University of Birmingham Self-incompatibility-induced programmed cell death in field poppy pollen involves dramatic acidification of the incompatible pollen tube cytosol

Self-incompatibility (SI) is an important genetically controlled mechanism to prevent inbreeding in higher plants. SI involves highly speci ﬁ c interactions during pollination, resulting in the rejection of incompatible (self) pollen. Programmed cell death (PCD) is an important mechanism for destroying cells in a precisely regulated manner. SI in ﬁ eld poppy ( Papaver rhoeas ) triggers PCD in incompatible pollen. During SI-induced PCD, we previously observed a major acidi ﬁ cation of the pollen cytosol. Here, we present measurements of temporal alterations in cytosolic pH ([pH] cyt ); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction and stabilizing by 60 min at pH 5.5. By manipulating the [pH] cyt of the pollen tubes in vivo, we show that [pH] cyt acidi ﬁ cation is an integral and essential event for SI-induced PCD. Here, we provide evidence showing the physiological relevance of the cytosolic acidi ﬁ cation and identify key targets of this major physiological alteration. A small drop in [pH] cyt inhibits the activity of a soluble inorganic pyrophosphatase required for pollen tube growth. We also show that [pH] cyt acidi ﬁ cation is necessary and suf ﬁ cient for triggering several key hallmark features of the SI PCD signaling pathway, notably activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci. Importantly, the actin binding proteins Cyclase-Associated Protein and Actin-Depolymerizing Factor are identi ﬁ ed as key downstream targets. Thus, we have shown the biological relevance of an extreme but physiologically relevant alteration in [pH] cyt and its effect on several components in the context of SI-induced events and PCD. using a Leica DM IRE2 Confocal Microscope under the following microscope set- tings: pH-dependent wavelength, excitation at 488 nm and 7% power; and pH-independent wavelength, excitation at 458 nm and 12% power. Emission was col- lected at510 to550 nmforbothimages.UsingImageJsoftware,a50- 3 50-pixelbox was used to measure the mean intensity of the area of each pair of images (488 and 458 nm). A measurement was taken from each pollen tube image (488- and 458-nm images) at the same position on each image. Four pairs of measurements were collected for each tube. Each pair of measurements was used to create a ratio (pH-dependent:pH-independent ratio of 488:458 nm). The mean ratio values of each pollen tube were then used to determine the [pH] cyt of the pollen tube using a reference calibration curve with a pseudocytoplasm calibration set. A calibration curve was carried out for each individual imaging session. In vitro calibration was performed using 40 m M BCECF-free acid (Invitrogen) in a pseudocytosol (100 m M KCl, 10 m M NaCl, 1 m M MgSO 4 , 10 m M MES, and 10 m M HEPES adjusted to desired pH). Images were taken sequentially using the same microscope settings as those used for in vitro pollen tube measurements (488 and 458 nm). A pair of images was taken at each of the following pH values to create a calibration curve: pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5, and pH 8. Using ImageJ software, intensities were measured for each pair of images, and a ratio value was calculated. These ratios were plotted to give a calibration curve to pH ratio, which was used to calculate the pH of individual pollen tubes after various treatments. Statistical analysis (Wilcoxon rank sum test) was carried out; where statistical comparisons are made, they are between samples at the same time point. Activity assays for sPPase activity at different pH were performed using re- combinant Pr-p26.1a and Pr-p26.1b proteins. The p26.1a and p26.1b constructs with an N-terminal His-Tag were transfected into Escherichia coli (BL21 strain), and expression was induced with 1 m M isopropylthio- b -galactoside and puri ﬁ ed using Ni-agarose following the manufacturer ’ s protocol (QIAGEN). To determine pyrophosphatase (sPPase) activity, a standard curve was constructed using different concentrations of NaH 2 PO 4 . For the assays, recombinant p261a and p261b were diluted to 10 m M into basic medium (50 m M HEPES KOH, pH 8.0, 50 m M EGTA, 2 m M MgCl 2 supplemented with 2 m M Mg ++ , and 50 m M EGTA). To de- termine activity at different pHs, the buffer pH was adjusted before assaying. Fiske-Subbarow reagent was added to both standard curve solution and enzyme assay solution and left for 20 min at room temperature. The absorbance of each sample was read at optical density at 691 nm, and activities were calculated from the standard curve using known concentrations of NaH 2 PO 4 . construct (a gift from Natasha Raikhel) and cloned be- hind the pollen-speci ﬁ c promoter NTP303. GFP was expressed as a C-terminal fusion protein to yield NTP303:: d -TIP::GFP. Microprojectile bombardment was performed using the helium-driven PDS-1000/He Biolistic System (Bio-Rad) using tungsten particles (1.3 m m) coated with plasmid DNA. Images were acquired using confocal microscopy as previously described. SI-induced pollen This approach revealed three activities (green box): DEVDase and VEIDase increase between 1–5 h post SI and LEVDase increases later (still increasing at 8 h). DEVDase activity peaks at 5 h and has been shown to be required for DNA fragmentation, which can be prevented by the DEVDase inhibitor Ac-DEVD-CHO. These activities are optimal at pH 5. The caspase-like activities, which do not have any activity at normal cytosolic pH, need the pollen cytosol to acidify. We previously showed this occured in the first few hours of SI; here we have shown it begins at ~10 min and stablizes at ~pH 5.5 at 60 min (rainbow box). in signaling to SI-mediated cell death. cytoskeleton (yellow SI-stimulates actin depolymerization, causes inhibition of pollen tube Latrunculin tube tip depolymerization Ac-DEVD-AMC DNA and not Ac-YVAD-CHO. actin pollen tube growth without affecting the cytoskeleton, does not trigger DNA fragmentation. Together, these data implicate actin dynamics in modulating the DEVDase activity. SI also triggers microtubule depolymerization. This can be mimicked by use of the microtubule depolymerizer, oryzalin. Actin depolymerization using LatB triggers microtubule depolymerization but not vice versa, suggesting that SI-induced F-actin depolymerization signals to microtubule depolymerization. The tubulin-stabilizing drug, taxol, alleviates SI-induced Ac-DEVDAMC cleavage, implicating a requirement for tubulin depolymerization for the DEVDase activation. Disrupting microtubule dynamics alone using oryzalin does not trigger increased cleavage of the DEVDase substrate Ac-DEVD-AMC: this suggests that both actin and microtubule depolymerization are required for DEVDase-mediated SI-induced PCD. Evidence for involvement of a SI-activated p56-MAPK comes from use of the MEK inhibitor U0126 and its negative analogue U0124. Although U0126 inhibits pollen tube growth and p56-MAPK activity, it does not affect pollen viability. These drugs provided evidence that p56-MAPK activation is required for the activation of a DEVDase, DNA fragmentation, and cell death. SI stimulates transient increases in ROS and later NO. Pretreatment of pollen tubes with either the NADPH oxidase inhibitor diphenyliodonium or the NO scavenger cPTIO and then stimulating SI showed that both were required to reduce the SI-induced DEVDase activity. This suggests that ROS and NO act in concert or tandem to signal to activate SI-induced PCD through the activation of a DEVDase activity. Moreover, these drugs alleviated the formation of the characteristic punctate F-actin foci. This implicates ROS and NO signaling for the formation of these F-actin foci. From: