Genomic Cloning of Ribonucleases inNicotiana glutinosaLeaves, as Induced in Response to Wounding or to TMV-Infection, and Characterization of Their Promoters

Abstract

We previously cloned two distinct cDNA clones, NGR1 and NGR3, encoding S-like ribonucleases (RNases) induced by wounding and tobacco mosaic virus (TMV) infection, respectively, in Nicotiana glutinosa leaves. To gain insight into the regulatory mechanism of the RNase genes, we analyzed nucleotide sequences of the genes ngr1 (4.1 kbp) and ngr3 (5.3 kbp), containing their structural genes as well as 5'-flanking regions. The ngr1 gene is organized in three exons with two intervening introns, and ngr3 has four exons interrupted by three introns. Primer extension analyses localized single transcription initiation sites at -32 and -99 upstream of the translation initiation codons ATG in the genes ngr1 and ngr3, respectively. The beta-glucuronidase (GUS) reporter gene analysis with serial 5'-deletion mutants as well as a gel shift assay defined the wound-responsive region at residues -509 to -288 in gene ngr1 and a TMV-responsive region at the residues -401 to -174 in ngr3, respectively. Sequence search using PLACE and PlantCARE data bases showed that a wound-responsive element: the WUN-motif, occurs within the wound-responsive region in ngr1, while ngr3 contains several potential cis-regulating elements, such as the elicitor responsiveness element: the W-box, a TMV responsive element: GT1, and the WUN-motif at positions between -401 and -174. These findings suggested that some of these cis-elements may be involved in inducible expressions of ngr1 and ngr3. Furthermore, the gel shift assay suggested that the dissociation of protein factor(s) upon TMV-infection from the regulatory region may cause an inducible expression of ngr3.

Terpenoids in the Essential Oil and Concentrated Aromatic Products Obtained from Nicotiana glutinosa L. Leaves

“…All parts of N. glutinosa plants were capable of accumulating large quantities of sclareol [11]. Still, higher concentrations were found in the upper, younger plant tissues (upper leaves and flower buds), exceeding 600 µg/g tissue.The economic importance of N. glutinosa is connected mainly to its use in tobacco hybridization and as a model plant in viral control studies [25]. N. glutinosa showed good potential as a source of biorational agents against whitefly (Hemiptera: Aleyrodidae) [17,26]; anthracnose, Colletotrichum lagenarium [27]; blue mold, Peronospora tabacina [9]; rust diseases, Alternaria brassicocola [10]; powdery mildew of tobacco, Erysiphe cichoracearum DC.…”

Suppression of LX Ribonuclease in Tomato Results in a Delay of Leaf Senescence and Abscission

“…According to these expression patterns and potential or identified localizations, various possible functions were attributed to these enzymes in plants. Based on the effects of pathogen attack or wounding, roles for RNases in defense were suggested: induction of the tobacco (Nicotiana tabacum) NE gene by a pathogen (Galiana et al, 1997), which could inhibit plant pathogen hyphal growth (Hugot et al, 2002); induction of the tobacco NGR1 and NGR3 RNases by wounding and Tobacco mosaic virus, respectively (Kurata et al, 2002;Hayashi et al, 2003); local and systemic wound induction of the Arabidopsis (Arabidopsis thaliana) RNS1 (LeBrasseur et al, 2002), and wound induction of the tomato (Lycopersicon esculentum) LE (Lers et al, 1998;Gross et al, 2004) and tobacco NW (Kariu et al, 1998) RNases. An effect on the activity of RNases has also been observed in plants, following abiotic stresses such as ozone and salinity (Gomes-Filho and Sodek, 1988;Booker, 2004).…”

Cloning and Characterization of an RNase-Related Protein Gene Preferentially Expressed in Rice Stems

“…6) RRPs share high homology in amino-acid sequence with S-and S-like RNases, but lack one 3) or two [4][5][6] histidine residues located in the conserved regions that are required for RNase activity. [7][8][9][10] While it is well established that S-RNases are involved in the control of self-incompatibility, [11][12][13][14][15] there is growing evidence that S-like RNase genes are associated with processes in which RNA turnover might be involved, e.g., phosphate starvation, [16][17][18][19] senescence, [19][20][21] wounding, [22][23][24][25] and defense against pathogens. [24][25][26][27] But the function of RRPs in plants remains largely unknown.…”