In the C4 plant maize, members of the rbcS gene family, encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, are not expressed in mesophyll cells (MC) but are expressed strongly in the adjacent bundle sheath cells (BSC). Expression of genes in an in situ transient expression assay indicates that the photostimulated expression seen in BSC during the first 24 h that leaves of dark-grown seedlings are illuminated requires rbcS-m3 sequences lying between -211 bp and +434 bp of the transcription start site. Photoregulated partial suppression of rbcS-m3 expression in MC, on the other hand, requires gene sequences that lie between -907 bp and -445 bp together with sequences that lie between +720 and +957 bp within the 3' transcribed region ofthe gene. Suppression in MC occurs during the second 24-h period that dark-grown seedlings have been illuminated, but not during the first 24 h. The 3' +720-to +957-bp region is also effective in lowering MC expression when it is relocated to a position >2 kbp upstream of the transcription start site. Thus, suppression of rbcS-m3 expression in MC has, at the least, a substantial transcriptional component. As reported earlier, a converse pattern of suppression in BSC and stimulation of expression in MC is seen in the control of cab-ml in maize leaves.In C4 plants, such (3,4), but within the first 24 h of illumination their abundance increases 2-to 3-fold in BSC and they are undetectable in MC (3, 4). Transcripts of the maize gene rbcS-m3 (5) follow this pattern of BSC-preferred accumulation upon illumination of dark-grown leaves and constitute about 35% of the total leaf rbcS mRNA in 24-h illuminated dark-grown maize (3). Using a 3-glucuronidase (GUS) in situ transient expression assay (6), we found that expression from a reporter gene containing 2.1 kbp upstream of the rbcS-m3 transcription start site [-2.1-kbp rbcS-m3 promoter (Pr):GUS:nopaline synthase (nos) terminator] is about the same in MC and BSC of 24-h illuminated leaves. GUS expression is promoted 2-to 3-fold in BSC by illumination, as expected from the behavior of rbcS-m3, but this reporter gene does not behave like rbcS-m3 in MC after illumination: GUS expression from the rbcS-m3 promoter is about the same in MC of unilluminated and illuminated leaves (3, 6).We have now found that inclusion of a 238-bp sequence from within the transcribed 3' end of rbcS-m3 (extending from +720 to +957 bp relative to the transcription start site) in a reporter gene containing 2.1 kbp of rbcS-m3 from upstream of the transcription start site preferentially reduces expression of GUS in MC in leaves of dark-grown seedlings that are greened (i.e., illuminated) for 24 h prior to bombardment. Furthermore, the 238-bp fragment provides MCpreferred suppression of expression through a transcriptional mechanism. Through a series of promoter deletion experi- (Fig. 1). The pMT211 plasmid was obtained by first digesting pMT444 with Kpn I and HindIII, then treating it with exonuclease III and mung bean nuclease, ...
Cell-specific and light-regulated expression of the .8-glucuronidase (GUS) reporter gene from maize cab-ml and rbcS-m3 promoter sequences was studied in maize leaf segments by using an in situ transient expression microprojectile bombardment assay. The cab-ml gene is known to be strongly photoregulated and to be expressed almost exclusively in mesophyll cells (MC) but not in bundle sheath cells (BSC).Expression of GUS from a 1026-base-pair 5' promoter fragment of cab-ml is very low in dark-grown leaves; GUS expression is increased about 10-fold upon illumination ofdark-grown leaves. In illuminated leaves, the ratio of GUS expression in MC vs. BSC is about 10:1. The cab-ml region between 868 and 1026 base pairs 5' to the translation start confers strong MC-preferred expression on the remainder of the chimeric gene in illuminated leaves, but a region between -39 and -359 from the translation start is required for photoregulated expression. Transcripts of rbcS-m3 are found in BSC but not in MC and are about double in BSC of greening dark-grown seedlings. In contrast to the behavior of the cab-ml-GUS construct, GUS expression driven by 2.1 kilobase pairs of the rbcS-m3 5' region was about twice as high in MC as in BSC of unilluminated dark-grown maize leaves. The number of BSC, but not MC, expressing GUS nearly doubled upon greening of bombarded etiolated leaves. These data suggest that the 5' region ofrbcS-m3 used here could be responsible for most ofthe light-dependent increase in rbcS-m3 transcripts observed in BSC of greening leaves and that transcriptional or posttranscriptional mechanisms are responsible for the lack of rbcS-m3 transcripts in MC. Although the recent generation of transgenic maize (11) carrying 8-glucuronidase (GUS) reporter gene constructs makes this possible in principle, maize transformation and regeneration procedures are lengthy and thus are not convenient for the analysis of numerous modified forms of genes.We have found that DNA precipitated on tungsten microprojectiles can be delivered into MC and BSC in situ in maize leaf segments and that MC-specific and light-induced GUS expression from the cab-ml promoter1 requires two widely separated sequences 5' to the translation start site. In contrast, GUS expression from the rbcS-m3 promoter is about the same in MC and BSC of 24-hr greened leaves, but expression of the rbcS chimeric gene is promoted by light in BSC. MATERIALS AND METHODSPlant Material. Seeds of maize (Zea mays; FR9cms x FR37; Illinois Foundation Seeds, Champaign, IL) were sown in damp Vermiculite and grown at 300C in darkness. Ten-dayold seedlings were harvested under a dim-green safelight, and their second leaves were used in the transient in situ expression assay described below.Transient in Situ Expression Assay. Four 3.5-cm-long segments of the upper halves of the second leaves of 10-day-old dark-grown maize seedlings, a total area of about 12 cm2, were flattened on 0.8% agar Murashige and Skoog medium (GIBCO) in a 5-cm Petri plate with the lower epidermis facing ...
The coding sequence for FLP recombinase, originally from the 2 mu plasmid of Saccharomyces cerevisiae, was introduced into Arabidopsis behind the cauliflower mosaic virus 35S promoter. FLP activity was monitored by the glucuronidase activity resulting from inversion of an antisense-oriented GUS reporter gene flanked by a pair of FRT target sites in inverted repeat. FLP-dependent Gus activity was observed in both transient assays and transgenic plants. The FLP system will be useful for a variety of in planta genetic manipulations.
The HO endonuclease promotes gene conversion between mating-type alleles in yeast by a DNA double-strand break at the site of conversion (the MATYR site). As a first step toward understanding the molecular basis of homologous recombination in higher plants, we demonstrate that expression of HO in Arabidopsis enhances intrachromosomal recombination between inverted repeats of two defective p-glucuronídase (gus) genes (GUS-test construct). One of these genes has the Y/Z site. The two genes share 2.5 kb of DNA sequence homology around the HO cut site. Somatic recombination between the two repeats was determined by using a histochemical assay of GUS activity. The frequency of GUS+ sectors in leaves of F1 plants from a cross between parents homozygous for the GUS-test construct and HO, respectively, was 10-fold higher than in F1 plants from a cross between the same plant containing the GUS-test construct and a wild-type parent. Polymerase chain reaction analysis showed restoration of the 5' end of the GUS gene in recombinant sectors.The induction of intrachromosomal gene conversion in Arabidopsis by HO reveals the general utility of site-specific DNA endonucleases in producing targeted homologous recombination in plant genomes.
The HO endonuclease promotes gene conversion between mating-type alleles in yeast by a DNA double-strand break at the site of conversion (the MAT-Y/Z site). As a first step toward understanding the molecular basis of homologous recombination in higher plants, we demonstrate that expression of HO in Arabidopsis enhances intrachromosomal recombination between inverted repeats of two defective beta-glucuronidase (gus) genes (GUS- test construct). One of these genes has the Y/Z site. The two genes share 2.5 kb of DNA sequence homology around the HO cut site. Somatic recombination between the two repeats was determined by using a histochemical assay of GUS activity. The frequency of Gus+ sectors in leaves of F1 plants from a cross between parents homozygous for the GUS- test construct and HO, respectively, was 10-fold higher than in F1 plants from a cross between the same plant containing the GUS- test construct and a wild-type parent. Polymerase chain reaction analysis showed restoration of the 5' end of the GUS gene in recombinant sectors. The induction of intrachromosomal gene conversion in Arabidopsis by HO reveals the general utility of site-specific DNA endonucleases in producing targeted homologous recombination in plant genomes.
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