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First published online October 9, 2003; 10.1104/pp.103.028183 Plant Physiology 133:1158-1169 (2003) © 2003 American Society of Plant Biologists Analysis of the Alternative Oxidase Promoters from Soybean1Plant Molecular Biology Group, Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley 6009, Western Australia, Australia
Alternative oxidase (Aox) is a nuclear-encoded mitochondrial protein. In soybean (Glycine max), the three members of the gene family have been shown to be differentially expressed during normal plant development and in response to stresses. To examine the function of the Aox promoters, genomic fragments were obtained for all three soybean genes: Aox1, Aox2a, and Aox2b. The regions of these fragments immediately upstream of the coding regions were used to drive  -glucuronidase (GUS) expression during transient transformation of soybean suspension culture cells and stable transformation of Arabidopsis. The expression patterns of the GUS reporter genes in soybean cells were in agreement with the presence or absence of the various endogenous Aox proteins, determined by immunoblotting. Deletion of different portions of the upstream regions identified sequences responsible for both positive and negative regulation of Aox gene expression in soybean cells. Reporter gene analysis in Arabidopsis plants showed differential tissue expression patterns driven by the three upstream regions, similar to those reported for the endogenous proteins in soybean. The expression profiles of all five members of the Arabidopsis Aox gene family were examined also, to compare with GUS expression driven by the soybean upstream fragments. Even though the promoter activity of the upstream fragments from soybean Aox2a and Aox2b displayed the same tissue specificity in Arabidopsis as they do in soybean, the most prominently expressed endogenous genes in all tissues of Arabidopsis were of the Aox1 type. Thus although regulation of Aox expression generally appears to involve the same signals in different species, different orthologs of Aox may respond variously to these signals. A comparison of upstream sequences between soybean Aox genes and similarly expressed Arabidopsis Aox genes identified common motifs.
The alternative oxidase (Aox) is one of the most studied components of the plant electron transport chain since the first reports of its activity in 1925 (Day et al., 1980  ; Moore and Siedow, 1991). It is encoded in the nucleus and imported into mitochondria via the general import pathway and is responsible for cyanide-insensitive respiration. Together with the rotenone-insensitive internal and external NAD(P)H dehydrogenases, it provides additional branch points for input and output in the plant mitochondrial electron transport chain.
With the development of an Aox monoclonal antibody (Elthon et al., 1989
In all species examined to date, Aox is encoded by a small multigene family that displays tissue, developmental, and environmental regulation (Whelan et al., 1996
The members of the Aox family are differentially expressed in plant tissues, with much of this work carried out in soybean. These studies have been aided by the ability to separate and identify the individual proteins by SDS-PAGE and by N-terminal protein sequencing (Finnegan et al., 1997
Multiple signaling pathways may be involved in the induction of Aox. Induction of Aox1 in soybean cell cultures by citrate is sensitive to the protein kinase inhibitor staurosporine, but induction by reactive oxygen species or antimycin A is not (Djajanegara et al., 2002
To gain further insight into the mechanism of Aox gene regulation, we have used regions upstream of the soybean Aox genes to drive the expression of the
Isolation of Soybean Aox Genomic Clones for Promoter Analysis
To carry out promoter analysis of all Aox genes from soybean, it was necessary to isolate the genomic clones for GmAox2a and 2b, previously called Aox2 and Aox3, respectively (Whelan et al., 1996
Soybean suspension culture cells were used initially to determine the promoter activity of the available GmAox1, GmAox2a, and GmAox2b upstream regions. The regions 1.5 kb upstream of the translational start site of GmAox1 and 2.1 and 2.2 kb upstream from the translational start site of GmAox2a and 2b were fused independently to the GUS reporter gene in pCAMBIA1301 (accession no. AF234297). The GUS constructs were then used to transiently transform soybean cells using a biolistic gun (Iida et al., 1990
Before undertaking these transient transformations, we determined which Aox proteins were present in the cultured soybean cells throughout a 9-d growth period. Previously, it has been shown that the proteins encoded by the three soybean genes have distinct mobilities on SDS-PAGE gels, and thus an Aox protein detected by immunoblotting can be assigned to a gene (Finnegan et al., 1997
Transformations using upstream regions of the three soybean genes showed that the fragments from GmAox1 and 2b, but not that from GmAox2a, had promoter activity in cells (Fig. 3). This is in agreement with the immunoblot analysis where endogenous GmAox2a protein was never observed (Fig. 2A). With the two active promoters, GmAox2b promoter strength was almost 10 times that of GmAox1 at maximal activity of both promoters (–242 construct for GmAox1 versus –1,444 construct for GmAox2b; Fig. 3). In addition, GUS activity driven by the GmAox1 upstream fragment did not display a significant difference when d-6 and -9 cells were transformed. With the GmAox2b upstream fragment, transforming d-9 cells resulted in promoter activity that was consistently 80% the strength obtained compared with transforming d-6 cells. GUS expression under the control of the CaMV 35S promoter was always slightly higher in older cells, indicating that the lower activity observed when transforming d-9 cells with the GmAox2b fragment was specific for the promoter activity of this fragment.
The GmAox1 1.5 kb upstream fragment showed no significant promoter activity above background until the sequences upstream of –470 with respect to the translational start were deleted (Fig. 3). A further 75-bp deletion to position –395 bp, doubled the promoter activity, and deletion of another 153 bp, to –242 bp, further increased activity again to the maximum observed. Removal of another 126 bp, to position –116, abolished activity to background levels. These results indicate that there are negative elements between –1,522 and –470 and between –470 and –242 that control the expression of GmAox1. Because no promoter activity was observed with the complete –1,522 upstream region of GmAox1 in d-6 cells but Aox1 protein was present (Fig. 2), it appears that additional positive elements exist either further upstream or in the 3' end of this gene. Analysis of the GmAox2b promoter also suggested the presence of repressor elements. Deleting 670 bp from the upstream end of the longest fragment created a construct with –1,444 bp upstream of the translational start, which consistently displayed 1.75 times the promoter activity of the full-length fragment. Removal of approximately 1 kb between –1,223 bp and –220 bp resulted in a large loss of GUS activity, indicating that positive elements driving expression were present in this region. Noticeably, the expression driven by the –1,223 fragment was up to 10-fold greater than that driven by the GmAox1 upstream fragments and 1.5- to 3-fold stronger than that of the CaMV 35S promoters.
To determine the tissue specificity of the soybean Aox upstream regions, we carried out stable transformation of Arabidopsis and analyzed the expression of GUS using histochemical staining. Although soybean and Arabidopsis diverged more than 100 million years ago, Arabidopsis has been extensively used in the analysis of promoter regions from a wide variety of plant species (Ermolaeva et al., 2003
Histochemical analysis of transgenic plants where GUS expression was driven by the entire upstream fragments outlined in Figure 3, yielded a whole-plant perspective of the promoter activity of the Aox fragments. The GmAox1 upstream fragment drove expression throughout the plant and was strongest in vascular tissue, especially that of the root (Fig. 4, i and ii). The vascular activity of this fragment seemed to increase in leaves with increasing age (Fig. 4, iii–v). The GmAox2a promoter only drove expression in aerial parts of the plant, and again this appeared strongest in vascular tissue (Fig. 4, vi). Message and protein derived from this gene in soybean are also found only in the shoot (Finnegan et al., 1997
Deletion analysis of the GmAox2a upstream fragment showed that deletion of approximately 2 kb allowed root expression (Fig. 5, vii–ix). The –278 bp of promoter showed weak GUS expression in Arabidopsis compared with the –2,268 and –814 constructs (Figs. 4, vi, and 5, vii versus viii). An additional deletion to produce the –192 construct resulted in strong activity in root vascular tissue (Fig. 5, ix).
Each of the three upstream fragments showed differential promoter activity throughout the flower. Although all three fragments showed some activity in sepals (Fig. 5, i–iii), only the GmAox2a fragment supported strong activity in the anthers (Fig. 5 ii). Cross section of the anthers indicated that the expression was not in the microspores but in the tapetal cells that surround the pollen sacs (Fig. 5, iv). This anther expression was developmentally regulated and did not appear in mature flowers. (Fig. 5, ii, inset). In contrast, GmAox2b promoter activity was observed in the sepals at all stages of floral development. In mature flowers, activity was detected in the filaments supporting the anthers, the tip of the style, and the stigma (Fig. 5, v). The GmAox1 upstream fragment drove expression throughout the whole of the carpel and in the filaments (Fig. 5, vi).
To aid the identification of upstream regions that control the expression of Aox, the soybean Aox upstream sequences were compared with those from Arabidopsis. These are the only dicot plants for which upstream sequences for all Aox genes are available. To ensure that comparisons were made between promoters driving the expression of genes with similar expression profiles, the expression patterns of the five Arabidopsis Aox genes was first analyzed in various tissues. Additionally, we analyzed the expression of three ribosomal genes: Rps1, a nuclear-encoded plastid ribosomal protein; Rps13, a nuclear-encoded mitochondrial ribosomal protein; and Rps8, a cytosolic ribosomal protein (Adams et al., 2002 The expression pattern of the various Arabidopsis Aox genes differed considerably from each other. AtAox1a was the most highly expressed isoform, being expressed in all tissues and showing a noticeable increase in cotyledons from d 4 to 10 (Fig. 6). For AtAox1b, significant expression was only detected in flower buds, at about 20% of the level observed for AtAox1a (Fig. 6). AtAox1c was the second most dominant isoform and was expressed in most tissues examined, albeit at significantly lower levels than AtAox1a. Its expression decreased in cotyledons from d 4 to 10, in a pattern opposite to that observed for AtAox1a. A similar pattern was also seen in leaves, where AtAox1c expression decreased over d 10 to 25. AtAox1c expression was highest in flowers and floral buds (Fig. 6). Expression of AtAox1d was barely detectable in any tissues, even in floral tissues, in contrast to the other genes (Fig. 6). AtAox2 expression was also very low in the tissues examined, being about 100-fold less than AtAox1a, -b, and -c (Fig. 6).
It is interesting that the expression of orthologous Aox genes in soybean and Arabidopsis (GmAox1 versus AtAox1a, -b, -c, and -d; GmAox2a and -2b versus AtAox2) display quite different expression patterns in planta, suggesting that their functional roles have diverged since these two plant lineages separated during evolution (Ermolaeva et al., 2003
We report here the genomic sequence encompassing the soybean GmAox2a and -2b genes. Together with the previously sequenced soybean GmAox1 gene, this allowed an initial characterization of the Aox promoter regions for the whole family. Differences between the previously published cDNA sequences and the genomic sequences were found that resulted in changes in one amino acid residue each in GmAox2a and -2b. These amino acids are at positions that are not conserved between plant Aox proteins and therefore appear to represent allelic variation only.
Soybean Aox2a and -2b represent a tandem duplication that, by sequence comparison with soybean bacterial artificial chromosome ends, maps to linkage group A2, near the RFLP marker A110 (accession no. AZ044777; Soybase, available at http://soybase.org). Arabidopsis Aox2 is located in a region represented by clone MSJ1, which is within 0.15 Mb of clone MBK5. This clone represents part of a region of known synteny between Arabidopsis chromosome 5 and soybean linkage group A2 (Grant et al., 2000 The genomic regions immediately upstream of all three of the soybean Aox coding sequences were examined for promoter activity in both soybean cells and Arabidopsis plants. All three upstream fragments had promoter activity and were able to drive expression of the GUS reporter gene in Arabidopsis. The GmAox1 and Aox2b upstream regions functioned in soybean cells in a manner consistent with the expression of the endogenous GmAox proteins. By making deletions to the upstream regions, functional regions could be assigned.
Both the GmAox1 and GmAox2b upstream sequences appear to possess negative elements that repress their expression. The GmAox1 promoter showed maximal activity with the fragment that only contained 242 bp immediately upstream of the coding region, whereas no GUS activity was detected with fragments extending more than 470 bp upstream. This suggests that repressor(s) of expression are present between positions –1,522 and –470 relative to the translational start. Because GUS activity continued to increase with subsequent deletions up to –242 bp, it is likely that further negative elements are present between –470 and –242. Further deletions inhibited reporter gene activity, indicating that positive element(s) exist between –242 and –116. Because the complete upstream region did not possess promoter activity, it appears that additional elements exist for this gene. Positive elements greater than 1.5 kb upstream have been previously characterized in soybean (Marsolier et al., 1995
It is interesting to note that GmAox1 is only detected in soybean tissues upon stress treatments (Djajanegara et al., 2002
The deletion of the distal 660 bp from the 2,104-bp fragment of GmAox2b resulted in 1.75 times greater GUS activity. However, even with the negative elements present, the GUS activity driven by this promoter was twice as high as from the CaMV 35S promoter, which is considered a strong constitutive promoter (Benfey et al., 1990
GUS activity driven by the GmAox2a upstream region was prominent in the anthers of Arabidopsis, perhaps indicative of a role for Aox in pollen development. Such a role has been suggested previously when an Aox antisense construct under the control of a tapetum-specific promoter resulted in reduced pollen viability in tobacco (Kitashiba et al., 1999
Overall, these results suggest that there is some divergence of gene responsiveness between Arabidopsis and soybean, as illustrated by the differences in the expression of the Aox orthologs in the two plants. Although the soybean Aox2 locus is orthologous with that of Arabidopsis Aox2, the expression patterns of the two GmAox2 genes are more closely related to the Arabidopsis Aox1 genes (especially Aox1a and Aox1c). For example, AtAox1a transcript levels increased with age in cotyledons, whereas the levels of AtAox1c transcripts decreased. A similar pattern has been demonstrated in soybean where the levels of GmAox2a mRNA is initially high and decreases over development while the initially low levels of GmAox2b increase (McCabe et al., 1998
Plant Material
Suspension cells developed from soybean (Glycine max cv Stevens) leaf tissue (Djajanegara et al., 2002 For transient expression analysis, soybean suspension cells of the appropriate age were aseptically coated onto filter paper (55-mm diameter, Whatman No. 1, Maidstone, UK), which was then placed onto solid media containing soybean culture media supplemented with 200 mM mannitol and incubated for 2 h at 28°C in the dark. Before transformation, 5 µg each of a GUS reporter construct and the luciferase calibration construct was precipitated onto 5 mg of 1-µm (average diameter) gold particles (Chempur, Karlsruhe, Germany) using 2.5 M CaCl2 (Sigma-Aldrich, St. Louis) and 100 mM spermidine (Sigma-Aldrich). For each bombardment, 0.5 mg of particles was used. Bombardment was carried out under vacuum using helium pressure of 1,400 kPa. After transformation the cells were incubated for 48 h at 28°C in the dark on the media-impregnated filter paper discs.
A soybean genomic library (BD Biosciences Clontech, Sydney) was screened by standard procedures using 32P-labeled probes synthesized from GmAox2a and Aox2b cDNA fragments (Sambrook et al., 1989
Total RNA was isolated from Arabidopsis tissue using a commercial kit (RNeasy Plant mini, Qiagen, Clifton Hill, Australia). Each batch of total RNA was treated with DNaseI (Roche Diagnostics) and then treated with DNAfree (Ambion, Austin, TX) to remove contaminating DNA and reverse transcribed using Expand Reverse Transcriptase according to the manufacturer's recommendations (Roche Diagnostics). Random primers (Roche Diagnostics) were used in the reverse transcription for the analysis of transcripts in different tissues, and oligo(dT) primer (Roche Diagnostics) was used for the cotyledon transcript analysis. The QIAquick PCR Purification Kit (Qiagen) was used to purify the cDNA before real-time PCR analysis.
Total RNA was reverse transcribed with the appropriate reverse primer (see below) using Expand Reverse Transcriptase according to the manufacturer's recommendations (Roche Diagnostics). Five microliters of the resulting cDNA was used for PCR with the appropriate forward and reverse primers (30 pmol each) and the Expand High Fidelity PCR system (Roche Diagnostics) according to the manufacturer's instructions. Fragments amplified by PCR were separated on agarose gels and purified (QIAquick Gel Extraction Kit, Qiagen) and ligated into the pCR2.1 vector (Invitrogen, Sydney) according to the manufacturer's instructions. The following primers were used: Aox1a.F, 5'-GGACCACGTTTGTTCTCGACG-3'; Aox1b.F, 5'-CTGCTGTGACTCACAGCCATC-3'; Aox1c.F, 5'-GCATCAAAGCAACCGACATCC-3'; Aox2.F, 5'-CGTGAGTTCTGTTTCCTCCAC-3'; Aox.R, 5'-CCTCCAACCATTCCWGGWACYG-3'; Aox1d.F, 5'-CCTACAGATCGATTTACCGC-3'; and Aox1d. R, 5'-GGTTTGTATGAATCCCATGG-3'.
Transcript levels were assayed by real time PCR using the iCycler and iQSupermix (Bio-Rad Laboratories, Hercules, CA). Reactions were carried out in a total volume of 25 µL with a final concentration of 0.008% (w/v) bovine serum albumin, 1 µM fluorescein, and 1x SYBR Green, under conditions optimized to minimize primer-dimer formation and maximize amplification efficiency. The iCycler program consisted of four cycles: denaturation, 95°C for 10 min; amplification, 15 cycles at 95°C for 15 s, touchdown annealing from 85°C to 55°C decreasing 2°C per cycle for 30 s, and extension 72°C for 30 s, followed by amplification without touchdown in the annealing phase: 30 cycles at 95°C for 15 s, 50°C for 30 s, and 72°C for 30 s with single data acquisition collected during the extension; melting curve analysis, 95°C for 0 s, 70°C for 60 s, and 95°C for 0 s with a transition rate of 0.1°C s–1 and continuous data acquisition; cooling to 4°C. Samples were analyzed as outlined previously (Considine et al., 2001
Assays for Luc, GUS, and Histochemical Staining and Sectioning
Histochemical localization of GUS was carried out in a solution containing 5 mg mL–1 5-bromo-4-chloro-3-indolyl-
Extracts from soybean cells used for immunoblot analysis were prepared by first disrupting the cells with a mortar and pestle under liquid nitrogen until a fine powder was produced. This was solubilized in 0.5 M Tris-Cl (pH 7.5), 10 mM EDTA, 1% (v/v) Triton X-100, and 2% (v/v) 2-mercaptoethanol. This was then centrifuged at 20,000g for 5 min, and the supernatant was kept. Soybean cotyledon mitochondria were isolated as described previously (Day et al., 1985
Immunoblot analysis was carried out using 40 µg of protein, which was resolved by SDS-PAGE and transferred onto a Hybond-C extra membrane (Amersham Biosciences) using a semidry blotting apparatus (Hoefer Semi-Phor, Amersham Biosciences). Aox proteins were labeled with the AOA monoclonal antibody (Elthon et al., 1989
Over-represented and previously identified motifs in the Aox promoter sequences were identified using publicly available software. The sequences of the regions upstream of the soybean Aox genes were compared with sequences extending approximately 2 kb upstream of the translational start sites of the Arabidopsis Aox genes identified as having similar patterns of expression using MotifSampler (Thijs et al., 2001
We appreciate the contribution of undergraduate students Helen Trend and Eric Chan to sequencing the Aox2b/Aox2a region. Received June 9, 2003; returned for revision August 1, 2003; accepted August 1, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.028183.
1 This work was supported by the Australian Research Council. * Corresponding author; e-mail seamus{at}cyllene.uwa.edu.au; fax 61–08–9380–1148.
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ABSTRACT
RESULTS
-32P]dCTP, using a commercial kit (High Prime, Roche Diagnostics, Sydney). Unincorporated nucleotides were removed using a spin column (ProbeQuant G50, Amersham Biosciences, Sydney). DNA inserts of the isolated clones were sequenced using a commercial kit (ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Mix, Applied Biosystems, Melbourne) and the ABI PRISM 310 Genetic Analyzer. 