Silencing approach using Poly (ADP-ribose) polymerase gene to improve drought stress tolerance in maize

Abstract

Current and predicted climatic conditions, such as prolonged drought and heat episodes affect plant growth and yield, cause annual losses estimated at billions of dollars and pose a serious challenge for agricultural production worldwide, (Boyer 1982, Mittler 2006). Progress in generating transgenic crops with enhanced tolerance to abiotic stress has nevertheless been slow. The complex field environment with its heterogenic conditions, abiotic stress combinations, and global climatic changes are but a few of the challenges of modern agriculture. A combination of approaches has contributed significantly towards improving abiotic stress tolerance in both the greenhouse and the field (Hirayama and Shinozaki 2010). However, the increasing unpredictability and vagaries of rainfall and temperature variations consequent to global climate change, the dwindling availability of irrigation water and phosphate fertilizer, the escalating population in developing countries indicate that new ways of improving maize yield stability and stress tolerance under suboptimal conditions must be found and employed. A novel strategy to improve abiotic stress tolerance in Arabidopsis and Brassica has been reported by De Block et al., (2005). In their study, poly(ADP-ribosyl) ation activity was downregulated and transgenic plants became tolerant to a broad range of abiotic stresses such as drought, high light and heat. The researchers noted that stress tolerance was obtained by maintaining energy homeostasis due to reduced stress-induced energy consumption by prevention of NADP breakdown. Under abiotic stress conditions, plants overexpressing hairpin RNAi against Arabidopsis APP gene preserve their energy homeostasis without an overactivation of the mitochondrial respiration and avoiding the production of reactive oxygen species. Hence, plants with a lowered PARP activity appear tolerant to multiple stresses. Furthermore, a genome-wide transcript analysis of stressed anti PARP2 transgenic Arabidopsis (hpAt-PARP2) plants revealed that the induction of specific ABA signalling pathways steered increased levels of the cyclic nucleotide ADP-ribose (cADPR) thereby contributing towards tolerance to abiotic stresses (Vanderauwera et al., 2007). Indeed, modulation of poly(ADPribosyl) ation (PAR) reaction by an Arabidopsis thaliana ADP-ribose/NADH pyrophosphohydrolase, AtNUDX7 led to plants becoming tolerant to oxidative stress (Ogawa et al., 2009). Under oxidative stress, AtNUDX7 serves to maintain NAD+ levels by supplying ATP via nucleotide recycling from free ADP-ribose molecules and thus regulates the defence mechanisms against oxidative DNA damage via modulation of the PAR reaction. Furthermore, energy use efficiency is characterized by an epigenetic component that can be directed through selection to increase yield on top of heterosis (Hauben et al., 2009). Arabidopsis findings have previously been found to be directly applicable to commercial crop improvement (Nelson et al., 2005). Therefore, engineering crop plants for high NAD+ regeneration by an efficient 8 upregulation of the NAD+ salvage pathway or by a reduced NAD+ consumption under stress conditions, is a valuable approach to enhance overall stress tolerance in crops. The aim of this study was to assess a similar approach in the model species Zea mays through silencing maize PARP1 gene, a homolog of the Arabidopsis APP gene. This was achieved by establishing tropical maize regeneration system from immature embryo through somatic embryogenesis as a prerequisite for effective genetic transformation mediated by Agrobacterium. Our next goal was to engineer two hairpin constructs targeting maize PARP1 gene within the specific region at the 5’-end of the gene because silencing of the Arabidopsis APP gene using hairpin RNA constructs proved to be a valuable approach to obtain drought tolerance in Arabidopsis and canola (De Block et al., 2005). However, hairpin constructs are not so stable in extreme temperatures in the greenhouse which results in problems with stability in subsequent progenies (Szittya et al., 2003). Therefore, we aimed at evaluating the utility of artificial micro- RNAs (amiRNA) to silence the maize PARP1 gene. This technology is expected to be more effective and stable than the hairpin RNA approach because temperature does not affect the accumulation of microRNA (Szittya et al., 2003). Three amiRNAs were designed targeting maize PARP1 gene. As a complementary approach to the amiRNA and RNAi transgene technology we sought for mutants in the maize PARP1 gene in the Uniform Mu collections mutagenized by the transposon Mutator. Transgene technology and efficient transformation are important to fully exploit a species as a model for functional genomics studies. We developed an efficient and standardized maize transformation method based on the following criteria: (1) Agrobacterium tumefaciens co-cultivation was preferred over particle bombardment because usually intact T-DNA’s are transferred at low copy number, (2) a maize inbred line was favoured over a hybrid line because homogeneous progenies allow transgene testing already in T1 or T2, (3) Gateway vectors were optimized for use in monocots and provide a toolbox for rapid gene cloning. And finally, the transgenic maize plants overexpressing amiRNA constructs against maize PARP1 gene were evaluated for drought and Methyl Viologen-induced oxidative stress tolerance.