SeagrassDB: An open-source transcriptomics landscape for phylogenetically profiled seagrasses and aquatic plants.

Seagrasses and aquatic plants are important clades of higher plants, significant for carbon sequestration and marine ecological restoration. They are valuable in the sense that they allow us to understand how plants have developed traits to adapt to high salinity and photosynthetically challenged environments. Here, we present a large-scale phylogenetically profiled transcriptomics repository covering seagrasses and aquatic plants. SeagrassDB encompasses a total of 1,052,262 unigenes with a minimum and maximum contig length of 8,831 bp and 16,705 bp respectively. SeagrassDB provides access to 34,455 transcription factors, 470,568 PFAM domains, 382,528 prosite models and 482,121 InterPro domains across 9 species. SeagrassDB allows for the comparative gene mining using BLAST-based approaches and subsequent unigenes sequence retrieval with associated features such as expression (FPKM values), gene ontologies, functional assignments, family level classification, Interpro domains, KEGG orthology (KO), transcription factors and prosite information. SeagrassDB is available to the scientific community for exploring the functional genic landscape of seagrass and aquatic plants at: http://115.146.91.129/index.php .

Molecular physiology reveals ammonium uptake and related gene expression in the seagrass Zostera muelleri.

Seagrasses are important marine foundation species, which are presently threatened by coastal development and global change worldwide. The molecular mechanisms that drive seagrass responses to anthropogenic stresses, including elevated levels of nutrients such as ammonium, remains poorly understood. Despite the evidence that seagrasses can assimilate ammonium by using glutamine synthetase (GS)/glutamate synthase (glutamine-oxoglutarate amidotransferase or GOGAT) cycle, the regulation of this fundamental metabolic pathway has never been studied at the gene expression level in seagrasses so far. Here, we combine (i) reverse transcription quantitative real-time PCR (RT-qPCR) to measure expression of key genes involved in the GS/GOGAT cycle, and (ii) stable isotope labelling and mass spectrometry to investigate 15N-ammonium assimilation in the widespread Australian species Zostera muelleri subsp. capricorni (Z. muelleri). We demonstrate that exposure to a pulse of ammonium in seawater can induce changes in GS gene expression of Z. muelleri, and further correlate these changes in gene expression with 15N-ammonium uptake rate in above- and below-ground tissue.

Microenvironment and phylogenetic diversity of Prochloron inhabiting the surface of crustose didemnid ascidians.

The cyanobacterium Prochloron didemni is primarily found in symbiotic relationships with various marine hosts such as ascidians and sponges. Prochloron remains to be successfully cultivated outside of its host, which reflects a lack of knowledge of its unique ecophysiological requirements. We investigated the microenvironment and diversity of Prochloron inhabiting the upper, exposed surface of didemnid ascidians, providing the first insights into this microhabitat. The pH and O2 concentration in this Prochloron biofilm changes dynamically with irradiance, where photosynthetic activity measurements showed low light adaptation (Ek ∼ 80 ± 7 µmol photons m(-2) s(-1)) but high light tolerance. Surface Prochloron cells exhibited a different fine structure to Prochloron cells from cloacal cavities in other ascidians, the principle difference being a central area of many vacuoles dissected by single thylakoids in the surface Prochloron. Cyanobacterial 16S rDNA pyro-sequencing of the biofilm community on four ascidians resulted in 433 operational taxonomic units (OTUs) where on average -85% (65-99%) of all sequence reads, represented by 136 OTUs, were identified as Prochloron via blast search. All of the major Prochloron-OTUs clustered into independent, highly supported phylotypes separate from sequences reported for internal Prochloron, suggesting a hitherto unexplored genetic variability among Prochloron colonizing the outer surface of didemnids.

Chlorophyll f-driven photosynthesis in a cavernous cyanobacterium.

Chlorophyll (Chl) f is the most recently discovered chlorophyll and has only been found in cyanobacteria from wet environments. Although its structure and biophysical properties are resolved, the importance of Chl f as an accessory pigment in photosynthesis remains unresolved. We found Chl f in a cyanobacterium enriched from a cavernous environment and report the first example of Chl f-supported oxygenic photosynthesis in cyanobacteria from such habitats. Pigment extraction, hyperspectral microscopy and transmission electron microscopy demonstrated the presence of Chl a and f in unicellular cyanobacteria found in enrichment cultures. Amplicon sequencing indicated that all oxygenic phototrophs were related to KC1, a Chl f-containing cyanobacterium previously isolated from an aquatic environment. Microsensor measurements on aggregates demonstrated oxygenic photosynthesis at 742 nm and less efficient photosynthesis under 768- and 777-nm light probably because of diminished overlap with the absorption spectrum of Chl f and other far-red absorbing pigments. Our findings suggest the importance of Chl f-containing cyanobacteria in terrestrial habitats.

Genome-wide survey of the seagrass Zostera muelleri suggests modification of the ethylene signalling network.

Seagrasses are flowering plants which grow fully submerged in the marine environment. They have evolved a range of adaptations to environmental challenges including light attenuation through water, the physical stress of wave action and tidal currents, high concentrations of salt, oxygen deficiency in marine sediment, and water-borne pollination. Although, seagrasses are a key stone species of the costal ecosystems, many questions regarding seagrass biology and evolution remain unanswered. Genome sequence data for the widespread Australian seagrass species Zostera muelleri were generated and the unassembled data were compared with the annotated genes of five sequenced plant species (Arabidopsis thaliana, Oryza sativa, Phoenix dactylifera, Musa acuminata, and Spirodela polyrhiza). Genes which are conserved between Z. muelleri and the five plant species were identified, together with genes that have been lost in Z. muelleri. The effect of gene loss on biological processes was assessed on the gene ontology classification level. Gene loss in Z. muelleri appears to influence some core biological processes such as ethylene biosynthesis. This study provides a foundation for further studies of seagrass evolution as well as the hormonal regulation of plant growth and development.

Photosynthetic acclimation of Nannochloropsis oculata investigated by multi-wavelength chlorophyll fluorescence analysis.

Multi-wavelength chlorophyll fluorescence analysis was utilised to examine the photosynthetic efficiency of the biofuel-producing alga Nannochloropsis oculata, grown under two light regimes; low (LL) and high (HL) irradiance levels. Wavelength dependency was evident in the functional absorption cross-section of Photosystem II (σII(λ)), absolute electron transfer rates (ETR(II)), and non-photochemical quenching (NPQ) of chlorophyll fluorescence in both HL and LL cells. While σII(λ) was not significantly different between the two growth conditions, HL cells upregulated ETR(II) 1.6-1.8-fold compared to LL cells, most significantly in the wavelength range of 440-540 nm. This indicates preferential utilisation of blue-green light, a highly relevant spectral region for visible light in algal pond conditions. Under these conditions, the HL cells accumulated saturated fatty acids, whereas polyunsaturated fatty acids were more abundant in LL cells. This knowledge is of importance for the use of N. oculata for fatty acid production in the biofuel industry.

The effect of Diel temperature and light cycles on the growth of nannochloropsis oculata in a photobioreactor matrix.

A matrix of photobioreactors integrated with metabolic sensors was used to examine the combined impact of light and temperature variations on the growth and physiology of the biofuel candidate microalgal species Nannochloropsis oculata. The experiments were performed with algal cultures maintained at a constant 20 °C versus a 15 °C to 25 °C diel temperature cycle, where light intensity also followed a diel cycle with a maximum irradiance of 1920 µmol photons m(-2) s(-1). No differences in algal growth (Chlorophyll a) were found between the two environmental regimes; however, the metabolic processes responded differently throughout the day to the change in environmental conditions. The variable temperature treatment resulted in greater damage to photosystem II due to the combined effect of strong light and high temperature. Cellular functions responded differently to conditions before midday as opposed to the afternoon, leading to strong hysteresis in dissolved oxygen concentration, quantum yield of photosystem II and net photosynthesis. Overnight metabolism performed differently, probably as a result of the temperature impact on respiration. Our photobioreactor matrix has produced novel insights into the physiological response of Nannochloropsis oculata to simulated environmental conditions. This information can be used to predict the effectiveness of deploying Nannochloropsis oculata in similar field conditions for commercial biofuel production.

A case of adaptation through a mutation in a tandem duplication during experimental evolution in Escherichia coli.

BACKGROUND: DNA duplications constitute important precursors for genome variation. Here we analyzed an unequal duplication harboring a beneficial mutation that may provide alternative evolutionary outcomes. RESULTS: We characterized this evolutionary event during experimental evolution for only 100 generations of an Escherichia coli strain under glucose limitation within chemostats. By combining Insertion Sequence based Restriction Length Polymorphism experiments, pulsed field gel electrophoresis and two independent genome re-sequencing experiments, we identified an evolved lineage carrying a 180 kb duplication of the 46' region of the E. coli chromosome. This evolved duplication revealed a heterozygous state, with one copy harboring a 2668 bp deletion that included part of the ogrK gene and both the yegR and yegS genes. By genetically manipulating ancestral and evolved strains, we showed that the single yegS inactivation was sufficient to confer a frequency dependent fitness increase under the chemostat selective conditions in both the ancestor and evolved genetic contexts, implying that the duplication itself was not a direct fitness contributor. Nonetheless, the heterozygous duplicated state was relatively stable in the conditions prevailing during evolution in chemostats, in striking contrast to non selective conditions in which the duplication resolved at high frequency into either its ancestral or deleted copy. CONCLUSIONS: Our results suggest that the duplication state may constitute a second order selection process providing higher evolutionary potential. Moreover, its heterozygous nature may provide differential evolutionary opportunities in alternating environments. Our results also highlighted how careful analyses of whole genome data are needed to identify such complex rearrangements.

Formyl group modification of chlorophyll a: a major evolutionary mechanism in oxygenic photosynthesis.

We discuss recent advances in chlorophyll research in the context of chlorophyll evolution and conclude that some derivations of the formyl side chain arrangement of the porphyrin ring from that of the Chl a macrocycle can extend the photosynthetic active radiation (PAR) of these molecules, for example, Chl d and Chl f absorb light in the near-infrared region, up to ∼750 nm. Derivations such as this confer a selective advantage in particular niches and may, therefore, be beneficial for photosynthetic organisms thriving in light environments with particular light signatures, such as red- and near-far-red light-enriched niches. Modelling of formyl side chain substitutions of Chl a revealed yet unidentified but theoretically possible Chls with a distinct shift of light absorption properties when compared to Chl a.

Reactive oxygen production induced by near-infrared radiation in three strains of the Chl d-containing cyanobacterium Acaryochloris marina.

Cyanobacteria in the genus Acaryochloris have largely exchanged Chl a with Chl d, enabling them to harvest near-infrared-radiation (NIR) for oxygenic photosynthesis, a biochemical pathway prone to generate reactive oxygen species (ROS). In this study, ROS production under different light conditions was quantified in three Acaryochloris strains (MBIC11017, HICR111A and the novel strain CRS) using a real-time ethylene detector in conjunction with addition of 2-keto-4-thiomethylbutyric acid, a substrate that is converted to ethylene when reacting with certain types of ROS. In all strains, NIR was found to generate less ROS than visible light (VIS). More ROS was generated if strains MBIC11017 and HICR111A were adapted to NIR and then exposed to VIS, while strain CRS demonstrated the opposite behavior. This is the very first study of ROS generation and suggests that Acaryochloris can avoid a considerable amount of light-induced stress by using NIR instead of VIS for its photosynthesis, adding further evolutionary arguments to their widespread appearance.

The identification of global patterns and unique signatures of proteins across 14 environments using outer membrane proteomics of bacteria.

We test the hypothesis that organisms sourced from different environments exhibit unique fingerprints in macromolecular composition. Experimentally, we followed proteomic changes with 14 different sub-lethal environmental stimuli in Escherichia coli at controlled growth rates. The focus was on the outer membrane sub-proteome, which is known to be extremely sensitive to environmental controls. The analyses surprisingly revealed that pairs of proteins belonging to very different regulons, such as Slp and OmpX or FadL and OmpF, have the closest patterns of change with the 14 conditions. Fe-limited and cold-cultured bacteria have the most distinct global patterns of spot changes, but the patterns with fast growth and oxygen limitation are the closest amongst the 14 environments. These unexpected but statistically robust results suggest that we have an incomplete picture of bacterial regulation across different stress responses; baseline choices and growth-rate influences are probably underestimated factors in such systems-level analysis. In terms of our aim of getting a unique profile for each of the 14 investigated environments, we find that it is unnecessary to compare all the proteins in a proteome and that a panel of five proteins is sufficient for identification of environmental fingerprints. This demonstrates the future feasibility of tracing the history of contaminating bacteria in hospitals, foods or industrial settings as well as for released organisms and biosecurity purposes.

A Novel Epiphytic Chlorophyll d-containing Cyanobacterium Isolated from a Mangrove-associated Red Alga.

A new habitat and a new chlorophyll (Chl) d-containing cyanobacterium belonging to the genus Acaryochloris are reported in this study. Hyperspectral microscopy showed the presence of Chl d-containing microorganisms in epiphytic biofilms on a red alga (Gelidium caulacantheum) colonizing the pneumato-phores of a temperate mangrove (Avicennia marina). The presence of Chl d was further proven by high performance liquid chromatography (HPLC)-based pigment analysis and by confocal imaging of cultured cells. Enrichment of mangrove biofilm samples under near-infrared radiation (NIR) yielded the new Acaryochloris sp. MPGRS1, which was closely related in terms of 16S rRNA gene sequence to an isolate from the hypertrophic Salton Sea, USA. The new isolate used Chl d as its major photopigment; Chl d and Chl a contents were ~98% and 1%-2% of total cellular chlorophyll, respectively. These findings expand the variety of ecological niches known to harbor Chl d-containing cyanobacteria and support our working hypothesis that such oxyphototrophs may be ubiquitous in habitats depleted of visible light, but with sufficient NIR exposure.

A red-shifted chlorophyll.

Chlorophylls are essential for light-harvesting and energy transduction in photosynthesis. Four chemically distinct varieties have been known for the past 60 years. Here we report isolation of a fifth, which we designate chlorophyll f. Its in vitro absorption (706 nanometers) and fluorescence (722 nanometers) maxima are red-shifted compared to all other chlorophylls from oxygenic phototrophs. On the basis of the optical, mass, and nuclear magnetic resonance spectra, we propose that chlorophyll f is [2-formyl]-chlorophyll a (C55H70O6N4Mg). This finding suggests that oxygenic photosynthesis can be extended further into the infrared region and may open associated bioenergy applications.

18O labeling of chlorophyll d in Acaryochloris marina reveals that chlorophyll a and molecular oxygen are precursors.

The cyanobacterium Acaryochloris marina was cultured in the presence of either H(2)(18)O or (18)O(2), and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H(2)(18)O, newly synthesized Chl a and d, both incorporated up to four isotopic (18)O atoms. Time course H(2)(18)O labeling experiments showed incorporation of isotopic (18)O atoms originating from H(2)(18)O into Chl a, with over 90% of Chl a (18)O-labeled at 48 h. The incorporation of isotopic (18)O atoms into Chl d upon incubation in H(2)(18)O was slower compared with Chl a with approximately 50% (18)O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of (18)O(2) gas, one isotopic (18)O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H(2)(18)O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic (18)O atoms derived from molecular oxygen ((18)O(2)) was observed in the extracted Chl d, and the percentage of double isotopic (18)O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3(1)-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.

A new chlorophyll d-containing cyanobacterium: evidence for niche adaptation in the genus Acaryochloris.

Chlorophyll d is a photosynthetic pigment that, based on chemical analyses, has only recently been recognized to be widespread in oceanic and lacustrine environments. However, the diversity of organisms harbouring this pigment is not known. Until now, the unicellular cyanobacterium Acaryochloris marina is the only characterized organism that uses chlorophyll d as a major photopigment. In this study we describe a new cyanobacterium possessing a high amount of chlorophyll d, which was isolated from waters around Heron Island, Great Barrier Reef (23° 26' 31.2″ S, 151° 54' 50.4″ E). The 16S ribosomal RNA is 2% divergent from the two previously described isolates of A. marina, which were isolated from waters around the Palau islands (Pacific Ocean) and the Salton Sea lake (California), suggesting that it belongs to a different clade within the genus Acaryochloris. An overview sequence analysis of its genome based on Illumina technology yielded 871 contigs with an accumulated length of 8 371 965 nt. Their analysis revealed typical features associated with Acaryochloris, such as an extended gene family for chlorophyll-binding proteins. However, compared with A. marina MBIC11017, distinct genetic, morphological and physiological differences were observed. Light saturation is reached at lower light intensities, Chl d/a ratios are less variable with light intensity and the phycobiliprotein phycocyanin is lacking, suggesting that cyanobacteria of the genus Acaryochloris occur in distinct ecotypes. These data characterize Acaryochloris as a niche-adapted cyanobacterium and show that more rigorous attempts are worthwhile to isolate, cultivate and analyse chlorophyll d-containing cyanobacteria for understanding the ecophysiology of these organisms.

Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana.

Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes.

Chromatic photoacclimation extends utilisable photosynthetically active radiation in the chlorophyll d-containing cyanobacterium, Acaryochloris marina.

Chromatic photoacclimation and photosynthesis were examined in two strains of Acaryochloris marina (MBIC11017 and CCMEE5410) and in Synechococcus PCC7942. Acaryochloris contains Chl d, which has an absorption peak at ca 710 nm in vivo. Cultures were grown in one of the three wavelengths (525 nm, 625 nm and 720 nm) of light from narrow-band photodiodes to determine the effects on pigment composition, growth rate and photosynthesis: no growth occurred in 525 nm light. Synechococcus did not grow in 720 nm light because Chl a does not absorb effectively at this long wavelength. Acaryochloris did grow in 720 nm light, although strain MBIC11017 showed a decrease in phycobilins over time. Both Synechococcus and Acaryochloris MBIC11017 showed a dramatic increase in phycobilin content when grown in 625 nm light. Acaryochloris CCMEE5410, which lacks phycobilins, would not grow satisfactorily under 625 nm light. The cells adjusted their pigment composition in response to the light spectral conditions under which they were grown. Photoacclimation and the Q (y) peak of Chl d could be understood in terms of the ecological niche of Acaryochloris, i.e. habitats enriched in near infrared radiation.

Gene expression profiling of single epidermal, basal and trichome cells of Arabidopsis thaliana.

Samples of single epidermal, basal and trichome cells were collected by glass microcapillaries from 7-week-old Arabidopsis thaliana leaves. Transcript amplification of these single-cell samples was performed by RT PCR. For gene expression profiling, we hybridized the amplified transcriptome of each individual cell type to nylon membranes spotted with 16,000 Arabidopsis expressed sequence tags (ESTs). Initial analysis of the array filter data enabled us to functionally categorize transcripts that were present in each individual cell type. In order to confirm the filter array data, we used RT PCR. Results of this RT PCR approach confirmed the presence of 12 selected candidate genes in agreement with array filter hybridization data. Further, transcripts involved in detoxification and sulfur metabolism could be identified in epidermal cell extracts. Together, the results of our study provide the localization of approximately 1000 expressed genes to either pavement, basal or trichome cells. To cluster transcripts with similar expression levels, we developed a novel mathematical algorithm. Based on the mean and standard deviation, ratios of expression levels of a transcript were defined for pairs of the three cell types. This numerical analysis enabled subdivision into 67 categories of genes differentially expressed in epidermal, basal and trichome cells. Transcripts in each category displayed similar ratios of expression levels in the three cell types. Examples of these clusters are presented and discussed in Appendix A.