Lace Plant Research Articles

The lace plant: a novel model system to study plant proteases during developmental programmed cell death in vivo

Programmed cell death (PCD) plays a major role in plant development and defense throughout the plant kingdom. Within animal systems, it is well accepted that caspases play a major role in the PCD process, although no true caspases have yet to be identified in plants. Despite this, vast amounts of evidence suggest the existence of caspase-like proteases in plants. The lace plant (Aponogeton madagascariensis) forms perforations in a predictable pattern between longitudinal and transverse veins over its entire leaf surface via PCD. Due to the thin nature of the leaf, allowing for long-term live cell imaging, a perfected method for sterile culture, as well as the feasibility of pharmacological experiments, the lace plant provides an excellent model to study developmental PCD. In this review, we report the suitability of the lace plant as a novel organism to study proteases in vivo during developmentally regulated cell death.

Do mitochondria play a role in remodelling lace plant leaves during programmed cell death?

BackgroundProgrammed cell death (PCD) is the regulated death of cells within an organism. The lace plant (Aponogeton madagascariensis) produces perforations in its leaves through PCD. The leaves of the plant consist of a latticework of longitudinal and transverse veins enclosing areoles. PCD occurs in the cells at the center of these areoles and progresses outwards, stopping approximately five cells from the vasculature. The role of mitochondria during PCD has been recognized in animals; however, it has been less studied during PCD in plants.ResultsThe following paper elucidates the role of mitochondrial dynamics during developmentally regulated PCD in vivo in A. madagascariensis. A single areole within a window stage leaf (PCD is occurring) was divided into three areas based on the progression of PCD; cells that will not undergo PCD (NPCD), cells in early stages of PCD (EPCD), and cells in late stages of PCD (LPCD). Window stage leaves were stained with the mitochondrial dye MitoTracker Red CMXRos and examined. Mitochondrial dynamics were delineated into four categories (M1-M4) based on characteristics including distribution, motility, and membrane potential (ΔΨm). A TUNEL assay showed fragmented nDNA in a gradient over these mitochondrial stages. Chloroplasts and transvacuolar strands were also examined using live cell imaging. The possible importance of mitochondrial permeability transition pore (PTP) formation during PCD was indirectly examined via in vivo cyclosporine A (CsA) treatment. This treatment resulted in lace plant leaves with a significantly lower number of perforations compared to controls, and that displayed mitochondrial dynamics similar to that of non-PCD cells.ConclusionsResults depicted mitochondrial dynamics in vivo as PCD progresses within the lace plant, and highlight the correlation of this organelle with other organelles during developmental PCD. To the best of our knowledge, this is the first report of mitochondria and chloroplasts moving on transvacuolar strands to form a ring structure surrounding the nucleus during developmental PCD. Also, for the first time, we have shown the feasibility for the use of CsA in a whole plant system. Overall, our findings implicate the mitochondria as playing a critical and early role in developmentally regulated PCD in the lace plant.

Regeneration of the aquatic monocot Aponogeton madagascariensis (lace plant) through callus induction

The lace plant ( Aponogeton madagascariensis) is an aquatic monocot native to Madagascar that forms perforated leaves via developmentally regulated programmed cell death (PCD). Although a technique for culturing the lace plant under axenic conditions through corm separation has been developed, it is less productive for mass propagation of this species. Thus, an alternative method was investigated using 27 plant growth regulator (PGR) combinations to induce callus and subsequent whole plant regeneration. Combinations of the auxin picloram and the cytokinin thidiazuron (TDZ) successfully induced callus in corm sections, but no shoot regeneration was observed. Successful results for both callus induction and shoot regeneration were achieved using immature inflorescences with the PGR combination of 2 mg/L of the cytokinin 6-benzyaminopurine (BAP) and 2 mg/L of the auxin 1-napthaleneacetic acid (NAA) under light conditions, resulting in the regeneration of over 70 plants. The protocol shows both reproducibility and consistency in results; therefore, it is concluded that a technique for mass propagation of the lace plant through callus has been established. This technique may be useful in the study of other aquatic plant species, as well as in the study of developmental PCD in the lace plant itself.

Environmentally induced programmed cell death in leaf protoplasts of Aponogeton madagascariensis

Within plant systems, two main forms of programmed cell death (PCD) exist: developmentally regulated and environmentally induced. The lace plant (Aponogeton madagascariensis) naturally undergoes developmentally regulated PCD to form perforations between longitudinal and transverse veins over its leaf surface. Developmental PCD in the lace plant has been well characterized; however, environmental PCD has never before been studied in this plant species. The results presented here portray heat shock (HS) treatment at 55 °C for 20 min as a promising inducer of environmental PCD within lace plant protoplasts originally isolated from non-PCD areas of the plant. HS treatment produces cells displaying many characteristics of developmental PCD, including blebbing of the plasma membrane, increased number of hydrolytic vesicles and transvacuolar strands, nuclear condensation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive nuclei, as well as increased Brownian motion within the vacuole. Results presented here for the first time provide evidence of chloroplasts in the vacuole of living protoplasts undergoing environmentally induced PCD. Findings suggest that the mitochondria play a critical role in the cell death process. Changes in mitochondrial dynamics were visualized in HS-treated cells, including loss of mitochondrial mobility, reduction in ΔΨ(m), as well as the proximal association with chloroplasts. The role of the mitochondrial permeability transition pore (PTP) was examined by pre-treatment with the PTP agonist cyclosporine A. Overall, HS is depicted as a reliable method to induce PCD within lace plant protoplasts, and proves to be a reliable technique to enable comparisons between environmentally induced and developmentally regulated PCD within one species of plant.

Calcium inhibition halts developmental programmed cell death in the lace plant, Aponogeton madagascariensis?

This study examined the effect of the calcium channel blocker ruthenium red (RR) on developmental programmed cell death (PCD) and perforation formation in leaves of the aquatic lace plant ( Aponogeton madagascariensis (Mirbel) H. Bruggen). Plant immersion experiments were conducted using various concentrations (0, 10, 20, 30, and 40 µmol·L–1) of RR applied to whole plants over a 3 to 4 week period. The ratio of number of leaf perforations per centimetre of leaf length along with leaf length were used to assess treatment effect. While the ratio of number of perforations per centimetre of leaf length was significantly reduced with RR treatment, leaf length was not, overall, significantly affected by RR. Therefore, it was concluded that RR was able to inhibit calcium movement and halt PCD, thus reducing perforation formation without having detrimental effects on leaf growth. The present research provides indirect evidence of the possible role of calcium in developmental PCD in vivo during leaf morphogenesis in the lace plant. Furthermore, it suggests the usefulness of the lace plant as a model system for pharmacological studies involving developmental PCD.

In vivo study of developmental programmed cell death using the lace plant (Aponogeton madagascariensis; Aponogetonaceae) leaf model system

Programmed cell death (PCD) is required for many morphological changes, but in plants it has been studied in much less detail than in animals. The unique structure and physiology of the lace plant (Aponogeton madagascariensis) is well suited for the in vivo study of developmental PCD. Live streaming video and quantitative analysis, coupled with transmission electron microscopy, were used to better understand the PCD sequence, with an emphasis on the chloroplasts. Dividing, dumbbell-shaped chloroplasts persisted until the late stages of PCD. However, the average size and number of chloroplasts, and the starch granules associated with them, declined steadily in a manner reminiscent of leaf senescence, but distinct from PCD described in the Zinnia tracheary element system. Remaining chloroplasts often formed a ring around the nucleus. Transvacuolar strands, which appeared to be associated with chloroplast transport, first increased and then decreased. Mitochondrial streaming ceased abruptly during the late stages of PCD, apparently due to tonoplast rupture. This rupture occurred shortly before the rapid degradation of the nucleus and plasma membrane collapse, in a manner also reminiscent of the Zinnia model. The presence of numerous objects in the vacuoles suggests increased macro-autophagy before cell death. These objects were rarely observed in cells not undergoing PCD.

Programmed cell death and tissue remodelling in plants: Fig. 1.

The use of programmed cell death (PCD) to remodel plants at the cellular, tissue, and organ levels is particularly fascinating and occurs in such processes as tracheary element differentiation, lysigenous aerenchyma formation, development of functionally unisexual flowers from bisexual floral primordia, and leaf morphogenesis. The formation of complex leaf shape through the use of PCD is a rare event across vascular plants and occurs only in a few species of Monstera and related genera, and in the lace plant (Aponogeton madagascariensis). During early development, the lace plant leaf forms a pattern of equidistantly positioned perforations across the surface of the leaf, giving it a lattice-like appearance. Due to the accessibility and predictability of this process, the lace plant provides highly suitable material for the study of developmentally regulated PCD in plants. A sterile lace plant culture system has been successfully established, providing material free of micro-organisms for experimental study. The potential role of ethylene and caspase-like activity in developmentally regulated PCD in the lace plant is currently under investigation, with preliminary results indicating that both may play a role in the cell death pathway.

Cell wall degradation and modification during programmed cell death in lace plant, Aponogeton madagascariensis (Aponogetonaceae)

An unusual form of leaf morphogenesis occurs in the aquatic, lace plant, Aponogeton madagascariensis (Aponogetonaceae). Early in development, discrete patches of cells undergo programmed cell death (PCD) and form perforations during leaf expansion. In addition to the protoplasts, walls of the dying cells are degraded during PCD. The cuticle of the perforation site is eroded first, followed by dissolution of cell wall matrix components, so that walls appear as loose fibrillar networks as perforations form. Gel diffusion assays of wall-degrading enzyme activity indicated that pectinases are active throughout leaf development, while cellulase activity was restricted to early stages of perforation formation. Alcian blue staining showed that degrading walls remain rich in pectin, and immunolocalization of pectin epitopes indicated that the proportions of esterified and de-esterifed pectins do not change significantly. Walls of perforation border cells are modified by suberin deposition late in development, and reactive oxygen species, thought to have a role in polymerization of phenolic suberin monomers, are present at the same stage. This timing suggests that suberization may limit the spread of PCD and provide an apoplastic barrier against microbial invasion but does not initiate PCD.

Programmed cell death remodels lace plant leaf shape during development.

Programmed cell death (PCD) functions in the developmental remodeling of leaf shape in higher plants, a process analogous to digit formation in the vertebrate limb. In this study, we provide a cytological characterization of the time course of events as PCD remodels young expanding leaves of the lace plant. Tonoplast rupture is the first PCD event in this system, indicated by alterations in cytoplasmic streaming, loss of anthocyanin color, and ultrastructural appearance. Nuclei become terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive soon afterward but do not become morphologically altered until late stages of PCD. Genomic DNA is fragmented, but not into internucleosomal units. Other cytoplasmic changes, such as shrinkage and degradation of organelles, occur later. This form of PCD resembles tracheary element differentiation in cytological execution but requires unique developmental regulation so that discrete panels of tissue located equidistantly between veins undergo PCD while surrounding cells do not.