JXB Advance Access originally published online on February 7, 2005
Journal of Experimental Botany 2005 56(413):909-920; doi:10.1093/jxb/eri083
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
The GUS reporter-aided analysis of the promoter activities of Arabidopsis ACC synthase genes AtACS4, AtACS5, and AtACS7 induced by hormones and stresses
1Department of Plant Biology and Ecology, Nankai University, Tianjin 300071, China
2Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, China
3Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242, USA
* To whom correspondence should be addressed. Fax: +852 2358 1559. E-mail: boningli{at}ust.hk
Received 11 August 2004; Accepted 15 November 2004
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ethylene biosynthesis in higher plants is regulated developmentally and environmentally. To investigate the regulation of ACC synthase gene expression, the promoters of Arabidopsis ACS genes, AtACS4, AtACS5, and AtACS7, were fused to a GUS reporter gene, and the recombinant transgenes were introduced into Arabidopsis to produce three groups of AtACS::GUS transgenic plants. Histochemic and fluorometric study of these transgenic plants revealed that promoters of AtACS4, AtACS, and AtACS7 are all active in dark-germinated seedlings. AtACS5 has the highest promoter activity in leaves of 2-week-old light-grown seedlings among the three AtACS genes studied. In the mature leaves, AtACS4 and AtACS7 genes are expressed in both veins and areoles, whereas AtACS5 is expressed at a higher level in the areoles and epidermal cells surrounding trichomes. The promoter activities of all these AtACS genes are found in the reproductive organs. AtACS5 and AtACS7 are highly expressed in petals, sepals, carpels, stamens, cauline leaves, inflorescence stems, and siliques, while AtACS4 expression is undetectable in the petals of open flowers. All three AtACS genes are expressed in root tissue. In the 2-week-old light-grown Arabidopsis, the AtACS4 promoter is responsive to the plant hormones IAA, ethylene, and ABA, and to darkness and wounding; the AtACS5 promoter to IAA, ABA, salt, high temperature, and wounding; and the AtACS7 promoter to GA3, ethylene, and ABA, and to darkness and salt. Low-temperature treatment abolishes the darkness-induced AtACS7 gene expression, but not that of AtACS4. Each AtACS gene has a unique expression profile during growth and development. It appears that at any developmental stage or any growth period of Arabidopsis, there is always a member of AtACS multigene family that is actively expressed.
Key words: ACC synthase, Arabidopsis, ethylene, gene expression, GUS histochemical staining, reporter, stress treatments
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ethylene, a two-carbon olefin, is a volatile hormone in higher plants. It has diverse regulatory functions in plant growth and development (Yang and Hoffman, 1984
; Abeles et al., 1992; Kende, 1993; Fluhr and Mattoo, 1996; Bleecker and Kende, 2000). As a senescing hormone, it promotes leaf-yellowing, climacteric fruit ripening, flower and leaf abscission. As a stress hormone, it is involved in biotic and abiotic stress responses of plants (Yang and Hoffman, 1984; Ge et al., 2000). In addition to its stimulatory effect on seed germination and root growth (Tanimoto et al., 1995; Clark et al., 1999; Petruzzelli et al., 2000), it also regulates gravitropism and phototropism (Harper et al., 2000; Lu et al., 2001, 2002). The production of ethylene in higher plants is from S-adenosyl-L-methionine (AdoMet) via the ACC-dependent pathway. The prevalent AdoMet is first converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase and then to ethylene by ACC oxidase (Adams and Yang, 1979). Sustainable production of ethylene through this pathway is accomplished by replenishing AdoMet through the methionine cycle at the expense of ATP. The regulation of the rate of ethylene biosynthesis in higher plants is at the step of ACC formation (Yang and Hoffmann, 1984). The key enzyme that regulates the level of ethylene production, including most cases of stress ethylene production, is ACC synthase (S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14
[EC]
; Boller et al., 1979; Yu et al., 1979). It is known that a higher level of ACC accumulation is stimulated by increasing chilling stress (Wang and Adams, 1982). Cadmium- and copper-elicited ethylene production was preceded or paralleled by enhanced ACC production (Yu and Yang, 1980). Wounding of winter squash mesocarp first triggers a rise in ACC synthase activity, followed by a surge in the ACC accumulation and ethylene production rate (Hyodo et al., 1985). IAA-induced ethylene biosynthesis (Abeles, 1966) is accompanied by an increase in ACC synthase activity and ACC content (Yu and Yang, 1979). Thus, the study of the environmental and developmental regulation of ACC synthase gene expression is of pivotal importance to the understanding of the molecular mechanisms by which ethylene regulates plant growth and development, as well as plant stress in response to environmental changes.
ACC synthase (ACS), the key enzyme in the ethylene biosynthesis pathway, is encoded by a multigene family (Theologis, 1992; Kende, 1993; Fluhr and Mattoo, 1996; Ge et al., 2000; Wang et al., 2002). It has been reported that there are eight ACS genes in tomato (Rottmann et al., 1991; Yip et al., 1992; Lincoln et al., 1993; Olson et al., 1995; Oetiker et al., 1997; Shiu et al., 1998), six in mung bean (Botella et al., 1992; Yoon et al., 1997), and five in potato (Destefano-Beltran et al., 1995; Schlagnhaufer et al., 1997). Complete sequencing of the rice genome (Yu et al., 2002) helped to identify five ACS genes from rice plants (van der Straeten et al., 2001). The expression of ACC synthase genes in higher plants is regulated developmentally and environmentally (Kende, 1993; Fluhr and Mattoo, 1996). They are induced either in such specific tissues as in the hypocotyl, leaf, root, tuber, petiole, flower petal, pistil, stamen, and fruit or in response to such biotic and/or abiotic environmental factors as radiation, Cu2+, Li+, ozone, wounding, amino-oxyacetic acid, cyclohexamide, EIX, protein kinase inhibitor, anaerobiosis (flooding), ethylene, IAA, benzyladenine, chilling, and pathogens (Ge et al., 2000; Wang et al., 2002). The expression of each member of the ACS multigene family is also differentially regulated in response to these factors. On the one hand, a member of the ACS gene family is able to respond to numerous different developmental and environmental signals (van der Straeten et al., 1990; Rottmann et al., 1991; Zarembinski and Theologis, 1993; Wang and Arteca, 1995; Clark et al., 1997; Oetiker et al., 1997; Shiu et al., 1998; Arteca and Arteca, 1999; Ge et al., 2000). On the other hand, a single developmental cue or environmental factor is able to induce the co-ordinated expression of several ACS genes (Olson et al., 1991; Subramaniam et al., 1996; Schlagnhaufer et al., 1997).
To address how ACS gene expression is regulated, the model plant Arabidopsis was selected as the subject of investigation for the reason that a wealth of information related to ethylene biosynthesis and action has already been accumulated about this plant. The complete sequencing of the Arabidopsis (Salanoubat et al., 2000) genome has revealed that there are 10 AtACS genes. All AtACS genes, except AtACS3, are known to be active and differentially expressed during Arabidopsis growth and development (Yamagami et al., 2003). Among these active AtACS genes, AtACS1 (At3g61510), AtACS2 (At1g01480), AtACS4 (At2g22810), AtACS5 (At5g65800), and AtACS6 (At4g11280) were best studied for their transcriptional activities with both RT-PCR and northern blot analysis (van der Straeten et al., 1992; Liang et al., 1992, 1996; Abel et al., 1995; Vahala et al., 1998; Miller et al., 1999; Smalle et al., 1999). Only the promoter activity of AtACS2 (At1g01480) was thoroughly analysed with a GUS reporter-aided approach (Rodrigues-Pousada et al., 1993). Thus, in the present study, a GUS reporter-aided approach was adopted to elucidate the promoter activities of AtACS4, AtACS5, and AtACS7. To that end, the 5' flanking regions of the three AtACS genes were cloned in order to perform the GUS reporter-aided histochemical and fluorometric study. It was found that there was always a member of AtACS gene family that remains active at any stage of Arabidopsis growth and development. AtACS5 had the highest promoter activity in the 2-week-old light-grown seedlings. At the reproductive stage, the expression AtACS5 gene was localized in areoles, whereas AtACS4 and AtACS7 were localized in both areoles and veins. The AtACS4 promoter became more active as plants grew older. All AtACS genes studied were expressed in root tissues. Treatment of these transgenic plants with hormones and stresses showed that AtACS genes were differentially regulated, suggesting that each AtACS gene had a unique expression programme during Arabidopsis growth and development.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant growth
Arabidopsis thaliana (L.) Heynh, Columbia (Col), seeds were obtained from the Arabidopsis Biological Resource Center (Columbus, OH). The growth of Arabidopsis has been described previously (Lu et al., 2001
, 2002). The transgenic seeds were surface-sterilized in a solution of 25% bleach plus 0.01% Triton X-100 for 10 min, then washed with sterilized water five times. These seeds were germinated and grown on the filter paper put on top of the selection media plate (0.5x Murashige and Skoog media containing 60 mg l–1 hygromycin, 0.8% agar, pH 5.7) for 2 weeks at 20±1 °C with cycles of 16 h light and 8 h darkness to produce Arabidopsis seedlings. The 2-week-old seedlings were transferred into soil and grown at 20±1 °C under continuous light for further experiments. Alternatively, they were subjected to different hormonal and stress treatments.
Hormone treatments
Ethylene treatment of the transgenic Arabidopsis seedlings was carried out in a sealed plastic box (8.8 l; Lu et al., 2001). Ethylene was injected to the box and diluted to a final concentration of 10 ppm. Two-week-old seedlings were exposed to 10 ppm ethylene for 48 h before they were harvested for protein extraction. Seedlings sealed in a box containing air were used as the control. In the cases of IAA, 6-BA, GA3, and ABA treatments, the filter papers, on which the transgenic seedlings were grown, were moistened with a solution containing either 100 µM ABA or 20 µM IAA or 20 µM GA3, or 20 µM 6-BA. The hormone treatments continued for 24 h before protein extracts were prepared from these seedlings. Seedlings grown on filter papers soaked with water or buffer without the addition of hormones were used as the control. All these treatments were carried out under a growth regime of 16/8 h light/darkness at 20±1 °C unless otherwise mentioned.
Abiotic stress treatments
For wounding treatment, a tweezer was used to squeeze leaves of the transgenic seedlings. The wounded seedlings were harvested 30 min later. Drought stress was carried out such that the transgenic seedlings were placed on the surface of a dry filter paper and kept in air for 3 h. The flooding stress was applied to transgenic seedlings by immersing seedlings in distilled water for 3 h. The high salt stress was achieved by wetting the filter papers with 0.3 M NaCl solution and transgenic seedlings were allowed to grow on it for 1 d. In the dark treatment, the transgenic seedlings grown on agar plates were wrapped with aluminium foil and kept at 20±1 °C for 2 d. As for the combined dark and low-temperature stress, seedlings were incubated in the dark for 2 d at 0 °C. To apply high-temperature stress to seedlings, the transgenic seedlings were exposed to 37 °C for 6 h in the dark. For all these stress treatments, plant samples were frozen in liquid nitrogen immediately following the treatments. Most of the treatments were carried out at 20±1 °C unless otherwise mentioned.
Construction of AtACS::GUS fusion genes
DNA fragments covering the 5' flanking regions of ACC synthase genes (ACS), –1211 to +9 of AtACS4 (Genbank accession number U23482, At2g22810), –1238 to –3 of AtACS5 (Genbank accession number L29260, At5g65800), and –1216 to –3 of AtACS7 (Genbank accession number AL161564, At4g26200), were amplified from Arabidopsis genomic DNA by polymerase chain reaction (PCR). The three pairs of primers used in the PCR reactions were ACS4S, 5'-ACG GAT CCG TTA CTT TTC AAA TCT TCC CTC-3'; ACS4R, 5'-TTG ACA ATT GAA CCA TGG CTT TTG TTC TTG-3'; ACS5S, 5'-ACG GAT CCA GGG AGC ATA AAT GGT CCT ATT-3'; ACS5R, 5'-TTC CAT GGT CTG TTT TTA AAG TCA AGA GAT-3'; ACS7S, 5'-ACG GAT CCA GTG TAA ATG GAT AGC CAC CCA-3'; ACS7R, 5'-ACC CAT GGT TTT CTT AGA GCT TCG AAC CTG-3'. The EcoRI site and the NcoI site was inserted into the 5' end forward primer and the 3' end reverse primer, respectively, to facilitate the cloning of the promoter sequence of the ACS gene. These PCR DNA fragments were first double-strand sequenced to confirm that there is no spontaneous mutation being introduced into these PCR products. These DNA fragments were then inserted into a binary vector pCAMBIA1301 and fused to a GUS reporter gene to create a recombinant transcription unit, AtACS::GUS. The transcript is terminated with a NOS terminator. The authenticity of each fusion construct was confirmed by DNA sequencing again.
Arabidopsis transformation
The recombinant AtACS::GUS fusion genes were introduced into Arabidopsis plants through Agrobacterium-mediated transformation using the floral infiltration method (Tague, 2001). The independent transformants were screened on 
MS media containing 60 mg l–1 hygromycin and 0.8% agar. The transformed seedlings of green and expanded leaves were screened out from the yellow-coloured non-transformed seedlings and transferred to soil after 2–3 weeks. The homozygous T2 plants were used for the GUS assay and histochemical staining. The PCR primers used to confirm the four recombinant transgenes in transgenic plants are PGUS1, 5'-TCG CGA TCC AGA CTG AAT GCC-3' and ACS4S, ACS5S, and ACS7S.
Histochemical GUS staining
GUS histochemical staining of transgenic Arabidopsis plants containing AtACS::GUS fusion constructs followed the previously described method (Crone et al., 2001). The image of blue-coloured whole plants of different developmental stages was recorded with a Canon scanner. The GUS-positive plant tissues were examined with a bright field microscope (Leica Q500MC, Cambridge, England) at a low magnification and photographed with a digital camera. GUS-stained tissues and plants shown in this paper represent the typical results of at least six independent lines for each construct.
Protein extraction and fluorometric GUS-assay
Plant protein extraction and assay for GUS activity were performed as previously described (Jefferson, 1989). The protein concentration of the extract was determined using the Bio-Rad Protein Assay kit. Fluorescence was measured in a Microplate Spectrofluorometer (SPECTRAmax GEMINI XS, Biocompare). The excitation wavelength was 360 nm and the emission wavelength 450 nm. Each assay was repeated three times. The data presented were collected from at least four independent lines for each construct. The standard curve was built using 1 to 10 µM 4-MU. Because 4-MU solution decays rapidly during storage, the 4-MU stock solution was only used for assay 2–3 times.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
GUS reporter-aided localization of AtACS gene expression during Arabidopsis growth and development
To investigate the promoter activities of AtACS4, AtACS5, and AtACS7 genes, three genomic DNA fragments containing the 5' flanking regions of these ACS genes from Arabidopsis were cloned using PCR. The promoter size of AtACS4, AtACS5, and AtACS7 is 1.2 kb, 1.23 kb, and 1.2 kb, respectively. These promoters of AtACS genes were fused to a commonly used E. coli reporter gene uidA encoding a ß-glucuronidase (GUS) reporter enzyme and introduced into Arabidopsis through Agrobacterium-mediated gene transfer (see Materials and methods). The T2 transgenic Arabidopsis plants containing the homozygous recombinant transgene were subjected to histochemical staining for GUS activity. The blue colour observed either in plant tissues or in cells indicates the active expression of an AtACS gene. Figure 1 shows the expression profiles of all three AtACS transgenes from vegetative to the reproductive stages. These GUS staining data are the representatives of at least six independent transgenic lines for each construct. In 3-d-old etiolated seedlings, GUS expressions were detected in both shoots and roots of AtACS4, AtACS5, and AtACS7 transgenic plants (Fig. 1, Aa, Ba, Ca). It appeared that the GUS-staining blue colours in the light-grown AtACS5 seedlings (1–2-weeks-old) were much deeper (Fig. 1, Bb, Bc) than those of AtACS4 and AtACS7 (Fig. 1 Ab, Ac, Cb, Cc), suggesting that the promoter activity for AtACS5 is the strongest among the three AtACS genes examined. However, as the transgenic plants mature to 4-weeks-old, older leaves of AtACS4::GUS and AtACS7::GUS transgenic plants appeared to have a deeper blue colour and clearly netted venation (Fig. 1, Ad, Cd, see the rosette leaf 1 and 2), whereas in AtACS5 leaves, there was no distinct netted venation exhibited by GUS staining, suggesting that the promoter activity of AtACS5 is largely restricted to areoles (Fig. 1, Ad, Bd, Cd; Fig. 2, Aa, Ba, Ca). Moreover, the three AtACS genes are all actively expressed in the root (Fig. 1, Ad, Bd, Cd). When the transgenic plants started to bolt and entered the reproductive stage, the promoter activities of all three AtACS genes were still recorded in leaf cells including both epidermal and guard cells (Fig. 2). Figure 2 (Aa, Ba, Ca) shows the close-up photos of leaves of the transgenic plants, which strongly support the differential regulation of three AtACS genes at the adult stage. One interesting observation was that in AtACS5::GUS plants, the epidermal cells surrounding the trichome had a higher level of gene expression (Fig. 2, Bb, Bc), implicating a novel role for the AtACS5 gene in the trichome formation.
|
|
GUS staining of the florets of AtACS4 plants showed no GUS expression (Fig. 3, Aa). By contrast, when the whole developing florets of both AtACS5::GUS and AtACS7::GUS plants were stained for GUS activity, it was found that these two genes were highly expressed in the fully opened older flowers (Fig. 3, Ba, Ca). Close-up examination of the fully opened flowers revealed that the expression profiles of AtACS5 and AtACS7 genes are similar. Both genes were highly expressed in sepals, the veins of flower petals, and sepals, stigmas, and filament (Fig. 3, Bb, Bc, Cb, Cc). GUS staining of anthers of both AtACS5 and AtACS7 (Fig. 3, Bd, Cd) indicated that only pollen of AtACS5 plants have GUS activity, suggesting that the AtACS5 gene is highly expressed in pollens.
|
GUS expression was detected in the stalk, inflorescence stem, and young siliques when the whole inflorescence of AtACS4 was histochemically stained (Fig. 4, Aa, Ab). Histochemical staining of the whole inflorescences of AtACS5 and AtACS7 transgenic plants showed that the stronger promoter activities of these two genes were localized primarily in the cauline leaves, silique cover, and the upper part of inflorescence stem (Fig. 4, Ba, Bb, Ca, Cb). AtACS4 promoter activity is much lower in all these tissues compared with those of AtACS5 and AtACS7. During the initial developmental stage of the silique, promoters of AtACS4, AtACS5, and AtACS7 are all active (Fig. 4, Ab, Bb, Cb; Fig. 5, Ab, Bb, Cb). As the silique matures, promoter activities of AtACS5 and AtACS7 still remain active and have relatively lower expression levels in the middle region of the inflorescence stems (Fig. 4, Bb, Cb). Figure 5 shows the close-up photographs of young and mature siliques of AtACS4, AtACS5, and AtACS7 plants. It appears that promoters of AtACS4, AtACS5, and AtACS7 are very active in silique covers, false septums and the suspensors (which connects the seed to the false septum in the young immature siliques; Fig. 5, Aa, Ab, Ba, Bb, Ca, Cb). As the seeds mature, the GUS staining in AtACS4 tissues decreases dramatically to an undetectable level (Fig. 4, Ac, Ad). Similarly, there is no GUS expression detected in the ageing suspensor of AtACS5 plant (Fig. 5, Bc, Bd). However, AtACS7 gene expression remains active in suspensor and seed as the silique becomes mature and turns yellow (Fig. 5, Cc, Cd).
|
|
Effects of plant hormones on AtACS gene expression in Arabidopsis seedlings
To investigate the subtle impact of plant hormones on the gene expression of members of the ACS family in Arabidopsis, a fluorometric assay for GUS activity was used. Without the hormone treatments, the specific activity of the GUS reporter enzyme in AtACS5, AtACS7, and AtACS4 seedlings was measured to be 351±44 pmol h–1 µg–1, 182±43 pmol h–1 µg–1, and 101±30 pmol h–1 µg–1, respectively. They are consistent with the histochemical staining results (Fig. 1). The promoter activity of AtACS5 gene is still the highest and is 2.0- and 3.5-fold of that of AtACS7 and AtACS4, respectively. When several major plant hormones were applied to 2-week-old light-grown Arabidopsis seedlings, GUS expression responded differently in these treated seedlings. Figure 6 shows the results of this study. GA3 enhances AtACS7 gene expression (Fig. 6). The promoters of both AtACS4 and AtACS5 respond to IAA treatment, and the GUS activity of AtACS4 and AtACS5 increases 25% and 33%, respectively (Fig. 6). Interestingly, AtACS4 and AtACS7 promoter activities are increased by 10 ppm ethylene, which strongly suggests that an autocatalytic regulation of ethylene biosynthesis by ethylene itself exists in Arabidopsis vegetative tissues. Application of exogenous ABA significantly increases the promoter activities of AtACS4, AtACS5, and AtACS7. No measurable increase was detected for all three genes upon 6-BA application. Due to the batch variation in Arabidopsis protein extracts, and the decay of the GUS substrate 4-MU, the basal GUS activities detected from the untreated control plants may vary (Figs 6, 7).
|
|
Effects of stresses on the expression of AtACS genes in Arabidopsis seedlings
Abiotic stresses are known to regulate ACC synthase gene expression. Seven abiotic stresses were therefore selected to treat 2-week-old seedlings. Again, a distinct gene expression profile was found for each of the three ACS isomers (Fig. 7). Among the three ACS promoters, only the AtACS7 promoter responds to a high salt treatment (0.3 M NaCl) stress. High-temperature stress (37 °C) triggers a slight increase in AtACS5 gene expression and has no measurable effect on other two AtACS promoter activities (Fig. 7). Wound stress enhances the expressions of both AtACS4 and AtACS5 genes (Fig. 7). Moreover, drought stress results in a slight decrease in the expression of AtACS5 and AtACS7 genes, whereas it does not have any effect on AtACS4 (Fig. 7). Submergence of these transgenic seedlings under water failed to induce GUS activities. Taken together, it is concluded that the AtACS4 promoter positively responds to darkness and wounding stresses, the AtACS5 promoter to high salt, high temperature, and wounding, and the AtACS7 promoter to high salt and darkness factors.
One more interesting finding from this study was that some AtACS genes could be induced by darkness (Fig. 7). Darkness is known to induce detaching leaf senescence and flower bud abscission (Becker and Apel, 1993;Weaver and Amasino, 2001; Fujiki et al., 2001). Ethylene, as a stress hormone, is also known to be involved in dark-induced and age-dependent leaf senescence in Arabidopsis (Yang and Hoffman, 1984; Oh et al., 1997). To address the question whether darkness is able to induce ethylene biosynthesis in the whole plant, the intact Arabidopsis seedlings were subjected to darkness treatment. To the authors' surprise, out of the three AtACS genes, both AtACS4 and AtACS7 were dark-inducible (Fig. 7A, C), but dark treatment had no effect on the AtACS5 promoter activity (Fig. 7B). When the darkness was supplemented with the low-temperature stress, no additive effect was observed on the enhancement of gene expression in all three AtACS isomers (Fig. 7). Instead, the low-temperature treatment abolished the darkness-induced AtACS7 promoter activity.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Phylogenetic analysis of the AtACS gene family has already shown that there exist two distantly related AtACS gene clusters in Arabidopsis (Ge et al., 2000
; Yamagami et al., 2003). It was decided to focus on one of the two AtACS gene clusters, which includes AtACS4, AtACS5, and AtACS7, to study the regulation of gene expression of these AtACS genes during plant growth and development, as well as in response to environmental stresses. Because the regulation of AtACS gene expression has been investigated at the steady-state transcript level using RT-PCR and northern blot analysis (van der Straeten et al., 1992; Liang et al., 1992, 1996; Abel et al., 1995; Arteca and Arteca, 1997; Vahala et al., 1998; Miller et al., 1999; Smalle et al., 1999; Yamagami et al., 2003) as well as the post-transcriptional level (Kim and Yang, 1992; Spanu et al., 1994; Li and Mattoo, 1994; Chae et al., 2003), the emphasis was on the investigation of AtACS gene expression at the transcriptional level using the GUS reporter gene-aided histochemical and fluorometric method. It is believed that this approach should complement what has been known for the regulation of AtACS gene expression in Arabidopsis. One exception in this regard is AtACS2 (At1g01840 previously known as AtACS1; Rodrigues-Pousada et al., 1993), whose gene expression has already been investigated with the GUS reporter-aided approach. In that experiment, a 1.4 kb-long promoter of AtACS2 (At1g01840) was fused to a GUS reporter and introduced into an Arabidopsis plant. The assay for GUS activities in Arabidopsis tissues has clearly shown that the AtACS2 gene is active in young leaf and flower tissue and its gene expression is dramatically reduced in older mature tissues (Rodrigues-Pousada et al., 1993). Thus, in the present study, 1.2 kb-long 5' flanking region DNA fragments of AtACS4, AtACS5, and AtACS7 were fused to a uidA gene to carry out the promoter analysis. These results have demonstrated that these DNA fragments possess diverse promoter functions. Some of the results obtained are consistent with the previously reported RT-PCR and RNA gel blot analysis data. For example, AtACS4 and AtACS5 have been shown to be auxin-inducible according to RNA gel blot analysis (Liang et al., 1992; Abel et al., 1995). Similarly, the GUS reporter-aided study conducted in this experiment also found these two genes to be auxin-inducible (Fig. 7). At the post-transcriptional level, cytokinin was reported to enhance the stability of the AtACS5 isozyme (Chae et al., 2003). In this GUS-mediated promoter analysis, it was also found that cytokinin was unable to induce AtACS5 gene expression (Fig. 6). Because the steady-state level of AtACS transcripts should be a consequence of the dynamic balance between transcription activity and transcript stability, the consistency found between the transcript's accumulation and GUS activity for AtACS4 and AtACS5 studied here strongly suggests that the transcriptional activity plays a predominant role in the regulation of AtACS gene expression. Thus, the histochemical and fluorometric data presented here should reveal the level and pattern of AtACS gene expression in vivo in Arabidopsis. Nonetheless, it is possible that there may exist additional cis-acting regulatory enhancers and repressors located either upstream of the 1.2 kb-long 5' flanking region or in the 3' flanking region of these AtACS genes. Exploration of these potential cis-acting elements may provide additional information about the regulation of AtACS4, AtACS5, and AtACS7 genes in Arabidopsis, which is beyond the scope of this study.
Mechanical wounding and the insect bites induce ethylene production and ACC synthase gene expression (Yang and Hoffmann, 1984; van der Straeten et al., 1990; Li et al., 1992; Liu et al., 1993). Ethylene, in turn, is required for mediating wound responses (O'Donnell et al., 1996), and for a maximal expression of defence genes (Weiss and Bevan, 1991) in the presence of JA (Xu et al., 1994). Numerous ACC synthase genes have been found to be wound-inducible (Ge et al., 2000). In the present study, it was found that AtACS4 and AtACS5 were responsive to wounding treatment while AtACS7 was not (Fig. 7). The data confirmed the previous finding that AtACS4 is wound-inducible (Liang et al., 1992). As wound induction of ACS transcript's accumulation is rapid (10–40 min; Olson et al., 1991; Huang et al., 1991), this wounding experiment was therefore designed to last for 30 min only.
In the literature, it has been well documented that ethylene inhibits its own biosynthesis in vegetative tissues. This so-called ethylene autoinhibition phenomenon (Kende, 1993) is supported by the findings that the ethylene-insensitive mutants etr1-1 and ein4-1 produce higher levels of ethylene (Bleecker et al., 1988; Guzman and Ecker, 1990). However, the phenomenon in which ethylene promotes ethylene biosynthesis, or so-called ethylene autocatalysis (Kende, 1993), has rarely been reported in Arabidopsis vegetative tissues. Van der Straeten et al. (1992) once reported that 2 h ethylene exposure transiently induced the steady-state level of the AtACS2 transcript (At1g01480) in Arabidopsis vegetative tissue. In the present study, it was found that ethylene stimulated ACC synthase gene expression in vegetative tissues for both AtACS4 and AtACS7. It strongly suggests that ethylene is able to stimulate AtACS promoter activities in vegetative tissues. Taken together, these data indicate that ethylene autocatalysis coexists in vegetative tissues with ethylene autoinhibition. Although it is difficult to calculate exactly how much ethylene is contributed from each ethylene-inducible AtACS isozyme to the total amount of ethylene released from the vegetative tissue, it is believed that the amount of ethylene that directly resulted from ethylene-increased AtACS4 and AtACS7 enzyme activities is relatively low compared with, for example, that from AtACS9 gene expression and enzyme activity because the kcat of AtACS9 isozyme is 10–25-fold that of AtACS4 and AtACS7 isozymes (Yamagami et al., 2003). AtACS5 and AtACS9 isozymes that have a higher turnover number may contribute more to overall ethylene production than the rest of the AtACS genes in vegetative tissue. Of course, the post-translational regulatory mechanism adds another layer of complexity to the precise calculation of each AtACS' contribution to overall ethylene production in vegetative tissue.
As AtACS4 and AtACS7 were found to be both dark-inducible and ethylene-inducible, it is therefore conceivable that the Arabidopsis leaf probably produces some ethylene upon darkness induction and, consequently, the dark-elicited ethylene stimulates more ethylene biosynthesis in the leaves. This situation appears to be the opposite of what was found for AtACS2, whose expression is light-inducible and ethylene-inducible (van der Straeten et al., 1992; Rodrigues-Pousada et al., 1993). Because the ethylene-insensitive Arabidopsis ein2-1 mutant has been shown to exhibit delayed dark-induced and age-dependent leaf senescence, it is possible that ethylene biosynthesis mediated by the dark-induced and ethylene-stimulated AtACS4 and AtACS7 gene expression may play an important role in promoting leaf senescence in the dark-treated tissues or in the senescing tissues. Interestingly, AtACS2 gene expression is switched off in these mature tissues or significantly reduced under darkness (Rodrigues-Pousada et al., 1993). Thus, a continuous and thorough investigation of gene expression of the AtACS family will help elucidate the role played by ethylene in regulating plant growth and development.
|
Acknowledgements |
|---|
The work was supported by grants from the Research Grant Council of Hong Kong (HKUST6105/01M and HKUST6102/02M) and the National Science Foundation of China (No. 30129001) awarded to Ning Li. It is partially supported by a grant from the National Science Foundation of China (No. 30070075 and 30470174) awarded to Ning Ning Wang. Ming-Che Shih was supported by a grant from the United States Department of Agriculture NRICGP No. 2003-00741. The authors would like to express their sincere thanks to Ms Di Guo for her contribution to the authentication of the transgenic AtACS::GUS plants.
|
Footnotes |
|---|
Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; AdoMet, S-adenosyl-L-methionine; ACS, ACC synthase; DTT, dithiothreitol; PCR, polymerase chain reaction, GUS, ß-glucuronidase.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Abel S, Nguyen MD, Chow W, Theologis A. 1995. ACS4, a primary indoleacetic acid-responsive gene encoding 1-aminocyclopropane-1-carboxylate synthase in Arabidopsis thaliana. Structural characterization, expression in Escherichia coli, and expression characteristics in response to auxin. Journal of Biological Chemistry 270, 19093–19099.
Abeles FB, Morgan PW, Saltveit ME. 1992. Ethylene in plant biology, 2nd edn. San Diego: Academic Press.
Abeles FB. 1966. Auxin stimulation of ethylene evolution. Plant Physiology 41, 585–588.
Adams DO, Yang SF. 1979. Ethylene biosynthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proceedings of the National Academy of Sciences, USA 76, 170–174.
Arteca JM, Arteca RN. 1997. A touch-induced ACC synthase in Arabidopsis. Plant Physiology 114, 1267–1267.[Abstract]
Arteca JM, Arteca RN. 1999. A multi-responsive gene encoding 1-aminocyclopropane-1-carboxylate synthase (ACS6) in mature Arabidopsis leaves. Plant Molecular Biology 39, 209–219.[CrossRef][ISI][Medline]
Becker W, Apel K. 1993. Differences in gene-expression between natural and artificially induced leaf senescence. Planta 189, 74–79.
Bleecker AB, Estelle MA, Somerville C, Kende H. 1988. Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science 241, 1086–1089.
Bleecker AB, Kende H. 2000. Ethylene: a gaseous signal molecule in plants. Annual Review of Cell and Developmental Biology 16, 1–18.[CrossRef][ISI][Medline]
Boller T, Herner RC, Kende H. 1979. Assay for and enzymatic formation of an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid in tomatoes. Planta 145, 293–303.[CrossRef]
Botella JR, Arteca JM, Schlagnhaufer CD, Arteca RN, Phillips AT. 1992. Identification and characterization of a full-length cDNA encoding for an auxin-induced 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments and expression of its mRNA in response to indole-3-acetic acid. Plant Molecular Biology 20, 425–436.[CrossRef][ISI][Medline]
Chae HS, Faure F, Kieber JJ. 2003. The eto1, eto2, and eto3 mutations and cytokinin treatment increase ethylene biosynthesis in Arabidopsis by increasing the stability of ACS protein. The Plant Cell 15, 545–559.
Clark DG, Gubrium EK, Barrett JE, Nell TA, Klee HJ. 1999. Root formation in ethylene-insensitive plants. Plant Physiology 121, 53–60.
Clark DG, Richards C, Hilioti Z, Lind-Iversen S, Brown K. 1997. Effect of pollination on accumulation of ACC synthase and ACC oxidase transcripts, ethylene production and flower petal abscission in geranium (Pelargonium hortorum L.H. Bailey). Plant Molecular Biology 34, 855–865.[CrossRef][ISI][Medline]
Crone D, Rueda J, Martin KL, Hamilton DA, Mascarenhas JP. 2001. The differential expression of a heat shock promoter in floral and reproductive tissues Plant, Cell and Environment 24, 869–874.[CrossRef]
Destefano-Beltran LJ, Van Caeneghem W, Gielen J, Richard L, Van Montagu M, Van der Straeten D. 1995. Characterization of three members of the ACC synthase gene family in Solanum tuberosum L. Molecular and General Genetics 246, 496–508.
Fluhr R, Mattoo AK. 1996. Ethylene: biosynthesis and perception. Critical Reviews in Plant Sciences 15, 479–523.[ISI]
Fujiki Y, Yoshikawa Y, Sato T, Inada N, Ito M, Nishida I, Watanabe A. 2001. Dark-inducible genes from Arabidopsis thaliana are associated with leaf senescence and repressed by sugars. Physiologia Plantarum 111, 345–352.[CrossRef][Medline]
Ge L, Liu JZ, Wong WS, Hsiao WLW, Chong K, Xu ZK, Yang SF, Kung SD, Li N. 2000. Identification of a novel multiple environmental factor-responsive 1-aminocyclopropane-1-carboxylate synthase gene, NT-ACS2, from tobacco. Plant, Cell and Environment 23, 1169–1182.[CrossRef]
Guzman P, Ecker JR. 1990. Exploiting the triple response of Arabidopsis to identify ethylene-related mutants. The Plant Cell 2, 513–523.
Harper RM, Stowe-Evans EL, Luesse DR, Muto H, Tatematsu K, Watahiki MK, Yamamoto K, Liscum E. 2000. The NPH4 locus encodes the auxin response factor ARF7, a conditional regulator of differential growth in aerial Arabidopsis tissue. The Plant Cell 12, 757–770.
Huang P, Parks JE, Rottman WH, Theologis A. 1991. Two genes encoding 1-aminocyclopropane-1-carboxylate synthase in zucchini (Cucurbita pepo) are clustered and similar but differentially regulated. Proceedings of the National Academy of Sciences, USA 88, 7021–7025.
Hyodo H, Tanaka K, Yoshisaka J. 1985. Induction of 1-aminocyclopropane-1-carboxylic acid synthase in wounded mesocarp tissue of Winter Squash fruit and the effects of ethylene. Plant and Cell Physiology 26, 161–167.
Jefferson RA. 1989. The GUS reporter gene system. Nature 342, 837–838.[CrossRef][Medline]
Kende H. 1993. Ethylene biosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 44, 283–307.[CrossRef][ISI]
Kim WT, Yang SF. 1992. Turnover of 1-aminocyclopropane-1-carboxylic acid synthase protein in wounded tomato fruit tissue. Plant Physiology 100, 1126–1131.
Li N, Parsons BL, Liu D, Mattoo AK. 1992. Accumulation of wound-inducible ACC synthase transcript in tomato fruit is inhibited by salicylic acid and polyamines. Plant Molecular Biology 18, 477–487.[CrossRef][ISI][Medline]
Li N, Mattoo AK. 1994. Deletion of the carboxyl-terminal region of 1-aminocyclopropane-1-carboxylic acid synthase, a key protein in the biosynthesis of ethylene, results in catalytically hyperactive, monomeric enzyme. Journal of Biological Chemistry 269, 6908–6917.
Liang XW, Abel S, Keller JA, Shen NF, Theologis A. 1992. The 1-aminocyclopropane-1-carboxylate synthase gene family of Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 89, 11046–11050.
Liang XW, Shen NF, Theologis A. 1996. Li+-regulated 1-aminocyclopropane-1-carboxylate synthase gene expression in Arabidopsis thaliana. The Plant Journal 10, 1027–1036.[CrossRef][ISI][Medline]
Lincoln JE, Campbell AD, Oetiker J, Rottmann WH, Oeller PW, Shen NF, Theologis A. 1993. LE-ACS4, a fruit ripening and wound-induced 1-aminocyclopropane-1-carboxylate synthase gene of tomato (Lycopersicon esculentum). Expression in Escherichia coli, structural characterization, expression characteristics, and phylogenetic analysis. Journal of Biological Chemistry 268, 19422–19430.
Liu DR, Li N, Dube S, Kalinsky A, Herman E, Mattoo AK. 1993. Molecular characterization of a rapidly and transiently wound-induced soybean (Glycine max L.) gene encoding 1-aminocyclopropane-1-carboxylate synthase. Plant Cell Physiology 34, 1121–1157.
Lu B, Yu HY, Pei LK, Wong MY, Li N. 2002. Prolonged exposure to ethylene stimulates the negative gravitropic responses of Arabidopsis inflorescence stems and hypocotyls. Functional Plant Biology 29, 987–997.
Lu BW, Pei LK, Chan WK, Zhang H, Zhu G, Li JY, Li N. 2001. The dual effects of ethylene on the negative gravicurvature of Arabidopsis inflorescence, an intriguing action model for the plant hormone ethylene. Chinese Science Bulletin 46, 279–283.
Miller JD, Arteca RN, Pell EJ. 1999. Senescence-associated gene expression during ozone-induced leaf senescence in Arabidopsis. Plant Physiology 120, 1015–1023.
O'Donnell PJ, Calvert C, Atzorn R, Wasternack C, Leyser HMO, Bowles DJ. 1996. Ethylene as a signal mediating the wound response of tomato plants. Science 274, 1914–1917.
Oetiker JH, Olson DC, Shiu OY, Yang SF. 1997. Differential induction of seven 1-aminocyclopropane-1-carboxylate synthase genes by elicitor in suspension cultures of the tomato (Lycopersicon esculentum). Plant Molecular Biology 34, 275–286.[CrossRef][ISI][Medline]
Oh SA, Park JH, Lee GI, Paek KH, Park SK, Nam HG. 1997. Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana. The Plant Journal 12, 527–535.[CrossRef][ISI][Medline]
Olson DC, Oetiker JH, Yang SF. 1995. Analysis of LE-ACS3, an ACC synthase gene expressed during flooding in roots of tomato plants. Journal of Biological Chemistry 270, 14056–14061.
Olson DC, White JA, Edelman L, Harkins RN, Kende H. 1991. Differential expression of two genes for 1-aminocyclopropane-1-carbolic acid synthase in tomato fruits. Proceedings of the National Academy of Sciences, USA 88, 5340–5344.
Petruzzelli L, Coraggio I, Leubner-Metzger G. 2000. Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase. Planta 211, 144–149.[CrossRef][ISI][Medline]
Rodrigues-Pousada RA, De Rycke R, Dedonder A, Van Caeneghem W, Engler G, Van Montagu M, Van Der Straeten D. 1993. The Arabidopsis 1-aminocyclopropane-1-carboxylate synthase gene 1 is expressed during early development. The Plant Cell 5, 897–911.
Rottmann WE, Petre GF, Oeller PW, Keller JA, Shen NF, Nagy BP, Tayler LP, Campbell AD, Theologis A. 1991. 1-aminocyclopropane-1-carboxylate acid synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence. Journal of Molecular Biology 222, 937–961.[CrossRef][ISI][Medline]
Salanoubat M, Lemcke K, Rieger M, et al. 2000. Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana. Nature 408, 820–822.[CrossRef][Medline]
Schlagnhaufer CD, Arteca RN, Pell EJ. 1997. Sequential expression of two 1-aminocyclopropane-1-carboxylase synthase genes in response to biotic and abiotic stresses in potato (Solanum tuberosum L.) leaves. Plant Molecular Biology 35, 683–688.[CrossRef][ISI][Medline]
Shiu OY, Oetiker JH, Yip WK, Yang SF. 1998. The promoter of LE-ACS7, an early flooding-induced 1-aminocyclopropane-1-carboxylate synthase gene of the tomato, is tagged by a Sol3 transposon. Proceedings of the National Academy of Sciences, USA 95, 10334–10339.
Smalle J, Haegman M, Mertens J, Vangronsveld J, Van Montagu M, Van Der Straeten D. 1999. The expression pattern of the Arabidopsis ACC synthase gene 1 during rosette leaf development. Journal of Experimental Botany 50, 1561–1566.
Spanu P, Grosskopf Dg, Felix G, Boller T. 1994. The apparent turnover of 1-aminocyclopropane-1-carboxylate synthase in tomato cells is regulated by protein-phosphorylation and dephosphorylation. Plant Physiology 106, 529–535.[Abstract]
Subramaniam K, Abbo S, Ueng PP. 1996. Isolation of two differentially expressed wheat ACC synthase cDNAs and the characterization of one of their genes with root-predominant expression. Plant Molecular Biology 31, 1009–1020.[CrossRef][ISI][Medline]
Tague BW. 2001. Germ-line transformation of Arabidopsis lasiocarpa. Transgenic Research 10, 259–267.[CrossRef][ISI][Medline]
Tanimoto M, Roberts K, Dolan L. 1995. Ethylene is a positive regulator of root hair development in Arabidopsis thaliana. The Plant Journal 8, 943–948.[ISI][Medline]
Theologis A. 1992. One rotten apple spoils the whole bushel: the role of ethylene in fruit ripening. Cell 70, 181–184.[CrossRef][ISI][Medline]
Vahala J, Schlagnhaufer CD, Pell EJ. 1998. Induction of an ACC synthase cDNA by ozone in light-grown Arabidopsis thaliana leaves. Physiologia Plantarum 103, 45–50.
Van der Straeten D, Rodrigues-Pousada RA, Villarroel R, Hanley S, Goodman HM, van Montagu M. 1992. Cloning, genetic-mapping, and expression analysis of an Arabidopsis thaliana gene that encodes 1-aminocyclopropane-1-carboxylate synthase. Proceedings of the National Academy of Sciences, USA 89, 9969–9973.
Van Der Straeten, D, Van Wiemeersch L, Goodman HM, Van Montagu M. 1990. Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-1-carboxylate synthase in tomato. Proceedings of the National Academy of Sciences, USA 87, 4859–4863.
Van der Straeten D, Zhou ZY, Prinsen E, Van Onckelen HA, Van Montagu MC. 2001. A comparative molecular-physiological study of submergence response in lowland and deepwater rice. Plant Physiology 125, 955–968.
Wang CY, Adams DO. 1982. Chilling-induced ethylene production in cucumbers (Cucumis sativus L.). Plant Physiology 69, 424–427.
Wang KLC, Li H, Ecker JR. 2002. Ethylene biosynthesis and signaling networks. The Plant Cell 14, S131–S151.
Wang TW, Arteca RN. 1995. Identification and characterization of cDNAs encoding ethylene biosynthetic enzymes from Pelargoniumxhortorum cv. Snow Mass leaves. Plant Physiology 109, 627–636.[Abstract]
Weaver LM, Amasino RM. 2001. Senescence is induced in individually darkened Arabidopsis leaves, but inhibited in whole darkened plants. Plant Physiology 127, 876–886.
Weiss C, Bevan M. 1991. Ethylene and a wound signal modulate local and systemic transcription of win2 genes in transgenic potato plants. Plant Physiology 96, 943–951.
Xu Y, Chang PFL, Liu D, Narasimhan ML, Raghothama KG, Hasegawa PM, Bressan RA. 1994. Plant defense genes are synergistically induced by ethylene and methyl jasmonate. The Plant Cell 6, 1077–1085.[Abstract]
Yamagami T, Tsuchisaka A, Yamada K, Haddon WF, Harden LA, Theologis A. 2003. Biochemical diversity among the 1-aminocyclopropane-1-carboxylate synthase isozymes encoded by the Arabidopsis gene family. Journal of Biological Chemistry 278, 49102–49112.
Yang SF, Hoffman NE. 1984. Ethylene biosynthesis and its regulation in higher plants. Annual Review of Plant Physiology 35, 155–189.[CrossRef][ISI]
Yip WK, Moore T, Yang SF. 1992. Differential accumulation of transcripts for four tomato 1-aminocyclopropane-1-carbolic acid synthase homologs under various conditions. Proceedings of the National Academy of Sciences, USA 89, 2475–2479.
Yoon IS, Mori H, Kim JH, Kang BG, Imaseki H. 1997. VR-ACS6 is an auxin-inducible 1-aminocyclopropane-1-carboxylate acid synthase gene in mungbean (Vigna radiata). Plant Cell Physiology 38, 217–224.
Yu J, Hu SN, Wang J et al., 2002. A draft sequence of the rice genome (Oryza sativa