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.

A survey of transcriptome complexity using full-length isoform sequencing in the tea plant Camellia sinensis.

Tea is one of the most popular beverages and its leaves are rich in catechins, contributing to the diverse flavor as well as beneficial for human health. However, the study of the post-transcriptional regulatory mechanism affecting the synthesis of catechins remains insufficient. Here, we sequenced the transcriptome using PacBio sequencing technology and obtained 63,111 full-length high-quality isoforms, including 1302 potential novel genes and 583 highly reliable fusion transcripts. We also identified 1204 lncRNAs with high quality, containing 188 known and 1016 novel lncRNAs. In addition, 311 mis-annotated genes were corrected based on the high-quality Isoseq reads. A large number of alternative splicing (AS) events (3784) and alternative polyadenylation (APA) genes (18,714) were analyzed, accounting for 8.84% and 43.7% of the total annotated genes, respectively. We also found that 2884 genes containing AS and APA features exhibited higher expression levels than other genes. These genes are mainly involved in amino acid biosynthesis, carbon fixation in photosynthetic organisms, phenylalanine, tyrosine, tryptophan biosynthesis, and pyruvate metabolism, suggesting that they play an essential role in the catechins content of tea polyphenols. Our results further improved the level of genome annotation and indicated that post-transcriptional regulation plays a crucial part in synthesizing catechins.

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.

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.

Chloroplast genome analysis of three Acanthus species reveal the adaptation of mangrove to intertidal habitats.

Acanthus is a distinctive genus that covers three species with different ecological niches including Acanthus mollis (arid terrestrial), Acanthus leucostachyus (damp forest) and Acanthus ilicifolius (coastal intertidal). It is an intriguing question how these species evolved from terrestrial to coastal intertidal. In the present study, we assembled chloroplast genomes of A. ilicifolius, A. leucostachyus and A. mollis, which exhibited typical quadripartite structures. The sizes were 150,758, 154,686 and 150,339 bp that comprised a large single copy (LSC, 82,963, 86,461 and 82,612 bp), a small single copy (SSC, 17,191, 17,511 and 17,019 bp), and a pair of inverted repeats (IRs, 25,302, 25,357 and 25,354 bp), respectively. Gene annotation revealed that A. ilicifolius, A. leucostachyus and A. mollis contained 113, 112 and 108 unique genes, each of which contained 79, 79 and 74 protein-coding genes, 30, 29 and 30 tRNAs, and 4 rRNA genes, respectively. Differential gene analysis revealed plenty of ndhs gene deletions in the terrestrial plant A. mollis. Nucleotide diversity analysis showed that the psbK, ycf1, ndhG, and rpl22 have the highest nucleotide variability. Compared to A. leucostachyus and A. mollis, seven genes in A. ilicifolius underwent positive selection. Among them, the atpF gene showed a strong positive selection throughout terrestrial to marine evolution and was important for adaptation to coastal intertidal habitats. Phylogenetic analysis indicated that A. ilicifolius has a closer genetic relationship with A. leucostachyus than A. mollis which further confirmed the evolutionary direction of Acanthus going from terrestrial to coastal intertidal zones.

Comparative transcriptome analysis on the mangrove Acanthus ilicifolius and its two terrestrial relatives provides insights into adaptation to intertidal habitats.

Acanthus is a unique genus covering both mangroves and terrestrial species, and thus is an ideal system to comparatively analyze the mechanisms of mangrove adaptation to intertidal habitats. We performed RNA sequencing of the mangrove plant Acanthus ilicifolius and its two terrestrial relatives, Acanthus leucostachyus and Acanthus mollis. A total of 91,125, 118,290, and 141,640 unigenes were obtained. Simple sequence repeats (SSR) analysis showed that A. ilicifolius had more SSRs, the highest frequency of distribution, and higher in polymorphism potential compared to the two terrestrial relatives. Phylogenetic analyses suggested a relatively recent split between A. ilicifolius and A. leucostachyus, i.e., about 16.76 million years ago (Mya), after their ancestor divergence with A. mollis (32.11 Mya), indicating that speciation of three Acanthus species occurred in the Early to Middle Miocene. Gene Ontology (GO) enrichment revealed that the unique unigenes in A. ilicifolius are predominantly related to rhythmic process, reproductive process and response to stimuli. The accelerated evolution and positive selection analyses indicated that the genus Acanthus migrated from terrestrial to intertidal habitats, where 311 pairs may be under positive selection. Functional enrichment analysis revealed that these genes associated with essential metabolism and biosynthetic pathways such as oxidative phosphorylation, plant hormone signal transduction, photosynthetic carbon fixation and arginine and proline metabolism, are related to the adaptation of A. ilicifolius to intertidal habitats, which are characterized by high salinity and hypoxia. Our results indicate the evolutionary processes and the mechanisms underlying the adaptability of Acanthus to various harsh environments from the arid terrestrial to intertidal habitats.

Identification, Characterization and Function of Orphan Genes Among the Current Cucurbitaceae Genomes.

Orphan genes (OGs) that are missing identifiable homologs in other lineages may potentially make contributions to a variety of biological functions. The Cucurbitaceae family consists of a wide range of fruit crops of worldwide or local economic significance. To date, very few functional mechanisms of OGs in Cucurbitaceae are known. In this study, we systematically identified the OGs of eight Cucurbitaceae species using a comparative genomics approach. The content of OGs varied widely among the eight Cucurbitaceae species, ranging from 1.63% in chayote to 16.55% in wax gourd. Genetic structure analysis showed that OGs have significantly shorter protein lengths and fewer exons in Cucurbitaceae. The subcellular localizations of OGs were basically the same, with only subtle differences. Except for aggregation in some chromosomal regions, the distribution density of OGs was higher near the telomeres and relatively evenly distributed on the chromosomes. Gene expression analysis revealed that OGs had less abundantly and highly tissue-specific expression. Interestingly, the largest proportion of these OGs was significantly more tissue-specific expressed in the flower than in other tissues, and more detectable expression was found in the male flower. Functional prediction of OGs showed that (1) 18 OGs associated with male sterility in watermelon; (2) 182 OGs associated with flower development in cucumber; (3) 51 OGs associated with environmental adaptation in watermelon; (4) 520 OGs may help with the large fruit size in wax gourd. Our results provide the molecular basis and research direction for some important mechanisms in Cucurbitaceae species and domesticated crops.

Effects of shrimp pond effluent on functional traits and functional diversity of mangroves in Zhangjiang Estuary.

In recent years, the scale of shrimp ponds has rapidly increased adjacent to mangrove forests. Discharge of shrimp pond effluent has led to degradation of the surrounding environment and reduction of biodiversity in the estuary. But it remains poorly understood how shrimp pond effluent affects functional traits and functional diversity of mangroves. We sampled roots, stems and leaves of Kandelia obovata and other mangrove plants, as well as sediments and pore water from shrimp pond effluent polluted area (P) and clean area (control area, C) in Zhangjiang Estuary in southeast coast of China. Twenty plant functional traits and six functional diversity indices were analyzed to explore the effects of shrimp pond effluent on individual plants and mangrove communities. The results showed that the discharge of shrimp pond effluent significantly affected the nutrient content in soils and pore water, for example, sediment NH4+ and NO3- concentration increased from 0.26 ± 0.06 to 0.77 ± 0.29 mg/g and from 0.05 ± 0.03 to 0.16 ± 0.05 mg/g, respectively, when comparing the C and P site. Furthermore, some mangrove plant functional traits such as plant height, diameter at breast height, canopy thickness and specific leaf area were significantly increased by the effluent discharge. Functional diversity in the polluted area reduced as a whole compared to the control area. In particular, ammonium and nitrate nitrogen input is the main reason to induce the changes of plant functional traits and functional diversity. Besides, the community structure changed from functional differentiation to functional convergence after shrimp pond effluent discharge. In addition, the long-term shrimp pond effluent discharge may lead to the ecological strategy shift of K. obovata, while different organs may adopt different ways of nutrient uptake and growth strategies in the face of effluent disturbance. In conclusion, pollution from shrimp pond does affect the functional traits of mangrove plants and functional diversity of mangrove community. These results provide strong evidence to assess the impact of effluent discharges on mangrove plants and provide theoretical basis for conservation and sustainable development of mangroves.

Exogenous hydrogen sulfide mediates Na+ and K+ fluxes of salt gland in salt-secreting mangrove plant Avicennia marina.

Hydrogen sulfide (H2S), is a crucial biological player in plants. Here, we primarily explored the interaction between sodium hydrosulfide (NaHS, a H2S donor) and the fluxes of Na+ and K+ from the salt glands of mangrove species Avicennia marina (Forsk.) Vierh. with non-invasive micro-test technology (NMT) and quantitative real-time PCR (qRT-PCR) approaches under salinity treatments. The results showed that under 400-mM NaCl treatment, the addition of 200-µM NaHS markedly increased the quantity of salt crystals in the adaxial epidermis of A. marina leaves, accompanied by an increase in the K+/Na+ ratio. Meanwhile, the endogenous content of H2S was dramatically elevated in this process. The NMT result revealed that the Na+ efflux was increased from salt glands, whereas K+ efflux was decreased with NaHS application. On the contrary, the effects of NaHS were reversed by H2S scavenger hypotaurine (HT), and DL-propargylglycine (PAG), an inhibitor of cystathionine-γ-lyase (CES, a H2S synthase). Moreover, enzymic assay revealed that NaHS increased the activities of plasma membrane and tonoplast H+-ATPase. qRT-PCR analysis revealed that NaHS significantly increased the genes transcript levels of tonoplast Na+/H+ antiporter (NHX1), plasma membrane Na+/H+ antiporter (SOS1), plasma membrane H+-ATPase (AHA1) and tonoplast H+-ATPase subunit c (VHA-c1), while suppressed above-mentioned gene expressions by the application of HT and PAG. Overall, H2S promotes Na+ secretion from the salt glands of A. marina by up-regulating the plasma membrane and tonoplast Na+/H+ antiporter and H+-ATPase.

Chromosome-level reference genome assembly provides insights into aroma biosynthesis in passion fruit (Passiflora edulis).

Passion fruit, native to tropical America, is an agriculturally, economically and ornamentally important fruit plant that is well known for its acid pulp, rich aroma and distinctive flavour. Here, we present a chromosome-level genome assembly of passion fruit by incorporating PacBio long HiFi reads and Hi-C technology. The assembled reference genome is 1.28 Gb size with a scaffold N50 of 126.4 Mb and 99.22% sequences anchored onto nine pseudochromosomes. This genome is highly repetitive, accounting for 86.61% of the assembled genome. A total of 39,309 protein-coding genes were predicted with 93.48% of those being functionally annotated in the public databases. Genome evolution analysis revealed a core eudicot-common γ whole-genome triplication event and a more recent whole-genome duplication event, possibly contributing to the expansion of certain gene families. The 33 rapidly expanded gene families were significantly enriched in the pathways of isoflavone biosynthesis, galactose metabolism, diterpene biosynthesis and fatty acid metabolism, which might be responsible for the formation of featured flavours in the passion fruit. Transcriptome analysis revealed that genes related to ester and ethylene biosynthesis were significantly upregulated in the mature fruit and the expression levels of those genes were consistent with the accumulation of volatile lipid compounds. The passion fruit genome analysis improves our understanding of the genome evolution of this species and sheds new lights into the molecular mechanism of aroma biosynthesis in passion fruit.

High-Quality Genome of the Medicinal Plant Strobilanthes cusia Provides Insights Into the Biosynthesis of Indole Alkaloids.

Strobilanthes cusia (Nees) Kuntze is an important plant used to process the traditional Chinese herbal medicines "Qingdai" and "Nanbanlangen". The key active ingredients are indole alkaloids (IAs) that exert antibacterial, antiviral, and antitumor pharmacological activities and serve as natural dyes. We assembled the S. cusia genome at the chromosome level through combined PacBio circular consensus sequencing (CCS) and Hi-C sequencing data. Hi-C data revealed a draft genome size of 913.74 Mb, with 904.18 Mb contigs anchored into 16 pseudo-chromosomes. Contig N50 and scaffold N50 were 35.59 and 68.44 Mb, respectively. Of the 32,974 predicted protein-coding genes, 96.52% were functionally annotated in public databases. We predicted 675.66 Mb repetitive sequences, 47.08% of sequences were long terminal repeat (LTR) retrotransposons. Moreover, 983 Strobilanthes-specific genes (SSGs) were identified for the first time, accounting for ~2.98% of all protein-coding genes. Further, 245 putative centromeric and 29 putative telomeric fragments were identified. The transcriptome analysis identified 2,975 differentially expressed genes (DEGs) enriched in phenylpropanoid, flavonoid, and triterpenoid biosynthesis. This systematic characterization of key enzyme-coding genes associated with the IA pathway and basic helix-loop-helix (bHLH) transcription factor family formed a network from the shikimate pathway to the indole alkaloid synthesis pathway in S. cusia. The high-quality S. cusia genome presented herein is an essential resource for the traditional Chinese medicine genomics studies and understanding the genetic underpinning of IA biosynthesis.

Source or sink? A study on the methane flux from mangroves stems in Zhangjiang estuary, southeast coast of China.

Mangrove ecosystems are an important component of "blue carbon". However, it is not clear whether the stems play roles in the CH4 budget of mangrove ecosystems. This study investigated the CH4 emission from mangrove stems and its potential driving factors. We set up six sample plots in the Zhangjiang Estuary National Mangrove Nature Reserve, where Kandelia obovata, Avicennia marina and Aegiceras corniculata are the main mangrove tree species. Soil properties such as total carbon content, redox potential and salinity were determined in each plot. The dynamic chamber method was used to measure mangrove stems and soil CH4 fluxes. Combined field survey results with Principal Component Analysis (PCA) of soil properties, we divided the six plots into two sites (S1 and S2) to perform statistical analyses of stem CH4 fluxes. Then the CH4 fluxes from mangrove tree stems and soil were further scaled up to the ecosystem level through the mapping model. Under different backgrounds of soil properties, salinity and microbial biomass carbon were the main factors modified soil CH4 fluxes in the two sites, and further affected the stem CH4 fluxes of mangroves. The soil of both sites are sources of CH4, and the soil CH4 emission of S2 was about twice higher than that of S1. Results of upscaling model showed that mangrove stems in S1 were CH4 sinks with -105.65 g d-1. But stems in S2 were CH4 sources around 1448.24 g d-1. Taken together, our results suggested that CH4 emission from mangrove soils closely depends on soils properties. And mangrove stems were found to act as both CH4 sources and CH4 sinks depend on soil CH4 production. Therefore, when calculating the CH4 budget of the mangrove ecosystem, the contribution of mangrove plant stems cannot be ignored.

Chromosome-level assembly of the mangrove plant Aegiceras corniculatum genome generated through Illumina, PacBio and Hi-C sequencing technologies.

Aegiceras corniculatum is a major mangrove plant species adapted to waterlogging and saline conditions, grows in the coastal intertidal zone of tropical and subtropical regions. Here, we present a chromosome-level genome assembly of A. corniculatum by incorporating PacBio long-read sequencing and Hi-C technology. The results showed that the PacBio draft genome size is 906.63 Mb. Hi-C scaffolding anchored 885.06 Mb contigs (97.62% of draft assembly) onto 24 pseudochromosomes. The contig N50 and scaffold N50 were 7.1 Mb and 37.74 Mb, respectively. Out of 40,727 protein-coding genes predicted in the study, 89% have functional annotations in public databases. We also showed that of the 603.93 Mb repetitive sequences predicted in the assembled genome, long terminal repeat retrotransposons constitute 41.52%. The genome evolution analysis showed that the A. corniculatum genome experienced two whole-genome duplication events and shared the ancient γ whole-genome triplication event. A comparative genomic analysis revealed an incidence of expansion in 1,488 gene families associated with essential metabolism and biosynthetic pathways, including photosynthesis, oxidative phosphorylation, phenylalanine, glyoxylate, dicarboxylate metabolism, and DNA replication, which probably constitute adaptation traits that allow the A. corniculatum to survive in the intertidal zone. Also, the systematic characterization of genes associated with flavonoid biosynthesis pathway and the AcNHX gene family conducted in this study will provide insight into the adaptation mechanism of A. corniculatum to intertidal environments. The high-quality genome reported here can provide historical insights into genomic transformations that support the survival of A. corniculatum under harsh intertidal habitats.

Spartina alterniflora invasion alters soil bacterial communities and enhances soil N2O emissions by stimulating soil denitrification in mangrove wetland.

Chinese mangrove, an important ecosystem in coastal wetlands, is sensitive to the invasive alien species Spartina alterniflora. However, the effects of the S. alterniflora invasion on mangrove soil N2O emissions and the underlying mechanisms by which emissions are affected have not been well studied. In this study, the N2O emitted from soils dominated by two typical native mangroves (i.e. Kandelia obovata: KO; Avicennia marina: AM), one invaded by S. alterniflora (SA), and one bare mudflat (Mud) were monitored at Zhangjiang Mangrove Estuary (where S. alterniflora is exotic). Together with soil biogeochemical properties, the potential denitrification rate and the composition of soil bacterial communities were determined simultaneously by 15NO3- tracer and high-throughput sequencing techniques, respectively. Our results showed that S. alterniflora invasion significantly (p < 0.05) increases soil N2O emissions by 15-28-fold. In addition, isotope results revealed that the soil potential denitrification rate was significantly (p < 0.05) enhanced after S. alterniflora invasion. Moreover, the S. alterniflora invasion significantly (p < 0.05) decreased soil bacterial α-diversity and strongly modified soil bacterial communities. Indicator groups strongly associated with S. alterniflora were Chloroflexia, Alphaproteobacteria, and Bacilli, each of which was abundant and acts as connector in the co-occurrence network. FAPROTAX analysis implied that the S. alterniflora invasion stimulated soil denitrification and nitrification while depressing anaerobic ammonium oxidation (anammox) and dissimilatory nitrate reduction to ammonium (DNRA). Redundancy analysis (RDA) found that soil organic matter (SOM) and pH were the most important environmental factors in altering soil bacterial communities. Taken together, our results imply that the S. alterniflora invasion in mangrove wetlands significantly stimulates soil denitrification and N2O emissions, thereby contributing N2O to the atmosphere and contributing to global climate change.