which is a well-used polar auxin transport (auxin influx phase) inhibitor. The results were similar to the auxin treatment (Fig. S2). 3.5. Expression analysis of CsARFs in response to the other signaling molecule treatments To investigate the relationship between the CsARFs and the other signaling molecules, the expression of CsARFs under salicylic acid (SA), gibberellic acid (GA) and 6-benzyladenine (6-BA) treatments was analyzed by qRT-PCR. As shown in Fig. 6, different genes generated different expression patterns in the shoots and roots after the 6 h treatment of different signaling molecules. Typically, CsARF1-1, 3-1, 3-2, 8-1 and 16-1 were up-regulated, while CsARF1-2 was down-regulated, in both the shoots and roots under all three treatments. CsARF2-1, 2-2, 9 and 19 were down-regulated in the shoots and up-regulated in roots under the 6-BA treatment and CsARF4 and 6 were down-regulated in the shoots and up-regulated

ARF proteins constitute a large and multigenic family. The availability of sequence information in the public domain, such as the ESTs and genome sequences and those in our laboratory, has provided an opportunity to identify the members of the ARF gene family in C. sinensis. In this work, 15 tea plant ARFs were identified, and the full-length ORF sequences of these genes were obtained. Because the genome of the tea plant has not been released, the number of CsARF family members of C. sinensis in this study may be not be complete. In the public literature, there were more than 15 ARFs in higher plants. For example, 22 maize ARFs, 23 Arabidopsis ARFs, and 39 Medicago ARFs have been identified based on genomic resources, respectively (Guilfoyle and Hagen, 2007; Shen et al., 2015; Xing et al., 2011). For woody plants, 39 poplar ARFs, 19 grapevine ARFs and 19 sweet orange ARFs were reported (Kalluri et al., 2007; Li et al., 2015b; Wan et al., 2014). Considering the high heterozygosity and large genome size of the tea plant, we have reason to believe that there are more than 15 ARF proteins in the tea plant. Especially, the members of class III were not identified in the tea plant, probably due to the spatial and temporal-specific expression of AtARF5-like genes. Additionally, the homologous genes of AtARF7 whose function was well characterized were also not identified in our study, due to their non-complete sequence information in the transcriptome data. Phylogenetic comparison of the ARF proteins has been widely conducted in many species, and the evolutionary relationships of these proteins within and among the different species have been widely reported (Kalluri et al., 2007; Li et al., 2015b; Shen et al., 2015; Wan et al., 2014; Xing et al., 2011). In this work, a phylogenetic tree with Arabidopsis, Populous and the tea plant showed that ARFs were distributed into six classes, class I to VI homology to AtARF1/2, AtARF3/4, AtARF5, AtARF7/19, AtARF6/8 and AtARF10/ 16/17, respectively (Fig. 1). This would not only help direct the

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Fig. 5. Expression analysis of CsARF genes under auxin treatments in shoots and roots. (A) IAA treatment, shoots. (B) IAA treatment, roots. (C) 2,4-D treatment, shoots. (D) 2,4-D treatment, roots. (E) NAA treatment, shoots. (F) NAA treatment, roots. “CK” represents untreated seedlings grown in water.

evolutionary relationships of these ARF proteins but also indicate the putative role for each CsARF, as the above Arabidopsis ARF proteins take part in different developmental processes, including those of the leaf, root, floral and seed development (Fahlgren et al., 2006; Marin et al., 2010; Schruff et al., 2006; Wilmoth et al., 2005; Yang et al., 2013). Similar distribution is found in many other species (Shen et al., 2015; Xing et al., 2011). All of the 15 CsARF proteins were predicted to be localized to the nucleus, suggesting their roles in transcriptional regulation (Table 1). All of the 15 CsARF proteins contained DBD, which is a B3like DNA-binding domain and ARF domain (Figs. 2 and S1; Table 1). Only 4 CsARF proteins lacked the CTD (Figs. 2 and 3; Table 1), which is in the C-terminus region and allows the interaction between the ARF proteins and Aux/IAA proteins. The percentage (26.7%) for members lacking the CTD was similar to other species, such as the sweet orange (21.1%), tomato (28.6%) and Brassica rapa (22.6%) (Ha

et al., 2013; Li et al., 2015b). Previous studies demonstrated that whether an ARF acts as an activator or repressor depends on the amino acid composition of MR (Guilfoyle and Hagen, 2007). In the current work, of the 15 CsARF proteins, 4 members (CsARF6, 8-1, 8-2 and 19) belonging to class IV and V contain the QSL-rich region, suggesting their roles in transcriptional activation. The other 11 members contain either a SP-rich or SG-rich region, suggesting their roles in transcriptional repression (Fig. 3). All of the predicted repressors are distributed in classes I, II and VI. Each class contains either an activator or repressor. 4.2. CsARF genes have diverse expression patterns Considering the vital roles that ARF transcription factors play during plant development, it is not surprising that CsARFs can be

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Fig. 6. Expression analysis of CsARF genes under SA, GA and 6-BA treatments in shoots and roots. (A) SA treatment, shoots. (B) SA treatment, roots. (C) GA treatment, shoots. (D) GA treatment, roots. (E) 6-BA treatment, shoots. (F) 6-BA treatment, roots. “CK” represents untreated seedlings grown in water.

detected in many tissues of the tea plant. Here the expression of 13 CsARFs (Fig. 4) could be detected in all of the tea tissues we used, which may indicate their fundamental roles in different tea plant cell-types. Two genes, CsARF1-1 and CsARF1-2 were not detected in the roots but were detected in other tissues, with the highest transcription level in the leaves, suggesting they may play roles in leaf development like their Arabidopsis homolog, AtARF1 (Ellis et al., 2005). However, the finding that the transcription levels of both genes were very low in the flowers is somewhat surprising because AtARF1 was also involved in flower development (Ellis et al., 2005). The relative expression levels of CsARF2-1, 2-2, 3-1, 3-2, 4 and 19 were ubiquitously high in many detected tissues, including the roots, flowers and leaves, which are consistent with the reported function of their homologous genes in Arabidopsis (Ellis et al., 2005; Fahlgren et al., 2006; Marin et al., 2010; Wilmoth et al., 2005; Zhou et al., 2007). CsARF8-1, 8-2, 9 and 11 were mainly expressed in the

roots and flowers, suggesting their function in root and flower development. However, a further investigation is required to discover how these CsARF genes participate in the regulation of development. 4.3. CsARF genes respond to various phytohormones and abiotic stresses Phytohormones are major modulators of plant development and plant responses to various environmental stimuli. As the core element in auxin signaling, ARF was detected to mediate the crosstalk of many pathways. For example, our previous studies showed that OsARF12 and OsARF16 mediated the crosstalk between auxin and mineral nutrition in rice (Qi et al., 2012; Shen et al., 2013). In the current work, we conducted a systematic expression profiling of the tea plant ARFs under various

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Fig. 7. Expression analysis of CsARF genes under salinity and drought treatments in shoots and roots. (A) NaCl treatment, shoots. (B) NaCl treatment, roots. (C) Drought treatment, shoots. (D) PEG treatment, shoots. “CK” represents untreated seedlings grown in water.

phytohormones and abiotic stresses. Extensive research has shown that ARFs respond to auxin. As is well known, auxin has many forms, of which, IAA, 2,4-D and NAA are the most-commonly used. However, the mechanism of action for each form of auxin is different. IAA is a natural auxin that is transported via plant cells either by a carrier or not; 2,4-D is a synthetic auxin that is transported into plant cells depending a uptake carrier, while NAA, also a synthetic auxin, depends on an export carrier to be transported out of plant cells (Xu et al., 2014b). It will be more meaningful to examine the ARF expression level under different types of auxin treatment. Nevertheless, during the different forms of auxin treatment explored in this study, most of the detected CsARF genes exhibited a similar response (Fig. 5). The expression of most CsARF genes decreased significantly in the shoots with each type of auxin treatment (Fig. 5A, C, E). Li et al. (2015b) reported that most CiARFs in the sweet orange callus were suppressed by IAA treatment and Yu et al. (2014) reported that most EgrARFs in the young tree stems of Eucalyptus grandis were suppressed by NAA treatment. Our results appear to be consistent with these published reports in woody plants. For herbaceous plants, previous reports indicate that most ARFs were induced by auxin (Shen et al., 2015; Wu et al., 2014; Xing et al., 2011). Therefore, these data also suggest that ARF response to auxin cannot simply be deduced by phylogenetic analyses, or that the responses are achieved differently in woody plants and herbaceous plants. The experimental conditions, including the sampled tissues, treatment times and concentrations will lead to different results. The mRNA levels of 5 genes (CsARF1-1, 3-1, 8-1, 9 and 16-1) were quite different under different forms of auxin, suggesting their different function mechanisms. Plants often adopt different strategies to respond to various

abiotic stresses for survival in adverse environmental conditions (Ahuja et al., 2010). Increasing evidence showed that many proteins at the auxin signal pathway play vital roles in dealing with environmental change. For instance, genetic and physiological evidence have been provided that auxin influx transporter, AUX1, mediated ethylene-inhibited root elongation under alkaline stress (Li et al., 2015a). OsIAA6, a member of the rice Aux/IAA gene family, was associated to drought tolerance because its overexpression in transgenic rice enhanced drought tolerance (Jung et al., 2015). We also reported that an auxin transporter, OsABCB14, was involved in auxin transport and iron-deficiency responses (Xu et al., 2014b), and OsARF12, regulated root elongation and plays an important role in iron homeostasis in rice (Qi et al., 2012). To our knowledge, there are few reports about ARF function mechanism in the tea plant. In this work, our results showed that many of the CsARF genes responded to SA, GA, 6-BA, salt and drought signaling (Figs. 6 and 7). The changes in expression of some CsARFs genes were especially intriguing and led us to speculate that they may play essential roles in the crosstalk between the auxin, SA, GA, 6-BA, salt and drought signaling pathways. Additionally, high salt and drought are the major causes of the changes in osmatic pressure in plant cells (Kreps et al., 2002), so the involvement of these genes in response to salt and drought may indicate their involvement in osmotic stress in the tea plant. It is worth mentioning that, in the previous studies of woody plant ARFs, almost none of the researchers detected the expression of ARFs in various treatments in the roots, maybe due to the particularity of woody plants and the sampling destructiveness. In fact, the plant root is the essential organ to detect environmental stimuli. Our data showed most CsARFs responded to various abiotic stresses in the roots, consistent with the previous reports for many

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herbaceous plants. 5. Conclusion In this study, we have provided comprehensive information about 15 identified CsARF genes in the tea plant, including phylogenetic relationships, conserved domains, the amino acid compositions of the MR domain, subcellular localization and the expression patterns under various tissues/organs and treatments. The responsiveness of the CsARF genes to a wide range of phytohormones and abiotic stresses suggest that CsARFs are involved in the tea plant's tolerance to environmental stresses. This information not only provides a basis for further functional characterization of CsARFs but also provides new insights into breeding stresstolerant tea varieties. Contributions Conceived and designed the experiment: Y-X X, LC. Performed the experiments: JM, WC, T-T Q. Analyzed the data: Y-X X, S-C L, W-J H, C-F L. Wrote the paper: Y-X X, LC. Acknowledgments We gratefully acknowledge Dr. Chen-Jia Shen, the College of Life and Environmental Sciences in Hangzhou Normal University, for technical support. This work was supported by the Earmarked Fund for China Agriculture Research System (CARS-023), by the Chinese Academy of Agricultural Sciences through the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2014-TRICAAS), by the National Natural Science Foundation of China (Nos. 30901159, 31170624, 31100504, 31400581), by National Science and Technology Support Program of China (2013BAD01B03), by the Science and Technology Major Project for New Crop Varieties Breeding of Zhejiang Province (2012C12905). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2015.11.014. References Ahuja, I., de Vos, R.C., Bones, A.M., Hall, R.D., 2010. Plant molecular stress responses face climate change. Trends Plant Sci. 15, 664e674. de Jong, M., Wolters-Arts, M., Schimmel, B.C., Stultiens, C.L., de Groot, P.F., Powers, S.J., Tikunov, Y.M., Bovy, A.G., Mariani, C., Vriezen, W.H., Rieu, I., 2015. Solanum lycopersicum auxin response factor 9 regulates cell division activity during early tomato fruit development. J. Exp. Bot. 66, 3405e3416. Ding, Z., Friml, J., 2010. Auxin regulates distal stem cell differentiation in Arabidopsis roots. Proc. Nat. Acad. Sci. U. S. A. 107, 12046e12051. Du, L., Bao, C., Hu, T., Zhu, Q., Hu, H., He, Q., Mao, W., 2015. SmARF8, a transcription factor involved in parthenocarpy in eggplant. Mol. Genet. Genom. http:// dx.doi.org/10.1007/s00438-015-1088-5. Ellis, C.M., Nagpal, P., Young, J.C., Hagen, G., Guilfoyle, T.J., Reed, J.W., 2005. Auxin response factor1 and auxin response factor2 regulate senescence and floral organ abscission in Arabidopsis thaliana. Development 132, 4563e4574. Fahlgren, N., Montgomery, T.A., Howell, M.D., Allen, E., Dvorak, S.K., Alexander, A.L., Carrington, J.C., 2006. Regulation of auxin response factor3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Curr. Biol. 16, 939e944. Garrett, J.J., Meents, M.J., Blackshaw, M.T., Blackshaw, L.C., Hou, H., Styranko, D.M., Kohalmi, S.E., Schultz, E.A., 2012. A novel, semi-dominant allele of monopteros provides insight into leaf initiation and vein pattern formation. Planta 236, 297e312. Goetz, M., Vivian-Smith, A., Johnson, S.D., Koltunow, A.M., 2006. Auxin response factor8 is a negative regulator of fruit initiation in Arabidopsis. Plant Cell 18, 1873e1886. Guilfoyle, T.J., Hagen, G., 2007. Auxin response factors. Curr. Opin. Plant Biol. 10, 453e460. Ha, C.V., Le, D.T., Nishiyama, R., Watanabe, Y., Sulieman, S., Tran, U.T., Mochida, K.,

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