Mol Biol Rep DOI 10.1007/s11033-014-3306-3

Cloning and molecular characterization of phospholipase D (PLD) delta gene from longan (Dimocarpus longan Lour.) Xiangrong You • Yayuan Zhang • Li Li • Zhichun Li • Mingjuan Li • Changbao Li • Jianhua Zhu • Hongxiang Peng • Jian Sun

Received: 29 August 2013 / Accepted: 14 February 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Longan (Dimocarpus longan Lour.) is a nonclimacteric fruit with a short postharvest life. The regulation of phospholipase D (PLD) activity closely relates to postharvest browning and senescence of longan fruit. In this study, a novel cDNA clone of longan PLDd (LgPLDd) was obtained and registered in GenBank (accession No. JF791814). The deduced amino acid sequence possessed all of the three typical domains of plant PLDs, a C2 domain and two catalytic HxKxxxxD motifs. The tertiary structure of LgPLDd was further predicted. The western blot result showed that the LgPLDd protein was specifically recognized by PLDd antibody. The Q-RT-PCR (real-time quantitative PCR) result showed that the level of LgPLDd mRNA expression was higher in senescent tissues than in developing tissues, which was also high in postharvest fruit. The western-blotting result further certified the different expression of LgPLDd. These results provided a scientific basis for further investigating the mechanism of postharvest longan fruit adapting to environmental stress. Keywords Longan  Postharvest  PLDd gene  Clone  Molecular characterization X. You  Y. Zhang  L. Li  Z. Li  M. Li  C. Li  J. Sun (&) Institute of Agro-food Science & Technology, Guangxi Academy of Agricultural Sciences, Nanning 530007, People’s Republic of China e-mail: [email protected] X. You  Y. Zhang  L. Li  Z. Li  M. Li  C. Li  J. Zhu  H. Peng  J. Sun Guangxi Crop Genetic Improvement Laboratory, Nanning 530007, People’s Republic of China J. Zhu  H. Peng Institute of Horticulture, Guangxi Academy of Agricultural Sciences, Nanning 530007, People’s Republic of China

Introduction Phospholipase D (PLD, EC 3.1.4.4) has been proposed to play a pivotal role in many cellular processes, such as signal transduction, membrane trafficking, cytoskeleton rearrangements and membrane degradation [1, 2]. PLD hydrolyzes phospholipids at the terminal phosphodiester bond, producing phosphatidic acid and free head group, such as choline. PLDs form the major family of phospholipases in plants [1]. In Arabidopsis thaliana, 12 PLD genes have been identified and grouped tentatively into five classes, i.e. PLDa (1, 2, 3, 4), PLDb (1, 2), PLDc (1, 2, 3), PLDd and PLDf (1, 2) [3, 4]. This classification was based on gene architecture, sequence similarity, domain structure, biochemical properties and cDNA cloning order [5]. The biochemical properties for PLDa1, b1, c1, d, and f1 have been characterized, and they display different requirements for Ca2?, phosphatidylinositol 4,5-bisphosphate (PIP2) and free fatty acids [4–9]. These distinguishable properties have provided insights into the unique functions for some of PLDs in plant [10–12]. Most of the PLDs cloned from other plant species belong to PLDa group, and some belong to PLDb and PLDc groups. PLDd group has much higher expression level than PLDb and PLDc groups in plant [13], but the protein structure and the catalysis of PLDd group still remain scanty. The previous research has shown that PLDd is associated with the plasma membrane [14] and is suggested to be the microtubule-binding PLD in Arabidopsis [15–17]. PLDd is activated in response to oleate [14], H2O2 [18], dehydration [19], cold [12], salt stress [20] and postharvest oxidative stress [21]. The mRNA level of PLDd in old leaves, stems, flowers and roots is higher than that in young leaves [14]. Longan is a non-climacteric fruit with a very short postharvest life of 3–4 days at ambient temperature [22,

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23]. This short postharvest life limits longan fruit consumption [22, 24]. It has been suggested that the regulation of PLD activity has an important impact on postharvest browning and senescence of some fruits in Sapindaceae family [21]. The present study is to presume the role of PLDd in response to oxidative stress and other senescence related signaling in postharvest longan fruit. The cDNA of PLDd gene in longan fruit was amplified using the reverse transcription-polymerase chain reaction (RT-PCR) method, and the analyses on structure and sequence of this gene was further conducted. The research results will provide a reference for innovation ways in maintaining postharvest quality and extending storage life of longan fruit through regulating specific expression of PLDd.

Materials and methods RNA extraction Total RNA was prepared using the SDS-phenol extraction method. One gram of longan tissues (floral buds, flowers, green and senescent leaves, developing and mature fruits, pericarp and pulp at different postharvest stages) was ground in a mortar with a pestle in the presence of liquid nitrogen. Ten milliliters of 70 % acetone (chilled at -20 °C) were added in the powder, mixed vigorously, and centrifuged for 10 min at 8,0009g at 4 °C. The supernatant was discarded. Then, 5.5 mL of extraction buffer [150 mM Tris Base, 575 mM boric acid, 50 mM EDTA (pH 8.0), 0.5 mM NaCl and 4 % (w/v) SDS] containing 0.2 mL of bME was added at 60 °C. Ethanol (1.375 mL) and potassium acetate (0.605 mL, 5 M, pH 4.8) were further added softly and mixed together. The equal volumes of CIA (chloroform:isoamyl alcohol, 24:1, v/v) was added, shaked vigorously, and centrifuged for 30 min at 11,0009g at 4 °C. The upper aqueous phase was transferred to 1.5-mL centrifuge tubes (1 mL in each tube). An aliquot (0.4 mL) of 9 M lithium chloride was added and incubated for 3 h at -20 °C. RNA pellet was precipitated by centrifuging for 30 min at 14,0009g at 4 °C. Lithium chloride (0.5 mL, 3 M) was added in the precipitate to wash the RNA pellet, and then centrifuged for 15 min at 14,0009g at 4 °C. The pellet was dried at room temperature and dissolved in 0.5 mL of RNase-free water. After the equal volume of CIA (24:1, v/v) was added, the mixture was centrifuged for 15 min at 11,0009g at 4 °C. The upper aqueous phase (0.4 mL) was transferred into another tube. One milliliter of ethanol and 30 lL of 5 M potassium acetate (pH 4.8) were added and incubated for 1 h at -20 °C. RNA was precipitated by centrifuging for 30 min at 14,0009g at 4 °C. The pellet was washed twice with 70 % ethanol by centrifuging for 10 min at 14,0009g at 4 °C. RNA

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precipitate dissolved in RNase-free water was used in RTPCR. Cloning of the full-length cDNA of PLDd A full-length cDNA encoding PLDd was obtained by a combination of RT-PCR and RACE cloning. Total RNA (5 lg) was used for the first-strand cDNA synthesis, using primers and reagents from the PowerscriptTM MMLV reverse transcriptase (Clontech, CA, USA). The specific primers of PLDd conservative region PD-cons1 (50 GTGAGAGTCTTGTTGTTGGTTT-30 ) and PD-cons1 (50 TTGAGGTAGTCTTGAGGATGTG-30 ) were designed on the basis of the homologue sequence of other plants with close relationship. The fragment of PLDd cDNA was cloned by RT-PCR. The PCR condition was as follows: 94 °C, 4 min; 94 °C, 30 s, 53 °C, 30 s, 72 °C, 90 s, 35 cycles; 72 °C, 6 min. The resulting PCR product was isolated, cloned and sequenced (Invitrogen, Shanghai, China). The 50 and 30 -RACE fragments were cloned according to the protocol of SMARTTM RACE cDNA Amplification Kit (Clontech, CA, USA). The cDNA generated by this Kit was directly used in 50 and 30 -RACE PCR, without second strand synthesis and adaptor ligation. The 50 and 30 -RACE PCR specific primer (GSP1: 50 -GCTGTTATCTTTCGGTTATTACCAAACGCTTG-30 , GSP2: 50 -GGCACGATGATT CTCTGATTAAAATAGAGCGCA-30 ) was designed based on the sequence of conservative region of PLDd as the forward primer. The temperature program of PCR amplification was as follows: 94 °C, 3 min; 94 °C, 30 s, 72 °C, 3 min, 5 cycles; 94 °C, 30 s, 70 °C, 3 min, 5 cycles; 94 °C, 30 s, 68 °C, 3 min, 25 cycles; 68 °C, 3 min. The 50 and 30 -RACE amplified PCR products were purified and cloned into pMD18-T vector (TaKaRa, Otsu, Shiga, Japan) for sequencing. By comparing and aligning the sequences of the conservative region and the 50 and 30 -RACE products, the full-length cDNA sequence of PLDd gene were obtained. Bioinformatic and cladistic analyses Sequence alignments, open reading frame (ORF) translation and molecular mass calculation of predicted protein were carried out with DNAMAN. full. version. v5.2.2 (http://www.lynnon.com/) and performed at NCBI server (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The secondary structural analysis of predicted PLDd was carried out on the website (http://www.expasy.org). The putative domains of LgPLDd were predicted by SWISS-MODEL (http://swissmodel.expasy.org/). The tertiary structure of LgPLDd was predicted by 3D-JIGSAW (http://bmm.can cerresearchuk.org/). SWISS-MODEL was used in first approach and project (optimize) modes with the default

Mol Biol Rep

parameters. Structures were visualized through SwissPDBViewer [25]. PLD gene family related to amino acid sequences were aligned using the program ClustalX v.2 (http://www.clus tal.org/) under the default settings and further refined by visual inspection. The alignment output was used to generate a phylogenetic tree based on the minimum evolution method [26], as implemented in the MEGA package v4.0.2 [27]. The Poisson correction metric was used together with the pairwise deletion option. The confidence of the tree branches was checked by bootstrap generated from 1,000 replicates. For sequences selected in Fig. 1, the alignment was visualized using the Boxshade v. 3.21 software (http:// www.ch.embnet.org/software/BOXform.html). Determination of mRNA levels by quantitative realtime PCR The cDNAs from floral buds, flower, green leaves, senescent leaves, developing fruits, maturing fruits, and postharvest fruits on different storage stage were obtained as described above. These cDNAs were subjected to Q-RT-PCR analysis. A sequence was designed for the LgPLDd gene-specific primers as follows: cLgPLDdF (50 -CAAGTCCGTGAAAG GGCAGGTC-30 ) and cLgPLDdR (50 -CGTCGCAGGCGG TAAAGCAG-30 ). Longan actin gene was used as an endogenous control with primers as cLgactF (50 -GTGG TGGTTCAACTATGTTCAATGGC-30 ) and cLgactR (50 TGGAAGGTGCTGAGGGATGCTAA-30 ). After RNA digestion with RQ1 RNase-Free DNase (Promega, Wisconsin, USA) to eliminate contaminating genomic DNA, the amplification was carried out on cDNA synthesized as indicated above. UltraSYBR Mixture (CWbio. Co. Ltd, Bejing, China) was used for Q-RT-PCR, using a SmartCycler system (Cepheid, Sunnyvale, CA, USA). Relative gene expression was determined using the comparative Ct method (DDCt) [28]. A validation of the quantization method was performed in a seriated template dilution experiment, and a calibration curve was generated. PCR products were analyzed by agarose gel electrophoresis. The expected size (ca. 130 bp) was shown for the amplicon. The mean value of five replicas was calculated besides the experimental error: 2-(DDCt ± SDt), where the total standard deviation (SDt) was calculated from the SD of each gene.

BamHI and XhoI and cloned in frame with the N-terminal (6xHis) tag of pET-30a (Qiagen, Hilden, Germany) using the corresponding sites in the pET-30a vector. The obtained correct construction was determined by sequencing. The newly constructed vector pETaPLDd was used to transform competent E. coli BL21. The resulting colonies were screened by kanamycin resistance. The predicted molecular mass of the recombinant protein was 99,040 Da. Escherichia coli cells harbouring the recombinant plasmid pETaPLDd were grown by continuous shaking at 37 °C in LB broth containing kanamycin. The production of the recombinant protein was induced with 0.2 mM isopropylb-D-thiogalactoside (IPTG) when the culture reached an OD600 value of 0.5. After 2 h of incubation with IPTG at 37 °C, cells were harvested by centrifugation for 10 min at 10,0009g. Bacterial pellets were analyzed by SDS-PAGE followed by Coomassie blue staining. Western blot analysis Total protein of competent E. coli BL21 with expressing PLDd recombinant protein were prepared and suspended in 1 mL of protein sample mixture [62.5 mL of Tris–HCl (pH 6.8), 2 % (w/v) SDS, 10 % (w/v) glycerol and 0.001 % (w/ v) bromphenol blue]. The mixture was centrifuged for 5 min at 13,4009g. The proteins (50 lg per lane) were separated with 12.5 % SDS-PAGE and subsequently electroblotted onto a nitrocellulose membrane using a blottransfer buffer [50 mM Tris-base, 40 mM Gly, 20 % (v/v) methanol and 1 % (w/v) SDS]. Duplicate blots were blocked for 2 h in TTBS [50 mM Tris–HCl (pH 8.2), 150 mM NaCl, and 0.1 % (v/v) Tween 20] containing 5 % (w/v) nonfat dry milk. The membrane was then incubated for 2 h with rabbit polyclonal antibody raised against PLDd and peroxiredoxin (Boster, Wuhan, China) at 1:5,000 dilution for 12 h at 4 °C. After washing thrice with TTBS buffer [10 mM TBS, 0.1 % (v/v) Tween-20, pH 7.6], the membranes were stained with horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit IgG at 1:5,000 dilution for 2 h at 25 °C. Positive signals were visualized with 3,30 -diaminobenzidine (DAB) (Boster, Wuhan, China). The immunodetection image was photographed using a Canon camera.

Expression of PLDd recombinant protein

Statistical analysis

PLDd cDNA was amplified with the following synthetic oligonucleotide primers: PLDdSrp (50 -CGCGGATCC ATGGCTGCCGCCGGCGAAGACAAGTC-30 ) and PLDdArp (50 -CCGCTCGAGGCCATATAGCCAATACCCTC TTATCGT-30 ). The PCR product was digested with

All experiments were performed in triplicate (n = 3). The results represented mean ± standard deviation (SD) of three biological replicated determinations. The comparison of differences among groups was carried out by using the SPSS 13.0 statistical software (SPSS Inc., Chicago, USA).

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C2 Domain LgPLDd VvPLDd AtPLDd GhPLDd RcPLDd

MAAAGEDKSVKGQVIYLHGDLDLKIIGARRLPNMDVVANSLRRCFTACDACTPPQPSSRSPSIDGDGYDKKS........ IG V L ...........MADDSETIVYLHGDLDLNIIEARYLPNMDLMSERIRRCFTAFDSCRAPFSGGRKKGRHH.......... S LHGDLDL I EAR LPNMDL S R CFTA ..........MAEKVSEDVMLLHGDLDLKIVKARRLPNMDMFSEHLRRLFTACNACARPTDTDDVDPRDKGEFGDKNIRS S LHGDLDL I AR LPNMDM S R FTA ...........MEEASKQQIYLHGDLDLTIVEARRLPNMDFMVNHLRSCLTCEPCKSPAQTAAKEGDSKIRG........ S LHGDLDL I EAR LPNMDF R CLT .......MAEISGSNDQQPIILHGDLDLYIIEARSLPNMDLVSTRIRGCFSACNCTKKSTSAASGGASTDEENEDQKLHH LHGDLDL I EAR LPNMDL S R CF A

72 59 70 61 73

LgPLDd VvPLDd AtPLDd GhPLDd RcPLDd

HRKIIT DPYVTVVVPQATVARTRVLKN Q P W EHF IPLAHPV VEFQVKDDDVFGA IG AKIPAS IA GE I HRKIITTDPYVTVVVPQATVARTRVLKNTQSPHWDEHFVIPLAHPVVDVEFQVKDDDVFGAELIGMAKIPASKIAAGEHI ..KIITSDPYVTVCLAGATVARTRVISNSQHPVWNEHLKIPLAHPVSCVEFQVKDNDVFGADMIGTATVSAERIRTGDSI KIITSDPYVTV LA ATVARTRVI NSQ P W EHL IPLAHPV VEFQVKDNDVFGA IGTA V A I GD I HRKVITSDPYVTVVVPQATLARTRVLKNSQEPLWDEKFNISIAHPFAYLEFQVKDDDVFGAQIIGTAKIPVRDIASGERI H RKVITSDPYVTVVVPQATLARTRVLKNSQ P W E F I IAHP LEFQVKDDDVFGA IGTAKIP IA GE I HRKIITSDPYVTVCLPQATVARTRVLKNSQNPKWNEHFIIPLAHPVTELDINVKDNDLFGADAIGTAKIPASRIATGEHI H RKIITSDPYVTV LPQATVARTRVLKNSQ P W EHF IPLAHPV LDI VKDNDLFGA IGTAKIPAS IA GE I HRNIITSDPYVTVVVPQATLARTRVVKNAKNPKWKQRFFIPLAHPVTNLEFHVKDNDLFGAEVMGIVKFPASKIASGESI H R IITSDPYVTVVVPQATLARTRVVKN P W QRF IPLAHPV LEFHVKDNDLFGA MG KFPAS IA GE I

152 137 150 141 153

LgPLDd VvPLDd AtPLDd GhPLDd RcPLDd

SGWFPII SGWFPIINAKGQPPKLDSAIRLEMKFTPCEENPLYRHGVAGDPEQSGVRRTYFPLRKGCQLKLYQDAHVKPGQLPEVKLN G PPK DSAI L MKFTPCE NPLYR GVAGDPE GVR TYFPLRKG QL LYQDAHV G LP V LN SDWFPILGFNGKPPKPDSAIYLKMRFISSEINPLYTRGITDPDHFGVKQS.YFPVRLGGSVTLYQDAHVPNGMLPELELD S DWFPILG GKPPKPDSAI L MRF E NPLY GI D YFPVR GG V LYQDAHV NG LP LELD SGWFPVLGASGKPPKAETAIFIDMKFTPFDQIHSYRCGIAGDPERRGVRRTYFPVRKGSQVRLYQDAHVMDGTLPAIGLD S GWFPVLG SGKPPKAE AI I MKFTP D YR GIAGDPE GVR TYFPVRKGSQV LYQDAHV DG LP IGLD TGWFPLIGPSGKPPKPDSAIYLDMKFTPCENNPLYKQGVASDPEQAGVRHTYFPLRKGSQVTLYQDAHVTDDLLPKIELD GWFPLIG SGKPPKPDSAI L MKFTPCE NPLYK GVASDPE GVR TYFPLRKGSQV LYQDAHV DD LP IELD AGWFSIIGSSGKPPKPDTALHLEMKFTPCEKNVLYRHGIAGDPEHKGVRNTYFPLRRGSRVKTYQDAHVTDGMLPNIELD GWF IIG SGKPPKPD AL L MKFTPCE N LYR GIAGDPE GVR TYFPLRRGS V YQDAHV DG LP IELD

232 216 230 221 233

LgPLDd VvPLDd AtPLDd GhPLDd RcPLDd

GHVDYTAGTCWEDICYAISEAHHLVYIVGWSVFYKIKLIREPTRELPRGGDLTLGELLKYKSEEGVRILLLVWDDKTSHD Y G CWEDICYAISEAHHLVYIVGWSVF KIKLIREPTR LPRGGDLTLGELLKYKSEEGVRILLLVWDDKTSHD DGVVYQHGKCWEDICHSILEAHHLVYIVGWSVYHKVKLVREPTRPLPSGGNLNLGELLKYKSQEGVRVLLLVWDDKTSHS G VY GKCWEDIC I EAHHLVYIVGWSVYHKVKLVREPTRPLP GGNL LGELLKYKSQEGVRVLLLVWDDKTSH NGKVYEHGKCWEDICYAISEAHHMIYIVGWSIFHKIKLVRETKVPRDKDM..TLGELLKYKSQEGVRVLLLVWDDKTSHD G VY GKCWEDICYAISEAHHMIYIVGWSIFHKIKLVRE P KD TLGELLKYKSQEGVRVLLLVWDDKTSHD DGKVYSPAKCWEDICYAISEAHHLVYIVGWSVFHKVKLVREPTRPFPRGGDLTLGELLKYKSEEGVRVLLLVWDDKTSHD G VY AKCWEDICYAISEAHHLVYIVGWSVFHKVKLVREPTRPFPRGGDLTLGELLKYKSEEGVRVLLLVWDDKTSHD NGMVYKQEKCWEDICYAISEAHHMIYIVGWSVFYKIKLIREPTKPLPRGGDLTLGELLKYKSEEGVRVLLLIWDDKTSRD G VY EKCWEDICYAISEAHHMIYIVGWSVF KIKLIREPTKPLPRGGDLTLGELLKYKSEEGVRVLLLIWDDKTSRD

312 296 308 301 313

LgPLDd VvPLDd AtPLDd GhPLDd RcPLDd

KFGI T GVM THDEET KYFKHSSV CVLAPRYASSKLG FKQQVVGT FTHHQKCVLVDTQA GNNRKITAF GGIDL KFGIKTGGVMGTHDEETLKYFKHSSVNCVLAPRYASSKLGIFKQQVVGTMFTHHQKCVLVDTQAFGNNRKITAFIGGIDL RFLVNTVGVMQTHDEETRKFFKHSSVLCVLSPRYASSKLSIFKQQVVGTLFTHHQKCVIVDTQASGNNRKITAFLGGLDL R F V T GVM THDEETRKFFKHSSV CVL PRYASSKLS FKQQVVGT FTHHQKCVIVDTQA GNNRKITAF GGLDL KFGIKTPGVMGTHDEETRKFFKHSSVICVLSPRYASSKLGLFKQQVVGTLFTHHQKCVLVDTQAVGNNRKVTAFIGGLDL K FGI T GVM THDEETRKFFKHSSV CVL PRYASSKLG FKQQVVGT FTHHQKCVLVDTQA GNNRKVTAF GGLDL KFGIRTAGVMQTHDEETLKFFKHSSVTCVLAPRYASSKLGYFKQQVVGTMFTHHQKCVLVDTQAAGNNRKITAFVGGIDL K FGI T GVM THDEET KFFKHSSV CVLAPRYASSKLG FKQQVVGT FTHHQKCVLVDTQA GNNRKITAF GGIDL IFGYQTVGLMDTHDEETRKFFKHSSVTCVLAPRYASSKTGLLKQKVVGTAFTHHQKFVLVDTQASGNNRKVTAFLGGIDL FG T GLM THDEETRKFFKHSSV CVLAPRYASSK G LKQ VVGT FTHHQK VLVDTQA GNNRKVTAF GGIDL

392 376 388 381 393

LgPLDd VvPLDd AtPLDd GhPLDd RcPLDd

CDGRYDTPEHRL RDLNTIF DFHNPTYP CDGRYDTPEHRLFRDLNTIFEGDFHNPTYPSTVKAPRQPWRDLHCRIDGPAAYDVLINFEQRWRKSTKWKEFSLKFKKVS KAPRQPWRDLHCRIDGPAAYDVLINFEQRWRKSTKWKEFSL FK CDGRYDTPEHRLCHDLDTVFQNDYHNPTFSAVSKGPRQPWHDLHCKIEGPAAYDVLTNFEQRWRKATKWSEFGRRFKRIT C DGRYDTPEHRL HDLDTVF DYHNPTF A KGPRQPWHDLHCKIEGPAAYDVL NFEQRWRK TKW EFG FK T CDGRYDTPEHRILHDLDTVFKDDFHNPTFPAGTKAPRQPWHDLHCRIDGPAAYDVLINFEQRWRKATRWKEFSLRLKGKT C DGRYDTPEHRI HDLDTVFK DFHNPTFPA KAPRQPWHDLHCRIDGPAAYDVLINFEQRWRK TRWKEFSL LK T CDGRYDTPEHRILRDLDTVFKDDFHNPTFPVGTMAPRQPWHDLHSKIEGPAAYDVLINFEQRWRKSTKWKEFSLLFKGKS C DGRYDTPEHRI RDLDTVFK DFHNPTFP APRQPWHDLH KIEGPAAYDVLINFEQRWRKSTKWKEFSL FK CDGRYDTPEHRLFHDLDTVFKGDFHNPTFSATLKVPRQPWHDLHCRIDGPAVYDVLINFEQRWRKSTRWSEFGLSFKRVT C DGRYDTPEHRL HDLDTVFK DFHNPTF A K PRQPWHDLHCRIDGPA YDVLINFEQRWRKSTRW EFGL FK T

472 456 468 461 473

LgPLDd VvPLDd AtPLDd GhPLDd RcPLDd

HW DD LIKIERISWILSP L HWHDDSLIKIERISWILSPELSSTREGTTIVPTDDRIVRVSDEKNPENWHVQVFRSIDSGSVKGFPKSINIKQIGEQNLL GT I P DD V VS E NPENWHVQVFRSIDSGSVKGFPK Q E HWHEDALIKLERISWILSPSPSVPY........DDPSLWVSEENDPENWHVQVFRSIDSGSLRGFPKDVPSAEAQNLV.. H W EDALIKLERISWILSP V DDP L VS ENDPENWHVQVFRSIDSGSLRGFPK AEAQNL HWQDDALIRIGRISWILSPVFKFLKDGTSIIPEDDPCVWVSKEDDPENWHVQIFRSIDSGSVKGFPKYEDEAEAQHLE.. H W DDALIRIGRISWILSP F GT I P DDP V VS EDDPENWHVQIFRSIDSGSVKGFPK AEAQHL HWSDDAMIRIERISWIQSPPLAVTDDGTTIVPDDDPKVHVLSKDNRENWNVQIFRSIDSGSLKGFPKYIKKAENQNFF.. H W DDAMIRIERISWI SP L V GT I P DDP V V DN ENWNVQIFRSIDSGSLKGFPK AE QNF HW.DDALIKIERISWILSPPLAVKDGVTVVPP.DDPTVHVSSEDDPENWHVQIFRSIDSGSLKGFPKNVHDCQAQNLI.. H W DDALIKIERISWILSP L V T V P DDP V VS EDDPENWHVQIFRSIDSGSLKGFPK QAQNL

552 526 546 539 549

LgPLDd VvPLDd AtPLDd GhPLDd RcPLDd

CAK LVIEKSIQ AYIQAIRSAQHYIYIENQYFLGSSYAWPSYK AGADNLIPMELALKIASKIRAKERFAVYIIIPMWP CAKDLVIEKSIQAAYIQAIRSAQHYIYIENQYFLGSSYAWPSYKFAGADNLIPMELALKIASKIRAKERFAVYIIIPMWP CAKNLVIDKSIQTAYIQAIRSAQHFIYIENQYFIGSSYAWPSYKNAGADNLIPMELALKIASKIRAKERFSVYVVIPMWP C AK LVIDKSIQTAYIQAIRSAQHFIYIENQYFIGSSYAWPSYKNAGADNLIPMELALKIASKIRAKERF VYVVIPMWP CAKRLVVDKSIQTAYIQTIRSAQHFIYIENQYFLGSSYAWPSYRDAGADNLIPMELALKIVSKIRAKERFAVYVVIPLWP C AK LVVDKSIQTAYIQ IRSAQHFIYIENQYFLGSSYAWPSYRDAGADNLIPMELALKI SKIRAKERFAVYVVIPLWP CAKNLVIDKSIQAAYIQAIRSAQHYIYIENQYFLGSSYAWPSYKNAGADNLIPMELALKVASKIRAGERFAVYIIIPLWP C AK LVIDKSIQ AYIQAIRSAQHYIYIENQYFLGSSYAWPSYKNAGADNLIPMELALKVASKIRA ERFAVYIIIPLWP SAKTQVIDRSIQTAYIQAIRSAQHFIYIENQYFLGSSYAWPSYENAGADNLIPMELALKIVSKIRANERFAVYIILPMWP AK VIDRSIQTAYIQAIRSAQHFIYIENQYFLGSSYAWPSY NAGADNLIPMELALKI SKIRA ERFAVYIILPMWP

632 606 626 619 629

LgPLDd VvPLDd AtPLDd GhPLDd RcPLDd

EGDPK TVQEILFWQSQTMQMMY IAQ LK MQ EGDPKDNTVQEILFWQSQTMQMMYSIIAQALKDMQMDTDSHPQDYLNFYCLGNRE...ELPDDASNTNGA.TVSESQKNR DYLNFYCLG RE E EGNPSCASVQEILFWQGQTMQMMYDIIAQELQSMQLEDAHPQDYLNFYCLGNREEPPKEVSSSNTQASDG.VSTSKKFH. E GNP VQEILFWQGQTMQMMYD IAQEL SMQ Y E E VS S KF EGDPKSGPVQEILYWQSQTMQMMYDVIAKELKAVQSDAHPL..DYLNFYCLGKRE...QLPDDMPATNGSVVSDSYNFQ. E GDPK VQEILYWQSQTMQMMYD IA ELK VQ DYLNFYCLG RE Q VSDS F EGDPKTATVQEILYWQSQTMQMMYDVVAQELKSMQIK.DSHPRDYLNFYCLGKRE...EVSQEMLSGKDS.VSDSAKFG. E GDPK TVQEILYWQSQTMQMMYD VAQELKSMQ DYLNFYCLG RE E VSDS KF EGDPKTETMQEILYWQSQTMQMMYDLVAREIKSMKLVDTHPQDYLNFYCLGNREE...NPQPSTNGET...VSDAYKNQ. E GDPK TMQEILYWQSQTMQMMYD VA EIKSM Y E N VSD K

708 684 700 693 702

LgPLDd VvPLDd AtPLDd GhPLDd RcPLDd

RFMIYVHAKGMIVDDEYVI GSANINQRSMAGTKDTEIAMGAYQPHHTW A KKKRP GQVYGYRMSLWAEHLG RFMIYVHAKGMIVDDEYVIVGSANINQRSMAGTKDTEIAMGAYQPHHTW.AKKKKRPFGQVYGYRMSLWAEHLGKLERHF F RFMIYVHAKGMIVDDEYVILGSANINQRSMAGSRDTEIAMGAYQPRHTWAK.KKKHPHGQIYGYRMSLWAEHLGMINNSF R FMIYVHAKGMIVDDEYVI GSANINQRSMAG RDTEIAMGAYQPRHTW KKKHPHGQIYGYRMSLWAEHLG F RFMIYVHAKGMIVDDEYVLMGSANINQRSMAGTKDTEIAMGAYQPNHTW.AHKGRHPRGQVYGYRMSLWAEHLGKTGDEF R FMIYVHAKGMIVDDEYVL GSANINQRSMAGTKDTEIAMGAYQPNHTW A K RHPRGQVYGYRMSLWAEHLG F RFMIYVHAKGMIVDDEYVIVGSANINQRSMAGTKDTEIAMGAYQPHYTW.A.KKKYPRGQVHGYRMSLWAEHLGELNKLF R FMIYVHAKGMIVDDEYVI GSANINQRSMAGTKDTEIAMGAYQPH TW A KKK PRGQV GYRMSLWAEHLG F RFMIYVHAKGMIVDDEYAIIGSANINQRSMAGSKDTEIALGAYQPHYTWAA.KKKHPRGQIYGYRMSLWAEHLGQKLIE. R FMIYVHAKGMIVDDEY I GSANINQRSMAG KDTEIALGAYQPH TW A KKKHPRGQIYGYRMSLWAEHLG

787 763 779 771 780

LgPLDd VvPLDd AtPLDd GhPLDd RcPLDd

EEPESLECVKRVNQIADENWKHFTAPDFTLLQGHLLMYPLHIRKDGKVEPLPGQENFPDAGGRVIGVHALRLPDVLT EPESLECVK VN IADENWK FT P FT LQGHLL YPLHI DGKV PLPG E FPD GGRVIG H LPD LT KEPQTLDCVKNVNKMAEENWKRFTSDAYTPLQGHLLKYPIQVDVDGKVRPLPGHETFPDFGGKVLGTRCNLPDALTT EPQ LDCVK VN MAEENWK FT YT LQGHLL YPIQVD DGKV PLPG ETFPD GGKVLG R T VEPSDLECLKKVNTISEENWKRFIDPKFSELQGHLIKYPLQVDVDGKVSPLPDYETFPDVGGKIIGAHSMALPDTLT EP LECLK VN I EENWK F DP F LQGHLI YPLQVD DGKV PLPD ETFPD GGKIIGAH LPD LT KEPESVECVKMVNSIAEENWKKFTDAEYSPLQGHLLMYPLQVDMDGKVNPLPEHENFPDVGGKVIGAHSIQLPDVLT EPESVECVK VN IAEENWK FTDA Y LQGHLL YPLQVD DGKV PLPE E FPD GGKVIGAH LPD LT .EPESLDCVKTVNNIAEENWKKYTDPDFTLLQGHLLRYPLQVDADGKVGPLPGYETFPDAGGRVLGAPAIKVPDILT EPESLDCVK VN IAEENWK YTDP FT LQGHLL YPLQVD DGKV PLPG ETFPD GGRVLGA VPD LT

864 840 856 848 856

C2 Domain

H K

H K

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D

D

Mol Biol Rep b Fig. 1 Multiple alignment of the deduced amino acid sequences of

LgPLDd and other closely related PLDds. Amino acid sequences were aligned as follows: LgPLDd (D. longan, JF791814), AtPLDd (A. thaliana, NP_849501.1), GhPLDd (G. hirsutum, AAN34820.1), RcPLDd (R. communis, XP_002530642.1), VvPLDd (V. vinifera, XP_002284764.1). The completely identical amino acids were indicated with black capital letters against grey background. Less conserved amino acids and non-conserved amino acids were indicated with black capital letters against white background. The putative domains of LgPLDd were predicted by SWISS-MODEL (http:// swissmodel.expasy.org/workspace/index.php?func=tools_sequences can1). The putative C2 domains were marked by under lines, two HKD motifs were boxed with solid lines, and an ‘IGSANINQR’ motif and an ‘IYIENQFF’ motif were boxed with dotted lines

Results Cloning and characterization of longan PLDd gene The conserved regions from available PLDd sequences were used to design specific primers of PLDd conservative region in this study. Using these primers, a 1246 bp fragment was amplified by RT-PCR from longan pericarp cDNA, which was corresponded to an internal region of PLDd mRNA. Subsequently, the full-length cDNA of LgPLDd was obtained by RACE. The full-length cDNA of LgPLDd was 2890 bp long, including a 295 bp 30 untranslated region, a polyA tail and a 2595 bp ORF (open reading frame). The protein encoded by LgPLDd comprised 865 residues, with a molecular mass of 98,261 Da and an isoelectric point of 7.42. Bioinformatic analysis The protein–protein BLAST analysis of the deduced LgPLDd amino acid sequence was compared with the related PLDds amino acid sequence from other plants (http://www.ncbi.nlm.nih.gov) (Fig. 1). The amino acid alignment revealed 74, 72, 70, and 68 % homology similarity between LgPLDd and PLDds from Gossypium hirsutum, A. thaliana, Ricinus communis, and Vitis vinifera, respectively. The deduced amino acid sequence of LgPLDd contained the Ca2?/phospholipid-binding C2 domain, the duplicated catalytic HxKxxxxD (HKD) motifs, and the IYIENQF/YF stretch that was highly conserved in all PLDs [29]. C2 domain is present in all cloned plant PLDs, but not in animal or yeast PLDs. C2 is a Ca2?-dependent phospholipid-binding structural fold and such binding is coordinated by 4–5 amino acid residues presented in two bipartite loops of this domain [30]. LgPLDd has a conserved C2 domain consisting of 119 amino acid residues (aa 21–140) near its N-terminus (Fig. 1). A highly conserved phospholipase superfamily-specific consensus sequence, HKD, was also found in both N- and

C-terminal regions of the peptides, located at the 366–373 and 715–722 amino acids, indicating their catalytic properties. The two HKD motifs were separated by 342 amino acids in the primary structure. Immediately following the second HKD motif, a highly conserved sequence of IGSANINQR was an invariant serine residue that was proposed to be the nucleophile attacking phosphorus atom of substrate phospholipids [31]. IYIENQFF region is found only in family members that exhibit bona fide PLD activity. Although mutagenesis experiments have demonstrated that the region is almost as critically important as the HKD domains, its role is not known. One intriguing possibility comes from the observation that the region of acetylcholine receptor binding choline is dominated by aromatic residues (Trp, Phe, or Tyr) [17]. In the present study, the IYIENQFF region was located at the 578–586 amino acids in LgPLDd with a Phe replaced by Leu. The secondary structural analysis of the predicted LgPLDd was carried out on the website (http://www. expasy.org). The results showed that the putative LgPLDd peptide consisted of 26.13 % alpha helix, 20.23 % extended strand, 5.66 % beta turn, and 47.98 % random coil. The random coil constituted the major part of the secondary structure, while alpha helix was the basic element of both N and C terminal parts. The tertiary structure of LgPLDd was predicted based on the segment crystal structure data of other PLDds from the known plants such as A. thaliana (AtPLDd) and V. vinifera (VvPLDd), whose structure fragments could be found in Swiss-Model. The sequence from LgPLDd protein showed the identity of 72.0 % and 68.4 % with AtPLDd and VvPLDd, respectively. The tertiary structural model of LgPLDd was built on amino acid residue sequence with a C-terminus 112 amino acid (753–865 aa) residue sequence deletion, using protein modeling software programs based on the homology with proteins of the known crystalline structure (Swiss-Model and 3D-JIGSAW) [32, 33]. N- and C- terminus, C2 domain, the active site of two HKD motifs and IYIENQFF region were indicated on the residues (Fig. 2). Phylogenetic tree was generated using LgPLDd sequence and other related PLDs deposited in NCBI. The cladistic analysis using the minimum evolution methodology [26] grouped LgPLDd in the same clade of PLDds from other plants. A relatively high similarity (0.102–0.152 branch lengths) was found for PLDd cDNA sequences of D. longan, G. hirsutum, A. thaliana, R. communis and V. vinifera. The clade of PLDds was sister to the group of other PLDs. In this analysis, the members of PLDds and PLDas groups lied apart from each other, while PLDbs and PLDcs were grouped in the same clade in the whole PLD family (Fig. 3).

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ABX83202_LtPLDa ADK60917_SiPLDa NP_001105686_ZmPLDa XP_002882959_AtPLDa ADP23922_LcPLDa ADY75750_DlPLDa ACA49723_CisPLDa ABN13537_CusPLDa ACV70146_GaPLDa AAG45485_SlPLDa

Fig. 2 Tertiary structure of LgPLDd predicted by 3D-JIGSAW (http:// bmm.cancerresearchuk.org/*3djigsaw/). Alpha helix, extended strand, beta turn, and random coil were indicated by red, yellow and gray. N- and C-terminus, C2 domain were indicated by straight line. The active site of HKD basic sequence showed the residues involved in the catalysis: H433, K435, D440, H715, K717, D722 by green. (Color figure online)

AAW83125_FaPLDa ACG80607_PpPLDa ADA72022_JcPLDa AAB37305_RcPLDa BAE79735_AhPLDa

Expression analysis of LgPLDd

ABC59316_VvPLDa AAL48261_PsPLDa

The expression pattern in different organs of longan plant was investigated for LgPLDd gene by semiquantitative RTPCR using gene-specific primers. The highest expression for LgPLDd was found in both green and senescent leaves, and the relatively low expression was detected in floral buds, flowers, mature fruits and fruits at different developing stage. A relatively high expression for LgPLDd mRNA were found in senescent or mature tissues compared to developing tissues in individual leaf, flower and fruit organs, respectively. The expression of LgPLDd mRNA in mature flowers was 2.86 times to those in floral buds, the expression of LgPLDd mRNA in senescent leaves was 1.29 times to those in green leaves, while the expression in mature fruits were 14.38, 2.00 and 0.88 times to those in developing fruits at zygotic embryo stage, heart-shape embryo stage and torpedo embryo stage, respectively (Fig. 4). In postharvest longan fruits, the expression for LgPLDd mRNA was found up-regulated with the extended storage time. It reached the highest expression peak at day 3 (4.99 times to control sample in day 0), and then decreased in day 4 (0.31 times to the highest expression in day 3) (Fig. 5).

ADY75749_LcPLDb ADV31547_DlPLDb XP_002511773_RcPLDb AAG45487_SlPLDb XP_002881817_AtPLDb1 NP_567160_AtPLDb2 AAD43343_GhPLDb NM_117255.2_AtPLDc1 EU591736_BoPLDc NM_117252.5_AtPLDc2 NM_117254.2_AtPLDc3 AAL78821_OsPLDb LgPLDd AEI99558_LcPLDd AAN34820_GhPLDd NP_849501_AtPLDd XP_002530642_RcPLDd XP_002284764_VvPLDd

Expression of PLDd recombinant protein and immunity analysis To verify the cloned cDNA encoding a PLD, the coding sequence of LgPLDd cDNA was cloned into the pET-30a

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Fig. 3 Minimum evolution tree depicted relationships among LgPLDd and related proteins of PLDa, b, c and d from other plant species. Amino acid sequences retrieved from the GenBank (accession numbers provided) were aligned and further analyzed using the MEGA software v4 [27] as described in ‘‘Materials and methods’’ section

Relative expression level of LgPLDδ

Mol Biol Rep

Fb

Flo

Gl

Sl

Df1

Df2

Mf

Df3

Different organs of longan

Relative expression level of LgPLDδ

Fig. 4 Expression of LgPLDd in different longan organs. Equivalent cDNA amounts from different longan organs, i.e. floral buds (Fb), flowers (Flo), green leaves (Gl), senescent leaves (Sl), developing fruits at zygotic embryo stage (Df1), heart-shape embryo stage (Df2), torpedo embryo stage (Df3) and mature fruits (Mf), were analyzed by qPCR as described in ‘‘Materials and methods’’, using gene-specific primers for LgPLDd and a housekeeping actin gene as endogenous control. Expression levels were represented relative to that of green leaves, besides standard deviation of the mean (n = 5)

16

Fig. 6 The 12 % SDS-PAGE analysis of LgPLDd protein expression in E. coli (coomassie blue staining). Lane M was protein marker, lane 1 was whole cell lysate of BL21 E. coli cells containing the empty vector pET-30a obtained at 6 h post-induction with 0.5 mM IPTG, lane 2 was whole cell lysate of noninduced BL21 E. coli cells containing plasmid pET-LgPLDd, lane 3 was whole cell lysate of the same cells obtained at 6 h post-induction with 0.5 mM IPTG, and lane 4 was purified LgPLDd protein

14 12 10 8 6 4 2 0

D0

D1

D2

D3

D4

Different postharvest stages of longan fruits

Fig. 5 Expression of LgPLDd in different longan organs. Equivalent cDNA amounts from different postharvest stages of longan fruits, i.e. day 0 (D0), day 1 (D1), day 2 (D2), day 3 (D3) and day 4 (D4), were analyzed by qPCR as described in ‘‘Materials and methods’’ section, using gene-specific primers for LgPLDd and a housekeeping actin gene as endogenous control. Expression levels were represented relative to that of the postharvest stages of longan fruits at day 0, besides standard deviation of the mean (n = 5)

expression vector to produce in E. coli LgPLDd recombinant protein with a His tag at the N-terminus. Figure 6 showed the recombinant (6His) LgPLDd protein band analyzed by SDS PAGE. His-LgPLDd protein (lane 3) was overexpressed in competent E. coli BL21, and had an apparent molecular mass about 105 kDa. A purified LgPLDd protein (lane 4) was collected and had an apparent molecular mass about 98.0 kDa.

Fig. 7 Western blotting profile of LgPLDd protein expressed in E. coli, immunity indentified with a polyclonal antibody raised against purified longan PLDd. Lane 1 was whole cell lysate of BL21 E. coli cells containing empty vector pET-30a immunized with a polyclonal PLDd antibody, lane 2 was whole cell lysate of noninduced BL21 E. coli cells containing plasmid pET-LgPLDd immunized with a polyclonal PLDd antibody, lane 3 was LgPLDb protein expressed in E. coli and post-induction with 0.5 mM IPTG immunized with a polyclonal PLDd antibody

LgPLDd protein expressed in E. coli was identified by western blot, with a polyclonal antibody raised against purified longan PLDd. Although LgPLDd had the domain features common to the other cloned plant PLDs, the western blot result showed that LgPLDd protein was specifically recognized by PLDd antibody (Fig. 7), but the same antibody did not react with PLDa or PLDb. LgPLDd proteins expressed in different organs and different postharvest stages were also identified, with a beta-tubulin as control protein. Western blotting analysis on LgPLDd protein showed a relative expression pattern as its mRNA. A relatively high expression for LgPLDd protein was found in senescent tissues compared to developing

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Fig. 8 Western blotting profile of LgPLDd protein expressed in different organs of longan. Different organs were identified with a polyclonal antibody raised against purified longan PLDd. Floral buds (Fb), flowers (Flo), green leaves (Gl), senescent leaves (Sl), developing fruits (Df) and mature fruits (Mf) were analyzed as described in ‘‘Materials and methods’’ section. Beta-tubulin was chosen as control protein

Fig. 9 Western blotting profile of LgPLDd protein expressed in different postharvest stages of longan fruits. Proteins in postharvest fruits from day 0 to day 4 were identified with a polyclonal antibody raised against purified longan PLDd. Day 0 (D0), day 1 (D1), day 2 (D2), day 3 (D3) and day 4 (D4) were analyzed as described in ‘‘Materials and methods’’. Beta-tubulin was chosen as control protein

tissues in individual leaf and fruit organs (Fig. 8). In postharvest longan fruits, the expression for LgPLDd protein was found up-regulated with the extended storage time (Fig. 9).

Discussion Phospholipase D, a major plant lipid-degrading enzyme, has been suggested to be involved in many important functions such as cellular signal transduction, membrane trafficking and membrane degradation, stress signaling, cell proliferation and cell defense response [29]. Great progress has recently been made toward understanding the roles of distinct PLD isoforms in their different functions in cellular processes. In this study, a distinct isoform of PLD gene family, LgPLDd, was cloned and analyzed for the first time. This LgPLDd had the domain features common to the other cloned plant PLDs. It contained two HKD motifs that were conserved in all cloned PLDs and formed catalytic triads responsible for the hydrolysis of phosphoester bonds. It also contained a Ca2?/phospholipid binding (C2) domain near the N terminus, and the acidic residues in the three

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Ca2?-binding loops were conserved [34]. It indicated that LgPLDs, like most other plant PLDs, required Ca2? for activity and was stimulated by PIP2 [14]. In the absence or at low levels of Ca2?, PLDs bound to PIP2 at the C2 domain. The higher concentrations of Ca2? inhibited this binding but enhanced the binding of PIP2 to the catalytic region, which promoted PC binding and catalysis. One PIP2-binding site near the catalytic region lied between two HKDs and contained many basic side chains (Lys, Arg and His), and these critical basic residues were conserved in PLDd. Although PIP2 binding itself was not required for PLDd activity, this conserved region could also be responsible for the PIP2 stimulation effect observed for PLDd. The different PIP2 requirements suggested that other unique factor(s) could help to activate PLDd [5]. Oleic acid was shown to be a specific stimulator for PLDd but not for other Arabidopsis PLDs [14]. The tertiary structure of LgPLDd was predicted based on the structure data of PLDds from A. thaliana and V. vinifera. The western blot result showed that LgPLDd protein was specifically recognized by PLDd antibody, but the same antibody did not react with PLDa or PLDb. These results revealed that the current LgPLDd encoded a newly characterized PLD which exhibited unique properties and played a distinct catalysis function in longan PLD gene family [14, 19]. The recently identified PLDds displayed unique biochemical properties. In Arabidopsis, PLDd was found playing an important role in mechanical wounding [14] and various environmental stresses. It was found that a designated PLDd was oleate-activated and associated tightly with plasma membrane. PLDd specifically served as a stabilizing anchor for microtubules at the cortex–plasma membrane interface [35]. The plasma membrane-bound PLDd was activated in response to H2O2 and the resulting phosphatidic acid (PA) functioned to decrease H2O2-promoted programmed cell death [18], indicating that the activation of oleate-stimulated PLDd was an important step for plant in response to H2O2 and increasing stress tolerance. The previous studies suggested that PA was likely to be involved in numerous physiological processes in plants, especially in stomatal movement [36] and senescence [37]. The roles of PA in plant senescence were demonstrated using PLD antisense plants; this mutant plant was delayed in abscisic acid (ABA) and ethylene-induced senescence [38]. A potential mechanism of PA action in plant stress responses was the activation of reactive oxygen species (ROS) [39]. A recent report showed that PA promoted ROS formation in Arabidopsis [40]. Pharmacological and biochemical studies suggested that the enzyme was responsible for ROS generation in plants [41]. It seemed that the formation of PA by the action of PLDd helped plant to acclimate to ROS stresses.

Mol Biol Rep

Research also showed that PLDd cooperated in ABA signaling in guard cells. A PLDd mutation Arabidopsis reduced ABA-induced phosphatidic acid production in epidermal tissues, and this PLDd mutants failed to close the stomata in response to nitric oxide (NO) [13]. ABA signal transduction in guard cells was mediated by signaling molecules including ROS and NO [42, 43]. PLDd guard cells produced similar NO and H2O2 levels in response to ABA, indicating that PLDd was downstream of NO and H2O2 in ABA-induced stomatal closure [44]. These research results indicated that plant PLDd had a defense mechanism in response to H2O2 stress, and the accumulation of H2O2 in plant was found to play important roles in postharvest senescence. In longan, the level of PLDd mRNA expression was higher in senescent tissues like old leaves, senescent flowers and mature fruits than that in developing tissues like young leaves, floral buds and developing fruits at zygotic and heartshape embryo stage. Similar circumstance was found in other plants, e.g. the expression level was higher in old leaves, stems, flowers and roots than that in young leaves and siliques in Arabidopsis [14]. Moreover, the level of PLDd mRNA expression was also higher in postharvest longan fruits, corresponding to the accumulation of H2O2 [45]. The up-regulation of LgPLDd could be seen as a response to ROS stress. These results indicated that LgPLDd had a positive function in response to H2O2 and the resulting PA functioned to decrease H2O2-promoted oxidation stress and reduce longan fruit postharvest senescence. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 31000927, 31160407, 31360494), the Foundation from China Litchi and Longan Industry Technology Research System (Grant No. nycytx-32-05, CARS-33-03), the New Century Guangxi Ten, Hundred and Thousand Talent Project (Grant No. Gui Ren She Ban Han [2012]92), the Construction Program of Guangxi Key Laboratory (Grant No. 12-071-09), the Guangxi Natural Science Foundation (Grant No. 2011GXNSFB018049) and the Fundamental Research Funds of GXAAS (Grant No. Gui Nong Ke 2011YZ24, Gui Nong Ke 2012YZ08).

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