JXB Advance Access originally published online on October 29, 2003
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Journal of Experimental Botany, Vol. 54, No. 393, pp. 2615-2622,
December 1, 2003
© 2003 Oxford University Press
Characterization of expression, and cloning, of ß-D-xylosidase and 
-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit
Received 15 April 2003; Accepted 24 July 2003
1 Laboratory of Horticultural Science, Faculty of Agriculture, Tottori University, Tottori, 680-8553 Japan
2 Department of Botany, University of Guelph, Ontario N1G 2W1, Canada
*To whom correspondence should be addressed. Fax: +81 857 31 6749. E-mail: itai{at}muses.tottori-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
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Modifications to the cell wall of developing and ripening tomato fruit are mediated by cell wall-degrading enzymes, including a ß-D-xylosidase or
-L-arabinofuranosidase, which participate in the breakdown of xylans and/or arabinoxylans. The activity of both enzymes was highest during early fruit growth, before decreasing during later development and ripening. Two ß-D-xylosidase cDNAs, designated LeXYL1 and LeXYL2, and an -L-arabinofuranosidase cDNA, designated LeARF1, were obtained. Accumulation of mRNAs for ß-D-xylosidase and -L-arabinofuranosidase was examined during fruit development and ripening. LeARF1 and LeXYL2 genes were relatively highly expressed during fruit development and decreased after the onset of ripening. By contrast, LeXYL1 was not expressed during fruit development, but was expressed later, particularly during over-ripening. The expression of all three genes was also followed in ripening-impaired mutants, Nr, Nr2, nor, and rin of cv. Ailsa Craig fruit. LeXYL2 mRNA was detected in the ripe fruits of all the mutants and its abundance was similar to that in mature green wild-type fruit. By contrast, LEXYL1 mRNA was expressed only in the ripe fruits of the Nr mutant, suggesting that the two ß-D-xylosidase genes are subject to distinct regulatory control during fruit development and ripening. LeARF1 mRNA was detected in ripe fruits of Nr2, nor and rin, and not in ripe fruit of the Nr mutant. The accumulation of LeARF1 in ripe fruit was restored by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, while 1-MCP had no effect on the expression of LeXYL1 or LeXYL2. This suggests that LeARF1 expression is subject to negative regulation by ethylene and that the two ß-D-xylosidase genes are independent of ethylene action.
Key words: -L-Arabinofuranosidase, cell walls, ethylene, 1-methylcyclopropene (1-MCP), tomato fruit ripening, ß-D-xylosidase.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Fruit development and ripening are complex processes that involve numerous modifications to the cell wall, a structure composed of pectins, hemicellulose, cellulose, and protein. These modifications may be mediated by hydrolases, of which polygalacturonase, pectin methylesterase and ß-galactosidase have extensively studied (Grierson, 1985; Tucker et al., 1982; Pressey, 1983; reviewed in Giovannoni, 2001). The participation of these enzymes in cell wall degradation during fruit development and ripening is reflected in changes in its physical properties, and molecular composition.
Among plant cell wall hemicellulose components, arabinoxylans and xylans are widely distributed (Carpita, 1987). The xylans consist of ß-D-xylopyranosyl residues that form a core backbone, which may be substituted with -L-arabinofuranosyl (arabinoxylans) and to a lesser extent -D-glucuronic acid (glucuronarabinoxylans) residues. Cell walls of the inner and outer pericarp of tomato fruits contain arabinose and xylose as prominent components (Huysamer et al., 1997), the latter including xyloglucans (Wakabayashi, 2000). The chemical structures of wall arabinoxylans and xylans are subject to modification during plant growth and development, including seed germination, fruit development and ripening and abscission (Beldman et al., 1996; Cleemput et al., 1995; Fincher, 1989; Sozzi et al., 2002a; Tateishi et al., 1996). -L-Arabinofuranosidase (EC 3.2.1.55
[EC]
) and ß-D-xylosidase (EC 3.2.1.37
[EC]
) are responsible for the hydrolysis of arabinoxylans and xylans liberating -L-arabinofuranosyl residues and ß-D-xylosyl residues, respectively. Little is known about these enzymes during fruit ripening, although a xylosidase-like gene in ripening Japanese pear (Itai et al., 1999), and three arabinofuranosidase isoforms in developing and ripening tomato fruits, have been identified recently. By contrast, these enzymes of microbial origin have received much attention, mainly because of their use in the paper and pulp industries (Poutanen and Puls, 1988). In this paper, the cloning and characterization of -L-arabinofuranosidase and ß-D-xylosidase enzymes from tomato fruit are reported, as are their activities during growth and ripening.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant material
Fruits of tomato (Lycopersicon esculentum Mill) cv. Ailsa Craig and its ripening impaired mutants (Nr, Nr2, rin, nor) were grown in the greenhouse of Tottori University, Japan or the Department of Botany, University of Guelph, Canada under natural daylight conditions. Some red-ripe fruits of cv. Ailsa Craig were treated with 2 ppm 1-methylcyclopropene (1-MCP), an ethylene-receptor inhibitor, for 48 h, before harvest.
-L-Arabinofuranosidase and ß-D-xylosidase extraction and assay
Tomato fruits were picked at the 1–2 cm, 2–3 cm and 3–4 cm diameter growth stages, when mature green, turning, red-ripe, and following overripening (4 d after harvesting of red-ripe fruit). Extraction of the enzymes was in 50 mM MES buffer (pH 6) (Cleemput et al., 1997); extraction in the presence or absence of 500 mM NaCl was equally efficient. Pericarp tissue (0.1 g) was ground in liquid nitrogen, extracted in MES buffer, and then centrifuged at 21 000 g for 15 min at 4 °C. The supernatant was used for -L-arabinofuranosidase and ß-D-xylosidase assays, adapted from Cleemput et al. (1997). The modified assays involved using 96-well microtitre-assay plates (Feurtado et al., 2001). The assay mixture consisted of 50 µl 50 mM MES buffer pH 6.0, 25 µl substrate (10 mM p-nitrophenyl-ß-D-xylopyranoside or p-nitrophenyl--L-arabinofuranoside, Sigma, St Louis, MO) dissolved in 50 mM MES buffer, and 75 µl pericarp extract. After incubation at 37 °C for 2 h, the reaction was stopped by adding 75 µl 1 M Na2CO3. Controls were made by adding the stop solution immediately after the enzyme extracts, before incubation, and were used for zero calibration. The release of p-nitrophenol from the glycoside substrate was determined colorimetrically at OD405 in a microplate reader (Molecular Devices Corp., Sunnyvale, CA). Calibration curves were obtained using p-nitrophenol (0–0.2 mM).
Isolation of cDNA clones
RNA was extracted by the hot borate method (Wan and Wilkins, 1994). cDNA was synthesized with an AMV reverse transcriptase first-strand cDNA synthesis kit (Life Sciences, St Petersberg, FL) using 5 µg of total RNA from mature green and ripe tomato fruit, followed by PCR with appropriate primers. Oligonucleotide primers were designed for -L-arabinofuranosidase and ß-D-xylosidase as follows. Degenerate primers were synthesized based on conserved regions obtained by searching Genbank databases for both plant and micro-organisms genes. For -L-arabinofuranosidase, the upstream primer (ARAU1) was 5'-TTYCCIGGIGGITGYTTYGTIGARGG-3' and the downstream primer (ARAD1) was 5'-TTIACRAAIARIGGIGCRTAISWIGCCAT-3'. For ß-D-xylosidase, the upstream primer (XYLU1) was 5'-TGGWVIGARGCIYTICAYGG-3' and the downstream primer (XYLD1) was 5'-GTYYVYTGICCICKICCCCA-3'. RT-PCR conditions for the -L-arabinofuranosidase RNA were 40 cycles of 94 °C for 1 min, 57 °C for 1 min, and 72 °C for 2 min. The conditions for PCR of the ß-D-xylosidase RNA were the same, except that the annealing temperature was 50 °C. To determine the full-length nucleotide sequences, 3' and 5' RACE-PCR were performed using 3' RACE and 5' RACE kits, according to the manufacturer’s instructions (Takara, Kyoto, Japan). The PCR products were ligated into pGEM easy vector (Promega, Madison, WI) and then introduced into E. coli XL1-Blue. After screening, target cDNAs were sequenced using an ALF express sequencer (Amersham Pharmacia, Uppsala, Sweden).
RNA extraction and northern blot analysis
Total RNA was prepared from 10 g skin and pericarp at different stages of tomato fruit growth and ripening using the cv. Ailsa Craig, its ripening-impaired mutants (Nr, Nr2, rin, nor) and 1-MCP-treated fruits, using the hot borate method (Wan and Wilkins, 1994) following initial grinding in liquid nitrogen. Ten µg of total RNA were separated by electrophresis on 1.2% agarose gels containing 0.66 M formaldehyde, transferred to Hybond N+ nylon membranes (Amersham Biosciences, Baie d’Urfé, PQ, Canada), and fixed by a UV cross-linker or baking at 80 °C. The membranes were prehybridized and probed with 32P-labelled full length cDNAs for -L-arabinofuranosidase and ß-D-xylosidase (LeARF1, LeXYL1, LeXYL2), obtained by RT-PCR, using a Rediprime kit (Amersham Biosciences) and hybridized in Church buffer, 1% (w/v) bovine serum albumin, 1 mM EDTA, 0.5 M phosphate buffer, 7% (w/v) SDS (Sambrook and Russell, 2001). Following hybridization, membranes were washed twice at 65 °C in 2x SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7), 0.1% (w/v) SDS for 15 min and then three times at 65 °C in 0.2x SSC, 0.1% (w/v) SDS for 15 min. Finally, membranes were exposed to an imaging plate (Fuji Film, Tokyo, Japan) or X-ray film.
DNA extraction and Southern blot analysis
Total DNA was extracted from immature leaves of cv. Ailsa Craig tomato plants by a modified SDS method (Itai et al., 2003). Five grams of fresh leaves were ground in liquid nitrogen to which 40 ml of washing buffer (0.1 M HEPES pH 8.0, 0.1% [w/v] polyvinylpyrolidone (K-30), 2% [v/v] 2-mercaptoethanol) were added, and homogenized. The homogenate was centrifuged at 10 000 g for 10 min, and the supernatant discarded. The pellet was resuspended in 10 ml extraction buffer (0.5 M NaCl, 100 mM TRIS-HCI pH 8.0, 50 mM EDTA-Na, 2% [w/v] SDS) and incubated at 70 °C for 10 min. After DNA was precipitated with 2-propanol, it was treated with RNase A, dissolved in TE buffer (10 mM TRIS-HCl pH 8, 1 mM EDTA), and used for Southern blot analysis. Ten µg of DNA was digested with EcoRI, EcoRV, HindIII, and DraI, fractionated by electrophoresis in 0.9% agarose gels, and transferred to Hybond N+ nylon membranes. DNA was fixed by UV crosslinking. Membranes were hybridized with 32P-labelled inserts from plasmids containing LeARF1, LeXYL1, LeXYL2 cDNAs at 65 °C overnight in Church buffer. After hybridization, membranes were washed as described above, and then exposed to an imaging plate.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Activities of ß-D-xylosidase and
-L-arabinofuranosidase during tomato fruit development and ripeningPrevious studies on ripening tomato fruits have shown that two hemicellulases, endo-ß-mannanase and ß-mannosidase are active in the outer tissues (exocarp) of the fruit, especially in the skin (Bewley et al., 2000; Banik et al., 2001). Both enzymes are associated with the cell walls, and require high-salt buffers for their extraction. While endo-ß-mannanase activity increases during ripening, that of ß-mannosidase decreases from the mature green to the red-ripe stages (Banik et al., 2001).
The activities of two more hemicellulases, ß-D-xylosidase and -L-arabinofuranosidase, are reported here; the presence of the latter during tomato fruit development and ripening was recently reported (Sozzi et al., 2002a). In contrast to the mannan-degrading enzymes, both of these enzymes were extractable in low-salt buffers, suggestive of a loose association with the cell walls, although their substrates are located therein. Both were present in the fruit at the earliest stage of its development, when its diameter was 1–2 cm (Fig. 1). Enzyme activity declined markedly by the time the fruit reached 2–3 cm in diameter, and was low during the remainder of fruit growth, and during ripening and over-ripening. Sozzi et al. (2002a) have shown that two isoforms of -L-arabinofuranosidase decline during tomato cv. VF36 fruit development and a third isoform increases slightly during ripening. No increase in -L-arabinofuranosidase activity was discernible during ripening of fruits of the cv. Ailsa Craig, a discrepancy with cv. VF36 fruit which may be due to cultivar differences, or else the minor changes in activities of all three isoforms when assayed together at these later stages (Sozzi et al., 2002a) was within the error of the extraction and assay techniques.
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The decline in activity of
-L-arabinofuranosidase is shown on a per gram fresh weight basis (Fig. 1), but expressing the data on a per gram dry weight basis also resulted in a similar decline (not shown), since the dry weight of the fruit was approximately 5.5% of fresh weight (4.4–7.24%) throughout growth and subsequent ripening. However, the fruit was increasing in size and weight throughout development (e.g. there was an 8-fold increase in fresh weight from the 1–2 cm diameter to the 2–3 cm diameter stage, a further 3.7-fold increase to the 3–4 cm stage, and a doubling of fresh weight to the mature green stage, after which it remained constant). The decline in enzyme activities was not strictly proportional to the changes in fresh weight; for example, activity of both enzymes decreased by 83% during fruit development from the 1–2 cm diameter to the mature green stage, while the increase in fresh weight was over 40-fold (i.e. the fresh weight of the fruit at the earliest stages of development was approximately 98% less than that of the mature green fruit).
More precise location of enzyme activity was possible when the fruit became large enough to be dissected, at the 2–3 cm stage of development. It is evident (Fig. 2) that the skin region of the exocarp contained the most ß-D-xylosidase and -L-arabinofuranosidase activity. The outer pericarp, which lies in the 1–2 mm region beneath the skin also contained activity, as did the inner pericarp (the next 0.5 cm of tissue), to about the same extent on a fresh weight basis. This is similar to the situation for endo-ß-mannanase, which is very high in activity in the skin, lower in the outer pericarp, and only slightly lower at most stages of ripening in the inner pericarp (Bewley et al., 2000).
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Cloning and characterization of ß-D-xylosidase and
-L-arabinofuranosidaseTo understand the expression and activities of ß-D-xylosidase and
-L-arabinofuranosidase further, cDNA clones for both were obtained by RT-PCR and subsequent 5' RACE- and 3' RACE-PCR. The amino acid composition of the mature -L-arabinofuranosidase protein from tomato fruit (data not shown) (LeARF1, GenBank accession number AB073310
[GenBank]
) has approximately 62% identity with three other arabinofuranosidases, one from Arabidopsis (AtARF1, GenBank accession number AF372949
[GenBank]
) and two from barley seedlings (AXAH1 and AXAH2, GenBank accession numbers AF320324
[GenBank]
and AF320325
[GenBank]
) (Lee et al., 2001). The deduced LeARF1 protein was predicted to have a hydrophobic region of 24 amino acids at the N-terminus which resembles an extracellular signal sequence as determined by the PSORT program (Bannai et al., 2002). Two ß-D-xylosidase proteins were identified from tomato fruit (data not shown) and the amino acid compositions of two proteins (LeXYL1, GenBank accession number AB041811
[GenBank]
and LeXYL2, Genbank accession number AB041812
[GenBank]
) are 74% similar to each other. LeXYL1 has a 65% and 57% identity with a putative Arabidopsis ß-D-xylosidase (ATXYL1, GenBank accession number T49925
[GenBank]
) and ß-D-xylosidase from Japanese pear fruit (PPXYL1, GenBank accession number AB007121) (Itai et al., 1999). Using the PSORT program as above, the deduced LeXYL1 protein was predicted to have a signal sequence of 28 amino acids at the N-terminus. By contrast, the deduced LeXYL2 protein was not predicted to have a signal sequence. Thus, the two isozymes have different features for their N-terminal sorting signals, suggesting different roles during fruit development and ripening.
A Southern blot analysis of tomato DNA following digestion by four restriction enzymes, for which there are no sites within the cDNA, showed the presence of several bands following hybridization with LeARF1 and the two LeXYL clones (Fig. 3). The possibility that the LeXYL1 probe cross-hybridized with the LeXYL2 gene was examined. LeXYL1 cDNA was used as a probe on gel blots of a plasmid containing LeXYL2 cDNA; however, cross-hybridization was not observed under the same high stringency conditions (data not shown). LeARF1 detected only one band following restriction by EcoRI and DraI, although three bands were evident following restriction of the genomic DNA with EcoRV and HindIII. This is indicative of there being only one gene for -L-arabinofuranosidase, with the possibility of their being restriction sites for EcoRV and HindIII in the intron regions. The LeXYL1 probe identifies one gene in the HindIII restricted DNA, and at least two following restriction by the other enzymes. Thus, again, it is possible that there is one gene for this xylosidase enzyme, with sites for three of the restriction enzymes in intron regions. The LeXYL2 probe recognized at least three bands of restricted DNA (with the exception of EcoRV, where the bands were less clear), which were different from those hybridizing with the LeXYL1 probe (Fig. 3). This is indicative of more than one gene for this isozyme of ß-D-xylosidase.
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Expression of the ß-D-xylosidase and
-L-arabinofuranosidase genes during fruit development and ripeningNorthern blot analysis using the three cDNA probes was used to determine the temporal expression of
-L-arabinofuranosidase and the two ß-D-xylosidase isoenzymes during fruit development and ripening. mRNA for -L-arabinofuranosidase was expressed most highly during fruit development up to the time of the formation of the mature green fruit. Thereafter, its expression declined markedly and was very low during the whole of fruit ripening (Fig. 4). A similar pattern of expression was evident for the ß-D-xylosidase encoded by LeXYL2, although its expression into fruit ripening was somewhat more persistent. By contrast, the encoding of ß-D-xylosidase mRNA by LeXYL1 did not occur during fruit development, and very little during the early stages of ripening; expression occurred only during the final stages of fruit ripening, and particularly strongly in the over-ripe fruit. Thus the fairly constant amount of ß-D-xylosidase activity observed during fruit ripening (Fig. 1) was presumably due to the sum of activities of the two isoforms of the enzyme; the synthesis of one increased as the other diminished. The almost total decline in mRNA for -L-arabinofuranosidase (LeARF1) (Fig. 4) is not easily reconciled to the persistence of activity of the enzyme in the ripening fruit (Fig. 1). In a recent study, three different -L-arabinofuranosidase isoforms, designated -Af I, II, and III, were identified by size exclusion chromatography of a protein extract from tomato fruit (Sozzi et al., 2002b). According to their report, -Af III activity increased with the onset of ripening, suggesting the existence of a new -L-arabinofuranosidase gene (LeARF2), not identified by this study, which is responsible for the persistence of activity of the enzyme in the fruit during ripening. They also reported that -Af II accounted for 80% of the total arabinofuranosidase activity in young fruits and declined during fruit ripening (Sozzi et al., 2002b), suggesting that the -Af II isoform coincides with the expression of LeARF1. It is also possible that some activity of -L-arabinofuranosidase persists long after its transcription and translation ceases, and that the third isoform, with low activity, is the result of post-translational modification of one of the earlier synthesized isoforms.
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Several non-ripening mutants of tomato are known, and the activities of
-L-arabinofuranosidase and ß-D-xylosidase, and expression of their genes, were monitored in Nr, Nr2, nor, and rin. Low activities of both enzymes in cv. Ailsa Craig, designated here as the wild type (wt), were present at the ripe-red stage, and were lower in activity than at the mature green stage, the end of fruit development (Figs 1, 5A). Activities of the enzymes were monitored in the non-ripening mutants at their maximum stage of ripeness; nor and rin fruit only reached the stage of turning to yellow and did not soften, Nr2 fruit turned red but did not soften (Kerr, 1982), whereas those of NR turned red and softened, but were delayed compared to wt fruit. There was only a small decline in enzyme activities in the fruits of the Nr2 mutant, with virtually no effect on either enzyme in the nor and rin mutants. In the Nr mutant, ß-D-xylosidase activity approached that in the mature green fruit of the wt, and that of -L-arabinofuranosidase was a little higher than in the other mutants, and the red-ripe wt fruits. These changes in enzyme activity were not reflected in the changes in expression of their genes, however. LeARF1 expression declined in the wt fruit during ripening (Fig. 5B), but the three mutants (Nr2, nor, rin) showing the same or slightly lower -L-arabinofuranosidase activities at maximum ripeness retained a high expression of the gene for this enzyme. Fruits of the Nr mutant, which exhibited the most activity of this enzyme showed the greatest decline in its transcript, to an undetectable level (Fig. 5A, B). This discrepancy may be due to the presence of an undetected gene for a different isoform, or else the enzyme is very stable after its synthesis has ceased, as suggested above.
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LeXYL1 expression was retained in the mature fruits of the wt and Nr mutant, with the latter exhibiting increased enzyme activity (Fig. 5A, B). But the activity of the enzyme was very similar in the nor and rin fruits to those of the wt, and yet the former two showed no LeXYLI expression. On the other hand, LeXYL2 expression was equally high in the mature green fruit of the wt and of Nr mutant which exhibited similar enzyme activities, and was less in the fruits of the mutants which had less activity than these two. Hence activity of ß-D-xylosidase appeared to be more related to expression of the LeXYL2 gene than that of LeXYL1. This inconsistency between enzyme activity and expression pattern may be because the the natural substrate for LEXYL1 is different from that for LEXYL2, and that LEXYL1 could not use the p-nitrophenyl-ß-D-xylopyranoside as efficiently as substrate.
Effect of 1-MCP on the expression of LeARF, LeXYL1 and LeXYL 2 genes in ripe tomato fruit
Many of the physiological changes that occur during fruit ripening are in response to ethylene production. The three genes identified in this study were classified as ethylene-dependent or ethylene-independent using 1-MCP, an inhibitor of the ethylene receptor. Expression of the 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene is considered to be subjected to positive feedback regulation (Nakatsuka et al., 1998; Itai et al., 2000). Therefore, a tomato ACC oxidase gene (LeACO1) was used as a control gene to monitor the response to 1-MCP treatment. The abundance of LeACO1 mRNA was high in control ripe fruit, and its expression was much reduced by 1-MCP treatment (Fig. 6), confirming that 1-MCP suppressed the expression of the LeACO1 gene during fruit ripening (Nakatsuka et al., 1998). By contrast, while almost no signal for LeARF1 gene expression was detected in ripe tomato fruits, accumulation of LeARF1 mRNA was high in the fruit treated with 1-MCP (Fig. 6). As shown in Fig. 4, the LeARF1 gene was expressed in wt fruit from the immature to before the turning stage, with very little expression later during ripening and over-ripening. However, since a strong signal for this gene was detected in the ripe fruit treated with 1-MCP (Fig. 6), it is likely that the expression of the LeARF1 gene is negatively regulated by ethylene. Nakatsuka et al. (1998) also demonstrated that among five members of the ACC synthase family (LeACS1A, 2, 3, 4, 6), LeACS2 and LeACS4 were highly regulated through a positive feedback mechanism by ethylene, whereas LeACS6 was under negative feedback regulation. LeARF1 appears to have the same regulatory mechanism as LeACS6. Sozzi et al. (2002b) investigated the effect of ethylene on the activity of three arabinofuranosidase isoforms (-Af I, II, and III); -Af I and II activity were not affected by ethylene treatment, whereas it enhanced -Af III activity. They suggested that -Af III is the only isoform directly influenced by ethylene and that ethylene has little to do with the ripening-related decrease in -Af II activity. However, these results clearly suggest the existence of an isoform regulated negatively by ethylene. LeXYL2 was expressed abundantly during fruit development in a manner similar to that of LeARF1, and LeXYL1 was expressed during fruit ripening (Fig. 4). The abundance of their mRNAs was not influenced by treatment with 1-MCP (Fig. 6), indicating that the expression of two ß-D-xylosidase genes is independent of ethylene action.
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The results obtained using 1-MCP (Fig. 6) are also consistent with those obtained using the tomato mutants (Fig. 5) with respect to the change in expression of the LeARF1 gene. Nor and rin mutants produce low amounts of ethylene during fruit ripening and fail to show a climacteric rise in this regulator, due to an inhibition upstream of the ethylene transduction pathway (Giovannoni, 2001). Hence they would be expected to increase in their LeARF1 expression in the absence of a negative regulation by ethylene, which was observed (Fig. 5B). It has been held that the Nr mutant is unresponsive to ethylene (reviewed in Moore et al., 2002), but this is questionable. Lanahan et al. (1994) reported that the Nr mutant in different genetic backgrounds appears to have different effects on the sensitivity of fruit to ethylene. For example, Nr fruit in an Ailsa Craig background ripened to a greater extent than a Pearson background, because the former retained residual ethylene responsiveness. It has also been observed that the amount of ethylene produced in fruit with lower Nr gene expression, reduced by antisense mRNA, is still approximately half that produced by wild-type fruits (Tieman et al., 2000). In the experiments reported here, Nr fruit in the Ailsa Craig background ripened with a delay of a few days. Hence it is likely that the Nr mutant fruit showed a reduced response to ethylene, rather than no response and, as suggested by Tieman et al. (2000), there is the possibility that another of the ethylene receptor genes, LeETR4, replaces the receptor which is lost from the Nr mutant. Hence LeARF1 expression might still be expected to be repressed in Nr mutant fruit, and this is what occurred (Fig. 6). The nature of the Nr2 mutation is unknown, but on the basis of LeARF1 expression it would appear to impose a similar behaviour to the nor and rin mutants.
In conclusion, the expression and activity of -L-arabinofuranosidase is high during early tomato fruit development, and has low activities during fruit ripening. This is consistent with another exo-hydrolase, ß-mannosidase, which exhibits high activity only during fruit development (Bewley et al., 2000). The major activity of ß-xylosidase, and the expression of its gene, LeXYL2, also exhibits a similar pattern. But the activity of a second, much less active ß-xylosidase, encoded by LeXYL1, is more similar to that of exo-(1,4)-ß-D-galactanase which increases in activity during fruit ripening (Carey et al., 1995), as does one of the isoforms of ß-galactosidase (Pressey, 1983). Hemicellulose-degrading endo-enzymes are high in activity during ripening and not during development, for example, endo-ß-mannanase (Bewley et al., 2000; Carrington et al., 2002), endo-1,4-ß-glucanase (Hall, 1964; Harpster et al., 2002) and polygalacturonases (Hobson, 1964; Tucker et al., 1980). This is indicative that the enzymes and changes that take place during the loosening of cell walls to permit tomato fruit growth are different from those associated with the degradation of the cell walls that occurs as the fruit ripens and softens. The expression of two ß-xylosidase genes (LeXYL1 and LeXYL2) differ during fruit development and ripening, and regulation of their activities would appear to be independent of each other, and of ethylene. On the other hand the gene for -L-arabinofuranosidase (LeARF1) is negatively regulated by ethylene, which may control its decline in expression during ripening.
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Acknowledgements |
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This research was supported by Grant-in-Aid number 14760019 to AI from the Ministry of Education, Science, Sports, and Culture of Japan, and the Natural Sciences and Engineering Research Council of Canada grant A2210 to JDB.
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Banik M, Bourgault R, Bewley JD. 2001. Endo-ß-mannanase is present in an inactive form in ripening tomato fruits of the cultivar Walter. Journal of Experimental Botany 52, 105–111.
Bannai H, Tamada Y, Maruyama O, Nakai K, Miyano S. 2002. Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18, 298–305.
Beldman G, Osuga D, Whitaker JR. 1996. Some characteristics of ß-D-xylopyranosidase, -L-arabinofuranosidases and arabin oxylan -L-arabinofuranosidase from wheat bran and germinated wheat. Journal of Cereal Science 23, 169–180.[CrossRef]
Bewley JD, Banik M, Bourgault R, Feurtado JA, Toorop P, Hilhorst HWM. 2000. Endo-ß-mannanase activity increases in the skin and outer pericarp of tomato fruits during ripening. Journal of Experimental Botany 51, 529–538.
Carey AT, Holt K, Picard S, Wilde R, Tucker GA, Bird CR, Schuch W, Seymour GB. 1995. Tomato exo-(1
4)-ß-D-galactanase. Isolation, changes during ripening in normal and mutant tomato fruit, and characterization of a related cDNA clone. Plant Physiology 108, 1099–1107.[Abstract]
Carpita NC. 1987. The biochemistry of ‘growing’ cell walls. In: Cosgrove DJ, Knievel DP, eds. Physiology of cell expansion during plant growth. Rockville, MD: American Society of Plant Physiologists, 28–45.
Carrington CMS, Vendrell M, Domínguez-Puigjaner E. 2002. Characterisation of an endo-(1,4)-ß-mannanase (LeMan4) expressed in ripening tomato fruit. Plant Science 163, 599–606.[CrossRef]
Cleemput G, Bleukx W, van Oort M, Grobet PJ, Delcour JA. 1995. Evidence for the presence of arabinoxylan hydrolyzing enzymes in European wheat flours. Journal of Cereal Science 22, 139–145.[CrossRef]
9 Cleemput G, Hessing M, van Oort M, Deconynck M, Delcour JA. 1997. Purification and charcterization of ß-D-xylosidase and an endo-xylanase from wheat flour. Plant Physiology 113, 377–386.[Abstract]
Feurtado JA, Banik M, Bewley JD. 2001. The cloning and characterization of -galactosidase present during and following germination of tomato (Lycopersicon esculentum Mill.) seed. Journal of Experimental Botany 52, 1239–1249.
Fincher GB. 1989. Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annual Review of Plant Physiology and Plant Molecular Biology 40, 305–346.[CrossRef][Web of Science]
Giovannoni J. 2001. Molecular biology of fruit maturation and ripening. Annual Review of Plant Physiology and Plant Molecular Biology 52, 725–749.[CrossRef][Web of Science][Medline]
Grierson D. 1985. Gene expression in ripening tomato fruit. Critical Reviews in Plant Science 3, 113–132.
Hall CB. 1964. Cellulase activity in tomato fruits according to portion and maturity. Botanical Gazette 125, 156–157.[CrossRef]
Harpster MH, Dawson DM, Nevins DJ, Dunsmuir P, Brummell DA. 2002. Constitutive overexpression of a ripening-related pepper endo-1,4-ß-glucanase in transgenic tomato fruit does not increase xyloglucan depolymerization or fruit softening. Plant Molecular Biology 50, 357–369.[CrossRef][Web of Science][Medline]
Hobson GE. 1964. Polygalacturonase in normal and abnormal tomato fruit. Biochemical Journal 92, 324–332.[Web of Science][Medline]
Huysamer M, Greve LC, Labavitch JM. 1997. Cell wall metabolism in ripening fruit. IX. Synthesis of pectic and hemicellulosic cell wall polymers in the outer pericarp of mature green tomatoes (cv. XMT-22). Plant Physiology 114, 1523–1521.[Abstract]
Itai A, Kotaki T, Tanabe K, Tamura F, Kawaguchi D, Fukuda M. 2003. Rapid identification of 1-aminocyclopropane-1-carboxylate (ACC) synthase genotypes in cultivars of Japanese pear (Pyrus pyrifolia Nakai) using CAPS markers. Theoretical and Applied Genetics 51, 1266–1272.
Itai A, Tanabe K, Tamura F, Tanaka T. 2000. Isolation of cDNA clones corresponding to the genes expressed during fruit ripening in Japanese pear (Pyrus pyrifolia Nakai): involvement of the ethylene signal transduction pathway in their expression. Journal of Experimental Botany 51, 1163–1166.
Itai A, Yoshida K, Tanabe K, Tamura F. 1999. A ß-D-xylosidase-like gene is expressed during fruit ripening in Japanese pear (Pyrus pyrifolia Nakai). Journal of Experimental Botany 50, 877–878.
Kerr EA. 1982. Never ripe-2 (Nr-2), a slow ripening mutant resembling Nr and Gr. Report of the Tomato Genetics Co-operative 32, 33.
Lanahan MB, Yen HC, Giovannoni JJ, Klee HJ. 1994. The Never ripe mutation blocks ethylene perception in tomato. The Plant Cell 6, 521–530.[Abstract]
Lee RC, Burton RA, Hrmova M, Fincher GB. 2001. Barley arabinoxylan arabinofuranohydrolases: purification, character ization and determination of primary structures from cDNA clones. Biochemical Journal 356, 181–189.[CrossRef][Web of Science][Medline]
Moore S, Vrebalov J, Payton P, Giovannoni J. 2002. Use of genomics tools to isolate key ripening genes and analyse fruit maturation in tomato. Journal of Experimental Botany 53, 2023–2030.
Nakatsuka A, Murachi S, Okunishi H, Shiomi S, Nakano R, Kubo Y, Inaba A. 1998. Differential expression and internal feedback regulation of 1-aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit ripening during development and ripening. Plant Physiology 118, 1295–1305.
Poutanen K, Puls J. 1988. Characterization of Trichoderma reesei ß-D-xylosidase and its use in the hydrolysis of solublised xylans. Applied Microbiolgy and Biotechnology 28, 425–432.
Pressey R. 1983. ß-Galactosidases in ripening tomatoes. Plant Physiology 71, 132–135.
Sambrook J, Russell DW. 2001. Molecular cloning—a laboratory manual, 3rd edn. New York: Cold Spring Harbor Laboratory Press.
Sozzi GO, Fraschina AA, Navarro AA, Cascone O, Greve LC, Labavitch JM. 2002a. -L-Arabinofuranosidase activity during development and ripening of normal and ACC synthase antisense tomato fruit. HortScience 37, 564–566.
Sozzi GO, Greve LC, Prody GA, Labavitch JM. 2002b. Gibberellic acid, synthetic auxins, and ethylene differentially modulate -L-arabinofuranosidase activities in antisense aminocyclopropane-1-carboxylic acid synthase tomato pericarp discs. Plant Physiology 129, 1330–1340.
Tateishi A, Kanayama Y, Yamaki S. 1996. -L-arabino furanosidase from cell wall of Japanese pear fruits. Phytochemistry 42, 295–299.[CrossRef]
Tieman DM, Taylor MG, Ciardi JA, Klee HJ. 2000. The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family. Proceedings of the National Academy of Sciences, USA 97, 5663–5668.
Tucker GA, Robertson NG, Grierson D. 1980. Changes in polygalacturonase isoenzymes during the ripening of normal and mutant tomato fruit. European Journal of Biochemistry 112, 119–124.[Web of Science][Medline]
Tucker GA, Robertson NG, Grierson D. 1982. Purification and changes in actvities of tomato pectinesterase isozymes. Journal of the Science of Food and Agriculture 33, 396–400.[CrossRef]
Wakabayashi K. 2000. Changes in cell wall polysaccharides during fruit ripening. Journal of Plant Research 113, 231–237.[CrossRef]
Wan CY, Wilkins TA. 1994. A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Analytical Biochemistry 223, 7–12.[CrossRef][Web of Science][Medline]
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