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BackgroundDevelopmentally regulated programmed cell death (PCD) is the controlled death of cells that occurs throughout the life cycle of both plants and animals. The lace plant (Aponogeton madagascariensis) forms perforations between longitudinal and transverse veins in spaces known as areoles, via developmental PCD; cell death begins in the center of these areoles and develops towards the margin, creating a gradient of PCD. This gradient was examined using both long- and short-term live cell imaging, in addition to histochemical staining, in order to establish the order of cellular events that occur during PCD.ResultsThe first visible change observed was the reduction in anthocyanin pigmentation, followed by initial chloroplast changes and the bundling of actin microfilaments. At this stage, an increased number of transvacuolar strands (TVS) was evident. Perhaps concurrently with this, increased numbers of vesicles, small mitochondrial aggregates, and perinuclear accumulation of both chloroplasts and mitochondria were observed. The invagination of the tonoplast membrane and the presence of vesicles, both containing organelle materials, suggested evidence for both micro- and macro-autophagy, respectively. Mitochondrial aggregates, as well as individual chloroplasts were subsequently seen undergoing Brownian motion in the vacuole. Following these changes, fragmentation of nuclear DNA, breakdown of actin microfilaments and early cell wall changes were detected. The vacuole then swelled, causing nuclear displacement towards the plasma membrane (PM) and tonoplast rupture followed closely, indicating mega-autophagy. Subsequent to tonoplast rupture, cessation of Brownian motion occurred, as well as the loss of mitochondrial membrane potential (ΔΨm), nuclear shrinkage and PM collapse. Timing from tonoplast rupture to PM collapse was approximately 20 minutes. The entire process from initial chlorophyll reduction to PM collapse took approximately 48 hours. Approximately six hours following PM collapse, cell wall disappearance began and was nearly complete within 24 hours.ConclusionResults showed that a consistent sequence of events occurred during the remodelling of lace plant leaves, which provides an excellent system to study developmental PCD in vivo. These findings can be used to compare and contrast with other developmental PCD examples in plants.

Main conclusion Antioxidants and reactive oxygen species are integral for programmed cell death signaling during perforation formation in the lace plant ( Aponogeton madagascariensis ). The lace plant is an excellent model system for studying developmentally regulated programmed cell death (PCD). During early lace plant leaf development, PCD systematically deletes cells resulting in a perforated leaf morphology that is unique in planta. A distinct feature in young lace plant leaves is an abundance of anthocyanins, which have antioxidant properties. The first sign of PCD induction is the loss of anthocyanin pigmentation in cells that are targeted for destruction, which results in a visible gradient of cell death. The cellular dynamics and time course of lace plant PCD are well documented; however, the signals involved in the pathway remain elusive. This study investigates the roles of antioxidants and ROS in developmental PCD signaling during lace plant perforation formation. The involvement of antioxidants and ROS in the pathway was determined using a variety of techniques including pharmacological whole plant experimentation, long-term live cell imaging, the 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid anti-radical activity assay, and western blot analysis. Results indicate that antioxidants and ROS are key regulators of PCD during the remodelling of lace plant leaves.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-017-2683-y) contains supplementary material, which is available to authorized users.

Lace plant leaves utilize programmed cell death (PCD) to form perforations during development. The role of heat shock proteins (Hsps) in PCD during lace plant leaf development is currently unknown. Hsp70 amounts were measured throughout lace plant leaf development, and the results indicate that it is highest before and during PCD. Increased Hsp70 amounts correlate with raised anthocyanin content and caspase-like protease (CLP) activity. To investigate the effects of Hsp70 on leaf development, whole plants were treated with either of the known regulators of PCD [reactive oxygen species (ROS) or antioxidants] or an Hsp70 inhibitor, chlorophenylethynylsulfonamide (PES-Cl). ROS treatment significantly increased Hsp70 2-fold and CLP activity in early developing leaves, but no change in anthocyanin and the number of perforations formed was observed. Antioxidant treatment significantly decreased Hsp70, anthocyanin, and CLP activity in early leaves, resulting in the fewest perforations. PES-Cl (25 μM) treatment significantly increased Hsp70 4-fold in early leaves, while anthocyanin, superoxide, and CLP activity significantly declined, leading to fewer perforations. Results show that significantly increased (4-fold) or decreased Hsp70 amounts lead to lower anthocyanin and CLP activity, inhibiting PCD induction. Our data support the hypothesis that Hsp70 plays a role in regulating PCD at a threshold in lace plant leaf development. Hsp70 affects anthocyanin content and caspase-like protease activity, and helps regulate PCD during the remodelling of leaves of lace plant, Aponogeton madagascariensis.

The lace plant (Aponogeton madagascariensis) is an aquatic monocot that utilizes programmed cell death (PCD) to form perforations throughout its mature leaves as part of normal development. The lace plant is an emerging model system representing a unique form of developmental PCD. The role of autophagy in lace plant PCD was investigated using live cell imaging, transmission electron microscopy (TEM), immunolocalization, and in vivo pharmacological experimentation. ATG8 immunostaining and acridine orange staining revealed that autophagy occurs in both healthy and dying cells. Autophagosome-like vesicles were also found in healthy and dying cells through ultrastructural analysis with TEM. Following autophagy modulation, there was a noticeable increase in vesicles and vacuolar aggregates. A novel cell death assay utilizing lace plant leaves revealed that autophagy enhancement with rapamycin significantly decreased cell death rates compared to the control, whereas inhibition of autophagosome formation with wortmannin or blocking the degradation of cargoes with concanamycin A had an opposite effect. Although autophagy modulation significantly affected cell death rates in cells that are destined to die, neither the promotion nor inhibition of autophagy in whole plants had a significant effect on the number of perforations formed in lace plant leaves. Our data indicate that autophagy predominantly contributes to cell survival, and we found no clear evidence for its direct involvement in the induction of developmental PCD during perforation formation in lace plant leaves.