Transcriptome-wide expression landscape and starch synthesis pathway co-expression network in sorghum.

The gene expression landscape across different tissues and developmental stages reflects their biological functions and evolutionary patterns. Integrative and comprehensive analyses of all transcriptomic data in an organism are instrumental to obtaining a comprehensive picture of gene expression landscape. Such studies are still very limited in sorghum, which limits the discovery of the genetic basis underlying complex agricultural traits in sorghum. We characterized the genome-wide expression landscape for sorghum using 873 RNA-sequencing (RNA-seq) datasets representing 19 tissues. Our integrative analysis of these RNA-seq data provides the most comprehensive transcriptomic atlas for sorghum, which will be valuable for the sorghum research community for functional characterizations of sorghum genes. Based on the transcriptome atlas, we identified 595 housekeeping genes (HKGs) and 2080 tissue-specific expression genes (TEGs) for the 19 tissues. We identified different gene features between HKGs and TEGs, and we found that HKGs have experienced stronger selective constraints than TEGs. Furthermore, we built a transcriptome-wide co-expression network (TW-CEN) comprising 35 modules with each module enriched in specific Gene Ontology terms. High-connectivity genes in TW-CEN tend to express at high levels while undergoing intensive selective pressure. We also built global and seed-preferential co-expression networks of starch synthesis pathways, which indicated that photosynthesis and microtubule-based movement play important roles in starch synthesis. The global transcriptome atlas of sorghum generated by this study provides an important functional genomics resource for trait discovery and insight into starch synthesis regulation in sorghum.

Chromosome-level Subgenome-aware de novo Assembly of Saccharomyces bayanus Provides Insight into Genome Divergence after Hybridization.

Interspecies hybridization is prevalent in various eukaryotic lineages and plays important roles in phenotypic diversification, adaption, and speciation. To better understand the changes that occurred in the different subgenomes of a hybrid species and how they facilitated adaptation, we completed chromosome-level de novo assemblies of all 16 pairs chromosomes for a recently formed hybrid yeast, Saccharomyces bayanus strain CBS380 (IFO11022), using Nanopore MinION long-read sequencing. Characterization of S. bayanus subgenomes and comparative analysis with the genomes of its parent species, S. uvarum and S. eubayanus, provide several new insights into understanding genome evolution after a relatively recent hybridization. For instance, multiple recombination events between the two subgenomes have been observed in each chromosome, followed by loss of heterozygosity (LOH) in most chromosomes in nine chromosome pairs. In addition to maintaining nearly all gene content and synteny from its parental genomes, S. bayanus has acquired many genes from other yeast species, primarily through the introgression of S. cerevisiae, such as those involved in the maltose metabolism. In addition, the patterns of recombination and LOH suggest an allotetraploid origin of S. bayanus. The gene acquisition and rapid LOH in the hybrid genome probably facilitated its adaption to maltose brewing environments and mitigated the maladaptive effect of hybridization.

An machine learning model to predict quality of life subtypes of disabled stroke survivors.

OBJECTIVE: Stroke causes serious physical disability with impaired quality of life (QoL) and heavy burden on health. The goal of this study is to explore the impaired QoL typologies and their predicting factors in physically disabled stroke survivors with machine learning approach. METHODS: Non-negative matrix factorization (NMF) was applied to clustering 308 physically disabled stroke survivors in rural China based on their responses on the short form 36 (SF-36) assessment of quality of life. Principal component analysis (PCA) was conducted to differentiate the subtypes, and the Boruta algorithm was used to identify the variables relevant to the categorization of two subtypes. A gradient boosting machine(GBM) and local interpretable model-agnostic explanation (LIME) algorithms were used to apply to interpret the variables that drove subtype predictions. RESULTS: Two distinct subtypes emerged, characterized by short form 36 (SF-36) domains. The feature difference between worsen QoL subtype and better QoL subtype was as follows: role-emotion (RE), body pain (BP) and general health (GH), but not physical function (PF); the most relevant predictors of worsen QoL subtypes were help from others, followed by opportunities for community activity and rehabilitation needs, rather than disability severity or duration since stroke. INTERPRETATION: The results suggest that the rehabilitation programs should be tailored toward their QoL clustering feature; body pain and emotional-behavioral problems are more crucial than motor deficit; stroke survivors with worsen QoL subtype are most in need of social support, return to community, and rehabilitation.

Origin and evolution of the triploid cultivated banana genome.

Most fresh bananas belong to the Cavendish and Gros Michel subgroups. Here, we report chromosome-scale genome assemblies of Cavendish (1.48 Gb) and Gros Michel (1.33 Gb), defining three subgenomes, Ban, Dh and Ze, with Musa acuminata ssp. banksii, malaccensis and zebrina as their major ancestral contributors, respectively. The insertion of repeat sequences in the Fusarium oxysporum f. sp. cubense (Foc) tropical race 4 RGA2 (resistance gene analog 2) promoter was identified in most diploid and triploid bananas. We found that the receptor-like protein (RLP) locus, including Foc race 1-resistant genes, is absent in the Gros Michel Ze subgenome. We identified two NAP (NAC-like, activated by apetala3/pistillata) transcription factor homologs specifically and highly expressed in fruit that directly bind to the promoters of many fruit ripening genes and may be key regulators of fruit ripening. Our genome data should facilitate the breeding and super-domestication of bananas.

Molecular and phylogenetic evidence of parallel expansion of anion channels in plants.

Aluminum-activated malate transporters (ALMTs) and slow anion channels (SLACs) are important in various physiological processes in plants, including stomatal regulation, nutrient uptake, and in response to abiotic stress such as aluminum toxicity. To understand their evolutionary history and functional divergence, we conducted phylogenetic and expression analyses of ALMTs and SLACs in green plants. Our findings from phylogenetic studies indicate that ALMTs and SLACs may have originated from green algae and red algae, respectively. The ALMTs of early land plants and charophytes formed a monophyletic clade consisting of three subgroups. A single duplication event of ALMTs was identified in vascular plants and subsequent duplications into six clades occurred in angiosperms, including an identified clade, 1-1. The ALMTs experienced gene number losses in clades 1-1 and 2-1 and expansions in clades 1-2 and 2-2b. Interestingly, the expansion of clade 1-2 was also associated with higher expression levels compared to genes in clades that experienced apparent loss. SLACs first diversified in bryophytes, followed by duplication in vascular plants, giving rise to three distinct clades (I, II, and III), and clade II potentially associated with stomatal control in seed plants. SLACs show losses in clades II and III without substantial expansion in clade I. Additionally, ALMT clade 2-2 and SLAC clade III contain genes specifically expressed in reproductive organs and roots in angiosperms, lycophytes, and mosses, indicating neofunctionalization. In summary, our study demonstrates the evolutionary complexity of ALMTs and SLACs, highlighting their crucial role in the adaptation and diversification of vascular plants.

A systems-level mass spectrometry-based technique for accurate and sensitive quantification of the RNA cap epitranscriptome.

Chemical modifications of transcripts with a 5' cap occur in all organisms and function in many aspects of RNA metabolism. To facilitate analysis of RNA caps, we developed a systems-level mass spectrometry-based technique, CapQuant, for accurate and sensitive quantification of the cap epitranscriptome. The protocol includes the addition of stable isotope-labeled cap nucleotides (CNs) to RNA, enzymatic hydrolysis of endogenous RNA to release CNs, and off-line enrichment of CNs by ion-pairing high-pressure liquid chromatography, followed by a 17 min chromatography-coupled tandem quadrupole mass spectrometry run for the identification and quantification of individual CNs. The total time required for the protocol can be up to 7 d. In this approach, 26 CNs can be quantified in eukaryotic poly(A)-tailed RNA, bacterial total RNA and viral RNA. This protocol can be modified to analyze other types of RNA and RNA from in vitro sources. CapQuant stands out from other methods in terms of superior specificity, sensitivity and accuracy, and it is not limited to individual caps nor does it require radiolabeling. Thanks to its unique capability of accurately and sensitively quantifying RNA caps on a systems level, CapQuant can reveal both the RNA cap landscape and the transcription start site distribution of capped RNA in a broad range of settings.

Evolution of a fatty acyl-CoA elongase underlies desert adaptation in Drosophila.

Traits that allow species to survive in extreme environments such as hot-arid deserts have independently evolved in multiple taxa. However, the genetic and evolutionary mechanisms underlying these traits have thus far not been elucidated. Here, we show that Drosophila mojavensis, a desert-adapted fruit fly species, has evolved high desiccation resistance by producing long-chain methyl-branched cuticular hydrocarbons (mbCHCs) that contribute to a cuticular lipid layer reducing water loss. We show that the ability to synthesize these longer mbCHCs is due to evolutionary changes in a fatty acyl-CoA elongase (mElo). mElo knockout in D. mojavensis led to loss of longer mbCHCs and reduction of desiccation resistance at high temperatures but did not affect mortality at either high temperatures or desiccating conditions individually. Phylogenetic analysis showed that mElo is a Drosophila-specific gene, suggesting that while the physiological mechanisms underlying desert adaptation may be similar between species, the genes involved in these mechanisms may be species or lineage specific.

mRNA decapping activators Pat1 and Dhh1 regulate transcript abundance and translation to tune cellular responses to nutrient availability.

We have examined the roles of yeast mRNA decapping-activators Pat1 and Dhh1 in repressing the translation and abundance of specific mRNAs in nutrient-replete cells using ribosome profiling, RNA-Seq, CAGE analysis of capped mRNAs, RNA Polymerase II ChIP-Seq, and TMT-mass spectrometry of mutants lacking one or both factors. Although the Environmental Stress Response (ESR) is activated in dhh1Δ and pat1Δ mutants, hundreds of non-ESR transcripts are elevated in a manner indicating cumulative repression by Pat1 and Dhh1 in wild-type cells. These mRNAs show both reduced decapping and diminished transcription in the mutants, indicating that impaired mRNA turnover drives transcript derepression in cells lacking Dhh1 or Pat1. mRNA degradation stimulated by Dhh1/Pat1 is not dictated by poor translation nor enrichment for suboptimal codons. Pat1 and Dhh1 also collaborate to reduce translation and protein production from many mRNAs. Transcripts showing concerted translational repression by Pat1/Dhh1 include mRNAs involved in cell adhesion or utilization of the poor nitrogen source allantoin. Pat1/Dhh1 also repress numerous transcripts involved in respiration, catabolism of non-preferred carbon or nitrogen sources, or autophagy; and we obtained evidence for elevated respiration and autophagy in the mutants. Thus, Pat1 and Dhh1 function as post-transcriptional repressors of multiple pathways normally activated only during nutrient limitation.

Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in dcp2Δ cells that appears to result directly from impaired decapping rather than elevated transcription. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Edc3, or Scd6; whereas most of the remaining transcripts utilize nonsense-mediated mRNA decay factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed that dcp2Δ confers widespread changes in relative translational efficiencies (TEs) that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased by dcp2Δ, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs in dcp2Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are upregulated, and both mitochondrial function and cell filamentation are elevated in dcp2Δ cells, suggesting that decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.

Factors Influencing Disabled Stroke Survivors' Quality of Life in Rural China: Based on the Structural Characteristics and Psychometric Properties of the SF-36 Assessment.

Many stroke survivors' quality of life is impaired. Few studies of factors influencing their quality of life have been based on the factors tested by the short form 36 instrument. This study did so with 308 physically disabled stroke survivors in rural China. Principal components analysis was applied to refine the dimension structure of the short form 36 assessment, followed by backward multiple linear regression analysis to determine the independent factors influencing quality of life. The structure revealed differed from the generic structure in showing that the mental health and vitality dimensions are not unidimensional. Subjects who reported access to the outdoors as convenient demonstrated better quality of life in all dimensions. Those who exercised regularly achieved better social functioning and negative mental health scores. Other factors influencing a better quality of life in terms of physical functioning were younger age and not being married. Being older and better educated predicted better role-emotion scores. Being female correlated with better social functioning scores, while men scored better on bodily pain. Being less educated predicted higher negative mental health, while being less disabled predicted better physical and social functioning. The results suggest that the SF-36's dimension structure should be re-evaluated before using it to assess stroke survivors.

Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in dcp2 Δ cells that appears to result directly from impaired decapping rather than elevated transcription, which was confirmed by ChIP-Seq analysis of RNA Polymerase II occupancies genome-wide. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Lsm2, Edc3 or Scd6; whereas most of the remaining transcripts utilize NMD factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed that dcp2 Δ confers widespread changes in relative TEs that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased by dcp2 Δ, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs in dcp2 Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are derepressed, and both mitochondrial function and cell filamentation (a strategy for nutrient foraging) are elevated by dcp2 Δ, suggesting that mRNA decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.

Assessing the risk of prenatal depressive symptoms in Chinese women: an integrated evaluation of serum metabolome, multivitamin supplement intake, and clinical blood indicators.

Background: Prenatal depressive symptoms (PDS) is a serious public health problem. This study aimed to develop an integrated panel and nomogram to assess at-risk populations by examining the association of PDS with the serum metabolome, multivitamin supplement intake, and clinical blood indicators. Methods: This study comprised 221 pregnant women, categorized into PDS and non-PDS groups based on the Edinburgh postnatal depression scale. The participants were divided into training and test sets according to their enrollment time. We conducted logistic regression analysis to identify risk factors, and employed liquid chromatography/high resolution mass spectrometry-based serum metabolome analysis to identify metabolic biomarkers. Multiple factor analysis was used to combine risk factors, clinical blood indicators and key metabolites, and then a nomogram was developed to estimate the probability of PDS. Results: We identified 36 important differential serum metabolites as PDS biomarkers, mainly involved in amino acid metabolism and lipid metabolism. Multivitamin intake works as a protective factor for PDS. The nomogram model, including multivitamin intake, HDL-C and three key metabolites (histidine, estrone and valylasparagine), exhibited an AUC of 0.855 in the training set and 0.774 in the test set, and the calibration curves showed good agreement, indicating that the model had good stability. Conclusion: Our approach integrates multiple models to identify metabolic biomarkers for PDS, ensuring their robustness. Furthermore, the inclusion of dietary factors and clinical blood indicators allows for a comprehensive characterization of each participant. The analysis culminated in an intuitive nomogram based on multimodal data, displaying potential performance in initial PDS risk assessment.

Large-scale analyses of angiosperm Flowering Locus T genes reveal duplication and functional divergence in monocots.

FLOWERING LOCUS T (FT) are well-known key genes for initiating flowering in plants. Delineating the evolutionary history and functional diversity of FT genes is important for understanding the diversification of flowering time and how plants adapt to the changing surroundings. We performed a comprehensive phylogenetic analysis of FT genes in 47 sequenced flowering plants and the 1,000 Plant Transcriptomes (1KP) database with a focus on monocots, especially cereals. We revealed the evolutionary history of FT genes. The FT genes in monocots can be divided into three clades (I, II, and III), whereas only one monophyletic group was detected in early angiosperms, magnoliids, and eudicots. Multiple rounds of whole-genome duplications (WGD) events followed by gene retention contributed to the expansion and variation of FT genes in monocots. Amino acid sites in the clade II and III genes were preferentially under high positive selection, and some sites located in vital domain regions are known to change functions when mutated. Clade II and clade III genes exhibited high variability in important regions and functional divergence compared with clade I genes; thus, clade I is more conserved than clade II and III. Genes in clade I displayed higher expression levels in studied organs and tissues than the clade II and III genes. The co-expression modules showed that some of the FT genes might have experienced neofunctionalization and subfunctionalization, such as the acquisition of environmental resistance. Overall, FT genes in monocots might form three clades by the ancient gene duplication, and each clade was subsequently subjected to different selection pressures and amino acid substitutions, which eventually led to different expression patterns and functional diversification. Our study provides a global picture of FT genes' evolution in monocots, paving a road for investigating FT genes' function in future.

TSSr: an R package for comprehensive analyses of TSS sequencing data.

Transcription initiation is regulated in a highly organized fashion to ensure proper cellular functions. Accurate identification of transcription start sites (TSSs) and quantitative characterization of transcription initiation activities are fundamental steps for studies of regulated transcriptions and core promoter structures. Several high-throughput techniques have been developed to sequence the very 5'end of RNA transcripts (TSS sequencing) on the genome scale. Bioinformatics tools are essential for processing, analysis, and visualization of TSS sequencing data. Here, we present TSSr, an R package that provides rich functions for mapping TSS and characterizations of structures and activities of core promoters based on all types of TSS sequencing data. Specifically, TSSr implements several newly developed algorithms for accurately identifying TSSs from mapped sequencing reads and inference of core promoters, which are a prerequisite for subsequent functional analyses of TSS data. Furthermore, TSSr also enables users to export various types of TSS data that can be visualized by genome browser for inspection of promoter activities in association with other genomic features, and to generate publication-ready TSS graphs. These user-friendly features could greatly facilitate studies of transcription initiation based on TSS sequencing data. The source code and detailed documentations of TSSr can be freely accessed at https://github.com/Linlab-slu/TSSr.

Transcriptome analysis provides genome annotation and expression profiles in the central nervous system of Lymnaea stagnalis at different ages.

BACKGROUND: The pond snail, Lymnaea stagnalis (L. stagnalis), has served as a valuable model organism for neurobiology studies due to its simple and easily accessible central nervous system (CNS). L. stagnalis has been widely used to study neuronal networks and recently gained popularity for study of aging and neurodegenerative diseases. However, previous transcriptome studies of L. stagnalis CNS have been exclusively carried out on adult L. stagnalis only. As part of our ongoing effort studying L. stagnalis neuronal growth and connectivity at various developmental stages, we provide the first age-specific transcriptome analysis and gene annotation of young (3 months), adult (6 months), and old (18 months) L. stagnalis CNS. RESULTS: Using the above three age cohorts, our study generated 55-69 millions of 150 bp paired-end RNA sequencing reads using the Illumina NovaSeq 6000 platform. Of these reads, ~ 74% were successfully mapped to the reference genome of L. stagnalis. Our reference-based transcriptome assembly predicted 42,478 gene loci, of which 37,661 genes encode coding sequences (CDS) of at least 100 codons. In addition, we provide gene annotations using Blast2GO and functional annotations using Pfam for ~ 95% of these sequences, contributing to the largest number of annotated genes in L. stagnalis CNS so far. Moreover, among 242 previously cloned L. stagnalis genes, we were able to match ~ 87% of them in our transcriptome assembly, indicating a high percentage of gene coverage. The expressional differences for innexins, FMRFamide, and molluscan insulin peptide genes were validated by real-time qPCR. Lastly, our transcriptomic analyses revealed distinct, age-specific gene clusters, differentially expressed genes, and enriched pathways in young, adult, and old CNS. More specifically, our data show significant changes in expression of critical genes involved in transcription factors, metabolisms (e.g. cytochrome P450), extracellular matrix constituent, and signaling receptor and transduction (e.g. receptors for acetylcholine, N-Methyl-D-aspartic acid, and serotonin), as well as stress- and disease-related genes in young compared to either adult or old snails. CONCLUSIONS: Together, these datasets are the largest and most updated L. stagnalis CNS transcriptomes, which will serve as a resource for future molecular studies and functional annotation of transcripts and genes in L. stagnalis.

The origin and evolution of a distinct mechanism of transcription initiation in yeasts.

The molecular process of transcription by RNA Polymerase II is highly conserved among eukaryotes ("classic model"). A distinct way of locating transcription start sites (TSSs) has been identified in a budding yeast Saccharomyces cerevisiae ("scanning model"). Herein, we applied genomic approaches to elucidate the origin of the scanning model and its underlying genetic mechanisms. We first identified TSSs at single-nucleotide resolution for 12 yeast species using the nAnT-iCAGE technique, which significantly improved the annotations of these genomes by providing accurate 5' boundaries for protein-coding genes. We then inferred the initiation mechanism of each species based on its TSS maps and genome sequences. We discovered that the scanning model likely originated after the split of Yarrowia lipolytica and the other budding yeasts. Species that use the scanning model showed an adenine-rich region immediately upstream of the TSS that might facilitate TSS selection. Both initiation mechanisms share a strong preference for pyrimidine-purine dinucleotides surrounding the TSS. Our results suggest that the purine is required to accurately recruit the first nucleotide, thereby increasing the chances of a messenger RNA of being capped during mRNA maturation, which is critical for efficient translation initiation during protein biosynthesis. Based on our findings, we propose a model for TSS selection in the scanning-model species, as well as a model for the stepwise process responsible for the origin and evolution of the scanning model.

Distinct roles of nucleosome sliding and histone modifications in controlling the fidelity of transcription initiation.

Regulation of gene expression starts from the transcription initiation. Regulated transcription initiation is critical for generating correct transcripts with proper abundance. The impact of epigenetic control, such as histone modifications and chromatin remodelling, on gene regulation has been extensively investigated, but their specific role in regulating transcription initiation is far from well understood. Here we aimed to better understand the roles of genes involved in histone H3 methylations and chromatin remodelling on the regulation of transcription initiation at a genome-scale using the budding yeast as a study system. We obtained and compared maps of transcription start site (TSS) at single-nucleotide resolution by nAnT-iCAGE for a strain with depletion of MINC (Mot1-Ino80C-Nc2) by Mot1p and Ino80p anchor-away (Mot1&Ino80AA) and a strain with loss of histone methylation (set1Δset2Δdot1Δ) to their wild-type controls. Our study showed that the depletion of MINC stimulated transcription initiation from many new sites flanking the dominant TSS of genes, while the loss of histone methylation generates more TSSs in the coding region. Moreover, the depletion of MINC led to less confined boundaries of TSS clusters (TCs) and resulted in broader core promoters, and such patterns are not present in the ssdΔ mutant. Our data also exhibits that the MINC has distinctive impacts on TATA-containing and TATA-less promoters. In conclusion, our study shows that MINC is required for accurate identification of bona fide TSSs, particularly in TATA-containing promoters, and histone methylation contributes to the repression of transcription initiation in coding regions.

Gap Junction Coding Innexin in Lymnaea stagnalis: Sequence Analysis and Characterization in Tissues and the Central Nervous System.

Connections between neurons called synapses are the key components underlying all nervous system functions of animals and humans. However, important genetic information on the formation and plasticity of one type, the electrical (gap junction-mediated) synapse, is understudied in many invertebrates. In the present study, we set forth to identify and characterize the gap junction-encoding gene innexin in the central nervous system (CNS) of the mollusk pond snail Lymnaea stagnalis. With PCR, 3' and 5' RACE, and BLAST searches, we identified eight innexin genes in the L. stagnalis genome, named Lst Inx1-Lst Inx8. Phylogenetic analysis revealed that the L. stagnalis innexin genes originated from a single copy in the common ancestor of molluskan species by multiple gene duplication events and have been maintained in L. stagnalis since they were generated. The paralogous innexin genes demonstrate distinct expression patterns among tissues. In addition, one paralog, Lst Inx1, exhibits heterogeneity in cells and ganglia, suggesting the occurrence of functional diversification after gene duplication. These results introduce possibilities to study an intriguing potential relationship between innexin paralog expression and cell-specific functional outputs such as heterogenic ability to form channels and exhibit synapse plasticity. The L. stagnalis CNS contains large neurons and functionally defined networks for behaviors; with the introduction of L. stagnalis in the gap junction gene field, we are providing novel opportunities to combine genetic research with direct investigations of functional outcomes at the cellular, synaptic, and behavioral levels.

Origins and Stepwise Expansion of R2R3-MYB Transcription Factors for the Terrestrial Adaptation of Plants.

The R2R3-MYB transcription factors play critical roles in various processes in embryophytes (land plants). Here, we identified genes encoding R2R3-MYB proteins from rhodophytes, glaucophytes, Chromista, chlorophytes, charophytes, and embryophytes. We classified the R2R3-MYB genes into three subgroups (I, II, and III) based on their evolutionary history and gene structure. The subgroup I is the most ancient group that includes members from all plant lineages. The subgroup II was formed before the divergence of charophytes and embryophytes. The subgroup III genes form a monophyletic group and only comprise members from land plants with conserved exon-intron structure. Each subgroup was further divided into multiple clades. The subgroup I can be divided into I-A, I-B, I-C, and I-D. The I-A, I-B, and I-C are the most basal clades that have originated before the divergence of Archaeplastida. The I-D with the II and III subgroups form a monophyletic group, containing only green plants. The II and III subgroups form another monophyletic group with Streptophyta only. Once on land, the subgroup III genes have experienced two rounds of major expansions. The first round occurred before the origin of land plants, and the second round occurred after the divergence of land plants. Due to significant gene expansion, the subgroup III genes have become the predominant group of R2R3-MYBs in land plants. The highly unbalanced pattern of birth and death evolution of R2R3-MYB genes indicates their important roles in the successful adaptation and massive radiation of land plants to occupy a multitude of terrestrial environments.