A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification

A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.     

A novel wound-responsive cis-element, VWRE, of the vascular system-specific expression of a tobacco peroxidase gene, tpoxN1

The wound-induced expression of tpoxN1, encoding a tobacco peroxidase, is unique because of its vascular system-specific expression and insensitivity to known wound-signal compounds such as jasmonic acid, ethylene, and plant hormones [Sasaki et al. (2002) Plant Cell Physiol 43:108–117]. To study the mechanism of expression, the 2-kbp tpoxN1 promoter region and successive 5′-deletion of the promoter were introduced as GUS fusion genes into tobacco plants. Analysis of GUS activity in transgenic plants indicated that a vascular system-specific and wound-responsive cis-element (VWRE) is present at the −239/−200 region of the promoter. Gel mobility shift assays suggested that a nuclear factor(s) prepared from wounded tobacco stems binds a 14-bp sequence (−229/−215) in the −239/−200 region in a sequence-specific manner. A mutation in this 14-bp region of the −239 promoter fragment resulted in a considerable decrease in wound-responsive GUS activity in transgenic plants. An 11-bp sequence, which completely overlaps with the 14-bp sequence, was found in the 5′ distal region (−420/−410) and is thought to contribute to the wound-induced expression together with the 14-bp. The −114-bp core promoter of the tpoxN1 gene was indispensable for wound-induced expression, indicating that the 14-bp region is a novel wound-responsive cis-element VWRE, which may work cooperatively with other factors in the promoter.     

Ntlim1, a PAL-box binding factor,controls promoter activity of the horseradish wound-inducible peroxidase gene

To understand molecular mechanisms underlying wound-induced expression of plant peroxidase genes, the promoter of a horseradish C2 peroxidase (prxC2) gene was analyzed. We had previously isolated a tobacco nuclear protein, Ntlim1, as a trans factor binding to a PAL-box motif of the prxC2 promoter; however, the function of the Ntlim1 trans factor and the PAL-box motif in wound-responsive expression of the prxC2 gene remains unclear. Here, we found that the prxC2 promoter without the intact PAL-box motif failed to direct a normal level of both the basal and the wound-induced expression of -glucuronidase (GUS) reporter gene in transgenic tobacco plants, indicating that the PAL-box motif functions as an essential cis element of the prxC2 promoter. We also found that antisense expression of Ntlim1 in transgenic plants carrying the prxC2 promoter::GUS chimeric construct decreased not only the level of the basal and the wound-induced expression of the GUSreporter gene but also the extent of wound inducibility of the prxC2 promoter itself. This result indicates that Ntlim1 is required for the basal level of prxC2 promoter activity as well as its up-regulation under wound stress. Moreover, consistent with the results obtained in planta, result from super-shift assay indicates that the Ntlim1 binds to the PAL-box motif independently of wound stress.     

A wound-inducible gene fromSalix viminalis coding for a trypsin inhibitor

A gene designatedswin1.1 has been isolated by screening aSalix viminalis genomic library with a heterologous probe,win3 fromPopulus. The region sequenced included the entire coding sequence for a protein with 199 amino acids plus the promoter and terminator. At the 5 end of the coding region is a sequence that encodes a hydrophobic region of 25–30 amino acids, that could form a signal peptide. A putative TATAA box and polyadenylator sequence were identified. Introns were absent. The gene product showed similarities with serine protease inhibitors from the Kunitz family and especially withwin3 from wounded leaves ofPopulus. Southern blot analysis indicated thatswin1.1 is a member of a clustered gene family,swin1. An oligonucleotide corresponding to the putative hypervariable region to-wards the carboxyl end when used as a probe in Southern hybridization showed high specificity forswin1.1. Expression of theswin1.1 gene was enhanced in wounded leaves. Theswin1.1 coding region without the signal sequence was highly expressed inEscherichia coli and the protein showed inhibitory activity against trypsin but at most slight activity against the other proteases tested. A systemically induced protein, SVTI, with inhibitor activity against trypsin, was isolated fromSalix leaves by affinity chromatography on a column of trypsin-Sepharose 4B and N-terminal sequenced. It corresponded with the translatedswin1.1 gene at 16 of the 19 amino acid sites, suggesting that SVTI is encoded by another member of theswin1 gene family.     

Strategies for the suppression of peroxidase gene expression in tobacco. II.In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs

Several strategies involving the use of antisense and ribozyme constructs in different expression vectors were investigated as methods of suppressing gene expressionin planta. We had previously identified an efficiently cleaving ribozyme (Rz), with two catalytic units and 60 nucleotide (nt) of complementary sequence, to the ligninforming peroxidase of tobacco (TPX). This Rz was cloned behind the 35S CaMV (35S) and nopaline synthase (NOS) promoters, and into a vector utilising the tobacco tyrosine tRNA for expression. For comparison with more traditional antisense strategies, full-length TPX antisense (AS) constructs were also constructed behind the NOS and 35S promoters. Populations of transgenic tobacco containing these constructs were produced and compared to control plants transformed with the vector only. Significant suppression of peroxidase expression in the range of 40–80% was seen in the T₀ and T₁ populations carrying 35S-AS, 35S-Rz and tRNA-Rz constructs. Co-segregation of the suppressed peroxidase phenotype and the tRNA-Rz transgenes was demonstrated. Northern blot analysis indicated that levels of TPX mRNA were lower in the Rz plants. No evidence of mRNA cleavage was observed and thus it was unclear if the Rz constructs were acting as Rzsin vivo. Transgenic plants containing the tRNA-Rz construct had significantly lower levels of peroxidase than the other transgenic plants. There was no significant difference in levels of suppression of TPX between the short Rz in the 35S vector and the full-length AS constructs. Although peroxidase levels were significantly reduced in transgenic plants carrying 35S-AS, 35S-Rz and tRNA-Rz constructs, no significant difference in lignin levels was observed.     

Strategies for the suppression of peroxidase gene expression in tobacco. I. Designing efficient ribozymes

Five short hammerhead ribozymes (Rzs) were constructed and tested, using a range ofin vitro reaction conditions, for catalytic activity against the mRNA encoding the lignin-forming peroxidase (TPX) of tobacco. Although all 5 Rzs were shown to be able to cleave the RNA substrate, percentage cleavage varied with pre-denaturation of Rz and substrate, incubation temperature, length of incubation and ribozyme (Rz)-to-substrate ratio. One Rz with two catalytic units and 60 nucleotides of complementary sequence in 3 regions was shown to most efficiently cleave the substrate under allin vitro conditions tested. This ribozyme cleaved better than the two single ribozymes from which it was made. The superior cleaving ability of this Rz was shown to be due to the accessibility of the chosen target site and to the increased length of the hybridizing arms spanning this accessible region of the RNA.     

A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)₄ repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.