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. 2022 Jul 23;236(5):1691–1707. doi: 10.1111/nph.18350

Fig. 9.

Fig. 9

Cartoon of self‐incompatibility (SI)‐ and ATP depletion‐induced events and their timing in Papaver and Arabidopsis PrpS‐expressing lines. SI in P. rhoeas pollen tubes triggers rapid increases in cytosolic free calcium ([Ca2+]cyt), reactive oxygen species (ROS) and decreases in cytosolic pH ([pH]cyt) which trigger increased caspase‐3‐like/DEVDase activities required for executing programmed cell death (PCD). Filamentous (F)‐actin depolymerization is observed within 10 min, whereas large F‐actin foci are apparent after 60 min. Cytosolic ATP ([ATP]i) decreases to 40% after 30 min, whereas [pH]cyt drops to 5.5 after 60 min. In the Arabidopsis thaliana ‘slow’ line co‐expressing PrpS1‐GFP and Lifeact‐mRuby2, the timing of SI‐induced ATP depletion and actin remodelling shows a similar pattern as in P. rhoeas pollen tubes. In the A. thaliana ‘rapid’ line co‐expressing PrpS1 and Lifeact‐mRuby2, pollen tube [ATP]i is depleted faster; within 10 min after SI induction. Cytosolic acidification occurs rapidly, reaching pH 5.5 after 12 min of SI induction, whereas F‐actin foci form within this timeframe, after a threshold of pH c. 5.8 has been reached. Plasma membrane integrity is maintained and no significant increase in caspase‐3/DEVDase activity is detected within the timeframe of dramatic cytosolic acidification and ATP depletion induced by SI, even after [ATP]i and [pH]cyt had plateaued. This demonstrates that PCD is the downstream result and not the cause of SI‐induced ATP depletion and cytosolic acidification. Treatment of the ‘rapid’ line with the ATP depletion drugs 2‐deoxyglucose (2‐DG) and antimycin A resulted in the rapid inhibition of apical tip growth and a major [ATP]i reduction within 10 min. This treatment induces much slower cytosolic acidification compared with SI responses, whereas F‐actin remodelling occurs with actin aggregation much later and only after [pH]cyt reached 5.8. This suggests that the decrease in cytosolic pH may play a more predominant role in actin reorganization than ATP depletion. Overall, based on the timing of SI‐induced ATP depletion, cytosolic acidification and actin remodelling in the pollen tube of P. rhoeas and A. thaliana ‘rapid’ and ‘slow’ lines, the ATP depletion occurs slightly earlier than cytosolic acidification, indicating that ATP depletion may contribute to the decrease in [pH]cyt after SI. This is further supported by the effect of ATP depletion drugs on [pH]cyt, although other factors are likely to be involved in orchestrating the significant cytosolic acidification and F‐actin reorganization during the SI response.