Response to Cacas and Diamond: is the autophagy machinery an executioner of programmed cell death in plants?

We are grateful for the interest that Jean-Luc Cacas and Mark Diamond have shown towards our <i>Trends in Plant Science</i> opinion article on the timing and co-ordinate regulation of plant cell death [1]. They identified a potential catachresis in our definition of autophagic programmed cell death (PCD), whereby they suggest that the autophagy observed in fatally committed cells might not directly induce cell death. Our initial definition of ‘autophagic cell death’ was more intended to highlight that autophagy was a predominant feature of this type of cell death, irrespective of its role in the death process. Although our opinion article was written from a broad signalling perspective, we are glad that it generated a response from those interested in autophagy. Autophagy is a tightly controlled process, and investigation of autophagy frequently produces conflicting results. For example, knocking out genes regulating autophagy might either promote or inhibit autophagic pathways; which in turn might inhibit or promote the cell death processes. This probably reflects the complexities of potential feedback regulation and the type of studies carried out. Here, we briefly reiterate the evidence for pro-life and pro-death roles of autophagy. Cacas and Diamond [2] quite correctly ingeminate that autophagy has a pro-life function. This has been well characterized in a plethora of different cell types, such as human haemopoietic cells [3], and organisms, such as <i>Caenorhabditis elegans</i> [4],<i>Dictyostelium discoideum</i> [5] and tobacco (<i>Nicotiana tabacum</i>) [6]. In these cases it was demonstrated that inhibition of autophagy by ablation or silencing of <i>AUTOPHAGY</i> (ATG) genes led to cell death during hormone and nutrient deprivation and pathogen attack. The pro-life function of autophagy is possibly due to its capacity to improve cellular fitness via a controlled vacuolar recycling and redistribution of nutrients. This process is known to inhibit apoptosis-like cell death in plants. Conversely, there is also considerable evidence to suggest that autophagy might have a ‘pro-death’ function. Cacas and Diamond [2] stated that ‘there are only two examples published thus far showing that the successful execution of PCD requires functional autophagy machinery <i>in vivo</i>: the work on degradation of salivary gland cells in <i>Drosophila</i> [7] and reports on cell death associated with spore germination of the rice blast fungus [8]’. In fact, there have been many studies carried out on <i>C. elegans</i> [9], HeLa cells [10], mouse embryonic fibroblasts [11] and murine fibrosarcoma cells [12] in which the genetic or chemical ablation of autophagy components suppresses cell death, thereby illustrating that in many situations autophagy is a requirement of PCD. Because autophagy has a pro-death role in many different organisms, it is compelling to assume that this also applies to plants, and indeed there is some evidence to support this. Knocking out <i>atg7</i> and <i>atg9</i> in <i>Arabidopsis</i> leads to either normal or enhanced levels of autophagy, with the concomitant development of premature senescence (see Ref. [2]). However, the mechanics behind this process are poorly understood at present. It is possible that the outcome of autophagy, whether it be pro-life or pro-death, is dependent on the method of influencing <i>ATG</i> gene regulation (i.e. genetic knockouts or gene silencing) and the particular treatment applied to the organism. In addition, it might be an oversimplification to assume that the pro-life or pro-death phenotypes that result from the knockout of <i>ATG</i> genes are caused by suppression of the autophagic process because <i>ATG</i> genes might have diverse roles in cellular regulation. For autophagy to be properly correlated to pro-life or -death functions, it has to be measured in an appropriate manner, which is often difficult because there are many different assays, each with their own caveats. Recently, a large consortium of experts published guidelines for accurately measuring autophagy, which should enable a more thorough interpretation of experimental results [13]. This, coupled with the identification of mediators and regulators of autophagy and their protein interactors, should enable an elaborate delineation from molecular switch to phenotype. Such data will hopefully ameliorate any current debates and will foster the development of more advanced plant autophagic models that can be used to finely dissect pro-life and -death functions. For autophagy to participate in seemingly contrasting biological functions in plants, there must be crosstalk and possibly some level of functional redundancy in the autophagic pathways. At present, these mechanisms remain elusive. Also, we must not just consider the downstream effects of autophagy – the upstream signals must also be considered because they induce the process and are likely to help determine whether pro-life or pro-death routes are taken. We touched on several of these possible modulators in our opinion article, and we look forward to understanding how they contribute to the autophagic crosstalk