Comparative Transcriptomics and Metabolomics Analyses of Avicennia marina and Kandelia obovata under Chilling Stress during Seedling Stage.

One of the most productive ecosystems in the world, mangroves are susceptible to cold stress. However, there is currently insufficient knowledge of the adaptation mechanisms of mangrove plants in response to chilling stress. This study conducted a comparative analysis of transcriptomics and metabolomics to investigate the adaptive responses of Kandelia obovata (chilling-tolerant) and Avicennia marina (chilling-sensitive) to 5 °C. The transcriptomics results revealed that differentially expressed genes (DEGs) were mostly enriched in signal transduction, photosynthesis-related pathways, and phenylpropanoid biosynthesis. The expression pattern of genes involved in photosynthesis-related pathways in A. marina presented a downregulation of most DEGs, which correlated with the decrease in total chlorophyll content. In the susceptible A. marina, all DEGs encoding mitogen-activated protein kinase were upregulated. Phenylpropanoid-related genes were observed to be highly induced in K. obovata. Additionally, several metabolites, such as 4-aminobutyric acid, exhibited higher levels in K. obovata than in A. marina, suggesting that chilling-tolerant varieties regulated more metabolites in response to chilling. The investigation defined the inherent distinctions between K. obovata and A. marina in terms of signal transduction gene expression, as well as phenylpropanoid and flavonoid biosynthesis, during exposure to low temperatures.

The Kandelia obovata transcription factor KoWRKY40 enhances cold tolerance in transgenic Arabidopsis.

BACKGROUND: WRKY transcription factors play key roles in plant development processes and stress response. Kandelia obovata is the most cold-resistant species of mangrove plants, which are the important contributors to coastal marine environment. However, there is little known about the WRKY genes in K. obovata. RESULTS: In this study, a WRKY transcription factor gene, named KoWRKY40, was identified from mangrove plant K. obovata. The full-length cDNA of KoWRKY40 gene was 1420 nucleotide bases, which encoded 318 amino acids. The KoWRKY40 protein contained a typical WRKY domain and a C2H2 zinc-finger motif, which were common signatures to group II of WRKY family. The three-dimensional (3D) model of KoWRKY40 was formed by one α-helix and five ß-strands. Evolutionary analysis revealed that KoWRKY40 has the closest homology with a WRKY protein from another mangrove plant Bruguiera gymnorhiza. The KoWRKY40 protein was verified to be exclusively located in nucleus of tobacco epidermis cells. Gene expression analysis demonstrated that KoWRKY40 was induced highly in the roots and leaves, but lowly in stems in K. obovata under cold stress. Overexpression of KoWRKY40 in Arabidopsis significantly enhanced the fresh weight, root length, and lateral root number of the transgenic lines under cold stress. KoWRKY40 transgenic Arabidopsis exhibited higher proline content, SOD, POD, and CAT activities, and lower MDA content, and H2O2 content than wild-type Arabidopsis under cold stress condition. Cold stress affected the expression of genes related to proline biosynthesis, antioxidant system, and the ICE-CBF-COR signaling pathway, including AtP5CS1, AtPRODH1, AtMnSOD, AtPOD, AtCAT1, AtCBF1, AtCBF2, AtICE1, AtCOR47 in KoWRKY40 transgenic Arabidopsis plants. CONCLUSION: These results demonstrated that KoWRKY40 conferred cold tolerance in transgenic Arabidopsis by regulating plant growth, osmotic balance, the antioxidant system, and ICE-CBF-COR signaling pathway. The study indicates that KoWRKY40 is an important regulator involved in the cold stress response in plants.

Cloning and characterization of KoOsmotin from mangrove plant Kandelia obovata under cold stress.

BACKGROUND: Low temperature is a major abiotic stress that seriously limits mangrove productivity and distribution. Kandelia obovata is the most cold-resistance specie in mangrove plants, but little is known about the molecular mechanism underlying its resistance to cold. Osmotin is a key protein associated with abiotic and biotic stress response in plants but no information about this gene in K. obovata was reported. RESULTS: In this study, a cDNA sequence encoding osmotin, KoOsmotin (GenBank accession no. KP267758), was cloned from mangrove plant K. obovata. The KoOsmotin protein was composed of 221 amino acids and showed a calculated molecular mass of 24.11 kDa with pI 4.92. The KoOsmotin contained sixteen cysteine residues and an N-terminal signal peptide, which were common signatures to most osmotins and pathogenesis-related 5 proteins. The three-dimensional (3D) model of KoOsmotin, contained one α-helix and eleven ß-strands, was formed by three characteristic domains. Database comparisons of the KoOsmotin showed the closest identity (55.75%) with the osmotin 34 from Theobroma cacao. The phylogenetic tree also revealed that the KoOsmotin was clustered in the branch of osmotin/OLP (osmotin-like protien). The KoOsmotin protein was proved to be localized to both the plasma membrane and cytoplasm by the subcellular localization analysis. Gene expression showed that the KoOsmotin was induced primarily and highly in the leaves of K. obovata, but less abundantly in stems and roots. The overexpressing of KoOsmotin conferred cold tolerance in Escherichia coli cells. CONCLUSION: As we known, this is the first study to explore the osmotin of K. obovata. Our study provided valuable clues for further exploring the function of KoOsmotin response to stress.

Fluorene degradation by Rhodococcus sp. A2-3 isolated from hydrocarbon contaminated sediment of the Pearl River estuary, China.

The pollution of polycyclic aromatic hydrocarbons was serious in sediments of the Pearl River estuary, China. A fluorene-degrading bacterium, strain A2-3, was isolated from hydrocarbon contaminated sediment of this estuary and identified as Rhodococcus sp. based on the analyses of 16S rRNA gene sequence and morphology. Rhodococcus sp. A2-3 can take naphthalene, p-Teropheny, fluorene, pyrene, salicylic acid, citric acid, acetic acid, diethyletheranhydrous, methanol or 4,4'-dibromodiphenyl ether as sole carbon source. 100% of 100 mg/L fluorene or 89% of 400 mg/L fluorene was removed in 7 days by strain A2-3 at 30 °C and pH 7.5. The strain A2-3 showed a high degradation efficiency of fluorene when pH values ranged from 5.5 to 8.5. The proposed pathway of fluorene catabolism by strain A2-3 was initially attacked by 3,4 dioxygenation. Our results suggested Rhodococcus sp. A2-3 can degrade PAHs under aerobic conditions and can function in bioremediation, particularly for weakly acid environment.

Comparative physiological and proteomic analyses of mangrove plant Kandelia obovata under cold stress.

Cold events had broadly affected the survival and geographic distribution of mangrove plants. Kandelia obovata, has an excellent cold tolerance as a true halophyte and widespread mangrove species. In this study, physiological characters and comparative proteomics of leaves of K. obovata were performed under cold treatment. The physiological analysis showed that K. obovata could alleviate its cold-stress injuries through increasing the levels of antioxidants, the activities of related enzymes, as well as osmotic regulation substances (proline). It was detected 184 differentially expressed protein spots, and of 129 (70.11%) spots were identified. These proteins have been involved in several pathways such as the stress and defense, photosynthesis and photorespiration, signal transduction, transcription factors, protein biosynthesis and degradation, molecular chaperones, ATP synthesis, the tricarboxylic acid (TCA) cycle and primary metabolisms. The protein post-translational modification may be a common phenomenon and plays a key role in cold-response process in K. obovata. According to our precious work, a schematic diagram was drawn for the resistance or adaptation strategy of mangrove plants under cold stress. This study provided valuable information to understand the mechanism of cold tolerance of K. obovata.

Assessing ecological health of mangrove ecosystems along South China Coast by the pressure-state-response (PSR) model.

The pressure-state-response (PSR) model was applied to establish a mangrove ecosystem health evaluation system combined with analytical hierarchy process (AHP) in this paper. The mangrove wetlands are divided into five ecological levels: excellent health, good health, health, sub-health and morbidity, which is based on the comprehensive health index (CHI) value. Twelve representative sites were selected for sampling to assess the ecological health condition of mangroves. As a result, the ecological health level of Gaoqiao mangrove area is excellent health; the ecological health level of Taiping mangrove area is good health; the ecological health level of Huguang and Qi'ao mangrove area is health; the ecological health level of Techeng and He'an mangrove area is sub-health; the ecological health level of Huidong mangrove area is morbidity. These results will give some advises for ecological protection and biological resource sustainable development of mangrove ecosystem in China.

Bacterial community variations in the South China Sea driven by different chemical conditions.

In this study, Illumina MiSeq sequencing of the 16 S rRNA gene was used to describe the bacterial communities in the South China Sea (SCS) during the southwest monsoon period. We targeted different regions in the SCS and showed that bacterial community was driven by the effects of the river, upwelling, and mesoscale eddy through changing the environmental factors (salinity, temperature, and nutrients). Distinct bacterial communities were observed among different chemical conditions, especially between the estuary and the open sea. The abundance of Burkholderiales, Frankiales, Flavobacteriales, and Rhodobacterales dominated the estuary and its adjacent waters. Bacteria in cyclonic eddy were dominated by Methylophilales and Pseudomonadales, whereas Prochlorococcus, SAR11 clade, and Oceanospirillales had relatively high abundance in the anticyclonic eddy. Overall, the abundance of specific phylotypes significantly varied among samples with different chemical conditions. Chemical conditions probably act as a driver that shapes and controls the diversity of bacteria in the SCS. This study suggests that the interaction between microbial and environmental conditions needs to be further considered to fully understand the diversity and function of marine microbes.

Effect of mangrove restoration on sediment properties and bacterial community.

Mangrove reconstruction is an efficient approach for mangrove conservation and restoration. The present study aimed to explore the effects of mangrove reconstruction on sediment properties and bacterial community. The results showed that mangrove restoration greatly promoted sediment fertility, whereas the improvements were more obvious induced by Kandelia obovata when compared to Avicennia marina. In all the samples, the dominant top5 bacterial group were Proteobacteria (48.31-54.52%), Planctomycetes (5.98-8.48%), Bacteroidetes (4.49-11.14%) and Acidobacteria (5.69-8.16%). As for the differences among the groups, the relative abundance of Chloroflexi was higher in the sediments of K. obovata, while Bacteroidetes was more abundant in A. marina group. Furthermore, the two bacterial genera (Rhodoplanes and Novosphingobium) were more dominant in the sediments of K. obovata, while the sediments of A. marina contained higher abundance of Vibrio and Marinobacterium. Besides, bacterial community was highly correlated with mangrove species and sediment property and nutrient status. The results of this study would provide a better understanding of the ecological benefits of mangroves and highlighted the information on biogeochemical processes driven by mangrove restoration and microorganisms.

Proline metabolism and molecular cloning of AmP5CS in the mangrove Avicennia marina under heat stress.

Proline is one of the most important compatible osmolyte in cells, which accumulates in response to various stresses, including salt, water deficit, heavy metal, pathogen infection and extreme temperature. In this study, a growth chamber was employed to simulate heat environment for Avicennia marina seedlings. We detected some physiological indices in the leaves of A. marina at 40 °C, including the activity of delta-1-pyrroline-5-carboxylate synthase (P5CS), the content of free proline and soluble protein, transpiration rate and membrane permeability, and discussed the relationship between these five indices and heat resistant ability. And then a P5CS gene was cloned from A. marina using homologous cloning and rapid amplification of cDNA ends methods. It was designated as AmP5CS, encoding a protein that contained a feedback inhibition site of proline, proA, proB, conserved Leu zipper, GSA-DH domain and other functional domains of P5CS protein in high plants. Expression analysis of AmP5CS gene indicated it was involved in heat stress response. It is the first time that P5CS from A. marina has been cloned and the findings laid the foundation of figuring out heat resistant mechanisms and relieving heat damage, which is significant during global warming.

Modeling the ecosystem response of the semi-closed Daya Bay to the thermal discharge from two nearby nuclear power plants.

A two-dimensional flow model coupled with a nutrient, phytoplankton, zooplankton, and detritus (NPZD) ecosystem model was applied to simulate the thermal discharge of two nuclear power plants near Daya Bay of South China Sea, and their impact on hydrodynamic conditions and ecosystem. The results show that the thermal discharge influence area of neap tide is much larger than spring tide, and the high and mid temperature rise area in winter is much larger than that in summer. More importantly, the present data further confirmed that the Daya Bay ecosystem has significant responses to the thermal discharge in nutrients, phytoplankton and zooplankton. In winter and early spring, the thermal discharge facilitates the growth of phytoplankton and their abundance often peak in March and April. In summer, the thermal discharge inhibits the growth of phytoplankton and their abundance keep at a low level from June to August. Although the abundance of zooplankton changed with phytoplankton, the characteristic of seasonal variation of zooplankton do not coincide with the phytoplankton, but are lagged in time, by nearly one month. Moreover, the concentration of nutrients and chlorophyll a were compared between thermal discharge and the nearby aquaculture, which has shown that the aquaculture contributed more to the eutrophication.

Molecular cloning and expression of AmCDPK from mangrove Avicennia marina under elevated temperature.

Considered as an essential calcium sensor, the calcium-dependent protein kinase (CDPK) family plays a critical part in terrestrial plants' responses to both biotic and abiotic stresses. In the study, Avicennia marina was proved to have better heat tolerance than other species. A CDPK gene was cloned from mangrove species A. marina using RACE-PCR and designated as AmCDPK. By predicting and analyzing its properties, structures and expression patterns, we found that the amino acid sequence, containing a kinase domain and four EF-hand Ca2+-binding sites, shared high identity with Handroanthus impetiginosus and Sesamum indicum. Quantitative real-time PCR data analysis suggested that AmCDPK demonstrated significant up-regulation under heat stress. It is likely that AmCDPK is a versatile gene involved in various stresses, including dehydration, cold, light, defense and ABA stress responses by analyzing cis-elements. It is the first time that CDPKs from mangroves have been cloned and our results brought evidence to the effect of AmCDPK on heat stress, which is particularly important under the background of global warming.

Marine coastal ecosystem and ecotoxicology.

Environmental pollution from industrialization, urbanization and agriculture is a serious issue facing all countries during economic development. The Pearl River Delta in Southern China offers a unique case for study and documentation on the ecosystem changes during the fast economic development and urbanization over the past 40 years. This collection of papers from a conference on marine coastal pollution and ecotoxicology held in Guangzhou of China provide the current status on the environmental and ecological processes from the intensive urbanization and development. The research information available from this special issue can be used for both a comparison over time and with other locations and countries, and, in addition, for policy making to serve the long-term sustainable development of the region and the world.

Phytoplankton diversity and community responses to physicochemical variables in mangrove zones of Guangzhou Province, China.

The phytoplankton diversity and community response to physicochemical variables in mangrove zones of Guangdong Province along the South China coast was investigated from October to December, 2017. This study was set to investigate the phytoplankton community structure in the mangrove zone and assess the relationship between the physicochemical variables and phytoplankton species diversity. Physicochemical variables such as water temperature, total dissolve solids (tds), pH, salinity, turbidity, electrical conductivity (EC) and nutrient salts were measured in situ across the 27 stations. A total of 451 species of phytoplankton were identified belonging to 10 groups (Bacillariophta > Cyanophyta > Chlorophyta > Euglenophyta > Dinoflagellate > Eubacteria > Ochrophyta > Crytophyta > Rhodophyta > Charophyta) and quantified to constitute a standing crop of 7.11 × 108 cells dm-3. The principal component analysis (PCA) reveals that reactive nitrate, phosphate, electrical conductive (EC) and turbidity were the best abiotic factors that controlled the phytoplankton community structure in the area. However, Cannon Corresponding Analysis and Pearson correlation have explicitly revealed the impact of reactive nitrate, phosphate, EC and turbidity on the phytoplankton community structure. For instance, the CCA ordination revealed that species richness and evenness were positively influenced by reactive nitrate but negatively affected by EC, turbidity and water temperature. Diatoms were mostly controlled by total dissolved solids (tds) and salinity, whereas Euglena, cyanobacteria and green algae were impacted EC and turbidity, apart from the general contribution of the nutrient salts as delineated by CCA ordination. The Shannon diversity index value exposed different levels of organic pollution across the mangrove zone of which GD37 was the most impacted station.

Isolation and expression analysis of two novel C-repeat binding factor (CBF) genes involved in plant growth and abiotic stress response in mangrove Kandelia obovata.

Kandelia obovata is one of the cold tolerant mangrove plants along the China coast. To reveal the cold tolerant mechanism of K. obovata, the present work isolated two CBF/DREB1 genes (designated KoCBF1 and KoCBF3) from cold-stressed K. obovata and characterized their expression profiles in various organs and in response to multiple abiotic stresses. The deduced proteins of KoCBF1 and 3 all contain specific features of CBFs, and show high similarity to AmCBF1 and 3 from Avicennia marina, respectively. Different expression patterns of the two CBF orthologous under various abiotic stresses and exogenous hormone suggested that they may have different regulators and be involved in different regulatory pathway. The high basal and cold induced expression of the two genes indicated that they may all play important roles in growth and cold resistance of plants. The significant induction of KoCBF3 after salt and lead (Pb2+) treatments suggested that this CBF gene may also participate in response to salinity and heavy metal stresses. This study will provide a better understanding of CBF-regulated stress-resistant mechanism, which may be benefit in mangrove biotechnological breeding, high-latitude transplanting, and bioremediation of heavy metal pollutions.

Nitrogen dynamics in the mangrove sediments affected by crabs in the intertidal regions.

Crab is an important benthonic animal in mangrove ecosystem, however, the potential function of crabs on nitrogen (N) transformation in mangrove ecosystems is still poorly understood. The present study aimed to explore the potential effect of crab burrows on nitrification/denitrification within the sediments. The results showed that the presence of crab burrows could directly promote soil nitrification, the regions within more crab burrows appeared to possess higher nitrification. Higher AOA and AOB gene copies were also observed in the sediments surrounding crab burrows than those in the sediments without crab burrow. On the contrary, lower nirS copies, a denitrification related gene, were detected in the sediments surrounding crab burrows. In summary, the present study proposed new evidences of nitrification enhancement deriving by crabs, the presence of crabs might be significant in alleviating nitrification inhibition and benefits the growth of mangroves under tidal flooding.

Distribution and risk of mercury in the sediments of mangroves along South China Coast.

The importance of mangrove was widely reported. However, the potential risks of pollutants (e.g., Hg) accumulated in the mangroves are often ignored. Thus, the present study aimed to explore the distribution and risk of mercury (Hg) in the sediments of mangroves along South China Coast. Results showed that concentrations of total Hg ranged from 0.0815 to 0.6377 mg/kg, with an arithmetic mean value of 0.2503 mg/kg. The contamination index (Pi) showed mild pollution toxicity risks in NS, slight toxicity risks in DZG, QZ, SY, ND, GQ, TLG, and free pollutions in BMW, SJ, ZJK and BLHK. NS, DZG and SY scored the highest values of Igeo among the eleven mangrove regions studied, indicating moderate to heavy pollution inputs in these regions. As for the distribution of Hg in the sediments along tidal gradient, concentrations of Hg in the sediments sharply increased from seaward mudflat to landward mangrove, corresponding with the increases of TOC. In summary, the present data indicated that mangrove ecosystem is efficient in Hg reservoir. However, the potential ecological risks of Hg, especially in some mangrove regions easily affected by human activities, should be noted.

Isolation and expression analysis of a CBF transcriptional factor gene from the mangrove Bruguiera gymnorrhiza.

The present work isolated a CBF/DREB1 gene from mangrove Bruguiera gymnorrhiza (BgCBF1) and compared its expression levels in various tissues under normal condition and cold stress, and in leaves exposed to various environmental stimuli. Results showed that the BgCBF1 deduced protein showed almost 100% similarities to that of AcCBF1 from Aegiceras corniculatum and AmCBF1 from Avicennia marina. Real-time quantitative PCR analysis showed that BgCBF1 gene displayed constitute expression in leaf, stem and root samples of plantlets under normal condition, but with different expression levels and tissue preference. When exposed to cold, BgCBF1 could be rapidly, slightly and transiently induced in all tissues. Furthermore, the BgCBF1 gene in leaves displayed a transient and small induction after salt and drought (PEG) exposure, while exhibited relatively high up-regulated expression after the phytohormone abscisic acid (ABA) treatment. These results suggest that the BgCBF1 gene may participate in the ABA mediated development and protection of plant against cold and drought. Further studies on its promoters and downstream genes will be needed to better understand its functions.

Salt tolerance and exclusion in the mangrove plant Avicennia marina in relation to root apoplastic barriers.

Salt tolerance and the possible functions of suberization on salt exclusion and secretion were examined in a dominant mangrove plant, Avicennia marina. The results showed that low salinities (10‰ and 20‰) almost has no negative effect on A. marina, however significant growth inhibitions were observed in the seedlings grown in higher salinities (30‰ and 40‰). With the increases of salinity, increased tissue Na+ content and enhanced salt secretion by glands were observed. Obvious suberization thickening were detected both in the exodermis and endodermis of the roots after salt pretreatment when compared to the roots without salt treatment. More importantly, the present data further confirmed that these root apoplastic barriers would directly decrease Na+ loading into xylem. Higher salt tolerance was observed in the seedlings pre-cultivated by salty tide when compared to fresh water cultivated A. marina. In summary, this study suggests a barrier property of suberization in dealing with salt exclusion in mangroves, a moderate salt pre-treatment may benefit plant withstanding high salinity.

Dynamics of radial oxygen loss in mangroves subjected to waterlogging.

Tidal flooding can directly result in oxygen (O2) shortage, however the functions of root aeration in flooding tolerance and O2 dynamics within mangroves are still poorly understood. Thus, in this study, the correlations among waterlogging tolerance, root porosity and O2 movement within the plants were investigated using two mangrove species (Aegiceras corniculatum and Bruguiera gymnorrhiza) and a semi-mangrove Heritiera littoralis. Based on the present data, the species A. corniculatum and B. gymnorrhiza, which possessed higher root porosity, exhibited higher waterlogging tolerance, while H. littoralis is intolerant. Increased root porosity, leaf stoma, and total ROL were observed in the roots of A. corniculatum and B. gymnorrhiza growing in stagnant solution when compared to respective aerated controls. As for ROL spatial pattern along roots, external anaerobic condition could promote ROL from apical root regions but reduce ROL from basal roots, leading to a 'tighter barrier'. In summary, the present study indicated that the plants (e.g., A. corniculatum and B. gymnorrhiza) prioritized to ensure O2 diffusion towards root tips under waterlogging by increasing aerenchyma formation and reducing O2 leakage at basal root regions.

Mixture of Pb, Zn and Cu on root permeability and radial oxygen loss in the mangrove Bruguiera gymnorrhiza.

A short term pot trail was employed to evaluate the exposure of mixed heavy metals (Cu, Pb and Zn) on growth, radial oxygen loss (ROL) and root anatomy in Bruguiera gymnorrhiza. The possible function of BgC4H, a cytochrome P450 gene, on root lignification was also discussed. The exposures of mixed Cu, Pb and Zn directly reduce O2 leakage at root surface. The reduced ROL inhibited by heavy metals was mainly ascribed by the changes in root anatomical features, such as decreased root porosity together with increased lignification within the exodermis. BgC4H was found to be up-regulated after 0.5-day metal exposure, and remained higher transcript levels within 3-day metal exposure when compared to control roots. Besides, the inhibited photosynthesis may also result in less oxygen can be transported to the underground roots. In summary, the mangrove B. gymnorrhiza appeared to react to external mixed metal contaminants by developing a lignified and impermeable exodermis, and such a root barrier induced by mixed Cu, Pb and Zn appeared to be an adaptive response to block metal ions enters into the roots.