Chloroplast genomic comparison provides insights into the evolution of seagrasses.

BACKGROUND: Seagrasses are a polyphyletic group of monocotyledonous angiosperms that have evolved to live entirely submerged in marine waters. Thus, these species are ideal for studying plant adaptation to marine environments. Herein, we sequenced the chloroplast (cp) genomes of two seagrass species (Zostera muelleri and Halophila ovalis) and performed a comparative analysis of them with 10 previously published seagrasses, resulting in various novel findings. RESULTS: The cp genomes of the seagrasses ranged in size from 143,877 bp (Zostera marina) to 178,261 bp (Thalassia hemprichii), and also varied in size among different families in the following order: Hydrocharitaceae > Cymodoceaceae > Ruppiaceae > Zosteraceae. The length differences between families were mainly related to the expansion and contraction of the IR region. In addition, we screened out 2,751 simple sequence repeats and 1,757 long repeat sequence types in the cp genome sequences of the 12 seagrass species, ultimately finding seven hot spots in coding regions. Interestingly, we found nine genes with positive selection sites, including two ATP subunit genes (atpA and atpF), three ribosome subunit genes (rps4, rps7, and rpl20), one photosystem subunit gene (psbH), and the ycf2, accD, and rbcL genes. These gene regions may have played critical roles in the adaptation of seagrasses to diverse environments. In addition, phylogenetic analysis strongly supported the division of the 12 seagrass species into four previously recognized major clades. Finally, the divergence time of the seagrasses inferred from the cp genome sequences was generally consistent with previous studies. CONCLUSIONS: In this study, we compared chloroplast genomes from 12 seagrass species, covering the main phylogenetic clades. Our findings will provide valuable genetic data for research into the taxonomy, phylogeny, and species evolution of seagrasses.

Molecular phylogenetic species delimitation in the aquatic genus Ottelia (Hydrocharitaceae) reveals cryptic diversity within a widespread species.

Ottelia, a pantropical genus of aquatic plants belonging to the family Hydrocharitaceae, includes several narrowly distributed taxa in Asia. Although the Asian species have received comparatively more research attention than congeners in other areas, various key taxonomic questions remain unaddressed, especially with regards to apparent cryptic diversity within O. alismoides, a widespread species complex native to Asia, northern Australia and tropical Africa. Here we test taxonomic concepts and evaluate species boundaries using a phylogenetic framework. We sampled five of the seven species of Ottelia in Asia as well as each species endemic to Africa and Australia; multiple samples of O. alismoides were obtained from across Asia. Phylogenetic trees based on five plastid DNA markers and the nuclear ITS region shared almost identical topologies. A Bayesian coalescent method of species delimitation using the multi-locus data set discerned one species in Africa, one in Australia and four in Asia with the highest probability. The results lead us to infer that a population sampled in Thailand represents a hitherto unrecognised cryptic taxon within the widespread species complex, although the apparent lack of unambiguous diagnostic characters currently precludes formal description. Conversely, no molecular evidence for distinguishing O. cordata and O. emersa was obtained, and so the latter is synonymised under the former. Two accessions that exhibit inconsistent positions among our phylogenetic trees may represent cases of chloroplast capture, however incomplete lineage sorting or polyploidy are alternative hypotheses that ought to be tested using other molecular markers.

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.

Blue-light-induced rapid chloroplast de-anchoring in Vallisneria epidermal cells.

In the outer periclinal cytoplasm of leaf epidermal cells of an aquatic angiosperm Vallisneria, blue light induces "chloroplast de-anchoring", a rapid decline in the resistance of chloroplasts against centrifugal force. Chloroplast de-anchoring is known induced within 1 min of irradiation with high-fluence-rate blue light specifically, preceding the commencement of chloroplasts migration toward the anticlinal cytoplasm. However, its regulatory mechanism has remained elusive, although pharmacological analysis suggested that a calcium release from intracellular calcium stores is necessary for the response. In search of the responsible photoreceptors, immunoblotting analysis using antibodies against phototropins demonstrated that cross-reactive polypeptides of 120-kDa exist in the plasma-membrane fraction prepared from the leaves. In vitro phosphorylation analysis revealed that 120-kDa polypeptides were phosphorylated by exposure to blue light in a fluence-dependent manner. The blue-light-induced phosphorylation activity was sensitive to a Ser/Thr kinase inhibitor, staurosporine, and unusually was retained at a high level for a long time in darkness. Furthermore, phototropin gene homologs (Vallisneria PHOTOTROPIN1 and PHOTOTROPIN2) expressed in leaves were isolated. We propose that calcium-regulated chloroplast de-anchoring, possibly mediated by phototropins, is an initial process of the blue-light-induced avoidance response of chloroplasts in Vallisneria.

Analysis of the Elodea nuttallii transcriptome in response to mercury and cadmium pollution: development of sensitive tools for rapid ecotoxicological testing.

Toxic metals polluting aquatic ecosystems are taken up by inhabitants and accumulate in the food web, affecting species at all trophic levels. It is therefore important to have good tools to assess the level of risk represented by toxic metals in the environment. Macrophytes are potential organisms for the identification of metal-responsive biomarkers but are still underrepresented in ecotoxicology. In the present study, we used next-generation sequencing to investigate the transcriptomic response of Elodea nuttallii exposed to enhanced concentrations of Hg and Cd. We de novo assembled more than 60 000 contigs, of which we found 170 to be regulated dose-dependently by Hg and 212 by Cd. Functional analysis showed that these genes were notably related to energy and metal homeostasis. Expression analysis using nCounter of a subset of genes showed that the gene expression pattern was able to assess toxic metal exposure in complex environmental samples and was more sensitive than other end points (e.g., bioaccumulation, photosynthesis, etc.). In conclusion, we demonstrate the feasibility of using gene expression signatures for the assessment of environmental contamination, using an organism without previous genetic information. This is of interest to ecotoxicology in a wider sense given the possibility to develop specific and sensitive bioassays.

Redox state and energetic equilibrium determine the magnitude of stress in Hydrilla verticillata upon exposure to arsenate.

Arsenic (As) is a potential hazard to plants' health, however the mechanisms of its toxicity are yet to be properly understood. To determine the impact of redox state and energetic in stress imposition, plants of Hydrilla verticillata (L.f.) Royle, which are known to be potential accumulator of As, were exposed to 100 and 500 µM arsenate (AsV) for 4 to 96 h. Plants demonstrated significant As accumulation with the maximum being at 500 µM after 96 h (568 µg g(-1) dry weight, dw). The accumulation of As led to a significant increase in the level of reactive oxygen species, nitric oxide, carbonyl, malondialdehyde, and percentage of DNA degradation. In addition, the activity of pro-oxidant enzymes like NADPH oxidase and ascorbate oxidase also showed significant increases. These parameters collectively indicated oxidative stress, which in turn caused an increase in percentage of cell death. These negative effects were seemingly linked to an altered energetic and redox equilibrium [analyzed in terms of ATP/ADP, NADH/NAD, NADPH/NADP, reduced glutathione/oxidized glutathione, and ascorbate/dehydroascobate ratios]. Although there was significant increase in the levels of phytochelatins, the As chelating ligands, a large amount of As was presumably present as free ion particularly at 500 µM AsV, which supposedly produced toxic responses. In conclusion, the study demonstrated that the magnitude of disturbance to redox and energetic equilibrium of plants upon AsV exposure determines the extent of toxicity to plants.

Characterization of the NADP malic enzyme gene family in the facultative, single-cell C4 monocot Hydrilla verticillata.

Hydrilla verticillata has a facultative single-cell system that changes from C3 to C4 photosynthesis. A NADP+-dependent malic enzyme (NADP-ME) provides a high [CO2] for Rubisco fixation in the C4 leaf chloroplasts. Of three NADP-ME genes identified, only hvme1 was up-regulated in the C4 leaf, during the light period, and it possessed a putative transit peptide. Unlike obligate C4 species, H. verticillata exhibited only one plastidic isoform that may perform housekeeping functions, but is up-regulated as the photosynthetic decarboxylase. Of the two cytosolic forms, hvme2 and hvme3, the latter exhibited the greatest expression, but was not light-regulated. The mature isoform of hvme1 had a pI of 6.0 and a molecular mass of 64 kD, as did the recombinant rHVME1m, and it formed a tetramer in the chloroplast. The recombinant photosynthetic isoform showed intermediate characteristics between isoforms in terrestrial C3 and C4 species. The catalytic efficiency of rHVME1m was four-fold higher than the cytosolic rHVME3 and two-fold higher than recombinant cytosolic isoforms of rice, but lower than plastidic forms of maize. The Km (malate) of 0.6 mM for rHVME1 was higher than maize plastid isoforms, but four-fold lower than found with rice. A comprehensive phylogenetic analysis of 25 taxa suggested that chloroplastic NADP-ME isoforms arose from four duplication events, and hvme1 was derived from cytosolic hvme3. The chloroplastic eudicot sequences were a monophyletic group derived from a cytosolic clade after the eudicot and monocot lineages separated, while the monocots formed a polyphyletic group. The findings support the hypothesis that a NADP-ME isoform with specific and unusual regulatory properties facilitates the functioning of the single-cell C4 system in H. verticillata.