The Tall Fescue Turf Grass Class I Chitinase Gene <Emphasis Type="Italic">FaChit1</Emphasis> Is Activated by Fungal Elicitors,Dehydration, Ethylene,and Mechanical Wounding

The cDNA, genomic DNA, and promoter sequence of FaChit1, a class I chitinase gene from Festuca arundinacea, were isolated and characterized in the present work. The deduced amino acid sequence of FaChit1 contains the chitin binding, catalytic, and proline and glycine-rich domains characteristic for most class I chitinases, but no C-terminal extension region. FaChit1 is induced effectively by fungal elicitors, dehydration, and ethylene, but only slightly by mechanical wounding. To identify potential stress-related cis-acting elements, 5′ sequences 935, 651, and 233 bp upstream of the FaChit1 start codon were fused to the GUS reporter gene and analyzed in transgenic tobacco. The results indicated that the 935 bp fragment closely mirrored endogenous gene expression and that the 651 bp fragment was sufficient to direct reporter the gene expression in response to fungal elicitors, ethylene, dehydration, or mechanical wounding due to both known and presently uncharacterized cis-acting elements. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ethylene- and ozone-induced regulation of a grapevine resveratrol synthase gene: different responsive promoter regions

Stilbene synthases (STSs) are enzymes that play a critical role in the biosynthesis of stilbenes, phytoalexins that are induced by various biotic and abiotic stressors like pathogen attack, UV radiation or ozone exposure. To investigate the molecular basis for ozone- and ethylene-induced plant stress responses, we analysed the full size promoter of the grapevine resveratrol synthase gene (Vst1). Transgenic tobacco plants harbouring a Vst1 promoter::β-glucuronidase (GUS) reporter construct showed an increased GUS activity upon ozone (0.3 μl l^–1, 8 h) or ethylene (10 μl l^–1, 8 h) treatment. Using the fluorimetric GUS assay, deletion analysis of the 5′-flanking sequence identified a minimal ethylene-responsive region between –280 and –40 bp upstream of the translation start codon. This region differs from the ozone-responsive Vst1 promoter region, suggesting that ethylene is not directly involved in signalling ozone-induced stilbene biosynthesis.     

Characterization of <Emphasis Type="Italic">VvPAL-like</Emphasis> promoter from grapevine using transgenic tobacco plants

A 2000-bp 5′-flanking region of VvPAL-like was isolated from ‘Summer Black’ grapevine by PCR amplification, named pVvPAL-like. To gain a better understanding of the expression and regulatory mechanism of VvPAL-like, a chimeric expression unit consisting of the β-glucuronidase (GUS) reporter gene under the control of a 2000-bp fragment of the VvPAL-like promoter was transformed into tobacco via Agrobacterium tumefaciens. Histochemical staining showed that the full-length promoter directs efficient expression of the reporter gene in cotyledons and hypocotyls, stigma, style, anthers, pollen, ovary, trichomes, and vascular bundles of transgenic plants. A series of 5′ progressive deletions of the promoter revealed the presence of a negative regulatory region (?424 to ?292) in the VvPAL-like promoter. Exposure of the transgenic tobacco plants to various abiotic stresses demonstrated that the full-length construct could be induced by light, copper (Cu), abscisic acid (ABA), indole-3-acetic (IAA), methyl jasmonate (MeJA) (N-1-naphthylphthalamic acid), ethylene, and drought. Furthermore, the ethylene-responsive region was found to be located in the ?1461/?930 fragment, while the element(s) for the MeJA-responsive expression may be present in the ?424/?292 region in the VvPAL-like promoter. These findings will help us to better understand the molecular mechanisms by which VvPAL-like participates in biosynthesis of flavonoids and stress responses.     

Isolation and Characterization of the Genes Encoding Basic and Acidic Chitinase in Arabidopsis thaliana

Plants synthesize a number of antimicrobial proteins in response to pathogen invasion and environmental stresses. These proteins include two classes of chitinases that have either basic or acidic isoelectric points and that are capable of degrading fungal cell wall chitin. We have cloned and determined the nucleotide sequence of the genes encoding the acidic and basic chitinases from Arabidopsis thaliana (L.) Heynh. Columbia wild type. Both chitinases are encoded by single copy genes that contain introns, a novel feature in chitinase genes. The basic chitinase has 73% amino acid sequence similarity to the basic chitinase from tobacco, and the acidic chitinase has 60% amino acid sequence similarity to the acidic chitinase from cucumber. Expression of the basic chitinase is organ-specific and age-dependent in Arabidopsis. A high constitutive level of expression was observed in roots with lower levels in leaves and flowering shoots. Exposure of plants to ethylene induced high levels of systemic expression of basic chitinase with expression increasing with plant age. Constitutive expression of basic chitinase was observed in roots of the ethylene insensitive mutant (etr) of Arabidopsis, demonstrating that root-specific expression is ethylene independent. Expression of the acidic chitinase gene was not observed in normal, untreated Arabidopsis plants or in plants treated with ethylene or salicylate. However, a transient expression assay indicated that the acidic chitinase promoter is active in Arabidopsis leaf tissue.     

Ozone- and ethylene-induced regulation of a grapevine resveratrol synthase promoter in transgenic tobacco

Stilbene synthases (STSs) are enzymes that play a critical role in the biosynthesis of stilbene, phytoalexins in a small number of unrelated plant species, and are induced by various biotic and abiotic stressors like pathogen attack, UV-irradiation or ozone exposure. To investigate the molecular basis for ozone-induced plant stress responses, we have examined the promoter of the grapevine resveratrol synthase (Vst1). In this report we summarize the influence of ozone on gene regulation. In transgenic tobacco a chimeric gene construct, containing the Vst1 promoter combined with the β-glucuronidase (GUS) reporter gene, is rapidly induced by ozone (0.1 μl·l⁻¹, 12 h). The same construct is also strongly induced by ethylene (20 μl·l⁻¹, 12 h). Promoter deletion analysis of the 5′ flanking sequence identified a positive regulatory element between −430 bp and −280 bp. This region contains ethylene-responsive enhancer elements, as well as an elicitor-responsive sequence in inverse orientation.     

Analysis of an osmotically regulated pathogenesis-related osmotin gene promoter

Osmotin is a small (24 kDa), basic, pathogenesis-related protein, that accumulates during adaptation of tobacco (Nicotiana tabacum) cells to osmotic stress. There are more than 10 inducers that activate the osmotin gene in various plant tissues. The osmotin promoter contains several sequences bearing a high degree of similarity to ABRE, as-1 and E-8 cis element sequences. Gel retardation studies indicated the presence of at least two regions in the osmotin promoter that show specific interactions with nuclear factors isolated from cultured cells or leaves. The abundance of these binding factors increased in response to salt, ABA and ethylene. Nuclear factors protected a 35 bp sequence of the promoter from DNase I digestion. Different 5 deletions of the osmotin promoter cloned into a promoter-less GUS-NOS plasmid (pBI 201) were used in transient expression studies with a Biolistic gun. The transient expression studies revealed the presence of three distinct regions in the osmotin promoter. The promoter sequence from –108 to –248 bp is absolutely required for reporter gene activity, followed by a long stretch (up to –1052) of enhancer-like sequence and then a sequence upstream of –1052, which appears to contain negative elements. The responses to ABA, ethylene, salt, desiccation and wounding appear to be associated with the –248 bp sequence of the promoter. This region also contains a putative ABRE (CACTGTG) core element. Activation of the osmotin gene by various inducers is discussed in view of antifungal activity of the osmotin protein.     

Cloning of a new LEA1 gene promoter from soybean and functional analysis in transgenic tobacco

LEA1 gene from Glycine max can be expressed in late-embryo stage of plants, and respond to salinity and dehydration stress. To elucidate the mechanism for stress tolerance and high expression in seeds, we isolated and characterized the promoter of LEA1 gene (EQ, 1997 bp) starting the 5′LEA1 coding region. A deletion mutant of EQ promoter (ED) and the full length promoter (EQ) were fused to GUS reporter gene and transformed into the tobacco leaf discs. The results indicated that expression of the reporter gene (GUS) could be regulated by EQ promoter, and was stronger than the mutant under the stress conditions. Also, the expression level of GUS gene driven by EQ promoter in transgenic tobacco seeds was significantly higher than that by the mutant promoter, which meant that it had a better tissue-specificity. Therefore, the active domain for the promoter was located between ?1997 and ?1000 bp. Additionally, the activity of EQ promoter was 2.1-, 3.3- and 0.4- times stronger than the activity of promoter CaMV35S under salt (24 h), drought (10 h) or ABA (24 h), respectively. Meanwhile, the GUS activity of EQ promoter in seeds was 1.8-fold stronger compared to the promoter CaMV35S. In summary, the new promoter (EQ) is bi-functional, stress-inducible and seed-specific. These findings provide a further understanding for the regulation of LEA1gene expression, and suggest a new way for improving seed quality under saline and alkaline land.