Gene expression and plant performance in oryzacystatin-I expressing transformed tobacco (Nicotiana tabacum L. cv Samsun) plants under abiotic stress

Abstract

Plant cysteine proteinase inhibitors or also called phytocystatins inhibit the action of cysteine proteinases in plants. These proteinases are involved in many developmental processes by degrading proteins. In this study possible effects of an exogenous oryzacystatin-I (OC-I) expressed in transformed tobacco has been investigated. By challenging OC-I expressing and non-expressing tobacco with drought and heat stress, OC-I transcription and translation were not affected in OC-I expressing plants and plant extracts from stressed plants containing the inhibitor inhibited papain activity in vitro. Further, plant growth and photosynthesis was not greatly different under the selected growth conditions in both plant types under stress and non-stress conditions. However, OC-I expressing plants showed slightly lower photosynthetic rate, were shorter and had a higher lower dry mass production under non-stress condition. By applying cDNA Representational Difference Analysis (cDNA-RDA) to detect differentially expressed genes in the two types of plants, a gene coding for the light harvesting chlorophyll a/b binding protein gene (lhcb1) of photosystem II (LHC II) was isolated from non-OCI expressing plants. Northern blot analysis showed lower transcript accumulation of the lhcb gene in OCI-expressing plants both under non-stress and stress conditions, which was accompanied by lower chlorophyll content in OC-I expressing plants. Furthermore, plants benefited from OC-I expression by protection of a variety of expressed proteins against degradation. Identification of possible target cysteine proteinases for OC-I in tobacco resulted in the isolation, cloning and characterization of two new papain-like cysteine proteinases from tobacco designated NtCP1 and NtCP2. NtCP1 was expressed only in senescent leaves and it was not induced in mature green leaves upon exposure to drought or heat stress. NtCP1 has therefore a possible potential as a developmental senescence marker in tobacco. In contrast, NtCP2, which was expressed in mature green leaves, has a high similarity to KDEL-tailed cysteine proteinases that are involved in programmed cell death. Both drought and heat decreased NtCP2 transcript abundance in mature green leaves. Overall, this study has provided evidence that expression of exogenous OC-I does not significantly improve plant performance in tobacco in terms of physiological traits under drought and heat stress but provides protection in terms of stability of protein expression by possibly interacting with endogenous tobacco cysteine proteinases. Further detailed studies are suggested on the interaction of endogenous cysteine proteinases and exogenous phytocystatins to elucidate in more detail the type of interaction. Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Beyene, G 2006, Gene expression and plant performance in oryzacystatin-I expressing transformed tobacco (Nicotiana tabacum L. cv Samsun) plants under abiotic stress, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-12052006-144409 / >