A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole.

Neuhaus, Jean‐Marc; Sticher, Liliane; Meins, Frederick; Böller, Thomas

doi:10.1073/pnas.88.22.10362

Cited by 306 publications

(247 citation statements)

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“…C-terminal extensions can function as vacuolar targeting signals, as has been shown, for example, in tobacco chitinases (Neuhaus et al, 1991) and barley lectin (Bednarek et al, 1990). Vacuolar targeting is clearly a candidate for the role of the C-terminal extension of horseradish peroxidase.…”

Section: Discussionmentioning

confidence: 99%

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An N-terminal Peptide Extension Results in Efficient Expression, but not Secretion, of a Synthetic Horseradish Peroxidase Gene in Transgenic Tobacco

Kis¹

2004

Annals of Botany

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d Background and Aims Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the speci®c functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. d Methods Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the Nand C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. d Key Results Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are re¯ected in transcript levels. d Conclusions There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to in¯uence transcript stability or translational ef®ciency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity. These will be valuable for exploring the role of these enzymes in stress amelioration and plant development.ã 2004 Annals of Botany Company

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Section: Discussionmentioning

confidence: 99%

“…However, other evidence suggests that the absence of retention signals does not necessarily lead to secretion (Neuhaus et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

An N-terminal Peptide Extension Results in Efficient Expression, but not Secretion, of a Synthetic Horseradish Peroxidase Gene in Transgenic Tobacco

Kis¹

2004

Annals of Botany

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“…This domain is also followed by a short C-terminal extension. The C-terminal extension in class I proteins has been postulated to be involved in vacuolar targeting (Neuhaus et al 1991b). Endochitinases ChiI24175, ChiI93771, ChiI11654 and ChiI201 have all the features of typical class I chitinolytic enzymes and possess all of the essential and important residues for catalytic activity, indicating they are active chitinases.…”

Section: Discussionmentioning

confidence: 99%

Genome-wide identification, expression and chromosomal location of the genes encoding chitinolytic enzymes in Zea mays

Shoresh

Harman

2008

Mol Genet Genomics

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Chitinolytic enzymes are important pathogenesis and stress related proteins. We identiWed 27 putative genes encoding endochitinases in the maize genome via in silico techniques and four exochitinases. Only seven of the endochitinases and segments of the exochitinases were heretofore known. The endochitinases included members of family 19 chitinases (classes I-IV of PR3, II of PR4) and members of family 18 chitinases (class III of PR8). Some similar enzymes were detected on adjacent regions of the same chromosome, and seem to result from duplication events. Most of the genes expressed were identiWed from EST libraries from plants exposed to biotic or abiotic stresses but also from libraries from tissues not exposed to stresses. We isolated proteins from seedlings of maize in the presence or absence of the symbiotic root colonizing fungus Trichoderma harzianum strain T22, and analyzed the activity of chitinolytic enzymes using an in-gel activity assay. The activity bands were identiWed by LC/MS/MS using the database from our in silico study. The identities of the enzymes changed depending on whether or not T22 was present. One activity band of about 95 kDa appeared to be a heterodimer between an exochitinase and any of several diVerent endochitinases. The identity of the endochitinase component appeared to be dependent upon treatment.

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“…The 33-kD class I ␤-1,3-glucanase and the 32-kD class I chitinase B were purified from wild-type tobacco cv Havana 425 leaves (Felix and Meins, 1985;Shinshi et al, 1987). The tobacco 34-kD class I chitinase A was purified from leaves of transgenic Nicotiana sylvestris that overproduces the tobacco chitinase (Neuhaus et al, 1991). For boiled enzyme treatments, the enzymes were placed in boiling water for 15 min before addition to assay tubes.…”

Section: Assays For Hydrolysis Of Tomato Endosperm Cap Cell Wallsmentioning

confidence: 99%

Class I β-1,3-Glucanase and Chitinase Are Expressed in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence

et al. 2001

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β-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. β-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. β-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1tomato seeds only when supplied with gibberellin. Accumulation of β-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 μM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both β-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed.

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A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole.

Cited by 306 publications

References 35 publications

An N-terminal Peptide Extension Results in Efficient Expression, but not Secretion, of a Synthetic Horseradish Peroxidase Gene in Transgenic Tobacco

An N-terminal Peptide Extension Results in Efficient Expression, but not Secretion, of a Synthetic Horseradish Peroxidase Gene in Transgenic Tobacco

Genome-wide identification, expression and chromosomal location of the genes encoding chitinolytic enzymes in Zea mays

Class I β-1,3-Glucanase and Chitinase Are Expressed in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence

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