JXB Advance Access originally published online on January 5, 2006
Journal of Experimental Botany 2006 57(3):633-643; doi:10.1093/jxb/erj048
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RESEARCH PAPER |
Cloning and characterization of two 9-cis-epoxycarotenoid dioxygenase genes, differentially regulated during fruit maturation and under stress conditions, from orange (Citrus sinensis L. Osbeck)
Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Apartado de Correos 73, 46100 Burjassot, Valencia, Spain
* To whom correspondence should be addressed. E-mail: lzacarias{at}iata.csic.es
Received 28 July 2005; Accepted 7 November 2005
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Abstract |
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There is now biochemical and genetic evidence that oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. The peel of Citrus fruit accumulates large amounts of ABA during maturation. To understand the regulation of ABA biosynthesis in Citrus, two full-length cDNAs (CsNCED1 and CsNCED2) encoding NCEDs were isolated and characterized from the epicarp of orange fruits (Citrus sinensis L. Osbeck). Expression of the CsNCED1 gene increased in the epicarp during natural and ethylene-induced fruit maturation, and in water-stressed leaves, in a pattern consistent with the accumulation of ABA. The second gene, CsNCED2, was not detected in dehydrated leaves and, in fruits, exhibited a differential expression to that of CsNCED1. Taken together, these results suggests that CsNCED1 is likely to play a primary role in the biosynthesis of ABA in both leaves and fruits, while CsNCED2 appears to play a subsidiary role restricted to chromoplast-containing tissue. Furthermore, analysis of 9-cis-violaxanthin and 9'-cis-neoxanthin, as the two possible substrates for NCEDs, revealed that the former was the main carotenoid in the outer coloured part of the fruit peel as the fruit ripened or after ethylene treatment, whereas 9'-cis-neoxanthin was not detected or was in trace amounts. By contrast, turgid and dehydrated leaves contained 9'-cis-neoxanthin but 9-cis-violaxanthin was absent. Based on these results, it is suggested that 9-cis-violaxanthin may be the predominant substrate for NCED in the peel of Citrus fruits, whereas 9'-cis-neoxanthin would be the precursor of ABA in photosynthetic tissues.
Key words: Abscisic acid, Citrus sinensis, 9-cis-epoxycarotenoid dioxygenase, epoxycarotenoid, gene expression, orange fruit
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The plant hormone abscisic acid (ABA) plays a crucial role in the adaptation of plants to different environmental stresses and in several physiological processes such as seed maturation and dormancy and fruit development, or senescence (Zeevaart and Creelman, 1988
). Thus, environmental and developmental signals may operate in the regulation of ABA biosynthesis in plant tissues. In higher plants, ABA is derived from C40-cis-epoxycarotenoids, either 9'-cis-neoxanthin or 9-cis-violaxanthin or both, which are cleaved by the 9-cis-epoxycarotenoid dioxygenase (NCED) to produce xanthoxin, the direct C15 precursor of ABA (Cutler and Krochko, 1999; Liotenberg et al., 1999; Taylor et al., 2000). An important advance in the understanding of the regulation of ABA biosynthesis was the molecular characterization of the gene responsible for the ABA deficiency in the Vp14 mutant of maize, which was the first NCED gene to be cloned (Tan et al., 1997). Subsequently, the ABA-deficient tomato mutant notabilis (not) was also shown to be defective in the Vp14-related gene (LeNCED1) (Burbidge et al., 1999). Recombinant NCED proteins have been shown to display in vitro dioxygenase activity cleaving specifically 9-cis-xanthophylls to form cis-xanthoxin (Schwartz et al., 1997; Chernys and Zeevaart, 2000; Schwartz et al., 2003a). Moreover, NCED cDNAs have been cloned and characterized in different plants, such as bean (Qin and Zeevaart 1999), cowpea (Iuchi et al., 2000), avocado (Chernys and Zeevaart, 2000), and Arabidopsis (Iuchi et al., 2001; Tan et al., 2003), and in all plant species NCEDs comprise a small multigene family.
Regulation of ABA biosynthesis has been mainly studied in vegetative tissues of several plant species in response to stress conditions. In water-stressed leaves, accumulation of ABA was well correlated with an increased expression of the NCED gene (for reviews, see Xiong and Zhu, 2003; Schwartz et al., 2003b; Nambara and Marion-Poll, 2005) and accumulation of the corresponding NCED protein (Qin and Zeevaart, 1999). Moreover, transgenic plants overexpressing the NCED gene accumulated large amounts of ABA and were more resistant to drought stress (Thompson et al., 2000; Iuchi et al., 2001; Qin and Zeevaart, 2002). Collectively, these results provide strong evidence for the regulatory role of NCED in ABA biosynthesis in leaves under stress conditions. However, the regulation of ABA biosynthesis in other tissues and developmental processes is less well known. Recently, an exhaustive expression analysis of the five NCED genes from Arabidopsis revealed that AtNCED3 is the major stress-induced isoform in leaves, while developmental control of ABA synthesis involves localized expression patterns of the five AtNCED genes (Tan et al., 2003). As far as is known, regulation of NCED gene expression in fruits has only been reported in avocado (Persea americana Mill cv. Lula) (Chernys and Zeevaart, 2000). Two different NCED genes, PaNCED1 and PaNCED3, were strongly induced during fruit ripening but only PaNCED1 was induced in leaves subjected to water stress.
Fruit ripening is a complex developmental process involving a number of physiological and biochemical changes which are thought to be under hormonal, nutritional, and environmental control (Giovannoni, 2004). Coloration of the peel is one of the most remarkable transformations accompanying fruit maturation. During natural maturation, the peel of Citrus fruits accumulates large amounts of coloured oxygenated carotenoids, mainly 9-cis-violaxanthin, and also of ABA (Goldschmidt et al., 1973; Gross, 1987; Rodrigo et al., 2003). Moreover, cell-free systems prepared from flavedo (the outer coloured part of the fruit peel) of mature orange fruits have ABA biosynthetic activity (Cowan and Richardson, 1997). Citrus fruits are non-climacteric and evolve minute amounts of ethylene during maturation, but exogenous ethylene treatment increases carotenoid content and also stimulates ABA synthesis in the fruit epicarp (Goldschmidt et al., 1973; Lafuente et al., 1997). Moreover, analysis of the maturation process in an ABA-deficient mutant of oranges revealed a role for ABA in the regulation of fruit coloration, since mutant fruit displayed a slow rate of fruit degreening (Rodrigo et al., 2003) and altered sensitivity to ethylene-induced fruit coloration (Alferez and Zacarias, 1999). Thus, the co-ordinated increase in carotenoids and ABA in the peel of Citrus fruits suggests that the cis-xanthophyll content is not a limiting factor for ABA synthesis but how their cleavage is regulated is currently unknown. Therefore, the peel of Citrus provides an excellent system to investigate the role of NCEDs in the regulation of ABA biosynthesis in fruit. To address this objective, in this study two NCEDs genes, named CsNCED1 and CsNCED2, have been isolated from the peel of orange (Citrus sinensis L. Osbeck). Expression of the CsNCED1 gene in flavedo during natural and ethylene-induced fruit maturation and water-stressed leaves was consistent with the accumulation of ABA but the CsNCED2 gene displayed a different and tissue-specific expression. The relative abundance of 9-cis-epoxycarotenoids in fruit and leaves was analysed and results suggest that 9-cis-violaxanthin may be the in vivo precursor of ABA in coloured fruit peel.
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Materials and methods |
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Plant material and treatments
Fruits and leaves from Citrus sinensis L. Osbeck (cv. Navelate) were collected from adult trees grafted on to Citrange carrizo rootstock. Trees were grown at The Citrus Germplasm Bank (Instituto Valenciano de Investigaciones Agrarias, IVIA, Moncada, Valencia, Spain). Fruits at different developmental and maturation stages were periodically harvested from September to February. Peel colour was measured at three locations around the equatorial plane of the fruit using a Minolta CR-330 colorimeter and was expressed as the a/b Hunter ratio (Stewart and Whitaker, 1972
; Rodrigo et al., 2003). At each harvest time, 30 fruits were used and the flavedo tissue (the outer coloured part of the fruit peel) was excised. Flavedo and leaves were frozen in liquid nitrogen, ground to a fine powder, and stored at –80 °C until analysis. Degreening experiments were conducted on mature-green fruits with an a/b ratio of –0.60±0.02. Fruits were exposed to an ethylene-free atmosphere or 10 µl l–1 ethylene for 3, 6, and 14 h and 1, 3, and 7 d at 20 °C in 25 l tanks. To avoid excess of respiratory CO2, lime powder was introduced in the tanks and fruits were ventilated every day. After each incubation period, the colour of the fruit was monitored and flavedo tissue excised from the whole fruit. Ten fruits were used for each treatment and incubation time.
To evaluate the effect of water stress on fruit ABA content and NCED gene expression, full-coloured fruits with an a/b ratio of 0.65±0.05 were used. Fruits were divided into two groups of 30 fruits. The first group was incubated in a storage room for 3 d at 20 °C under high relative humidity (95% RH, control fruits) and the second group was stored at the same temperature for 3 d under low relative humidity (45% RH, dehydrated fruits).
Water stress experiments in leaves were carried out with detached mature leaves. Leaves were collected, weighed, and allowed to dehydrate by placing them on filter paper under continuous light at room temperature. Control non-stressed leaves were kept with their petioles in distilled water and maintained under the same conditions as dehydrated leaves. The weight of the leaves was monitored every hour and tissue was collected after 2, 4, and 6 h. Four replicate samples of four leaves were used for each time period.
ABA analysis
Quantification of ABA in flavedo and leaf tissue was performed by indirect enzyme-linked immunosorbent assay as reported previously (Zacarías et al., 1995; Lafuente et al., 1997).
Carotenoid analysis
Carotenoids from tissues were extracted as previously described by Rodrigo et al. (2003). Briefly, freeze ground material (500 mg) of flavedo or (250 mg) of leaves was extracted with a mixture of methanol and 50 mM TRIS-HCl buffer (pH 7.5) containing 1 M NaCl, and partitioned against chloroform until the colour was removed from the plant material. Pooled organic phases were dried under vacuum and saponified overnight using a KOH methanolic solution. The carotenoids were subsequently re-extracted with diethyl ether. An aliquot of the ethereal extract was used to quantify the total carotenoid content. Absorption spectra of saponified extracts were recorded with a diode array spectrophotometer (model 8452A, Hewlett Packard, Germany). The maximum absorbance peaks were registered and the total carotenoid content calculated by measuring the absorbance at 450 nm according to Davies (1976), using the extinction coefficient of ß-carotene, E1%=2500.
The extracts were reduced to dryness by rotary evaporation and prepared for HPLC analysis by dissolving them in MeOH:acetone (2:1 v:v). Chromatography was carried out with a Waters liquid chromatography system, equipped with a 600E pump and a 996 photodiode array detector, and Millenium Chromatography Manager (version 2.0). Carotenoid pigments were separated by HPLC using a C30 carotenoid column (250x4.6 mm, 5 µm) coupled to a C30 guard column (20x4.0 mm, 5 µm) (YMC Europe GMBH, Germany) with a ternary gradient elution of MeOH, water, and methyl tert-butyl ether (Rouseff et al., 1996; Rodrigo et al., 2003). Carotenoids were identified by their retention time, absorption, and fine spectra (Rouseff et al., 1996; Britton, 1998). The 9'-cis-neoxanthin and 9-cis-violaxanthin peaks were integrated at their individual maximum wavelengths, and their content was calculated using a calibration curve of lutein standard (Sigma-Aldrich) because pure standards of 9'-cis-neoxanthin and 9-cis-violaxanthin were unavailable and absorption coefficients are very similar (Britton, 1998). The limit of detection was estimated to be 7 ng.
Samples were extracted at least twice and each analytical determination was replicated. All operations were carried out on ice under dim light to prevent photodegradation, isomerizations, and structural changes to the carotenoids.
Isolation of NCED genes from Citrus sinensis and sequence analysis
First-strand cDNAs were synthesized by reverse transcription (RT) from 1 µg of total RNA from flavedo tissue of orange fruits with an a/b ratio of 0.24 (cDNA-1) or mature green fruits treated for 1 d with ethylene (cDNA-2), using 200 units of Superscript II Reverse Transcriptase (Gibco BRL, Karlsruhe, Germany) and 500 ng oligo-dT primer. The cDNA-1 was used as the template for PCR using the degenerated primers MJ19 and MJ20 (Table 1) for the amplification of CsNCED1. cDNA-2 was used with degenerated primers MJ41 and MJ42 (Table 1) for the amplification of CsNCED2. These degenerated primers were designed considering conserved sequences from plant NCEDs or related carotenoid cleavage dioxygenases (CCDs) available in the database. RT conditions were: 70 °C for 10 min, followed by 42 °C for 1 h, followed by 15 min at 70 °C. PCR amplification was performed as follows: 5 min 94 °C, 35 cycles of 94 °C 30 s, 52 °C for 1 min and 72 °C for 1 min, and 72 °C for 10 min. Sequence information of PCR products permitted rapid amplification of cDNA ends by RACE-PCR (rapid amplification of cDNAs ends by polymerase chain reaction) strategy, using the 5'/3' RACE Kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. The 5' end of the CsNCED1 was amplified using the primers MJ37SP1, MJ38SP2, and MJ39SP3, and the 3' end with MJ40SP5 (Table 1). The 5' end of the CsNCED2 was amplified using the primers MJ45SP1, MJ46SP2, MJ47SP3, MJ53SP1, MJ54SP2, and MJ55SP3, and the 3' end with primer MJ48SP5 (Table 1). The PCR products were ligated into pGEM-T Easy vector (Promega, Madison, USA) and sequenced.
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Prediction of transit peptide of CsNCED1 and CsNCED2 proteins was carried out using ChloroP (Emanuelsson et al., 1999
) and PSORT (Nakai and Kanehisa, 1992).
Sequences encoding plant NCEDs were recovered by homology search in sequence databanks using the program BLAST (Altschul et al., 1990) at the NCBI (Bethesda, USA) and only full-length amino acid sequences were used for phylogenetic analysis. The phylogenetic tree was generated using the Neighbor–Joining method (Saitou and Nei, 1987) included in the ClustalW program (Thompson et al., 1994), and bootstrap re-sampling analysis (1000 replicates) was performed.
Probe synthesis and labelling
A CsNCED1 fragment of 193 bp and a CsNCED2 fragment of 137 bp were amplified with specific-primers (MJ51 and MJ52 for CsNCED1, and MJ48SP5 and MJ45SP1 for CsNCED2) and used as probes. Both CsNCED1 and CsNCED2 fragments were labelled with [
-32P]dATP by linear PCR amplification with MJ52 and MJ45SP1 primers, respectively, using the Strip-EZ PCR Kit (Ambion, Huntingdon, UK) following the manufacturer's instructions. The sequences of primers used are given in Table 1.
Northern, Southern, and dot-blot analysis
The plant material used for total RNA isolation was the same as that used for ABA and carotenoid analysis. Total RNA was isolated from the flavedo and leaves as previously described (Rodrigo et al., 2004). Northern blot analysis was carried out by electrophoresis of denatured total RNA (10 µg) in 1% (w/v) agarose-formaldehyde/MOPS [3-(N-morpholino)-propanesulphonic acid] gels and blotted onto nylon membranes (Hybond-N, Amersham-Bioscience, Spain) essentially as described by Sambrook et al. (1989). Loading and transferring equal amounts of RNA were checked by staining with ethidium bromide and methylene blue, respectively.
DNA was extracted from young leaves as described by Taylor et al. (1993). Samples of genomic DNA (10 µg) were digested with selected restriction enzymes, electrophoresed on 1% (w/v) agarose gel, and transferred as above.
Dot-blots were prepared by applying, directly to a nylon membrane, 4 ng of empty pGEM-T Easy vector, 4 ng of pGEM-T harbouring the CsNCED1 or CsNCED2 probe fragment and 4 ng of the CsNCED1 or CsNCED2 full-length cDNAs. Nucleic acids were cross-linked to membrane using a Stratalinker UV Crosslinker (Stratagene, La Jolla, CA, USA).
Northern, Southern and dot-blots were prehybridized for 3 h and hybridized overnight at 42 °C to the CsNCED1 and CsNCED2 probes with ULTRAhyb hybridization buffer (Ambion, Huntingdon, UK) following the manufacturer's instructions. Southern-blot (low-stringency) was washed for 40 min with 2x SSC (150 mM NaCl and 15 mM trisodium citrate, pH 7.0) and 0.1% (w/v) SDS (sodium dodecyl sulphate) at 42 °C and 30 min with 2x SSC and 0.1% (w/v) SDS at 50 °C. Southern (high-stringency), northern, and dot-blots were washed twice for 10 min with 2x SSC and 0.1% (w/v) SDS at 42 °C, once for 15 min with 0.1x SSC, 0.1% (w/v) SDS at 42 °C, and once for 15 min with 0.1x SSC, 0.1% (w/v) SDS at 47 °C. Membranes were stripped using the Strip-EZ PCR Kit (Ambion, Huntingdon, UK) following the manufacturer's instructions. Membranes were then exposed to Kodak X-Omat SX film for the periods of time indicated.
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Results |
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Isolation and sequence analysis of two NCED genes from Citrus sinensis
To isolate NCED genes from orange fruits, a reverse-transcription–polymerase chain reaction (RT-PCR)-based strategy was adopted. Using cDNA of flavedo from coloured fruits and degenerated primers designed in conserved regions of NCEDs, a 580 bp fragment was amplified. The DNA sequence showed high homology to other plant NCEDs and was designated as CsNCED1. Using cDNA of flavedo from fruits treated with ethylene for 1 d and a second pair of degenerated primers, a 170 bp fragment was obtained, and the sequencing revealed that corresponded to a second Citrus NCED gene which was named CsNCED2. The full-length sequences of CsNCED1 and CsNCED2 were obtained by 5' and 3' RACE-PCR strategy (GenBank accession numbers DQ028471 and DQ028472, respectively). The CsNCED1 sequence contained an open reading frame of 1818 bp, with a 5' leader region of 169 bp and a 3'-untranslated region of 264 bp, and the CsNCED2 gene contained an open reading frame of 1827 bp with 3'- and 5'-untranslated regions of 97 and 389 bp, respectively. CsNCED1 and CsNCED2 encoded predicted proteins of 606 and 609 amino acids, respectively. Similar to other plant NCEDs, CsNCED1 and CsNCED2 showed a putative chloroplast signal peptide at the amino terminus and immediately downstream a predicted amphipathic
-helix involved in protein membrane binding (Fig. 1; Tan et al., 2001, 2003). Both CsNCEDs contain four conserved His residues required for iron sequestration and therefore important for catalytic activity (Fig. 1; Schwartz et al., 1997; Tan et al., 1997). The CsNCED1 and CsNCED2 sequences are 70% identical and most of the variability occurred in the putative plastid-targeting transit peptide (Fig. 1). The CsNCED1 and CsNCED2 proteins are highly homologous to other plant NCED (77–69% identity) and are more distantly related to other CCDs (30–40% identity) not involved in ABA biosynthesis (Giuliano et al., 2003). Alignment of the CsNCED1 and CsNCED2, 13 additional NCEDs, and seven plant CCDs reported to be expressed in fruits was performed using the ClustalW program, generating a phylogenetic tree (Fig. 2). The phylogenetic tree showed two different classes, one including NCED proteins involved in ABA biosynthesis, and a clearly distinct class including CCDs. Within the NCED cluster, CsNCED1 is more closely related to NCED isoforms which play a major role in stress-induced ABA biosynthesis in vegetative tissue such as Arabidopsis NCED3 or tomato NCED1 (Tan et al., 2003; Thompson et al., 2004), while CsNCED2 is grouped with Pisum sativum NCED3, which has not yet been characterized, and Arabidopsis NCED2 and NCED5, which are mainly expressed in non-photosynthetic tissues (Tan et al., 2003).
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Dot- and Southern-blot analysis of CsNCEDs
Due to the high homology of the two Citrus NCED genes, the specificity of the corresponding probes was first checked by dot-blot analysis. Probes of CsNCED1 and CsNCED2 did not cross-hybridize with full-length cDNAs of CsNCED2 and CsNCED1, respectively, under the hybridization and washing conditions used (data not shown). Therefore, these probes and conditions were further used in Southern (high-stringency) and northern blot analysis.
Southern blot analysis was performed to estimate the complexity of the Citrus NCED gene family and the gene copy number of CsNCED1 and CsNCED2. A DNA-blot of EcoRV, HindIII, and SacI DNA-digested samples was allowed to hybridize with the CsNCED1 probe and several bands were detected under low stringency conditions (Fig. 3), indicating that there is a small NCED gene family in C. sinensis. Under high stringency conditions, the CsNCED1 probe showed one hybridized band for EcoRV and HindIII and two bands for SacI since this enzyme has an internal restriction site. For the CsNCED2 probe only one hybridized band was obtained for the three restriction enzymes (Fig. 3). These results suggest that CsNCED1 and CsNCED2 are single copy genes in the Citrus genome.
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Up-regulation of CsNCED1 and CsNCED2 genes expression in the flavedo of orange during fruit maturation
The expression of CsNCED1 and CsNCED2 genes in the flavedo during maturation of orange fruit in relation to its changes in ABA content was investigated (Fig. 4). The ABA content was low (below 0.05 µg g–1 FW), and the expression of CsNCED1 and CsNCED2 genes was barely detectable in the flavedo of immature green fruits (stages 1 and 2). The initial increase in ABA was coincident with the onset of fruit degreening (stage 3) and progressively increased during development of fruit coloration to reach the highest value (0.51 µg g–1 FW) when fruit achieved full coloration. Expression of the CsNCED1 gene was detectable after the initiation of fruit degreening and the transcript progressively accumulated to reach its maximum in coloured fruits (stage 8), with the exception of a significant decrease in fruits at colour break (stage 6). The signal corresponding to CsNCED2 mRNA was not detectable during fruit development and coloration (stages 1–7) and a moderate transcript signal was only distinguished at later stages of maturation (stages 8 and 9) (Fig. 4). A similar gene expression profile was consistently observed in fruits harvested in other seasons (data not shown).
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Ethylene stimulates ABA content and CsNCED1 and CsNCED2 expression in the flavedo of orange fruit
The ABA content in the flavedo of Citrus fruits has been shown previously to be enhanced by ethylene (Goldschmidt et al., 1993
; Lafuente et al., 1997). Treatment of mature green orange fruit (a/b=–0.60±0.02) with ethylene greatly accelerated fruit coloration and after 7 d flavedo tissue developed the characteristic yellow-orange coloration (a/b=0.23±0.05), while air-treated fruits did not show significant changes in fruit colour (a/b=–0.55±0.03). The ABA content in the flavedo of ethylene-treated fruits increased as early as 6 h upon application, and by 3 d a 15-fold increase in ABA was observed (Fig. 5). In fruits incubated in an ethylene-free atmosphere a transient increase in ABA was detected between 14 and 24 h after detachment. Expression of CsNCED1 and CsNCED2 genes was differentially up-regulated by ethylene. Accumulation of CsNCED1 mRNA preceded the increase in ABA content and was detected as soon as 3 h after the initiation of the experiment. Interestingly, the pattern of CsNCED1 mRNA accumulation was parallel to the ABA changes in both air- and ethylene-treated fruits. CsNCED2 mRNA was only detected after 14 h of ethylene treatment, to reach a maximum after 1 d and declined thereafter (Fig. 5).
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Expression of CsNCED1, but not CsNCED2, is strongly induced and precedes ABA accumulation in water-stressed Citrus sinensis leaves
To examine whether the expression of CsNCED1 and CsNCED2 is induced by environmental stress in vegetative tissues, expression of CsNCED1 and CsNCED2 genes was analysed in water-stressed citrus leaves and related to the changes in ABA content. During a dehydration experiment lasting 6 h, detached mature Citrus leaves lost 15% of their water content. Figure 6A shows that ABA concentration significantly increased after 4 h of water stress and by 6 h it was 18 times higher that at the beginning. CsNCED1 mRNA was hardly detectable in freshly harvested leaves and only 2 h after water stress and before a noticeable increase in ABA, transcript accumulation was clearly detected. Progressive dehydration of the leaves strongly induced accumulation of CsNCED1 mRNA. It is important to notice that the timing of accumulation of the CsNCED1 transcript was earlier than that of ABA. Transcript of CsNCED2 was undetectable in leaves throughout the whole water-stress experiment (Fig. 6A). In non-stressed leaves, ABA content did not increase and CsNCED1 and CsNCED2 transcripts were not detected (data not shown).
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Expression of CsNCED1 and CsNCED2 genes and ABA content are enhanced in flavedo of orange fruits stored under low humidity conditions
To test whether the Citrus NCED genes isolated might also be up-regulated in flavedo tissue during fruit dehydration, a comparative analysis of transcript accumulation and ABA content in the flavedo of mature fruit stored for 3 d under low (45%) or high RH (95%) was performed (Fig. 6B). Storage at low RH produced a 38% increase in ABA content, while no significant differences were detected in the flavedo of control fruits (stored under 95% RH). The CsNCED1 gene showed a higher expression level than CsNCED2 in the flavedo of freshly harvested fruits. Expression of both CsNCED1 and CsNCED2 was significantly stimulated in the flavedo of fruits maintained under low RH, in good agreement with the changes in ABA (Fig. 6B).
9-cis-Violaxanthin as the primary substrate for cleavage of NCEDs in the peel of Citrus fruit
Biochemical studies have revealed that two 9-cis-epoxycarotenoids, 9-cis-violaxanthin and 9'-cis-neoxanthin, are substrates for recombinant NCEDs (Schwartz et al., 2003b). In order to investigate the potential in vivo substrate/s for Citrus NCEDs, the abundance of these two putative ABA precursors, as well as total carotenoid content, was analysed in selected flavedo and leaf tissues. Concentration of total carotenoids was much higher in leaves than in the flavedo of green fruits, and in fruits increased during maturation (Table 2; Rodrigo et al., 2003, 2004). The concentration of 9-cis-violaxanthin in flavedo increased dramatically during the transformation from chloroplasts to chromoplasts, becoming the main carotenoid in this tissue (59% of the total content), while the concentration of 9'-cis-neoxanthin in fruits after the mature green stage was below the limit of HPLC–PDA (photodiode array) detection (see Materials and methods). During the ethylene-induced degreening, only traces of 9'-cis-neoxanthin were found and the concentration of 9-cis-violaxanthin was increased by ethylene and reduced in the flavedo of air-treated fruits. By contrast, 9-cis-violaxanthin was undetectable in leaves and the concentration of 9'-cis-neoxanthin of turgid leaves was halved after 4 h of water stress (Table 2). This reduction in 9'-cis-neoxanthin is consistent with the increase of ABA observed during this period (Fig. 6A).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Biochemical and genetic studies have demonstrated that the cleavage reaction of cis-epoxycarotenoids into xanthoxin, catalysed by NCEDs, is the main regulatory step in ABA biosynthesis in higher plants (for reviews, see Schwartz et al., 2003b
; Nambara and Marion-Poll, 2005). In the present work two novel cDNAs encoding NCED, named CsNCED1 and CsNCED2, have been isolated from the flavedo of orange fruit (Fig. 1). The predicted CsNCED1 and CsNCED2 proteins share high sequence identity to other plant NCEDs and contain structural features conserved in all NCEDs. Genomic Southern-blot analysis suggests that Citrus NCEDs are encoded by a small gene family (Fig. 3) as has been reported for other plant species (Tan et al., 1997, 2003; Burbidge et al., 1999; Chernys and Zeevaart, 2000; Iuchi et al., 2000) and both CsNCED1 and CsNCED2 are single copy genes in the Citrus genome (Fig. 3).
It is noteworthy that CsNCED1 and CsNCED2 do not cluster together in the phylogenetic tree (Fig. 2) in spite of the high sequence identity. CsNCED1 is more closely related to AtNCED3 and LeNCED1, which play a major role in stress-induced ABA synthesis in Arabidopsis and tomato leaves (Iuchi et al., 2001; Tan et al., 2003). Expression of CsNCED1 was also induced by dehydration of fruits and leaves, and it is likely to be the orange orthologue of AtNCED3 and LeNCED1. By contrast, CsNCED2 aligns more closely to the Arabidopsis NCED2 and NCED5 isoforms. These genes are mainly expressed in non-photosynthetic tissues (Tan et al., 2003), in agreement with the chromoplast-specific expression of the CsNCED2 gene.
A striking characteristic of the CsNCED1 and CsNCED2 genes is their differential tissue-specific expression and distinctive responses under different developmental and environmental signals. CsNCED1 was up-regulated in the flavedo during natural fruit maturation and by exposure to ethylene, and induced in dehydrated leaves and fruits. However, accumulation of the CsNCED2 transcript was only detected in the flavedo of fruits at later stages of maturation, transiently in response to ethylene and in dehydrated flavedo (Figs 4–6
). During natural fruit pigmentation and with ethylene treatment, flavedo tissue accumulates large amounts of oxygenated carotenoids that may serve as precursors for the synthesis of ABA. Whether ABA accumulates as an end-product of a stimulated carotenoid biosynthetic pathway or its biosynthesis is specifically regulated by the enzymatic cleavage of cis-xanthophylls is not known. The present study shows that the evolution of endogenous ABA is well correlated with CsNCED1 and CsNCED2 transcript accumulation, suggesting a key role for NCEDs in the regulation of ABA synthesis in Citrus fruits and leaves. Moreover, the distinctive temporal and tissue-specific expression of CsNCED1 and CsNCED2 reveals a differential contribution of both genes in the regulation of ABA content. The present results suggest that CsNCED1 gene expression is the rapid and central mechanism by which ABA biosynthesis is regulated in both flavedo tissue and water-stressed Citrus leaves. The presence of a second isoform, CsNCED2, in the flavedo of mature fruits may contribute to an additional co-ordination mechanism of ABA production in fruits. The expression of two isoforms in fruits appears to be redundant, but it can be suggested that, at least in fruit tissues accumulating large amounts of ABA, such as flavedo of Citrus during development and maturation, a second fruit-specific isoform may be required to adjust and to sustain ABA levels to specific developmental stages (Fig. 4) or to stress-signalling responses, such as fruit dehydration (Fig. 6B). In avocado fruits also, two different NCED isoforms, PaNCED1 and PaNCED3, were up-regulated in the mesocarp during ripening, but only PaNCED1 was induced in water-stressed leaves (Chernys and Zeevaart, 2000). Recently, ethylene- and dehydration-responsible elements have been identified in the promoter region of the tomato LeNCED1 gene (Thompson et al., 2004). It is likely that promoters of CsNCED1 and CsNCED2 may also contain similar regulatory regions, and the combination of these elements with fruit-specific motifs could explain the differential expression observed for each gene. Additionally, the overlapping expression of CsNCED1 and CsNCED2 genes observed in the flavedo may also indicate differences in substrate specificity or in membrane-binding affinity of the corresponding proteins which could regulate enzyme activity, as it has been suggested for the different Arabidopsis isoforms (Tan et al., 2003).
Unexpectedly, CsNCED1 gene expression temporarily declined in the flavedo of fruits at the breaker stage (Fig. 4). This stage is characterized by the transition from chloroplasts to chromoplasts, and the disorganization and breakdown of the thylakoid membranes (Gross, 1987). Different NCEDs contain chloroplast-targeting signals and are associated with the thylakoid membrane (Qin and Zeevaart, 1999; Iuchi et al., 2000; Tan et al., 2001, 2003). Disintegration of the internal plastid membrane at the breaker stage of fruit maturation could be a reason for the transitory decrease of CsNCED1 mRNA levels and, once the internal membranous system is restored in the chromoplasts, CsNCED1 transcript levels would rise up again.
In-vitro studies with recombinant NCEDs have shown that they can cleave 9-cis-violaxanthin and 9'-cis-neoxanthin, although with different efficiencies (Schwartz et al., 1997, 2003a; Qin and Zeevaart, 1999; Chernys and Zeevaart, 2000; Iuchi et al., 2000). The low relative abundance of 9-cis-violaxanthin in green tissues and the high Km for the cleavage have led to the suggestion that 9'-cis-neoxanthin is the main precursor of xanthoxin in plants (Schwartz et al., 2003a). However, analysis of the content and composition of 9-cis-xanthophylls in the flavedo of orange fruit suggests that 9-cis-violaxathin may be the major in vivo substrate for CsNCEDs in this tissue. First, 9'-cis-neoxanthin was undetectable in the peel of orange during natural maturation, whereas 9-cis-violaxanthin was the main carotenoid in the flavedo once coloration is initiated (Table 2; Gross, 1987; Rodrigo et al., 2003, 2004). Secondly, the presence of apo-12-violaxanthal, a C25 epoxy-apocarotenal, has been reported in flavedo of orange and other coloured Citrus fruits (Curl, 1967; Molnar and Szabolcs, 1980; Gross, 1987). This C25 apocarotenal is, together with xanthoxin, the predicted cleavage product of 9-cis-violaxanthin by NCED, indicating that the cleavage of 9-cis-violaxanthin is occurring in vivo in the flavedo of Citrus. Thirdly, during the ethylene-induced degreening that substantially stimulated CsNCEDs gene expression, 9-cis-violaxanthin was present in relatively high amounts but only traces of 9'-cis-neoxanthin were identified (Table 2). By contrast, 9-cis-violaxanthin was not detected in either turgid or dehydrated leaves, in agreement with previous results (Norman et al., 1990). Taken together, these results suggest that, in the peel of orange fruit, 9-cis-violaxanthin appears to be the main cis-xanthophyllic precursor of ABA, whereas 9'-cis-neoxanthin would be the main substrate for the NCED cleavage reaction in vegetative tissue, as has been proposed for other plant species (Iuchi et al., 2000; Schwartz et al., 2003a).
In conclusion, two different genes, CsNCED1 and CsNCED2, have been isolated from the epicarp of orange fruits. Expression of the CsNCED1 gene increased in the epicarp during natural and ethylene-induced fruit maturation, and in water-stressed leaves in a pattern consistent with the accumulation of ABA. The second gene, CsNCED2, was not detected in dehydrated leaves and, in fruits, exhibited a weak and differential expression to that of CsNCED1. CsNCED1 may play a key role in ABA biosynthesis induced by water stress and during natural and artificial fruit maturation, while CsNCED2 may be involved in a fine adjusting of ABA content in chromoplast-containing tissue. There is evidence to suggest that 9-cis-violaxanthin is the putative in vivo epoxy-carotenoid substrate for Citrus NCED in coloured flavedo while 9'-cis-neoxanthin may be the ABA precursor in photosynthetic tissues.
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Acknowledgements |
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We acknowledge Dr MT Lafuente (IATA-CSIC) for the synthesis of the ABA conjugate used for ABA quantification and for her help during the course of this work. We thank Dr JF Marcos (IATA-CSIC) for the preparation of Southern-blot and Dr L Navarro (IVIA, Moncada, Valencia) for the use of The Citrus Germoplasm Bank. The technical assistance of Amparo Beneyto and Beatriz Octavio is gratefully acknowledged. This work has been supported by research grants AGL2003-01304 (CICyT, Ministerio de Educacion y Ciencia, Spain) and GV-CAPA-00-15 (Generalitat Valenciana). MJR was the recipient of an I3P (CSIC) and ‘Programa Ramon y Cajal’ (MEC) post-doctoral contract.
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Footnotes |
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Abbreviations: ABA, abscisic acid; CCD, carotenoid cleavage dioxygenase; NCED, 9-cis-expoxycarotenoid dioxygenase; RACE, rapid amplification of cDNAs ends by polymerase chain reaction; RH, relative humidity; RT-PCR, reverse-transcription–polymerase chain reaction; SDS, sodium dodecyl sulphate.
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