The Root Hair Assay Facilitates the Use of Genetic and Pharmacological Tools in Order to Dissect Multiple Signalling Pathways That Lead to Programmed Cell Death

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.

Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley

“…Moreover, 3-MA treatment resulted in the reduction of cell death levels of microspores after stress. Secondary effects of 3-MA have been reported in some systems, such as in Arabidopsis root hairs, where 3-MA could inhibit mitochondrial-activated PCD rather than autophagy ( Kacprzyk et al , 2014 ). Although possible secondary effects of 3-MA in microspores cannot be completely ruled out, there is no evidence in microspores of mitochondrial activation of PCD, and our results indicate that in microspores 3-MA inhibits autophagy and leads to a reduction of cell death.…”

AtCSLD3 and GhCSLD3 mediate root growth and cell elongation downstream of the ethylene response pathway in Arabidopsis

“…In Arabidopsis, a root hair develops in a stepwise manner divided into three phases: cell specification, initiation, and elongation (tip growth) ( Cho and Cosgrove, 2002 ). Root hairs are thus important for studies of cell differentiation, development, and elongation ( Kacprzyk et al , 2014 ; Li et al , 2014 ).…”

Stressed to Death: The Role of Transcription Factors in Plant Programmed Cell Death Induced by Abiotic and Biotic Stimuli

“…Detailed PCD phenotyping, in both abiotic and biotic context, is required for plants with reduced/enhanced expression of these TFs in order to further elucidate their role in modulation of cell death pathways, ideally in combination with monitoring of mitochondrial retrograde signaling. Methods such as root hair assay ( Kacprzyk et al., 2014 ; Kacprzyk et al., 2016 ) or measurements of aerenchyma formation may provide useful tools to easily obtain quantitative information on the rates of PCD induced by numerous environmental stimuli in such mutants/transgenes. Finally, it remains to be established if the touch signaling, involving rapid upregulation of cell death promoting OM66 , and activation regulatory network that mediates the responses to abiotic and biotic stresses, has an effect on plant’s susceptibility to subsequent PCD triggers by environmental stimuli.…”