The promotors of the single-copy genes for subunit 6 of the chloroplast ATP synthase (atpD) and plastocyanin (Pc> from spinach have been sequenced, dissected and analysed in transgenic Fo and F1 tobacco plants using the bacterial GUS gene as a reporter for promotor activity. The transcription of these genes is photo-controlled. The results have been compared with those obtained for the spinach rbcS-1 gene, one of the light-regulated genes encoding the small subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase, and for the cauliflower mosaic virus (CaMV) 35s RNA promotor. We find that the 5' upstream regions of about 1200 nucleotides contain all the sequences required for light regulation, organ-, tissue-and development-specific expression, and that they are structurally diverse. Their cis-acting elements are functionally defined. The proximal regions of the spinach promotors contain potential TATA, CAAT and T-cyt boxes at appropriate positions, but only sequence elements with low similarity to published light-responsive elements. Positive light-stimulated regions, regions with constitutive, light-independent enhancing effects and with 'silencer'-like activity in complete darkness are found in proximal and far upstream promotor segments. Highest activity of these promotors is correlated with the presence of chloroplasts but is not confined to photosynthetic tissue. Surprisingly, expression is observed in the phloem regions of transgenic leaves, leaf and floral stems, in the vascular area of anthers and in pollen. No histochemical staining has been detected in roots. The distal region of atpD located between -1137 and -590 contains elements for expression in the outer phloem, the region from -590 to -185 for activity in the inner phloem of floral stems. Similar tissue-specific patterns are observed with a fusion between the caufliflower mosaic virus 35s RNA promotor and the GUS gene.Light is one of the most important exogenous factors for harmoniously balanced growth and development of a plant [I]. It provides the energy for photosynthesis and controls the expression of a large number of genes during ontogeny (for reviews see [2,31). Its profound influence is quite obvious in the biogenesis of chloroplasts which develop from small, more or less undifferentiated organelle forms, the colorless, elementary proplastid in vegetation tips or the pale-yellow etioplast in dark-grown material, into the green photosynthetically competent organelle. This morphogenetic process is accompanied by the differentiation of the complex internal (thylakoid) membrane system which catalyses the fundamental light reactions in photosynthesis.
The nucleotide sequence of the entire nuclear-encoded precursor for subunit delta of the ATP synthase from spinach thylakoid membranes was determined by cDNA sequencing. Appropriate recombinant DNAs were selected from pBR322 and lambda gt11 libraries made from polyadenylated RNA of greening spinach seedlings. The mature protein consists of 187 amino acid residues corresponding to a molecular weight of 20468. The precursor protein (257 amino acid residues; M r=27676) is probably processed between a Met-Val bond. The predicted secondary structure of the transit sequence (70 residues; 7.2 kDa) resembles that of the Rieske Fe/S polypeptide, but shows little similarity with those of stromal or luminal proteins. The comparison of the chloroplast delta amino acid sequence with the published delta sequences from respiratory ATP synthases of bacterial and mitochondrial sources and from the thylakoid ATP synthase of the cyanobacterium Synechococcus suggests substantial divergence at the genic level although structural elements appear to be remarkably conserved.
Abstract. Deletion analysis of the nitrite-reductase (NiR) promoter from spinach (Spinacia oleracea L.) fused to the fl-glucuronidase (GUS) reporter gene and introduced into tobacco (Nicotiana tabacum L., cv. Coker 176) indicates that basic elements required for light-and nitratedependent expression of the reporter are located within the promoter sequence -200/+ 131 relative to the transcription-initiation site. Detailed analysis indicates that positive regulatory elements exist between -200 and -330 as well as between -1450 and -1730, stimulating the level of GUS gene expression under all experimental conditions. Induction/reversion light-pulse experiments show that the promoter sequence -200/+ 131 suffices for phytochrome-mediated expression of the reporter gene. The observation that the NiR promoter from spinach exhibits full reversibility in transgenic tobacco confirms the previous conclusion that the NiR promoter from spinach fused to a GUS reporter gene and introduced into tobacco responds to nitrate and phytochrome as would be expected for tobacco (host) and not as would be expected for spinach (donor). When the plastids were damaged by photooxidation in the presence of Norflurazon, GUS activity levels were reduced to the same extent for all NiR-promoter/GUS fusions tested, indicating that the promoter region involved in the action of the 'plastidic factor' is between -200 and + 131. The GUS gene expression under the control of the CaMV-35S promoter is not affected by light, nitrate or the 'plastidic factor'.
In the present study the question was addressed of whether the nitrite-reductase (NIR-)promoter from spinach (Spinacia oleracea L.), fused to a reporter gene (bacterial β-glucuronidase, GUS) and introduced into tobacco (Nicotiana tabacum L.) responds to nitrate and light in accordance with spinach (donor) or in accordance with tobacco (host). The data obtained at the GUS enzyme level as well as at the transcript level allow an unambiguous answer to this question: GUS gene expression under the control of the NIR-promoter from spinach responds to nitrate and light in accordance with the host (tobacco). Expression of the promoter-less GUS gene was not induced by any treatment.
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