GENE-40208; No. of pages: 8; 4C: Gene xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

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Article history: Received 30 October 2014 Received in revised form 10 January 2015 Accepted 12 January 2015 Available online xxxx

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Keywords: Heat shock factor Ponkan Heat stress Fruit development

Laboratory of Fruit Quality Biology/The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Zhejiang University, Zijingang Campus, Hangzhou 310058, PR China b Quzhou Academy of Agricultural Science, Quzhou 324000, Zhejiang Province, PR China

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Heat shock transcription factors (Hsfs) play a role in plant responses to stress. Citrus is an economically important fruit whose genome has been fully sequenced. So far, no detailed characterization of the Hsf gene family is available for citrus. A genome-wide analysis was carried out in Citrus clementina to identify Hsf genes, named CcHsfs. Eighteen CcHsfs were identified and classified into three main clades (clades A, B and C) according to the structural characteristics and the phylogenetic comparison with Arabidopsis and tomato. MEME motif analysis highlighted the conserved DBD and HR-A/B domains, which were similar to Hsf protein structures in other species. Gene expression analysis in Ponkan (Citrus reticulata Blanco cv. Ponkan) fruit identified 14 Hsf genes, named CrHsf, as important candidates for a role in fruit development and ripening, and showed seven genes to be expressed in response to hot air stress. CrHsfB2a and CrHsfB5 were considered to be important regulators of citrate content and showed variation in both developmentally-related and hot air-triggered citrate degradation processes. In summary, the data obtained from this investigation provides the basis for further study to dissect Hsf function during fruit development as well as in response to heat stress and also emphasizes the potential importance of CrHsfs in regulation of citrate metabolism in citrus fruit. © 2015 Published by Elsevier B.V.

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1. Introduction

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Heat shock transcription factors (Hsfs) are important components in sensing and signaling different environmental stresses (von KoskullDoring et al., 2007). Arabidopsis, which served as the prototype for the Hsf family, has a set of 21 Hsf encoding genes with 15 members belonging to class A, five members to class B and one to class C (Nover et al., 2001; Baniwal et al., 2004). Genome wide identification of Hsf family members has been fully described in rice, tomato, apple, Chinese cabbage and other species (Nover et al., 2001; Baniwal et al., 2004; Wang et al., 2009; Giorno et al., 2012; Ma et al., 2014). Commonly, all

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Qiong Lin a,1, Qing Jiang a,1, Juanying Lin a, Dengliang Wang b, Shaojia Li a, Chunrong Liu b, Chongde Sun a,⁎, Kunsong Chen a

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Heat shock transcription factors expression during fruit development and under hot air stress in Ponkan (Citrus reticulata Blanco cv. Ponkan) fruit

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Abbreviations:Hsf,heatshock transcription factor; DBD, DNA binding domains; TF,transcription factor; DAF, days after flowering; RH, relative humidity; GC–MS, gas chromatography–mass spectrometry; qRT-PCR, quantitative real-time PCR; H2-T-H3, helix-turn-helix motif; HSE, heat stress element ⁎ Corresponding author. E-mail addresses: [email protected] (Q. Lin), [email protected] (Q. Jiang), [email protected] (J. Lin), [email protected] (D. Wang), [email protected] (S. Li), [email protected] (C. Liu), [email protected] (C. Sun), [email protected] (K. Chen). 1 These authors contributed equally to this work.

plant Hsfs have DNA binding domains (DBD) at the N-terminal and an oligomerization domain (HR-A/B) (Nover et al., 2001). Other Hsf functional modules include a nuclear localization signal (NLS) essential for nuclear import, leucine-rich export sequences important for nuclear export (NES), and a less conserved C-terminal activator domain, the so-called AHA motifs (Doring et al., 2000; Nover et al., 2001). The functions of Hsf genes have been widely studied in model plants, such as Arabidopsis and tomato. For example, HsfA1a has been known as a master regulator of heat response in tomato, since it cannot be replaced by any other Hsf proteins (Mishra et al., 2002); Arabidopsis plants overexpressing HsfA2 showed not only higher levels of thermotolerance but also increased resistance to salt or osmotic stress (Ogawa et al., 2007), oxidative stress (Zhang et al., 2009) and anoxia (Banti et al., 2010). Despite structural similarities, HsfA4 acted as a potent activator of heat shock gene expression, whereas HsfA5 was inactive and inhibited HsfA4 activity (Baniwal et al., 2007). In contrast to class A Hsfs, a considerable number of Hsfs assigned to classes B and C have no evident function as transcription activators on their own (Czarnecka-Verner et al., 2000), but a highly conserved -LFGVtetrapeptide forms the core of a repressor domain in class B Hsfs (Ikeda and Ohme-Takagi, 2009). However, under certain conditions of

http://dx.doi.org/10.1016/j.gene.2015.01.024 0378-1119/© 2015 Published by Elsevier B.V.

Please cite this article as: Lin, Q., et al., Heat shock transcription factors expression during fruit development and under hot air stress in Ponkan (Citrus reticulata Blanco cv. Ponkan) fruit, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.01.024

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Reverse primer

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CrHsfA1a CrHsfA1b CrHsfA2 CrHsfA3 CrHsfA4a CrHsfA4b CrHsfA5a CrHsfA6 CrHsfA7 CrHsfA8 CrHsfA9b CrHsfB1 CrHsfB2a CrHsfB2b CrHsfB3 CrHsfB4 CrHsfB5 CrHsfC1 Actin

GAAAGCACTATTAGCATCCCTG GGCAATCCTCTTCACGTTCC CACCTGTGGGTCTGGATTG GGGGAAACCTCACCATACCT TGTTGATGCTCGCCCTAAAT GTGGCGGTAGTTTCATTGTC ATGGAGCAGAGGCAGGAGAAT AATTAGCCCAGCAGAAGGAT CTGGTAGCGAAGTGGGTGAT CCAGCAGCAGATGTTGTCATT CAAAGCGTGCCTGAGTCTGT TTATCGGACGAGAATGCG GAACTTTCTGGAGGCCGAGT CGACGGATCGACCTTCATAG CCTAAGGGATGAGAATAAGCGT GCACCCACATTCACATTCTC AGCCAGTCGTCTCCAAGAAC TTTTGTCAGTCATCTCCGTCAC CATCCCTCAGCACCTTCC

CCATCTGGCGAAACATCC ACTTTCGGGCATTATTTCTGG ACCAGCATTTGGGTTGTCG TACCTGATCCGATGCCTACC GCTTGCACAGGAATGGTGGT AGATGCCTCTGTCCTCGTATG CCACGATTGGCTTTGATTGAT AAGTTCAGCCAGGTCACCAT AACTGATGCCGAATGCTGAT AACTTCTTCGAGCATAGTGCC AAGCCATCGTCATCCATAAGAG GACCCACAGCTTCCTTGC CTGCCATTTCCATCCAAGC TCTCACCTCTTCGGAAACAATC AGCCACTAAATCAAGCAACTCC CAGCCAGCACCACTACTCAT AACTCAGCCGGTGACCATAC CATCACTTGCCTGCTTTGC CCAACCTTAGCACTTCTCC

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2.2. Identification and classification of Hsfs in the citrus genome

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Hsf genes were isolated from the Citrus clementina genome (Wu et al., 2014) (http://www.citrusgenomedb.org) based on annotation and BLAST. Firstly, the sequences indicated as belonging to Hsfs were downloaded and assembled with the CAP3 Sequence Assembly

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Gene name

Genome number

Scafford

Start

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Size (aa)

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CcHsfA1a CcHsfA1b CcHsfA2 CcHsfA3 CcHsfA4a CcHsfA4b CcHsfA5 CcHsfA6 CcHsfA7 CcHsfA8 CcHsfA9 CcHsfB1 CcHsfB2a CcHsfB2b CcHsfB3 CcHsfB4 CcHsfB5 CcHsfC1

Ciclev10020928m Ciclev10015269m Ciclev10008617m Ciclev10011531m Ciclev10015472m Ciclev10015413m Ciclev10005059m Ciclev10006902m Ciclev10020718m Ciclev10015541m Ciclev10008116m Ciclev10026058m Ciclev10005403m Ciclev10021287m Ciclev10005649m Ciclev10001482m Ciclev10016707m Ciclev10008768m

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40240644 1187218 4143948 21074422 34516499 26097343 17075598 4098428 44141518 26890335 10527419 3182426 28757274 7047024 23409097 40075099 33354368 6358030

40244399 1192976 4146608 21078686 34518924 26099320 17077856 4100155 44144453 26894409 10529892 3186292 28759075 7049430 23411093 40076671 33355106 6359568

497 516 384 505 403 412 358 360 367 390 478 329 330 309 264 383 208 355

55.32 56.29 43.09 56.20 46.00 46.69 40.31 41.85 41.71 44.96 53.53 36.55 36.00 34.08 30.39 42.77 24.08 39.67

5.31 4.91 4.8 4.96 5.09 5.17 5.92 5.48 4.94 4.65 4.68 6.95 6.61 4.94 5.81 8.09 9.56 6.02

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Table 2 List of Hsf genes in the Citrus clementina genome.

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Ponkan fruit (C. reticulata Blanco cv. Ponkan) of uniform size were collected for the developmental series from orchards in Quzhou, Zhejiang, China. Nine fruits were selected from three different trees at each sampling point at 60 (S1), 90 (S2), 120 (S3), 150 (S4), 180 (S5) and 200 (S6) days after flowering (DAF) in 2012. S1 and S2 were characterized by extensive cell division, while S3 and S4 were characterized by cell expansion. S5 was the fruit ripening stage when fruit growth slowed down and the pulp reached its final size. S6 was the harvest stage when fruit had reached commercial maturity. For heat treatment, mature Ponkan fruits picked in 2011 were subjected to the following treatments: (1) Hot air treatment: the fruits were kept in a chamber at 40 °C, N 90% relative humidity (RH) for two days followed by storage at 10 °C, 85%–95% RH; and (2) control fruits were stored at 10 °C with 85%–95% RH. The fruits were sampled at 0, 2, 10, 20 days after storage. The initiation of the treatments is referred to as 0 day in storage. Each sample consisted of nine fruits for each treatment, separated into three replicates with three fruits in each. Fruit at each sampling point were transported to the laboratory as soon as possible, the flesh was taken and immediately frozen in liquid nitrogen and stored at −80 °C.

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appropriate promoter architecture, the heat shock-induced tomato 70 HsfB1 can act as co-activator cooperating with HsfA1a (Kotak et al., 71 2004). With the exception of tomato, the Hsf gene function has rarely 72 been reported in fruit. 73 Organic acids are also involved in many stress processes (Marschner 74 and Marschner, 2012), especially in fruit storage under heat treatment, 75 which has been widely studied in various fruits. For example, organic 76 acids content was observed to decrease in apples under heat treatments 77 (Klein and Lurie, 1990); lower organic acid content was detected in 78 peach fruit exposed to hot air (Lara et al., 2009); lower titratable acid 79 was observed in navel oranges treated with hot air (Shellie and 80 Mangan, 1998); citrate content decreased under hot air treatment in 81 citrus fruit (Chen et al., 2012; Yun et al., 2013). Also, expression of 82 some genes related to citrate metabolism has been identified in 83 response to stress. For example, increased levels of citrate synthase 84 gene expression enhanced Al tolerance (delaFuente et al., 1997; 85 Anoop et al., 2003); lack of Aco1 enzymatic activity in mitochondria 86 Q10 increased zinc tolerance in Saccharomyces cerevisiae (Guirola et al., 87 2014); the CitAco3–CitIDH2/3–CitGAD4 cascade was enhanced under 88 hot air treatment in Ponkan fruit (Chen et al., 2012). However, the 89 regulation of organic acid responses to environmental stress and the 90 role of related transcription factors have rarely been investigated and 91 are not understood.

2. Materials and methods

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Citrus, which is one of the most important horticultural crops, is characterized by citrate accumulation and degradation during different fruit developmental stages. It has been shown previously that fruit citrate content can also be influenced by heat stress (Chen et al., 2012; Yun et al., 2013). However, the possible relationship between Hsf genes, the transcription factors (TFs) most closely related to heat shock, and citrate metabolism has not been reported. In the present study, sequences of citrus Hsf genes were isolated based on genomic information, and phylogenetic and structural analysis performed. A developmental series of Ponkan (Citrus reticulata Blanco cv. Ponkan) fruit was collected for temporal expression analysis of Hsf genes, and the expression of Hsf genes in response to hot air treatment were characterized in mature Ponkan fruit. The possible role of Hsf genes in citrate metabolism is discussed.

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Table 1 Primers used to analyze gene expression patterns by quantitative real-time PCR (qRT-PCR) in the present research.

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Please cite this article as: Lin, Q., et al., Heat shock transcription factors expression during fruit development and under hot air stress in Ponkan (Citrus reticulata Blanco cv. Ponkan) fruit, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.01.024

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Fig. 1. Neighbor-joining phylogeny of Hsfs from Citrus clementina, Solanum lycopersicum and Arabidopsis thaliana. The phylogenetic tree was obtained using the ClustalX program and visualized by TreeView on the basis of amino acid sequences. The black, blue and red branches represent the divided clades A, B and C, respectively. The abbreviations of species names are as follows: SI, S. lycopersicum; AT, A. thaliana. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

250 °C and the helium carrier gas had a flow rate of 1.0 ml/min. The column temperature was held at 100 °C for 1 min, increased to 185 °C with a temperature gradient of 3 °C/min, increased to 250 °C at 15 °C/min and then held for 2 min. The significant MS operating parameters were as follows: ionization voltage was 70 eV, ion source temperature was 230 °C and the interface temperature was 280 °C.

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143 point at pH 8.2 according to the method described by Chen et al. (2012). 144 Q12 The citrate content was measured according to previous reports (Lin

Total RNA was prepared according to the method used in our previous reports (Yin et al., 2008). Contaminating genomic DNA was removed with TURBO DNA free kit (Ambion). First strand cDNA was synthesized from 1.0 μg DNA-free RNA, using iScriptTM cDNA Synthesis Kit (Bio-Rad). Three biological replicates were used for RNA extraction and subsequent cDNA synthesis.

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2.5. Oligonucleotide primers and quantitative real-time PCR (qRT-PCR)

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Oligonucleotide primers for real-time PCR analysis were designed with primer 3 (http://frodo.wi.mit.edu/primer3/). The specificity of primers was determined by melting curves and PCR products re-sequencing. The sequences of oligonucleotide primers are described in Table 1. The qRT-PCR was carried out with Ssofast EvaGreen Supermix kit (Bio-Rad) and CFX96 instrument (Bio-Rad) for gene expression studies. The PCR reaction mixture (20 μl total volume) comprised 10 μl 2 × realtime PCR mix (Bio-Rad), 1 μl of each primer (10 μM), 2 μl diluted cDNA, and 6 μl DEPC H2O. The PCR program was initiated for 30 s at 95 °C,

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Titratable acid of juice sacs was titrated with 0.1 N NaOH to the end

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Program (http://pbil.univ-lyon1.fr/cap3.php) to avoid the inclusion of redundant sequences. Non-redundant citrus Hsf sequences, tomato Hsfs and Arabidopsis Hsfs homologs were aligned using the ClustalX program (Thompson et al., 1997) and were named according to the phylogenetic tree, which was constructed with TreeView. The motif identification of citrus Hsf protein sequences was carried out using a motif based sequence analysis tool, MEME Suite version 4.8.0 (Bailey and Gribskov, 1998). The optimum width of amino acid sequence was set from 6 to 50. The maximum number of motifs was set to 30.

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et al., 2015; Osorio et al., 2012). Mixed samples of 0.1 g were ground in liquid nitrogen and extracted with 1 ml of methanol. The mixture was extracted at 70 °C for 15 min, and centrifuged at 10,000 g. Aliquots of 100 μl from the upper phase were dried in a vacuum. The residue was dissolved in 40 μl of 20 mg/ml pyridine methoxyamine hydrochloride, and incubated for 1.5 h at 37 °C. The sample was then treated with 60 μl Bis (trimethylsilyl) trifluoroacetamide (1% trimethylchlorosilane) for 30 min at 37 °C. In this analysis, 10 μl ribitol (0.2 mg/ml) was added to each sample as an internal standard. A volume of 1 μl for each sample was absorbed with a split ratio 25:1 and injected into a gas chromatography–mass spectrometry (GC–MS) fitted with a fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 μm DB-5 MS stationary phase). The injector temperature was

Please cite this article as: Lin, Q., et al., Heat shock transcription factors expression during fruit development and under hot air stress in Ponkan (Citrus reticulata Blanco cv. Ponkan) fruit, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.01.024

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Best possible match

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FIVWDPHEFARDLLPKYFKHNNFSSFVRQLNTY GFRKIDPDRWEFANECFRRGQKHLLKNIHRRKHWHSHQAQ CLHRNGPPPFLTKTYEMVDDPSTDHIISW QRIQCMEQRQQQMMSFLAKAMQNPGFLQQ HYGLWEEIERLKRDKNVLMMELVRLRQQC VNDVFWEQFLTENP KFWWNMQNMDHLTEQMGHLTSA GQIVKYQPSMNEAAKAMLHKIIKM KKRRRPI ACVEVG ATCGGGH RLFGVPIGAKRAR PGIECI HHHHQHPH QNTREYLQAME IDYFYD IFCSPC MNPYYC NNIFALMTNY ISPSNSGEEQVISCNSSP FVKHEPHEYGD CGPPVDYTRQDQYCVGVMEI VQQQNDSNKHI CDGHVLCH NGQMGY IGKRHEWGGYV QATDHQLH TVTYAAV KLFGVW SHHRPN

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Least significant difference (LSD) at the 0.05 level was calculated by 190 DPS 7.05 (Zhejiang University, Hangzhou, China). Figures were drawn 191 using Origin 8.0 (Microcal Software Inc., Northampton, MA, USA). 192 3. Results

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3.1. Identification and classification of Hsf genes in the citrus genome

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CDS sequences corresponding to putative Hsf genes from citrus (CcHsfs) were searched in the C. clementina genome database, and 29 genes encoding putative CcHsf proteins were identified. All candidate CcHsf proteins were surveyed, and incomplete sequences were removed. This resulted in the selection of 18 complete sequences. These CcHsf genes were distributed on seven of the nine putative citrus chromosomes, excluding chromosomes 4 and 8, with the largest number, comprised of five CcHsf genes, located on chromosome 2. The molecular weight of the deduced proteins varied from 24.08 to 56.29 kDa (Table 2). To investigate the evolution of CcHsfs, an unrooted phylogenetic tree was generated by using the 18 C. clementina Hsfs, 23 Solanum lycopersicum Hsfs (SIHsfs) and 21 Arabidopsis thaliana Hsfs (ATHsfs) (Fig. 1). These Hsf members were clearly grouped into three different clades corresponding to the main Hsf classes A, B and C. Within the A-type clade, nine distinct sub-clades were resolved, all of which comprised the citrus Hsf sequences. Five sub-clades were resolved within the B-type clade, each sub-clade containing at least one citrus Hsf sequence. The C-type Hsfs from the three plant species also

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followed by 44 cycles of 95 °C for 5 s, 60 °C for 5 s, and completed with a melting curve analysis program. The efficiency of amplification of different primers is rather similar and close to 100%, and was assumed

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Numbers in the first column indicate the motifs represented in Fig. 2.

2.6. Statistical analysis

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to be 100% in this study. The actin gene was included as an internal control (Pillitteri et al., 2004), and was considered to be stable in the conditions of the present study (data not shown). △ Ct was used to calculate the relative expression level of genes in Ponkan fruit.

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Table 3 Motif sequences identified by MEME tools in citrus Hsfs.

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Fig. 2. Distribution of conserved motifs in CcHsf deduced protein sequences. The motifs (1–30) identified using the MEME search tool were marked on the protein sequences. The length and order of each motif correspond to the actual length and the position in the protein sequences.

Please cite this article as: Lin, Q., et al., Heat shock transcription factors expression during fruit development and under hot air stress in Ponkan (Citrus reticulata Blanco cv. Ponkan) fruit, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.01.024

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Fig. 3. Expression analyses of CrHsfs in Ponkan fruit during development and ripening. The development stages from S1 to S6 represent 60, 90, 120, 150, 180 and 200 days after flowing. Error bars represent SEs from three biological replicates.

constituted one distinct clade which appeared more closely related to the A-type group. As expected, the citrus Hsfs were clustered and distributed in all the sub-clades, and were named accordingly (Table 2).

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3.2. Analysis of conserved domains in the citrus Hsf proteins

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The MEME motif search tool was used to identify the conserved domains in CcHsf proteins. Thirty corresponding consensus motifs were detected (Table 3). The majority of CcHsfs, contained motifs 1, 2, 3, 4 and 5, which correspond to the highly conserved regions including the DBD and HR-A/B region domains. Motif 4 was detected in all A and C-type group members, but did not appear in B-type group members. In addition, the analysis revealed that some motifs were only present in specific classes of the CcHsf family. For example, motif 7 was representative of A-type Hsf members such as CcHsfA1a, CcHsfA1b, CcHsfA4a, CcHsfA4b, CcHsfA6 and CcHsfA8, and it contained the signature domains corresponding to NES sequences. Similarly, motif 6 containing the AHA sequence was detected in the C-terminal parts of many A-type CcHsf proteins. Furthermore, 12 members, namely CcHsfA1a, CcHsfA1b, CcHsfA2, CcHsfA4a, CcHsfA4b, CcHsfA5, CcHsfA6, CcHsfA7, CcHsfB1, CcHsfB3, CcHsfB4 and CcHsfC1, were characterized by the presence of motif 9, which contained the NLS domains (Fig. 2).

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3.3. Expression patterns of Hsf genes in Ponkan fruit (CrHsfs) during fruit development and ripening

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In order to investigate the temporal expression patterns of CrHsf genes, samples in a developmental series were collected from Ponkan fruit at 60 (S1), 90 (S2), 120 (S3), 150 (S4), 180 (S5) and 200 (S6) days after flowing, respectively. Of the CrHsf genes, six members,

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including CrHsfA1a, CrHsfA1b, CrHsfA3, CrHsfA4a, CrHsfA8 and CrHsfB3, were highly expressed at S1, and then decreased continuously from S1 to S3, when citrate accumulated in the Ponkan fruit. The CrHsfB4 member was expressed in S1 and S2, but not detected from S3 to S6. During the citrate degradation stage, from S3 to S6, nine gene members increased continuously, CrHsfA1a, CrHsfA2, CrHsfA4b, CrHsfA6, CrHsfA7, CrHsfA8, CrHsfA9, CrHsfB2a and CrHsfB5 were included in this pattern. The other genes, CrHsfA5, CrHsfB1, CrHsfB2b and CrHsfC1, were constantly or irregularly expressed during Ponkan fruit development and ripening (Fig. 3).

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3.4. Effects of hot air treatment on Ponkan fruit citrate degradation and 250 CrHsf genes expression 251 To further characterize the expression of CrHsf genes in Ponkan fruit, we applied hot air treatment to mature fruits, which causes an accelerated reduction in titratable acid and citrate content (Fig. 4). Compared with the control fruits, titratable acid content was substantially lower in hot air-treated fruit (reduced by 11%–17%), and the citrate content was significantly decreased at 2 and 20 days after storage. The marked reduction in citrate content was accompanied by the expressions of seven CrHsf genes, including CrHsfA1b, CrHsfA3, CrHsfA5, CrHsfB2a, CrHsfB2b, CrHsfB3 and CrHsfB5, which were immediately induced under hot air treatment. Of these seven genes, CrHsfB3 was induced 3-fold under hot air treatment, and showed high expression continuously thereafter, while CrHsfA3 was very strongly induced, by 42-fold, but decreased to low levels later in the treatment period. In addition, CrHsfA1a, CrHsfA4a and CrHsfA7 were highly expressed at 20 days after storage of fruits treated with hot air. CrHsfB4 mRNA was not detected in the control fruit, although induced under hot air

Please cite this article as: Lin, Q., et al., Heat shock transcription factors expression during fruit development and under hot air stress in Ponkan (Citrus reticulata Blanco cv. Ponkan) fruit, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.01.024

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heat stress elements (HSEs) (Damberger et al., 1994; Schultheiss et al., 1996). The HR-A/B region, represented by motifs 4 and 5, is connected to the C-terminus of the DBD by a flexible linker, which is of different length in different clades. The HR-A/B region of class B Hsfs is similar to all non-plant Hsfs, whereas all class A and class C Hsfs have an extended HR-A/B region, which was represented by motif 4. These results were consistent with previous Hsf structure analysis in Arabidopsis (Nover et al., 2001) and apple (Giorno et al., 2012). In addition, all class B Hsfs, except HsfB5, are characterized by the tetrapeptide -LFGV- in the C-terminal domain, which is assumed to function as repressor motif in the transcription machinery (Czarnecka-Verner et al., 2004; Ikeda and Ohme-Takagi, 2009). Temporal expression analysis indicated that most CrHsf genes were changing at the mRNA level during Ponkan fruit development, similar to the findings from apple (Giorno et al., 2012). The expression patterns of 15 CrHsf genes were different from each other with mRNA for five genes decreasing and six genes increasing continuously during fruit development, indicating these genes were important regulators during fruit development. Those genes strongly expressed at early stages may be involved in early citrus fruit development, while the genes with increased expression during fruit development until commercially maturity could be selected for further investigation of their putative roles in fruit ripening. The duplicated genes, such as CrHsfA1a/b, CrHsfA4a/b and CrHsfB2a/b, were differentially expressed. A similar situation was found in other plants, such as Arabidopsis (von Koskull-Doring et al., 2007), which suggested that the duplicated pairs may respond to different conditions. During citrus fruit development, one of the most important changes is citrate accumulation and degradation. Considering the citrate content variation, the five genes with decreased expression pattern may be related to citrate accumulation, while the six continuously increased genes may be related to citrate degradation. In the present investigation, the expression of seven Hsf genes, including three A-type and four B-type genes was immediately induced under hot air treatment and accompanied by a marked reduction in citrate content. Effects of heat stress on Hsf genes have been examined in several plant species, but no data is available for citrus fruit. It was shown that AtHsfA1a and AtHsfA1b regulate the early response to heat shock in Arabidopsis (Lohmann et al., 2004; Busch et al., 2005). MdHsfA2a/b and MdHsfA3b/c were strongly induced in apple leaf tissue exposed to naturally increased temperature (Giorno et al., 2012). Members of the B class were shown to act mainly as repressors of the expression of heat shock inducible genes (Czarnecka-Verner et al., 2000; Ikeda et al., 2011). Some of them form a complex with Hsf A-types to maintain housekeeping gene expression during heat shock regimes (Bharti et al., 2004). Therefore, the strong transcription activation in citrus fruit may indicate that some of them are up-regulated in response to high temperature and may be involved in hot air-triggered citrate degradation in citrus fruit. CrHsfB2a and CrHsfB5 were induced during both the developmentally-related and hot air-triggered citrate degradation in Ponkan fruit. According to sequence searches, CrsfB2a contained the previously mentioned repressor domain -LFGV- in the C-terminal region. Similar -LFGV- motifs were found also in other plant TFs known to have repressor functions, e.g. ABI3/VP1, AP2/ERF, MYB and GRAS (Ikeda and Ohme-Takagi, 2009), which indicated the potential repressor activity of CrHsfB2a. However, it was reported that HsfB1 acted as co-activator cooperating with HsfA1a under certain conditions of promoter architecture in tomato (Czarnecka-Verner et al., 2000; Bharti et al., 2004; Kotak et al., 2004). The role of HsfB1 as co-activator has been observed with a given set of constitutively active promoters, providing the basis for the maintenance or even enhancement of transcription of certain housekeeping or viral genes during heat shock (Bharti et al., 2004). There is a possibility that other CrHsf genes also participated in the process of regulation of citrate metabolism, and the citrate accumulation level was a consequence of the regulation balance among several genes. Thus, it is emphasized that the CrHsfs plays a role

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Fig. 4. Changes in titratable acid and citric acid content of Ponkan fruit in response to hot air treatment. Mature fruits were treated with 40 °C hot air for 2 days, and then transferred to 10 °C for storage. The control fruits were kept at 10 °C throughout. Error bars represent SEs from three biological replicates.

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Recently, as a result of innovation in sequencing technology, many fruits, including grape (Jaillon et al., 2007), apple (Velasco et al., 2010), strawberry (Shulaev et al., 2011), tomato (Sato et al., 2012), banana (D'Hont et al., 2012), and citrus (Xu et al., 2013), have had their entire genomes sequenced, which has enable more in-depth evaluation of gene function and networking in relation to fruit development and ripening. The present study investigated the Hsf gene family in the C. clementina genome and showed that it contains 18 full length Hsf genes. This number is the same as previously profiled for the Citrus sinensis Hsf gene (CsHsf) family, but is smaller than well characterized species, such as tomato, Arabidopsis, rice, and apple, and similar to the Hsf members in grape, carpa, etc. (Giorno et al., 2012; Scharf et al., 2012). Unlike the other genes, which contained more than two members in most sub-clades, there was only one member in all of the sub-clades in citrus, except for sub-clades A1, A4 and B2, as in carpa, prupe and grape (Scharf et al., 2012). Despite considerable variability in size and sequence, the basic structure of the Hsf is conserved among eukaryotes. Close to the N-terminus, the highly structured DBD is the most conserved region of Hsfs, which contained motifs 1, 2 and 3 in citrus Hsfs. The hydrophobic core of this domain ensures the precise positioning of the central helix-turn-helix motif (H2-T-H3) required for specific recognition of the palindromic

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treatment, was only expressed at a very low level. The other CrHsf members, including CrHsfA2, CrHsfA4b, CrHsfA6, CrHsfA7, CrHsfA8, CrHsfA9, CrHsfB1 and CrHsfC1, were constantly or irregularly expressed under hot air treatment of Ponkan fruit (Fig. 5). These results showed that, CrHsfB2a and CrHsfB5 were induced during both developmentallyrelated and hot air-triggered citrate degradation in Ponkan fruit.

Please cite this article as: Lin, Q., et al., Heat shock transcription factors expression during fruit development and under hot air stress in Ponkan (Citrus reticulata Blanco cv. Ponkan) fruit, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.01.024

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Fig. 5. Expression of CrHsf genes in response to hot air treatment in flesh of Ponkan fruit during storage. Mature fruits were treated with 40 °C hot air for 2 days, and then transferred to 10 °C for storage. The control fruits were kept at 10 °C throughout. Error bars represent SEs from three biological replicates.

in citrate metabolism, but the mechanism of regulation still needs further research.

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In the present research, 18 CcHsf genes were identified from the genome database of C. clementina. Based on structural characteristics of the proteins and the comparison with homologs from other species, the CcHsfs were grouped into three different clades. MEME motif analysis highlighted the conserved domains similar to Hsf protein structures in other species. Temporal expression studies identified 14 CrHsf genes as potential candidates for a role in fruit development and ripening, while seven CrHsf genes were expressed in response to hot air stress. CrHsfB2a and CrHsfB5 play roles in both developmentally-related and hot air-triggered citrate degradation processes, but the mechanism of regulation needs further research.

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Declaration of conflicting interests

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The authors declare no conflict of interest.

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Acknowledgments

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We thank Prof. Donald Grierson from the University of Nottingham (UK) for his kind suggestions and efforts in language editing. This research was supported by the National Basic Research Program of China (973 program) (2011CB100602), Program of International Science and Technology Cooperation (2011DFB31580) and the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2012BAD38B03).

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Please cite this article as: Lin, Q., et al., Heat shock transcription factors expression during fruit development and under hot air stress in Ponkan (Citrus reticulata Blanco cv. Ponkan) fruit, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.01.024

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