Agrobacterium- mediated transformation of tomato with yeast halotolerance genes

Abstract

Tomato cv Rio Grande plants were transformed with yeast halotolerance genes (HAL I or HAL II) by using pPM7 HAL I or pJRM16 HAL II or p35GUSINT as control. Transformation efficiencies varied with the three constructs with the highest transformation rate shown by p35GUSINT vector. Final selection of the transgenic plants was made on the basis of PCR. Transgene integration and copy number were assessed by Southern hybridization. The primary transformants were allowed to self- pollinate and the expected Mendelian ratios were studied in second generation progenies. Five independent homozygous lines of each HAL I and HAL II gene along with control were characterized for inter- transformant expression variability. The transformants showed considerable variability in the expression of the respective genes as shown by salt tolerant assays, chlorophyll contents and peroxidase activity. The transgene expressions in transgenic lines were analyzed by semi- quantitative RT-PCR. In response to different salt concentrations tested, the transgenic plants overexpressing HAL I and HAL II showed significantly (α = 0.05) better performance in comparison with the control. After analyzing the halotolerant transgenic lines of tomato cv Rio Grande for their response to salt stress under in-vitro conditions, three independent homozygous lines of each HAL I or HAL II genes with highest expression level were selected for exploring their physiological responses against different salt stresses in the field. These transgenic plants showed significant growth, biomass and improved water content in comparison with control under 100 mM and 150 mM salt stress conditions. The HAL I lines showed improved K+/Na+ selectivity as compared to HAL II and control plants whereas both the HAL I and HAL II transgenic lines showed better Ca2+ content than their control counterpart. Moreover, lower values of relative electrical conductivity in the transgenic tomato lines suggest the possible role of these yeast genes in enhancing the salt tolerance by protecting the cell membranes from damage along with the other already known mechanisms. Furthermore, the transgenic lines exhibited improved resistance against the fungal pathogens Fusarium oxysporum and Alternaria solani when tested by the detached leaf assay. The leaf extracts from the transgenic lines also corroborated the same behaviour. In-vitro and in-vivo salt tolerant analyses of the transgenic tomato lines were then followed by the metabolomic studies of the respective transformants. Changes in metabolism occurring in homozygous lines of transgenic tomatoes with the highest expression level of HAL I and HAL II compared to control plants were selected and studied with Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry techniques. Principal component analysis (PCA) of the metabolic profiles obtained with ESI-MS successfully distinguished the control tomato cv Rio Grande seeds from the transgenic HAL I and HAL II population. Increased biomass was observed for the transgenic halotolerant tomato lines in comparison to control when germinated under 0, 50 and 100 mM salt stress conditions for a period of seven days. Multivariate partial least square-discriminant analysis (PLS-DA) revealed metabolic phenotypes and differences in seedlings before and after salt stress for each plant population. Based on mass, sugars (sucrose, glucose), organic acids (cinnamic acid, malonic acid, chorismic acid) and fatty acids (linolenic acid and palmitic acid) were putatively identified as the metabolites responsible for discriminating the transgenic from the wild type populations with or without salt stress. Tandem mass spectrometry confirmed the identity of the metabolites varying in amount between samples. MALDI-mass spectrometry was used to analyze the distribution of metabolites direct within the plant tissue of transgenic and wild type tomato seeds. The MALDI-MS procedure was optimized for matrix application, laser frequency and machine quadrapole settings. Only a few masses with insufficient counts to map as images across tomato seed sections were obtained. This study represents the comparative responses of the three constructs used under the same transformation conditions and speculates about the possible mechanisms governed by yeast HAL I and HAL II genes which seem to work in a coordinated manner by decreasing osmotic and oxidative shocks relatively at different rates. Our results suggest that the yeast HAL I gene has a better functional role than the HAL II gene in improving the salt tolerance of the local tomato cultivar Rio Grande, grown in Pakistan. Physiological analyses carried out in this study suggest involvement of multiple mechanisms in transgenic tomato plants harbouring yeast genes to confer biotic and abiotic tolerances under stress conditions. Moreover, metabolomic studies revealed distinct metabolic phenotypes of the halotolerant transgenic tomato lines and provided biochemical indicators possibly involved in protecting the transgenic salt tolerant lines from the salt damaging effects.