Gene expression in opening and senescing petals of morning glory (Ipomoea nil) flowers
Yamada, T. ; Ichimura, K. ; Kanekatsu, M. ; Doorn, W.G. van - \ 2007
Plant Cell Reports 26 (2007)6. - ISSN 0721-7714 - p. 823 - 835.
programmed cell-death - senescence-associated genes - leaf senescence - arabidopsis-thaliana - cysteine proteinase - postharvest senescence - alstroemeria petals - caffeoyl-coenzyme - identification - disease
We isolated several senescence-associated genes (SAGs) from the petals of morning glory (Ipomoea nil) flowers, with the aim of furthering our understanding of programmed cell death. Samples were taken from the closed bud stage to advanced visible senescence. Actinomycin D, an inhibitor of transcription, if given prior to 4 h after opening, suppressed the onset of visible senescence, which occurred at about 9 h after flower opening. The isolated genes all showed upregulation. Two cell-wall related genes were upregulated early, one encoding an extensin and one a caffeoyl-CoA-3-O-methyltransferase, involved in lignin production. A pectinacetylesterase was upregulated after flower opening and might be involved in cell-wall degradation. Some identified genes showed high homology with published SAGs possibly involved in remobilisation processes: an alcohol dehydrogenase and three cysteine proteases. One transcript encoded a leucine-rich repeat receptor protein kinase, putatively involved in signal transduction. Another transcript encoded a 14-3-3 protein, also a protein kinase. Two genes have apparently not been associated previously with senescence: the first encoded a putative SEC14, which is required for Golgi vesicle transport, the second was a putative ataxin-2, which has been related to RNA metabolism. Induction of the latter has been shown to result in cell death in yeast, due to defects in actin filament formation. The possible roles of these genes in programmed cell death are discussed.
Selection by phage display of a mustard chymotrypsin inhibitor toxic to pea aphid.
Ceci, L.R. ; Volpicella, M. ; Conti, S. ; Gallerani, R. ; Beekwilder, M.J. ; Jongsma, M.A. - \ 2003
The Plant Journal 33 (2003). - ISSN 0960-7412 - p. 557 - 566.
serine proteinase-inhibitor - amino-acid-sequence - protease inhibitors - cysteine proteinase - acyrthosiphon-pisum - transgenic plants - reactive-site - alba l. - adaptation - resistance
The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 ?g ml1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.