Return of the Lemnaceae: duckweed as a model plant system in the genomics and postgenomics era.

The aquatic Lemnaceae family, commonly called duckweed, comprises some of the smallest and fastest growing angiosperms known on Earth. Their tiny size, rapid growth by clonal propagation, and facile uptake of labeled compounds from the media were attractive features that made them a well-known model for plant biology from 1950 to 1990. Interest in duckweed has steadily regained momentum over the past decade, driven in part by the growing need to identify alternative plants from traditional agricultural crops that can help tackle urgent societal challenges, such as climate change and rapid population expansion. Propelled by rapid advances in genomic technologies, recent studies with duckweed again highlight the potential of these small plants to enable discoveries in diverse fields from ecology to chronobiology. Building on established community resources, duckweed is reemerging as a platform to study plant processes at the systems level and to translate knowledge gained for field deployment to address some of society's pressing needs. This review details the anatomy, development, physiology, and molecular characteristics of the Lemnaceae to introduce them to the broader plant research community. We highlight recent research enabled by Lemnaceae to demonstrate how these plants can be used for quantitative studies of complex processes and for revealing potentially novel strategies in plant defense and genome maintenance.

Genomics of turions from the Greater Duckweed reveal its pathways for dormancy and re-emergence strategy.

Over 15 families of aquatic plants are known to use a strategy of developmental switching upon environmental stress to produce dormant propagules called turions. However, few molecular details for turion biology have been elucidated due to the difficulties in isolating high-quality nucleic acids from this tissue. We successfully developed a new protocol to isolate high-quality transcripts and carried out RNA-seq analysis of mature turions from the Greater Duckweed Spirodela polyrhiza. Comparison of turion transcriptomes to that of fronds, the actively growing leaf-like tissue, were carried out. Bioinformatic analysis of high confidence, differentially expressed transcripts between frond and mature turion tissues revealed major pathways related to stress tolerance, starch and lipid metabolism, and dormancy that are mobilized to reprogram frond meristems for turion differentiation. We identified the key genes that are likely to drive starch and lipid accumulation during turion formation, as well as those in pathways for starch and lipid utilization upon turion germination. Comparison of genome-wide cytosine methylation levels also revealed evidence for epigenetic changes in the formation of turion tissues. Similarities between turions and seeds provide evidence that key regulators for seed maturation and germination were retooled for their function in turion biology.

Identification of an oleosin-like gene in seagrass seeds.

OBJECTIVE: To investigate the oil body protein and function in seeds of mature seagrass, Thalassia hemprichii. RESULTS: Seeds of mature seagrass T. hemprichii when stained with a fluorescent probe BODIPY showed the presence of oil bodies in intracellular cells. Triacylglycerol was the major lipid class in the seeds. Protein extracted from seagrass seeds was subjected to immunological cross-recognition with land plant seed oil body proteins, such as oleosin and caleosin, resulting in no cross-reactivity. An oleosin-like gene was found in seagrass seeds. Next generation sequencing and sequence alignment indicated that the deduced seagrass seed oleosin-like protein has a central hydrophobic domain responsible for their anchoring onto the surface of oil bodies. Phylogenetic analysis showed that the oleosin-like protein was evolutionarily closer to pollen oleosin than to seed oleosins. CONCLUSION: Oil body protein found in seagrass seeds represent a distinct class of land seed oil body proteins.

Nutrient limitation in Auxenochlorella protothecoides induces qualitative changes of fatty acid and expression of caleosin as a membrane protein associated with oil bodies.

Oil bodies formed in Auxenochlorella protothecoides induced during limited nutrition had a coating of caleosin. The total lipid content obtained from A. protothecoides in unstressed cultures (first week) was ~210 mg/g compared to the 231 mg/g obtained in the third week (nutrient limited) and 290 mg/g obtained in the fourth week (nutrient limited). The proportion of total saturated fatty acids increased from 28 to 46 %, whereas that of total polyunsaturated fatty acids decreased from 52 to 35 %. The expression levels of the 28 kDa caleosin protein in A. protothecoides rose to a maximum up to 4 weeks; immunolocalization studies showed that caleosin was predominantly associated with the membranes of oil bodies.

Lipid Droplets in Endosymbiotic Symbiodiniaceae spp. Associated with Corals.

Symbiodiniaceae species is a dinoflagellate that plays a crucial role in maintaining the symbiotic mutualism of reef-building corals in the ocean. Reef-building corals, as hosts, provide the nutrition and habitat to endosymbiotic Symbiodiniaceae species and Symbiodiniaceae species transfer the fixed carbon to the corals for growth. Environmental stress is one of the factors impacting the physiology and metabolism of the corals-dinoflagellate association. The environmental stress triggers the metabolic changes in Symbiodiniaceae species resulting in an increase in the production of survival organelles related to storage components such as lipid droplets (LD). LDs are found as unique organelles, mainly composed of triacylglycerols surrounded by phospholipids embedded with some proteins. To date, it has been reported that investigation of lipid droplets significantly present in animals and plants led to the understanding that lipid droplets play a key role in lipid storage and transport. The major challenge of investigating endosymbiotic Symbiodiniaceae species lies in overcoming the strategies in isolating lesser lipid droplets present in its intercellular cells. Here, we review the most recent highlights of LD research in endosymbiotic Symbiodiniaceae species particularly focusing on LD biogenesis, mechanism, and major lipid droplet proteins. Moreover, to comprehend potential novel ways of energy storage in the symbiotic interaction between endosymbiotic Symbiodiniaceae species and its host, we also emphasize recent emerging environmental factors such as temperature, ocean acidification, and nutrient impacting the accumulation of lipid droplets in endosymbiotic Symbiodiniaceae species.

Interactions between marine megafauna and plastic pollution in Southeast Asia.

Southeast (SE) Asia is a highly biodiverse region, yet it is also estimated to cumulatively contribute a third of the total global marine plastic pollution. This threat is known to have adverse impacts on marine megafauna, however, understanding of its impacts has recently been highlighted as a priority for research in the region. To address this knowledge gap, a structured literature review was conducted for species of cartilaginous fishes, marine mammals, marine reptiles, and seabirds present in SE Asia, collating cases on a global scale to allow for comparison, coupled with a regional expert elicitation to gather additional published and grey literature cases which would have been omitted during the structured literature review. Of the 380 marine megafauna species present in SE Asia, but also studied elsewhere, we found that 9.1 % and 4.5 % of all publications documenting plastic entanglement (n = 55) and ingestion (n = 291) were conducted in SE Asian countries. At the species level, published cases of entanglement from SE Asian countries were available for 10 % or less of species within each taxonomic group. Additionally, published ingestion cases were available primarily for marine mammals and were lacking entirely for seabirds in the region. The regional expert elicitation led to entanglement and ingestion cases from SE Asian countries being documented in 10 and 15 additional species respectively, highlighting the utility of a broader approach to data synthesis. While the scale of the plastic pollution in SE Asia is of particular concern for marine ecosystems, knowledge of its interactions and impacts on marine megafauna lags behind other areas of the world, even after the inclusion of a regional expert elicitation. Additional funding to help collate baseline data are critically needed to inform policy and solutions towards limiting the interactions of marine megafauna and plastic pollution in SE Asia.

Identification and characterization of caleosin in Cycas revoluta pollen.

Oil bodies are essential energy storage organelles that are generally present in the seeds of plants. Caleosin protein has been identified in the seed oil bodies of Cycas revolutaseed. In this study, mature Cycas revoluta pollen grains were collected from cycad elliptical cones. However, the isolation and identification of oil bodies protein from mature Cycas revoluta grains have never been experimentally determined. Ultrastructural studies have shown that the oil bodies were present in pollen Cycas revoluta grains. Lipid analysis showed that oil bodies are predominantly composed of triacylglycerol. Complete cDNA fragments encoding the caleosin were obtained by PCR cloning. Phylogenetic tree analyzes showed that cycad pollen caleosin is closely related to the caleosin of Cycas revoluta seeds. Fresh Cycas revoluta pollen grains were allowed to germinate for 48 h in a germination medium containing 2% sucrose and 0.01% H3BO3. Pollen germination were recorded periodically from day one to day 10 and the results showed that the Cycas revoluta pollen elongate the tube and increasing of triacylglycerol(TAG) after 4 days.

Identification of caleosin and oleosin in oil bodies of pine pollen.

Unique proteins including steroleosin, caleosin, oleosin-L, and oleosin-G have been identified in seed oil bodies of pine (Pinus massoniana). In this study, mature pollen grains with wing-like bladders were collected from pine (Pinus elliottii). Ultrastructural studies showed that oil bodies were present in pollen grains, but not the attached bladders, and the presence of oil bodies was further confirmed by fluorescent staining with BODIPY 493/503. Stable oil bodies were successfully purified from pine pollen grains, and analyzed to be mainly composed of triacylglycerols. Putative oleosin and caleosin in pine pollen oil bodies were detected by immunoassaying with antibodies against sesame seed caleosin and lily pollen oleosin. Complete cDNA fragments encoding these two pollen oil-body proteins were obtained by PCR cloning. Sequence alignment showed that pine pollen caleosin (27 kDa) was highly homologous to pine seed caleosin (28 kDa) except for the lack of an appendix of eight residues at the C-terminus in accord with the 1 kDa difference in their molecular masses. Pine pollen oleosin (15 kDa) was highly homologous to pine seed oleosin-G (14 kDa) except for an insertion of eight residues at the N-terminus in accord with the 1 kDa difference in their molecular masses.

Identification of steroleosin in oil bodies of pine megagametophytes.

Three classes of integral proteins termed oleosin, caleosin and steroleosin have been identified in seed oil bodies of diverse angiosperm species. Recently, two oleosin isoforms and one caleosin were identified in megagametophyte oil bodies of pine (Pinus massoniana), a representative gymnosperm species. In this study, a putative steroleosin of approximately 41 kDa was observed in isolated oil bodies of pine megagametophytes, and its corresponding cDNA fragment was obtained by PCR cloning and further confirmed by mass spectrometric analysis. Phylogenetic tree analysis showed that pine steroleosin was evolutionarily more closely-related to steroleosin-B than steroleosin-A found in angiosperm seed oil bodies. As expected, artificial oil bodies constituted with recombinant steroleosin over-expressed in Escherichia coli were less stable and larger than native pine oil bodies. Filipin staining of artificial oil bodies sheltered by recombinant steroleosin with or without its sterol binding domain showed that the sterol binding domain was responsible for the sterol binding capability of steroleosin. Sterol-coupling dehydrogenase activity was demonstrated in artificial oil bodies constituted with recombinant steroleosin as well as in purified pine oil bodies.

Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella.

Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates.

Nitrogen-deprivation elevates lipid levels in Symbiodinium spp. by lipid droplet accumulation: morphological and compositional analyses.

Stable cnidarian-dinoflagellate (genus Symbiodinium) endosymbioses depend on the regulation of nutrient transport between Symbiodinium populations and their hosts. It has been previously shown that the host cytosol is a nitrogen-deficient environment for the intracellular Symbiodinium and may act to limit growth rates of symbionts during the symbiotic association. This study aimed to investigate the cell proliferation, as well as ultrastructural and lipid compositional changes, in free-living Symbiodinium spp. (clade B) upon nitrogen (N)-deprivation. The cell proliferation of the N-deprived cells decreased significantly. Furthermore, staining with a fluorescent probe, boron dipyrromethane 493/503 (BODIPY 493/503), indicated that lipid contents progressively accumulated in the N-deprived cells. Lipid analyses further showed that both triacylglycerol (TAG) and cholesterol ester (CE) were drastically enriched, with polyunsaturated fatty acids (PUFA; i.e., docosahexaenoic acid, heneicosapentaenoic acid, and oleic acid) became more abundant. Ultrastructural examinations showed that the increase in concentration of these lipid species was due to the accumulation of lipid droplets (LDs), a cellular feature that have previously shown to be pivotal in the maintenance of intact endosymbioses. Integrity of these stable LDs was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Proteomic analyses of these LDs identified proteins putatively involved in lipid metabolism, signaling, stress response and energy metabolism. These results suggest that LDs production may be an adaptive response that enables Symbiodinium to maintain sufficient cellular energy stores for survival under the N-deprived conditions in the host cytoplasm.

Nitrogen deprivation induces lipid droplet accumulation and alters fatty acid metabolism in symbiotic dinoflagellates isolated from Aiptasia pulchella.

The stability of cnidarian-dinoflagellate (genus Symbiodinium spp.) endosymbioses depends on the regulation of nutrient transport between Symbiodinium populations and their hosts. Previously, we successfully induced the production of lipid droplets in the free-living cultured Symbiodinium (clade B) under the nitrogen-deprivation condition for 5 days. Therefore, the present study aimed at understanding the disruption of the endosymbiotic relationship between the cnidarians and dinoflagellates by nitrogen deprivation using Aiptasia pulchella as an example. Transmission electron micrographs revealed the formation of lipid droplets induced by nitrogen deprivation, and the lipid analyses further showed that polyunsaturated fatty acids were drastically enriched in Symbiodinium after 30 days of nitrogen deprivation, although these were unaffected after 5 days of nitrogen starvation. The present study also suggested that the host provided nitrogen to the symbiotic cells during short-term environmental stress. However, the relationship started to deteriorate after 30 days. These findings provide a more detailed understanding of the mechanisms of the symbiotic relationship between the symbiotic dinoflagellates in terms of the nitrogen source, which might provide more information for the explanation of the regulatory mechanism underlying endosymbiotic associations.

Identification of caleosin and two oleosin isoforms in oil bodies of pine megagametophytes.

Numerous oil bodies of 0.2-2 µm occupied approximately 80% of intracellular space in mature pine (Pinus massoniana) megagametophytes. They were stably isolated and found to comprise mostly triacylglycerols as examined by thin layer chromatography analysis and confirmed by both Nile red and BODIPY stainings. Fatty acids released from the triacylglycerols of pine oil bodies were mainly unsaturated, including linoleic acid (60%), adrenic acid (12.3%) and vaccenic acid (9.7%). Proteins extracted from pine oil bodies were subjected to immunological cross-recognition, and the results showed that three proteins of 28, 16 and 14 kDa were detected by antibodies against sesame seed caleosin, sesame oleosin-L and lily pollen oleosin-P, respectively. Complete cDNA fragments encoding these three pine oil-body proteins, tentatively named caleosin, oleosin-L and oleosin-G, were obtained by PCR cloning and further confirmed by mass spectrometric analysis. Consistently, phylogenetic tree analyses showed that pine caleosin was closely-related to the caleosin of cycad megagametophyte among known caleosin sequences. While pine oleosin-L was found clustered with seed oleosin isoforms of angiosperm species, oleosin-G was distinctively grouped with the oleosin-P of lily pollen. The oleosin-G identified in pine megagametophytes seems to represent a new class of seed oleosin isoform evolutionarily close to the pollen oleosin-P.

SLDP: a novel protein related to caleosin is associated with the endosymbiotic Symbiodinium lipid droplets from Euphyllia glabrescens.

Intracellular lipid droplets (LDs) have been proposed to play a key role in the mutualistic endosymbiosis between reef-building corals and the dinoflagellate endosymbiont Symbiodinium spp. This study investigates and identifies LD proteins in Symbiodinium from Euphyllia glabrescens. Discontinuous Percoll gradient centrifugation was used to separate Symbiodinium cells from E. glabrescens tentacles. Furthermore, staining with a fluorescent probe, Nile red, indicated that lipids accumulated in that freshly isolated Symbiodinium cells and lipid analyses further showed polyunsaturated fatty acids (PUFA) was abundant. The stable LDs were purified from endosymbiotic Symbiodinium cells. The structural integrity of the Symbiodinium LDs was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Protein extracts from the purified LDs revealed a major protein band with a molecular weight of 20 kDa, which was termed Symbiodinium lipid droplet protein (SLDP). Interestingly, immunological cross-recognition analysis revealed that SLDP was detected strongly by the anti-sesame and anti-cycad caleosin antibodies. It was suggested that the stable Symbiodinium LDs were sheltered by this unique structural protein and was suggested that SLDP might be homologous to caleosin to a certain extent.