Plant Mol Biol Rep (2009) 27:292–297 DOI 10.1007/s11105-008-0082-z
Identification and Expression Pattern of a Novel NAM, ATAF, and CUC-Like Gene from Citrus sinensis Osbeck Yong-Zhong Liu & M. N. R. Baig & Rui Fan & Jun-Li Ye & Yin-Chuan Cao & Xiu-Xin Deng
Published online: 15 January 2009 # Springer-Verlag 2009
Abstract A citrus NAM, ATAF, and CUC (NAC)-like gene (CitNAC) was isolated from fruit tissues of Citrus sinensis Osbeck using complementary DNA (cDNA) amplified fragment length polymorphism and rapid amplification of cDNA ends techniques. Its full length was 988 bp in which 781 bp form the open reading frame, coding for a protein of 264 amino acids. Sequence comparison revealed that CitNAC possesses the general structural features at the N terminus of the NAC domains. Phylogenetic analysis results showed that CitNAC was closely related to AtNAP and PeNAP, which are involved in plant organ senescence. Gene expression analysis showed that the messenger RNA level of CitNAC was just detected in fruit peel and pulp during fruit ripening or senescence stage. The observed expression pattern of CitNAC along with the result of phylogenetic analysis suggested that CitNAC is related to fruit development and senescence. Keywords Citrus sinensis . Fruit development . Fruit senescence . NAC domain . Rapid amplification of cDNA ends (RACE) Y.-Z. Liu (*) : M. N. R. Baig : R. Fan : J.-L. Ye : Y.-C. Cao : X.-X. Deng (*) National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China e-mail:
[email protected] e-mail:
[email protected] Y.-Z. Liu Key Laboratory of Horticultural Plant Biology (Ministry of Education), Huazhong Agricultural University, Wuhan 430070, People’s Republic of China
Abbreviations CitNAC Citrus NAC gene FJ72-1 ‘Fengjie72-1’ navel orange OR Citrus sinensis Osbeck cv.“Fengjie72-1” ORF Open reading frame RACE Rapid amplification of cDNA ends TDF Transcript-derived fragment
Introduction NAM, ATAF, and CUC (NAC) proteins, derived from proteins of Petunia NAM, Arabidopsis ATAF1, ATAF2, and CUC2 (Aida et al. 1997), are plant-specific transcriptional regulators and comprise one of the largest transcription factor families (Olsen et al. 2005). NAC genes, firstly identified by mutations (Souer et al. 1996; Aida et al. 1997), are abundant in plant genomes. There are at least 75 NAC genes predicted in full-length complementary DNA (cDNA) data sets of Oryza sativa (Kikuchi et al. 2003) and 105 in Arabidopsis thaliana genome (Riechmann et al. 2000). Up to date, many NAC genes have been identified from different plant species, such as tomato (Selth et al. 2005), wheat (Uauy et al. 2006), soybean (Meng et al. 2007), and citrus (Fan et al. 2007). So far, different members of the plant NAC family have been found to be involved in diverse processes, including maintenance of the shoot apical meristem, flower formation, responses to different abiotic/biotic stresses (Olsen et al. 2005), plant organ senescence (Guo and Gan 2006; Uauy et al. 2006), and formation of secondary walls in woody tissues (Mitsuda et al. 2007; Zhong et al. 2007).
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from the end of October to the late of December. Eight fruit samples were collected at random on 10/11, 11/03, 11/23, 12/12, and 01/09. Peel and pulp were separated and ground into fine powder in liquid nitrogen and then stored at −80°C until use. Fruit senescence may begin in the middle of ripening or after maturation. In this research, the sample of 01/09 was considered as a senescence stage of FJ72-1.
Citrus fruit is an important fruit commodity in the world (Ladaniya 2007). Knowledge of citrus NAC proteins is still rare except that a citrus NAC-like gene response to postharvest stresses was recently reported (Fan et al. 2007). Using cDNA-AFLP, we isolated some transcript-derived fragments (TDFs) of transcription factors from ripening navel orange (Citrus sinensis Osbeck) in which TDF fjfw18 (Accession no. EH117789) is related to NAC protein. Here, we succeeded in cloning a full-length citrus NAC gene based on TDF fjfw18 using rapid amplification of cDNA ends (RACE) technique, and found that it is involved in citrus fruit development and senescence.
RNA Extraction and Molecular Cloning Total RNAs of fruit peel and pulp of FJ72-1 collected at different time points were isolated according to the protocol described before (Liu et al. 2006a). RNA pool was made by equal mixture of peel and pulp RNA (50 μg) from different samples. In order to clone the full length of citrus NAC gene, 5′ RACE and 3′ RACE were performed respectively according to the RLM-RACE protocol (FirstChoice® RLMRACE, Ambion, USA). Gene-specific primers (NAC-L: GGATTCAGATTCCACCCAAC; NAC-R:GCCAGGGGT CAAACTTGTAA) were designed based on the sequence of EH117789. To confirm the assembled full-length sequence of citrus NAC gene, primer NF (CCATTATATTGAGAGCT
Materials and Methods Plant Materials Fruits of ‘Fengjie72-1’ navel orange (Citrus sinensis Osbeck cv.“Fengjie72-1”, FJ72-1) were collected from the Fengjie county, Chongqing, People’s Republic of China. FJ72-1 is the main cultivar in this region where fruit ripens Fig. 1 Isolation of the fulllength cDNA of CitNAC gene in ripening citrus fruit. a A simple amplification scheme of the full length of CitNAC. b 3′ RACE (3R), 5′ RACE (5R), and confirming PCR (cP). The product length of confirming PCR was not the same of the full cDNA length of CitNAC, but included the open reading frame of CitNAC. c Nucleotide and deduced amino acid sequence of CitNAC. The start codon ATG and the stop codon TGA are represented in frame. Gray shadow indicated the position of similar sequence with the EST of EH117789. Gene-specific primers for RACE or confirming amplification are presented in arrow (→ or ←)
a
EST- EH117789
c
1 caccttcatttctcttttctcttagcaaacaataatgacccccttcaacctcaagatttta 62
TTTTATTTTTTGTACCATTATATTGAGAGCTGATTAAGCTTAACAATGGAAGCACAAGCC NF NAC-L NAC-R NR
Gene-specific primers (NAC-L, NAC-R) designed for RACE
(1)
M E A Q A
122 AGCACCGAGCTGCCACCCGGATTCAGATTCCACCCAACTGACGAAGAACTAATTGTTCAT (6) S T E L P P G F R F H P T D E E L I V H 182 TACCTTCGTAACCAAGCCACGTCGAGACCCTGCCCCGTGTCAATAATCCCGGAAGTGGA T
5’ RACE
(26) Y L R N Q A T S R P C P V S I I P E V D
3’ RACE
242 ATTTACAAGTTTGACCCCTGGCAACTTCCTGAGAAAGCTGAATTCGGGGAGAAGGAATGG
Primer NF designed
Primer NR designed
Confirming PCR and Sequence assemble Full length of CitNAC
(46) I Y K F D P W Q L P E K A E F G E K E W 302 TACTTTTTCAGCCCACGAGACAGGAAGTATCCCAATGGGACGAGGCCTAACAGGGCAAC T (66) Y F F S P R D R K Y P N G T R P N R A T 362 GTATCGGGATATTGGAAGGCCACGGGGACTGATAAGGCAATATATGGTGGGTCTAAATA T (86) V S G Y W K A T G T D K A I Y G G S K Y 422 CTTGGTGTCAAGAAAGCTCTTGTTTTTTATAAGGGCAGGCCCCCCAAGGGGATCAAGACT (106) L G V K K A L V F Y K G R P P K G I K T
b
M
3R
5R
cP
482 GATTGGATCATGCATGAATATCGGCTAAATGACCCAACAAGACAACCCTACAAACATAAT (126) D W I M H E Y R L N D P T R Q P Y K H N 542 GGTTCCATGAAACTGGATGATTGGGTACTTTGTAGGATTTACAAAAAGAGGCAAACGGGA
1kb
(146) G S M K L D D W V L C R I Y K K R Q T G 602 TCAAGATCAGTTTTGGATGCAAAAGTCGAGGAAGATCAATCATGTGTTGATCAATTAGGC
500bp
(166) S R S V L D A K V E E D Q S C V D Q L G 662 AAAACAGGAGGCTATGTTGAGCATGCTAATGCTAGTGATGAACAGAAATTGATGGTGAAA (186) K T G G Y V E H A N A S D E Q K L M V K 722 TTTCCAAGGACATGTTCACTTGCTCATCTAGTGGAACTGGAATACTTTGCTCCGATTTCA (206) F P R T C S L A H L V E L E Y F A P I S 782 CAACTTCTCAATGACAACACTTACAATTTTAACTATGATTTCCAGAACGGCATTAACAAT (226) Q L L N D N T Y N F N Y D F Q N G I N N 842 AATGCTGCCTCCGATGACCAATTCGAAATAAACTTCAGCCAAGTGACATGCACCAAGTGA (246) N A A S D D Q F E I N F S Q V T C T K * 902 accacagtgactcgttaaaccagcagtcattgtttgtgaacccaacggtgtacgaatttc 962 agtgattttggatcttcgaaaaaaaaa
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GAT) and NR (GTACACCGTTGGGTTCACAA) were designed based on the sequences of 5′ and 3′ RACE fragment, respectively, to amplify the full-length fragment with the following PCR condition: 35 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min. The simple cloning scheme of the full length of CitNAC was shown in Fig. 1a. RACE products and subsequent full-length clones were cloned into pMD18-T vector (TaKaRa, Dalian, China) and sequenced. The sequencing of the cloned fragments was single strand with at least two replicates. Bioinformatics Analysis The open reading frame (ORF) of CitNAC was found by ORF Finder analysis (http://www.ncbi.nlm.nih.gov/gorf/ gorf.html). Similarity search was done with the BLASTX program (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The NAC domain of CitNAC was queried against InterPro (http://www.ebi.ac.uk/InterProScan/). Multiple sequence alignment of CitNAC with other NAC proteins was conducted using the CLUSTAL X (version 1.81) program. The phylogenetic trees were constructed using the MEGA software 4.0 (Tamura et al. 2007). Sequences used here for phylogenetic analysis were selected according to their reported functions. GenBank Accession nos. of such sequences are as follows: ATAF1 (NP_171677), ATAF2 (NP_680161), AtNAC2 (NP_188170), AtNAP (NP_564966), CUC1 (BAB20598), CUC2 (BAA19529), HvNAC6 (CAM57978), NAM-B1 (ABI94353), NAC1 (AAF21437), NST1 (NP_182200), NST2 (NP_191750), NST3 (NP_174554), OsNAC6 (BAA89800), PeNAM (CAA63102), PvNAP (AAK84884), SINAC1, SNAC1 (ABD52007), and TIP (AAF87300). The sequence of SINAC1 from tomato has not been registered in GenBank, but it was reported by Selth et al. (2005). RNA Gel Blot Hybridization Total RNAs were isolated from fruit peel and pulp of FJ721 at five sampling points by using Liu’s modified protocol (Liu et al. 2006a), respectively. Twenty micrograms of them was fractionated on a 1.0% (w/v) agarose gel containing formaldehyde in MOPS buffer and then transferred onto Hybond-N+ membranes (Amersham) with 20× SSC. Membranes were fixed by baking at 120°C for 30 min. A CitNAC fragment of 188 bp was amplified with specific primers (NAC-L and NAC-R) and was used as probe. The subsequent procedures including probe labeling, hybridization, washing steps, and detection were performed strictly according to the manufacturer’s instruction of DIG High Prime DNA Labeling and Detection Starter Kit I (Cat. No.1 745 832, Roche, Germany) with the hybridization temperature of 45°C.
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Results Cloning the Full-Length of CitNAC and Sequence Analysis Blastx search results of EH117789 showed that it shared relatively high identities (maximum=81%) and low e-value (minimum=1e−22) with other plant NAC genes, implying that it is a partial sequence of a citrus NAC-like gene. Primers NAC-L and NAC-R were designed based on the sequence of EH117789. Using two rounds of PCR with specific primer sets resulted in a band of 304 bp for 5′ RACE and 884 bp for 3′ RACE, respectively. The fulllength cDNA for CitNAC was obtained by aligning and assembling the 3′ and 5′ RACE products’ sequence. To confirm its authenticity, we designed primer NF at the 5′end of 5′ RACE sequence (before codon ATG) and primer NR at the 3′-end of 3′ RACE sequence (after the stop codon TGA), respectively. We succeeded in amplifying a specific band using this primer pair of NF and NR (lane cP in Fig. 1b). Sequencing results showed that the amplified fragment (cP) had similar sequence with the assembled sequence in the range between NF and NR. This confirming result showed that we succeeded in cloning a full-length cDNA of CitNAC with the length of 988 bp. Using the ORF Finder, CitNAC has a maximal ORF of 781 bp (including stop codon TGA), a 106-bp leader sequence (5′ UTR) and a 101-bp 3′ UTR (Fig. 1b). CitNAC is predicted to encode a 264 amino acid protein with an estimated molecular mass of 30.03 kDa and a calculated isoelectric point of 7.62. The GenBank Accession no. of CitNAC is EF185419. Homology search results in GenBank (NCBI) showed that the predicted amino acid sequence of CitNAC had a high identity and low e-value (the lowest is 1e−99) with other plant NAC proteins. The mean identity of the first 10 Blast Hits is 66%. NAC proteins have a remarkably conserved region at their N-terminal ends (Aida et al. 1997; Ooka et al. 2003). InterPro inquiry found that this gene did contain a InterPro NAM domain (IPR003441). The NAC domain of the predicted CitNAC was further analyzed with predicted NAC domains of some other typical NAC proteins, including Petunia NAM (X92205), Arabidopsis NAP (AJ222713), AtNAM (AF123311), ATAF1 (X74755), ATAF2 (X74756), and CUC2 (AB002560) using the CLUSTAL X (version 1.81) program. Results showed that CitNAC had high similarity with other typical NAC proteins in the N-terminal region and obviously owned five subdomains (A–E; Fig. 2) according to a previous report (Ooka et al. 2003). Phylogenetic Analysis of CitNAC Protein To predict the possible function(s) of CitNAC protein, a phylogenetic tree was constructed using the MEGA 4.0
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Fig. 2 Sequence alignment of CitNAC with other NAC domain proteins including Petunia NAM (X92205), Arabidopsis NAP (AJ222713), AtNAM (AF123311), CUC2 (AB002560), ATAF1 (X74755), and ATAF2 (X74756). The five core subgroups of NAC gene family described by Ooka et al. (2003) are presented on the upper line. The GenBank accession number for CitNAC is EF185419
software with the neighbor-joining method (Tamura et al. 2007). Results showed that most of NAC proteins with similar functions could be classified into the same subgroups on the basis of similarities in NAC domains (Fig. 3). The CitNAC protein reported here was clustered into the subgroup senescence-related II which included AtNAP and PvNAP.
was increased thereafter. The transcripts of CitNAC reached its maximum level on 12/12, followed by a moderate decrease on 01/09. Expression of CitNAC in fruit peel was different from that in fruit pulp. The hybridization signal corresponding to CitNAC messenger RNA in fruit peel was not detected at the early stages of fruit development (10/11 and 11/03) and clearly observed at fruit maturation phases, with peak on 12/12 (Fig. 4).
Expression Patterns of CitNAC Northern blot analysis was undertaken to investigate the expression pattern of CitNAC in the fruit of FJ72-1 during fruit development or senescence. In fruit pulp, the expression of CitNAC gene was negligible on 10/11 and 11/03 and Fig. 3 Phylogenetic tree of the predicted CitNAC protein and other known NAC proteins as created using the neighborjoining method in MEGA 4.0. The NAC protein sequences were used for the analysis as described in “Materials and Methods”. The numbers at the nodes indicate the bootstrap values obtained from 100 bootstrap replicates. The arrow indicates the position of CitNAC. The functional definition of subgroup depended on the reported functions of NAC proteins in the same subgroup
Discussion NAC transcription factors, which were firstly described a decade ago (Souer et al. 1996), are specific to and abundant Functional subgroup Abiotic/biotic stress related
Senescencerelated I Senescencerelated II
Secondary walls formation related Shoot apical meristem related and organ development
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Fig. 4 RNA gel blot hybridization of CitNAC in peel and pulp tissues of Fengjie 72-1 navel orange during fruit ripening and senescence
in plants. NAC proteins were characterized by a remarkably conserved region at their N-terminal ends and play important roles in diverse processes of plant development (Aida et al. 1997; Ooka et al. 2003). According to their reported functions, NAC genes are involved in at least four kinds of processes, including (1) plant developmental processes such as the maintenance of shoot apical meristem (Souer et al. 1996; Takada et al. 2001; Kim et al. 2007), cotyledon development(Aida et al. 1997; Taoka et al. 2004), lateral root development (Xie et al. 2000; Guo et al. 2005; He et al. 2005), and flower formation (Sablowski and Meyerowitz 1998); (2) responses to different abiotic/ biotic stresses (Olsen et al. 2005; Selth et al. 2005; Fan et al. 2007; Nakashima et al. 2007); (3) plant organ senescence (Guo and Gan 2006; Uauy et al. 2006); and (4) formation of secondary walls (Mitsuda et al. 2007; Zhong et al. 2007). In this study, we succeeded in isolating of a full-length cDNA of a NAC-like gene from fruit tissues of navel orange (Fig. 1). Homology search, InterPro inquiry, and multiple sequence alignment showed that the CitNAC had high similarity with other NAC proteins and presented the five typical NAC subdomains (Fig. 2), indicating that CitNAC belongs to the citrus NAC gene family. To predict the possible function(s) of CitNAC, we selected some NAC family protein sequences according to the functions described above for constructing a phylogenetic tree. Results showed that NAC proteins with similar functions could be classified into the same subgroups, though the NAC proteins involved in the regulation of senescence were divided into two subgroups (Fig. 3). AtNAC2 and NAM-B1 belonged to senescence-related group I of which AtNAC2 could be induced by salt stress, abscisic acid, 1aminocyclopropane-1-carboxylate, and naphthalene acetic acid and is involved in different bioprocesses including senescence (He et al. 2005), while NAM-B1 is involved in senescence and increasing nutrient remobilization from leaves to developing grains (Uauy et al. 2006). The citrus NAC protein reported here was clustered into the subgroup II of senescence-related proteins. This subgroup included AtNAP and PvNAP which are associated with leaf senes-
cence (Guo and Gan 2006). Therefore, we can speculate that citrus CitNAC might be associated with plant organ senescence in this species. To further identify the role of CitNAC in citrus, RNA gel blot hybridization was performed. Results indicated that the CitNAC showed different expression patterns in fruit peel and pulp (Fig. 4). However, we could not detect its expression in fully developed leaves and fully bloomed flowers (data not shown). The citrus fruit arises through the late development of the ovary. Its development can be divided into three phases: cell division, rapid cell enlargement, and fruit maturation. The cell division stage (phase I) lasts about 1 to 2 months. In the rapid growth period (phase II), the fruit experiences a huge increase in size by cell enlargement and water accumulation which lasts 4 to 6 months. Finally, in phase III, growth is mostly arrested and fruits undergo a non-climateric ripening process such as the conversion of chloroplast to chromoplast (color break), sugar accumulation, and acid decrease (Reuther et al. 1968). Based on the color break of the flavedo and sugar change in the juice sac (Liu et al. 2006b, 2007), on 10/11, the collected fruits were at the end of rapid cell enlargement phase of fruit development; while on 11/03, 11/23, and 12/12, they were at the fruit maturation phase. Also, by these same criteria, fruits collected on the last sampling date (01/09) could be considered as being at the senescence stage. Hence, the expression patterns of CitNAC showed that it is involved in at least phases II and III of citrus fruit development. Based on phylogenetic and expression analysis, the CitNAC might play a role in fruit development and senescence. Although we did not detect the CitNAC expression in mature leaves, we cannot exclude that CitNAC is involved in leaf senescence. In the few years since the discovery of the NAC transcription factor family, considerable knowledge has been gained about the physiological and molecular functions of NAC proteins. Nevertheless, this area of research in citrus fruit development and senescence is still in its infancy. The results of this research will therefore trigger the interest in how the CitNAC gene might be involved in the regulation of citrus fruit development and senescence.
Plant Mol Biol Rep (2009) 27:292–297 Acknowledgments The National Natural Science Foundation of China (No. 30700551), Ministry of Education of China (IRT0548), and University funds (No. 52204-07024) supported this work.
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