An in vivo root hair assay for determining rates of apoptotic-like programmed cell death in plants

Programmed cell death (PCD) can be described as the organised destruction of a cell [1,2] and plays an important role in many plant developmental pathways including xylogenesis, embryogenesis, root and leaf development and senescence [3,4]. During the hypersensitive response it provides a defence response against invading pathogens [5] and has been shown to be part of the plants response to environmental stresses [6].While there are several different forms of plant PCD [reviewed [7,8]] and differences between plant PCD and apoptosis, the type of PCD which we observe in cell suspension cultures, apoptotic-like PCD (AL-PCD), has some shared features with apoptosis [2]. For example, in response to biotic and abiotic stress, suspension culture cells have been shown to undergo nuclear condensation and internucleosomal DNA cleavage [6], activate caspase-like activity [9] and release cytochrome c from the mitochondria [10]. In addition, both animal and plant cells demonstrate condensation of the cytoplasm. In plant cells the retraction and condensation of the cytoplasm leaves a visible gap between the cell wall and the plasma membrane resulting in a specific corpse morphology [11], a hallmark feature that has been a useful tool in quantifying rates of AL-PCD in plant suspension cultures from a wide variety of species. For example, the effects of chemically induced cell death in sycamore cell cultures [12] and in soybean cells [13], the effect of HR elicitors on PCD rates in tobacco [14] and Arabidopsis thaliana [15] and the role of hydrogen peroxide on the induction of PCD in Arabidopsis thaliana [15], Glycine max [16] and Nicotiana plumbaginifolia [17] have all been investigated in cell suspension cultures. More recently, in transgenic lines, Burbridge et al. [18] showed that AL-PCD morphology could be used to investigate the effects of elevated levels of peroxidase on the AL-PCD induction threshold in transgenic cell suspension cultures of Nicotiana tabacum.The AL-PCD as