Abstract
The activation of programmed cell death (PCD) is often a result of complex signalling pathways whose relationship and intersection are not well understood. We recently described a PCD root hair assay and proposed that it could be used to rapidly screen genetic or pharmacological modulators of PCD. To further assess the applicability of the root hair assay for studying multiple signalling pathways leading to PCD activation we have investigated the crosstalk between salicylic acid, autophagy and apoptosis-like PCD (AL-PCD) in Arabidopsis thaliana. The root hair assay was used to determine rates of AL-PCD induced by a panel of cell death inducing treatments in wild type plants treated with chemical modulators of salicylic acid synthesis or autophagy, and in genetic lines defective in autophagy or salicylic acid signalling. The assay demonstrated that PCD induced by exogenous salicylic acid or fumonisin B1 displayed a requirement for salicylic acid signalling and was partially dependent on the salicylic acid signal transducer NPR1. Autophagy deficiency resulted in an increase in the rates of AL-PCD induced by salicylic acid and fumonisin B1, but not by gibberellic acid or abiotic stress. The phenylalanine ammonia lyase-dependent salicylic acid synthesis pathway contributed only to death induced by salicylic acid and fumonisin B1. 3-Methyladenine, which is commonly used as an inhibitor of autophagy, appeared to influence PCD induction in all treatments suggesting a possible secondary, non-autophagic, effect on a core component of the plant PCD pathway. The results suggest that salicylic acid signalling is negatively regulated by autophagy during salicylic acid and mycotoxin-induced AL-PCD. However, this crosstalk does not appear to be directly involved in PCD induced by gibberellic acid or abiotic stress. This study demonstrates that the root hair assay is an effective tool for relatively rapid investigation of complex signalling pathways leading to the activation of PCD.
Highlights
Programmed cell death is a crucial component of development and defence responses [1]
The characteristic apoptosis-like programmed cell death (AL-PCD) morphology induced by Salicylic acid (SA) was accompanied by early mitochondrial swelling, which has been shown to occur during plant programmed cell death in numerous studies, including Arabidopsis protoplasts following application of chemical or heat stimuli [45], UV-C treatment [50], as well as during PCD related senescence in Medicago truncatula cell suspension cultures [51]
The rate of SA-induced AL-PCD in Arabidopsis root hairs increased in autophagy defective atg5 and atg7 seedlings, supporting the view that SA signalling leading to cell death is negatively regulated by autophagy
Summary
Programmed cell death is a crucial component of development and defence responses [1]. We reported that a similar corpse morphology can be scored by a root hair assay, a novel technique for quantitative determination of AL-PCD rates in plants in vivo [3]. We investigated whether the root hair assay can be used for dissecting multiple signalling pathways leading to ALPCD by studying the putative crosstalk between SA, autophagy and AL-PCD in Arabidopsis thaliana. It has been shown to play a role in growth, senescence and seed production, and the regulation of plant development relies on a coordinated crosstalk between SA and other phytohormones [4]. Genetic studies employing salicylic acid induction-deficient mutants provide further evidence of a role for SA during HR [10,11]
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