Over-expression of PR-10a leads to increased salt and osmotic tolerance in potato cell cultures
Introduction
PR proteins (pathogenesis-related proteins) have been defined as plant proteins that are induced during pathogen infection or wounding. The major families of PR proteins have been grouped at least into 14 different classes, primarily on the basis of their amino acid sequence identity (Van Loon and Van Strien, 1999). The group of PR-10 proteins was defined by Van Loon et al. (1994). This group included the PR-10a protein (Marineau et al., 1987) from potato, proteins isolated from parsley after elicitor treatment (Somssich et al., 1986), the asparagus PR1 protein, the birch Betv I protein, pea 149 and the bean PR1 and PR2 proteins. All these proteins show similar sizes of app. 17 kDa, acidic isoelectric points and are located in the cytosol (Van Loon et al., 1994, Ziadi et al., 2001). Already Matton and Brisson (1989) demonstrated the strong homology of the amino acid sequences and homology of the hydropathic patterns of the PR-10a (formerly STH-2) protein, the PR1 protein from parsley and the pea I49 protein (Matton and Brisson, 1989). Warner et al. (1992) later showed a sequence homology of app. 30% between the pea disease resistance protein 149, the parsley PR protein PcPRI-1, the birch pollen allergen Betv l, the bean induced transcripts PvPR1 and PvPR2, the AoPR1 isolated by the authors from Asparagus officinalis and the potato PR-10a (STH-2). Finally Osmark et al. (1998) compared PR-10 proteins to major latex proteins (MLPs) found in poppy, arabidopsis, bell pepper, melon, strawberry and tobacco. Similarity of proteins was only weak (25%), but from a detailed analysis, after sequence alignment, the authors concluded, that these protein groups are related.
In 1994 Moiseyev et al. (1994) isolated a ribonuclease from ginseng callus, which showed high sequence homology with the PR proteins from parsley (Somssich et al., 1986). From these results they concluded ribonuclease activity for the parsley proteins. Ribonuclease activity has been demonstrated also for other PR-10 proteins like for the Betv I protein (Bufe et al., 1996) and for the AmPR-10 protein from Astragalus mongholicus (Yan et al., 2008).
Although ribonuclease activity may serve as a possible defence mechanism against pathogen attack, the function of these proteins remains unclear. Constabel et al. (1993) carried out over-expression of the Pr-10a protein in transgenic potato, and did not find an enhanced resistance against Phytophthora infestans. Furthermore the PR-10 proteins, unlike classical virally induced “pathogenesis-related” proteins, occur intracellular and are not excreted (Constabel and Brisson, 1995). Constabel and Brisson (1995) analyzed in detail the expression pattern of the Pr-10a protein in transgenic potato plants of the cultivar Desiree, expressing a gus gene under the control of a pr-10a regulatory sequence. No major organ showed constitutive expression of PR-10a, but only induced formation after infection, elicitor treatment or, to lower extent, after wounding. Unexpectedly, the strongest expression was observed in vascular bundles and roots. Leaves, which would be the primary target for virus attack, showed a much lower induction (Constabel and Brisson, 1995). Constitutive expression of PR-10a under developmental control in healthy uninfected plants was found only in the stigma (Constabel and Brisson, 1995). Crowell et al. (1992) previously found genes for a PR-10a homologous protein, expressed under developmental control in the late stationary phase of cultured soybean cells, as well as in roots and senescent tissue of intact soybean plants. A developmental function of PR-10 proteins may result from a cytokinin binding capacity. Proteins of the PR-10 family with high cytokinin affinity have been isolated from mung bean (Fujimoto et al., 1998) and even moss Physcomitrella patens (Gonneau et al., 2001). Other binding capacities for PR-10 proteins were observed for fatty acids, flavonoids (Fujimoto et al., 1998, Mogensen et al., 2002) and brassinosteroids (BRs) (Markovic-Housely et al., 2003).
Apart from pathogen attack, PR-10 proteins are induced also by abiotic stresses like salinity, drought, copper, oxidative stress or ultraviolet radiation (Liu and Ekramoddoullah, 2006). Evidence has especially emerged for an involvement of PR-10 proteins in salt tolerance. In pea (Pisum sativum L.) roots results, obtained by two dimensional electrophoresis and mass spectrometry (MS), revealed that proteins, belonging to the PR-10 family (ABA-17, ABA-18, p I49, p I176), showed a 10 fold increase under salt stress (Kav et al., 2004). Proteomic analysis of saline-tolerant peanut callus (Arachis hypogaea) displayed an elevated level of a PR-10 protein compared to non-tolerant callus (Jain et al., 2006).
In Brassica napus a pea PR-10 protein was found to enhance the germination of seeds under saline conditions (Srivastava et al., 2004). Srivastava et al. (2006) could show that a recombinant Pr-10.1 protein from pea, expressed in Escherichia coli, showed ribonuclease activity. They also demonstrated lowered ABA levels and increased cytokinin levels in B. napus plants, over-expressing the PR protein, and discussed a possible mechanism on salt tolerance by influencing the hormone balance in plants. Recently it has been demonstrated by Krishnaswamy et al. (2008) in a transcriptional study, that in Arabidopsis plants the over-expression of a PR-10 protein from pea (ABR17) induced changes of transcript abundance of many ABA and cytokinin responsive genes.
In the present study, a comparison of the protein pattern of a potato cell culture of the cultivar Desiree under normal growth conditions and salt, as well as osmotic challenge showed the PR-10a protein as one of several spots occurring de novo. For proteome analysis, potato cell cultures have been used, since heterotrophic cell cultures, grown in the dark, lack photosynthetic pigments and the otherwise dominating photosynthetic proteins. Therefore the analysis of the residual proteome is often facilitated, compared to the use of intact green plant tissue. Concerning salt challenge, Dobranszki et al. (2003) showed, that potato cell cultures, established from different potato cultivars, showed the same range of sensitivity than in vitro plants of the corresponding cultivars. In the present study, homologous over-expression of the pr-10a gene was carried out in potato cell cultures. For monitoring the over-expression, special dicistronic transformation vectors were constructed. In these vectors the pr-10a gene is linked by an IRES sequence to the luc reporter gene. This construct leads to the formation of a unique mRNA for both genes, but the formation of two physically independent proteins, assuring full physiological functionality of both, target as well as reporter gene (Dorokhov et al., 2002). The target gene expression can be simply monitored by measuring the reporter gene expression, which is especially helpful when working with undifferentiated cell cultures, consisting of heterogeneous cell populations (Ali et al., 2010). The effect of the PR-10a over-expression on osmotic and salt tolerance in the transformed cell cultures was investigated.
Section snippets
Plant material and osmotic challenge
A none-embryogenic suspension culture of Solanum tuberosum cv. Desiree (DSMZ No. PC-1182) was sub-cultured weekly by transferring 40 ml of suspension to 60 ml of fresh 4× medium (Gamborg et al., 1968) containing 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4 D), 0.5 mg l−1 indole-3-acetic acid (IAA), 0.5 mg l−1 1-naphtylacetic acid (NAA) and 0.4 mg l−1 kinetin, pH 5.6 and incubated in a 300 ml Erlenmeyer flask on a gyratory shaker (TR-250, Infors AG, Basel, Switzerland) with 50 mm orbit (100 rpm) at 25 °C. For
Protein identification
The PR-10a protein appeared in the proteome of S. tuberosum suspension cultures cv ‘Desiree’ after challenge with either NaCl or sorbitol under equiosmolal conditions (740 mOs/kg). NaCl was chosen as a substance, providing salt stress, and sorbitol as an osmotic not, or only in small amounts, entering the cells. Six spots, formed de novo after salt and osmotic stress in high amounts, were selected for identification. These spots were identified as: STH-2 (PR-10a, gi|169551), STH-21 (P17641),
Discussion
In the present study, a PR-10a protein was detected after osmotic and salt challenge as the most prominent de novo induced protein spot in the proteome of cell cultures, established from potato plants of the cultivar Desiree. cDNA of the pr-10a gene was cloned into a dicistronic transformation vector, carrying the luc gene as second cistron, linked to the first cistron by a viral IRES element. The initial plant cell culture was transformed and the resulting transformed cell line characterized
Acknowledgments
The first author expresses his gratitude to the Egyptian government for providing Ph.D. scholarship. We are also thankful to Dr. Yoshiharu Y. Yamamoto (RIKEN FRS, Japan) who provided the vector yy376 harboring the respective Tobamo virus IRES element.
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Present address: Genetics Department, Faculty of Agriculture, Kafrelsheikh University, 33516 Kafrelsheikh, Egypt.