Promoters from genes for plastid proteins possess regions with different sensitivities toward red and blue light.

The light-regulated expression of eight nuclear-encoded genes for plastid proteins from spinach (Spinacia oleracea) (RBCS-1 and CAB-1; ATPC and ATPD, encoding the subunits gamma and delta of the ATP synthase; PC and FNR; PSAD and PSAF, encoding the subunits II and III of photosystem I reaction center) was analyzed with promoter/beta-glucuronidase (GUS) gene fusions in transgenic tobacco (Nicotiana tabacum and Nicotiana plumbaginifolia) seedlings and mature plants under standardized light and growth conditions. Unique response patterns were found for each of these promoters. GUS activities differed more than 30-fold. Strong promoters were found for the PC and PSAD genes. On the other hand, the ATPC promoter was relatively weak. Expression of the CAB/GUS gene fusion in etiolated material was at the detection limit; all other chimeric genes were expressed in the dark as well. Light stimulation of GUS activities ranged from 3- (FNR promoter) to more than 100-fold (CAB-1 promoter). The FNR promoter responded only to red light (RL) and not significantly to blue light (BL), whereas the PC promoter contained regions with different sensitivities toward RL and BL. Furthermore, different RNA accumulation kinetics were observed for the PSAF, CAB, FNR, and PC promoter/GUS gene fusions during de-etiolation, which, at least in the case of the PSAF gene, differed from the regulation of the corresponding endogenous genes in spinach and tobacco. The results suggest either that not all cis elements determining light-regulated and quantitative expression are present on the spinach promoter fragments used or that the spinach cis-regulatory elements respond differently to the host (tobacco) regulatory pathway(s). Furthermore, as in tobacco, but not in spinach, the trans-gene hardly responds to single light pulses that operate through phytochrome. Taken together, the results suggest that the genes have been independently translocated from the organelle to the nucleus during phylogeny. Furthermore, each gene seems to have acquired a unique set of regulatory elements.     

Developmental and pathogen-induced activation of an msr gene,str246C,from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5′ flanking DNA sequence from the str246C gene fused to the β-glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5′ deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.     

Isolation and Characterization of a Curcin Promoter from <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. and Its Regulation of Gene Expression in Transgenic Tobacco Plants

Ribosome-inactivating proteins (RIPs) represent those proteins that universally depurinate conserved α-sarcin loops of large rRNAs. In this study, a 0.6-kb fragment of a 5′ flanking region preceding a curcin gene, encoding a type I RIP curcin, of Jatropha curcas L. endosperm was cloned, and its regulation of expression of the β-glucuronidase (GUS) reporter gene was investigated in transgenic tobacco. Analysis of GUS activities showed that the 0.6-kb flanking fragment of the curcin gene was sufficient to drive the GUS reporter gene expression in tobacco seed. The activity of this flanking fragment was analyzed at different stages of seed development. Histochemical localization of GUS activity indicated that the promoter was specifically active in the endosperm tissue of the dicotyledonous tobacco embryo. Moreover, this activity was first initiated at the heart-shaped embryonic stage during seed development.     

Developmental and pathogen-induced activation of an msr gene, str246C, from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5 flanking DNA sequence from the str246C gene fused to the -glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5 deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.Joint first authors     

Characterization of a Novel Pollen-Specific Promoter from Wheat (Triticum Aestivum L.)

PSG076 is a pollen-specific gene isolated from wheat. The 1.4-kb promoter upstream of the ATG start codon was isolated by inverse-PCR (IPCR). To determine its activity, the PSG076 promoter was fused with the ??-glucuronidase (GUS) reporter gene and introduced into tobacco. Histochemical analysis in transgenic tobacco showed that GUS activity was detected in late bicellular pollen grains and increased rapidly in mature pollen. GUS activity was also detected in pollen tubes of transgenic tobacco. No GUS activity was found in other floral and vegetable tissues. These results indicate that the PSG076 promoter directs pollen-specific activity at late stages of pollen development and pollen tube growth. Deletion analysis showed that a 0.4?kb fragment of the promoter was enough to confer pollen-specific expression.     

Anaerobic induction of the maize<Emphasis Type="Italic"> GapC4</Emphasis> promoter in poplar leaves requires light and high CO<Subscript>2</Subscript>

The maize (Zea mays L.) glyceraldehyde-3-phosphate dehydrogenase gene 4 (GapC4) promoter confers anaerobic gene expression in tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and Arabidopsis thaliana (L.) Heynh. Here we have investigated its expression in hybrid poplar (Populus tremula × P. alba). Our results show that the promoter is not expressed in leaves and stems under normoxic conditions while anaerobiosis induces reporter gene expression in leaves up to a level observed for the STLS-1 promoter from potato that is shown to confer leaf-specific gene expression in transgenic poplar. Anaerobic induction is cell autonomous and requires a CO₂ atmosphere and light. As in tobacco, the GapC4 promoter in poplar is wound inducible. The induction by CO₂ and light may reflect a natural situation because flooding, a natural cause of anaerobiosis, is often accompanied by high CO₂ concentrations in the floodwater. Our results show that the GapC4 promoter is suitable as an anaerobic reporter and as an inducible gene expression system in poplar.Abbreviations CaMV cauliflower mosaic virus - GapC4 glyceraldehyde-3-phosphate dehydrogenase gene 4 - GUS -glucuronidase - 4-MU methylumbelliferone - STLS-1 stem- and leaf-specific promoter 1