NADPH oxidase-dependent H2O2 production mediates salicylic acid-induced salt tolerance in mangrove plant Kandelia obovata by regulating Na+/K+ and redox homeostasis.

Salicylic acid (SA) is known to enhance salt tolerance in plants. However, the mechanism of SA-mediated response to high salinity in halophyte remains unclear. Using electrophysiological and molecular biological methods, we investigated the role of SA in response to high salinity in mangrove species, Kandelia obovata, a typical halophyte. Exposure of K. obovata roots to high salinity resulted in a rapid increase in endogenous SA produced by phenylalanine ammonia lyase pathway. The application of exogenous SA improved the salt tolerance of K. obovata, which depended on the NADPH oxidase-mediated H2O2. Exogenous SA and H2O2 increased Na+ efflux and reduced K+ loss by regulating the transcription levels of Na+ and K+ transport-related genes, thus reducing the Na+/K+ ratio in the salt-treated K. obovata roots. In addition, exogenous SA-enhanced antioxidant enzyme activity and its transcripts, and the expressions of four genes related to AsA-GSH cycle as well, then alleviated oxidative damages in the salt-treated K. obovata roots. However, the above effects of SA could be reversed by diphenyleneiodonium chloride (the NADPH oxidase inhibitor) and paclobutrazol (a SA biosynthesis inhibitor). Collectively, our results demonstrated that SA-induced salt tolerance of K. obovata depends on NADPH oxidase-generated H2O2 that affects Na+/K+ and redox homeostasis in response to high salinity.

Trehalose along with ABA promotes the salt tolerance of Avicennia marina by regulating Na+ transport.

Mangroves grow in tropical/subtropical intertidal habitats with extremely high salt tolerance. Trehalose and trehalose-6-phosphate (T6P) have an alleviating function against abiotic stress. However, the roles of trehalose in the salt tolerance of salt-secreting mangrove Avicennia marina is not documented. Here, we found that trehalose was significantly accumulated in A. marina under salt treatment. Furthermore, exogenous trehalose can enhance salt tolerance by promoting the Na+ efflux from leaf salt gland and root to reduce the Na+ content in root and leaf. Subsequently, eighteen trehalose-6-phosphate synthase (AmTPS) and 11 trehalose-6-phosphate phosphatase (AmTPP) genes were identified from A. marina genome. Abscisic acid (ABA) responsive elements were predicted in AmTPS and AmTPP promoters by cis-acting elements analysis. We further identified AmTPS9A, as an important positive regulator, that increased the salt tolerance of AmTPS9A-overexpressing Arabidopsis thaliana by altering the expressions of ion transport genes and mediating Na+ efflux from the roots of transgenic A. thaliana under NaCl treatments. In addition, we also found that ABA could promote the accumulation of trehalose, and the application of exogenous trehalose significantly promoted the biosynthesis of ABA in both roots and leaves of A. marina. Ultimately, we confirmed that AmABF2 directly binds to the AmTPS9A promoter in vitro and in vivo. Taken together, we speculated that there was a positive feedback loop between trehalose and ABA in regulating the salt tolerance of A. marina. These findings provide new understanding to the salt tolerance of A. marina in adapting to high saline environment at trehalose and ABA aspects.

Brassinosteroid enhances salt tolerance via S-nitrosoglutathione reductase and nitric oxide signaling pathway in mangrove Kandelia obovata.

Brassinosteroid (BR) has been shown to modulate plant tolerance to various stresses. S-nitrosoglutathione reductase (GSNOR) is involved in the plant response to environment stress by fine-turning the level of nitric oxide (NO). However, whether GSNOR is involved in BR-regulated Na+ /K+ homeostasis to improve the salt tolerance in halophyte is unknown. Here, we firstly reported that high salinity increases the expression of BR-biosynthesis genes and the endogenous levels of BR in mangrove Kandelia obovata. Then, salt-induced BR triggers the activities and gene expressions of GSNOR and antioxidant enzymes, thereafter decrease the levels of malondialdehyde, hydrogen peroxide. Subsequently, BR-mediated GSNOR negatively regulates NO contributions to the reduction of reactive oxygen species generation and induction of the gene expression related to Na+ and K+ transport, leading to the decrease of Na+ /K+ ratio in the roots of K. obovata. Finally, the applications of exogenous BR, NO scavenger, BR biosynthetic inhibitor and GSNOR inhibitor further confirm the function of BR. Taken together, our result provides insight into the mechanism of BR in the response of mangrove K. obovata to high salinity via GSNOR and NO signaling pathway by reducing oxidative damage and modulating Na+ /K+ homeostasis.

Genome-wide identification of Avicennia marina aquaporins reveals their role in adaptation to intertidal habitats and their relevance to salt secretion and vivipary.

Aquaporins (AQPs) regulate the transport of water and other substrates, aiding plants in adapting to stressful environments. However, the knowledge of AQPs in salt-secreting and viviparous Avicennia marina is limited. In this study, 46 AmAQPs were identified in A. marina genome, and their subcellular localisation and function in transporting H2 O2 and boron were assessed through bioinformatics analysis and yeast transformation. Through analysing their expression patterns via RNAseq and real-time quantitative polymerase chain reaction, we found that most AmAQPs were downregulated in response to salt and tidal flooding. AmPIP (1;1, 1;7, 2;8, 2;9) and AmTIP (1;5, 1;6) as salt-tolerant candidate genes may contribute to salt secretion together with Na+ /H+ antiporters. AmPIP2;1 and AmTIP1;5 were upregulated during tidal flooding and may be regulated by anaerobic-responsive element and ethylene-responsive element cis-elements, aiding in adaptation to tidal inundation. Additionally, we found that the loss of the seed desiccation and dormancy-related TIP3 gene, and the loss of the seed dormancy regulator DOG1 gene, or DOG1 protein lack heme-binding capacity, may be genetic factors contributing to vivipary. Our findings shed light on the role of AQPs in A. marina adaptation to intertidal environments and their relevance to salt secretion and vivipary.

Genome-Wide Identification of Pentatricopeptide Repeat (PPR) Gene Family and Multi-Omics Analysis Provide New Insights Into the Albinism Mechanism of Kandelia obovata Propagule Leaves.

Pentatricopeptide repeat (PPR) gene family constitutes one of the largest gene families in plants, which mainly participate in RNA editing and RNA splicing of organellar RNAs, thereby affecting the organellar development. Recently, some evidence elucidated the important roles of PPR proteins in the albino process of plant leaves. However, the functions of PPR genes in the woody mangrove species have not been investigated. In this study, using a typical true mangrove Kandelia obovata, we systematically identified 298 PPR genes and characterized their general features and physicochemical properties, including evolutionary relationships, the subcellular localization, PPR motif type, the number of introns and PPR motifs, and isoelectric point, and so forth. Furthermore, we combined genome-wide association studies (GWAS) and transcriptome analysis to identify the genetic architecture and potential PPR genes associated with propagule leaves colour variations of K. obovata. As a result, we prioritized 16 PPR genes related to the albino phenotype using different strategies, including differentially expressed genes analysis and genetic diversity analysis. Further analysis discovered two genes of interest, namely Maker00002998 (PLS-type) and Maker00003187 (P-type), which were differentially expressed genes and causal genes detected by GWAS analysis. Moreover, we successfully predicted downstream target chloroplast genes (rps14, rpoC1 and rpoC2) bound by Maker00002998 PPR proteins. The experimental verification of RNA editing sites of rps14, rpoC1, and rpoC2 in our previous study and the verification of interaction between Maker00002998 and rps14 transcript using in vitro RNA pull-down assays revealed that Maker00002998 PPR protein might be involved in the post-transcriptional process of chloroplast genes. Our result provides new insights into the roles of PPR genes in the albinism mechanism of K. obovata propagule leaves.

PlantC2U: deep learning of cross-species sequence landscapes predicts plastid C-to-U RNA editing in plants.

In plants, C-to-U RNA editing mainly occurs in plastid and mitochondrial transcripts, which contributes to a complex transcriptional regulatory network. More evidence reveals that RNA editing plays critical roles in plant growth and development. However, accurate detection of RNA editing sites using transcriptome sequencing data alone is still challenging. In the present study, we develop PlantC2U, which is a convolutional neural network, to predict plastid C-to-U RNA editing based on the genomic sequence. PlantC2U achieves >95% sensitivity and 99% specificity, which outperforms the PREPACT tool, random forests, and support vector machines. PlantC2U not only further checks RNA editing sites from transcriptome data to reduce possible false positives, but also assesses the effect of different mutations on C-to-U RNA editing based on the flanking sequences. Moreover, we found the patterns of tissue-specific RNA editing in the mangrove plant Kandelia obovata, and observed reduced C-to-U RNA editing rates in the cold stress response of K. obovata, suggesting their potential regulatory roles in plant stress adaptation. In addition, we present RNAeditDB, available online at https://jasonxu.shinyapps.io/RNAeditDB/. Together, PlantC2U and RNAeditDB will help researchers explore the RNA editing events in plants and thus will be of broad utility for the plant research community.

In silico analysis of NAC gene family in the mangrove plant Avicennia marina provides clues for adaptation to intertidal habitats.

NAC (NAM, ATAF1/2, CUC2) transcription factors (TFs) constitute a plant-specific gene family. It is reported that NAC TFs play important roles in plant growth and developmental processes and in response to biotic/abiotic stresses. Nevertheless, little information is known about the functional and evolutionary characteristics of NAC TFs in mangrove plants, a group of species adapting coastal intertidal habitats. Thus, we conducted a comprehensive investigation for NAC TFs in Avicennia marina, one pioneer species of mangrove plants. We totally identified 142 NAC TFs from the genome of A. marina. Combined with NAC proteins having been functionally characterized in other organisms, we built a phylogenetic tree to infer the function of NAC TFs in A. marina. Gene structure and motif sequence analyses suggest the sequence conservation and transcription regulatory regions-mediated functional diversity. Whole-genome duplication serves as the driver force to the evolution of NAC gene family. Moreover, two pairs of NAC genes were identified as positively selected genes of which AmNAC010/040 may be imposed on less constraint toward neofunctionalization. Quite a few stress/hormone-related responsive elements were found in promoter regions indicating potential response to various external factors. Transcriptome data revealed some NAC TFs were involved in pneumatophore and leaf salt gland development and response to salt, flooding and Cd stresses. Gene co-expression analysis found a few NAC TFs participates in the special biological processes concerned with adaptation to intertidal environment. In summary, this study provides detailed functional and evolutionary information about NAC gene family in mangrove plant A. marina and new perspective for adaptation to intertidal habitats.

Combined metabolome and transcriptome analysis reveals a critical role of lignin biosynthesis and lignification in stem-like pneumatophore development of the mangrove Avicennia marina.

MAIN CONCLUSION: Transcriptional and metabolic regulation of lignin biosynthesis and lignification plays crucial roles in Avicennia marina pneumatophore development, facilitating its adaptation to coastal habitats. Avicennia marina is a pioneer mangrove species in coastal wetland. To cope with the periodic intertidal flooding and hypoxia environment, this species has developed a complex and extensive root system, with its most unique feature being a pneumatophore with a distinct above- and below-ground morphology and vascular structure. However, the characteristics of pneumatophore lignification remain unknown. Studies comparing the anatomy among above-ground pneumatophore, below-ground pneumatophore, and feeding root have suggested that vascular structure development in the pneumatophore is more like the development of a stem than of a root. Metabolome and transcriptome analysis illustrated that the accumulation of syringyl (S) and guaiacyl (G) units in the pneumatophore plays a critical role in lignification of the stem-like structure. Fourteen differentially accumulated metabolites (DAMs) and 10 differentially expressed genes involved in the lignin biosynthesis pathway were targeted. To identify genes significantly associated with lignification, we analyzed the correlation between 14 genes and 8 metabolites and further built a co-expression network between 10 transcription factors (TFs), including 5 for each of MYB and NAC, and 23 enzyme-coding genes involved in lignin biosynthesis. 4-Coumarate-CoA ligase, shikimate/quinate hydroxycinnamoyl transferase, cinnamyl alcohol dehydrogenase, caffeic acid 3-O-methyltransferase, phenylalanine ammonia-lyase, and peroxidase were identified to be strongly correlated with these TFs. Finally, we examined 9 key candidate genes through quantitative real-time PCR to validate the reliability of transcriptome data. Together, our metabolome and transcriptome findings reveal that lignin biosynthesis and lignification regulate pneumatophore development in the mangrove species A. marina and facilitate its adaptation to coastal habitats.

Hydrogen sulfide upregulates the alternative respiratory pathway in mangrove plant Avicennia marina to attenuate waterlogging-induced oxidative stress and mitochondrial damage in a calcium-dependent manner.

Hydrogen sulfide (H2 S) is considered to mediate plant growth and development. However, whether H2 S regulates the adaptation of mangrove plant to intertidal flooding habitats is not well understood. In this study, sodium hydrosulfide (NaHS) was used as an H2 S donor to investigate the effect of H2 S on the responses of mangrove plant Avicennia marina to waterlogging. The results showed that 24-h waterlogging increased reactive oxygen species (ROS) and cell death in roots. Excessive mitochondrial ROS accumulation is highly oxidative and leads to mitochondrial structural and functional damage. However, the application of NaHS counteracted the oxidative damage caused by waterlogging. The mitochondrial ROS production was reduced by H2 S through increasing the expressions of the alternative oxidase genes and increasing the proportion of alternative respiratory pathway in the total mitochondrial respiration. Secondly, H2 S enhanced the capacity of the antioxidant system. Meanwhile, H2 S induced Ca2+ influx and activated the expression of intracellular Ca2+ -sensing-related genes. In addition, the alleviating effect of H2 S on waterlogging can be reversed by Ca2+ chelator and Ca2+ channel blockers. In conclusion, this study provides the first evidence to explain the role of H2 S in waterlogging adaptation in mangrove plants from the mitochondrial aspect.

Integration of eQTL and GWAS analysis uncovers a genetic regulation of natural ionomic variation in Arabidopsis.

KEY MESSAGE: This study provided important insights into the genetic architecture of variations in A. thaliana leaf ionome in a cell-type-specific manner. The functional interpretation of traits associated variants by expression quantitative trait loci (eQTL) analysis is usually performed in bulk tissue samples. While the regulation of gene expression is context-dependent, such as cell-type-specific manner. In this study, we estimated cell-type abundances from 728 bulk tissue samples using single-cell RNA-sequencing dataset, and performed cis-eQTL mapping to identify cell-type-interaction eQTL (cis-eQTLs(ci)) in A. thaliana. Also, we performed Genome-wide association studies (GWAS) analyses for 999 accessions to identify the genetic basis of variations in A. thaliana leaf ionome. As a result, a total of 5,664 unique eQTL genes and 15,038 unique cis-eQTLs(ci) were significant. The majority (62.83%) of cis-eQTLs(ci) were cell-type-specific eQTLs. Using colocalization, we uncovered one interested gene AT2G25590 in Phloem cell, encoding a kind of plant Tudor-like protein with possible chromatin-associated functions, which colocalized with the most significant cis-eQTL(ci) of a Mo-related locus (Chr2:10,908,806:A:C; P = 3.27 × 10-27). Furthermore, we prioritized eight target genes associated with AT2G25590, which were previously reported in regulating the concentration of Mo element in A. thaliana. This study revealed the genetic regulation of ionomic variations and provided a foundation for further studies on molecular mechanisms of genetic variants controlling the A. thaliana ionome.

The genome of a mangrove plant, Avicennia marina, provides insights into adaptation to coastal intertidal habitats.

MAIN CONCLUSION: Whole-genome duplication, gene family and lineage-specific genes analysis based on high-quality genome reveal the adaptation mechanisms of Avicennia marina to coastal intertidal habitats. Mangrove plants grow in a complex habitat of coastal intertidal zones with high salinity, hypoxia, etc. Therefore, it is an interesting question how mangroves adapt to the unique intertidal environment. Here, we present a chromosome-level genome of the Avicennia marina, a typical true mangrove with a size of 480.43 Mb, contig N50 of 11.33 Mb and 30,956 annotated protein-coding genes. We identified 621 Avicennia-specific genes that are mainly related to flavonoid and lignin biosynthesis, auxin homeostasis and response to abiotic stimulus. We found that A. marina underwent a novel specific whole-genome duplication, which is in line with a brief era of global warming that occurred during the paleocene-eocene maximum. Comparative genomic and transcriptomic analyses outline the distinct evolution and sophisticated regulations of A. marina adaptation to the intertidal environments, including expansion of photosynthesis and oxidative phosphorylation gene families, unique genes and pathways for antibacterial, detoxifying antioxidant and reactive oxygen species scavenging. In addition, we also analyzed salt gland secretion-related genes, and those involved in the red bark-related flavonoid biosynthesis, while significant expansions of key genes such as NHX, 4CL, CHS and CHI. High-quality genomes in future investigations will facilitate the understand of evolution of mangrove and improve breeding.

PlantPhoneDB: A manually curated pan-plant database of ligand-receptor pairs infers cell-cell communication.

Ligand-receptor pairs play important roles in cell-cell communication for multicellular organisms in response to environmental cues. Recently, the emergence of single-cell RNA-sequencing (scRNA-seq) provides unprecedented opportunities to investigate cellular communication based on ligand-receptor expression. However, so far, no reliable ligand-receptor interaction database is available for plant species. In this study, we developed PlantPhoneDB (https://jasonxu.shinyapps.io/PlantPhoneDB/), a pan-plant database comprising a large number of high-confidence ligand-receptor pairs manually curated from seven resources. Also, we developed a PlantPhoneDB R package, which not only provided optional four scoring approaches that calculate interaction scores of ligand-receptor pairs between cell types but also provided visualization functions to present analysis results. At the PlantPhoneDB web interface, the processed datasets and results can be searched, browsed, and downloaded. To uncover novel cell-cell communication events in plants, we applied the PlantPhoneDB R package on GSE121619 dataset to infer significant cell-cell interactions of heat-shocked root cells in Arabidopsis thaliana. As a result, the PlantPhoneDB predicted the actively communicating AT1G28290-AT2G14890 ligand-receptor pair in atrichoblast-cortex cell pair in Arabidopsis thaliana. Importantly, the downstream target genes of this ligand-receptor pair were significantly enriched in the ribosome pathway, which facilitated plants adapting to environmental changes. In conclusion, PlantPhoneDB provided researchers with integrated resources to infer cell-cell communication from scRNA-seq datasets.

Genome-wide identification and characterization of aquaporins in mangrove plant Kandelia obovata and its role in response to the intertidal environment.

Aquaporins (AQPs) play important roles in plant growth, development and tolerance to environmental stresses. To understand the role of AQPs in the mangrove plant Kandelia obovata, which has the ability to acquire water from seawater, we identified 34 AQPs in the K. obovata genome and analysed their structural features. Phylogenetic analysis revealed that KoAQPs are homologous to AQPs of Populus and Arabidopsis, which are evolutionarily conserved. The key amino acid residues were used to assess water-transport ability. Analysis of cis-acting elements in the promoters indicated that KoAQPs may be stress- and hormone-responsive. Subcellular localization of KoAQPs in yeast showed most KoAQPs function in the membrane system. That transgenic yeast with increased cell volume showed that some KoAQPs have significant water-transport activity, and the substrate sensitivity assay indicates that some KoAQPs can transport H2 O2 . The transcriptome data were used to analyze the expression patterns of KoAQPs in different tissues and developing fruits of K. obovata. In addition, real-time quantitative PCR analyses combined transcriptome data showed that KoAQPs have complex responses to environmental factors, including salinity, flooding and cold. Collectively, the transport of water and solutes by KoAQPs contributed to the adaptation of K. obovata to the coastal intertidal environment.

Continental-scale plant invasions reshuffle the soil microbiome of blue carbon ecosystems.

Theory and experiments support that plant invasions largely impact aboveground biodiversity and function. Yet, much less is known on the influence of plant invasions on the structure and function of the soil microbiome of coastal wetlands, one of the largest major reservoirs of biodiversity and carbon on Earth. We studied the continental-scale invasion of Spartina alterniflora across 2451 km of Chinese coastlines as our model-system and found that S. alterniflora invasion can largely influence the soil microbiome (across six depths from 0 to 100 cm), compared with the most common microhabitat found before invasion (mudflats, Mud). In detail, S. alterniflora invasion was not only positively associated with bacterial richness but also resulted in important biotic homogenization of bacterial communities, suggesting that plant invasion can lead to important continental scale trade-offs in the soil microbiome. We found that plant invasion changed the community composition of soil bacterial communities across the soil profile. Moreover, the bacterial communities associated with S. alterniflora invasions where less responsive to climatic changes than those in native Mud microhabitats, suggesting that these new microbial communities might become more dominant under climate change. Plant invasion also resulted in important reductions in the complexity and stability of microbial networks, decoupling the associations between microbes and carbon pools. Taken together, our results indicated that plant invasions can largely influence the microbiome of coastal wetlands at the scale of China, representing the first continental-scale example on how plant invasions can reshuffle the soil microbiome, with consequences for the myriad of functions that they support.

Identification, characterization and expression analysis of lineage-specific genes within mangrove species Aegiceras corniculatum.

Lineage-specific genes (LSGs) are the genes that have no recognizable homology to any sequences in other species, which are important drivers for the generation of new functions, phenotypic changes, and facilitating species adaptation to environment. Aegiceras corniculatum is one of major mangrove plant species adapted to waterlogging and saline conditions, and the exploration of aegiceras-specific genes (ASGs) is important to reveal its adaptation to the harsh environment. Here, we performed a systematic analysis on ASGs, focusing on their sequence characterization, origination and expression patterns. Our results reveal that there are 4823 ASGs in the genome, approximately 11.84% of all protein-coding genes. High proportion (45.78%) of ASGs originate from gene duplication, and the time of gene duplication of ASGs is consistent with the timing of two genome-wide replication (WGD) events that occurred in A. corniculatum, and also coincides with a short period of global warming during the Paleocene-Eocene Maximum (PETM, 55.5 million years ago). Gene structure analysis showed that ASGs have shorter protein lengths, fewer exons, and higher isoelectric point. Expression patterns analysis showed that ASGs had low levels of expression and more tissue-specific expression. Weighted gene co-expression network analysis (WGCNA) revealed that 86 ASGs co-expressed gene modules were primarily involved in pathways related to adversity stress, including plant hormone signal transduction, phenylpropanoid biosynthesis, photosynthesis, peroxisome and pentose phosphate pathway. This study provides a comprehensive analysis of the characteristics and potential functions of ASGs and identifies key candidate genes, which will contribute to the subsequent further investigation of the adaptation of A. corniculatum to intertidal coastal wetland habitats.

Proteomic analysis reveals the protective role of exogenous hydrogen sulfide against salt stress in rice seedlings.

Hydrogen sulfide (H2S) is an important gaseous signal molecule which participates in various abiotic stress responses. However, the underlying mechanism of H2S associated salt tolerance remains elusive. In this study, sodium hydrosulfide (NaHS, donor of H2S) was used to investigate the protective role of H2S against salt stress at the biochemical and proteomic levels. Antioxidant activity and differentially expressed proteins (DEPs) of rice seedlings treated by NaCl or/and exogenous H2S were investigated by the methods of biochemical approaches and comparative proteomic analysis. The protein-protein interaction (PPI) analysis was used for understanding the interaction networks of stress responsive proteins. In addition, relative mRNA levels of eight selected identified DEPs were analyzed by quantitative real-time PCR. The result showed that H2S alleviated oxidative damage caused by salt stress in rice seedling. The activities of some antioxidant enzymes and glutathione metabolism were mediated by H2S under salt stress. Proteomics analyses demonstrated that NaHS regulated antioxidant related proteins abundances and affected related enzyme activities under salt stress. Proteins related to light reaction system (PsbQ domain protein, plastocyanin oxidoreductase iron-sulfur protein), Calvin cycle (phosphoglycerate kinase, sedoheptulose-1,7-bisphosphatase precursor, ribulose-1,5-bisphosphate carboxylase/oxygenase) and chlorophyll biosynthesis (glutamate-1-semialdehyde 2,1-aminomutase, coproporphyrinogen III oxidase) are important for NaHS against salt stress. ATP synthesis related proteins, malate dehydrogenase and 2, 3-bisphosphoglycerate-independent phosphoglycerate mutase were up-regulated by NaHS under salt stress. Protein metabolism related proteins and cell structure related proteins were recovered or up-regulated by NaHS under salt stress. The PPI analysis further unraveled a complicated regulation network among above biological processes to enhance the tolerance of rice seedling to salt stress under H2S treatment. Overall, our results demonstrated that H2S takes protective roles in salt tolerance by mitigating oxidative stress, recovering photosynthetic capacity, improving primary and energy metabolism, strengthening protein metabolism and consolidating cell structure in rice seedlings.

Physiological and Proteomic Analyses of Two Acanthus Species to Tidal Flooding Stress.

The mangrove plant Acanthus ilicifolius and its relative, Acanthus mollis, have been previously proved to possess diverse pharmacological effects. Therefore, evaluating the differentially expressed proteins of these species under tidal flooding stress is essential to fully exploit and benefit from their medicinal values. The roots of A. ilicifolius and A. mollis were exposed to 6 h of flooding stress per day for 10 days. The dry weight, hydrogen peroxide (H2O2) content, anatomical characteristics, carbon and energy levels, and two-dimensional electrophoresis coupled with MALDI-TOF/TOF MS technology were used to reveal the divergent flooding resistant strategies. A. ilicifolius performed better under tidal flooding stress, which was reflected in the integrity of the morphological structure, more efficient use of carbon and energy, and a higher percentage of up-regulated proteins associated with carbon and energy metabolism. A. mollis could not survive in flooding conditions for a long time, as revealed by disrupting cell structures of the roots, less efficient use of carbon and energy, and a higher percentage of down-regulated proteins associated with carbon and energy metabolism. Energy provision and flux balance played a role in the flooding tolerance of A. ilicifolius and A. mollis.

Comparative Proteomic Analysis Reveals the Regulatory Effects of H2S on Salt Tolerance of Mangrove Plant Kandelia obovata.

As a dominant mangrove species, Kandelia obovata is distributed in an intertidal marsh with an active H2S release. Whether H2S participates in the salt tolerance of mangrove plants is still ambiguous, although increasing evidence has demonstrated that H2S functions in plant responses to multiple abiotic stresses. In this study, NaHS was used as an H2S donor to investigate the regulatory mechanism of H2S on the salt tolerance of K. obovata seedlings by using a combined physiological and proteomic analysis. The results showed that the reduction in photosynthesis (Pn) caused by 400 mM of NaCl was recovered by the addition of NaHS (200 µM). Furthermore, the application of H2S enhanced the quantum efficiency of photosystem II (PSII) and the membrane lipid stability, implying that H2S is beneficial to the survival of K. obovata seedlings under high salinity. We further identified 37 differentially expressed proteins by proteomic approaches under salinity and NaHS treatments. Among them, the proteins that are related to photosynthesis, primary metabolism, stress response and hormone biosynthesis were primarily enriched. The physiological and proteomic results highlighted that exogenous H2S up-regulated photosynthesis and energy metabolism to help K. obovata to cope with high salinity. Specifically, H2S increased photosynthetic electron transfer, chlorophyll biosynthesis and carbon fixation in K. obovata leaves under salt stress. Furthermore, the abundances of other proteins related to the metabolic pathway, such as antioxidation (ascorbic acid peroxidase (APX), copper/zinc superoxide dismutase (CSD2), and pancreatic and duodenal homeobox 1 (PDX1)), protein synthesis (heat-shock protein (HSP), chaperonin family protein (Cpn) 20), nitrogen metabolism (glutamine synthetase 1 and 2 (GS2), GS1:1), glycolysis (phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI)), and the ascorbate-glutathione (AsA-GSH) cycle were increased by H2S under high salinity. These findings provide new insights into the roles of H2S in the adaptations of the K. obovata mangrove plant to high salinity environments.

The reduced state of the plastoquinone pool is required for chloroplast-mediated stomatal closure in response to calcium stimulation.

Besides their participation in photosynthesis, leaf chloroplasts function in plant responses to stimuli, yet how they direct stimulus-induced stomatal movement remains elusive. Here, we showed that over-reduction of the plastoquinone (PQ) pool by dibromothymoquinone (DBMIB) was closely associated with stomatal closure in plants which required chloroplastic H2O2 generation in the mesophyll. External application of H2 O2 reduced the PQ pool, whereas the cell-permeable reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) reversed the DBMIB-induced over-reduction of the PQ pool and stomatal closure. Mesophyll chloroplasts are key players of extracellular Ca(2+) (Ca(2+)o)-induced stomatal closure, but when treated with either 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) or NAC they failed to facilitate Ca(2+)o-induced stomatal closure due to the inhibition of chloroplastic H2 O2 synthesis in mesophyll. Similarly, the Arabidopsis electron transfer chain-related mutants npq4-1, stn7 and cas-1 exhibited diverse responses to Ca(2+)o or DBMIB. Transcriptome analysis also demonstrated that the PQ pool signaling pathway shared common responsive genes with the H2 O2 signaling pathway. These results implicated a mechanism for chloroplast-mediated stomatal closure involving the generation of mesophyll chloroplastic H2O2 based on the reduced state of the PQ pool, which is calcium-sensing receptor (CAS) and LHCII phosphorylation dependent.

Comparative Proteomic Analysis Reveals the Effects of Exogenous Calcium against Acid Rain Stress in Liquidambar formosana Hance Leaves.

Acid rain (AR) impacts forest health by leaching calcium (Ca) away from soils and plants. Ca is an essential element and participates in various plant physiological responses. In the present study, the protective role of exogenous Ca in alleviating AR stress in Liquidambar formosana Hance at the physiological and proteomic levels was examined. Our results showed that low Ca condition resulted in the chlorophyll content and photosynthesis decreasing significantly in L. formosana leaves; however, these effects could be reversed by high Ca supplementation. Further proteomic analyses successfully identified 81 differentially expressed proteins in AR-treated L. formosana under different Ca levels. In particular, some of the proteins are involved in primary metabolism, photosynthesis, energy production, antioxidant defense, transcription, and translation. Moreover, quantitative real time polymerase chain reaction (qRT-PCR) results indicated that low Ca significantly increased the expression level of the investigated Ca-related genes, which can be reversed by high Ca supplementation under AR stress. Further, Western blotting analysis revealed that exogenous Ca supply reduced AR damage by elevating the expression of proteins involved in the Calvin cycle, reactive oxygen species (ROS) scavenging system. These findings allowed us to better understand how woody plants respond to AR stress at various Ca levels and the protective role of exogenous Ca against AR stress in forest tree species.