Filling in the gaps: a road map to establish a model system to study developmental programmed cell death

Only a handful of model systems for studying programmed cell death (PCD) exist. The model Arabidopsis thaliana has generated a plethora of knowledge, but it is essential to introduce new models to broaden our understanding of the commonalities of PCD. This review focuses on Aponogeton madagascariensis (the lace plant) as a choice model to study PCD in vivo. PCD plays a key role in plant development and defence. Thus, identifying key regulators across plants is a priority in the field. The formation of perforations in lace plant leaves in areas called areoles is a striking example of PCD. Cells undergoing PCD within areoles can be easily identified from a loss of their anthocyanin pigmentation. In contrast, cells adjacent to veins, non-PCD cells, retain anthocyanins, creating a gradient of cell death. The spatiotemporal pattern of perforation formation, a gradient of cell death within areoles, and the availability of axenic cultures provide an excellent in vivo system to study mechanisms of developmental PCD. The priorities to further develop this model involve sequencing the genome, establishing transformation protocols, and identifying anthocyanin species to determine their medicinal properties. We discuss practical methodologies and challenges associated with developing the lace plant as a model to study PCD

The pathway of cell dismantling during programmed cell death in lace plant (Aponogeton madagascariensis) leaves

Abstract Background Developmentally 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. Results The 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. Conclusion Results 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.</p

A comparison of the early developmental morphologies of Aponogeton madagascariensis and A. boivinianus

Aponogeton madagascariensis is an aquatic monocot that develops perforations in its leaves through developmentally regulated programmed cell death (PCD). Aponogeton boivinianus is a close relative found in comparable environments with leaves that have a similar shape with no perforations. Little is known about the early developmental morphology of the family. This study characterized and compared these two species via scanning electron microscopy (SEM) and confocal laser scanning microscopy using both fresh and fixed specimens. The shoot apical meristem (SAM) of A. madagascariensis was significantly larger than that of A. boivinianus, but there was no difference in phyllotaxy observed, as both exhibited a 2/5 spiral pattern. A novel technique using serial dissections and SEM of fresh, hydrated specimens revealed that there are 16 plastochrons before perforation formation in A. madagascariensis and 34 until a similar developmental stage in A. boivinianus. The effects on early development of A. madagascariensis plants with supressed ethylene production were also analyzed. Ethylene inhibition alters leaf development by blocking PCD, but had no significant effect on the SAM morphology or early leaf development in A. madagascariensis

Unveiling interactions among mitochondria, caspase-like proteases, and the actin cytoskeleton during plant programmed cell death (PCD).

Aponogeton madagascariensis produces perforations over its leaf surface via programmed cell death (PCD). PCD begins between longitudinal and transverse veins at the center of spaces regarded as areoles, and continues outward, stopping several cells from these veins. The gradient of PCD that exists within a single areole of leaves in an early stage of development was used as a model to investigate cellular dynamics during PCD. Mitochondria have interactions with a family of proteases known as caspases, and the actin cytoskeleton during metazoan PCD; less is known regarding these interactions during plant PCD. This study employed the actin stain Alexa Fluor 488 phalloidin, the actin depolymerizer Latrunculin B (Lat B), a synthetic caspase peptide substrate and corresponding specific inhibitors, as well as the mitochondrial pore inhibitor cyclosporine A (CsA) to analyze the role of these cellular constituents during PCD. Results depicted that YVADase (caspase-1) activity is higher during the very early stages of perforation formation, followed by the bundling and subsequent breakdown of actin. Actin depolymerization using Lat B caused no change in YVADase activity. In vivo inhibition of YVADase activity prevented PCD and actin breakdown, therefore substantiating actin as a likely substrate for caspase-like proteases (CLPs). The mitochondrial pore inhibitor CsA significantly decreased YVADase activity, and prevented both PCD and actin breakdown; therefore suggesting the mitochondria as a possible trigger for CLPs during PCD in the lace plant. To our knowledge, this is the first in vivo study using either caspase-1 inhibitor (Ac-YVAD-CMK) or CsA, following which the actin cytoskeleton was examined. Overall, our findings suggest the mitochondria as a possible upstream activator of YVADase activity and implicate these proteases as potential initiators of actin breakdown during perforation formation via PCD in the lace plant

The role of Atg16 in autophagy, anthocyanin biosynthesis, and programmed cell death in leaves of the lace plant (Aponogeton madagascariensis).

Aponogeton madagascariensis, commonly known as the lace plant, produces leaves that form perforations by programmed cell death (PCD). Leaf development is divided into several stages beginning with "pre-perforation" furled leaves enriched with red pigmentation from anthocyanins. The leaf blade is characterized by a series of grids known as areoles bounded by veins. As leaves develop into the "window stage", anthocyanins recede from the center of the areole towards the vasculature creating a gradient of pigmentation and cell death. Cells in the middle of the areole that lack anthocyanins undergo PCD (PCD cells), while cells that retain anthocyanins (non-PCD cells) maintain homeostasis and persist in the mature leaf. Autophagy has reported roles in survival or PCD promotion across different plant cell types. However, the direct involvement of autophagy in PCD and anthocyanin levels during lace plant leaf development has not been determined. Previous RNA sequencing analysis revealed the upregulation of autophagy-related gene Atg16 transcripts in pre-perforation and window stage leaves, but how Atg16 affects PCD in lace plant leaf development is unknown. In this study, we investigated the levels of Atg16 in lace plant PCD by treating whole plants with either an autophagy promoter rapamycin or inhibitors concanamycin A (ConA) or wortmannin. Following treatments, window and mature stage leaves were harvested and analyzed using microscopy, spectrophotometry, and western blotting. Western blotting showed significantly higher Atg16 levels in rapamycin-treated window leaves, coupled with lower anthocyanin levels. Wortmannin-treated leaves had significantly lower Atg16 protein and higher anthocyanin levels compared to the control. Mature leaves from rapamycin-treated plants generated significantly fewer perforations compared to control, while wortmannin had the opposite effect. However, ConA treatment did not significantly change Atg16 levels, nor the number of perforations compared to the control, but anthocyanin levels did increase significantly in window leaves. We propose autophagy plays a dual role in promoting cell survival in NPCD cells by maintaining optimal anthocyanin levels and mediating a timely cell death in PCD cells in developing lace plant leaves. How autophagy specifically affects anthocyanin levels remained unexplained

Plant Programmed Cell Death Methods

Programmed cell death (PCD) is a critical component of plant development, defense against invading pathogens, and response to environmental stresses. In this chapter, we provide detailed technical methods for studying PCD associated with plant development or induced by abiotic stress. A root hair assay or electrolyte leakage assay are excellent techniques for the quantitative determination of PCD and/or cellular injury induced in response to abiotic stress, whereas the lace plant provides a unique model that facilitates the study of genetically regulated PCD during leaf development.Department of Agriculture, Food and the MarineEngineering Research Council of Canada (NSERC)Killam TrustsCanadian Foundation for Innovation (CFI; Leaders Opportunity Fund

Rearrangement of the actin cytoskeleton during leaf morphogenesis over five stages of leaf development in the lace plant.

<p>Each image depicts a piece of a single corner of an areole (A–E). Coloured differential interference contrast (DIC) images of (A) pre perforation (B) window (C) perforation formation (D) perforation expansion and (E) mature stage leaf areoles. (F–J) Black and white DIC images of (F) pre perforation (G) window (H) perforation formation (I) perforation expansion and (J) mature stage leaves. (K–O) Fluorescent images of Alexa Fluor 488 phalloidin (green) stained areoles counterstained with propidium iodide (PI; red) of (K) pre perforation (L) window (M) perforation formation (N) perforation expansion and (O) mature stage leaves. Please note that images A-E do not correspond with fluorescent images F-O. These images are representative micrographs to illustrate the five stages of leaf development. DIC images F-J are corresponding to K-O. There is a consistent gradient of actin microfilament staining over the pre perforation areole; conversely there is variation (bundling followed by breakdown) in actin microfilament dynamics over the gradient of PCD (NPCD-LPCD) found within the single areole of the window stage leaf. Note the complete degradation and disappearance of actin microfilament staining as the perforation becomes larger, from perforation formation to the mature stage of leaf development. Scale bars = 70 µm.</p