Journal of Experimental Botany, Vol. 52, No. 358, pp. 949-959,
May 1, 2001
© 2001 Oxford University Press
Original Papers |
Characterization of position-induced spatial and temporal regulation of transgene promoter activity in plants
Laboratory of Plant Physiology, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
Received 30 August 2000; Accepted 5 December 2000
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Abstract |
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Quantitative differences in transgene expression between independent transformants are generally ascribed to different integration sites of the transgene (position effect). The contribution of spatial and temporal changes in transgene promoter activity to these position-induced differences in transgene expression in planta are characterized, using the firefly luciferase (luc) reporter system. The activity of three different promoters (Cauliflower Mosaic Virus (CaMV) 35S, modified CaMV 35S and the promoter of an Arabidopsis thaliana Lipid Transfer Protein gene) was shown to vary not only among independent transformants, but also between leaves on the same plant and within a leaf. The differences in local LUC activity between leaves and within a leaf correlated with differences in local luc mRNA steady-state levels. Imaging of LUC activity in the same leaves over a 50 d period, shows that individual transformants can show different types of temporal regulation. Both the spatial and the temporal type of luc transgene expression pattern are inherited by the next generation. It is concluded that previously reported position-induced quantitative differences in transgene expression are probably an accumulated effect of differences in spatial and temporal regulation of transgene promoter activity.
Key words: Cauliflower Mosaic Virus 35S promoter, luciferase reporter system, position effect, transgene expression, variegation.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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The expression of plant genes is usually characterized by quantifying mRNA or protein steady-state levels in different tissues. Gene expression analysis was greatly facilitated by the use of plant transformation techniques and the introduction of reporter genes like Chloramphenicol Acetyl Transferase (CAT, Gorman et al., 1982
), ß-Glucuronidase (GUS, Jefferson et al., 1987), Green Fluorescent Protein (GFP, Niedz et al., 1995) and firefly luciferase (luc, Ow et al., 1986). However, it has been shown that the level of transgene expression varies among individual transformants with the same transgene copy number. Apparently this quantitative variation depends on the site of integration and it is referred to as the ‘position effect’ (Blundy et al., 1991; Dean et al., 1988; Mlynárová et al., 1994; Peach and Velten, 1991). When the character of the promoter driving the transgene expression is known (e.g. its tissue specificity), often no detailed information is available on the distribution of transgene expression throughout a plant, the distribution of transgene activity within a tissue, the distribution of transgene activity in the same tissue over prolonged periods of time, or possible differences in these distributions between independent transformants. There are several possible origins of the position-dependent quantitative differences in transgene expression. Independent transgenic lines can show (1) differences in the level of promoter activity, but the same spatial and/or developmental regulation or (2) the same level of promoter activity, but differences in spatial and/or developmental regulation or (3) a combination of these two possibilities. With the introduction of the firefly luc reporter gene all these aspects of transgene expression can now be imaged in planta (Gould and Subramani, 1988).
The luc gene encodes a protein that catalyses the oxidative decarboxylation of firefly luciferin using Mg2+-ATP and oxygen. A photon (562 nm) is released in 90% of the catalytic cycles (DeLuca and McElroy, 1974). The substrate luciferin is an amphipathic molecule that easily penetrates most plant tissues. Therefore a transgenic luc plant, sprayed with luciferin will emit photons where and when a luc reporter gene is active. These photons can be visualized with a sensitive CCD camera (2D-luminometer). The luc transgene expression can be monitored in vivo in the same tissue throughout plant development and under different physiological conditions. The LUC enzyme activity is only very slowly regenerated after reacting with the substrates, because the product of the reaction, oxyluciferin, is only very slowly released from the AMP-oxyluciferin-luciferase complex (Denburg et al., 1969). Therefore, after pre-incubation with luciferin, continuous light production in vivo is mostly caused by newly synthesized LUC and not by previously accumulated LUC. Under these conditions, luciferase activity is closely related to the promoter activity of the reporter gene. This feature allows for the identification of changes in luc transgene activity within a tissue, enhancing the temporal resolution of the gene expression study. An extensive report on the features of luciferase activity measurements in planta has recently been published (Van Leeuwen et al., 2000).
Here, the spatial and temporal aspects of gene expression have been compared among individual transgenic lines, carrying the same luc reporter gene construct. For these studies the Cauliflower Mosaic Virus (CaMV) 35S promoter, a modified CaMV 35S promoter (m35S) and the promoter of an A. thaliana Lipid Transfer Protein gene (LTP1; Thoma et al., 1994) was used to drive luc expression in transgenic Petunia hybrida (Vilm.) plants. The CaMV 35S promoter is often used as a ‘constitutively active’ promoter for ectopic expression of foreign genes (Benfey et al., 1989). The m35S promoter was designed to increase the level of transgene expression by optimization and multimerization of DNA binding-sites within the CaMV 35S promoter and has been used for ectopic expression of floral homeotic genes (van der Krol et al., 1993). The LTP promoter has been shown to be active in the L1-layer, both in Arabidopsis (Thoma et al., 1994) as well as in Daucus carota (Toonen et al., 1997).
This study's analyses show for all three luc reporter gene constructs, (1) that the LUC activity is variegated, occasionally showing a more than 100-fold difference within a leaf tissue, (2) that the type of variegated LUC activity differs between transformants carrying the same reporter construct, indicating that the pattern of variegation is not related to the developmental stages of the cells within a leaf, (3) that a different temporal regulation might occur in different transformants carrying the same transgene, and (4) that the variegated LUC activity correlates with variegated luc mRNA steady-state levels. The differences in variegation and in temporal regulation of transgene promoter activity, can account for the previously reported position-dependent quantitative differences in transgene expression (Dean et al., 1988). The factors contributing to this variegated transgene promoter activity are speculated upon and the implications for gene expression studies are discussed.
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Materials and methods |
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Introduction of luc reporter gene constructs in Petunia hybrida plants
Agrobacterium tumefaciens (A. tum. strain ABI) was transformed with the binary vector pMON721 containing one of the following gene constructs: CaMV 35S promoter-luc (pGM46); CaMV m35S promoter-luc+ (pGM107); LTP promoter-luc (pMT520).
The CaMV promoter used in these constructs consists of the -343 to +8 sequence (Benfey et al., 1989; Gardner et al., 1981). The modified CaMV 35S (m35S) promoter, contains the -90 to +8 fragment of the CaMV 35S promoter, with four copies of the B3 domain and four copies of an optimized AS-1 binding site placed upstream (van der Krol et al., 1993), thereby increasing potential binding of B-ZIP transcription factors. The luciferase gene that is used in the pGM46 and in the pMT520 construct is the original luciferase coding sequence cloned by deWet et al. (deWet et al., 1985). For the pGM107 construct a modified firefly luciferase gene was used (luc+, without the peroxisomal protein import signal, Promega, Madison, WI, USA; Sherf and Wood, 1994), which shows increased expression in plant cells (Lonsdale et al., 1998). In the pGM46 and pGM107 constructs an N-terminal SV40 Nuclear Localization Signal (NLS) was present in front of the luc coding sequence, which had no apparent effect on its activity (van der Krol and Chua, 1991). Petunia hybrida (Vilm.) plants (cv. V26) were transformed by A. tumefaciens clones containing either pGM46, pGM107 or pMT520, and grown on agar plates containing selective antibiotics (100 µg ml-1 kanamycin to select for the transformed shoots) (Murashige and Skoog, 1962). Transformed shoots were, after rooting, transferred to soil and grown in growth chambers with a 16 h light period (50 W m-2, 22 °C, and 70% RH) and an 8 h dark period (20 °C, and 65% RH). For the analyses of the pGM46 transformed plants, the F1 progeny plants of a back-cross with wild-type V26 were used. These plants are coded as: 35S-‘primary transformant code "b"' F1 progeny code’ (e.g. 35S-1b4).
In vivo luciferase activity measurement with the 2D-luminometer
Petunia luc reporter plants were sprayed with a luciferin solution (1 mM firefly D-luciferin, sodium-salt, Molecular Probes, Eugene, OR, USA, 0.01% Tween 80) 48 h, 40 h, 24 h, 16 h, and 2 h before measurement as described previously (van Leeuwen et al., 2000). Plants were analysed by measuring the excised leaves of the main shoot. Plants or excised leaves were imaged with a 2D-luminometer, consisting of an intensified CCD camera (C2400–77, Hamamatsu Photonics, Japan). Photon emission by luc-expressing plants was quantified by computer (shown as relative light units per pixel (rlu pixel-1), Argus-50 Image Processor, Hamamatsu Photonics, Japan) and depicted with false colour scales (blue indicating low activity, red indicating high activity). Integration intervals varied from 5 min to 30 min.
RNA isolation
Petunia leaf material was ground in a liquid nitrogen cooled 2.2 ml microtube containing two 0.25 inch vanadium bullets in a Braun Biotech Micro-dismembrator for 90 s at 1600 rpm. Subsequently, 300 µl RNA extraction buffer (4 M guanidinthiocyanate (GuSCN), 25 mM sodium citrate, 0.5% lauroyl sarcosine) per 100 mg sample was added and the samples were thawed on ice. After addition of 0.1 vol. 2 M NaAc, 1 vol. acidic phenol and 0.2 vols chloroform/isoamylalcohol (24:1, v:v), the mixture was vortexed vigorously for at least 1 min and, subsequently put on ice for 5–15 min. After vortexing the mixture was centrifuged for 20 min at 16000 g at 4 °C. RNaid MATRIX glass beads (BIO 101 inc., Carlsbad, CA, USA) were, subsequently added to the aqueous phase (20 µl per 100 mg leaf material) and mixed and incubated for 10–15 min at RT. After 30 s centrifugation at 16000 g at RT, the pelleted glass beads were resuspended in 1 ml 6 M GuSCN. This step was repeated and the beads were again centrifuged for 30 s at 16000 g at RT. The pellet was now washed 2–3 times with 0.5–0.8 ml 60% ethanol-T10E1 by resuspending and centrifuging. The pellet was resuspended in RNase-free water in a volume equal to the original volume of beads added and incubated at 60–65 °C for 5 min to wash the RNA from the RNaid beads. The mixture was centrifuged for 2 min at maximum speed and the supernatant was again centrifuged. These two steps were repeated to elute another 10% of the RNA from the beads. The RNA was quantified in a GeneQuant RNA/DNA Calculator (Pharmacia, Peapack, NJ, USA/LKB Biochrom Ltd. model 80-2103-98) and stored at -80 °C.
Reverse transcriptase PCR and hybridization
Ten microgram total RNA was DNase treated in 60 µl with 2U DNase (Boehringer-Mannheim, Germany) and 20 U RNAsin (Gibco BRL, Paisley, UK) (Sambrook et al., 1989). One microgram was again quantified with the GeneQuant as well as on 1.5% agarose formaldehyde gel. First strand cDNA was then synthesized of 2.5 µg RNA using reverse transcriptase with Oligo(dT) primers (SuperscriptTM Preamplification System, Gibco BRL, Paisley, UK). Two µl of the obtained 20 µl was then used in a PCR, using ubiquitin specific primers (UBIQ-f and UBIQ-r, Geurts et al., 1997). When the ubiquitin PCR showed comparable levels of total RNA as determined after 25 cycles on a 1.0% w/v agarose (ethidium bromide stained, 150 µg l-1) 1xTAE gel, 2 µl of the cDNA was used in a PCR using luc specific primers. Of each luc PCR a 5 µl sample was taken after 16, 18, 20, 22, and 24 cycles. Five µl of the luc samples was size-fractionated by electrophoresis through a 1.0% w/v agarose gel and transferred to positively charged nylon membrane according to manufacturers instructions (Genescreen Plus, NENTM Life Science Products, Boston, MA, USA). Blots were prehybridized in 1% w/v BSA, 1 mM EDTA, 0.5 M NaHPO4, pH 7.2, 7% SDS at 60 °C for 90 min. Hybridization was carried out in the same mixture in the hybridization oven at 60 °C for 16 h, after addition of approximately 100 ng 
[32P]dATP (Amersham, Didcot, UK) radiolabelled probe prepared by random priming (Boehringer-Mannheim, Germany) of gel-purified DNA or PCR-products. Filters were washed with 2xSSC, 0.1% SDS at 60 °C and exposed to Kodak X-Omat AR films at -70 °C with intensifying screens or exposed to a Molecular Dynamics Phosphor Screen and subsequently, scanned with a Phosphor Imager (Molecular Dynamics, Sunnyvale, CA, USA). Intensity of the bands was quantified with the ImageQuant program (Molecular Dynamics, Sunnyvale, CA, USA).
Primers used for RT-PCR
Ubiquitin primers: UBIQ-f: ATG CAG ATY TTT GTG AAG AC; UBIQ-r: ACC ACC ACG RAG ACG GAG. Luc primers: (forward) SK333: ATG GAA GAC GCC AAA AAC ATA AAG; (reverse) SK305: GGC GGA TCC TAT ATG AGG ATC TCT CTG ATT TTT C.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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Differences in the level of luciferase activity between independent transgenic lines
Petunia hybrida (Vilm.) plants (cv. V26) were transformed by Agrobacterium with a luciferase (luc) reporter gene, driven by either the viral CaMV 35S promoter, a modified version of the CaMV 35S-promoter (m35S), or the A. thaliana Lipid Transfer Protein (LTP) promoter. For each of the chimeric genes, several independent transformants were obtained which contained 1–8 copies of the transgene (as determined by Southern analysis, data not shown).
For each luc reporter gene construct, the individual transformants were analysed for in planta LUC activity in leaf tissue, after equilibration with luciferin. The average LUC activity per Petunia plant was calculated by quantifying LUC activity in excised, fully expanded leaves 5 through 11 of the main shoot (numbering starting at the first visible leaf at the apex). For the CaMV 35S promoter the average transgene activity in leaves (averaged per total leaf area) varied between plants from 0.1–61 rlu pixel-1, for the m35S promoter the transgene activity varied from 0.1–51 rlu pixel-1 and for the LTP promoter from 0.5–27 rlu pixel-1. Relative differences between single copy transformants were as large as between multiple copy transformants (e.g. in seven single copy m35S plants LUC activity varied from 0.3 to 51, while six m35S plants with three copies showed LUC activity varying between 0.1 and 11). These results confirm previous reports, which show that the level of expression of a transgene can vary among independent transformants (Dean et al., 1988).
Differences in the level of luciferase activity between leaves on the same plant
For each luc reporter gene construct, the LUC activity was quantified in individual excised, fully expanded leaves from the main shoot of three independent single locus transgenic lines. Figure 1 shows the average LUC activity per leaf measured in 30 min in leaves 5 through 11 (rlu pixel-1), in three single locus plants. In general, the LUC activity decreases upon ageing of the leaf. However, in, for example, line 35S-2b10, line m35S-6 and line LTP-7, the highest average LUC activity occurs in leaf 9, leaf 9 and leaf 10, respectively, instead of in leaf 5. The differences in LUC activity between the leaves of a single plant can range from a factor 1.9 (in line 35S-2b10) to a factor 22 (in line LTP-7). The variation in average expression level of a plant can be characterized by the coefficient of variation (CV=[standard deviation/ average]x100%) of the average LUC activity in leaves 5 to 11. The average LUC activity and CV are shown for each individual transformant below each graph in Fig. 1. The CV for these seven subsequent leaves in all analysed plants varies per plant from 17–49% in the 35S-luc plants (n=12), from 34–169% in the m35S-luc plants (n=16) and from 23–100% in the LTP-luc plants (n=8).
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Although there are differences in the range of CV per plant between the different transgenes, it was conclude that for each of the three different transgenes the change in average LUC activity in leaves from one plant does not seem to follow a distinct pattern that can be related to either the intrinsic properties of the transgene promoter or to the developmental stage of the leaves.
The average in vivo LUC activity correlates with the average luc mRNA steady-state levels in leaves
Under these conditions, the observed variation in LUC activity is not related to differences in substrate availability (described in van Leeuwen et al., 2000). The alternative explanation for the observed variation is that there is local variation in the amount of LUC protein. This can be caused by variations either in translation efficiency or by differential promoter activity within a leaf. It was therefore verified whether the (average) photon production in leaves relates to the steady-state level of luc mRNA in these leaves.
A low, a medium, and a high LUC active leaf from plant 35S-1b4 were used as samples. Total RNA was extracted from each leaf and the amount of luc mRNA in each pool was semi-quantified by reverse transcriptase PCR, using ubiquitin expression levels as an internal control (Fig. 2). Figure 2A shows the result of the reverse transcriptase PCR reaction, which was quantified after 24 cycles and plotted against the average LUC activity per leaf (Fig. 2B). The additional lower band that is visible in Fig. 2A is caused by a small percentage of single stranded DNA in each sample. This percentage is the same in each sample. Figure 2C-E show that for all of the three different reporter constructs the imaged in vivo LUC activity in individual leaves correlates with the relative luc mRNA levels. The lower values of average luciferase activity per leaf in Fig. 2 (compared to Fig. 1
; e.g. 35S-1b4) are caused by the shorter measuring time and by the fact that older leaves are used (leaf 8 and higher). The data in Fig. 2 indicate that the observed differences in average LUC activity in leaves as measured by photon production, are a true reflection of differences in average transgene transcription rate.
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Different variegated patterns of LUC activity between transformants and between leaves of the same plant
A large variation was observed in luciferase activity within a single leaf (referred to as variegation) for each construct. In Fig. 3, the seventh, excised leaf of three different single locus lines for each of the three luc reporter constructs is shown as an example of this variegation. Similar variations were also observed in other plant species transformed with these and other luc constructs (e.g. tobacco, Arabidopsis, tomato and potato, data not shown), indicating that variegated promoter activity in leaves is intrinsic to many plant gene promoters. An indication of the degree of variegation can be obtained by calculating the CV of the LUC activity within each leaf. Below each image in Fig. 3, the average LUC activity and CV of the leaf are shown, as well as the maximum LUC activity (measured in 30 min, rlu pixel-1). When the CV of all fully expanded leaves (5–12) of all independent transformants is compared it is seen that the leaves of the 35S plants have an average CV of 89%, the leaves of the m35S plants have an average CV of 162% and the leaves of the LTP plants have an average CV of 99%. The percentage of leaves with a CV below 100% is for the 35S, the m35S and the LTP population, 81%, 31% and 69%, respectively.
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In Fig. 1
it is shown that the average level of LUC activity varies in different ways between individual leaves of a shoot. In Fig. 3 differences in the type and level of variegation between leaves (in the same developmental stage) of different plants are shown. It was, therefore, investigated whether there are only differences in the level of gene expression between leaves of the same shoot, or whether there are also differences in the degree of variegation in those leaves.
Like the average LUC activity (Fig. 1), the leaf to leaf variation of the maximum LUC activity per leaf does not seem to follow a distinct pattern that can be related to either the transgene promoter or to the developmental stage of the leaves (data not shown). Differences in average LUC activity per leaf are therefore not caused by a different number of ‘cells with maximum activity’ that are active within a leaf. The ratio of the maximum luciferase activity over the average luciferase activity (max/avg) can also be used to characterize the degree of variegation within a leaf. A linear relation is observed when the CV is plotted against the max/avg value (data not shown). In Table 1 this max/avg ratio is shown for each leaf of three single locus lines per construct. In leaves with a more or less even distribution of LUC activity, the maximum activity that was measured within a leaf was no more than 2–3 times the average value within that leaf. In highly variegated leaves this ratio can be as large as 40 (e.g. line m35S-6; Table 1). The degree of variegation differed between the three reporter gene constructs. The max/avg ratio for leaves 5 to 11 varied in 35S-luc plants from 2 to 8, in m35S-luc plants this ratio varied from 7 to 41 and in LTP luc plant the ratio varied from 3 to 14 (Table 1).
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Although both the average luciferase activity (Fig. 1
) as well as the maximum luciferase activity vary from leaf to leaf (and therefore the variegation varies, Table 1), there seems to be no obvious relation between these two quantitative aspects of transgene activity. This is illustrated, for example, in leaves 8 and 11 of plant m35S-6 which have comparable average activity (Fig. 1), but have a max/avg ratio of 9 and 27, respectively (Table 1). The substantial differences in the degree of variegation that can occur between subsequent leaves on the same plant make it difficult to assign a single pattern of expression to a plant. This complicates the comparison of transgene expression between individual transformants or between primary transformants and progeny plants.
In order to investigate whether the local luciferase light emission within a leaf correlates with local mRNA steady-state levels, several leaves with highly variegated luciferase activity were analysed. Figure 4 gives an example of this analysis for a highly variegated m35S-luc leaf. RNA was isolated from the low and high LUC active half of the leaf (as shown in Fig. 4A) and luc mRNA levels were semi-quantified by RT-PCR (Fig. 4B). Figure 4C shows that the luc mRNA level in the higher active part was higher than the luc mRNA level in the lower active part. Small deviations in the mRNA isolation of the small leaf samples as well as in the cDNA synthesis, RT-PCR and background of hybridization, might disturb the absolute correlation between LUC activity and luc mRNA. This figure indicates, however, that not only between leaves but also within leaves, the results from imaging LUC activity in vivo does give information about the local activity of the promoter driving the luciferase transgene.
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Developmental regulation of variegated luc transgene activity in leaves
To examine the temporal variation in the pattern of luciferase expression during the development of a single leaf, the luc transgene activity in the same leaf was imaged over a period of 50 d. The imaging started at a developmental stage where the size of the leaf was only a few millimetres and continued up to the stage of a fully expanded leaf. Several leaves of Petunia plants containing either one of the three promoter driven luc constructs were imaged every day in planta. The LUC activity images of line m35S-3 are shown as an example in Fig. 5 from day 1 to 43. As an indication of the degree of variegation, the max/avg ratio for two lines of each promoter-luc construct is shown in Table 2. With the continuous application of luciferin, a renewed application of luciferin directly after the measurement had no effect on the variegated pattern of LUC activity (Van Leeuwen et al., 2000). From Fig. 5 and Table 2, it can be seen that the degree of variegation within a leaf is not the same, but varies between subsequent days. Although the activity generally decreases after several weeks, the degree of variegation still can be very high. Eventually the leaves senesced and at the same time showed extinction of luciferase activity, mostly between 44 and 50 d (data not shown).
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Since individual transgenic lines show distinct and different types of variegated expression patterns, it is unlikely that the variegated patterns of LUC activity are strictly related to different developmental stages of the leaf. It would then be expected that clonal patterns related to leaf development would appear more often (Poethig and Sussex, 1985
). The pattern of LUC activity within a leaf is not stable, but slowly changes from day to day (Fig. 5). This indicates that there is a temporal regulation to the spatial distribution of transgene promoter activity. These day-to-day variations in gene expression within a leaf may also contribute to the observed variation in gene expression between leaves of the same shoot (Fig. 1).
Stable inheritance of the patterns of LUC activity
The luciferase activity pattern was compared in progeny plants of transformants carrying a single locus of the luc reporter gene. The primary transformants were back-crossed with wild-type Petunia plants and F1 progeny plants were selected on basis of LUC activity. In genetically identical F1 progeny plants, approximately the same global level of LUC activity was observed, as shown in Fig. 6A for three progeny plants for each of the three luc reporter constructs. The inheritance of easily recognizable developmental patterns of LUC activity (different levels between leaves) can also be shown. In LTP line 7 both the primary transformant as well as the genetically identical progeny plants showed a striking increase in LUC activity in the seventh leaf from the top of the shoot, directly followed by a decrease in LUC activity in older leaves (Fig. 6B). The temporal regulation of the level of LUC activity is, therefore, also inherited. Inheritance of the spatial distribution of LUC activity (within a leaf) is difficult to examine, because variegation may vary within a plant from leaf to leaf or within a leaf from day to day (see Fig. 5). However, Fig. 6C shows three leaves from m35S line 3 (primary transformant and two back-cross progeny plants, respectively) with a comparable pattern of variegation. Specific for these plants is the presence of higher LUC activity at the edges of the leaf. Since the different types of spatial expression patterns can be stably inherited to progeny plants, variegation can not only be caused by local physiological conditions, but must also be determined by the integration site of the transgene.
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Factors that possibly cause variegated luciferase activity
The observed variegated LUC activity might be intrinsic to the used transgene promoters. However, each of the three reporter gene constructs shows a variety of different variegated expression patterns in individual transformed lines. The m35S promoter was designed to increase the level of expression. Although very high active patches of luciferase gene expression exist in m35S leaves (Fig. 3), the overall level of average LUC activity per plant was (in the population of 16 independent transformants: 9.3±14.1 rlu pixel-1) lower than that of the 35S-luc population (12 plants: 24.0±22.1 rlu pixel-1), but clearly higher than that of the LTP-luc population (8 plants: 5.8±7.2 rlu pixel-1).
The LTP promoter is active in the L1 cell layer (epidermis) of the shoot. It was noted that in all these LTP-luc transformants this general tissue and cell layer specificity was retained (data not shown). Apparently, within this tissue and cell layer specific expression an extensive variation in spatial and temporal regulation of transgene expression still occurred. Imaging of leaf sections has shown that the 35S promoter and m35S promoter both are active in all cell layers of a leaf (Van Leeuwen et al., 2000). Sectioning through high and low active LUC patches in leaves of plants expressing a 35S or m35S luc gene showed that the observed local LUC activity was present in all cell layers (either all cell layers high or all cell layers low, Van Leeuwen et al., 2000). In all plants variegated LUC activity is thus caused by variegated promoter activity that locally extends to all cell layers of the leaf tissue.
Variegated gene expression has been observed and described before in relation to gene silencing phenomena (Depicker and Van Montagu, 1997; Flavell, 1994; Matzke and Matzke, 1998; Stam et al., 1997). It has also been shown that gene silencing can occur in distinct different patterns within a tissue (Jorgensen et al., 1996; Van der Krol et al., 1988). However, gene silencing phenomena rarely show a range of levels of gene expression, but only ‘on’ or ‘off’ gene expression (Jorgensen et al., 1998). Patterns caused by (trans-) gene silencing are fixed within the tissue and may only undergo reversion in newly synthesized organs. The observed day-to-day varying patterns of LUC activity in the same leaf (Fig. 5) in this study's experiments are therefore different from such gene silencing phenomena.
The chromatin structure around each transgene locus may differ and may result in a variable accessibility for transcription factors (resulting in the position effect, Dean et al., 1988). The results of this study would then indicate that this DNA accessibility might not only vary quantitatively between individual transformants, but also may vary differently within a plant in time and place. In that case, a variegated transgene expression pattern might occur with an even distribution of transcription factors; such a variegated pattern will not (or not necessarily) occur for endogenous gene expression.
Alternatively, the variegated transgene promoter activity may be caused by true local differences in amount and/or activity of transcription factors within cells of a tissue. Since these transcription factors also act on endogenous plant genes, the prediction would be that some plant genes would also show variegated expression patterns. Such a heterogeneous promoter activity of, for example, the endogenous chalcone synthase (chs) gene has already been reported in in situ experiments (Nick et al., 1993). The results of this present study indicate that at least for the ubiquitin gene, expression does not seem to be comparably variegated within a leaf (the luc mRNA levels were plotted relative to comparable ubiquitin mRNA levels).
It has been established that the variegation of LUC activity in plants can be attributed to differences in local mRNA steady-state levels, and it is currently being investigated whether this variegation is a feature only of the transgene(s) or whether some endogenous plant genes also show such varied patterns of promoter activity, possibly related to local differences in transcription factor availability and hormone signalling.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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Transgene promoter activity can only be characterized by the distribution of the different expression levels within a plant, each level occurring with its own frequency. Sampling of single leaves might lead to different conclusions about the level of gene expression per plant (compare, for example, leaf 6 of plant m35S-6 and m35S-12 in Fig. 1
). From Fig. 3 it is also clear that sampling leaf discs in order to compare the level of gene expression per plant, is even more imprecise, because the variations within a leaf can be as large as the variations between plants. Therefore, instead of describing the level of gene expression as one value per plant, one has to describe the range and frequency of gene expression levels per leaf and per plant. Only the general spatial and temporal expression features of a transgene in different independent transgenic lines must therefore be intrinsic to the transgene promoter. Since every independent transformant shows minor or major differences in spatial and temporal regulation of the transgene, apparently in every transformant there is a different influence from flanking plant DNA sequences. These results show that the promoter driving the transgene specifies the cell type(s) in which the transgene is expressed and defines the global temporal regulation of the transgene promoter activity within these cells. Superimposed on this are the effects of differences in transgene integration site, which may result in different local modulations of the temporal regulation of transgene activity. Local differences in temporal regulation may thus result in different variegated patterns of transgene activity (both within a leaf as well as between leaves), as were observed by the imaging of in planta transgene (luciferase) activity.
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Acknowledgments |
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We would like to thank D Postma-Haarsma and R Kuijpers for creating the 35S-luc and m35S-luc constructs, M Toonen for providing the LTP-luc construct and R Geurts for providing the ubiquitin primers. We thank O Vorst for his contributions to the initial set-up of the luciferase reporter system in our laboratory. This work is supported by the Dutch Organization for Scientific Research (NWO).
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Notes |
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1 Present address: Department of Plant Physiology, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands.
2 Present address: Laboratory of Molecular Biology, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
3 To whom correspondence should be addressed. Fax: +31 317 484740. E-mail: sander.vanderkrol{at}pph.dpw.wau.nl
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Abbreviations |
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CaMV, Cauliflower Mosaic Virus; CV, coefficient of variation; LTP, Arabidopsis thaliana Lipid Transfer Protein; luc, luciferase; max/avg, maximum luciferase activity/average luciferase activity; m35S, modified CaMV 35S; rlu, relative light units..
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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