Plant Cell Rep DOI 10.1007/s00299-014-1622-7
ORIGINAL PAPER
Genome-wide identification, characterization and expression analysis of the auxin response factor gene family in Vitis vinifera Sibao Wan • Weili Li • Yueying Zhu • Zhanmin Liu • Weidong Huang • Jicheng Zhan
Received: 24 January 2014 / Revised: 9 April 2014 / Accepted: 15 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Key message Our study has identified and analyzed the VvARF gene family that may be associated with the development of grape berry and other tissues. Abstract Auxin response factors (ARFs) are transcription factors that regulate the expression of auxin responsive genes through specific binding to auxin response elements (AuxREs). The ARF genes are represented by a large multigene family in plants. Until now, many ARF families have been characterized based on genome resources. However, there is no specialized research about ARF genes in grapevine (Vitis vinifera). In this study, a comprehensive bioinformatics analysis of the grapevine ARF gene family is presented, including chromosomal locations, phylogenetic relationships, gene structures, conserved domains and expression profiles. Nineteen VvARF genes were identified and categorized into four groups (Classes 1, 2, 3 and 4). Most of VvARF proteins contain B3, AUX_RESP and AUX_IAA domains. The VvARF genes were widely expressed in a range of grape tissues, and fruit had higher transcript levels for most VvARFs
Communicated by R. J. Rose.
Electronic supplementary material The online version of this article (doi:10.1007/s00299-014-1622-7) contains supplementary material, which is available to authorized users.
detected in the EST sources. Furthermore, analysis of expression profiles indicated some VvARF genes may play important roles in the regulation of grape berry maturation processes. This study which provided basic genomic information for the grapevine ARF gene family will be useful in selecting candidate genes related to tissue development in grapevine and pave the way for further functional verification of these VvARF genes. Keywords ARF gene Domain analysis Expression profiling Vitis vinifera Abbreviations Aux/IAA Auxin/indole-3-acetic acid ARF Auxin response factor AuxRE Auxin response element BLAT BLAST-like alignment tool CDD Conserved domain database CTD Carboxy-terminal domain DAB Days after full bloom DBD DNA binding domain EST Expressed sequence tag GH3 Gretchen hagen 3 kDa Kilodalton NJ Neighbour joining pI Isoelectric point SAUR Small auxin-up RNA
S. Wan W. Li Y. Zhu Z. Liu Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, People’s Republic of China W. Huang J. Zhan (&) College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China e-mail: [email protected]
Introduction The phytohormone auxin plays a critical role in regulating plant growth and development by altering expression of
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early response genes, such as Aux/IAA, GH3 and SAUR gene family members (Abel and Theologis 1996). Through promoter analysis of these early auxin response genes, a number of putative auxin response elements (AuxREs) have been identified, and it has been demonstrated that one or more copies of a conserved motif, TGTCTG, are often present in the AuxREs (Ulmasov et al. 1997a; Guilfoyle et al. 1998). Auxin response factors (ARFs) are transcription factors that bind specifically to TGTCTG AuxREs and mediate auxin response. Auxin response factor 1 (ARF1) was the first auxin transcription factor gene isolated from Arabidopsis, using a yeast one-hybrid system with the TGTCTC AuxRE as a bait sequence (Ulmasov et al. 1997a). Since cloning of AtARF1, 23 members of this family from Arabidopsis, 25 from rice, and 39 from Populus trichocarpa have been identified through genome-wide data mining (Kalluri et al. 2007; Wang et al. 2007). A typical ARF protein contains two conserved domains, namely, an N-terminal DNA binding domain (DBD) and a C-terminal dimerization domain (CTD). The DNA binding domain is sufficient to target at least some ARFs to AuxREs (Ulmasov et al. 1999a; Tiwari et al. 2003). The ARF CTD is related in amino acid sequence to domains III and IV found in C-terminal of Aux/IAA proteins, and facilitates protein–protein interactions among the members of both the ARF and Aux/IAA families (Kim et al. 1997; Ulmasov et al. 1997a, b). Whether a given ARF acts as a transcription activator or repressor is determined by the amino acid composition of the variable middle region (MR) between the DNA binding domain and domains III/IV of the protein (Ulmasov et al. 1999a; Tiwari et al. 2003). Grapevine is one of the most important fruit crops and is consumed in many ways, including wine, table grape, juice and raisins (Doligez et al. 2006; This et al. 2006). Grapevine is also considered a good model for studying berry development in perennial fruit plants. A number of reports have indicated that auxin levels are high early in development, after which they decline steadily to be very low at veraison (Baydar and Harmankaya 2005; Bottcher et al. 2010). With the accomplishments of grapevine (Vitis vinifera L.) genome sequencing (Jaillon et al. 2007; Velasco et al. 2007), it becomes possible and feasible to isolate the functional gene families from grapevine by in silico cloning. As key players in plant development, the ARF family genes in grapevine have not yet been analyzed in detail. Identification and analysis of VvARF genes will aid in the understanding of cellular responses to the plant hormone auxin. Here, we have undertaken a comprehensive bioinformatics-based analysis of the grapevine ARF gene family. The work involved the identification of putative VvARFs
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by genome-wide searches, analysis of their phylogenetic relationship, chromosomal distribution, gene structures and domain organizations. Also, the expression patterns of VvARFs have been investigated by searching against EST sequences and microarray data and real-time PCR method.
Materials and methods Database search for ARF gene family of grapevine The grapevine genome sequence data were downloaded from the NCBI ftp site (ftp://ftp.ncbi.nlm.nih.gov/genomes/ Vitis_vinifera/). For the identification of possible ARF genes in grapevine, the Hidden Markov Model-based HMMER program (2.3.2) (Eddy 1998) was initially used to perform local search in the genome sequence dataset with the HMM profile of the ARF domain (PF06507) as a query. Additional members of grapevine ARF family were searched by NCBI BLASTp for Vitis vinifera (http://blast.ncbi. nlm.nih.gov/Blast.cgi) using proteins identified in the initial HMM search as query sequences. Invalid and redundant sequences were verified based on the conserved domains and the chromosomal locations of the candidates. Location of ARF genes on grapevine chromosomes Chromosomal locations of ARF genes were assigned by running BLAT queries against Genoscope grape genome (129 coverage) (http://www.genoscope.cns.fr/externe/Gen omeBrowser/Vitis/) (Jaillon et al. 2007). Phylogenetic analysis Multiple sequence alignments were done using the Clustal X (version 1.83) program with default parameters (Thompson et al. 1997). Phylogenetic analysis was performed with MEGA 4 software (Tamura et al. 2007) by neighbor-joining method (Saitou and Nei 1987) and the bootstrap test was carried out with 1,000 iterations. Exon–intron structure and domain analysis The number and positions of exons and introns for individual grapevine ARF genes were determined by alignment of the cDNAs against their corresponding genomic DNA sequences using the online gene structure display server (http://gsds.cbi.pku.edu.cn/) (Guo et al. 2007). The NCBI conserved domain search tool (http://www.ncbi.nlm.nih. gov/Structure/cdd/cdd.shtml) was utilized to predict the conserved motifs on all identified VvARF protein sequences.
Plant Cell Rep
Expression analysis of VvARF genes Using the locus_tags of VvARF genes as queries, in silico analysis of EST number was performed against NCBI’s Unigene Database (http://www.ncbi.nlm.nih.gov/unigene). Meanwhile, the gene expression patterns in different tissues were profiled based on the EST cDNA libraries. The expression data for grapevine (Vitis vinifera cv. Cabernet Sauvignon) fruit development acquired with the Affymetrix GeneChipÒ Vitis oligonucleotide microarray was obtained from Deluc et al. (2007). The microarray data analysis was also based on Deluc et al.’s study. The IDs of probe sets present on the array corresponding to the VvARF genes were extracted using the VMatch tool at PLEXdb (http://www.plantgdb.org) (Wise et al. 2007). Nine VvARF genes found in the microarray data were also analyzed by quantitative real-time RT-PCR experiments using the Cabernet Sauvignon berries as materials. Total RNA was extracted from small pea-size grape berries (20 and 40 days after bloom), veraison berries (70 days after bloom) and mature berries (90 and 100 days after bloom) using a modified CTAB method (Wan et al. 2009). After treated with RNase-free DNase (TaKaRa, Japan), 1 lg total RNA was reverse-transcribed to cDNA using AMV Strand cDNA Synthesis Kit (BBI Company). At least three different RNA isolations and cDNA syntheses were used as replicates for the qPCR. The amplification was performed on an ABI StepOnePlus Real-Time PCR System with fluorescent intercalating dye SYBR Green. The primer sequences used in real-time PCR for VvARFs were shown in Supplementary file 1. VvUbiquitin cDNA (GenBank accession number BN000705) was used as an internal control. Every quantitative real-time PCR was performed in triplicate. The comparative cycle threshold method (DDCT) was used to evaluate the relative quantities of each amplified product in the samples.
Results and discussion Identification of ARF gene family of grapevine In plants, auxin response factors are encoded by a multigene family. The availability of genomic resources such as ESTs and genome sequences has provided an opportunity to look for the members of ARF gene family. Until now, 23 Arabidopsis ARFs, 25 rice ARFs, 39 poplar ARFs, 13 Physcomitrella patens ARFs, 17 tomato ARFs, 10 Selaginella moellendorfiri ARFs and 22 maize ARFs have been deduced based on genomic resources, respectively (Hagen and Guilfoyle 2002; Wang et al. 2007; Kalluri et al. 2007; Rensing et al. 2008; Kumar et al. 2011; Banks et al. 2011; Wang et al. 2012). Finet et al. (2013) also reported the
information on ARF gene families in other 15 plant species including 19 ARF genes in grapevine. In this present study, the HMM profile search and BLASTp search followed by domain analysis using the NCBI’s CDD tool resulted in the primary identification of 43 potential ARF proteins in the grapevine genome sequences. Subsequently, the redundant sequences with the same chromosome locations were removed from the candidates. In the end, a total of 19 grapevine ARFs (VvARFs) were extracted and named by their chromosomal locations, listed in Table 1. The deduced polypeptide included four fields: NCBI accession number, number of amino acids (length), molecular weight (MW), and isoelectric point (pI) (Table 1). According to the prediction, the length of most VvARF proteins ranges from 560 to 1,133 amino acids. The molecular weights of these deduced VvARF proteins ranged from 62.18 kDa (VvARF17) to 125.21 kDa (VvARF11), and the isoelectric points ranged from 5.09 to 8.16. Chromosomal locations of VvARF genes The family of 19 VvARFs identified is distributed on 13 of the 19 grapevine chromosomes (Fig. 1). Three genes are located on chromosome 18, two on chromosomes 6, 10, 11 and 12, and one on chromosomes 1, 2, 4, 7, 8, 13, 15 and 17. The position (in bp) and direction of transcription (arrows) of each gene were determined on grapevine chromosomes available at GENOSCOPE (129 coverage). There is a tandem gene cluster (VvARF4–VvARF5) located on chromosome 6. In addition, further investigation showed that the distribution of each class of VvARF genes was significantly irregular and most of the VvARF genes located on the same chromosome belonged to different class except VvARF10 and 11 on chromosome 11 composed a pair of sister paralogs of Class 3 in the phylogenetic tree of VvAFR genes (Fig. 2). We suspect that the functions of the VvARF genes on the same chromosome are complementary. It is noteworthy that the nomenclature system for VvARFs used in this work, a generic name from VvARF1 to VvARF19, was provisionally given to distinguish each of the ARF genes according to its position from the top to the bottom on the grapevine chromosomes 1–19. This numbering system has been broadly used in genome-wide studies to provide a unique identifier for each member of a given gene family, such as the ARF, Aux/IAA or SAUR gene family in rice (Jain et al. 2006a, b; Wang et al. 2007). Phylogenetic analysis of VvARF genes To study the phylogenetic relationships between the members of VvARF gene family, an unrooted tree was constructed from an alignment of their protein sequences
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Plant Cell Rep Table 1 The ARF gene family in grapevine Gene namea
Locus_tag
Fl-cDNA accession no.b
ORF length (bp)
Chr.c
Positionc
Strand
Deduced polypeptided Protein accession no.
Length (aa)
MW (kDa)
pI
VvARF1
LOC100250592
XM_002268813
2,310
1
21717978..21724422
–
XP_002268849
769
86.08
6.47
VvARF2
LOC100247833
XM_002265126
2,325
2
1652957..1657554
?
XP_002265162
774
86.66
6.85
VvARF3 VvARF4
LOC100258129 LOC100256989
XM_002266642 XM_002282401
2,547 2,136
4 6
10403355..10448235 3443778..3447902
1 ?
XP_002266678 XP_002282437
848 711
94.43 77.95
5.95 8.16
VvARF5
LOC100243320
XM_002284983
2,400
6
3879882..3888267
?
XP_002285019
799
88.30
5.85
VvARF6
LOC100246055
XM_002270250
2,421
7
2256656..2263483
–
XP_002270286
806
89.50
5.54
VvARF7
LOC100251645
XM_002281450
2,106
8
12924872..12928912
?
XP_002281486
701
77.49
6.85
VvARF8
LOC100245251
XM_002273365
2,361
10
1695112..1704509
–
XP_002273401
786
85.89
7.33
VvARF9
LOC100242923
XM_002279772
2,727
10
6956354..6964043
–
XP_002279808
908
100.51
6.19
VvARF10
LOC100257618
XM_002276601
3,255
11
629738..639662
?
XP_002276637
1,084
120.84
6.12
VvARF11
LOC100263801
XM_002266567
3,402
11
13985443..13992869
?
XP_002266603
1,133
125.21
6.20
VvARF12
LOC100260866
XM_002282794
2,538
12
1747340..1765195
–
XP_002282830
845
93.99
6.23
VvARF13
LOC100264303
XM_002268312
2,037
12
21940697..21948769
–
XP_002268348
678
75.39
5.75
VvARF14
LOC100265118
XM_002273554
2,052
13
5744374..5747220
–
XP_002273590
683
75.27
6.43
VvARF15
LOC100265555
XM_002264036
2,349
15
17300361..17306429
–
XP_002264072
782
87.17
8.07
VvARF16
LOC100268072
XM_002284507
2,589
17
225273..230747
?
XP_002284543
862
96.13
6.20
VvARF17
LOC100255673
XM_002284292
1,683
18
3748918..3754816
?
XP_002284328
560
62.18
6.00
VvARF18 VvARF19
LOC100254074 LOC100263592
XM_003634334 XM_002266911
2,844 1,938
18 18
11920486..11928772 28749580..28756733
– ?
XP_003634382 XP_002266947
947 645
104.25 71.88
5.09 6.23
NF not found a
Systematic designation given to grapevine ARF genes
b
Corresponding full-length cDNA is available in NCBI’s Genbank database or not
c
Chromosomal location of the VvARF genes in the Vitis vinifera genome (currently at 912 coverage)
d
The theoretical isoelectric points (pI) and molecular weights (MW) of the deduced polypeptides were calculated using the ExPASy Compute pI/Mw tool (http://expasy.org/)
Fig. 1 Genomic distribution of VvARF genes on grapevine chromosomes. Only those chromosomes bearing VvARF genes are represented. The chromosome numbers and sizes (Mb) are indicated at the top and bottom of each bar, respectively. The arrows next to gene
names show the direction of transcription. The numbers on the right side of the bars designated the approximate physical position of the first exon of corresponding VvARF gene on grapevine chromosomes available at GENOSCOPE (912 coverage)
and viewed in MEGA 4 program by the N-J method. It was showed that all the VvARFs fell into four broad groups (Classes 1, 2, 3 and 4) with well-supported bootstrap values (Fig. 2). There were 7 VvARFs in Class 1, 2 VvARFs in Class 2, 6 VvARFs in Class 3 and 4 VvARFs in Class 4.
All 19 VvARFs were distributed into 7 sister pairs of paralogous ARFs (VvARF2/15, VvARF13/19, VvARF1/ 16, VvARF5/8, VvARF9/12, VvARF10/11, VvARF4/14), 6 of which had very strong bootsrap support ([99 %), while the remaining VvARFs were not matched.
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Fig. 2 Phylogenetic relationship among the grapevine ARF proteins. Amino acid sequences of full-length predicted ARF proteins were aligned using ClustalX program. Tree was generated using MEGA4 program by neighbor-joining method. Bootstrap values from 1,000 replicates are indicated at each branch
A phylogenetic relationship between Arabidopsis and grapevine was further investigated by aligning protein sequences of 23 AtARFs and 19 VvARFs. Forty-two ARFs were divided into 5 groups named Classes 1, 2, 3, 4 and 5 containing 12, 4, 11, 7 and 8 members, respectively (Fig. 3). This classification is largely similar to the VvARFs (Fig. 2). Classes 1, 2, 3 and 5 were divided from the same branch, while Class 4 belongs to the other branch. In total, 12 sister pairs containing 6 VvARF-AtARF, 3 VvARF-VvARF and 3 AtARF-AtARF, were confirmed based on bootstrap values (above 50 %) in the phylogenetic tree. It is noteworthy that Classes 1–4 included both VvARFs and AtARFs while the Class 5 had only AtARFs without VvARFs. Other plant species, such as rice, maize and tomato, also appear to lack the Class 5 ARFs found in Arabidopsis (Wang et al. 2007; Kumar et al. 2011; Liu et al. 2011). Gene structure analysis of VvARFs A comparison of the full-length cDNA sequences with the corresponding genomic DNA sequences was made to determine the numbers and positions of exons and introns of each individual VvARF genes. It was showed that the coding sequences of all the ARF genes are disrupted by introns, the number of introns varied from 2 to 15 (Fig. 4). Previous studies have revealed that the coding sequences of
Fig. 3 Phylogenetic relationship of grapevine and Arabidopsis ARF proteins. The unrooted tree was generated using MEGA4 program by neighbor-joining method. Bootstrap values from 1,000 replicates are indicated at each branch
all 25 OsARF genes are interrupted by 2–14 introns (Wang et al. 2007). The VvARF genes of Class 1 and Class 3 in the phylognetic tree had 13–15 introns. In Class 2, VvARF genes had 10–11 introns. However, the VvARF genes of Class 4 only harbored 2–3 introns. All members of the same VvARF gene class also exhibited similar gene structures of intron position and length, and the intron phases were conserved (Fig. 4). These results provide important evidence to support the reliability of the phylogenetic analysis. In other words, the positions, lengths and phase of
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Fig. 4 Exon–intron organization of grapevine ARF genes. The coding and untranslated regions are represented by grey and black boxes, respectively. Lines represent the introns. The sizes of the boxes and lines are proportionally mapped. The numbers 0, 1 and 2
represent phase 0, 1, and 2 introns, respectively. The length of the sixth intron within VvARF3 was more than 30 kb, which was cut 24 kb in this figure
Fig. 5 Conserved domains in VvARF proteins. Domains were determined through searching the VvARF protein sequences in NCBI Conserved Domain Database. B3 plant-specific B3-DNA binding
domain, Auxin_resp a conserved region of auxin responsive factor, AUX_IAA C-terminal AUX_IAA domain
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introns can represent the phylogenetic relationships of the gene family. This phenomenon has also been reported in bHLH and SBP-box gene families (Li et al. 2006; Guo et al. 2008). Interestingly, the length of the sixth intron within VvARF3 was more than 30 kb (Fig. 4), which was not observed in other AtARF and OsARF genes. Analysis of functional domains of VvARF proteins Domains of VvARF protein sequences were identified and analyzed using NCBI Conserved Domain Database (Marchler-Bauer et al. 2009). Three conserved domains (B3, AUX_RESP and AUX_IAA) were identified and their distribution in the proteins of respective classes in the phylogenetic tree has been presented in Fig. 5. Seventeen VvARF protein sequences contained the B3, AUX_RESP and AUX_IAA domains. Two protein sequences, VvARF8 and VvARF17, contained the B3 and AUX_RESP domains. Most ARF proteins contain two well-conserved domains, one of which is N-terminal B3-type DNA binding domain (DBD) and the other is C-terminal AUX_IAA domain (CTD). The DNA binding domain that includes an ARF family-specific domain (referred to as AUX_RESP domain), is essential for the interaction between ARFs and the TGTCTC AuxREs (Ulmasov et al. 1999a; Tiwari et al. 2003). In this study, the conserved N-terminal regions of VvARFs were divided into B3 and AUX_RESP domains, and the AUX_RESP domain has been used to identify VvARFs from protein database. The AUX_IAA domain, also known as domains III and IV, are similar to those found in the C-terminus of Aux/IAAs, which is a protein– protein interaction domain that allows the homo- and hetero-dimerization of ARFs and the hetero-dimerization of ARF and Aux/IAA proteins (Ulmasov et al. 1997a, b, 1999b; Kim et al. 1997; Ouellet et al. 2001). The AUX_IAA domain in some ARF proteins can also increase the in vitro binding of these ARFs to TGTCTC AuxREs by facilitating the formation of dimers that occupy the two half-sites in palindromic AuxREs (Ulmasov et al. 1999b). The middle region (MR) of ARFs, which separates DBD from CTD, is critical in determining whether an ARF protein will act as an activator or repressor (Guilfoyle et al. 1998; Ulmasov et al. 1999b, Guilfoyle and Hagen 2001; Ouellet et al. 2001; Tiwari et al. 2003). Transfection assays with carrot protoplasts indicated that Arabidopsis ARF1, 2, 3, 4 and 9, among which ARF1 contain middle region rich in proline (P), serine (S) and threonine (T) residues, are repressors and ARF5, 6, 7 and 8, which contain middle regions rich in glutamine (Q) residues, are activators (Ulmasov et al. 1999a; Tiwari et al. 2003). The detailed sequence analysis of all 19 VvARF proteins revealed that S-rich middle regions were found in VvARF1, 2, 4, 6, 7,
13, 14, 15, 16, 17 and 19, indicating that these proteins are more likely to act as repressors of transcription (Supplementary file 2 and 3). While Q-rich middle regions were found in VvARF3, 9, 10, 11 and 12, implying that these proteins are probable transcriptional activators (Supplementary file 3). The middle regions of VvARF5, 8 and 18 contain no obvious biased amino acid sequences (Supplementary file 3). All VvARF proteins with Q-rich middle regions belong to Class 3, while S-rich VvARFs are present in Classes 1 and 4. Thus, phylogenetic analysis of VvARF proteins also showed that activators and repressors were appropriately placed in separate classes. Expression analysis of VvARF genes in grapevine The expression pattern of a gene often has a correlation with its function. ESTs and cDNAs widely available in different databases have been considered as useful resources for preliminary analysis of gene expression (Adams et al. 1995). In this paper, NCBI’s Unigene Database was used to test the existence of VvARF genes and to predict their tissue-specific expression profiles (Table 2). The number of ESTs for individual VvARF genes varies from 0 to 20. There are 20 ESTs corresponding to VvARF16 in the database, while the transcriptions of VvAFR6, 10 and 12 remained unclear, as no corresponding EST could be found. This lack of detection may be due to the temporal or spatial expression patterns of these genes. In grapevine, most of the VvARF genes detected in the EST sources had higher transcript levels in the tissue of fruit compared to their expression in other tissues, suggesting that these VvARF genes may be involved in control of a series of physiological and biochemical processes including fruit growth and development. In addition, the VvARF genes were widely expressed in buds, roots, leaves, inflorescences and flowers, indicating that the VvARF family genes may perform a variety of physiological functions in grapevine. The expression profile of VvARF genes during Cabernet Sauvignon berry development was analyzed using the Affymetrix GeneChipÒ Vitis oligonucleotide microarray ver. 1.0 (Deluc et al. 2007). Grape berry development is a dynamic process that involves a complex series of physiological and biochemical changes. These changes can be divided into three major phases from small pea-size berries (21, 42 and 49 DAB known as E-L stages 31, 32 and 33 in the modified E-L system), through veraison (56 and 63 DAB known as E-L stages 34 and 35), to mature berries (84 and 112 DAB known as E-L stages 36 and 38) (Deluc et al. 2007). The microarray data showed 9 VvARF genes were represented on the GeneChip (Fig. 6). The expression of 3 genes (VvARF 8, 11 and 19) did not change significantly during fruit development. However, VvARF1, 16
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Plant Cell Rep Table 2 The tissue distribution profile of grapevine ARF genes based on the number of expressed sequence tags (ESTs) present in NCBI’s Unigene Database Gene
Bud
Root
VvARF1 VvARF2
Stem
Leaf
Inflorescence
1 1
Flower
Fruit
Seed
Cell culturea
1
5
1
1
2
VvARF3
1
2 1
9 1
7
6
4
13
1 1
1
1
2
1
4
VvARF6 VvARF7 VvARF8
0 1
1
1
2
VvARF9
1
3
2 3
1
6 5
6
5
15
VvARF10
0
VvARF11
1
1
2
2
4
13
VvARF12
0
VvARF13 VvARF14
4 1
4
VvARF15 VvARF16
4 1
6
2
20
4 5
3
VvARF17
1
3
2
4
1 2 1
4
3
VvARF18 VvARF19
Total EST number
3
VvARF4 VvARF5
Mixedb
4
2
7
3
19
8
4
5
a
Carbernet Sauvignon cell culture
b
Carbernet Sauvignon flower, leaf and root non-normalized
10
2
Log2 intensity value observed by microarray
14
12
VvARF1 VvARF3 VvARF5 VvARF8 VvARF11 VvARF15 VvARF16 VvARF18 VvARF19
10
8
6
4
2
0
31 32 33 42 49 21 small pea-size berries
34 35 63 56 veraison
36 84
38 112 mature berries
39 E-L Stage DAB
Developmental stage
Fig. 6 Expression profile of nine grapevine ARF genes in berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChipÒ Vitis oligonucleotide microarray ver. 1.0 spanning seven developmental stages from small pea-size berries (E-L stages 31, 32 and 33 or
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21, 42 and 49 DAB), through veraison (E-L stages 34 and 35 or 56 and 63 DAB), to mature berries (E-L stages 36 and 38 or 84 and 112 DAB). Error bars showed standard errors of the means of the results from three replicates
Relative expression to VvUbiquitin
Plant Cell Rep 8 7 6 5 4 3 2 1
Relative expression to VvUbiquitin
10
2
5
1 0 40
70
90
VvARF8 3 2 1 0 40
70
90
40
1.6 1.4 1.2 1.0 .8 .6 .4 .2 0.0
90
100
20
0.0
70
90
100
20 40 70 90 100 small pea veraison mature berries size berries
Days after full bloom
4.0 3.5 3.0 2.5 2.0 1.5 1.0 .5 70
40
VvARF19
100
VvARF3
20
20 2.2 2.0 1.8 1.6 1.4 1.2 1.0 .8 .6 .4 .2 0.0
4
20
0
100
1.30
Relative expression to VvUbiquitin
15
3
5
Relative expression to VvUbiquitin
20
4
20
.90
VvARF18
VvARF16
5
0
1.25 1.20 1.15 1.10 1.05 1.00 .95
25
6
VvARF1
40
70
90
100
70
90
100
VvARF11
0
20
40
1.4
VvARF5
VvARF15
1.2 1.0 .8 .6 .4 .2 0.0
20
40
70
90
100
20
40
70
90
100
small pea veraison mature berries size berries
small pea veraison mature berries size berries
Days after full bloom
Days after full bloom
Fig. 7 Relative mRNA expression levels of VvARF genes in the whole grape berries during development. Quantitative RT-PCR was performed with gene-specific primers. For each sample, transcript levels were normalized with those of VvUbiquitin, and the relative
expression levels of each gene were obtained using the DDCT method. Error bars showed standard errors of the means of the results from three replicates
and 18 were highly expressed at the three phases and the expression of VvARF3, 5 and 15 gradually decreased during fruit development indicated that these 6 genes may play more important roles in grapevine berry development. In order to validate the expression profiles of VvARF genes as revealed from microarray analysis, quantitative real-time RT-PCR analysis was performed for these VvARFs at different developmental stages. The results of qPCR showed that the expression patterns of VvARF1, 5, 15, 18 were in good agreement with the microarray data. However, the remaining 5 VvARF genes appeared to have different expression results between microarray and qPCR data which may be mainly caused by different
environmental growth conditions of the grapes. Though Cabernet Sauvignon grape berries were used in both experiments, miroarray data were obtained from berries harvested from the Shenandoah Vineyard, Plymouth, California, in 2004 and berries used in qPCR analysis were sampled from a vineyard in the western suburbs of Beijing in 2012. As shown in Fig. 7, the expression of VvARF1, 16 and 18 was significantly increased at 70 DAB (veraison) and kept on high levels until the end of berry development. VvARF8 and 19 reached the largest transcription levels at veraison and then decreased. VvARF3 and 11 exhibited a peak of expression at the end of mature stage, while VvARF5 and 15 decreased and showed the minimum at the
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grape berry development stages of veraison and mature. The high levels of VvARF1, 16 and 19 which have S-rich middle regions and act as transcriptional repressors provided evidence in support of a decrease in auxin levels as veraison approaches. However, the high levels of transcriptional activators, VvARF3 and 11, at mature berries and VvARF5 and 15 at small pea-size berries indicated these genes may be involved in berry ripening and cell division, respectively. The distinct expression profiles indicated the potential functional differences among VvARFs which should be further investigated during grape berry development.
Conclusions ARF genes have been implicated in many development processes and stress responses. It is important to understand their regulation for increasing plant growth and development. In this study, 19 AFR genes were identified in grapevine, and a comprehensive account of this gene family has been performed. The information generated from analysis of the structure of VvARF proteins indicated repressor VvARFs which contained S-rich middle regions were present in Classes 1 and 4, while activator proteins with Q-rich middle regions belonged to Class 3. Expression analysis showed that the VvARF genes may be involved in control of the development of many tissues and some VVARFs that may play important roles during grape berry development were detected. These data would be useful in selecting candidate genes related to tissue development in grapevine and will pave the way for further functional verification of these VvARF genes. Acknowledgments This work was supported by the National Natural Science Foundation of China (30871699, 30901012 and 31171942), Leading Academic Discipline Project of Shanghai Municipal Education Commission-Molecular Physiology (J50108) and Shanghai Science and Technology Committee No. 10DZ2271800. Conflict of interest of interest.
The authors declare that they have no conflict
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ORIGINAL PAPER
Genome-wide identification, characterization and expression analysis of the auxin response factor gene family in Vitis vinifera Sibao Wan • Weili Li • Yueying Zhu • Zhanmin Liu • Weidong Huang • Jicheng Zhan
Received: 24 January 2014 / Revised: 9 April 2014 / Accepted: 15 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Key message Our study has identified and analyzed the VvARF gene family that may be associated with the development of grape berry and other tissues. Abstract Auxin response factors (ARFs) are transcription factors that regulate the expression of auxin responsive genes through specific binding to auxin response elements (AuxREs). The ARF genes are represented by a large multigene family in plants. Until now, many ARF families have been characterized based on genome resources. However, there is no specialized research about ARF genes in grapevine (Vitis vinifera). In this study, a comprehensive bioinformatics analysis of the grapevine ARF gene family is presented, including chromosomal locations, phylogenetic relationships, gene structures, conserved domains and expression profiles. Nineteen VvARF genes were identified and categorized into four groups (Classes 1, 2, 3 and 4). Most of VvARF proteins contain B3, AUX_RESP and AUX_IAA domains. The VvARF genes were widely expressed in a range of grape tissues, and fruit had higher transcript levels for most VvARFs
Communicated by R. J. Rose.
Electronic supplementary material The online version of this article (doi:10.1007/s00299-014-1622-7) contains supplementary material, which is available to authorized users.
detected in the EST sources. Furthermore, analysis of expression profiles indicated some VvARF genes may play important roles in the regulation of grape berry maturation processes. This study which provided basic genomic information for the grapevine ARF gene family will be useful in selecting candidate genes related to tissue development in grapevine and pave the way for further functional verification of these VvARF genes. Keywords ARF gene Domain analysis Expression profiling Vitis vinifera Abbreviations Aux/IAA Auxin/indole-3-acetic acid ARF Auxin response factor AuxRE Auxin response element BLAT BLAST-like alignment tool CDD Conserved domain database CTD Carboxy-terminal domain DAB Days after full bloom DBD DNA binding domain EST Expressed sequence tag GH3 Gretchen hagen 3 kDa Kilodalton NJ Neighbour joining pI Isoelectric point SAUR Small auxin-up RNA
S. Wan W. Li Y. Zhu Z. Liu Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, People’s Republic of China W. Huang J. Zhan (&) College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China e-mail: [email protected]
Introduction The phytohormone auxin plays a critical role in regulating plant growth and development by altering expression of
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early response genes, such as Aux/IAA, GH3 and SAUR gene family members (Abel and Theologis 1996). Through promoter analysis of these early auxin response genes, a number of putative auxin response elements (AuxREs) have been identified, and it has been demonstrated that one or more copies of a conserved motif, TGTCTG, are often present in the AuxREs (Ulmasov et al. 1997a; Guilfoyle et al. 1998). Auxin response factors (ARFs) are transcription factors that bind specifically to TGTCTG AuxREs and mediate auxin response. Auxin response factor 1 (ARF1) was the first auxin transcription factor gene isolated from Arabidopsis, using a yeast one-hybrid system with the TGTCTC AuxRE as a bait sequence (Ulmasov et al. 1997a). Since cloning of AtARF1, 23 members of this family from Arabidopsis, 25 from rice, and 39 from Populus trichocarpa have been identified through genome-wide data mining (Kalluri et al. 2007; Wang et al. 2007). A typical ARF protein contains two conserved domains, namely, an N-terminal DNA binding domain (DBD) and a C-terminal dimerization domain (CTD). The DNA binding domain is sufficient to target at least some ARFs to AuxREs (Ulmasov et al. 1999a; Tiwari et al. 2003). The ARF CTD is related in amino acid sequence to domains III and IV found in C-terminal of Aux/IAA proteins, and facilitates protein–protein interactions among the members of both the ARF and Aux/IAA families (Kim et al. 1997; Ulmasov et al. 1997a, b). Whether a given ARF acts as a transcription activator or repressor is determined by the amino acid composition of the variable middle region (MR) between the DNA binding domain and domains III/IV of the protein (Ulmasov et al. 1999a; Tiwari et al. 2003). Grapevine is one of the most important fruit crops and is consumed in many ways, including wine, table grape, juice and raisins (Doligez et al. 2006; This et al. 2006). Grapevine is also considered a good model for studying berry development in perennial fruit plants. A number of reports have indicated that auxin levels are high early in development, after which they decline steadily to be very low at veraison (Baydar and Harmankaya 2005; Bottcher et al. 2010). With the accomplishments of grapevine (Vitis vinifera L.) genome sequencing (Jaillon et al. 2007; Velasco et al. 2007), it becomes possible and feasible to isolate the functional gene families from grapevine by in silico cloning. As key players in plant development, the ARF family genes in grapevine have not yet been analyzed in detail. Identification and analysis of VvARF genes will aid in the understanding of cellular responses to the plant hormone auxin. Here, we have undertaken a comprehensive bioinformatics-based analysis of the grapevine ARF gene family. The work involved the identification of putative VvARFs
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by genome-wide searches, analysis of their phylogenetic relationship, chromosomal distribution, gene structures and domain organizations. Also, the expression patterns of VvARFs have been investigated by searching against EST sequences and microarray data and real-time PCR method.
Materials and methods Database search for ARF gene family of grapevine The grapevine genome sequence data were downloaded from the NCBI ftp site (ftp://ftp.ncbi.nlm.nih.gov/genomes/ Vitis_vinifera/). For the identification of possible ARF genes in grapevine, the Hidden Markov Model-based HMMER program (2.3.2) (Eddy 1998) was initially used to perform local search in the genome sequence dataset with the HMM profile of the ARF domain (PF06507) as a query. Additional members of grapevine ARF family were searched by NCBI BLASTp for Vitis vinifera (http://blast.ncbi. nlm.nih.gov/Blast.cgi) using proteins identified in the initial HMM search as query sequences. Invalid and redundant sequences were verified based on the conserved domains and the chromosomal locations of the candidates. Location of ARF genes on grapevine chromosomes Chromosomal locations of ARF genes were assigned by running BLAT queries against Genoscope grape genome (129 coverage) (http://www.genoscope.cns.fr/externe/Gen omeBrowser/Vitis/) (Jaillon et al. 2007). Phylogenetic analysis Multiple sequence alignments were done using the Clustal X (version 1.83) program with default parameters (Thompson et al. 1997). Phylogenetic analysis was performed with MEGA 4 software (Tamura et al. 2007) by neighbor-joining method (Saitou and Nei 1987) and the bootstrap test was carried out with 1,000 iterations. Exon–intron structure and domain analysis The number and positions of exons and introns for individual grapevine ARF genes were determined by alignment of the cDNAs against their corresponding genomic DNA sequences using the online gene structure display server (http://gsds.cbi.pku.edu.cn/) (Guo et al. 2007). The NCBI conserved domain search tool (http://www.ncbi.nlm.nih. gov/Structure/cdd/cdd.shtml) was utilized to predict the conserved motifs on all identified VvARF protein sequences.
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Expression analysis of VvARF genes Using the locus_tags of VvARF genes as queries, in silico analysis of EST number was performed against NCBI’s Unigene Database (http://www.ncbi.nlm.nih.gov/unigene). Meanwhile, the gene expression patterns in different tissues were profiled based on the EST cDNA libraries. The expression data for grapevine (Vitis vinifera cv. Cabernet Sauvignon) fruit development acquired with the Affymetrix GeneChipÒ Vitis oligonucleotide microarray was obtained from Deluc et al. (2007). The microarray data analysis was also based on Deluc et al.’s study. The IDs of probe sets present on the array corresponding to the VvARF genes were extracted using the VMatch tool at PLEXdb (http://www.plantgdb.org) (Wise et al. 2007). Nine VvARF genes found in the microarray data were also analyzed by quantitative real-time RT-PCR experiments using the Cabernet Sauvignon berries as materials. Total RNA was extracted from small pea-size grape berries (20 and 40 days after bloom), veraison berries (70 days after bloom) and mature berries (90 and 100 days after bloom) using a modified CTAB method (Wan et al. 2009). After treated with RNase-free DNase (TaKaRa, Japan), 1 lg total RNA was reverse-transcribed to cDNA using AMV Strand cDNA Synthesis Kit (BBI Company). At least three different RNA isolations and cDNA syntheses were used as replicates for the qPCR. The amplification was performed on an ABI StepOnePlus Real-Time PCR System with fluorescent intercalating dye SYBR Green. The primer sequences used in real-time PCR for VvARFs were shown in Supplementary file 1. VvUbiquitin cDNA (GenBank accession number BN000705) was used as an internal control. Every quantitative real-time PCR was performed in triplicate. The comparative cycle threshold method (DDCT) was used to evaluate the relative quantities of each amplified product in the samples.
Results and discussion Identification of ARF gene family of grapevine In plants, auxin response factors are encoded by a multigene family. The availability of genomic resources such as ESTs and genome sequences has provided an opportunity to look for the members of ARF gene family. Until now, 23 Arabidopsis ARFs, 25 rice ARFs, 39 poplar ARFs, 13 Physcomitrella patens ARFs, 17 tomato ARFs, 10 Selaginella moellendorfiri ARFs and 22 maize ARFs have been deduced based on genomic resources, respectively (Hagen and Guilfoyle 2002; Wang et al. 2007; Kalluri et al. 2007; Rensing et al. 2008; Kumar et al. 2011; Banks et al. 2011; Wang et al. 2012). Finet et al. (2013) also reported the
information on ARF gene families in other 15 plant species including 19 ARF genes in grapevine. In this present study, the HMM profile search and BLASTp search followed by domain analysis using the NCBI’s CDD tool resulted in the primary identification of 43 potential ARF proteins in the grapevine genome sequences. Subsequently, the redundant sequences with the same chromosome locations were removed from the candidates. In the end, a total of 19 grapevine ARFs (VvARFs) were extracted and named by their chromosomal locations, listed in Table 1. The deduced polypeptide included four fields: NCBI accession number, number of amino acids (length), molecular weight (MW), and isoelectric point (pI) (Table 1). According to the prediction, the length of most VvARF proteins ranges from 560 to 1,133 amino acids. The molecular weights of these deduced VvARF proteins ranged from 62.18 kDa (VvARF17) to 125.21 kDa (VvARF11), and the isoelectric points ranged from 5.09 to 8.16. Chromosomal locations of VvARF genes The family of 19 VvARFs identified is distributed on 13 of the 19 grapevine chromosomes (Fig. 1). Three genes are located on chromosome 18, two on chromosomes 6, 10, 11 and 12, and one on chromosomes 1, 2, 4, 7, 8, 13, 15 and 17. The position (in bp) and direction of transcription (arrows) of each gene were determined on grapevine chromosomes available at GENOSCOPE (129 coverage). There is a tandem gene cluster (VvARF4–VvARF5) located on chromosome 6. In addition, further investigation showed that the distribution of each class of VvARF genes was significantly irregular and most of the VvARF genes located on the same chromosome belonged to different class except VvARF10 and 11 on chromosome 11 composed a pair of sister paralogs of Class 3 in the phylogenetic tree of VvAFR genes (Fig. 2). We suspect that the functions of the VvARF genes on the same chromosome are complementary. It is noteworthy that the nomenclature system for VvARFs used in this work, a generic name from VvARF1 to VvARF19, was provisionally given to distinguish each of the ARF genes according to its position from the top to the bottom on the grapevine chromosomes 1–19. This numbering system has been broadly used in genome-wide studies to provide a unique identifier for each member of a given gene family, such as the ARF, Aux/IAA or SAUR gene family in rice (Jain et al. 2006a, b; Wang et al. 2007). Phylogenetic analysis of VvARF genes To study the phylogenetic relationships between the members of VvARF gene family, an unrooted tree was constructed from an alignment of their protein sequences
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Plant Cell Rep Table 1 The ARF gene family in grapevine Gene namea
Locus_tag
Fl-cDNA accession no.b
ORF length (bp)
Chr.c
Positionc
Strand
Deduced polypeptided Protein accession no.
Length (aa)
MW (kDa)
pI
VvARF1
LOC100250592
XM_002268813
2,310
1
21717978..21724422
–
XP_002268849
769
86.08
6.47
VvARF2
LOC100247833
XM_002265126
2,325
2
1652957..1657554
?
XP_002265162
774
86.66
6.85
VvARF3 VvARF4
LOC100258129 LOC100256989
XM_002266642 XM_002282401
2,547 2,136
4 6
10403355..10448235 3443778..3447902
1 ?
XP_002266678 XP_002282437
848 711
94.43 77.95
5.95 8.16
VvARF5
LOC100243320
XM_002284983
2,400
6
3879882..3888267
?
XP_002285019
799
88.30
5.85
VvARF6
LOC100246055
XM_002270250
2,421
7
2256656..2263483
–
XP_002270286
806
89.50
5.54
VvARF7
LOC100251645
XM_002281450
2,106
8
12924872..12928912
?
XP_002281486
701
77.49
6.85
VvARF8
LOC100245251
XM_002273365
2,361
10
1695112..1704509
–
XP_002273401
786
85.89
7.33
VvARF9
LOC100242923
XM_002279772
2,727
10
6956354..6964043
–
XP_002279808
908
100.51
6.19
VvARF10
LOC100257618
XM_002276601
3,255
11
629738..639662
?
XP_002276637
1,084
120.84
6.12
VvARF11
LOC100263801
XM_002266567
3,402
11
13985443..13992869
?
XP_002266603
1,133
125.21
6.20
VvARF12
LOC100260866
XM_002282794
2,538
12
1747340..1765195
–
XP_002282830
845
93.99
6.23
VvARF13
LOC100264303
XM_002268312
2,037
12
21940697..21948769
–
XP_002268348
678
75.39
5.75
VvARF14
LOC100265118
XM_002273554
2,052
13
5744374..5747220
–
XP_002273590
683
75.27
6.43
VvARF15
LOC100265555
XM_002264036
2,349
15
17300361..17306429
–
XP_002264072
782
87.17
8.07
VvARF16
LOC100268072
XM_002284507
2,589
17
225273..230747
?
XP_002284543
862
96.13
6.20
VvARF17
LOC100255673
XM_002284292
1,683
18
3748918..3754816
?
XP_002284328
560
62.18
6.00
VvARF18 VvARF19
LOC100254074 LOC100263592
XM_003634334 XM_002266911
2,844 1,938
18 18
11920486..11928772 28749580..28756733
– ?
XP_003634382 XP_002266947
947 645
104.25 71.88
5.09 6.23
NF not found a
Systematic designation given to grapevine ARF genes
b
Corresponding full-length cDNA is available in NCBI’s Genbank database or not
c
Chromosomal location of the VvARF genes in the Vitis vinifera genome (currently at 912 coverage)
d
The theoretical isoelectric points (pI) and molecular weights (MW) of the deduced polypeptides were calculated using the ExPASy Compute pI/Mw tool (http://expasy.org/)
Fig. 1 Genomic distribution of VvARF genes on grapevine chromosomes. Only those chromosomes bearing VvARF genes are represented. The chromosome numbers and sizes (Mb) are indicated at the top and bottom of each bar, respectively. The arrows next to gene
names show the direction of transcription. The numbers on the right side of the bars designated the approximate physical position of the first exon of corresponding VvARF gene on grapevine chromosomes available at GENOSCOPE (912 coverage)
and viewed in MEGA 4 program by the N-J method. It was showed that all the VvARFs fell into four broad groups (Classes 1, 2, 3 and 4) with well-supported bootstrap values (Fig. 2). There were 7 VvARFs in Class 1, 2 VvARFs in Class 2, 6 VvARFs in Class 3 and 4 VvARFs in Class 4.
All 19 VvARFs were distributed into 7 sister pairs of paralogous ARFs (VvARF2/15, VvARF13/19, VvARF1/ 16, VvARF5/8, VvARF9/12, VvARF10/11, VvARF4/14), 6 of which had very strong bootsrap support ([99 %), while the remaining VvARFs were not matched.
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Fig. 2 Phylogenetic relationship among the grapevine ARF proteins. Amino acid sequences of full-length predicted ARF proteins were aligned using ClustalX program. Tree was generated using MEGA4 program by neighbor-joining method. Bootstrap values from 1,000 replicates are indicated at each branch
A phylogenetic relationship between Arabidopsis and grapevine was further investigated by aligning protein sequences of 23 AtARFs and 19 VvARFs. Forty-two ARFs were divided into 5 groups named Classes 1, 2, 3, 4 and 5 containing 12, 4, 11, 7 and 8 members, respectively (Fig. 3). This classification is largely similar to the VvARFs (Fig. 2). Classes 1, 2, 3 and 5 were divided from the same branch, while Class 4 belongs to the other branch. In total, 12 sister pairs containing 6 VvARF-AtARF, 3 VvARF-VvARF and 3 AtARF-AtARF, were confirmed based on bootstrap values (above 50 %) in the phylogenetic tree. It is noteworthy that Classes 1–4 included both VvARFs and AtARFs while the Class 5 had only AtARFs without VvARFs. Other plant species, such as rice, maize and tomato, also appear to lack the Class 5 ARFs found in Arabidopsis (Wang et al. 2007; Kumar et al. 2011; Liu et al. 2011). Gene structure analysis of VvARFs A comparison of the full-length cDNA sequences with the corresponding genomic DNA sequences was made to determine the numbers and positions of exons and introns of each individual VvARF genes. It was showed that the coding sequences of all the ARF genes are disrupted by introns, the number of introns varied from 2 to 15 (Fig. 4). Previous studies have revealed that the coding sequences of
Fig. 3 Phylogenetic relationship of grapevine and Arabidopsis ARF proteins. The unrooted tree was generated using MEGA4 program by neighbor-joining method. Bootstrap values from 1,000 replicates are indicated at each branch
all 25 OsARF genes are interrupted by 2–14 introns (Wang et al. 2007). The VvARF genes of Class 1 and Class 3 in the phylognetic tree had 13–15 introns. In Class 2, VvARF genes had 10–11 introns. However, the VvARF genes of Class 4 only harbored 2–3 introns. All members of the same VvARF gene class also exhibited similar gene structures of intron position and length, and the intron phases were conserved (Fig. 4). These results provide important evidence to support the reliability of the phylogenetic analysis. In other words, the positions, lengths and phase of
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Fig. 4 Exon–intron organization of grapevine ARF genes. The coding and untranslated regions are represented by grey and black boxes, respectively. Lines represent the introns. The sizes of the boxes and lines are proportionally mapped. The numbers 0, 1 and 2
represent phase 0, 1, and 2 introns, respectively. The length of the sixth intron within VvARF3 was more than 30 kb, which was cut 24 kb in this figure
Fig. 5 Conserved domains in VvARF proteins. Domains were determined through searching the VvARF protein sequences in NCBI Conserved Domain Database. B3 plant-specific B3-DNA binding
domain, Auxin_resp a conserved region of auxin responsive factor, AUX_IAA C-terminal AUX_IAA domain
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introns can represent the phylogenetic relationships of the gene family. This phenomenon has also been reported in bHLH and SBP-box gene families (Li et al. 2006; Guo et al. 2008). Interestingly, the length of the sixth intron within VvARF3 was more than 30 kb (Fig. 4), which was not observed in other AtARF and OsARF genes. Analysis of functional domains of VvARF proteins Domains of VvARF protein sequences were identified and analyzed using NCBI Conserved Domain Database (Marchler-Bauer et al. 2009). Three conserved domains (B3, AUX_RESP and AUX_IAA) were identified and their distribution in the proteins of respective classes in the phylogenetic tree has been presented in Fig. 5. Seventeen VvARF protein sequences contained the B3, AUX_RESP and AUX_IAA domains. Two protein sequences, VvARF8 and VvARF17, contained the B3 and AUX_RESP domains. Most ARF proteins contain two well-conserved domains, one of which is N-terminal B3-type DNA binding domain (DBD) and the other is C-terminal AUX_IAA domain (CTD). The DNA binding domain that includes an ARF family-specific domain (referred to as AUX_RESP domain), is essential for the interaction between ARFs and the TGTCTC AuxREs (Ulmasov et al. 1999a; Tiwari et al. 2003). In this study, the conserved N-terminal regions of VvARFs were divided into B3 and AUX_RESP domains, and the AUX_RESP domain has been used to identify VvARFs from protein database. The AUX_IAA domain, also known as domains III and IV, are similar to those found in the C-terminus of Aux/IAAs, which is a protein– protein interaction domain that allows the homo- and hetero-dimerization of ARFs and the hetero-dimerization of ARF and Aux/IAA proteins (Ulmasov et al. 1997a, b, 1999b; Kim et al. 1997; Ouellet et al. 2001). The AUX_IAA domain in some ARF proteins can also increase the in vitro binding of these ARFs to TGTCTC AuxREs by facilitating the formation of dimers that occupy the two half-sites in palindromic AuxREs (Ulmasov et al. 1999b). The middle region (MR) of ARFs, which separates DBD from CTD, is critical in determining whether an ARF protein will act as an activator or repressor (Guilfoyle et al. 1998; Ulmasov et al. 1999b, Guilfoyle and Hagen 2001; Ouellet et al. 2001; Tiwari et al. 2003). Transfection assays with carrot protoplasts indicated that Arabidopsis ARF1, 2, 3, 4 and 9, among which ARF1 contain middle region rich in proline (P), serine (S) and threonine (T) residues, are repressors and ARF5, 6, 7 and 8, which contain middle regions rich in glutamine (Q) residues, are activators (Ulmasov et al. 1999a; Tiwari et al. 2003). The detailed sequence analysis of all 19 VvARF proteins revealed that S-rich middle regions were found in VvARF1, 2, 4, 6, 7,
13, 14, 15, 16, 17 and 19, indicating that these proteins are more likely to act as repressors of transcription (Supplementary file 2 and 3). While Q-rich middle regions were found in VvARF3, 9, 10, 11 and 12, implying that these proteins are probable transcriptional activators (Supplementary file 3). The middle regions of VvARF5, 8 and 18 contain no obvious biased amino acid sequences (Supplementary file 3). All VvARF proteins with Q-rich middle regions belong to Class 3, while S-rich VvARFs are present in Classes 1 and 4. Thus, phylogenetic analysis of VvARF proteins also showed that activators and repressors were appropriately placed in separate classes. Expression analysis of VvARF genes in grapevine The expression pattern of a gene often has a correlation with its function. ESTs and cDNAs widely available in different databases have been considered as useful resources for preliminary analysis of gene expression (Adams et al. 1995). In this paper, NCBI’s Unigene Database was used to test the existence of VvARF genes and to predict their tissue-specific expression profiles (Table 2). The number of ESTs for individual VvARF genes varies from 0 to 20. There are 20 ESTs corresponding to VvARF16 in the database, while the transcriptions of VvAFR6, 10 and 12 remained unclear, as no corresponding EST could be found. This lack of detection may be due to the temporal or spatial expression patterns of these genes. In grapevine, most of the VvARF genes detected in the EST sources had higher transcript levels in the tissue of fruit compared to their expression in other tissues, suggesting that these VvARF genes may be involved in control of a series of physiological and biochemical processes including fruit growth and development. In addition, the VvARF genes were widely expressed in buds, roots, leaves, inflorescences and flowers, indicating that the VvARF family genes may perform a variety of physiological functions in grapevine. The expression profile of VvARF genes during Cabernet Sauvignon berry development was analyzed using the Affymetrix GeneChipÒ Vitis oligonucleotide microarray ver. 1.0 (Deluc et al. 2007). Grape berry development is a dynamic process that involves a complex series of physiological and biochemical changes. These changes can be divided into three major phases from small pea-size berries (21, 42 and 49 DAB known as E-L stages 31, 32 and 33 in the modified E-L system), through veraison (56 and 63 DAB known as E-L stages 34 and 35), to mature berries (84 and 112 DAB known as E-L stages 36 and 38) (Deluc et al. 2007). The microarray data showed 9 VvARF genes were represented on the GeneChip (Fig. 6). The expression of 3 genes (VvARF 8, 11 and 19) did not change significantly during fruit development. However, VvARF1, 16
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Plant Cell Rep Table 2 The tissue distribution profile of grapevine ARF genes based on the number of expressed sequence tags (ESTs) present in NCBI’s Unigene Database Gene
Bud
Root
VvARF1 VvARF2
Stem
Leaf
Inflorescence
1 1
Flower
Fruit
Seed
Cell culturea
1
5
1
1
2
VvARF3
1
2 1
9 1
7
6
4
13
1 1
1
1
2
1
4
VvARF6 VvARF7 VvARF8
0 1
1
1
2
VvARF9
1
3
2 3
1
6 5
6
5
15
VvARF10
0
VvARF11
1
1
2
2
4
13
VvARF12
0
VvARF13 VvARF14
4 1
4
VvARF15 VvARF16
4 1
6
2
20
4 5
3
VvARF17
1
3
2
4
1 2 1
4
3
VvARF18 VvARF19
Total EST number
3
VvARF4 VvARF5
Mixedb
4
2
7
3
19
8
4
5
a
Carbernet Sauvignon cell culture
b
Carbernet Sauvignon flower, leaf and root non-normalized
10
2
Log2 intensity value observed by microarray
14
12
VvARF1 VvARF3 VvARF5 VvARF8 VvARF11 VvARF15 VvARF16 VvARF18 VvARF19
10
8
6
4
2
0
31 32 33 42 49 21 small pea-size berries
34 35 63 56 veraison
36 84
38 112 mature berries
39 E-L Stage DAB
Developmental stage
Fig. 6 Expression profile of nine grapevine ARF genes in berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChipÒ Vitis oligonucleotide microarray ver. 1.0 spanning seven developmental stages from small pea-size berries (E-L stages 31, 32 and 33 or
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21, 42 and 49 DAB), through veraison (E-L stages 34 and 35 or 56 and 63 DAB), to mature berries (E-L stages 36 and 38 or 84 and 112 DAB). Error bars showed standard errors of the means of the results from three replicates
Relative expression to VvUbiquitin
Plant Cell Rep 8 7 6 5 4 3 2 1
Relative expression to VvUbiquitin
10
2
5
1 0 40
70
90
VvARF8 3 2 1 0 40
70
90
40
1.6 1.4 1.2 1.0 .8 .6 .4 .2 0.0
90
100
20
0.0
70
90
100
20 40 70 90 100 small pea veraison mature berries size berries
Days after full bloom
4.0 3.5 3.0 2.5 2.0 1.5 1.0 .5 70
40
VvARF19
100
VvARF3
20
20 2.2 2.0 1.8 1.6 1.4 1.2 1.0 .8 .6 .4 .2 0.0
4
20
0
100
1.30
Relative expression to VvUbiquitin
15
3
5
Relative expression to VvUbiquitin
20
4
20
.90
VvARF18
VvARF16
5
0
1.25 1.20 1.15 1.10 1.05 1.00 .95
25
6
VvARF1
40
70
90
100
70
90
100
VvARF11
0
20
40
1.4
VvARF5
VvARF15
1.2 1.0 .8 .6 .4 .2 0.0
20
40
70
90
100
20
40
70
90
100
small pea veraison mature berries size berries
small pea veraison mature berries size berries
Days after full bloom
Days after full bloom
Fig. 7 Relative mRNA expression levels of VvARF genes in the whole grape berries during development. Quantitative RT-PCR was performed with gene-specific primers. For each sample, transcript levels were normalized with those of VvUbiquitin, and the relative
expression levels of each gene were obtained using the DDCT method. Error bars showed standard errors of the means of the results from three replicates
and 18 were highly expressed at the three phases and the expression of VvARF3, 5 and 15 gradually decreased during fruit development indicated that these 6 genes may play more important roles in grapevine berry development. In order to validate the expression profiles of VvARF genes as revealed from microarray analysis, quantitative real-time RT-PCR analysis was performed for these VvARFs at different developmental stages. The results of qPCR showed that the expression patterns of VvARF1, 5, 15, 18 were in good agreement with the microarray data. However, the remaining 5 VvARF genes appeared to have different expression results between microarray and qPCR data which may be mainly caused by different
environmental growth conditions of the grapes. Though Cabernet Sauvignon grape berries were used in both experiments, miroarray data were obtained from berries harvested from the Shenandoah Vineyard, Plymouth, California, in 2004 and berries used in qPCR analysis were sampled from a vineyard in the western suburbs of Beijing in 2012. As shown in Fig. 7, the expression of VvARF1, 16 and 18 was significantly increased at 70 DAB (veraison) and kept on high levels until the end of berry development. VvARF8 and 19 reached the largest transcription levels at veraison and then decreased. VvARF3 and 11 exhibited a peak of expression at the end of mature stage, while VvARF5 and 15 decreased and showed the minimum at the
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grape berry development stages of veraison and mature. The high levels of VvARF1, 16 and 19 which have S-rich middle regions and act as transcriptional repressors provided evidence in support of a decrease in auxin levels as veraison approaches. However, the high levels of transcriptional activators, VvARF3 and 11, at mature berries and VvARF5 and 15 at small pea-size berries indicated these genes may be involved in berry ripening and cell division, respectively. The distinct expression profiles indicated the potential functional differences among VvARFs which should be further investigated during grape berry development.
Conclusions ARF genes have been implicated in many development processes and stress responses. It is important to understand their regulation for increasing plant growth and development. In this study, 19 AFR genes were identified in grapevine, and a comprehensive account of this gene family has been performed. The information generated from analysis of the structure of VvARF proteins indicated repressor VvARFs which contained S-rich middle regions were present in Classes 1 and 4, while activator proteins with Q-rich middle regions belonged to Class 3. Expression analysis showed that the VvARF genes may be involved in control of the development of many tissues and some VVARFs that may play important roles during grape berry development were detected. These data would be useful in selecting candidate genes related to tissue development in grapevine and will pave the way for further functional verification of these VvARF genes. Acknowledgments This work was supported by the National Natural Science Foundation of China (30871699, 30901012 and 31171942), Leading Academic Discipline Project of Shanghai Municipal Education Commission-Molecular Physiology (J50108) and Shanghai Science and Technology Committee No. 10DZ2271800. Conflict of interest of interest.
The authors declare that they have no conflict
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