Aponogeton madagascariensis

Rowarth, Nathan M., Tattrie, Sophie B., Dauphinee, Adrian N., Lacroix, Christian R. & Gunawardena, Arunika H. L. A. N., 2023, Filling in the gaps: a road map to establish a model system to study developmental programmed cell death, Botany 101 (8), pp. 301-317 : 304-308

Aponogeton madagascariensis is one of the 57 species of

Aponogetonaceae but the only one that forms perforations

during leaf development ( van Bruggen 1985, 1998). Although a few species with perforated leaves in Araceae also belong to the Alismatales , it is unknown if perforation formation has a common evolutionary origin ( Gunawardena 2008). Therefore, the evolutionary advantage of leaf perforations in aquatic plants is unclear. However, several hypotheses have been proposed, such as promoting thermoregulation, camouflage, defence from herbivores, and mechanical protection from water drag (Gunawardena et al. 2006).

The lace plant model system is ideal for studying PCD due to the accessibility and predictability of PCD and the visible gradient of cell death in developing adult leaves. Lace plant leaves are also thin and translucent, making them ideal for live cell imaging. Finally, the sterile propagation of whole plants in axenic environments creates an opportunity for pharmacological studies ( Gunawardena 2008).

Predictability of perforation formation

The predictable spatiotemporal nature of PCD within lattice-like veins of mature lace plant leaves presents a unique opportunity to develop a model system to study PCD ( Gunawardena et al. 2004). The first three to four leaves that emerge from the corm do not produce perforations and are observed to senesce relatively quickly after maturity. The leaves that follow (known as adult leaves) are enriched with a red pigment from the antioxidant anthocyanin and form perforations . The developmental stages of leaves are subdivided into pre-perforation, window, perforation formation, perforation expansion, and mature ( Fig. 1). Newly emerged leaves are in the pre-perforation stage; they are furled, and the mesophyll cells of the lamina are full of anthocyanin. The initiation of PCD in window stage leaves is visible as a loss of anthocyanin pigmentation. In the perforation formation stage, the deletion of cells begins to progress outwards from the center of the areole, a region framed by longitudinal and transverse veins. During perforation expansion, the hole formed by PCD continues to expand until it reaches four to five cells from the veins. At maturity, the perforation is complete, and PCD has ceased ( Fig. 1). Mesophyll cells at the perforation border transdifferentiate into epidermal cells protected from water loss or infection by a suberin layer ( Gunawardena 2007).

Live cell imaging of lace plant PCD

Aquatic lace plant leaves are ideal for live cell imaging due to their near-translucent nature, which has been useful for characterizing the chronological subcellular events that take place during PCD ( Fig. 2; Lord et al. 2013; Dauphinee et al. 2017, 2019; Lord and Gunawardena 2013). Wertman et al. (2012) previously detailed the chronological order of lace plant developmental PCD using a combination of conventional light microscopy, transmission electron microscopy, and laser scanning confocal microscopy. Cells central to the areole undergo the first sign of PCD differentiation with the loss of anthocyanin pigment, but the cellular signalling that controls this change is unknown. This change in pigment is also observed in the senescence of petals in Arabidopsis , theorized to be a result of changes in selective permeability or pH of the vacuole ( van Doorn 2004; Wertman et al. 2012). Next, in early-PCD cells (EPCD cells, Fig. 2C, Video S1) comes the loss of chlorophyll along with a decrease in chloroplast size and number ( Wright et al. 2009; Wertman et al. 2012), also observed in Arabidopsis leaf senescence ( Lim et al. 2007). Actin microfilaments re-organized from thin and organized to thicker and disorganized in arrangement before degradation, a standard feature found in early plant PCD cells, including Norway spruce suspensor deletion ( Filonova et al. 2000; Smertenko and Franklin-Tong 2011). This feature is believed to occur to prime microfilaments for being targeted by upstream caspase-like proteases ( Wertman et al. 2012). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive PCD cell nuclei indicate deoxyribonucleic acid fragmentation is detectable in lace plant PCD cells during actin degradation, followed by tonoplast changes ( Wertman et al. 2012). Tonoplast rupture is common in plant PCD processes like tracheary element differentiation ( Iakimova and Woltering 2017), suspensor deletion ( Reza et al. 2018), and aerenchyma formation ( Ni et al. 2014). Once the tonoplast ruptures, vacuolar aggregates cease their Brownian movement ( Wertman et al. 2012), the nucleus condenses, the mitochondrial membrane potential is lost, and the plasma membrane retracts (∼20 min after tonoplast rupture). Early- and late-PCD cells (LPCD, Fig. 2C, Video S2) have aggregates in their vacuole. There is evidence that chloroplasts are brought to the vacuole by autophagy ( Wright et al. 2009; Dauphinee et al. 2019). In addition, an interesting observation in early and LPCD cells is the formation of perinuclear chloroplast aggregations that are common during developmental PCD ( Wright et al. 2009; Lord et al. 2013; Dauphinee et al. 2014). The exact process is also observed in tobacco protoplasts and leaf aerenchyma formation in Typha angustifolia ( Wright et al. 2009; Lord et al. 2011; Wertman et al. 2012; Ni et al. 2014). However, it is unknown if this morphological manifestation is required for coordinated genetic expression for timely PCD execution or a consequence of vacuolar swelling.

Cells proximal to the vasculature retain their anthocyanin (in the mesophyll layer) and chlorophyll pigmentation, maintaining homeostasis throughout the formation of perforations (non-PCD cells (NPCD), Fig. 2C, Video S3). NPCD cells maintain function during perforation formation providing a suitable control group to test and observe intracellular changes in EPCD and LPCD cells. This natural gradient of PCD is accessible in a single field of view, a distinct advantage over other plant PCD systems.

Sterile culture system

Although lace plants can be maintained in aquariums, they are propagated in sterile, controlled environments to avoid environmental disturbances and maintain a consistent leaf morphology and PCD pattern ( Dauphinee and Gunawardena 2015). This transfer to a controlled sterile environment represents a trade-off where lace plant leaves are generally smaller than aquarium plants. Lace plants are propagated as cleaned corms under established protocols in G47 magenta box containers ( Fig. 3) or 1 L glass jar vessels ( Fig. 4 ). Cultures are supplemented with Murashige and Skoog (MS) media containing 1% agar and 3% sucrose ( Fig. 3) and grown at 24 ◦ C un- der 12 h light:12 h dark cycle without additional hormones to promote growth. Pharmacological whole-plant treatments in axenic cultures make for consistent conditions for optimal (i) protein and RNA extractions and (ii) monitoring leaf growth and morphological changes ( Fig. 3). Whole plants can, therefore, be propagated and subjected to different types of treatment for weeks without infection, signs of necrosis, or accumulation of biproducts. In comparison, embryos of Norway spruce used to study suspensor deletion PCD need to be stimulated for development with growth regulators, a limitation the lace plant system does not experience ( Högberg et al. 1998). In addition, the constant recycling of removing overgrown shoots and cleaning lace plant mother corms before transplanting to a new culture magenta box provides highly repeatable experiments using plants with little genetic variation.

Summary of molecular lace plant PCD findings to date

Twenty years of lace plant research have provided the plant PCD community with a series of observations of events that characterize lace plant leaf remodelling on a morphological, biochemical, and molecular level. The inhibition of ethylene biosynthesis produces leaves with fewer perforations ( Dauphinee et al. 2012; Rantong et al. 2015). Downstream of ethylene, caspase-1-like activities triggered by the release of a mitochondrial signal have been postulated but not identified (Lord et al. 2013). The inhibition and promotion of lace plant heat shock protein 70 (Hsp70) in early developing leaves also affect anthocyanin levels, caspase-like protease activity, and the formation of perforations. However, the exact mechanism of its connection to PCD has not been elucidated ( Rowarth et al. 2020). The morphological and cellular changes during lace plant leaf development are well categorized. However, the genetic control underpinning lace plant PCD remains elusive ( Rantong and Gunawardena 2015; Rantong et al. 2016), partly due to a lack of genetic information for the Aponogetonaceae family. However, advancements in comparative RNA sequencing (RNA-Seq) analysis between PCD and NPCD-like cells in other plants have helped profile key differentially expressed genes (DEGs) that resemble PCD regulators ( Rowarth et al. 2021).

To date, lace plant experiments have taken advantage of sterile culture systems, long-term live cell imaging, protoplast extractions, successful Western blotting protocols, and RNA extractions from whole leaves or cell populations using laser capture microdissection ( Fig. 5, Video S4; Rowarth et al. 2020, 2021). Currently, the lace plant model system is limited from reaching its full potential due to the lack of genomic data, mutants, and a robust protocol for genetic transformation. For example, Agrobacterium tumefaciens strain GV2260 has been used to transform eudicots and monocots and, in optimal environments, was ∼25% successful in transforming lace plant shoot apical meristem (SAM) explants (Gunawardena lab (unpublished data, 2017)). On the other hand, callus tissue transformation produced a limited number of regenerated leaves, making it a less viable process than SAM transformation.

Figures

Fig. 4. Tracking newly furled leaf growth in 1 L glass culture jars. Furled adult or pre-perforation leaves (black arrows) emerge from the corm successively after three to four imperforate leaves have already emerged (Day 1). The laminar tissue of preperforation leaves has a red pigmentation from anthocyanin. Pre-perforation leaves grow and develop into window stage leaves (Day 4; black arrow) when leaves have unfurled; anthocyanins in the middle portion of the areoles start to fade, and a visible gradient of red pigment remains. Scale bars: 2 cm.