JXB Advance Access originally published online on May 7, 2004
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Journal of Experimental Botany, Vol. 55, No. 401, pp. 1315-1323, June 1, 2004
© 2004 Oxford University Press
RESEARCH PAPER |
Isolation of cucumber CsARF cDNAs and expression of the corresponding mRNAs during gravity-regulated morphogenesis of cucumber seedlings
Received 9 December 2003; Accepted 2 March 2004
1 Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan
2 Department of Biochemistry, University of Missouri, 117 Schweitzer Hall, Columbia, Missouri 65211, USA
* Present address: Fukuoka University of Education, 1-1 Akamabunkyomachi, Munakata, Fukuoka 811-4192, Japan. To whom correspondence should be addressed. Fax: +81 22 723 8218. E-mail: nobuharu{at}ige.tohoku.ac.jp
Abbreviations: ARF, auxin response factor; ORF, open reading frame; DBD, DNA-binding domain; CTD, carboxyl-terminal domain.
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Abstract |
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Cucumber seedlings show positive gravitropism and bend in the transition zone between the hypocotyl and the root. The peg, a specialized protuberance, develops on the concave side of the bending transition zone. Auxin and the mRNA of an auxin-inducible gene (CsIAA1) isolated from cucumber are differentially accumulated across the transition zone during the gravity-regulated peg formation. In this study, five cDNAs of Auxin Response Factors (ARFs) from cucumber were isolated and their mRNA accumulation was compared with that of CsIAA1. The tissue specificity of CsARF2 mRNA accumulation was similar to that of CsIAA1. Because the structural character of CsARF2 predicts that it is a transcriptional activator, CsARF2 may be involved in the activation of CsIAA1 transcription, which plays a role in gravity-regulated peg formation. Neither gravity nor auxin affected mRNA accumulation of five CsARFs including CsARF2, suggesting that CsARF2 may be regulated at a post-transcriptional level to induce the asymmetric expression of the CsIAA1 gene in response to gravistimulation and auxin in cucumber seedlings.
Key words: Aux/IAA, auxin response factors (ARFs), Cucumis sativus, gravity, peg.
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Introduction |
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Cucumber seedlings show tropistic curvature and the lateral placement of a peg in response to gravity (Witztum and Gersani, 1975; Takahashi and Scott, 1994; Takahashi, 1997; Fig. 1). When cucumber seeds are placed in a horizontal position and germinate, the transition zone between hypocotyl and root curves downward as the result of gravitropism. Cortical cells in the concave side of the transition zone elongate perpendicularly to the hypocotyl axis and cause peg formation, whereas cells in the convex side elongate longitudinally along the hypocotyl axis and do not develop a peg (Witztum and Gersani, 1975; Takahashi and Scott, 1994). The attachment of the seed coat to the peg at the transition zone and elongation of the hypocotyl facilitate removal of the seed coat from the cotyledons. When cucumber seeds germinate under microgravity conditions, seedlings develop two pegs that are formed symmetrically on each side of the transition zone (Takahashi et al., 2000). These results suggest that peg formation itself does not require gravity (Takahashi et al., 2000). It is therefore implied that cucumber seedlings in a horizontal position on the ground have the potential of developing two pegs, but the peg on the convex side is suppressed in response to gravity (Takahashi et al., 2000). The phytohormone auxin is implicated as a regulator of gravity response in peg formation in cucumber (Witztum and Gersani, 1975; Takahashi and Suge, 1988; Kamada et al., 2000) as well as in gravitropism of plants (Trewavas, 1992; Friml et al., 2002).
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It is known that auxin induces rapid gene expression (reviewed by Abel and Theologis, 1996; Hagen and Guilfoyle, 2002). A variety of early/primary auxin-inducible genes, such as the Aux/IAA gene family, SAUR (small auxin up-regulated RNAs), GH3, and ACC synthase (ACS) genes, have been shown to have increased expression levels within several minutes after exposure to exogenous auxin (reviewed by Abel and Theologis, 1996; Hagen and Guilfoyle, 2002). Aux/IAA genes encode nuclear proteins that are short-lived (Abel et al., 1994; Abel and Theologis, 1995). Transfection assays with plant protoplasts suggest that Aux/IAA proteins function as active repressors of early/primary auxin-inducible genes (Ulmasov et al., 1997b; Tiwari et al., 2001). Mutations in Aux/IAA genes alter auxin response and auxin-related phenotypes in Arabidopsis (reviewed by Liscum and Reed, 2002). Recently, it has been shown that auxin-dependent degradation of Aux/IAA proteins is essential for auxin response and is regulated by the ubiquitin-mediated proteolytic pathway (Gray et al., 2001; Dharmasiri and Estelle, 2002). ARFs are transcription factors that bind with specificity to TGTCTC auxin-response elements (AuxREs), which are found in promoters of early/primary auxin-inducible genes (Ulmasov et al., 1997a, 1999b). Transient expression assays demonstrated that some ARFs are activators and other ARFs are repressors of auxin-inducible genes (Ulmasov et al., 1999a; Tiwari et al., 2003). The nph4/msg1/arf7 mutations impair auxin-inducible gene expression (Stowe-Evans et al., 1998) and affect auxin-dependent differential growth, including phototropism, gravitropism, and hook formation (Liscum and Briggs, 1996; Watahiki and Yamamoto, 1997; Stowe-Evans et al., 1998; Harper et al., 2000). ARFs contain a C-terminal domain related to motifs III and IV found in the C-terminal domain of Aux/IAA proteins, which allow ARFs to interact with other ARFs or with Aux/IAA proteins (Ulmasov et al., 1997a; Kim et al., 1997).
Although it has been shown that the expression of Aux/IAA genes is greater in the lower side of gravistimulated organ (Wyatt et al., 1993; Luschnig et al., 1998), expression of Aux/IAA genes has not been compared with the expression of ARF genes. It is therefore important to demonstrate the co-localization of Aux/IAA and ARF transcripts for understanding the mechanism of auxin action and the graviresponse in plants. It has previously been shown that, at the stage of peg initiation in horizontally grown cucumber seedlings, the mRNA of CsIAA1, a member of the Aux/IAA family, accumulates asymmetrically across the transition zone (Fujii et al., 2000; Kamada et al., 2000). To unravel the regulatory mechanism of auxin-inducible gene expression during gravity-regulated peg formation, five ARF cDNAs were isolated from cucumber and their mRNA accumulation pattern was compared with those of Aux/IAA mRNAs in cucumber seedlings.
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Materials and methods |
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Plant materials
Cucumber seeds (Cucumis sativus L. cv. Shinfushinari-jibai) 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 (40x30x10 mm) attached to the inner surface of a plastic cap of a Petri dish (60x60x60 mm). After supplying the block with 10 ml of distilled water, the plastic cap with the plastic foam holding the cucumber seeds was placed in a 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 position in darkness at 25 °C so that the seedlings could be grown in a horizontal position. For a comparison between the concave side and the convex side of the transition zone, the transition zone was excised from 24-h-old seedlings, cut in half longitudinally, frozen in liquid nitrogen, and stored at –80 °C until needed. To examine the effects of exogenous auxin on gene expression in the transition zone of cucumber seedlings, transition zones excised from 24-h-old seedlings were treated with 10–4 M IAA according to Theologis et al. (1985). To deplete endogenous auxin, the sections of excised transition zone were incubated in 15 mM sucrose containing 50 µg ml–1 chloramphenicol for 90 min, and then in incubation buffer (1 mM citrate, 1 mM PIPES, 15 mM sucrose, 50 µg ml–1 chloramphenicol, pH 6.0) for 30 min. They were then kept for 2 h in incubation buffer with or without IAA. IAA was added to the medium from a 50 mM stock solution in DMSO to a final concentration of 10–4 M. For control experiments, the same amount of DMSO was added to the medium. After incubation, the sections of excised transition zone were frozen in liquid nitrogen and stored at –80 °C until needed.
Cloning of cDNAs of CsARFs
A Cucumis sativus (cv. Shinfushinari-jibai) 
ZAPII cDNA library was derived from auxin-treated hypocotyls of 72-h-old cucumber seedlings that did not include the transition zone (Fujii et al., 2000). Four hundred thousand clones from the library were screened by using an Arabidopsis ARF6 cDNA probe that encoded the DBD (Ulmasov et al., 1999a). Filters were screened by using low-stringency hybridization (5x SSC, 5x Denhardt, 1% SDS, 0.1 mg ml–1 denatured herring testes DNA, 57 °C). After the first screening, more than 100 positive clones were obtained. The second screening resulted in the purification of 12 positive clones, and DNA sequencing identified 11 clones encoding CsARFs.
Genomic DNA gel blot analysis
Genomic DNA gel blot analysis was performed according to Yamasaki et al. (2000). To construct plasmids for preparing RNA probes, cDNA containing a 3' non-coding region were amplified and inserted into the BamHI–XhoI site of pBluescript KS. The primers for the construction are as follows: CsARF1, CS2BF 5'-TTGATGGATccACACCAGATACCTTATTGTCG-3', CS2XR 5'-TCGGTCTCgAgATTCGCAGCATTGCACTGGC-3'; CsARF2, CS12BF 5'-ACCTAgGATcCACAG AAAGATCCT CAACAGG-3', CS12XR 5'-CCACCcTCgAgGACTTGACAACT CAGAAACCC-3'; CsARF3, CS6BF 5'-AATGAGGATcCAGG TTTCTTGCAGTCTCC-3', CS6XR 5'-GCTATCTCgAGTC ATACAAAGCTG TCC-3'; CsARF4, CS5BF 5'-ATTCTggATCC CTTCTAATGCAAAATGGG-3', CS5XR 5'-AGGTActcGAGTCA CTCAGT AGTCAAGAGGC-3'; CsARF5, CS1BF 5'-AACAA GGATCCTTGGTGGGAAGAGCTATCG-3', CS1XR 5'-ATGAT CTCGAgTCCAATTACAAGCAGCTGC-3'.
Quantitative RT-PCR analysis
Total RNA was isolated from the transition zone of 24-h-old etiolated cucumber seedlings with TRI reagent (Sigma, MO, USA). Quantitative RT-PCR analyses were performed according to Yamasaki et al. (2001). The PCR conditions were 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The specific primers for PCR were designed as follows: ARF1-F, 5'-ACAGAGTTGT CAACTGCTGC-3' and ARF1-R, 5'-ACTTGCATGGATGAC TTCCCC-3' for CsARF1; ARF2-F, 5'-CACCAAGGTTTAC AAACGT GG-3' and ARF2-R, 5'-AATATAACACTACG GCGTGG-3' for CsARF2; ARF3-F, 5'-TCAGGTTCCTT GCATTCTCC-3' and ARF3-R, 5'-CCAGTCATACAAA GCTGTCC-3' for CsARF3; ARF4-F, 5'-CCCTTCTAATGC AAAATGGG-3' and ARF4-R, 5'-ACGATGATGTTGTAACCAG G-3' for CsARF4; ARF5-F, 5'-CTTGGTGGGAAGAGCTATCG-3' and ARF5-R, 5'-TTCCAATTACAAGCAGCTGC-3' for CsARF5; IAA1-F, 5'-ACCTCGAGATCACCGAGCTT-3' and IAA1-R, 5'-CATCTTCCAAGGCACATCCC-3' for CsIAA1; IAA2-F, 5'-GGCTACAGAACTGAGGCTTGG-3' and IAA2-R, 5'-GGAGC ACCATCCATGCTG-3' for CsIAA2; actin-F, 5'-GACATTC AATGTGCCTGCTATG-3' and actin-R, 5'-CATACCGATGA GAGATGGCTG-3' for CsActin. For hybridization, RNA probes of CsARFs were prepared using the plasmids described above. The RNA probe of CsActin was prepared using a pGEM-T vector containing the RT-PCR product amplified using actin-F and actin-R primers.
In situ hybridization
Cucumber seedlings were fixed with 0.05 M sodium phosphate buffer (pH 7.2) containing 4% paraformaldehyde and 0.25% glutaraldehyde overnight, maintaining the direction of cucumber seedlings against gravity. Infiltration of the excised sections with the fixative was achieved under aspiration for 5 min twice, with a subsequent secondary fixation for 90 min. After fixation, the samples were dehydrated with an ethanol series, replaced by butanol and finally embedded in Paraplast Plus (Oxford, Labware, MO, USA). In situ hybridization was performed according to Kamada et al. (2003), except that sections of 10 µm thick were used and hybridization was carried out at 55 °C.
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Results |
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Isolation of ARF cDNA clones from cucumber
The amino-terminal DNA binding domain (DBD) is highly conserved among ARFs in Arabidopsis (Ulmasov et al., 1999b). Therefore, an 1125 bp cDNA fragment encoding the DNA binding domain of ARF6 from Arabidopsis was used as a probe to screen a cDNA library of cucumber (Fujii et al., 2000) with reduced stringency. The same probe, under the same reduced stringency condition, recognized at least eight bands in Southern blot analysis of genomic DNA (data not shown). Twelve positive clones were isolated. Sequence analysis revealed that 11 of these clones were divided into five different ARFs of cucumber. These ARFs from cucumber are referred to as CsARF1, CsARF2, CsARF3, CsARF4, and CsARF5, with GenBank accession numbers from AB112671 [GenBank] to AB112675 [GenBank] .
With the exception of CsARF4, each CsARF contains a conserved amino-terminal region spanning about 300 amino acids (Fig. 2B). This region was shown to function as a DBD in ARF1 (Ulmasov et al., 1997a). In addition, the central region within the DBD is related to the B3 domain of VP1/ABI3, RAV1, and RAV2 (Ulmasov et al., 1997a; Kagaya et al., 1999; Guilfoyle and Hagen, 2001). By contrast, CsARF4 does not contain a start codon at the typical position, so that the conserved DBD is truncated by one fifth (Fig. 2A, B). Because it was not possible to detect the mRNA accumulation of CsARF4 (data not shown), it is likely to be a pseudogene. Alternatively, CsARF4 cDNA was not derived from mature mRNA and may not contain an entire ORF. In the carboxyl-terminal region, ARFs, except ARF3 and ARF17 (Guilfoyle and Hagen, 2001) in Arabidopsis, contain a carboxyl-terminal domain (CTD) related to domains III and IV found in Aux/IAA proteins (Ulmasov et al., 1999b). Each CsARF contains the CTD (Fig. 2C). Therefore, each CsARF identified in cucumber except for CsARF4 has the typical structure of ARF proteins.
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To compare cucumber CsARFs with ARFs of other plants, a multiple alignment of amino acid sequences in the conserved B3 domain of ARF proteins (Ulmasov et al., 1997a; Kagaya et al., 1999) was produced, and this alignment was used to construct the phylogenetic tree shown in Fig. 3. CsARF1 and CsARF2 fall into one clade which includes Arabidopsis ARF5 and ARF7. The middle regions between the DBD and CTD of transcriptional activators ARF5 to ARF8 in Arabidopsis are rich in Q, and Q residues represent from 10–23% of the amino acid residues in the middle regions (Ulmasov et al., 1999a). The middle regions from CsARF1 to CsARF4 are also rich in Q; namely, the Q content of the CsARF1, CsARF2, CsARF3, and CsARF4 middle region is 18%, 15%, 15%, and 14%, respectively. These characteristics of CsARF1, CsARF2, and CsARF3 are consistent with transcriptional activators of ARFs (i.e. while CsARF4 fits into this class, it is predicted to be a pseudogene; see above). The phylogenetic tree also shows that CsARF5 is closely related to the Arabidopsis transcriptional repressor ARF4 (Tiwari et al., 2003). CsARF5 as well as Arabidopsis ARF4 has an SP-rich middle region; namely, the SP content of CsARF5 and Arabidopsis ARF4 is 23% and 22%, respectively.
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The number of genes corresponding to CsARF1, CsARF2, CsARF3, CsARF4, and CsARF5 in cucumber was estimated by genomic DNA gel blot analysis (Fig. 4). When the CsARF1 gene was used as a probe, a single band was detected in the XbaI restriction fragments, although two major bands were observed in the HindIII restriction fragments. When the CsARF2, CsARF3, and CsARF4 genes were used as a probe, a single band was detected in each lane. When the CsARF5 gene was used as a probe, a single band was detected in the XbaI restriction fragments, although one major band accompanied by a minor one was detected in the HindIII restriction fragments. The band pattern of the CsARF4 fragment was similar to that of the CsARF5 fragment, but a minor band was not detected in the HindIII restriction fragments when the CsARF4 gene was used as a probe. These results indicate that the CsARF4 and CsARF5 gene probes hybridized to different DNA fragments. These results also suggest that genes corresponding to CsARF1, CsARF2, CsARF3, CsARF4, and CsARF5 are present as single copies in the genome of cucumber (Fig. 4).
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The mRNA accumulation pattern of ARF genes in response to gravity and auxin
Twenty-four hours after imbibition, the radicle of cucumber seedlings pointed downward, and the peg began to appear in the concave side of the bending transition zone (Fig. 1). To compare the mRNA accumulation of CsARFs with that of CsIAAs in the transition zone, quantitative RT-PCR analysis was used because the signals of CsARFs in northern blot analysis with total RNA were not clear. Signals of CsIAA1 mRNA in the concave side of the transition zone were greater than those in the convex side, especially at 20 and 22 cycles (Fig. 5A), as described previously (Kamada et al., 2000). In addition, signals of CsIAA2 mRNA in the concave side were greater than those in the convex side, especially at 18 and 20 cycles (Fig. 5A). However, there were no significant differences in mRNA levels of CsARF1, CsARF2, and CsARF3 between the concave side and the convex side of the transition zone. With regard to CsARF5, repeated experiments indicated that mRNA accumulation between the concave side and the convex side was not significantly different (data not shown).
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In the gravity response of cucumber seedlings, the asymmetric distribution of auxin in the transition zone may cause the asymmetric accumulation of CsIAA1 mRNA (Fujii et al., 2000; Kamada et al., 2000). To examine whether the expression of ARF genes in the segments of the transition zone is induced by auxin treatments or not, RT-PCR analyses was carried out with fewer cycle numbers so that signal intensities were less saturated than in Fig. 5A (Fig. 5B). The mRNA accumulation of CsIAA1 increased with applied IAA, while the mRNA abundance of CsIAA2 was less affected by IAA treatment than that of CsIAA1. The mRNA abundance of four CsARF genes did not increase with IAA treatment.
Comparison of mRNA accumulation between CsARF and CsIAA by in situ hybridization
To compare the spatial patterns of expression between CsARFs and CsIAAs in cucumber seedlings, their mRNA accumulations were analysed by in situ hybridization (Figs 6, 7). Eighteen hours after imbibition, the protuberance of the peg and bending had not yet appeared in the transition zone of seedlings grown in a horizontal position (Fig. 1). The mRNAs of CsIAA1 and CsIAA2 in 18-h-old seedlings were detected in epidermal cells, outer cells of the cortex, and vascular cells, and their mRNAs accumulated symmetrically (Fig. 6A, B). In the root, their mRNAs also accumulated in lateral root primordia. Twenty-four hours after imbibition, peg formation began on the concave side of the transition zone in horizontally grown seedlings (Fig. 1). In the transition zone of 24-h-old seedlings that were grown in a horizontal position, mRNAs of CsIAA1 and CsIAA2 in epidermal and cortical cells of the concave side accumulated more abundantly than those of the convex side (Fig. 7A, B, G, H). In the root, their mRNAs accumulated in vascular tissues and lateral root primordia.
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In 18-h-old seedlings grown horizontally, the mRNA accumulation of CsARF2 was similar to that of CsIAA1 and CsIAA2; i.e. CsARF2 mRNA was detected in epidermal cells, outer cells of the cortex, vascular cells, and lateral root primordia (Fig. 6C). In 24-h-old seedlings, the mRNA accumulation of CsARF2 was similar to that of CsIAA1 and CsIAA2; namely, it was detected mainly in vascular tissues and lateral root primordia, and weakly in epidermal and cortical cells (Fig. 7C, I). CsARF1 mRNA was detected in epidermal cells in 18- and 24-h-old seedlings and CsARF5 mRNA was detected in all tissues of the transition zone in 24-h-old seedlings although CsARF3 mRNA did not show a clear tissue-specific signal (data not shown). In addition, CsARF2 mRNA (Fig. 7I) and the other CsARF mRNAs (data not shown) examined by in situ hybridization did not show a clear asymmetric mRNA accumulation across the transition zone.
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Discussion |
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It has been suggested that auxin is a regulator of the response to gravity in peg formation in cucumber, i.e. the application of exogenous auxin to cucumber seedlings induces peg formation (Witztum and Gersani, 1975; Takahashi and Suge, 1988; Kamada et al., 2000), and when cucumber seedlings are grown in a horizontal position, auxin accumulates in the lower side of the transition zone where the peg develops (Kamada et al., 2003). It has previously been shown that the mRNA of CsIAA1, a cucumber auxin-inducible gene, accumulates more abundantly in the lower side than in the upper side of the transition zone (Fujii et al., 2000; Kamada et al., 2000). To clarify the regulatory mechanism for the expression of auxin-inducible genes, which may play a role in the graviresponse of cucumber seedlings, cDNAs of cucumber auxin response factors (CsARFs) were isolated and the tissue specificity and the gravity- or auxin-induction of their mRNA accumulation were compared with those of the mRNA of auxin-inducible genes (CsIAA1 and CsIAA2).
These results indicate that the tissue specificity of CsARF2 mRNA accumulation is similar to those of CsIAA1 and CsIAA2 mRNAs in cucumber seedlings, although gravistimulation or auxin treatment affects mRNA accumulation of CsIAA1 and CsIAA2, but not CsARF2. Transient assays in plant protoplasts have indicated that ARFs containing a Q-rich middle region between the DBD and CTD act as transcriptional activators of auxin-inducible genes (Ulmasov et al., 1999a; Tiwari et al., 2003). CsARF2 contains a Q-rich middle region and falls into the clade including the Arabidopsis ARF activators, ARF5 and ARF7, in phylogenetic analysis (Fig. 3), suggesting that CsARF2 in cucumber seedlings is a transcriptional activator of auxin-inducible genes including CsIAA1 and CsIAA2. Because only five cucumber ARFs were examined, it is possible that other CsARFs are also involved in the transcriptional activation of CsIAA1 and CsIAA2 in the transition zone.
In dry seeds, CsIAA1 and CsIAA2 mRNAs were not detected, but in 12-h-old seedlings, their mRNAs could be detected by northern hybridization (Fujii et al., 2000). Eighteen hours after imbibition, before the initiation of peg formation, CsIAA1 and CsIAA2 mRNAs could be detected, but their asymmetric accumulation was not observed by in situ hybridization (Fig. 6A, B). These results indicate that CsIAA1 and CsIAA2 mRNAs are initially transcribed symmetrically after germination. When cucumber seedlings initiate peg formation, auxin levels in the concave side of the transition zone become higher than in the convex side (Kamada et al., 2003). The asymmetric distribution of auxin may explain the asymmetric accumulation of the CsIAA1 and CsIAA2 mRNAs across the transition zone (Fujii et al., 2000; Kamada et al., 2000; Figs 5, 7).
None of the CsARF genes examined here, including CsARF2, showed auxin- or gravity-responsive expression in the transition zone of cucumber seedlings (Figs 5, 7C, I). It was previously reported that several ARF genes which were tested in Arabidopsis were not responsive to auxin (Ulmasov et al., 1999b). Asymmetric mRNA accumulations of CsIAA1 and CsIAA2, therefore, cannot be explained by differential accumulation of CsARF2 mRNA. However, it remains possible that post-transcriptional events could result in asymmetric distribution or activity of the CsARF2 protein as described below.
CsIAA1 and CsIAA2 are members of the Aux/IAA family (Fujii et al., 2000). Aux/IAA proteins act as repressors on auxin-inducible genes, including Aux/IAA genes (Ulmasov et al., 1997b; Tiwari et al., 2003), although auxin increases the abundance of Aux/IAA mRNAs (Abel and Theologis, 1996). It has been shown that auxin facilitates the degradation of Aux/IAA proteins which leads to the activation of auxin-inducible genes (Gray et al., 2001). In addition, Aux/IAA proteins interact with ARFs (Kim et al., 1997). When ARF activation domains and CTDs are fused to a heterologous DNA-binding domain (e.g. yeast GAL4 DBD) and cotransfected with reporter genes that contain GAL4 binding sites, there is a small induction of reporter genes by auxin (Ulmasov et al., 1999a; Tiwari et al., 2003). Their cotransfection together with Aux/IAA genes results in a significant increase of auxin responsiveness of reporter genes (Tiwari et al., 2003), implying that the co-expression of ARF activators and Aux/IAA repressors in a cell is important for the auxin-inducible gene expression. This idea is consistent with this study’s results that the tissue specificity of CsARF2 mRNA accumulation is similar to those of CsIAA1 and CsIAA2 mRNAs in cucumber seedlings (Figs 6, 7). It is therefore possible that before the transition zone senses gravity and initiates peg formation, the co-expression of CsARF2, CsIAA1, and CsIAA2 genes confers the ability to respond to auxin in the transition zone. As a result of the response of the transition zone to gravity, the auxin content in the convex side of the transition zone decreases, although the auxin content in the concave side is maintained at a higher level (Kamada et al., 2003). A decrease of auxin content in the convex side may suppress the degradation of CsIAA1 and CsIAA2 proteins, so that CsIAAs proteins may suppress the transcriptional activity of CsARF2. By contrast, the maintenance of higher auxin content for peg formation may facilitate the continuous degradation of CsIAA proteins and may permit CsARF2 transcriptional activity in the concave side of the transition zone. Thus, differential activity of CsARF2 could cause asymmetric morphogenesis in the transition zone of cucumber seedlings.
In summary, these results show that an ARF activator, together with Aux/IAA repressors, is expressed in the same tissues in which both factors could interact for transcriptional regulation. This co-expression might be required for the response to auxin, which may regulate the graviresponse in cucumber seedlings. Although the hypothesis still needs to be verified, the present study supports the idea that interaction between an ARF activator and Aux/IAA repressors is involved in the auxin response for the gravimorphogenesis of cucumber seedlings.
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Acknowledgements |
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This work was financially supported by grants from the Japan Space Forum and NASDA, the Institute of Space and Astronautical Science (Sagamihara, Japan), Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science to HT, and by a Fellowship Program for Japanese Scholars and Researchers to Study Abroad from the Ministry of Education, Culture, and Sports of Japan to NF. Financial support was also obtained from NSF grants to TG and GH.
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