Aquatic Monocot Research Articles

Discovery, structural characterization, and functional insights into a novel apiosidase from the GH140 family, isolated from a lignocellulolytic-enriched mangrove microbial community.

Apiosidases are enzymes that cleave the glycosidic bond between the monosaccharides linked to apiose, a branched chain furanose found in the cell walls of vascular plants and aquatic monocots. There is biotechnological interest in this enzyme group because apiose is the flavor-active compound of grapes, fruit juice, and wine, and the monosaccharide is found to be a plant secondary metabolite with pharmaceutical properties. However, functional and structural studies of this enzyme family are scarce. Recently, a glycoside hydrolase family member GH140 was isolated fromBacteroides thetaiotaomicron and identified as an endo-apiosidase. The structural characterization and functional identification of a second GH140 family enzyme, termed MmApi, discovered through mangrove soil metagenomic approach, are described. Among the various substrates tested, MmApi exhibited activity on an apiose-containing oligosaccharide derived from the pectic polysaccharide rhamnogalacturonan-II. While the crystallographic model of MmApi was similar to the endo-apiosidase fromBacteroides thetaiotaomicron, differences in the shape of the binding sites indicated that MmApi could cleave apioses within oligosaccharides of different compositions. This enzyme represents a novel tool for researchers interested in studying the physiology and structure of plant cell walls and developing biocatalytic strategies for drug and flavor production.

Micropropagation of the Ornamental Aquatic Plant, Aponogeton ulvaceus, from Immature Tuber Explants.

The aquatic monocot, Aponogeton ulvaceus Baker, is endemic to Madagascar and is a commercially valuable ornamental aquarium plant. Members of the genus Aponogeton contain a spectrum of phytochemicals associated with a broad range of biological activities. However, much remains to be known about this genus, and the A. ulvaceus population is declining due to anthropogenic activities and climate change. To address these challenges, adopting plant tissue culture technology will be a viable solution for the sustainable production of pest- and pathogen-free plants to meet the demands of the ornamental aquatic plant trade, for conservation and research purposes. A simple micropropagation protocol for A. ulvaceus is described here, starting with seeds to establish sterile stock plants, from which immature tubers were acquired as explants for indirect organogenesis.

Loss of ancestral function in duckweed roots is accompanied by progressive anatomical reduction and a re-distribution of nutrient transporters

Organ loss occurs frequently during plant and animal evolution. Sometimes, non-functional organs are retained through evolution. Vestigial organs are defined as genetically determined structures that have lost their ancestral (or salient) function.1,2,3 Duckweeds, an aquatic monocot family, exhibit both these characteristics. They possess a uniquely simple body plan, variably across five genera, two of which are rootless. Due to the existence of closely related species with a wide diversity in rooting strategies, duckweed roots represent a powerful system for investigating vestigiality. To explore this, we employed a panel of physiological, ionomic, and transcriptomic analyses, with the main goal of elucidating the extent of vestigiality in duckweed roots. We uncovered a progressive reduction in root anatomy as genera diverge and revealed that the root has lost its salient ancestral function as an organ required for supplying nutrients to the plant. Accompanying this, nutrient transporter expression patterns have lost the stereotypical root biased localization observed inother plant species. While other examples of organ loss such as limbs in reptiles4 or eyes in cavefish5 frequently display a binary of presence/absence, duckweeds provide a unique snapshot of an organ with varying degrees of vestigialization in closely related neighbors and thus provide a unique resource for exploration of how organs behave at different stages along the process of loss.

DNA barcoding and biomass accumulation rates of native Iranian duckweed species for biotechnological applications.

The Lemnaceae family (duckweed) consists of at least three recognized genera with six reported species in Iran that are distributed in wetlands. Duckweeds are the simplest and smallest flowering aquatic monocots with free-floating fronds that can reproduce asexually every 2-3 days. Duckweed could be a major source of balanced amino acids and high protein content, which is increasingly promising for biotechnological applications. For molecular classification and species identification of the collected samples, DNA barcoding was performed using two standard chloroplast markers, the spacer region between the ATP synthase subunits F and H (atpF-atpH) and the intron region of the ribosomal protein S16 (rps16). The results confirm the presence of four species belonging to the two genera Lemna and Spirodela. In addition, L. turionifera was detected for the first time in Iran. Due to the high growth rates of duckweed, measurement of biomass accumulation and doubling time are important factors in determining growth potential, especially for native species. The relative growth rates (RGR), doubling times (DT), biomass accumulation, and relative weekly yields (RY) of 40 distinct duckweed clones were determined under standard cultivation conditions. The dry weight-based RGR ranged from 0.149 to more than 0.600 per day, DT from 1.12 to 9 days, and RY from 7 to 108.9 per week. All values are comparable with previous studies. RGR and RY of selected clones are higher than the growth potential for a wide range of wild plants and common crops. These data support that native duckweed has high productivity value and should be further investigated as a potentially rich protein source for alternative human food, livestock feed, and recombinant protein production.

The genome and preliminary single-nuclei transcriptome of Lemna minuta reveals mechanisms of invasiveness.

The ability to trace every cell in some model organisms has led to the fundamental understanding of development and cellular function. However, in plants the complexity of cell number, organ size, and developmental time makes this a challenge even in the diminutive model plant Arabidopsis (Arabidopsis thaliana). Duckweed, basal nongrass aquatic monocots, provide an opportunity to follow every cell of an entire plant due to their small size, reduced body plan, and fast clonal growth habit. Here we present a chromosome-resolved genome for the highly invasive Lesser Duckweed (Lemna minuta) and generate a preliminary cell atlas leveraging low cell coverage single-nuclei sequencing. We resolved the 360 megabase genome into 21 chromosomes, revealing a core nonredundant gene set with only the ancient tau whole-genome duplication shared with all monocots, and paralog expansion as a result of tandem duplications related to phytoremediation. Leveraging SMARTseq2 single-nuclei sequencing, which provided higher gene coverage yet lower cell count, we profiled 269 nuclei covering 36.9% (8,457) of the L. minuta transcriptome. Since molecular validation was not possible in this nonmodel plant, we leveraged gene orthology with model organism single-cell expression datasets, gene ontology, and cell trajectory analysis to define putative cell types. We found that the tissue that we computationally defined as mesophyll expressed high levels of elemental transport genes consistent with this tissue playing a role in L. minuta wastewater detoxification. The L. minuta genome and preliminary cell map provide a paradigm to decipher developmental genes and pathways for an entire plant.

RNA-Seq analysis reveals potential regulators of programmed cell death and leaf remodelling in lace plant (Aponogeton madagascariensis)

BackgroundThe lace plant (Aponogeton madagascariensis) is an aquatic monocot that develops leaves with uniquely formed perforations through the use of a developmentally regulated process called programmed cell death (PCD). The process of perforation formation in lace plant leaves is subdivided into several developmental stages: pre-perforation, window, perforation formation, perforation expansion and mature. The first three emerging “imperforate leaves” do not form perforations, while all subsequent leaves form perforations via developmentally regulated PCD. PCD is active in cells called “PCD cells” that do not retain the antioxidant anthocyanin in spaces called areoles framed by the leaf veins of window stage leaves. Cells near the veins called “NPCD cells” retain a red pigmentation from anthocyanin and do not undergo PCD. While the cellular changes that occur during PCD are well studied, the gene expression patterns underlying these changes and driving PCD during leaf morphogenesis are mostly unknown. We sought to characterize differentially expressed genes (DEGs) that mediate lace plant leaf remodelling and PCD. This was achieved performing gene expression analysis using transcriptomics and comparing DEGs among different stages of leaf development, and between NPCD and PCD cells isolated by laser capture microdissection.ResultsTranscriptomes were sequenced from imperforate, pre-perforation, window, and mature leaf stages, as well as PCD and NPCD cells isolated from window stage leaves. Differential expression analysis of the data revealed distinct gene expression profiles: pre-perforation and window stage leaves were characterized by higher expression of genes involved in anthocyanin biosynthesis, plant proteases, expansins, and autophagy-related genes. Mature and imperforate leaves upregulated genes associated with chlorophyll development, photosynthesis, and negative regulators of PCD. PCD cells were found to have a higher expression of genes involved with ethylene biosynthesis, brassinosteroid biosynthesis, and hydrolase activity whereas NPCD cells possessed higher expression of auxin transport, auxin signalling, aspartyl proteases, cysteine protease, Bag5, and anthocyanin biosynthesis enzymes.ConclusionsRNA sequencing was used to generate a de novo transcriptome for A. madagascariensis leaves and revealed numerous DEGs potentially involved in PCD and leaf remodelling. The data generated from this investigation will be useful for future experiments on lace plant leaf development and PCD in planta.

Flowering and Seed Production across the Lemnaceae.

Plants in the family Lemnaceae are aquatic monocots and the smallest, simplest, and fastest growing angiosperms. Their small size, the smallest family member is 0.5 mm and the largest is 2.0 cm, as well as their diverse morphologies make these plants ideal for laboratory studies. Their rapid growth rate is partially due to the family’s neotenous lifestyle, where instead of maturing and producing flowers, the plants remain in a juvenile state and continuously bud asexually. Maturation and flowering in the wild are rare in most family members. To promote further research on these unique plants, we have optimized laboratory flowering protocols for 3 of the 5 genera: Spirodela; Lemna; and Wolffia in the Lemnaceae. Duckweeds were widely used in the past for research on flowering, hormone and amino acid biosynthesis, the photosynthetic apparatus, and phytoremediation due to their aqueous lifestyle and ease of aseptic culture. There is a recent renaissance in interest in growing these plants as non-lignified biomass sources for fuel production, and as a resource-efficient complete protein source. The genome sequences of several Lemnaceae family members have become available, providing a foundation for genetic improvement of these plants as crops. The protocols for maximizing flowering described herein are based on screens testing daylength, a variety of media, supplementation with salicylic acid or ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) (EDDHA), as well as various culture vessels for effects on flowering of verified Lemnaceae strains available from the Rutgers Duckweed Stock Cooperative.

Improved Spirodela polyrhiza genome and proteomic analyses reveal a conserved chromosomal structure with high abundance of chloroplastic proteins favoring energy production.

Duckweeds are a monophyletic group of rapidly reproducing aquatic monocots in the Lemnaceae family. Given their clonal, exponentially fast reproduction, a key question is whether genome structure is conserved across the species in the absence of meiotic recombination. Here, we studied the genome and proteome of Spirodela polyrhiza, or greater duckweed, which has the largest body plan yet the smallest genome size in the family (1C=150 Mb). Using Oxford Nanopore sequencing combined with Hi-C scaffolding, we generated a highly contiguous, chromosome-scale assembly of S. polyrhiza line Sp7498 (Sp7498_HiC). Both the Sp7498_HiC and Sp9509 genome assemblies reveal large chromosomal misorientations relative to a recent PacBio assembly of Sp7498, highlighting the need for orthogonal long-range scaffolding techniques such as Hi-C and BioNano optical mapping. Shotgun proteomics of Sp7498 verified the expression of ~2250 proteins and revealed a high abundance of proteins involved in photosynthesis and carbohydrate metabolism among other functions. In addition, a strong increase in chloroplast proteins was observed that correlated to chloroplast density. This Sp7498_HiC genome was generated cheaply and quickly with a single Oxford Nanopore MinION flow cell and one Hi-C library in a classroom setting. Combining these data with a mass spectrometry-generated proteome illustrates the utility of duckweed as a model for genomics- and proteomics-based education.

High Saccharification, Low Lignin, and High Sustainability Potential Make Duckweeds Adequate as Bioenergy Feedstocks

Duckweeds are the smallest free-floating aquatic monocots. They have a unique cell wall containing pectin polymers named apiogalacturonan and xylogalacturonan. Knowing that the cell wall composition is essential for duckweeds as a bioenergy feedstock, notably ethanol production, this work reports the five duckweed species’ (Spirodela polyrhiza, Landoltia punctata, Lemna gibba, Wolffiella caudata, and Wolffia borealis) composition and saccharification potential. Nonstructural carbohydrates were, on average, 42% of the dry weight. The cell wall comprises 20.1% pectin and glucomannan, 35.2% hemicelluloses, 30% cellulose, and 5% lignin, and the fermentable sugars in duckweed walls are glucose, galactose, and xylose. Together, these monosaccharides constitute 51.4% of the cell wall. Duckweeds displayed low recalcitrance to hydrolysis, probably due to the low lignin and cellulose contents. Furthermore, the saccharification of the duckweeds was higher than sugarcane, a primary bioethanol feedstock. Results indicate that duckweed biomass displays a high potential as a feedstock for bioethanol production.

Role of Proton Motive Force in Photoinduction of Cytoplasmic Streaming in Vallisneria Mesophyll Cells.

In mesophyll cells of the aquatic monocot Vallisneria, red light induces rotational cytoplasmic streaming, which is regulated by the cytoplasmic concentration of Ca2+. Our previous investigations revealed that red light induces Ca2+ efflux across the plasma membrane (PM), and that both the red light-induced cytoplasmic streaming and the Ca2+ efflux are sensitive to vanadate, an inhibitor of P-type ATPases. In this study, pharmacological experiments suggested the involvement of PM H+-ATPase, one of the P-type ATPases, in the photoinduction of cytoplasmic streaming. We hypothesized that red light would activate PM H+-ATPase to generate a large H+ motive force (PMF) in a photosynthesis-dependent manner. We demonstrated that indeed, photosynthesis increased the PMF and induced phosphorylation of the penultimate residue, threonine, of PM H+-ATPase, which is a major activation mechanism of H+-ATPase. The results suggested that a large PMF generated by PM H+-ATPase energizes the Ca2+ efflux across the PM. As expected, we detected a putative Ca2+/H+ exchange activity in PM vesicles isolated from Vallisneria leaves.

Morphological and anatomical plasticity of a rare amphibious species of Eriocaulaceae (Poales, Monocotyledons)

Phenotypic plasticity is common in aquatic monocots, but is sometimes mistaken with heteroblasty. In Eriocaulaceae, aquatic and amphibious species may present interesting adaptive morphological features. However, Paepalanthus saxicola var. pilosus is unusual by having two leaf morphotypes and scapes that can be partially submerged or completely exposed. This taxon also presents an uncertain identity due to its incomplete morphological description. Here, we studied the morphology and anatomy of both leaf morphotypes and of scapes of P. saxicola var. pilosus to understand if the morphological differences in leaves correspond to phenotypic plasticity or heteroblasty; and the ecological and systematic significance of its leaves and scapes. Besides, we describe its morphology to evaluate its identity. The short curved leaves show supporting tissue and three vascular bundles, while the long filiform ones lack supporting tissue and have a single vascular bundle. All specimens share anatomical characteristics of scape. We raised this taxon to the species level, giving it a replacing name, Paepalanthus amphibius. We observed that P. amphibius is the first confirmed report of heterophylly in Eriocaulaceae. The anatomy of its short leaves and scapes reflects adaptations to a xeric environment, while the anatomy of the long leaves shows characteristics adapted to an aquatic habit. Possibly, such degree of plasticity is underreported in the family, so we stress the importance of seasonal studies to understand the morphology and taxonomy of the amphibious species.

The Function of Autophagy in Lace Plant Programmed Cell Death.

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.

Loss of Wood Formation Genes in Monocot Genomes.

Woodiness (secondary xylem derived from vascular cambium) has been gained and lost multiple times in the angiosperms, but has been lost ancestrally in all monocots. Here, we investigate the conservation of genes involved in xylogenesis in fully sequenced angiosperm genomes, hypothesizing that monocots have lost some essential orthologs involved in this process. We analyzed the conservation of genes preferentially expressed in the developing secondary xylem of two eudicot trees in the sequenced genomes of 26 eudicot and seven monocot species, and the early diverging angiosperm Amborella trichopoda. We also reconstructed a regulatory model of early vascular cambial cell identity and differentiation and investigated the conservation of orthologs across the angiosperms. Additionally, we analyzed the genome of the aquatic seagrass Zostera marina for additional losses of genes otherwise essential to, especially, secondary cell wall formation. Despite almost complete conservation of orthology within the early cambial differentiation gene network, we show a clear pattern of loss of genes preferentially expressed in secondary xylem in the monocots that are highly conserved across eudicot species. Our study provides candidate genes that may have led to the loss of vascular cambium in the monocots, and, by comparing terrestrial angiosperms to an aquatic monocot, highlights genes essential to vasculature on land.

Correlation of Apiose Levels and Growth Rates in Duckweeds.

The carbon assimilated by photosynthesis in plants can be partitioned into starch, soluble sugars, and cell wall polymers. Higher levels of starch accumulation in leaves are usually correlated with a lower growth capacity. Duckweeds are fast-growing aquatic monocot plants that can accumulate high levels of starch. They are an unusual group because their cell wall has very low levels of lignin while accumulating apiogalacturonan, a pectic polysaccharide that could be involved with boron assimilation. In this work, five duckweed species from different genera (Spirodela polyrhiza, Landoltia punctata, Lemna gibba, Wolffiella caudata, and Wolffia borealis) were cultivated under two light intensities (20 and 500 μmoles of photons m−2 s−1) to evaluate the effects of growth rate on carbohydrate metabolism. A comparative analysis was performed by measuring their relative growth rates (RGR), and their content for starch, as well as soluble and cell wall carbohydrates. We found that the faster-growing species (the Lemnoideae) accumulate lower starch and higher soluble sugars than the slower-growing species within the Wolffioideae. Interestingly, analysis of the cell wall monosaccharides revealed that the slower-growing species displayed lower content of apiose in their walls. Our results indicate that higher accumulation of apiose observed in cell walls of the Lemnoideae species, which likely correlates with a higher proportion of apiogalacturonan, may lead to higher efficiency in the assimilation of boron. This is consistent with the increased RGR observed under conditions with higher apiose in the cell wall, such as higher light intensity. Consistent with their lower growth capacity, the Wolffioideae species we studied shows higher starch accumulation in comparison with the Lemnoideae species. We suggest that apiose levels could be good biomarkers for growth capacity of duckweeds and suggest that boron uptake could be an important factor for growth control in this aquatic plant family.

Changes in the abundance of cell wall apiogalacturonan and xylogalacturonan and conservation of rhamnogalacturonan II structure during the diversification of the Lemnoideae.

The diversification of the Lemnoideae was accompanied by a reduction in the abundance of cell wall apiogalacturonan and an increase in xylogalacturonan whereas rhamnogalacturonan II structure and cross-linking are conserved. The subfamily Lemnoideae is comprised of five genera and 38 species of small, fast-growing aquatic monocots. Lemna minor and Spirodela polyrhiza belong to this subfamily and have primary cell walls that contain large amounts of apiogalacturonan and thus are distinct from the primary walls of most other flowering plants. However, the pectins in the cell walls of other members of the Lemnoideae have not been investigated. Here, we show that apiogalacturonan decreased substantially as the Lemnoideae diversified since Wolffiella and Wolffia walls contain between 63 and 88% less apiose than Spirodela, Landoltia, and Lemna walls. In Wolffia, the most derived genus, xylogalacturonan is far more abundant than apiogalacturonan, whereas in Wolffiella pectic polysaccharides have a high arabinose content, which may arise from arabinan sidechains of RG I. The apiose-containing pectin rhamnogalacturonan II (RG-II) exists in Lemnoideae walls as a borate cross-linked dimer and has a glycosyl sequence similar to RG-II from terrestrial plants. Nevertheless, species-dependent variations in the extent of methyl-etherification of RG-II sidechain A and arabinosylation of sidechain B are discernible. Immunocytochemical studies revealed that pectin methyl-esterification is higher in developing daughter frond walls than in mother frond walls, indicating that methyl-esterification is associated with expanding cells. Our data support the notion that a functional cell wall requires conservation of RG-II structure and cross-linking but can accommodate structural changes in other pectins. The Lemnoideae provide a model system to study the mechanisms by which wall structure and composition has changed in closely related plants with similar growth habits.

Roles of actin cytoskeleton for regulation of chloroplast anchoring

ABSTRACTChloroplasts are known to maintain specific intracellular distribution patterns under specific environmental conditions, enabling the optimal performance of photosynthesis. To this end, chloroplasts are anchored in the cortical cytoplasm. In leaf epidermal cells of aquatic monocot Vallisneria, we recently demonstrated that the anchored chloroplasts are rapidly de-anchored upon irradiation with high-intensity blue light and that the process is probably mediated by the blue-light receptor phototropins. Chloroplast de-anchoring is a necessary step rendering the previously anchored chloroplasts mobile to allow their migration. In this article, based on the results obtained in Vallisneria together with those in other plant species, we briefly discussed possible modes of regulation of chloroplast anchoring and de-anchoring by actin cytoskeleton. The topics include roles of photoreceptor systems, actin-filament-dependent and -independent chloroplast anchoring, and independence of chloroplast de-anchoring from actomyosin and microtubule systems.

Comprehensive definition of genome features in Spirodela polyrhiza by high-depth physical mapping and short-read DNA sequencing strategies.

Spirodela polyrhiza is a fast-growing aquatic monocot with highly reduced morphology, genome size and number of protein-coding genes. Considering these biological features of Spirodela and its basal position in the monocot lineage, understanding its genome architecture could shed light on plant adaptation and genome evolution. Like many draft genomes, however, the 158-Mb Spirodela genome sequence has not been resolved to chromosomes, and important genome characteristics have not been defined. Here we deployed rapid genome-wide physical maps combined with high-coverage short-read sequencing to resolve the 20 chromosomes of Spirodela and to empirically delineate its genome features. Our data revealed a dramatic reduction in the number of the rDNA repeat units in Spirodela to fewer than 100, which is even fewer than that reported for yeast. Consistent with its unique phylogenetic position, small RNA sequencing revealed 29 Spirodela-specific microRNA, with only two being shared with Elaeis guineensis (oil palm) and Musa balbisiana (banana). Combining DNA methylation data and small RNA sequencing enabled the accurate prediction of 20.5% long terminal repeats (LTRs) that doubled the previous estimate, and revealed a high Solo:Intact LTR ratio of 8.2. Interestingly, we found that Spirodela has the lowest global DNA methylation levels (9%) of any plant species tested. Taken together our results reveal a genome that has undergone reduction, likely through eliminating non-essential protein coding genes, rDNA and LTRs. In addition to delineating the genome features of this unique plant, the methodologies described and large-scale genome resources from this work will enable future evolutionary and functional studies of this basal monocot family.

Quercetin feeding protects plants against oxidative stress

Background: Flavonoids are a complex group of plant-made phenolic compounds that are considered of high nutraceutical value. Their beneficial impacts on human health relate predominantly to their capacity to serve as antioxidants, thus protecting cells against the damaging impact of reactive oxygen species. Recent studies have also pointed at an essential role for flavonoids as antioxidants in plants. Results: Here we show that the flavonoid quercetin, which is known to protect human cells from oxidative stress, has the same effect on plant cells. Under oxidative stress conditions, Arabidopsis plants grown on quercetin-supplemented media grew better than controls and contained less oxidized proteins. This protection was also observed in the dicot Nicotiana tabacum and the aquatic monocot Lemna gibba. Conclusion: Quercetin can be used as a general antioxidant stress protectant for plants.

A comparison of the early developmental morphologies ofAponogeton madagascariensisandA.boivinianus

Aponogeton madagascariensis (Mirb.) H. Bruggen is an aquatic monocot that develops perforations in its leaves through developmentally regulated programmed cell death (PCD). Aponogeton boivinianus Baill. ex Jum. 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 plastochrons 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.

Lace plant ethylene receptors, AmERS1a and AmERS1c, regulate ethylene-induced programmed cell death during leaf morphogenesis.

The lace plant, Aponogeton madagascariensis, is an aquatic monocot that forms perforations in its leaves as part of normal leaf development. Perforation formation occurs through developmentally regulated programmed cell death (PCD). The molecular basis of PCD regulation in the lace plant is unknown, however ethylene has been shown to play a significant role. In this study, we examined the role of ethylene receptors during perforation formation. We isolated three lace plant ethylene receptors AmERS1a, AmERS1b and AmERS1c. Using quantitative PCR, we examined their transcript levels at seven stages of leaf development. Through laser-capture microscopy, transcript levels were also determined in cells undergoing PCD and cells not undergoing PCD (NPCD cells). AmERS1a transcript levels were significantly lower in window stage leaves (in which perforation formation and PCD are occurring) as compared to all other leaf developmental stages. AmERS1a and AmERS1c (the most abundant among the three receptors) had the highest transcript levels in mature stage leaves, where PCD is not occurring. Their transcript levels decreased significantly during senescence-associated PCD. AmERS1c had significantly higher transcript levels in NPCD compared to PCD cells. Despite being significantly low in window stage leaves, AmERS1a transcripts were not differentially expressed between PCD and NPCD cells. The results suggested that ethylene receptors negatively regulate ethylene-controlled PCD in the lace plant. A combination of ethylene and receptor levels determines cell fate during perforation formation and leaf senescence. A new model for ethylene emission and receptor expression during lace plant perforation formation and senescence is proposed.