JXB Advance Access originally published online on August 1, 2005
Journal of Experimental Botany 2005 56(419):2563-2571; doi:10.1093/jxb/eri250
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
The leader intron of Arabidopsis thaliana genes encoding cytochrome c oxidase subunit 5c promotes high-level expression by increasing transcript abundance and translation efficiency
Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, CC 242 Paraje El Pozo, 3000 Santa Fe, Argentina
* To whom correspondence should be addressed. Fax: +54 342 4575219. E-mail: dhgonza{at}fbcb.unl.edu.ar
Received 4 March 2005; Accepted 20 June 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The involvement of regions located upstream of the translation start site in the expression of two Arabidopsis thaliana nuclear COX5c genes encoding subunit 5c of mitochondrial cytochrome c oxidase has been analysed. It was observed that these regions, which include a leader intron, direct the tissue-specific expression of the gus reporter gene, mainly in root and shoot meristems, actively growing tissues and vascular strands. Expression was also observed in flowers, specifically localized in anthers, stigma, and the receptacle, and in developing seeds. GUS activity measurements in protein extracts from transformed plants indicated that expression levels are higher than those observed with the constitutive CaMV 35S promoter. Removal of the leader intron produced a significant decrease in expression to values only slightly higher than those observed with a promoterless gus gene. Histochemical staining of plants transformed with the intronless construct revealed expression only in pollen, suggesting that regulatory elements capable of directing pollen-specific expression are present upstream of the intron. The COX5c-2 intron also increased GUS expression levels when fused in the correct orientation with the promoter of the unrelated COX5b-1 gene. Comparison of GUS activity values with the transcript levels suggests that the intron also increases translation efficiency of the corresponding mRNA. The results obtained point to an essential role of the intron present in the 5'-non-coding region of all known COX5c genes in directing the expression of these genes in plants.
Key words: Cytochrome c oxidase, gene expression, leader intron, mitochondrion, promoter analysis
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cytochrome c oxidase (COX) is a multimeric complex composed of several different subunits, two or three of them encoded by the mitochondrial genome and the rest encoded in the nucleus (Capaldi, 1990
; Jänsch et al., 1996). Three different nuclear-encoded subunits, COX5b, COX6a, and COX6b, have been identified in plants through sequence comparisons with yeast and animal counterparts (Kadowaki et al., 1996; Ohtsu et al., 2001; Curi et al., 2003). A fourth subunit, COX5c, is the smallest plant COX subunit and has been recognized by protein purification studies (Nakagawa et al., 1987, 1990). Recent studies using 2D gel electrophoresis combined with mass spectrometry indicated the presence of additional plant-specific subunits (Millar et al., 2004).
It is generally assumed that the expression of components of the plant mitochondrial respiratory chain must somehow be co-ordinated. It is now well established that most mitochondrial components show enhanced expression in flowers (Huang et al., 1994; Landschütze et al., 1995; Felitti et al., 1997; Heiser et al., 1997; Zabaleta et al., 1998). Expression in flowers is mainly localized in anthers as indicated by in situ hybridization experiments (Smart et al., 1994; Ribichich et al., 2001; Elorza et al., 2004). Expression studies in Arabidopsis thaliana have shown similar responses for the nuclear genes encoding cytochrome c and COX subunits 5b, 6a, and 6b (Welchen et al., 2002; Curi et al., 2003). Notably, a different behaviour has been observed for genes encoding COX subunit 5c (COX5c), at least in sunflower (Curi et al., 2002).
The first COX5c cDNA and gene were isolated from sweet potato (Nakagawa et al., 1990, 1993). However, no functional studies have been performed on the cis-acting sequences required for the expression of COX5c genes from any species. In the present study, it has been demonstrated that sequences located upstream of the transcription start site of two Arabidopsis COX5c genes direct specific expression in pollen and that an intron located within the 5'-non-coding region of all known COX5c genes is responsible for high-level expression throughout the plant.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material and growth conditions
Arabidopsis thaliana Heyhn. ecotype Columbia (Col-0) was purchased from Lehle Seeds (Tucson, AZ). Plants were grown on soil in a growth chamber at 22–24 °C under long-day photoperiods (16 h of illumination by a mixture of cool-white and GroLux fluorescent lamps) at an intensity of approximately 200 µE m–2 s–1. Plants used for the different treatments were grown in Petri dishes containing 0.5xMurashige and Skoog medium and 0.8% agar. The dishes were kept at 4 °C for 2 d and then transferred to growth chamber conditions and kept in complete darkness for 7 d.
Isolation of genomic clones
Arabidopsis EST clones encoding COX5c-1 and COX5c-2 (clones 245H10T7, accession no. N97140, and 248L23T7, accession no. AA713295) were obtained from ABRC. For the isolation of genomic clones, a mixture of these cDNAs was used to screen 1x105 pfu from an Arabidopsis genomic library (Voytas et al., 1990). Phage DNA was transferred to Hybond-N and, after overnight hybridization, filters were washed and exposed to X-ray films. Positive clones were purified through successive rounds of plating and hybridization. Isolated phage DNA from purified clones was characterized by restriction analysis and hybridization. A 1.8 kbp EcoRI fragment from COX5c-1 and a 3.2 kbp EcoRI/NheI fragment from COX5c-2, comprising the entire transcribed region and upstream sequences, were subcloned into pBluescript SK– digested with EcoRI or EcoRI and XbaI, respectively. Positive clones were checked by partial sequencing and named VCAT1 and VCAT2.
RNA isolation and analysis
Total RNA was isolated as described by Carpenter and Simon (1998). For northern blot analysis, specific amounts of RNA were electrophoresed through 1.5% (w/v) agarose/6% formaldehyde gels. The integrity of the RNA and equality of RNA loading were verified by ethidium bromide staining. RNA was transferred to Hybond-N nylon membranes (Amersham Corporation) and hybridized overnight at 68 °C to a 32P-labelled cDNA probe, comprising the entire GUS coding region isolated from vector pBI101.3, in buffer containing 6x SSC, 0.1% (w/v) polyvinylpyrrolidone, 0.1% (w/v) BSA, 0.1% (w/v) Ficoll, 0.2% (w/v) SDS, and 10% (w/v) polyethylene glycol 8000. Filters were washed with 2x SSC plus 0.1% (w/v) SDS at 68 °C (4 times, 15 min each), 0.1x SSC plus 0.1% (w/v) SDS at 37 °C for 15 min, dried, and exposed to Kodak BioMax MS films. To check the amount of total RNA loaded in each lane, filters were then re-probed with a 25S rDNA from Vicia faba under similar conditions as those described above, except that hybridization was performed at 62 °C and the wash with 0.1x SSC was omitted.
Reporter gene constructs and plant transformation
A 1.3 kbp BglII/SalI fragment, comprising sequences upstream of the ATG initiation codon (i.e. exon 1, the intron and part of exon 2, plus non-transcribed upstream sequences) from COX5c-1, was amplified from clone VCAT1 using oligonucleotide COXC32: 5'-GGCAGATCTCTCTTCTTTCTTCTTCTC-3' (BglII site underlined) and universal primer –40 and cloned in plasmid pBI101.3 digested with BamHI and SalI. A similar construct for COX5c-2 was made by amplifying a 2.2 kbp fragment from VCAT2 with primers COXC41: 5'-GCGTCTAGATTCTTCTCAACCTAGCAC-3' (XbaI site underlined) and –40 and cloning in pBI101.3 digested with XbaI and HindIII. Constructs containing exon 1 and upstream sequences were obtained in a similar way by amplification with either COXC33: 5'-GGCGGATCCCAAGTCGGAGTGTGGAGG-3' (COX5c-1) or COXC42: 5'-GGCGGATCCCGAGTCAGATTGTGTAGA-3' (COX5c-2), followed by cloning in the SalI/BamHI or HindIII/BamHI sites of pBI101.3, respectively. A construct containing the entire COX5c-2 5'-non-coding region without the intron was obtained by amplification with primers COXC45: 5'-GCGTCTAGATTCTTCTCAACCGAGTCAGATTGTGTAGA-3' and –40 followed by cloning into the XbaI and HindIII sites of pBI101.3. To test the effect of the intron on an exogenous promoter, the COX5c-2 intron and transcribed 5'-non-coding sequences were amplified with primers COXC44: 5'-GGCTCTAGAGTTTTCGTCGTGAGCTTC-3' and COXC41 and cloned in both orientations into the XbaI site of a construct containing a 609 bp promoter fragment from the COX5b-1 gene fused to gus (Welchen et al., 2004). In this way, the intron was placed between the COX5b-1 promoter and the gus coding region. The different constructs were introduced into Agrobacterium tumefaciens strain GV2260, and transformed bacteria were used to obtain transgenic Arabidopsis plants by the floral dip procedure (Clough and Bent, 1998). Transformed plants were selected on the basis of kanamycin resistance and positive PCR carried out on genomic DNA with primers specific for COX5c-1 or -2 and the gus-specific primer 5'-TTGGGGTTTCTACAGGAC-3'. Five to ten independent lines (depending on the construct) were further reproduced and homozygous T3 and T4 plants were used to analyse gus expression. Plants transformed with pBI101.3 or pBI121 were obtained in a similar way and used as negative or positive controls of expression, respectively.
ß-glucuronidase assays
ß-glucuronidase (GUS) activity of transgenic plants was analysed by histochemical staining using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid (X-gluc) as described by Hull and Devic (1995). Whole plants or separated organs were immersed in a 1 mM X-gluc solution in 100 mM sodium phosphate, pH 7.0, and 0.1% Triton X-100 and, after applying vacuum for 5 min, they were incubated at 37 °C until satisfactory staining was observed. Tissues were cleared by immersing them in 70% ethanol.
Specific GUS activity in protein extracts was measured using the fluorogenic substrate 4-methylumbelliferyl ß-D-glucuronide (MUG) essentially as described by Jefferson et al. (1987). Total protein extracts were prepared by grinding the tissues in extraction buffer (50 mM sodium phosphate, pH 7.0, 10 mM EDTA, 10 mM ß-mercaptoethanol) containing 0.1% (w/v) SDS and 1% Triton X-100, followed by centrifugation at 13 000 g for 10 min. GUS activity in supernatants was measured in extraction buffer containing 1 mM MUG and 20% methanol. Reactions were stopped with 0.2 M Na2CO3 and the amount of 4-methylumbelliferone was calculated by relating relative fluorescence units with those of a standard of known concentration. The protein concentration of extracts was determined as described by Sedmak and Grossberg (1977).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
COX5c related sequences in the Arabidopsis genome
A search for COX5c coding regions in the Arabidopsis genome using sunflower COX5c protein sequences (Curi et al., 2002
) and the program TBLASTN revealed the existence of four genomic regions from which proteins with COX5c-related sequences could be deduced. For three of them (At2g47380, At3g62400, and At5g61310) mRNA sequences are also deposited in data banks, indicating that they are expressed. The fourth region, located in chromosome 5, encodes a protein more distantly related for which no transcripts have been detected, suggesting that it may be a pseudogene. Accordingly, the genes present in chromosomes 2, 3 and 5 have arbitrarily been named COX5c-1, COX5c-2, and COX5c-3, respectively. Upon comparing the corresponding genomic and cDNA sequences, it becomes evident that the three Arabidopsis COX5c genes contain a single intron located within the 5'-non-coding region, at variable distances with respect to the ATG start codon (Fig. 1A). An intron in the same location is also present in the other COX5c genes for which sequences are available, the single genes from rice (Oryza sativa; BAC clone accession number AB027123), sweet potato (Ipomoea batatas; Nakagawa et al., 1993), and Lotus corniculatus (BAC clone accession number AP006137).
|
Sequences upstream of the translation start site of COX5c-1 and COX5c-2 promote high-level expression of a reporter gene in specific cell-types
Two Arabidopsis thaliana clones containing the COX5c-1 and -2 genes and additional genomic regions were isolated by direct screening of a library using a mixture of ESTs derived from these genes. Subclones of these lambda clones were used to amplify fragments containing sequences located upstream of the translation start site which were introduced in vector pBI101.3 in front of the gus coding region (Fig. 1B). These constructs (pBI5c1 and pBI5c2 for COX5c-1 and -2, respectively) were introduced into Arabidopsis by Agrobacterium-mediated transformation. An initial screening of kanamycin-resistant lines was carried out by histochemistry to define expression characteristics common to most of them. Plants from at least five independent representative transgenic lines carrying each construct were then analysed in detail. The results were essentially the same for all plants analysed, carrying either pBI5c1 or pBI5c2.
Seedlings grown on Petri dishes on MS medium showed strong staining along roots and hypocotyls, while activity in cotyledons was detected only in plants older than 3 d post-germination (Fig. 2A, B). Activity in roots was progressively localized to the vascular cylinder and the root meristem upon growth (Fig. 2B). After 15 d, strong expression was also detected in developing secondary roots (Fig. 2C). In hypocotyls, activity was also progressively localized to vascular tissues upon development (Fig. 2D). Cotyledons displayed GUS activity in the lamina and specially in vascular tissues (Fig. 2D, E). A similar expression pattern was evident in developing leaves (Fig. 2D, E). Strong activity was also observed in the shoot apical meristem (Fig. 2F).
|
Adult (45-d-old) plants grown on soil displayed activity in roots, leaves, and flowers. Expression in roots was similar to that described for younger plants. In leaves, vascular tissues and, to a lesser extent, mesophyll tissues were stained (Fig. 2G). In flowers, strong expression was detected in anthers, specially in reproductive tissues and pollen grains when these were formed (Fig. 2H, I). Activity was also detected in the stigma, receptacle, and petal and sepal veins, and in siliques and developing seeds (Fig. 2I, J).
To estimate the relative expression levels produced by both constructs, fluorometric assays of GUS activity in protein extracts from transformed 20-d-old plants were performed. Plants transformed with pBI5c1 and pBI5c2 displayed activities of 35 000 pmol min–1 mg–1 and 66 000 pmol min–1 mg–1, respectively. These values are even higher than those observed with plants transformed with pBI121 (i.e. the gus gene under the control of the strong constitutive CaMV 35S promoter), 18 000 pmol min–1 mg–1, indicating that the sequences contained within the constructs direct high-level expression. Activity measurements using extracts from different organs indicated that highest expression was attained in leaves followed by flowers, siliques, and roots (not shown).
Removal of the leader intron originates plants with pollen-specific expression
The presence of a conserved intron in the leader region led to the investigation of its role in expression of COX5c genes. Seedlings of plants transformed with constructs in which the respective leader introns from COX5c-1 or COX5c-2 and downstream sequences were removed (5c1-I/E2 and 5c2-I/E2 in Fig. 1B) showed no GUS activity when analysed by histochemical staining (Fig. 2K). Analysis of adult plants revealed the presence of GUS activity only in pollen grains (Fig. 2M, N), while no staining was evident in leaves, siliques, or flower organs other than anthers (Fig. 2L–O). Similar results were observed for both genes under study, indicating that the leader intron is essential to direct high-level expression throughout the plant. Indeed, activity in pollen was also reduced in these plants, since longer incubation times were required to reach similar staining: 18 h with plants transformed with the intronless constructs versus 3–5 h with plants transformed with the entire fragments. The levels of GUS activity present in protein extracts from plants transformed with the intronless constructs were extremely low: 800 and 1400 pmol min–1 mg–1, respectively, for COX5c-1 and COX5c-2, that is 40–50 times lower than those observed when the intron was present.
Untranslated exon sequences influence gus expression
The effect of 5' untranslated exon regions on expression was also analysed. For this purpose, plants transformed with a construct in which the COX5c-2 leader intron was removed, but the promoter and 5'-non-coding sequences from exons 1 and 2 were conserved (5c2-I in Fig. 1B), were used. These plants showed considerably lower GUS activity levels than plants carrying the construct with the intron (Fig. 3). They consistently showed, however, slightly higher activities than plants bearing only the promoter and exon 1 fused to gus (Fig. 3, inset). The different expression produced by the inclusion of exon 2 non-coding sequences was also observed by histochemical staining. Indeed, activity, although low, was detected in cotyledon veins and tips and also in leaf veins, trichomes, and hydathodes (Fig. 2P–R). In reproductive tissues, expression was only detected in pollen, as with the construct in which the intron and exon 2 were removed (Fig. 2S, T). These results, on one side, confirm the importance of the leader intron in determining high level expression and, on the other side, indicate that non-coding exon sequences, though slightly, also influence gene expression.
|
The COX5c-2 leader intron and adjacent regions increase expression from an unrelated promoter
The effect of the inclusion of the COX5c-2 intron between the promoter of an unrelated gene and the gus coding region was also tested. For this purpose, a region covering the entire intron and surrounding untranslated transcribed sequences from COX5c-2 was inserted between the promoter of the Arabidopsis gene COX5b-1 and the gus coding region, either in the sense or antisense orientation (5b+Is and 5b+Ias, respectively, in Fig. 1B). The portion of the COX5b-1 promoter used (–1 to –609 respective to the translation start site) directs relatively low expression localized in meristems, root, and cotyledon vascular tisssues, the leaf central vein, and in anthers (Welchen et al., 2004
). Inclusion of the COX5c-2 intron in the correct orientation resulted in considerably higher expression levels throughout the plant, in a pattern similar to those observed with the COX5c-1 and COX5c-2 promoter plus intron fragments (Fig. 4). As a consequence, GUS activity was extended from vascular tissues and anthers, as observed in plants bearing only the COX5b-1 promoter fragment without the intron (Fig. 4A, B, G, J, M, N), to the lamina of cotyledons and leaves (Fig. 4D, E, H, K) and to petals and siliques (Fig. 4O, P). GUS activity measurements in protein extracts from transformed plants indicated that the intron produces a 6-fold increase in expression levels from the COX5b-1 promoter (Fig. 5).
|
|
When the intron was placed in the same location, but in reverse orientation, GUS activity was not detected in any tissue or developmental stage by either the histochemical (Fig. 4C, F, I, L) or the fluorometric method (Fig. 5). It is logical to assume that intron splicing does not take place when the intron is present in the reverse orientation. The presence of a large non-coding sequence in the 5' region of the transcript may either affect its stability or its translation efficiency. Six spurious ATG codons are introduced when the intron is placed in the reverse orientation, thus probably affecting the recognition of the correct start codon by the translation machinery. In addition, it is well-known that transcripts with premature termination stop codons in phase with a start codon are degraded by a process termed nonsense-mediated mRNA decay (Baker and Parker, 2004
).
COX5c leader introns and adjacent regions increase translation efficiency
The increase in expression promoted by the presence of the intron was also observed when analysing transcript levels of the gus gene under the control of the respective fragments from COX5c-1 and COX5c-2. Figure 6 shows a northern hybridization using a gus probe and total RNA from plants carrying different portions of the COX5c-2 gene. While no or extremely faint signals were obtained in northern blots with total RNA from plants carrying the intronless constructs (Fig. 6, lanes 2, 3), a distinct band was visible in the sample from plants bearing the construct with the COX5c-2 intron (Fig. 6, lane 1). Transcript levels in these plants, however, were considerably lower than those observed in plants transformed with the T-DNA region of plasmid pBI121, which contains the gus gene under the control of the CaMV 35S promoter (Fig. 6, lane 9). Densitometric analysis of the respective bands, compared with the signals obtained with the rRNA probe, indicated that gus transcript levels are seven times higher in plants transformed with the CaMV 35S promoter fusion. Since plants transformed with the entire COX5c fragments showed 3.5 times higher GUS activities than the latter (see above), this suggests that transcripts containing COX5c 5'-non-coding sequences are more efficiently translated, producing a 25-fold increase in the protein/transcript ratio. Although protein levels were not directly quantified, it can be assumed that enzyme activity measurements constitute an appropriate estimation, since all constructs must produce proteins with the same amino acid sequence. An effect of the intron on translation could also be observed when analysing transcript levels of plants transformed with the COX5b-1 gene promoter with or without the COX5c-2 intron (Fig. 6, lanes 4, 6). In this case, the increase in transcript levels promoted by the presence of the intron was very low compared with the 6-fold increase in GUS activity observed in plants with the intron in the correct orientation. On the other hand, plants with the intron in the antisense orientation showed undetectable levels of gus transcripts (Fig. 6, lane 5). Although this may be due to a lower transcription efficiency, a more likely explanation is that the unspliced RNA is rapidly degraded.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
COX5c is a polypeptide of about 63 amino acids with sequence similarity to yeast COX VIIa and mammalian COX VIII (Nakagawa et al., 1990
). COX5c cDNAs have been isolated from sweet potato, rice, and sunflower (Nakagawa et al., 1990; Hamanaka et al., 1999; Curi et al., 2002), and ESTs from several species are available. The first COX5c gene was also isolated from sweet potato (Nakagawa et al., 1993), and related sequences can be detected in the totally or partially sequenced genomes from Arabidopsis, rice, and Lotus corniculatus. Expression studies in rice and sunflower indicated that COX5c genes are expressed at different levels throughout the plant (Hamanaka et al., 1999; Curi et al., 2002). However, no detailed analysis on the tissue specificity of expression or on the gene sequences involved in directing this expression have been performed for any plant COX5c gene.
In the present work, it has been determined that sequences from two Arabidopsis COX5c genes located upstream of the respective translation start sites, including an intron present in the 5'-non-coding region, direct similar tissue-specific expression patterns. Expression is observed throughout development, specially in vascular and meristematic tissues, and in pollen grains and siliques. The tissue-specific patterns of COX5c expression may be the consequence of responses to cell-specific factors or to the metabolic status of these tissues, which undergo constant cell proliferation. COX5b-1, a gene encoding a different COX subunit, shows a similar expression pattern, although more strictly localized to young proliferating tissues (Welchen et al., 2004).
It is noteworthy that removal of the leader intron produces a pronounced decrease in expression levels for both genes, making reporter gene activity barely detectable, except in pollen grains. Enhancement of expression by introns has been reported for several genes from monocot (Callis et al., 1987; McElroy et al., 1990; Christensen et al., 1992; Xu et al., 1994; Jeon et al., 2000; Morello et al., 2002) and dicot plants (Norris et al., 1993; Gidekel et al., 1996; Rose and Last, 1997; Plesse et al., 2001; Mun et al., 2002). Introns that influence expression are more frequently located near the translation start site within non-coding regions, as is the case for COX5c genes. The exact role of introns in promoting an increase in expression levels is not clear. Some introns seem to contain transcriptionally active regulatory elements (Gidekel et al., 1996), while others seem to act post-transcriptionally (Rose and Last, 1997), suggesting the existence of different mechanisms of action. It has recently been proposed that many introns would act by increasing the processivity of the transcription machinery (Rose, 2004). Besides the quantitative enhancement of expression, some introns direct tissue-specific patterns of expression (Bolle et al., 1996; Jeon et al., 2000). In some cases, like those of the Petunia actin-depolymerizing factor (Mun et al., 2002), the rice 
-tubulin OstubA1 (Jeon et al., 2000) and the Arabidopsis polyubiquitin Ubi.U4 genes (Plesse et al., 2001), expression is specifically observed in vascular tissues and/or metabolically active dividing cells. These expression patterns are similar to those observed here for the COX5c genes, probably indicating that these introns operate by similar mechanisms or respond to similar factors. To test if the COX5c introns contain sequences that direct tissue-specific expression or transcriptional enhancers, plants carrying fusions to a minimal CaMV 35S promoter will be obtained and analysed.
Comparison of GUS activities with the respective transcript levels indicate that COX5c 5'-non-coding sequences also increase translation efficiency. This observation could be made with constructs that possess the COX5c leader introns, but not with those that only carry non-coding exon sequences, due to the low expression levels produced by the latter. It should be emphasized that the context of the start codon, which could affect translation efficiency, is the same in all constructs analysed, since they use the ATG provided by the pBI101.3 vector which is placed several nucleotides downstream of the cloning sites. The differences in translation efficiency are then produced by the intron itself or by the presence of translational enhancers in the 5'-non-coding region. So far, similarities with other known translational enhancers (Yamamoto et al., 1995; Dickey et al., 1998) within COX5c untranslated regions have not been observed. Although the involvement of introns in translation seems an unexpected effect, similar observations have been made in animal and plant systems (Le Hir et al., 2003; Rose, 2004). It has been proposed that the elevation of translational efficiency by introns is related to the deposition of proteins near the exon–exon junctions during splicing that subsequently increase the interaction of ribosomes with the mRNA (Wiegand et al., 2003; Nott et al., 2004).
In addition, removal of the intron highlights the existence of elements that direct pollen-specific expression within the non-transcribed upstream region of both COX5c genes. This is not unexpected, since transcript levels for a set of mitochondrial genes are considerably higher in flowers and, specifically, in anther tissues (Smart et al., 1994; Zabaleta et al., 1998; Ribichich et al., 2001). Mascarenhas and Hamilton (1992) reported that GUS expression in pollen may be an artefact originated in the diffusion of dye produced in other parts of the anther. This kind of artefact arises when high-level expression is present and produces uneven pollen staining (i.e. both stained and unstained pollen grains are visible even when homozygous plants are used). Since staining of all pollen grains and very low expression levels was observed, it is highly unlikely that GUS expression in pollen is an artefact in this case. In addition, GUS staining of pollen isolated from anthers before performing the histochemical assay was observed. Zabaleta et al. (1998) have studied the promoter regions involved in pollen/anther expression of three genes that encode components of the NADH dehydrogenase (Complex I). Within these regions, they have identified conserved GT-rich elements similar to those found in other genes expressed in pollen. They have postulated that these motifs are involved in the co-ordinated expression of the three genes. Analysis of the promoter regions of both COX5c genes showed the presence of GT-rich elements (TGTGGTT and TGTGTTG for COX5c-1 and -2), located at –208 and –210, respectively, from the putative transcription start site. The first of these elements is identical to those observed in one of the Complex I genes and in the tomato LAT52 and LAT56 genes, specifically expressed in pollen (Twell et al., 1991). Both genes also possess two close copies of site II elements (TGGGCC/T), located at –109/–90 and –84/–71, respectively, known to be present in genes preferentially expressed in cycling cells (Kosugi et al., 1995; Trémousaygue et al., 2003). The functional significance of these sequences must be assessed by mutagenesis experiments.
The relevance of the elements directing pollen-specific expression in the COX5c genes is not clear, since the presence of the intron seems to abolish this specificity. It may well be that these elements help to respond to pollen-specific factors, coupled with intron sequences that promote high-level expression. Alternatively, acquisition of the leader intron by a gene that was predominantly expressed in pollen may have occurred later in evolution. The fact that these elements remain functional in both genes suggests, however, that they play an active role in determining the expression characteristics of present-day COX5c genes.
|
Acknowledgements |
|---|
We thank the Arabidopsis Biological Resource Center for providing the COX5c EST clones. This work was supported by grants from CONICET, ANPCyT (Agencia Nacional de Promoción Científica y Tecnológica), Fundación Antorchas, and Universidad Nacional del Litoral. RLC and DHG are members of CONICET (Argentina).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Baker KE, Parker R. 2004. Nonsense-mediated mRNA decay: terminating erroneous gene expression. Current Opinion in Cell Biology 16, 293–299.[CrossRef][Web of Science][Medline]
Bolle C, Hermann RG, Oelmüller R. 1996. Intron sequences are involved in the plastid and light-dependent expression of the spinach PsaD gene. The Plant Journal 10, 919–924.[CrossRef][Web of Science][Medline]
Callis J, Fromm M, Walbot V. 1987. Introns increase gene expression in cultured maize cells. Genes and Development 1, 1183–1200.
Capaldi RA. 1990. Structure and function of cytochrome c oxidase. Annual Review of Biochemistry 59, 569–596.[CrossRef][Web of Science][Medline]
Carpenter CD, Simon AE. 1998. Preparation of RNA. In: Martinez-Zapater J, Salinas J, eds. Methods in molecular biology, Vol. 82. Arabidopsis protocols. Totowa, NJ: Humana Press Inc, 85–89.
Christensen AH, Sharrock RA, Quail PH. 1992. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Molecular Biology 18, 675–689.[CrossRef][Web of Science][Medline]
Clough SJ, Bent AF. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16, 735–743.[CrossRef][Web of Science][Medline]
Curi GC, Chan RL, Gonzalez DH. 2002. Genes encoding cytochrome c oxidase subunit 5c from sunflower (Helianthus annuus L.) are regulated by nitrate and oxygen availability. Plant Science 163, 897–905.
Curi GC, Welchen E, Chan RL, Gonzalez DH. 2003. Nuclear and mitochondrial genes encoding cytochrome c oxidase subunits respond differently to the same metabolic factors. Plant Physiology and Biochemistry 41, 689–693.[CrossRef]
Dickey LF, Petracek ME, Nguyen TT, Hansen ER, Thompson WF. 1998. Light regulation of Fed-1 mRNA requires an element in the 5' untranslated region and correlates with differential polyribosome association. The Plant Cell 10, 475–484.
Elorza A, León G, Gómez I, Mouras A, Holuigue L, Araya A, Jordana X. 2004. Nuclear SDH2-1 and SDH2-2 genes, encoding the iron-sulfur subunit of mitochondrial complex II in Arabidopsis, have distinct cell-specific expression patterns and promoter activities. Plant Physiology 136, 4072–4087.
Felitti SA, Chan RL, Gago G, Valle EM, Gonzalez DH. 1997. Expression of sunflower cytochrome c mRNA is tissue-specific and controlled by nitrate and light. Physiologia Plantarum 99, 342–347.
Gidekel M, Jimenez B, Herrera-Estrella L. 1996. The first intron of the Arabidopsis thaliana gene coding for elongation factor 1 beta contains an enhancer-like element. Gene 170, 201–206.[CrossRef][Web of Science][Medline]
Hamanaka S, Ohtsu K, Kadowaki K, Nakazono M, Hirai A. 1999. Identification of cDNA encoding cytochrome c oxidase subunit 5c (COX5c) from rice: comparison of its expression with nuclear-encoded and mitochondrial-encoded COX genes. Genes and Genetic Systems 74, 71–75.
Heiser V, Brennicke A, Grohmann L. 1997. The plant mitochondrial 22 kDa (PSST) subunit of respiratory chain complex I is encoded by a nuclear gene with enhanced transcript levels in flowers. Plant Molecular Biology 31, 1195–1204.
Huang J, Struck F, Matzinger DF, Levings III CS. 1994. Flower-enhanced expression of a nuclear-encoded mitochondrial respiratory protein is associated with changes in mitochondrion number. The Plant Cell 6, 439–448.[Abstract]
Hull GA, Devic M. 1995. The beta-glucuronidase (gus) reporter gene system. Gene fusions; spectrophotometric, fluorometric, and histochemical detection. In: Jones H, ed. Methods in plant molecular biology, Vol. 49. Plant gene transfer and expression protocols. Totowa, NJ: Humana Press Inc, 125–141.
Jänsch L, Kruft V, Schmitz UK, Braun H-P. 1996. New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria. The Plant Journal 9, 357–368.[CrossRef][Web of Science][Medline]
Jefferson RA, Kavanagh TA, Bevan MW. 1987. GUS fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 20, 3901–3907.
Jeon J-S, Lee S, Jung K-H, Jun S-H, Kim C, An G. 2000. Tissue preferential expression of a rice -tubulin gene, OstubA1, mediated by the first intron. Plant Physiology 123, 1005–1014.
Kadowaki K, Kubo N, Ozawa K, Hirai A. 1996. Targeting presequence acquisition after mitochondrial gene transfer to the nucleus occurs by duplication of existing targeting signal. EMBO Journal 15, 6652–6661.[Web of Science][Medline]
Kosugi S, Suzuka I, Ohashi Y. 1995. Two of three promoter elements identified in a rice gene for proliferating cell nuclear antigen are essential for meristematic tissue-specific expression. The Plant Journal 7, 877–886.[CrossRef][Web of Science][Medline]
Landschütze V, Müller-Röber B, Willmitzer L. 1995. Mitochondrial citrate synthase from potato: predominant expression in mature leaves and young flower buds. Planta 196, 756–764.[CrossRef][Web of Science][Medline]
Le Hir H, Nott A, Moore MJ. 2003. How introns influence and enhance eukaryotic gene expression. Trends in Biochemical Sciences 28, 215–220.[CrossRef][Web of Science][Medline]
Mascarenhas JP, Hamilton DA. 1992. Artifacts in the localization of GUS activity in anthers of petunia transformed with a CaMV 35S–GUS construct. The Plant Journal 2, 405–408.[CrossRef]
McElroy D, Zhang J, Cao J, Wu R. 1990. Isolation of an efficient actin promoter for use in rice transformation. The Plant Cell 2, 163–171.[Medline]
Millar AH, Eubel H, Jänsch L, Kruft V, Heazlewood JL, Braun H-P. 2004. Mitochondrial cytochrome c oxidase and succinate dehydrogenase complexes contain plant specific subunits. Plant Molecular Biology 56, 77–90.[CrossRef][Web of Science][Medline]
Morello L, Bardini M, Sala F, Breviario D. 2002. A long leader intron of the Ostub16 rice ß-tubulin gene is required for high-level gene expression and can autonomously promote transcription both in vivo and in vitro. The Plant Journal 29, 33–44.[CrossRef][Web of Science][Medline]
Mun JH, Lee SY, Yu HJ, Jeong YM, Shin MY, Kim H, Lee I, Kim SG. 2002. Petunia actin-depolymerizing factor is mainly accumulated in vascular tissue and its gene expression is enhanced by the first intron. Gene 292, 233–243.[CrossRef][Web of Science][Medline]
Nakagawa T, Maeshima M, Muto H, Kajiura H, Hattori H, Asahi T. 1987. Separation, amino-terminal sequence and cell-free synthesis of the smallest subunit of sweet potato cytochrome c oxidase. European Journal of Biochemistry 165, 303–307.[Web of Science][Medline]
Nakagawa T, Maeshima M, Nakamura K, Asahi T. 1990. Molecular cloning of a cDNA for the smallest nuclear-encoded subunit of sweet potato cytochrome c oxidase. Analysis with the cDNA of the structure and import into mitochondria of the subunit. European Journal of Biochemistry 191, 557–561.[Web of Science][Medline]
Nakagawa T, Maeshima M, Nakamura K, Asahi T. 1993. The nuclear gene for subunit Vc of sweet potato cytochrome c oxidase. Plant and Cell Physiology 34, 621–626.
Norris SR, Meyer SE, Callis J. 1993. The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Molecular Biology 21, 895–906.[CrossRef][Web of Science][Medline]
Nott A, Le Hir H, Moore MJ. 2004. Splicing enhances translation in mammalian cells: an additional function of the exon junction complex. Genes and Development 18, 210–222.
Ohtsu K, Nakazono M, Tsutsumi N, Hirai A. 2001. Characterization and expression of the genes for cytochrome c oxidase subunit VIb (COX6b) from rice and Arabidopsis thaliana. Gene 264, 233–239.[CrossRef][Web of Science][Medline]
Plesse B, Criqui MC, Durr A, Parmentier Y, Fleck J, Genschik P. 2001. Effects of the polyubiquitin gene Ubi.U4 leader intron and first ubiquitin monomer on reporter gene expression in Nicotiana tabacum. Plant Molecular Biology 45, 655–667.[CrossRef][Web of Science][Medline]
Ribichich K, Tioni MF, Chan RL, Gonzalez DH. 2001. Cell-type specific expression of plant cytochrome c mRNA in developing flowers and roots. Plant Physiology 125, 1603–1610.
Rose AB. 2004. The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis. The Plant Journal 40, 744–751.[CrossRef][Web of Science][Medline]
Rose AB, Last RL. 1997. Introns act post-transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1. The Plant Journal 11, 455–464.[CrossRef][Web of Science][Medline]
Sedmak J, Grossberg S. 1977. A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G-250. Analytical Biochemistry 79, 544–552.[CrossRef][Web of Science][Medline]
Smart C, Monéger F, Leaver CJ. 1994. Cell-specific regulation of gene expression in mitochondria during anther development in sunflower. The Plant Cell 6, 811–825.[Abstract]
Trémousaygue D, Garnier L, Bardet C, Dabos P, Hervé C, Lescure B. 2003. Internal telomeric repeats and ‘TCP-domain’ protein binding sites co-operate to regulate gene expression in Arabidopsis thaliana cycling cells. The Plant Journal 33, 957–966.[CrossRef][Web of Science][Medline]
Twell D, Yamaguchi J, Wing R, Ushiba J, McCormick S. 1991. Promoter analysis of genes that are co-ordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Genes and Development 5, 496–507.
Voytas DF, Konieczny A, Cummings MP, Ausubel FM. 1990. The structure, distribution and evolution of the Ta1 retrotransposable element family of Arabidopsis thaliana. Genetics 126, 713–721.[Abstract]
Welchen E, Chan RL, Gonzalez DH. 2002. Metabolic regulation of genes encoding cytochrome c and cytochrome c oxidase subunit Vb in Arabidopsis. Plant, Cell and Environment 25, 1605–1615.
Welchen E, Chan RL, Gonzalez DH. 2004. The promoter of the Arabidopsis nuclear gene COX5b-1, encoding subunit 5b of the mitochondrial cytochrome c oxidase, directs tissue-specific expression by a combination of positive and negative regulatory elements. Journal of Experimental Botany 55, 1997–2004.
Wiegand HL, Lu S, Cullen BR. 2003. Exon junction complexes mediate the enhancing effect of splicing on mRNA expression. Proceedings of the National Academy of Sciences, USA 100, 11327–11332.
Xu Y, Yu H, Hall TC. 1994. Rice triosephosphate isomerase gene 5' sequence directs ß-glucuronidase activity in transgenic tobacco but requires an intron for expression in rice. Plant Physiology 106, 459–467.[Abstract]
Yamamoto YY, Tsuji H, Obokata J. 1995. 5' Leader of a photosystem I gene in Nicotiana sylvestris, psaDb, contains a translational enhancer. The Journal of Biological Chemistry 270, 12466–12470.
Zabaleta E, Heiser V, Grohmann L, Brennicke A. 1998. Promoters of nuclear-encoded respiratory chain complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen-specific expression. The Plant Journal 15, 49–59.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. B. Rose, T. Elfersi, G. Parra, and I. Korf Promoter-Proximal Introns in Arabidopsis thaliana Are Enriched in Dispersed Signals that Elevate Gene Expression PLANT CELL, March 1, 2008; 20(3): 543 - 551. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||