JXB Advance Access originally published online on October 10, 2006
Journal of Experimental Botany 2006 57(14):3825-3836; doi:10.1093/jxb/erl151
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RESEARCH PAPER |
Differential expression within the LOX gene family in ripening kiwifruit
1Laboratory of Fruit Molecular Physiology and Biotechnology/The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Biotechnology, Zhejiang University, Huajiachi Campus, Hangzhou 310029, China
2The Horticulture and Food Research Institute of New Zealand, Private Bag 92169, Auckland, New Zealand
* To whom correspondence should be addressed. E-mail: akun{at}zju.edu.cn or iferguson{at}hortresearch.co.nz
Received 26 May 2006; Accepted 1 August 2006
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Abstract |
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Real-time quantitative PCR was used to study lipoxygenase (LOX) gene expression patterns in kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) during fruit ripening, and in response to ethylene and low temperature during post-harvest storage. Six LOX genes were identified and cloned from a kiwifruit EST database. All were expressed in vegetative tissues and in the fruit. Expression of AdLox1 and AdLox5 increased markedly as fruit developed to the climacteric stage and were up-regulated by ethylene treatment, following a similar pattern to LOX enzyme activity. By contrast, AdLox2, AdLox3, and AdLox4 transcripts were negatively associated with ethylene accumulation, and ethylene application enhanced the decline in transcript levels. Transcripts of AdLox6 declined with fruit ripening. The fruit showed no ripening changes at low temperature, where transcripts of AdLox1 and AdLox6 were slightly induced about 72 h after harvest, suggesting an adaptive response to low temperature. Transient expression of the ethylene-responsive AdLox1 gene in tobacco leaves led to significant degradation of chlorophyll and promoted tissue senescence, whereas AdLox2 had no such effect. The results showed that the six LOX genes were differentially regulated during kiwifruit ripening and senescence, forming two groups, one active in ripening and responsive to ethylene and the other more constitutively expressed. The possible roles of individual LOX isoforms in kiwifruit are discussed.
Key words: Ethylene, fruit ripening, gene expression, kiwifruit, lipoxygenase, low temperature, senescence
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Introduction |
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Lipoxygenases (LOX; EC 1.13.11.12 [EC] ) are non-haem iron-containing dioxygenases widely distributed in the plant kingdom and with diverse functions (Porta and Rocha-Sosa, 2002). These include a role as storage proteins in seeds (Siedow, 1991), regulatory functions in some aspects of plant development such as potato tuber growth (Kolomiets et al., 2001), and mediation of lipid peroxidation in signal transduction cascades under biotic and abiotic stress (Blokhina et al., 2003), including low temperature response in fruit (González-Aguilar et al., 2004). The most important features of LOX action are the metabolic end-products, the products of LOX-mediated fatty acid oxidation being known as oxylipins. Such products have specific roles in signalling and plant defence responses.
Among the roles associated with plant development, is an association of LOX with plant senescence and fruit ripening. LOX has long been associated with membrane deterioration in plant tissues through peroxidation of polyunsaturated fatty acids, and such membrane dysfunction has been seen as a key process in senescence, resulting in loss of compartmentation and cell breakdown (Thompson, 1988; Rogiers et al., 1998). This activity has been associated with increases in general oxidative activity in senescing tissue, and in fruit, for example, there is a positive relationship between lipid peroxidation and increasing oxidative levels in avocados, pears, tomatoes, and saskatoons during ripening and senescence (Brennan and Frenkel, 1977; Brennan et al., 1979; Rogiers et al., 1998). Superoxide free radicals and hydroperoxides produced via the LOX pathway not only take part in peroxidation of cell membrane lipids, but also might be involved in other aspects of cell degradation. LOX activity has been shown to increase in conjunction with ripening processes such as loss of firmness in kiwifruit (Chen et al., 1999; Zhang et al., 2003) and peach fruit (Wu et al., 1999).
While LOX activity is an intrinsic part of the mosaic of cell degradative processes occurring in fruit ripening, the products of its action have consequences of particular importance in fruit. LOX is involved in the generation of C6 alcohols and aldehydes which constitute major volatile flavour components in ripening fruit such as tomato (Baldwin et al., 1991; Chen et al., 2004). These are principally formed from the 13-LOX group of enzymes which produce 13-HPOs (fatty acid hydroperoxides), which are in turn metabolized to hexanal, hexenals, and their alcohols. LOX activity has also been associated with production of apple (Echeverría et al., 2004; Defilippi et al., 2005), strawberry (Peréz et al., 1999), and pear fruit volatiles and, in the latter, varying levels of oxygen availability have been correlated with LOX activity and volatile production (Lara et al., 2003).
Multiple isoforms of LOX have been detected in a wide range of plants (Siedow, 1991; Feussner and Wasternack, 2002). In Arabidopsis thaliana, at least six LOX genes have been characterized. Expression of AtLox1 is stimulated by the stress-related hormones abscisic acid and methyl jasmonate (Melan et al., 1993) and AtLox2 has been identified as specifically involved in jasmonate biosynthesis stimulated by wounding (Bell et al., 1995). To date, at least five tomato LOX genes have been cloned: TomloxA expression declines during tomato fruit ripening and the decline is delayed in ACO1 transgenic low-ethylene and Nr fruit (Griffiths et al., 1999); TomloxB expression is present in mature green fruit and enhanced by ethylene (Ferrie et al., 1994; Griffiths et al., 1999); TomloxC is not detectable until the onset of ripening and has been identified as a specific isoform involved in synthesis of fruit flavour compounds (Heitz et al., 1997; Chen et al., 2004); TomloxD shows very low expression levels in fruit, but is rapidly and transiently induced by wounding (Heitz et al., 1997); TomloxE transcripts are present at the breaker and 4 d post-breaker stages of fruit ripening (Chen et al., 2004). Antisense approaches in potato have shown that PotLX1 is a key isoform involved in tuber development (Kolomiets et al., 2001); LoxH1 is detectable in leaves and involved in the generation of volatile defence and signalling compounds (León et al., 2002); LoxH3 is induced transiently after wounding (Royo et al., 1996). Three LOX genes have also been identified in tobacco, with NtLox2- and NtLox3-encoded enzymes putatively possessing 13-LOX activity (Halitschke and Baldwin, 2003). In addition, there is substantial LOX sequence information for solanaceous species in the SGN database (http://sgn.cornell.edu), including that for tomato, potato, eggplant, pepper, and petunia.
Previous work on LOX activity and expression of one member of the LOX gene family in kiwifruit, suggests that LOX activity and expression increases with ethylene-induced fruit ripening (Chen, 1998; Chen et al., 1999; Zhang et al., 2003). The increase in activity appears to precede an increase in endogenous ethylene associated with the climacteric (Zhang et al., 2003). Furthermore, LOX expression was stimulated by low temperature; transcripts increased about 24 h after transfer of kiwifruit to 0 °C (Chen, 1998). LOX may also be involved in aroma and flavour generation during kiwifruit ripening (Bartley and Schwede, 1989; Boyes et al., 1992), as in the other fruit species described above. These suggested roles for LOX in kiwifruit ripening and development of fruit-quality characteristics have prompted a more detailed analysis of the kiwifruit LOX gene family and the response of individual members to post-harvest conditions, including ethylene induction of ripening and low temperature response.
In this study, six LOX genes from a kiwifruit EST (expressed sequence tag) database have been identified and cloned, and expression of these genes has been followed by real-time quantitative PCR (qPCR) during fruit ripening and low temperature storage. Also tissue specificity and expression has been identified during fruit development for the family members and a phylogenetic analysis undertaken to show the relationship of the kiwifruit genes with those of other plant species.
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Materials and methods |
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Plant materials and treatments
Kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) were obtained from the HortResearch orchard in Te Puke, New Zealand. Medium-sized (93–126 g) fruit, free from visible defects or decay, were selected for treatments. Kiwifruit were harvested at commercial maturity, at about 7.89% soluble solids concentration (SSC). There were three ripening regimes in the experiment. One batch of fruit was exposed to ethylene (100 µl l–1, 24 h) and stored at 20 °C with the aim of hastening fruit ripening. A second batch of fruit was packed into boxes with polypropylene liners and placed at 0 °C for 7 d, followed by ripening at 20 °C without ethylene treatment. A third batch of fruit was stored at 20 °C without ethylene treatment. Each ripening regime consisted of three replicates of 100 fruit. At each sampling time, using three replicates of 10 fruit, an equatorial slice (
2.5 cm thick) was taken from each fruit, and skin, seeds, and core tissue excluded. The 10 slices of fruit flesh were combined and frozen in liquid nitrogen and stored at –80 °C until use. A separate experiment was conducted in China using the ‘Bruno’ cultivar of the same species. Although there may be qualifications in using data from a different cultivar, it seems useful to present data on the expression of the LOX gene family in different plant tissues and during fruit development. For this, various plant tissues were sampled from ‘Bruno’ vines growing in Wuyi, Zhejiang, China. Plant material was stored in liquid nitrogen and transported to the laboratory at Zhejiang University, Hangzhou. Samples of root, stem, leaf, petal at anthesis, and fruit at different developmental stages were taken at 20 daily intervals after anthesis over the growing season. Vegetative tissue samples were taken from young tissue, and petals were harvested at full bloom. At each time point, 10 fruit were harvested. For gene expression analysis in various plant tissues and developing fruit, at least three replicate RNA isolations and cDNA syntheses were performed.
Fruit ripening
For each ripening regime, nine jars (1.0 l) each containing a total of 30 fruit were used to measure ethylene production. Ethylene was measured by withdrawing 1 ml of head-space gas from each jar and, using gas chromatography (Hewlett-Packard 5890 Series II), fitted with an FID (Philips PU4500, Unicam) and an alumina F1 column (1.5 mx6 mm). The injector, detector, and oven temperatures were 160, 200, and 130 °C, respectively.
Fruit firmness was measured at two positions 90° to each other at the equator of the fruit with an Effegi FT-327 penetrometer with a 7.9-mm-diameter head, after the removal of a 1-mm-thick slice of skin. SSC was measured after expression of juice from two slices from opposite sides of each fruit on to an Atago N20 hand-held refractometer.
LOX activity and lipid peroxidation
Frozen samples were ground in liquid nitrogen and homogenized using a PolytronTM (Kinematica GmbH) in 50 mM TRIS-HCl buffer (pH 7.0), with 2% (w/v) polyvinylpolypyrrolidone. The homogenate was centrifuged (15 000 g, 30 min), and the supernatant was used for LOX enzyme activity determinations. LOX activity was measured by the method of Boyes et al. (1992), following the oxidation of linoleic acid by measuring absorbance at 234 nm. The stock linoleic acid substrate was prepared according to the method of Axelrod et al. (1981), and the substrate concentration used in the assay was 0.6 mM. One unit of LOX activity was defined as a change in absorbance of 0.001 min–1. The enzyme activity was measured using a SPECTRAmax PLUS384 high-throughput microplate spectrophotometer (Molecular Devices). Protein measurements were performed according to Bradford (1976), using bovine serum albumin as a standard. All samples had three replicates and each measurement was in triplicate.
Lipid peroxidation was assayed by measuring the malondialdehyde (MDA) content in a modification of the method of Rogiers et al. (1998). Fruit tissue (3 g) was extracted in 6 ml of 0.1% (w/v) TCA and blended in a PolytronTM for 20 s. The homogenate was centrifuged and the supernatant diluted 100-fold in 20% (w/v) TCA containing 0.5% (w/v) TBA. After incubating at 95 °C for 30 min, the samples were cooled on ice and centrifuged at 15 000 g for 20 min at 4 °C. Absorbance at 532 nm and 600 nm was recorded. MDA concentration was calculated using a molar extinction coefficient of 156 mM–1 cm–1.
Sequence analysis and bioinformatic methods
Candidate LOX sequences were found in the HortResearch EST database of Actinidia spp. using the BLAST algorithm. In silico analysis was used to identify LOX sequences in the kiwifruit EST database. 3'-RACE was then performed to obtain the 3'-untranslated region (UTR) based on the initial sequences, which offered more sequence information for both CLUSTAL analysis and PCR primer design. Finally, 5'-RACE was used to get full-length cDNA sequences. Alignment for the phylogenetic analysis was calculated with ClustalW1.82 (http://ibi.zju.edu.cn/clustalw/). The protein alignments were then exported to GeneDoc (v2.6.02; http://www.bioon.com/Soft/Class1/Class17/200408/165.html). The phylograms were drawn using Tree-View with default parameters (http://www.bioon.com/Soft/Class1/Class13/200408/127.html).
Oligonucleotide primers
The oligonucleotide primer sets used for real-time qPCR analysis were designed on the basis of the 3'-UTR of individual genes. The primers were designed using Primer Express software (Perkin-Elmer Applied Biosystems) according to the strategies set up by Yokoyama and Nishitani (2001) with little modification. The length of all PCR products ranged from 100 to 150 bp. The gene-specificity of these primer sets was tested using the following four independent procedures: (i) BLAST searches of Actinidia EST databases were performed for each primer to confirm that no other sequence in the kiwifruit genome was similar to any primer; (ii) the specificity of PCR amplification was examined by monitoring the dissociation curves during qPCR reactions using the ABI PRISM 7500 sequence detection system (Applied Biosystems); (iii) individual PCR products were separated on 1.5% agarose gels after staining with ethidium bromide to examine the size; (iv) the PCR products were cloned into pMD18-T vectors (TaKaRa) and sequence analysis confirmed the correct amplicons produced from each pair of primers. The six primer sets were finally used for qPCR as given in Table 1.
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Real-time qPCR analysis
Total RNA was extracted from kiwifruit plants according to the method described by Chang et al. (1993). Four micrograms of total RNA was pretreated with RQ1 DNase I (Promega) to remove contaminating genomic DNA. The concentration of total RNA was measured using a spectrophotometer. First-strand cDNA was synthesized using 2.0 µg of treated total RNA, Superscript III (Invitrogen), and oligo d(T)20 to a total volume of 20 µl. The cDNA was diluted 1:10 with water, and 2 µl of the diluted cDNA was used as a template for qPCR analysis. PCR reactions were performed in a total volume of 20 µl, 250 µM for each primer, and 10 µl of 2x SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7500 sequence detection system (Applied Biosystems). The qPCR programme included a preliminary step of 10 min at 94 °C, followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min. No-template controls for each primer pair were included in each run. Kiwifruit actin was used as an internal control to normalize small differences in template amounts with the forward primer 5'-TGCATGAGCGATCAAGTTTCAAG-3' and reverse primer 5'-TGTCCCATGTCTGGTTGATGACT-3'. At least three different RNA isolations and cDNA syntheses were used as replicates for the qPCR. Expression levels produced by qPCR were expressed as a ratio relative to the fruit harvest time point, which was set to 1.
Analysis of transcripts using end-point PCR analysis
Oligonucleotide primers used for qPCR were also used for end-point PCR analysis. Total RNA was treated with DNase I (Fermentas), and 2.0 µg of treated RNA was used for first-strand cDNA synthesis (Fermentas) following the manufacturer's instructions. PCR amplification was performed with TaKaRa TaqTM (TaKaRa) following the manufacturer's instructions, with the exception that the final concentration of each primer was 250 µM. A 10-fold dilution series of the cDNA was used as a template. The PCR regime consisted of an initial denaturation (94 °C for 5 min), 30 cycles each consisting of 94 °C denaturing (30 s), 60 °C annealing (30 s), and 72 °C elongation (20 s). At the end of the run, a final extension period was included (72 °C for 6 min). Ubiquitin was used in PCR reactions on all RNA templates as a positive control with the forward primer 5'-TGCAGATCTTCGTGAAAACC-3' and reverse primer 5'-CCACCACGGAGACGGAGCAC-3'. PCR products were visualized by electrophoresis using 1.5% agarose gels and staining with ethidium bromide. Electrophoresis results were recorded by photography.
Transient expression assay
Tobacco plants Nicotiana tabacum cv. Samsun were germinated on soil in a glasshouse with daylight extension to 16 h, temperature 25 °C, and PPFD 100 µmol m–2 s–1. Mature preflowering plants consisting of 11 leaves were selected for infiltration with Agrobacterium and maintained in a greenhouse for the duration of the experiments. Agrobacterium tumafaciens transformed with a binary vector pSAK778, a derivative of pART27 (Gleave, 1992), containing a LOX gene, was grown overnight in LB medium supplemented with 100 µg ml–1 spectinomycin and incubated at 28 °C. The overnight mixture was injected with a needle-less syringe into the tobacco leaves as described by Hellens et al. (2005). Tobacco leaves infiltrated with Agrobacterium containing an empty vector were used as negative controls.
The injected leaves were observed and sampled 8 d after injection. Chlorophyll fluorescence was measured on leaf tissues using a Mini-PAM (Waltz). Chlorophyll content was measured on the same leaves used for chlorophyll fluorescence measurements. Leaf discs 6 mm in diameter were used for chlorophyll content determination by the method of Tanaka et al. (1999). At least three replicates were performed.
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Results |
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Sequence analysis of the LOX gene family
From the HortResearch Actinidia EST database, six expressed gene sequences were identified as putative members of the LOX gene family. The six sequences were designated as AdLox1–6 (Genbank accession numbers: DQ497792, DQ497797, DQ497795, DQ497793, DQ497796, DQ497794, respectively). Phylogenetic analysis of translated amino acid sequences from different members of the known plant LOX families generated clusters which could be structured into the two main groups, 9-LOX and 13-LOX (Fig. 1A). AdLox2 and AdLox5 were clustered in the 9-LOX class together with the tomato LOX genes TomLoxA, TomLoxB, and TomLoxE and the potato gene PotLX1. The second group, 13-LOX, contains AdLox1, AdLox3, AdLox4, and AdLox6, together with the tomato genes, TomLoxC and TomLoxD, and potato genes StLoxH1 and StLoxH3. Five of the six kiwifruit LOX genes (excluding AdLox6 because of sequence truncation) display a motif of 38 amino acid residues conserved among plant and animal LOX sequences, which is involved in binding the iron essential for enzyme activity (Fig. 1b).
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Tissue specificity and temporal expression pattern in fruit development
End-point PCR was used to examine the expression pattern of the six LOX genes in root, stem, leaf, and petal tissues, and during fruit development in the ‘Bruno’ cultivar (Fig. 2). AdLox1 and AdLox2 had similar expression patterns in kiwifruit plant tissues, but AdLox1 appeared to have higher transcript abundance in petal tissue. Transcripts of AdLox3 and AdLox5 were barely detectable in the various tissues, whilst AdLox4 was expressed mainly in stem, leaf, and petal tissues. AdLox6 followed the expression pattern of AdLox4 but at a lower abundance. During fruit development, young fruit at 20 d after anthesis (daa) had relatively high expression levels of LOX genes, similar to those observed in stem tissues. AdLox1, AdLox2, and AdLox6 exhibited similar expression patterns throughout fruit development, with transcript abundance decreasing from 20 daa to 80 daa and increasing again at about 100 daa (Fig. 2, column 9). AdLox4 showed a relatively constant expression level during fruit development, and produced the strongest bands among the LOX gene family. No visible PCR band from AdLox5 was detected during fruit development.
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Fruit ripening and LOX gene expression
Fruit held at 20 °C for 216 h softened only to a small extent (Fig. 3A). When fruit were treated with ethylene (100 µl l–1, 24 h, 20 °C), the fruit softened rapidly from 67.7 N at harvest to <9.8 N at about 72 h after harvest (Fig. 3B). Fruit held at 0 °C maintained firmness, and did not significantly soften after transfer to 20 °C for 72 h (Fig. 3C). Another indicator of ripening, an increase in SSC, only occurred in fruit ripening after exposure to ethylene or when fruit were rewarmed after storage at 0 °C (Fig. 3).
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After exposure of fruit to ethylene at 20 °C, ethylene production was initiated at about 72 h after harvest, peaking at about 144 h, and then declining as fruit became overripe (Fig. 3D). By contrast, control fruit did not produce ethylene over the 216 h storage period (data not shown). Table 2 shows data for ethylene production, firmness, and SSC for fruit sampled 288 h after harvest and held at 20 °C. The fruit were separated according to their ethylene production rate and firmness into three stages: preclimacteric (P), climacteric (C), and post-climacteric (PC).
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qPCR was used to monitor changes in transcript levels of the six LOX genes during kiwifruit ripening at 20 °C. Gene-specific primers were designed to the divergent 3'-UTR of each gene (Table 1). The primers showed no cross-amplification to other members of the family (data not shown). End-point PCR results showed that AdLox2 and AdLox4 produced relatively strong bands during the earlier stages of kiwifruit ripening (Fig. 4A), and qPCR showed that AdLox1 and AdLox5 had the greatest increase in mRNA abundance compared with the harvest-time sample (Fig. 4C, D).
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For the fruit stored at 20 °C without ethylene treatment, transcripts of AdLox1 and AdLox5 increased with kiwifruit ripening (Fig. 4A, C). By contrast, expression of AdLox2, AdLox3, and AdLox4 decreased when kiwifruit ripening progressed to the climacteric stage. End-point PCR results matched the expression pattern produced by qPCR (Fig. 4A, B).
To determine whether ethylene treatment could influence kiwifruit LOX gene expression, fruit were exposed to ethylene treatment (100 µl l–1, 24 h, 20 °C; Fig. 4B, D). The relative qPCR results showed that transcript abundance of AdLox2, AdLox3, and AdLox4 decreased about 5-, 20-, and 10-fold, respectively, within 24 h of exposure to ethylene. The levels of these transcripts tended to increase immediately after exogenous ethylene treatment, and then decrease again as fruit internal ethylene accumulated (Fig. 4D). However, expression of AdLox1 and AdLox5 was stimulated by ethylene treatment within 24 h. Expression of AdLox6 appeared to be slightly induced when fruit became overripe.
Expression patterns of LOX genes in response to low temperature
The expression profiles were examined in kiwifruit stored at 0 °C for 168 h. Transcripts of AdLox1 were strongly induced by the low-temperature treatment, peaking at about 72 h after harvest, and this expression declined during rewarming (Fig. 5A, B). AdLox5 and AdLox6 had similar expression patterns to those of AdLox1 during the low-temperature treatment. However, AdLox2, AdLox3, and AdLox4 showed no significant changes in transcript levels at 0 °C (Fig. 5A, B). No visible band from AdLox5 was detected by end-point PCR at any time in the low-temperature experiment (Fig. 5A). In general, relative transcript abundance declined or did not change during the warming period.
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LOX enzyme activity and MDA contents
For the fruit at 20 °C, LOX activity was maintained at a constant level from harvest to 144 h after harvest with an average activity of 1.71±0.02 U mg–1 protein. Activity increased with ripening, with an activity of 2.25±0.24 U mg–1 protein in the pre-climacteric stage, 2.72±0.28 U mg–1 protein in the climacteric stage, and 3.02±0.04 U mg–1 protein in the post-climacteric stage. Exogenous ethylene treatment induced LOX activity at 20 °C (Fig. 6A). During ethylene treatment, there was an increase in total LOX enzyme activity. After the ethylene treatment, LOX activity returned to harvest levels within 24 h and subsequently increased again as fruit ripened further. MDA accumulated gradually with fruit ripening (Fig. 6C). When the fruit were exposed to low temperature, LOX activity was slightly stimulated at 72 h after harvest (Fig. 6B). MDA contents remained constant during the low-temperature treatment (Fig. 6D).
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Transient expression of the LOX genes in tobacco leaves
To study further the role of the LOX genes in plant senescence, transient expression of two LOX genes in Nicotiana tobacum leaves, one ethylene responsive (AdLox1) and one not (AdLox2), were carried out. Over-expression of AdLox1 in tobacco leaves significantly accelerated chlorophyll degradation (P <0.05) and decreased chlorophyll fluorescence (P <0.05) (Table 3). However, over-expression of AdLox2 had no effect on senescence as measured by these two properties.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Six kiwifruit LOX genes which are differentially expressed in the developing and ripening fruit were identified and studied. According to the classification of plant genes belonging to the LOX family (Feussner and Wasternack, 2002), AdLox2 and AdLox5 are grouped into the 9-LOX family, and AdLox1, AdLox3, AdLox4, and AdLox6 are proposed to have 13-LOX activity. It is the latter which have been suggested to take part in the conversion of lipid hydroperoxides to jasmonic acid cyclic precursors and volatile compounds such as aldehydes and alcohols (Porta and Rocha-Sosa, 2002). There is a characteristic shared N-terminal extension among members of the 13-LOX group, which is proposed as a chloroplast transit peptide, but no similar sequence is found in the 9-LOX genes (Royo et al., 1996; Feussner and Wasternack, 2002). Within the 13-LOX group, Arabidopsis AtLox2 (Bell et al., 1995), tomato TomLoxC and TomLoxD (Heitz et al., 1997), and rice LOX RCI-1 (Schaffrath et al., 2000) have been identified as chloroplastic isoforms. The LOX gene family in kiwifruit also resembles that in potato described by Royo et al. (1996), with three classes identified on the basis of sequence similarity. The Lox1 class includes the kiwifruit genes AdLox2 and AdLox5, while the Lox2 class is represented by AdLox4 and AdLox6 and Lox3 by AdLox1 and AdLox3. Both the Lox2 and Lox3 groups in potato possess chloroplast transit peptides and 13-LOX activity (Royo et al., 1996).
The present sequence data suggest that AdLox1, AdLox3, AdLox4, and AdLox6 might be chloroplastic, whereas AdLox2 and AdLox5 may have a cytoplasmic location. This is supported by classification of the five tomato LOX genes. TomloxC and TomloxD contain putative chloroplast transit peptides with 13-LOX activity and cluster phylogenetically with AdLox1, 3, 4, and 6, and TomloxC has been identified in the chloroplast (Chen et al., 2004). TomloxA, B, and E show high sequence homology and were classified in the 9-LOX group (Ferrie et al., 1994; Heitz et al., 1997; Chen et al., 2004), clustering with AdLox2 and AdLox5.
The six LOX genes had different expression levels in kiwifruit tissues, and are regulated differentially during fruit development and post-harvest ripening. Expression was variable in vegetative tissue and, based on PCR results, AdLox1, AdLox2, and AdLox4 had relatively higher transcript abundance than the other three isoforms, with the most consistent expression being in the leaf tissue. The present data show that all six LOX genes are expressed in the fruit, and analysis of the five tomato genes has also shown that all are expressed in the fruit under varying conditions (Ferrie et al., 1994; Heitz et al., 1997; Griffiths et al., 1999; Chen et al., 2004). In Arabidopsis, AtLox1 is expressed in various organs, with highest levels in young seedlings followed by root and leaf tissues, while AtLox2 transcripts have been detected in leaves, flowers, and during early seedling development (Melan et al., 1993; Bell et al., 1995). These data again confirm the widespread distribution of the gene family within plant organs and tissues of a species.
Most of the LOX genes showed relatively high abundance in young kiwifruit, with levels declining as the fruit developed. This matches published data on LOX enzyme activity. Boyes et al. (1992) showed that LOX activity declined throughout kiwifruit development from petal fall to fruit maturity. An important role for LOX in early tissue and organ development is supported by data from other species. Suppression of potato PotLX1 reduced tuber yield, decreased average tuber size, and caused a disruption in tuber formation (Kolomiets et al., 2001). In almond, a LOX gene involved in early seed development was characterized with maximal transcript levels and enzyme activity about 30 d after flowering and then declining (Mital et al., 2001), and a LOX gene from hazelnut has been associated with early seed development (Santinol et al., 2003). Transcripts of TomloxA declined during later stages of tomato fruit development and factors influencing development regulated its expression (Griffiths et al., 1999). These results suggest a greater requirement for LOX activity during fruit cell division and enlargement in these early developmental periods.
There were considerable differences amongst the genes in expression patterns during fruit ripening, and they tend to form two groups. AdLox2, AdLox3, AdLox4, and AdLox6 transcript levels generally declined with ripening and had little relationship with LOX activity and MDA content. This group exhibits a pattern similar to that of TomLoxA in ripening tomato fruit, which was down-regulated by ethylene during tomato fruit ripening (Griffiths et al., 1999). AdLox1 and AdLox5, which initially had negligible transcript levels, did show a response to external ethylene at the pre- and climacteric stages. This response seems related to the increases in LOX activity and the MDA contents after ethylene treatment. Previous northern analysis using a probe with close sequence homology to AdLox5 also showed transcript levels increasing with kiwifruit ripening and in response to external ethylene application (KS Chen, SL Zhang, GS Ross, unpublished data). The ethylene responsiveness of AdLox1 and AdLox5 is similar to that of TomLoxB (Ferrie et al., 1994; Heitz et al., 1997; Griffiths et al., 1999), and the present data suggest that these latter genes are those most involved in LOX-based metabolism in kiwifruit ripening.
The involvement of LOX genes in ethylene production is likely to be complex. The increase in AdLox1 and AdLox5 transcripts clearly followed the increases in autocatalytic ethylene production resulting from external ethylene treatment. These genes may be responding to disruption of cellular membranes (perhaps in chloroplasts) during fruit ripening and senescence. It is possible that release of linolenic and linoleic acids from membranes induces LOX gene expression; stimulation of gene levels and enzyme activity would then be a consequence of such earlier senescence events. Provision of LOX substrates to tomato fruit slices resulted in increased ethylene production (Sheng et al., 2000), confirming suggestions that LOX or its products, particularly reactive oxygen species, may stimulate ethylene synthesis. Thus LOX may be involved in feed-back loops of ethylene stimulation and production.
Transient expression of AdLox1 in tobacco leaves significantly lowered total chlorophyll contents and accelerated tissue senescence (Table 3). These data are preliminary since LOX activity and transgene levels were not measured. However, it is notable that it was the ethylene-responsive AdLox1 and not the insensitive AdLox2 that induced chlorophyll breakdown. LOX has been shown to be involved in co-oxidation of chlorophyll in French bean pericarp (Abbas et al., 1989) and cucumber leaves (Su et al., 1996). The present data suggest that endogenous substrates were available for stimulation of LOX activity, triggering further chlorophyll-based senescence changes. A more detailed study would show whether this involves ethylene or an autocatalytic loop in LOX expression and activity. A possible ethylene involvement is suggested by the differences in response to the two genes.
It is notable that AdLox1 and 5, which were ethylene-sensitive, were also particularly responsive to low temperature. There was no detectable ethylene increase with low temperature, and when the fruit were transferred to 20 °C, transcript levels declined. Although there was some fruit softening over this period, there was no substantial ethylene increase either. AdLox6, which also responded to low temperature, showed very little ripening or ethylene responsive behaviour. This suggests that there are specific low-temperature events triggering LOX gene transcription. However, it was not possible to find any marked response in enzyme activity to low temperature or in MDA contents over the time period studied. Although the transfer of fruit to 20 °C was followed by a reduction in transcript level (Fig. 5), it is possible that the biochemical consequence of up-regulation would appear at later post-harvest stages of fruit ripening. Xu et al. (2003) showed that LOX activity increased in kiwifruit at 20 °C after low-temperature storage, but only after 20 d at 0 °C. In a different Actinidia species (A. chinensis) it has been found that AdLox1 and 6 were substantially elevated in fruit tissues damaged by low temperature breakdown after long-term storage (B Zhang, KS Chen, JH Bowen, IB Ferguson, unpublished data). Low temperature resulted in increased LOX activity in guava fruit (González-Aguilar et al., 2004), and LOX-mediated lipid peroxidation has been regarded as a source of active oxygen species, particularly under stress conditions (Blokhina et al., 2003). In vegetative tissues such as roots, increased LOX activity and up-regulation of transcripts in response to low temperature have been associated with chilling tolerance (Lee et al., 2005).
An important role of plant LOX enzymes is an involvement in aroma and flavour generation during fruit ripening, this being very sensitive to the stage of ripening. Fatty acid-derived C6 aldehydes, which are derived from a LOX-mediated pathway, give the fruit a green aroma character. These decrease with post-harvest fruit softening while the ester levels increase (Young and Paterson, 1985; Bartley and Schwede, 1989). Kiwifruit with 8% SSC at harvest (similar to fruit used in the present study) showed a marked decline in aldehydes from an early (fruit firmness 7.9–10.8 N) to late (2.9–3.9 N) stage of ripening (Young and Paterson, 1985), and such a decline may be a reflection of LOX activity. In the present study, the ethylene climacteric rise began in fruit with a firmness of 5.8–7.4 N, at the time when there was a decrease in expression levels of AdLox2, AdLox3, AdLox4, and AdLox6. In tomato fruit, the specific depletion of TomLoxC in transgenic lines resulted in a significant reduction in the levels of C6 aldehydes, and this isoform has been identified as the LOX gene family member involved in tomato fruit flavour formation (Chen et al., 2004). AdLox3, AdLox4, and AdLox6, which are classed in the 13-LOX group might possess chloroplast transit peptide sequences similar to those in the TomLoxC, and thus be possible candidates for regulation of the synthesis of kiwifruit volatile compounds.
In summary, the present analysis of the LOX family in kiwifruit has identified members that are specifically responsive to ethylene and low temperature, and LOX family members that can be similarly classed with tomato and potato genes, and their putative functions. This information suggests that specific LOX genes in kiwifruit may be involved in fruit ripening, with consequences for flavour or aroma development, and in response to low temperature.
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We wish to thank Andrew Gleave for his work developing the binary vector used in the transient expression studies, and Sean Bulley, Rong Mei Wu, and Karen Bolitho for guidance and assistance. The work was supported by the National Natural Science Foundation of China (30571284), The University Doctoral Foundation of China (20040335022) and the III project (B06014 [GenBank] ), and a HortResearch NSOF programme, as part of a co-operative programme between The Horticulture and Food Research Institute of New Zealand and Zhejiang University.
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