AOBPreview originally published online on December 7, 2004
Annals of Botany 2005 95(3):413-422; doi:10.1093/aob/mci045
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Annals of Botany 95/3 © Annals of Botany Company 2004; all rights reserved
Possible Involvement of CS-ACS1 and Ethylene in Auxin-induced Peg Formation of Cucumber Seedlings
Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan
* For correspondence. E-mail nobuharu{at}ige.tohoku.ac.jp
Received: 21 June 2004 Returned for revision: 13 August 2004 Accepted: 26 October 2004 Published electronically: 7 December 2004
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ABSTRACT |
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 TOPFOOTNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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• Background and Aims Cucumber (Cucumis sativus) seedlings develop a peg on the concave side of the gravitropically bending transition zone between the hypocotyl and the root after seed germination. Peg initiation occurs in response to auxin when its levels in the concave side of the transition zone exceed a particular threshold through the graviresponse. Ethylene also plays an important role in peg formation, but its relationship to auxin in this event is not understood. Here, the role ethylene plays in auxin-induced peg formation is studied.
• Methods Peg formation of cucumber seedlings exposed to ethylene at different stages of growth or during exogenous auxin treatment was observed. In addition, ethylene evolution from the concave and convex sides of the transition zone was compared and their transcription of CS-ACS (1-aminocyclopropane-1-carboxylic acid synthase) genes was analysed by RT-PCR and in situ hybridization.
• Key Results Seedlings treated with ethylene after peg initiation produced an enlarged peg, whereas ethylene treatment before peg initiation inhibited peg formation. Ethylene also promoted the development of the peg in the auxin-treated seedlings. Furthermore, the concave side of the transition zone at peg initiation produced more ethylene and CS-ACS1 mRNA than the convex side.
• Conclusions Since CS-ACS1 is an auxin-inducible gene, the greater abundance of auxin in the concave side of the transition zone causes peg initiation and increases CS-ACS1-mediated ethylene biosynthesis, which then facilitates peg development.
Key words: ACS, auxin, Cucumis sativus, ethylene, gravity, peg
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INTRODUCTION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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When cucumber (Cucumis sativus) seeds germinate in a horizontal position, the transition zone between the root and the hypocotyl of the seedlings bends downward due to positive gravitropism. Thereafter, a protuberance called a peg forms on the concave side of the bending transition zone (Witztum and Gersani, 1975
; reviewed by Takahashi, 1997). The peg holds the seed coat during hypocotyl elongation and helps the cotyledons and the plumule to pull themselves out from the seed coat and to emerge from the soil. The response of the transition zone to gravity appears to regulate the lateral positioning of the peg in cucumber (Witztum and Gersani, 1975; reviewed by Takahashi, 1997) since, when cucumber seeds are germinated in a vertical position with the radicle pointing down or under microgravity conditions in space, the seedlings develop a peg on each side of the transition zone (Takahashi et al., 2000). Thus, peg formation on the convex side of the transition zone is suppressed in response to gravity when cucumber seedlings are grown in a horizontal position on the ground (Takahashi et al., 2000).
It has been suggested that auxin is a factor that regulates peg formation (Witztum and Gersani, 1975; Takahashi and Scott, 1994; Kamada et al., 2000) as application of exogenous auxin induces peg formation on the convex side of the transition zone (Witztum and Gersani, 1975; Kamada et al., 2000). It has also been found that, in cucumber seedlings grown in a horizontal position, the levels of free indole-3-acetic acid (IAA) are more abundant in the concave side than in the convex side (Kamada et al., 2003). Moreover, when the expression of the auxin-inducible gene CsIAA1 in cucumber seedlings was investigated, it was found that CsIAA1 mRNA accumulation was also more abundant in the concave side than in the convex side of the transition zone (Fujii et al., 2000; Kamada et al., 2000). In addition, cucumber seedlings treated with 2,3,5-triiodobenzoic acid, an inhibitor of auxin transport, develop a peg on each side of the transition zone even when seedlings are grown in a horizontal position (Kamada et al., 2003). Thus, auxin is thought to be an essential regulator for the induction of peg formation.
Ethylene has also been suggested to be required for peg formation because inhibitors of ethylene biosynthesis or activity inhibit not only the gravitropism of cucumber seedlings but also their peg formation (Takahashi and Suge, 1988). However, the relationship between ethylene and auxin in peg formation has not been investigated. Auxin is known to promote ethylene biosynthesis, and the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the first regulatory step in the ethylene biosynthesis pathway (reviewed by Kende, 1993; McKeon et al., 1995). Auxin can increase the expression of specific ACS genes (Huang et al., 1991; Nakagawa et al., 1991). Four ACS cDNAs have been identified in the cucumber (Kamachi et al., 1997; Trebitsh et al., 1997; Shiomi et al., 1998). However, while the accumulation of their mRNAs in shoot apices including floral buds (Kamachi et al., 1997; Trebitsh et al., 1997; Yamasaki et al., 2001, 2003) and fruits (Shiomi et al., 1998; Mathooko et al., 1999) has been investigated, their expression in cucumber seedlings has not been analysed.
To understand the relationship between ethylene and auxin in the peg formation of cucumber seedlings, it is important to determine the precise effect of ethylene on peg formation and to investigate whether the transition zone of horizontally placed cucumber seedlings asymmetrically biosynthesizes ethylene and asymmetrically expresses auxin-inducible CS-ACS genes. This study examined (a) the effect on peg formation of ethylene treatment at different stages of seedling development; (b) the effect of ethylene treatment on auxin-induced peg formation; and (c) the biosynthesis of ethylene and the expression of the CS-ACS genes during peg formation in cucumber seedlings.
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MATERIALS AND METHODS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Plant materials and growth conditions
Cucumber (Cucumis sativus L. ‘Shinfushinari-jibai’) seeds were purchased from Watanabe Seed Co., Miyagi, Japan. Fourteen cucumber seeds were placed vertically in a fissure within a block of water-absorbent plastic foam (40 mm x 30 mm x 10 mm) attached to the inner surface of a plastic cap of a Petri dish (60 mm x 60 mm x 60 mm). After supplying the block with 8·1 mL of distilled water, the plastic cap was placed in the Petri dish so that the seedlings could be suspended in the air space of the container after germination and grown aeroponically. This Petri dish was placed in a horizontal or a vertical position under darkness at 25°C so that the seedlings could be grown in either a horizontal or a vertical position. To compare the gene expression of the concave and convex sides of the transition zone, sections of the transition zone of 24-h-old seedlings were excised and then halved longitudinally, frozen in liquid nitrogen and stored at –80°C until use.
To observe the effect of ethylene on peg formation at the early developmental stage of cucumber seedlings, seed coats were removed prior to the experiments because the seed coat covers the transition zone and would prevent the transition zone from being exposed to the exogenous ethylene. Thus, seeds lacking a coat were germinated in a horizontal or a vertical position. For ethylene treatment, 1 mL L–1 ethylene gas was added to the glass chambers so that the final concentrations were 1, 5 and 20 mL L–1. Ethylene gas was removed after incubation for different time periods, after which the seedlings were grown in ambient air. Local IAA application was accomplished according to Reinhardt et al. (2000). In brief, stock solutions of 20 mM and 200 mM of IAA in DMSO were dissolved in pre-warmed (50°C) lanolin containing 2·5 % Paraplast Plus (Oxford Labware, MO, USA) so that the final concentrations of IAA were 0·6 mM and 6 mM, respectively. As a control, a lanolin paste containing 3 % DMSO was used. The lanolin paste was applied to either side of the transition zone of 24-h-old cucumber seedlings grown in the presence or absence of 20 mL L–1 ethylene. Then cucumber seedlings were grown in the same ethylene conditions as described above. Peg formation was observed in 72-h-old cucumber seedlings. Differences in the effect of ethylene on peg formation were tested with the chi-square test.
To examine the effects of exogenous auxin on gene expression in the transition zone of cucumber seedlings, the transition zone sections of 24-h-old seedlings were treated with 10–4 M IAA according to Theologis et al. (1985). After incubation, the segments were frozen in liquid nitrogen and stored at –80°C until use.
Ethylene quantification
The whole cucumber seedlings or the longitudinally halved transition zones excised from 24-h-old cucumber seedlings were enclosed in a flask and sealed with a rubber stopper to examine ethylene evolution. After incubation at 25°C for 6 h, 1 mL of head gas was taken from each vial using a gas-tight syringe and injected into a gas chromatograph (GC-4CMPF/Chromatopac C-R4A; Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and an activated alumina column for the measurement of ethylene. The instrument was calibrated with an ethylene gas standard and the amount of ethylene released from the samples per 1 g f. wt per hour was calculated. The means and standard deviations of triplicate samples are shown. Statistical difference of ethylene evolution was determined by Student's t-test.
Quantitative RT-PCR and northern blot analysis
Total RNA was isolated from the transition zone of 24-h-old etiolated cucumber seedlings with TRI reagent (Sigma, MO, USA). Four ACS cDNAs have been identified in the cucumber (Kamachi et al., 1997; Trebitsh et al., 1997; Shiomi et al., 1998). Their names have not been standardized, and thus in this study they have been denoted according to the nomenclatures in Yamasaki et al. (2003) as follows: CS-ACS1 (accession number U59813
[GenBank]
and AB006805
[GenBank]
), CS-ACS2 (Kamachi et al., 1997), CS-ACS3 (accession number AB006803
[GenBank]
) and CS-ACS4 (accession number AB006804
[GenBank]
). To analyse these CS-ACSs and CS-actin gene expressions, RT-PCR was performed as described previously (Yamasaki et al., 2003).
Northern blot analysis was carried out according to Mizuno et al. (2002). A CS-ACS1 cDNA fragment (corresponds to nucleotides 379–979 of GenBank accession number AB006805
[GenBank]
) was inserted into plasmid pGEM-T vector and was used to make probes (Yamasaki et al., 2003). After digestion with SalI, antisense probes were synthesized with DIG RNA labelling mix (Roche, Basel, Switzerland) and T7 polymerase. The preparation of the CsIAA1 RNA probes was performed as described by Fujii et al. (2000).
Microscopy and in situ hybridization
To observe cellular morphology, the segments of the transition zone of 72-h-old cucumber seedlings were fixed overnight with 0·05 M sodium phosphate buffer (pH 7·2) containing 4 % paraformaldehyde and 0·25 % glutaraldehyde while maintaining the orientation of the seedlings relative to gravity. Infiltration of the digested segments with the described fixative was achieved by aspiration for 5 min twice and subsequent secondary fixation for 90 min. After fixation, these samples were dehydrated by an ethanol series, after which the ethanol was replaced by butanol. Finally, the samples were embedded in Paraplast Plus (Oxford Labware). Sections, 10 µm thick, were placed on silicon-coated glass slides (Matsunami Glass Ind., Osaka, Japan) and baked at 50°C for 12 h. The paraffin was removed from the slides by immersion in xylene. After dehydration by an ethanol series, the sections were treated with 0·05 % toluidine blue O for 10 min and 0·04 % potassium iodide. The slides were then dehydrated with an ethanol series, replaced by xylene, and DIATEX (Matsunami Glass Ind.) was applied, after which the slides were covered with a glass coverslip. Photographs were taken with an Olympus C-35AD-4 camera attached to an Olympus SZH stereomicroscope using Fujicolor 100 film.
For in situ hybridization, the sections of 24-h-old cucumber seedlings were prepared as described above. Preparation of antisense probes was described above, and sense probes were synthesized with SP6 RNA polymerase after digestion of the plasmid with NcoI. In situ hybridization was performed according to Kamada et al. (2003) except that 10-µm-thick sections were used.
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RESULTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Effect of ethylene on the peg formation of cucumber seedlings
Previously it had been shown that, when ethylene was applied to cucumber seedlings with 3- to 5-mm-long radicles, the peg was enlarged (Takahashi and Suge, 1988
). To determine the period of development during which ethylene affects peg formation in cucumber seedlings, cucumber seedlings were grown in the horizontal or vertical position and ethylene was added at different stages of growth (Fig. 1 and Table 1). When the seedlings are grown in a horizontal position in ambient air, the cortical cells of the concave side of the transition zone begin to protuberate (i.e. peg initiation occurs) 24 h after imbibition, and peg initiation is apparent 36 h after imbibition (data not shown). When seedlings grown in a horizontal position were exposed to 5 µL L–1 or 20 µL L–1 of ethylene throughout the 72 h of seedling development, about half did not form a peg and instead showed a swollen transition zone (Fig. 1B). The other half of these seedlings formed an enlarged peg (Table 1). When the seedlings were grown in ethylene from 24 h to 72 h, most of seedlings formed an enlarged peg (Fig. 1C), although some of the seedlings did not form a peg (Table 1). All seedlings exposed to ethylene from 36 h to 72 h formed an enlarged peg (Table 1). By contrast, when the seedlings were treated with ethylene during the early development of the peg (0–36 h after imbibition), peg formation was markedly inhibited (Table 1). When the vertically grown seedlings were treated with ethylene the number of seedlings that did not form a peg was greater than that of the horizontally grown seedlings (Table 1). In addition, a lower concentration of ethylene (1 µL L–1) was also able to inhibit peg formation (Table 1). In the vertically grown seedlings, ethylene treatment during the early development of the peg (0–24 h or 0–36 h after imbibition) markedly inhibited peg formation (Table 1). On the other hand, all seedlings treated with ethylene showed normal positive gravitropism in the transition zone (Fig. 1B and C).
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Next, the effect of ethylene treatment on the cellular morphology of the transition zone was examined. With regard to control seedlings grown horizontally in air, the cortical cells in the concave side of the transition zone appeared to elongate perpendicularly to the hypocotyl axis and form a peg (Fig. 1D). By contrast, in seedlings treated with ethylene for the entire developmental period, the cortical cells in the concave side of transition zone did not elongate perpendicularly and were instead round shaped (Fig. 1E). However, when cucumber seedlings formed an enlarged peg by ethylene treatment after peg initiation (24–72 h), the cortical cells of the transition zone appeared to expand in all directions (Fig. 1F). Thus, the presence of ethylene early during peg formation appears to block the perpendicular elongation of cortical cells that results in the peg, while its presence after peg initiation enhances the expansion of the cortical cells.
Effect of ethylene treatment on IAA-induced peg formation
It has been suggested that auxin plays a key role in peg initiation since application of exogenous auxin to horizontally grown seedlings induces peg formation on the convex side of the transition zone (Witztum and Gersani, 1975; Kamada et al., 2000). To determine whether exogenous ethylene treatment can affect such auxin-induced peg formation, seedlings were grown in the presence or absence of 20 µL L–1 ethylene, and 24 h after imbibition auxin-containing lanolin paste was applied to the convex side of the transition zone. The seedlings then continued to grow in the presence (or absence) of 20 µL L–1 ethylene, and peg formation was observed 72 h after imbibition. As expected, in the absence of exogenous ethylene, local IAA application to the convex side of the transition zone of control seedlings grown in a horizontal position, induced the formation of an additional peg on the side that bore the IAA application (Fig. 2A and Table 2). In addition, the transition zone of these seedlings bent toward the side of IAA application (Fig. 2A). Thus, IAA application inhibited the growth of the side on which it was applied. In the presence of exogenous ethylene, local IAA application still induced a peg on both the upper and the lower sides of the transition zone (Fig. 2B and F and Table 2). However, the peg was larger and swollen compared with the peg induced by IAA in the absence of exogenous ethylene (Fig. 2A, B, E and F). In addition, the transition zone of these seedlings bent downward but not toward the side of IAA application (Fig. 2B).
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Also examined was the effect of growing the seedlings in a vertical position in the presence or absence of ethylene for 24 h, and then applying IAA to one side of the transition zone. The seedlings were then grown under the same air/ethylene conditions until 72 h after imbibition. As expected, when the plants were grown without exogenous ethylene, the transition zone formed a peg on both sides and bent toward the side of IAA application (Fig. 2C and Table 3). In contrast, the seedlings treated with ethylene developed an enlarged peg on the side of IAA application only, and the transition zone bent downward but not toward the side of IAA application (Fig. 2D).
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Ethylene evolution and ACS mRNA accumulation in cucumber seedlings
The evolution of ethylene from cucumber seedlings over time was measured and it was found that, just after imbibition, the seedlings produced little ethylene (Fig. 3A). However, as the seedlings grew, the ethylene evolution increased (Fig. 3A). The concave and convex sides of the transition zone of 24-h-old seedlings that were just starting to develop a peg were then subjected to ethylene evolution analysis. This revealed that 1·7 times more ethylene evolved from the concave side compared to that coming from the convex side on a fresh weight basis (Fig. 3B). Ethylene evolution based on dry weight, protein content or cell number was not determined.
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ACS encodes the key enzyme that acts in the first regulatory step in the ethylene biosynthesis pathway. Four ACS cDNAs have been identified in the cucumber (Kamachi et al., 1997
; Trebitsh et al., 1997; Shiomi et al., 1998). To examine the involvement of the cucumber ACS genes in peg formation, the mRNA accumulations of the four CS-ACS genes were compared in the concave and convex sides of the transition zone of 24-h-old seedlings (Fig. 4A). To do this, quantitative RT-PCR Southern blot analysis was employed rather than northern blot analysis due to the low amounts of RNA that could be isolated from the seedling samples. The specificity of the probes used in RT-PCR Southern blot analysis was examined, and the probe of each CS-ACS1, CS-ACS2 and CS-ACS4 gene hybridized a corresponding RT-PCR product but did not hybridize those of the other three genes (data not shown). The CS-ACS1 mRNA accumulation in the concave side were greater than in the convex side, regardless of the number of PCR cycles used (Fig. 4). By contrast, the CS-ACS2 and CS-ACS4 mRNA accumulation in the two sides did not differ significantly. It was not possible to detect signals for CS-ACS3 mRNA in this analysis, which suggests that the CS-ACS3 mRNA accumulation in the transition zone of young cucumber seedlings may be extremely low.
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To reveal CS-ACS1 mRNA accumulation at tissue level, in situ hybridization was performed in 24-old-seedlings. It was confirmed that the CS-ACS1 probes detect in vitro synthesized CS-ACS1 mRNA but not those of other CS-ACS mRNA such as CS-ACS2, CS-ACS3 and CS-ACS4 by northern blotting analysis (data not shown). In situ hybridization experiments revealed that CS-ACS1 mRNA accumulated asymmetrically across the transition zone in 24-h-old seedlings grown in a horizontal position, since the CS-ACS1 mRNA signals in the epidermal and cortical cells in the concave side of the transition zone were greater than those in the convex side (Fig. 5).
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Auxin increases the CS-ACS1 mRNA accumulation in the shoot apices and fruits of the cucumber (Trebitsh et al., 1997
; Shiomi et al., 1998). It has also been shown that CsIAA1 mRNA accumulates asymmetrically across the transition zone of cucumber seedlings grown in a horizontal position (Fujii et al., 2000; Kamada et al., 2000) and that incubation of transition zone sections in IAA-containing buffer increases their CsIAA1 mRNA accumulation (Kamada et al., 2003). To determine whether the CS-ACS1 mRNA in the transition zone is induced by auxin, transition zone sections were incubated in a buffer containing IAA for different lengths of time and then the accumulation of CS-ACS1 and CsIAA1 mRNA was compared. The CS-ACS1 and CsIAA1 mRNA signals both began to increase within 45 min of the start of IAA treatment and kept increasing until 2 h later (Fig. 6). Thus, auxin increases CS-ACS1 expression in the transition zone of cucumber seedlings.
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DISCUSSION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Effect of ethylene treatment on the graviresponse of cucumber seedlings
Cucumber seedlings showed two different responses to exogenous ethylene treatment. First, when ethylene was applied before peg initiation, peg formation was inhibited. Secondly, when ethylene was applied after peg initiation, the peg became enlarged (Fig. 1 and Table 1). Thus, when ethylene is applied before peg initiation, it appears to prevent the cortical cells from changing their direction of growth (which leads to the protuberance of the peg) but when applied after this event it appears to facilitate the expansion of the peg (Fig. 1).
It has been suggested that peg initiation in the transition zone requires the accumulation of auxin levels above a particular threshold (Kamada et al., 2000). As shown in Fig. 2 and Table 2, when seedlings were grown with ethylene for 24 h (which would normally inhibit the development of a peg) and then lanolin paste containing auxin was placed on the one side of the transition zone, the presence of ethylene did not prevent the formation of the peg. Indeed, the presence of ethylene actually facilitated the auxin-induced elongation of the cortical cells perpendicular to the hypocotyl axis, thus resulting in enlarged pegs. These results indicate that ethylene alone cannot induce peg formation, and indeed its presence in large amounts before the peg is initiated blocks peg formation. After peg initiation by auxin, ethylene works together with auxin in enlarging the peg. Ethylene treatment during early developmental stage would inhibit peg initiation by affecting any mechanisms, such as polar auxin transport, auxin biosynthesis, auxin responses and/or developmental cell differentiation.
It was found that the effect of ethylene differs between the horizontally and vertically grown seedlings. The ethylene treatment with the vertically grown seedlings was more effective in the inhibition of peg formation compared with that with the horizontally grown seedlings. It is likely that the formation of the peg in the vertically grown seedlings does not initiate at the same time as that of the peg in the horizontally grown seedlings because the mRNA accumulation of CsIAA1 and CsIAA3 in the transition zone in the vertically grown seedlings increased more slowly compared with the horizontally grown seedlings (Fujii et al., 2000). In addition, the commencement of peg initiation in seedlings grown in a vertical position appears to be delayed compared with that of the horizontally grown seedlings (data not shown). This difference in the developmental stage of the seedlings could cause the difference in the effectiveness of ethylene in seedlings grown in a vertical and horizontal position.
It has been shown that ethylene modulates various growth and developmental events of plants in cooperation with auxin, including root growth (Rahman et al., 2001), root hair development (Pitts et al., 1998; Rahman et al., 2002), and apical hook formation (Lehman et al., 1996). With regard to the root hair development of Arabidopsis, Rahman et al. (2002) have suggested that auxin plays a critical role in root hair initiation in the absence of an ethylene response but that ethylene affects the auxin-derived elongation of the root hair. In addition, the ethylene resistance of Arabidopsis mutants such as aux1 and eir1 appears to be due to the decreased level of auxin caused by impaired auxin transport, which suggests that the response of the roots to ethylene requires auxin (Rahman et al., 2001). These observations are consistent with the authors' conclusion that the effect of ethylene on peg formation of cucumber seedlings depends upon auxin in the transition zone.
Roles of ethylene and auxin in the graviresponse of the cucumber seedlings
Although the application of exogenous ethylene during early seedling development inhibits peg formation, it is likely that endogenous ethylene levels before peg initiation are too low to generate this inhibitory effect. Supporting this is the fact that it has been shown, in Arabidopsis pea and turnip seedlings, that ethylene evolution, ACC levels, and ACC oxidase activity are low in the first 24 h of germination and that these levels only begin to increase after the emergence of radicles (Woeste et al., 1999; Petruzzelli et al., 2000; Rodriguez-Gacio and Matilla, 2001). The present analysis of the ethylene evolution of whole cucumber seedlings also indicates that ethylene biosynthesis early in seedling development is low (Fig. 3A). In addition, CS-ACS1 mRNA signals could not be detected in 18-h-old seedlings (data not shown). Thus, cucumber seedlings in early development do not seem to synthesize enough endogenous ethylene, and thus peg initiation is not inhibited.
mRNA accumulation of CS-ACS1, CS-ACS2 and CS-ACS4 was detected in the transition zone of 24-h-old seedlings and it was found that CS-ACS1 mRNA accumulates more abundantly in the concave side than in the convex side (Figs 4 and 5). It has been shown that when seedlings are grown in a horizontal position the concave side of the transition zone contains more auxin than the convex side (Kamada et al., 2003). Moreover, the mRNA accumulation of the auxin-inducible gene CsIAA1 are greater in the concave side of the transition zone than in the convex side (Fujii et al., 2000; Kamada et al., 2000). It was also found, in this study, that the concave side evolves more ethylene compared with the convex side (Fig. 3). In addition, it was shown that IAA treatment of the transition zone increases its mRNA expression of CS-ACS1 as well as CsIAA1 (Fig. 6). These results suggest that asymmetric accumulation of CS-ACS1 mRNA across the transition zone is due to the gravistimulation-induced asymmetric redistribution of auxin and would induce asymmetric ethylene biosynthesis. The asymmetric ethylene biosynthesis induced by CS-ACS1 would participate in the development of the peg. By contrast, ACC synthase genes such as CS-ACS2 and CS-ACS4 that do not differentially express between the concave and the convex sides would mediate the basal level of ethylene evolution in both sides. The possibility cannot be ruled out that cucumber ACS genes other than the CS-ACS1 gene might also contribute to the evolvution of ethylene differentially between the concave and the convex sides, because ACS genes consist of a multi-gene family; for example, Arabidopsis has 12 ACS genes (Chae et al., 2003), and cucumber may have more than four CS-ACS genes used in the present study.
Although it has been proposed that ethylene is involved in gravitropism (Wheeler et al., 1986), it is suggested that ethylene is not involved in the primary response to gravity (Lee et al., 1990; Madlung et al., 1999). This is supported by the observation that, while ethylene treatment of maize roots does increase the latent period of the gravitropic curvature of the roots, it also exaggerates the curvature of the roots beyond 90° (Lee et al., 1990). Furthermore, while inhibitors of ethylene activity or biosynthesis shorten the apparent latent period, they also reduce the final gravitropic curvature (Lee et al., 1990). Lee et al. (1990) concluded that ethylene does not mediate the primary differential growth response that causes curvature. Moreover, the minor reduction of the gravitropic responses of both the ethylene-insensitive never-ripe mutant of the tomato and the ethylene-overproducing epinastic mutant suggests that ethylene does not play a primary role in the gravitropic response, although a low level of ethylene is necessary for a full gravitropic response (Madlung et al., 1999). These observations together thus suggest that ethylene does not play a primary role in gravitropism but that it facilitates gravitropic responses as well as gravimorphogenic events such as peg formation. Thus, it appears that graviresponses and several other growth-regulating mechanisms, such as those described above that take place in root growth, root hair growth and hook formation share a common or similar mechanism that involves auxin and ethylene.
It has been shown previously that the lateral placement of the peg on the transition zone of a cucumber seedling grown in a horizontal position is regulated by the asymmetric distribution of auxin (Kamada et al., 2003). In this study, the relationship between ethylene and auxin in the formation of the peg was investigated. The results suggest that, before peg formation is initiated, ethylene biosynthesis is low (too low to inhibit peg formation). Once the auxin level exceeds a particular threshold, it initiates peg formation and induces the expression of CS-ACS1. This causes ethylene biosynthesis to increase. The biosynthesized ethylene, together with auxin, then facilitates peg development. Thus, it appears that, in cucumber seedlings, the relationship of peg initiation and development to the graviresponse is due to the redistribution of auxin and the consequent ethylene biosynthesis.
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ACKNOWLEDGEMENTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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We thank Drs D. Kim and M. Kamada of our laboratory for helpful discussions. This work was financially supported by grants from the Japan Space Forum (JSF) and the National Space and Development Agency (NASDA), the Institute of Space and Astronautical Science (ISAS), and by a Grant-in-Aid for Scientific Research (B) from the Japanese Society for the Promotion of Science to H.T., and Research Fellowships of the Japanese Society for the Promotion of Science for Young Scientists to Y.S.
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FOOTNOTES |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Present address: Fukuoka University of Education, 1-1 Akamabunkyomachi, Munakata, Fukuoka 811-4192, Japan 
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LITERATURE CITED |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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