JXB Advance Access originally published online on March 3, 2003
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Journal of Experimental Botany, Vol. 54, No. 385, pp. 1183-1191,
April 1, 2003
© 2003 Oxford University Press
Isolation and characterization of a cDNA encoding a lipid transfer protein expressed in ‘Valencia’ orange during abscission
Received 14 October 2002; Accepted 9 December 2002
Citrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, 700 Experiment Station Road, Lake Alfred, Florida 33850-2299, USA
1 To whom correspondence should be addressed. Fax: +1 863 956 4631. E-mail: jkbu{at}lal.ufl.edu
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Abstract |
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The genetics and expression of a lipid transfer protein (LTP) gene was examined during abscission of mature fruit of ‘Valencia’ orange. A cDNA encoding an LTP, CsLTP, was isolated from a cDNA subtraction library constructed from mature fruit abscission zones 48 h after application of a mature fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-pyrazole (CMN-pyrazole). A full-length cDNA clone of 652 nucleotides was isolated using 5' and 3' RACE followed by cDNA library screening and PCR amplification. The cDNA clone encoded a protein of 155 amino acid residues with a molecular mass and isoelectric point of 9.18 kDa and 9.12, respectively. A partial genomic clone of 505 nucleotides containing one intron of 101 base pairs was amplified from leaf genomic DNA. Southern blot hybridization demonstrated that at least two closely related CsLTP genes are present in ‘Valencia’ orange. Temporal expression patterns in mature fruit abscission zones were examined by northern hybridization. Increased expression of CsLTP mRNA was detected in RNA of mature fruit abscission zones 6, 24, 48, and 72 h after application of a non-specific abscission agent, ethephon. Low expression of CsLTP transcripts was observed after treatment of CMN-pyrazole until 24 h after application. After this time, expression markedly increased. The results suggest that CsLTP has a role in the abscission process, possibly by assisting transport of cutin monomers to the fracture plane of the abscission zone or through its anti-microbial activity by reducing the potential of microbial attack.
Key words: Abscission zone, 5-chloro-3-methyl-4-nitro-pyrazole, ethephon, ethylene.
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Introduction |
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Abscission is a developmentally controlled process that results in the shedding of plant organs such as leaves, flowers, and fruit and occurs in a distinct layer of cells called the abscission zone. The process is regulated by plant growth regulators such as ethylene and auxin and involves active mobilization of cell wall hydrolases such as cellulase and polygalacturonase with subsequent cell wall breakdown along the fracture plane of the abscission zone (Brown, 1997; Patterson, 2001; Roberts et al., 2002). The study of some genes involved directly or indirectly in abscission has given additional insight into the various metabolic processes activated or suppressed as abscission proceeds. For example, cellulase and polygalacturonase gene expression was associated with fruit abscission in citrus and peach (Bonghi et al., 1992; Burns et al., 1998). In general, detectable expression of cell wall hydrolase genes preceded measurable enzyme activity. Other genes, such as MADS-box and pathogen-related transcripts, were differentially expressed both spatially and temporally during abscission (Kubigsteltig et al., 1999; Wu and Burns, 2000; Mao et al., 2000; Burns, 2002; Ruperti et al., 2002).
We were interested in the characterization of abscission-related gene expression in ‘Valencia’ orange for the purpose of exploring potential points of control during abscission. One gene that was isolated from the mature fruit abscission zone subtraction library encoded a lipid transfer protein (CsLTP; Burns, 2002). Lipid transfer proteins (LTPs) have been isolated from plants, animals, bacteria, and fungi (Kader, 1996). Plant LTPs are encoded by small multigene families in most plant species. The LTP-encoded proteins share many common features, including a high isoelectric point, a low molecular mass and eight highly conserved cysteine residues involved in the formation of intramolecular disulphide bonds (Kader, 1996, 1997). Numerous LTP genes have been isolated from plants, including Arabidopsis thaliana (Arondel et al., 2000), tomato (Terres-Schumann et al., 1992; Treviño and O’Connell, 1998), maize (Sossountzov et al., 1991), pepper (Jung and Hwang, 2000; Park et al., 2002), Euphorbia lagascae (Edqvist and Farbos, 2002), cabbage (Hincha et al., 2001), barley (Molina and Garcia-Olmedo, 1993; Molina et al., 1996), carrot (Sterk et al., 1991), and rice (Vignols et al., 1994). The function of plant LTPs remains unknown. However, LTPs have been shown to catalyse the transfer of phospholipids between membranes in vitro (Bourgis and Kader, 1997; Guerbette et al., 1999). It has been suggested that plant LTPs may be involved in epicuticular wax or cuticle biosynthesis (Sterk et al., 1991; Pyee et al., 1994; Pyee and Kolattukudy, 1995), somatic embryogenesis (Colmenero-Flores et al., 1997), defence reactions (Nielsen et al., 1996; Tassin et al., 1998), and responses to various environmental conditions (Kader, 1996; Ouvrard et al., 1996; Pearce et al., 1998; Romo et al., 2001; Blein et al., 2002). This work reports on the isolation, characterization and expression of CsLTP during abscission in ‘Valencia’ orange. As far as is known, this is the first report of an LTP markedly up-regulated during abscission.
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Materials and methods |
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Plant materials and abscission material treatment
Fifteen-year-old Citrus sinensis (L.) Osbeck cv. ‘Valencia’ trees between 3–5 m in height and located at the University of Florida’s Citrus Research and Education Center at Lake Alfred, Florida, USA, were used in this study during the 2000 and 2001 growing seasons. Approximately 48 trees were sprayed with a solution of either 200 mg l–1 5-chloro-3-methyl-4-nitro-pyrazole (CMN-pyrazole) and 0.125% kinetic adjuvant, 200 mg l–1 ethephon and 0.125% kinetic adjuvant, or 0.125% adjuvant alone. In general, trees of this size were sprayed with 5.0 l spray materials using a gas-powered single nozzle handgun sprayer until the solution began to run-off the foliage. Mature leaf blade, leaf abscission zone, young and mature fruit abscission zone tissues were collected from trees at 0, 6, 24, 48, and 72 h after application of CMN-pyrazole, ethephon, or adjuvant alone. Leaf blades and leaf abscission zones were excised from at least 300 randomly selected leaves in each treatment. Leaf tissue (2.5 cm) was removed from the centre of the blade and did not include major midrib vascular tissues. For leaf abscission zones, 4 mm of tissue, containing the laminar abscission zone located at the petiole/blade junction, was excised. Young fruit abscission zones were removed from at least 250 randomly selected fruit between 8 and 10 weeks old, whereas mature fruit abscission zones were removed from at least 250 randomly selected fruit that were approximately 12 months old. For mature and young fruit, the abscission zones were removed using a sharpened 4 mm diameter cork borer. The borer was slipped over the pedicel and pushed through the calyx and fruit peel. The location of the fruit abscission zone was visually determined in this cylinder of tissue and further trimmed to 6x4 mm. The harvested tissues were immediately frozen in liquid nitrogen and stored at –80 °C for future use.
DNA extraction
Genomic DNA was extracted from leaves by using Plant DNAZOL Reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s recommendation. Briefly, 2 g of fresh leaves were powdered in liquid nitrogen with a mortar and pestle and mixed with 6 ml DNAZOL ES containing 1% (v:v) ß-mercaptoethanol. The homogenate was incubated at room temperature for 5 min with agitation and then supplemented with 6 ml of chloroform. After incubation for an additional 5 min, the mixture was centrifuged at 12 000 g for 10 min. The upper aqueous phase was transferred to a clean tube and precipitated by mixing 1 vol. of supernatant with 0.75 vols of ethanol at room temperature for 5 min. The precipitated DNA was centrifuged at 5000 g for 4 min and washed with 6 ml DNAZOL ES-ethanol solution and 6 ml of 75% ethanol. The pellet was resuspended in 1 ml of 8 mM NaOH and stored at 4 °C.
Total RNA extraction
Total RNA was extracted following the protocol of phenol/SDS method for plant RNA preparation (Ausubel et al., 1989) with minor modifications. In brief, approximately 3 g tissue was ground to a fine powder in liquid nitrogen with a mortar and pestle. The powder was transferred into a centrifuge tube containing 9 ml of cold extraction buffer (100 mM TRIS–HCl, pH 8.0; 500 mM LiCl; 10 mM EDTA, pH 8.0; 1% SDS; and 5 mM DTT) and 3 ml of phenol saturated with 0.1 M citrate buffer (pH 4.3) (Sigma Chemical, St Louis, MO, USA). After vortexing, 3 ml of chloroform were added. The homogenate was centrifuged at 10 000 g for 30 min after incubating at 50 °C for 20 min. The aqueous phase was transferred to a new centrifuge tube, mixed with 1 vol. of phenol:chloroform (1:1, v:v), and centrifuged at 10 000 g for 10 min. The phenol/chloroform extractions were repeated until the milky interface disappeared after centrifugation. The upper aqueous phase was precipitated in 2 M lithium chloride (LiCl) at 4 °C overnight and then centrifuged at 10 000 g for 15 min. The pellet was washed twice with 2 M LiCl and resuspended in water. Total RNA samples were stored at –80 °C.
cDNA subtraction library
Polyadenylated mRNA was purified from total RNA samples extracted from either control or CMN-pyrazole-treated mature fruit abscission zones 48 h after application using Oligotex mRNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s recommendations. mRNA populations were subtracted using cDNAs synthesized from CMN-pyrazole-treated tissues as ‘driver’ and cDNAs from control tissues as ‘tester’ according to the protocol of PCR-SelectTM cDNA subtraction kit (Clontech, Palo Alto, CA, USA). The subtracted cDNAs were cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). cDNA clones obtained from the library were sequenced by the DNA Sequencing Core, University of Florida, Gainesville, FL, USA.
Cloning and sequencing of the LTP gene
One cDNA clone of 313 nucleotides (nt) from the subtraction library was identified as a CsLTP gene fragment by sequence comparison. Based on its sequence, primer LTP1 (5'-CGGATCAATCCCTAA CCTCA-3') was synthesized. This primer was used with primer ASBT (5'-CCTGGCCAGGGCCCGTCGACGGATCCTTTTTTT TTTTTTTTTTTV-3', where V is either G, C, or A) to amplify the 3' end of the CsLTP cDNA by polymerase chain reaction (PCR) from first-strand cDNA synthesized from total mature fruit abscission zone RNA extracted 48 h after CMN-pyrazole application. To obtain the 5' end of the CsLTP cDNA, a primer LTP2 (5'-GAATGCTGACTCCACAAGCTC-3') was designed to an internal sequence of the subtraction library clone. This primer was used with the vector T3 primer to amplify an LTP fragment from an ethylene-induced fruit abscission zone library previously described (Burns et al., 1998). A 324 nt PCR fragment was isolated, cloned and identified as a 5' end CsLTP fragment by sequence comparison. To confirm the presence of this sequence in CMN-pyrazole-treated mature fruit abscission zone RNA, a primer LTP3 (5'-AATC TCAGGAGAAATCTCAAATGGCTGCCC-3') identical to the 5'-most end of the 324 nt fragment clone was synthesized. This primer was used with primer ASBT to amplify a putative CsLTP full-length cDNA. A set of gene-specific primers LTP4 (5'-GCCATAACA TGTGGGCAGGTG-3') and LTP5 (5'-CGAACGAGACTCGCA GTACA-3'), that corresponded to the cDNA clone nucleotide positions 90–111 and 494–475, respectively, was used to amplify a DNA fragment containing the CsLTP intron from genomic DNA obtained from leaf blades. PCR products were cloned into vector pGEM-T Easy (Promega, Madison, WI). Plasmids containing the PCR fragments of interest were sent to the DNA sequencing core laboratory, University of Florida, Gainesville, FL, USA for sequencing.
Sequence analysis, alignment and comparison
Nucleotide sequences from RT-PCR clones were identified by NCBI BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/; Altschul et al., 1997). Open reading frame and protein predictions were made using the NCBI ORF Finder program (http://www3.ncbi.nlm.nih. gov/gorf/gorf.html). Signal peptides and their cleavage sites were predicted using the SignalP program (http://www.cbs.dtu.dk/ services/SignalP/; Nielsen et al., 1997). The theoretical isoelectric point (pI) and molecular mass values for mature peptides were calculated using the PeptideMass program (http://us.expasy.org/tools/peptide-mass.html). Amino acid alignments and phylogenetic tree were generated under default parameters using the ClustalW program (http://www.ebi.ac.uk/clustalw/; Thompson et al., 1994). A phylogenetic tree was generated using the Neighbor–Joining method (Saitou and Nei, 1987) included in the ClustalW program. Bootstrap re-sampling analysis with 1000 replicates was performed to assess branch support. The TreeView program, version 1.6.6 (Page, 1996) was used to draw the phylogenetic tree.
Southern and northern blot hybridization
For Southern blot hybridization, 10 µg of genomic DNA were digested with either single restriction enzyme BamHI, HindIII, or XbaI or double restriction enzymes BamHI and HindIII, or BamHI and XbaI at 37 °C for 5 h. The digested genomic DNA was separated on a 0.8% agarose gel at 23 mA overnight, transferred onto positively charged nylon membrane (Roche Biochemicals, Indianapolis, IN) with 10x SSC overnight using the Rapid Downward Transfer system (Schleicher and Schuell, NH), and then cross-linked using a Stratalinker UV Crosslinker (Stratagene, La Jolla, CA). A DIG-labelled specific DNA probe was synthesized by PCR with CsLTP-specific primer LTP3 and primer ASBT. Blots were hybridized in PerfectHybTM Plus Hybridization buffer (Sigma, St Louis, MO) at 65 °C for at least 2 h. Washing and detection were performed under high stringency conditions according to the DIG-high prime DNA labelling and detection starter kit II protocol (Roche Biochemicals, Indianapolis, IN) with the following modifications: Tris-buffer solution (20 mM Tris-HCl, pH 7.5; 150 mM NaCl) and non-fat dried milk powder (Carnation Milk Recipes, Young America, MN) were used instead of maleic acid solution and the block agent supplied by the manufacturer. For northern blot hybridization, 10 µg of total RNA were separated on a 1.2% formaldehyde/MOPS [3-(N-morpholino)-propanesulphonic acid] agarose gel. Hybridization and detection were the same as described above. Both northern and Southern blot hybridizations were replicated at least twice.
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Results |
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A cDNA fragment of 313 nt was isolated from the subtraction library prepared from mature fruit abscission zones 48 h after application of CMN-pyrazole. DNA sequence comparison with the BLAST program (Altschul et al., 1997) indicated that this fragment shared significant similarity with other plant LTPs. Based on the sequence of the 313 nt CsLTP fragment, primers were designed to amplify the 3' or 5' ends of the full length cDNA using a rapid amplification of cDNA ends (RACE) approach. A 396 nt fragment coding for the 3' end of the CsLTP cDNA was isolated and its identity confirmed by sequence comparison. However, a 5' RACE approach, using several primers designed to internal nucleotide sequences of the 313 nt fragment, failed to amplify the 5'-most end of the cDNA. To obtain the 5'-most end of the nucleotide sequence, primer LTP2 was used with vector primer T3 to amplify a 324 nt CsLTP cDNA fragment from a cDNA library prepared from ethylene-induced mature fruit abscission zone tissue (Burns et al., 1998). Using a 5'-most primer LTP3 designed to this sequence with our poly(T) primer ASBT, a full-length CsLTP cDNA was amplified from first-strand cDNA synthesized from CMN-pyrazole-treated mature fruit abscission zone tissue. In addition, this same strategy was used to amplify an identically sized nucleotide fragment from the ethylene-induced cDNA library. Sequence comparison determined that the two full-length clones were identical. The nucleotide sequence of full-length CsLTP gene was deposited in the GenBank under the accession number AF369931.
The nucleotide and encoded protein sequences of the citrus mature fruit abscission zone CsLTP are shown in Fig. 1. CsLTP contained a 345 nt open reading frame that encoded a putative protein of 115 amino acid residues. Analysis with the SignalP program (Nielsen et al., 1997) indicated that the N-terminus contained a eukaryotic signal sequence with a cleavage site between amino acid residues 24 and 25. After removal of the signal peptide, the mature protein consisted of 91 amino acid residues with an isoelectric point of 9.12 and a molecular mass of 9.18 kDa. The polyadenylation signal sequence (AATAAA) of CsLTP was located 220 nt upstream of the poly(A) tail.
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Alignment of the deduced amino acid sequences of CsLTP and 22 additional plant LTPs was performed and a phylogenetic tree was generated by using the ClustalW program (Fig. 2). Four major groups were distinguished within the tree. Group I included CsLTP and contained encoded proteins isolated and expressed in fruit. Group II contained sequences expressed in seeds. Group III included LTPs expressed in leaf tissues and was largely induced by environmental factors such as drought conditions and pathogen infection. Finally, group IV contained LTP sequences isolated and expressed in flowers.
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Southern blot hybridization under high stringency conditions demonstrated that at least two closely related LTP genes were contained in the ‘Valencia’ orange genome (Fig. 3). However, the possibility could not be discounted that only a single gene containing an intron(s) capable of being digested with the restriction enzymes used in the Southern blot analysis was present. Since plant LTPs contain a single conserved intron of small size (Kader, 1996), genomic DNA was isolated from ‘Valencia’ orange leaves and the intron amplified using primers LTP4 and LTP5 that flanked the reported intron insertion site. A 505 nt fragment was amplified that contained an intron of 101 nt and a portion of the 3' end of exon 1 and 5'end of exon 2 (Fig. 4). The intron was positioned two codons before the stop codon as found in other plant LTPs such as cotton (Liu et al., 2000; Orford and Timmis, 2000), rice (Vignols et al., 1994), sorghum (Pelese-Siebenbourg et al., 1994), Arabidopsis thaliana (Thoma et al., 1994), and barley (Skriver et al., 1992). The intron sequence contained GT/AG border junctions typical of plant introns (Brown and Simpson, 1998) and did not contain restriction digestion sites used in the Southern blot analysis. This suggested that at least two closely related LTP genes are found in the ‘Valencia’ orange genome. Comparison of the ORFs of the genomic DNA with the cDNA clone sequence indicated that they differed by only 2 nt.
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Northern blot hybridization indicated that CsLTP mRNA was constitutively expressed in leaf blades, leaf abscission zones and young fruit abscission zones (Fig. 5A, B, C). Application of ethephon or CMN-pyrazole had little effect on CsLTP expression in these tissues 48 h after treatment. Expression of CsLTP mRNA was followed at different time periods after ethephon or CMN-pyrazole application in mature fruit abscission zones of ‘Valencia’ orange (Fig. 5D). There was little or no hybridization detected in adjuvant-treated controls. Ethephon treatment induced CsLTP expression in mature fruit abscission zones at all time points examined. CsLTP expression was low 6 h and 24 h after CMN-pyrazole application. After this time, however, expression increased markedly.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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LTPs have been shown to transfer membranes in vitro without specificity, and have thus been called ‘non-specific’ LTPs (Kader, 1996). Recently, new insights have emerged regarding their role in the formation and reinforcement of plant surface layers and defence against pathogens. LTPs bind a wide variety of hydrophobic fatty acids and lipids, potentially providing an array of monomers for cutin and suberin synthesis (Blein et al., 2002; Kader, 1996). LTPs have also been shown to respond to biotic and abiotic stresses, indicating that they can be classified as pathogen-related (PR)-like proteins. LTPs can also evoke similar responses in plants as elicitin, a compound that binds to a plasma membrane receptor and activates defence responses (Buhot et al., 2001). In some cases the LTP protein itself was reported to have anti-microbial activity (Blein et al., 2002).
It has been demonstrated that the expression of the CsLTP gene isolated from ‘Valencia’ orange was very low or undetectable in mature fruit abscission zones, but strikingly up-regulated during abscission. The presence and induction of the CsLTP transcripts in mature fruit abscission zones is consistent with reported in vivo roles of LTPs. Abscission is a highly co-ordinated event that culminates in the removal of the organ from the parent plant, presumably as a result of the action of cell wall hydrolytic enzymes. As the abscission zone digests, the exposed cells along the fracture plane across the stem and abscising organ differentiate into suberized scar tissue (Bleecker and Patterson, 1997). It is likely that LTPs participate in this event by transporting hydrophobic monomers to the plasmalemma–cell wall interface where LTPs have been experimentally localized (Kader, 1996). Formation of the suberized layer may prevent water loss in the abscised organ and stem of the parent plant (Bleecker and Patterson, 1997; Roberts et al., 2002).
LTPs could have a role in protecting the abscised organ and parent plant from microbial attack. The formation of a suberized barrier physically excludes micro-organisms from living tissues. Additionally, LTPs were shown to bind the elicitin receptor of the plasma membrane (Buhot et al., 2001). Binding elicitin initiates a cascade of events leading to the hypersensitive response. Although the binding of LTP alone inhibits this response, binding of an LTP–lipid complex to the elicitin receptor could act as an agonist, triggering plant defence reactions (Blein et al., 2002). Increased gene expression of varied PR proteins during abscission has been reported in citrus (Wu and Burns, 2000; Burns, 2002) and peach (Ruperti et al., 2002), and some, such as chitinase, ß-1, 3-glucanase and ß-xylosidase, could restrict the growth of pathogens. Other PR proteins induced during abscission such as phenylalanine ammonia lyase, caffeic acid O-methyltransferase, and cinnamic acid 4-hydroxylase (Burns, 2002; Kostenyuk and Burns, 2002), may provide the monomers necessary for lignin deposition as well as secondary product biosynthesis with associated antimicrobial properties. There is no evidence to suggest that LTPs are directly involved in the induction of PR protein genes and their protein products. However, the induction of LTP suggests that this gene is one component of an array of PR-related genes induced during the abscission process.
CsLTP was constitutively expressed in young fruit abscission zones, leaf abscission zones and leaf blades; however, application of ethephon or CMN-pyrazole did not markedly alter CsLTP expression. CMN-pyrazole caused ethylene production and mature fruit abscission in citrus but did not induce abscission or cause ethylene production in leaves or young fruit (Yuan et al., 2001; Burns, 2002), whereas ethephon caused ethylene release by chemical degradation (Cooke and Randle, 1968). The response of young fruit and leaves to ethephon is determined by the sensitivity of the tissues to ethylene, as well as the intensity and duration of exposure. The low and variable abscission response to ethephon may indicate that these factors influence the abscission response in these tissues. Nevertheless, the lack of LTP gene induction in leaf blades treated with ethephon suggests that factors other than ethylene alone influenced expression in mature fruit abscission zones.
The CsLTP gene shared many structural similarities with other plant LTPs. After removal of the eukaryotic signal sequence, CsLTP encoded a 9.18 kDa basic protein with eight conserved cysteine residues. CsLTP also contained the LTP consensus signature pattern located between residues 92 and 113 of the encoded protein. Analysis of the genomic clone indicated that CsLTP contained a single, positionally conserved intron found in plant LTP genes (Kader, 1996). Phylogenetic analysis indicated that CsLTP was grouped with encoded proteins of genes isolated from or reported to be expressed in fruit. Unfortunately, detailed information about the expression of Group I genes in other tissues is lacking. Overall, these results reinforce the notion that the phylogenetic distribution of plant LTPs is based on functional relationships.
In conclusion, an LTP gene has been isolated that is highly up-regulated in mature fruit abscission zones during abscission. The CsLTP gene was constitutively expressed in other tissues examined, and little induction was observed in young fruit or leaf abscission zones after the application of abscission materials. The lack of expression in uninduced mature fruit abscission zones compared with young fruit abscission zones suggests that CsLTP may be developmentally regulated. Alternatively, CsLTP expression may be suppressed in uninduced mature fruit abscission zones, and the application of ethephon or CMN-pyrazole may release the suppression. Further work will be necessary to clarify these points. The CsLTP gene was one of at least two closely-related LTP genes in the ‘Valencia’ orange genome; and as such, hybridization with this gene may represent patterns of expression of a group of homologous LTP genes in citrus. Nevertheless, CsLTP may play an important role in the abscission process, possibly by transporting the necessary monomers for cutin and suberin biosynthesis at the fracture plane and retarding microbial attack by physical or chemical means.
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Acknowledgements |
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The authors wish to thank Tony Trieu for excellent technical assistance. This research was supported by the Florida Agricultural Experiment Station, the Institute of Food and Agricultural Sciences, University of Florida, and a grant from the Florida Department of Citrus (01-31) to JKB, and approved for publication as Journal Series No. R-09118.
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References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402.
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman, JG, Smith JA, Struhl K. 1989. Preparation and analysis of RNA. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman, JG, Smith JA, Struhl K. eds. Current protocols in molecular biology, Vol. 1. New York, NY: John Wiley & Sons, 4.3.1–4.3.4.
Arondel V, Vergnolle C, Cantrel C, Kader J-C. 2000. Lipid transfer proteins are encoded by a small multigene family in Arabidopsis thaliana. Plant Science 157, 1–12.
Bleecker AB, Patterson SE. 1997. Last exit: senescence, abscission and meristem arrest in Arabidopsis. The Plant Cell 9, 1169–1179.[CrossRef][Web of Science][Medline]
Blein J-P, Coutos-Thevenot P, Marion D, Ponchet M. 2002. From elicitins to lipid-transfer proteins: a new insight in cell signaling involved in plant defence mechanisms. Trends in Plant Science 7, 293–296.[CrossRef][Web of Science][Medline]
Bonghi C, Rascio N, Ramina A, Casadoro G. 1992. Cellulase and polygalacturonase involvement in the abscission of leaf and fruit explants of peach. Plant Molecular Biology 20, 839–848.[CrossRef][Web of Science][Medline]
Bourgis F, Kader J-C. 1997. Lipid-transfer proteins: tools for manipulating membrane lipids. Physiologia Plantarum 100, 78–84.[CrossRef]
Brown JWS, Simpson CG. 1998. Splice selection in plant pre-mRNA splicing. Annual Review of Plant Physiology and Plant Molecular Biology 49, 77–95.[CrossRef][Web of Science]
Brown KM. 1997. Ethylene and abscission. Physiologia Plantarum 100, 567–576.[CrossRef]
Buhot N, Douliez J-P, Jacquemard A, Marion D, et al. 2001. A lipid transfer protein binds to a receptor involved in the control of plant defence responses. FEBS Letters 509, 27–30.[CrossRef][Web of Science][Medline]
Burns JK. 2002. Using molecular biology tools to identify abscission materials for citrus. HortScience 37, 459–464.
Burns JK, Lewandowski DJ, Nairn CJ, Brown GE. 1998. Endo-1, 4-ß-glucanase gene expression and cell wall hydrolase activities during abscission in Valencia orange. Physiologia Plantarum 102, 217–225.[CrossRef]
Colmenero-Flores JM, Campos F, Garciarrubio A, Covarrubias AA. 1997. Characterization of Phaseolus vulgaris cDNA clones responsive to water deficit: identification of a novel late embryogenesis abundant-like protein. Plant Molecular Biology 35, 393–405.[CrossRef][Web of Science][Medline]
Cooke AR, Randle DI. 1968. 2-Haloethanephosphonic acids as ethylene-releasing agents for induction of flowering in pineapples. Nature 218, 974–975.[CrossRef][Medline]
Edqvist J, Farbos I. 2002. Characterization of germination-specific lipid transfer proteins from Euphorbia lagascae. Planta 215, 41–50.[CrossRef][Web of Science][Medline]
Guerbette F, Grosbois M, Jolliot-Croquin A, Kader J-C, Zachowski A. 1999. Lipid-transfer proteins from plants: structure and binding properties. Molecular and Cellular Biochemistry 192, 157–161.[CrossRef][Web of Science][Medline]
Hincha DK, Neukamm B, Sror HAM, Sieg F, Weckwarth W, Ruckels M, Lullien-Pellerin V, Schroder W, Schmitt JM. 2001. Cabbage cryprotein is a member of the nonspecific plant lipid transfer protein gene family. Plant Physiology 125, 835–846.
Jung HW, Hwang BK. 2000. Isolation, partial sequencing, and expression of pathogenesis-related cDNA genes from pepper leaves infected by Xanthomonas campestris pv. vesicatoria. Molecular Plant–Microbe Interactions 13, 136–142.
Kader J-C. 1996. Lipid-transfer protein in plants. Annual Review of Plant Molecular Biology 47, 627–254.[CrossRef][Web of Science]
Kader J-C. 1997. Lipid-transfer proteins: a puzzling family of plant proteins. Trends in Plant Science 2, 66–70.
Kostenyuk IA, Burns JK. 2002. Phenylalanine ammonia lyase gene expression during abscission in citrus. Physiologia Plantarum 116, 106–112.[CrossRef][Medline]
Kubigsteltig I, Laudert D, Weiler EW. 1999. Structure and regulation of the Arabidopsis thaliana allene oxide synthase gene. Planta 208, 463–471.[CrossRef][Web of Science][Medline]
Liu H-C, Creech RG, Jenkins JN, Ma D-P. 2000. Cloning and promoter analysis of the cotton lipid transfer protein gene LTP3. Biochimica et Biophysica Acta 1487, 106–111.[Medline]
Mao L, Begum D, Chuang H-W, Budiman MA, Szymkowiak EJ, Irish EE, Wing RA. 2000. JOINTLESS is a MADS-box gene controlling tomato flower abscission zone development. Nature 406, 910–913.[CrossRef][Medline]
Molina A, Garcia-Olmedo F. 1993. Developmental and pathogen induced expression of three barley genes encoding lipid transfer proteins. The Plant Journal 4, 983–991.[CrossRef][Web of Science][Medline]
Molina A, Diaz I, Vasil IK, Carbonero P, Garcia-Olmedo F. 1996. Two cold-inducible genes encoding lipid transfer protein LTP4 from barley show differential responses to bacterial pathogens. Molecular and General Genetics 252, 163–168.
Nielsen H, Engelbrecht J, Brunak S, von Heijne G. 1997. Identification of prokaryotic and eukaryotic peptides and prediction of their cleavage sites. Protein Engineering 10, 1–6.
Nielsen KK, Nielsen JE, Madrid SM, Mikkelsen JD. 1996. New antifungal proteins from sugar beet (Beta vulgaris L.) showing homology to non-specific lipid transfer proteins. Plant Molecular Biology 31, 539–552.[CrossRef][Web of Science][Medline]
Orford SJ, Timmis JN. 2000. Expression of a lipid transfer protein gene family during cotton fibre development. Biochimica et Biophysica Acta 1483, 275–284.[Medline]
Ouvrard O, Cellier F, Ferrare K, Toousch D, Lamaze T, Dupuis JM, Casse-Delbart F. 1996. Identification and expression of water stress- and abscisic acid-regulated genes in a drought-tolerant sunflower genotype. Plant Molecular Biology 31, 819–829.[CrossRef][Web of Science][Medline]
Page RDM. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12, 357–358.
Park C-J, Shin R, Park JM, Lee G-J, You J-M, Paek K-H. 2002. Induction of pepper cDNA encoding a lipid transfer protein during the resistance response to tobacco mosaic virus. Plant Molecular Biology 48, 243–254.[CrossRef][Web of Science][Medline]
Patterson SE. 2001. Cutting loose. Abscission and dehiscence in Arabidopsis. Plant Physiology 126, 494–500.
Pearce RS, Houlston CE, Atherton KM, Rixon JE, Harrison P, Hughes MA, Dun MA. 1998. Localization of expression of three cold-induced genes, blt101, blt4.9, and blt14, in different tissues of the crown and developing leaves of cold-acclimated cultivated barley. Plant Physiology 117, 787–795.
Pelese-Siebenbourg F, Caelles C, Kader JC, Delseny M, Puigdomenech P. 1994. A pair of genes coding for lipid –transfer proteins in Sorghum vulgare. Gene 148, 305–308.[CrossRef][Web of Science][Medline]
Pyee J, Kolattukudy PE. 1995. The gene for the major cuticular wax-associated protein and three homologous genes from broccoli (Brassica oleracea) and their expression patterns. The Plant Journal 7, 49–59.[CrossRef][Web of Science][Medline]
Pyee J, Yu H, Kolattukudy PE. 1994. Identification of a lipid transfer protein as the major protein in the surface wax of broccoli (Brassica oleracea) leaves. Archives of Biochemistry and Biophysics 311, 460–468.[CrossRef][Web of Science][Medline]
Roberts JA, Elliot, KA, Gonzalez-Carranza, ZH. 2002. Abscission, dehiscence, and other cell separation processes. Annual Review of Plant Biology 53, 131–158.[CrossRef][Medline]
Romo S, Labrador E, Dopico B. 2001. Water stress-regulated gene expression in Cicer arietinum seedlings and plants. Plant Physiology and Biochemistry 39, 1017–1026.[CrossRef]
Ruperti B, Cattivelli L, Pagni S, Ramina A. 2002. Ethylene-responsive genes are differentially regulated during abscission, organ senescence and wounding in peach (Prunus persica). Journal of Experimental Botany 53, 429–437.
Saitou N, Nei M. 1987. The neighbor–joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406–425.[Abstract]
Skriver K, Leah R, Muller-Uri F, Olsen FL, Mundy J. 1992. Structure and expression of the barley lipid transfer protein gene Ltp1. Plant Molecular Biology 18, 585–589.[CrossRef][Web of Science][Medline]
Sossountzov L, Ruiz-Avila L, Vignols F, Jolliot A, Aroundel V, Tchang F, Grosbois M, Guerbette F, Miginiac E, Delseny M. 1991. Spatial and temporal expression of a maize lipid transfer protein gene. The Plant Cell 3, 923–933.
Sterk P, Booij H, Schellekens GA, van Kammen A, De Vries SC. 1991. Cell-specific expression of the carrot EP2 lipid transfer protein gene. The Plant Cell 3, 907–921.
Tassin S, Broekaert WF, Marion D, Acland DP, Ptak M, Vovelle F, Sodano P. 1998. Solution structure of Ace AMP1, a potent anti-microbial protein extracted from onion seeds: structural analogies with plant nonspecific lipid transfer proteins. Biochemistry 37, 3623–3637.[CrossRef][Medline]
Terres-Schumann S, Godoy JA, Pintor-Toro JA. 1992. A probable lipid transfer protein gene is induced by NaCl in stems of tomato plants. Plant Molecular Biology 18, 749–757.[CrossRef][Web of Science][Medline]
Thoma S, Hecht U, Kippers A, Botella J, De Vries S, Somerville CR. 1994. Tissue-specific expression of a gene encoding a cell wall-localizes lipid transfer protein from Arabidopsis. Plant Physiology 105, 35–45.[Abstract]
Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–4680.
Treviño MB, O’Connell MA. 1998. Three drought-response members of the non-specific lipid transfer protein gene family in Lycopersicon pennellii show different development patterns of expression. Plant Physiology 116, 1461–1468.
Vignols F, Lund G, Pammi S, Tremousaygue D, Grellet F, Kader J-C, Puigdomenech P, Delseny M. 1994. Characterization of a rice gene coding for a lipid transfer protein. Gene 142, 265–270.[CrossRef][Web of Science][Medline]
Yuan R, Hartmond U, Kender WJ. 2001. Physiological factors affecting response of mature ‘Valencia’ orange to CMN-Pyrazole. II. Endogenous concentrations of indole-3-acetic acid, abscisic acid, and ethylene. Journal of the American Society for Horticultural Science 126, 420–426.
Wu Z, Burns JK. 2000. Expression of polygalacturonase, ß-galactosidase, chitinase, ß-1,3-glucanase and expansin during mature fruit abscission of Valencia orange. Proceedings of the International Society of Citriculture (in press).
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