ClusTrast: a short read de novo transcript isoform assembler guided by clustered contigs.

BACKGROUND: Transcriptome assembly from RNA-sequencing data in species without a reliable reference genome has to be performed de novo, but studies have shown that de novo methods often have inadequate ability to reconstruct transcript isoforms. We address this issue by constructing an assembly pipeline whose main purpose is to produce a comprehensive set of transcript isoforms. RESULTS: We present the de novo transcript isoform assembler ClusTrast, which takes short read RNA-seq data as input, assembles a primary assembly, clusters a set of guiding contigs, aligns the short reads to the guiding contigs, assembles each clustered set of short reads individually, and merges the primary and clusterwise assemblies into the final assembly. We tested ClusTrast on real datasets from six eukaryotic species, and showed that ClusTrast reconstructed more expressed known isoforms than any of the other tested de novo assemblers, at a moderate reduction in precision. For recall, ClusTrast was on top in the lower end of expression levels (<15% percentile) for all tested datasets, and over the entire range for almost all datasets. Reference transcripts were often (35-69% for the six datasets) reconstructed to at least 95% of their length by ClusTrast, and more than half of reference transcripts (58-81%) were reconstructed with contigs that exhibited polymorphism, measuring on a subset of reliably predicted contigs. ClusTrast recall increased when using a union of assembled transcripts from more than one assembly tool as primary assembly. CONCLUSION: We suggest that ClusTrast can be a useful tool for studying isoforms in species without a reliable reference genome, in particular when the goal is to produce a comprehensive transcriptome set with polymorphic variants.

De novo transcriptome assembly and global analysis of differential gene expression of aphid tolerant wild mustard Rorippa indica (L.) Hiern infested by mustard aphid Lipaphis Erysimi (L.) Kaltenbach.

Rapeseed-mustard, the oleiferous Brassica species are important oilseed crops cultivated all over the globe. Mustard aphid Lipaphis erysimi (L.) Kaltenbach is a major threat to the cultivation of rapeseed-mustard. Wild mustard Rorippa indica (L.) Hiern shows tolerance to mustard aphids as a nonhost and hence is an important source for the bioprospecting of potential resistance genes and defense measures to manage mustard aphids sustainably. We performed mRNA sequencing of the R. indica plant uninfested and infested by the mustard aphids, harvested at 24 hours post-infestation. Following quality control, the high-quality reads were subjected to de novo assembly of the transcriptome. As there is no genomic information available for this potential wild plant, the raw reads will be useful for further bioinformatics analysis and the sequence information of the assembled transcripts will be helpful to design primers for the characterization of specific gene sequences. In this study, we also used the generated resource to comprehensively analyse the global profile of differential gene expression in R. indica in response to infestation by mustard aphids. The functional enrichment analysis of the differentially expressed genes reveals a significant immune response and suggests the possibility of chitin-induced defense signaling.

Integrated transcriptomic and WGCNA analyses reveal candidate genes regulating mainly flavonoid biosynthesis in Litsea coreana var. sinensis.

Litsea coreana Levl. var. sinensis (Allen) Yang et P. H. Huang is a popular ethnic herb and beverage plant known for its high flavonoid content, which has been linked to a variety of pharmacological benefits and crucial health-promoting impacts in humans. The progress in understanding the molecular mechanisms of flavonoid accumulation in this plant has been hindered due to the deficiency of genomic and transcriptomic resources. We utilized a combination of Illumina and Oxford Nanopore Technology (ONT) sequencing to generate a de novo hybrid transcriptome assembly. In total, 126,977 unigenes were characterized, out of which 107,977 were successfully annotated in seven public databases. Within the annotated unigenes, 3,781 were categorized into 58 transcription factor families. Furthermore, we investigated the presence of four valuable flavonoids-quercetin-3-O-ß-D-galactoside, quercetin-3-O-ß-D-glucoside, kaempferol-3-O-ß-D-galactoside, and kaempferol-3-O-ß-D-glucoside in 98 samples, using high-performance liquid chromatography. A weighted gene co-expression network analysis identified two co-expression modules, MEpink and MEturquoise, that showed strong positive correlation with flavonoid content. Within these modules, four transcription factor genes (R2R3-MYB, NAC, WD40, and ARF) and four key enzyme-encoding genes (CHI, F3H, PAL, and C4H) emerged as potential hub genes. Among them, the R2R3-MYB (LcsMYB123) as a homologous gene to AtMYB123/TT2, was speculated to play a significant role in flavonol biosynthesis based on phylogenetic analysis. Our findings provided a theoretical foundation for further research into the molecular mechanisms of flavonoid biosynthesis. Additionally, The hybrid transcriptome sequences will serve as a valuable molecular resource for the transcriptional annotation of L. coreana var. sinensis, which will contribute to the improvement of high-flavonoid materials.

Day-night gene expression reveals circadian gene disco as a candidate for diel-niche evolution in moths.

Temporal ecological niche partitioning is an underappreciated driver of speciation. While insects have long been models for circadian biology, the genes and circuits that allow adaptive changes in diel-niches remain poorly understood. We compared gene expression in closely related day- and night-active non-model wild silk moths, with otherwise similar ecologies. Using an ortholog-based pipeline to compare RNA-Seq patterns across two moth species, we find over 25 pairs of gene orthologs showing differential expression. Notably, the gene disco, involved in circadian control, optic lobe and clock neuron development in Drosophila, shows robust adult circadian mRNA cycling in moth heads. Disco is highly conserved in moths and has additional zinc-finger domains with specific nocturnal and diurnal mutations. We propose disco as a candidate gene for the diversification of temporal diel-niche in moths.

Development of EST-SSR markers in Bergenia ciliata using de novo transcriptome sequencing.

Bergenia ciliata (Haw.) Sternb. is an important herb predominantly found in the Indian Himalayan Region. It is widely used in medicines, healthcare systems, cosmetics, fodder, and ornamental purposes. The Illumina sequencing and de novo transcriptome assembly were carried out in B. ciliata to develop and identify simple sequence repeat markers. A total of 18 226 simple sequence repeats (SSRs) were identified wherein di-nucleotides were found to be abundant (47.88%), followed by mono-nucleotide (35.03%) and tri-nucleotide (15.88%) repeats. A total of 11 839 EST-SSR primers were designed, of which 96 primer pairs were commercially synthesized. Finally, 17 primer pairs revealed clear, distinct polymorphic bands, and these primers were validated with 40 diverse B. ciliata accessions. The present study revealed moderate level of genetic diversity (Ho = 0.389, He = 0.542, and PIC = 0.513). Furthermore, the transcriptome data and EST-SSR markers generated during the present investigation could be an important genetic resource for functional genomics, population studies, and conservation genetics of the genus Bergenia.

transXpress: a Snakemake pipeline for streamlined de novo transcriptome assembly and annotation.

BACKGROUND: RNA-seq followed by de novo transcriptome assembly has been a transformative technique in biological research of non-model organisms, but the computational processing of RNA-seq data entails many different software tools. The complexity of these de novo transcriptomics workflows therefore presents a major barrier for researchers to adopt best-practice methods and up-to-date versions of software. RESULTS: Here we present a streamlined and universal de novo transcriptome assembly and annotation pipeline, transXpress, implemented in Snakemake. transXpress supports two popular assembly programs, Trinity and rnaSPAdes, and allows parallel execution on heterogeneous cluster computing hardware. CONCLUSIONS: transXpress simplifies the use of best-practice methods and up-to-date software for de novo transcriptome assembly, and produces standardized output files that can be mined using SequenceServer to facilitate rapid discovery of new genes and proteins in non-model organisms.

Transcriptomic landscape of posterior regeneration in the annelid Platynereis dumerilii.

BACKGROUND: Restorative regeneration, the capacity to reform a lost body part following amputation or injury, is an important and still poorly understood process in animals. Annelids, or segmented worms, show amazing regenerative capabilities, and as such are a crucial group to investigate. Elucidating the molecular mechanisms that underpin regeneration in this major group remains a key goal. Among annelids, the nereididae Platynereis dumerilii (re)emerged recently as a front-line regeneration model. Following amputation of its posterior part, Platynereis worms can regenerate both differentiated tissues of their terminal part as well as a growth zone that contains putative stem cells. While this regeneration process follows specific and reproducible stages that have been well characterized, the transcriptomic landscape of these stages remains to be uncovered. RESULTS: We generated a high-quality de novo Reference transcriptome for the annelid Platynereis dumerilii. We produced and analyzed three RNA-sequencing datasets, encompassing five stages of posterior regeneration, along with blastema stages and non-amputated tissues as controls. We included two of these regeneration RNA-seq datasets, as well as embryonic and tissue-specific datasets from the literature to produce a Reference transcriptome. We used this Reference transcriptome to perform in depth analyzes of RNA-seq data during the course of regeneration to reveal the important dynamics of the gene expression, process with thousands of genes differentially expressed between stages, as well as unique and specific gene expression at each regeneration stage. The study of these genes highlighted the importance of the nervous system at both early and late stages of regeneration, as well as the enrichment of RNA-binding proteins (RBPs) during almost the entire regeneration process. CONCLUSIONS: In this study, we provided a high-quality de novo Reference transcriptome for the annelid Platynereis that is useful for investigating various developmental processes, including regeneration. Our extensive stage-specific transcriptional analysis during the course of posterior regeneration sheds light upon major molecular mechanisms and pathways, and will foster many specific studies in the future.

A reference transcriptome for walnut anthracnose pathogen, Ophiognomonia leptostyla, guides the discovery of candidate virulence genes.

Despite the economic losses due to the walnut anthracnose, Ophiognomonia leptostyla is an orphan fungus with respect to genomic resources. In the present study, the transcriptome of O. leptostyla was assembled for the first time. RNA sequencing was conducted for the fungal mycelia grown in a liquid media, and the inoculated leaf samples of walnut with the fungal conidia sampled at 48, 96 and 144 h post inoculation (hpi). The completeness, correctness, and contiguity of the de novo transcriptome assemblies generated with Trinity, Oases, SOAPdenovo-Trans and Bridger were compared to identify a single superior reference assembly. In most of the assessment criteria including N50, Transrate score, number of ORFs with known description in gene bank, the percentage of reads mapped back to the transcript (RMBT), BUSCO score, Swiss-Prot coverage bin and RESM-EVAL score, the Bridger assembly was the superior and thus used as a reference for profiling the O. leptostyla transcriptome in liquid media vs. during walnut infection. The k-means clustering of transcripts resulted in four distinct transcription patterns across the three sampling time points. Most of the detected CAZy transcripts had elevated transcription at 96 hpi that is hypothetically concurrent with the start of intracellular growth. The in-silico analysis revealed 103 candidate effectors of which six were members of Necrosis and Ethylene Inducing Like Protein (NLP) gene family belonging to three distinct k-means clusters. This study provided a complex and temporal pattern of the CAZys and candidate effectors transcription during six days post O. leptostyla inoculation on walnut leaves, introducing a list of candidate virulence genes for validation in future studies.

Morphological, phytochemical, and transcriptome analyses provide insights into the biosynthesis of monoterpenes in Monarda citriodora.

MAIN CONCLUSION: Morphological, phytochemical, and transcriptome analyses revealed candidate genes involved in the biosynthesis of volatile monoterpenes and development of glandular trichomes in Monarda citriodora. Monarda citriodora Cerv. ex Lag. is a valuable aromatic plant due to the presence of monoterpenes as major constituents in its essential oil (EO). Thus, it is of sheer importance to gain knowledge about the site of the biosynthesis of these terpenoid compounds in M. citriodora, as well as the genes involved in their biosynthesis. In this study, we studied different types of trichomes and their relative densities in three different developmental stages of leaves, early stage of leaf development (L1), mid-stage of leaf development (L2), and later stage of leaf development (L3) and the histochemistry of trichomes for the presence of lipid and terpenoid compounds. Further, the phytochemical analysis of this plant through GC-MS indicated a higher content of monoterpenes (thymol, thymoquinone, γ-terpinene, p-cymene, and carvacrol) in the L1 stage with a substantial decrease in the L3 stage of leaf development. This considerable decrease in the content of monoterpenes was attributed to the decrease in the trichome density from L1 to L3. Further, we developed a de novo transcriptome assembly by carrying out RNA sequencing of different plant parts of M. citriodora. The transcriptome data revealed several putative unigenes involved in the biosynthesis of specialized terpenoid compounds, as well as regulatory genes involved in glandular trichome development. The data generated in the present study build a strong foundation for further improvement of M. citriodora, in terms of quantity and quality of its essential oil, through genetic engineering.

RNA Viruses Are Prevalent and Active Tenants of the Predatory Mite Phytoseiulus persimilis (Acari: Phytoseiidae).

Many arthropod species harbor a diverse range of viruses. While much is known about pathogenic viruses of some economically important insects and arthropods involved in disease transmission, viruses associated with mites have rarely been studied. The main objective of this study was to characterize the virome of Phytoseiulus persimilis (Phytoseiidae), a predatory mite commercially used worldwide for the biological control of the key pest Tetranychus urticae (Tetranichidae). A combination of de novo transcriptome assembly and virion sequencing, revealed that RNA viruses are highly prevalent and active tenants of commercial populations of P. persimilis, comprising on average 9% of the mite's total mRNA. Seventeen RNA viruses dominated the mite's virome (i.e., were highly transcribed) with over half (n = 10) belonging to the order Picornavirales, + ssRNA viruses that infect a large range of hosts, including arthropods. Screening of the 17 dominant virus sequences in P. persimilis and T. urticae revealed that three viruses (two Picornavirales of the families Iflaviridae and Dicistroviridae, and one unclassified Riboviria) are unique to P. persimilis and three others (two unclassified Picornavirales and one unclassified Riboviria) are present in both mite species. Most of the sequences were related to viruses previously documented in economically important arthropods, while others have rarely been documented before in arthropods. These findings demonstrate that P. persimilis, like many other arthropods, harbors a diverse RNA virome, which might affect the mite's physiology and consequently its efficiency as a biological control agent.

Cross-species transcriptomes reveal species-specific and shared molecular adaptations for plants development on iron-rich rocky outcrops soils.

BACKGROUND: Canga is the Brazilian term for the savanna-like vegetation harboring several endemic species on iron-rich rocky outcrops, usually considered for mining activities. Parkia platycephala Benth. and Stryphnodendron pulcherrimum (Willd.) Hochr. naturally occur in the cangas of Serra dos Carajás (eastern Amazonia, Brazil) and the surrounding forest, indicating high phenotypic plasticity. The morphological and physiological mechanisms of the plants' establishment in the canga environment are well studied, but the molecular adaptative responses are still unknown. To understand these adaptative responses, we aimed to identify molecular mechanisms that allow the establishment of these plants in the canga environment. RESULTS: Plants were grown in canga and forest substrates collected in the Carajás Mineral Province. RNA was extracted from pooled leaf tissue, and RNA-seq paired-end reads were assembled into representative transcriptomes for P. platycephala and S. pulcherrimum containing 31,728 and 31,311 primary transcripts, respectively. We identified both species-specific and core molecular responses in plants grown in the canga substrate using differential expression analyses. In the species-specific analysis, we identified 1,112 and 838 differentially expressed genes for P. platycephala and S. pulcherrimum, respectively. Enrichment analyses showed that unique biological processes and metabolic pathways were affected for each species. Comparative differential expression analysis was based on shared single-copy orthologs. The overall pattern of ortholog expression was species-specific. Even so, we identified almost 300 altered genes between plants in canga and forest substrates with conserved responses in the two species. The genes were functionally associated with the response to light stimulus and the circadian rhythm pathway. CONCLUSIONS: Plants possess species-specific adaptative responses to cope with the substrates. Our results also suggest that plants adapted to both canga and forest environments can adjust the circadian rhythm in a substrate-dependent manner. The circadian clock gene modulation might be a central mechanism regulating the plants' development in the canga substrate in the studied legume species. The mechanism may be shared as a common mechanism to abiotic stress compensation in other native species.

Comparative transcriptomic analysis and antioxidant defense mechanisms in clusterbean (Cyamopsis tetragonoloba (L.) Taub.) genotypes with contrasting drought tolerance.

To understand drought tolerance mechanism(s) in clusterbean (Cyamopsis tetragonoloba), we conducted physiological, biochemical, and de novo comparative transcriptome analysis of drought-tolerant (RGC-1002) and drought-sensitive (RGC-1066) genotypes subjected to 30 days of drought stress. Relative water content (RWC) was maintained in tolerant genotype but was reduced in sensitive genotype. Leaf pigment concentrations were higher in tolerant genotype. Net photosynthesis was significantly decreased in sensitive genotype but insignificant reduction was found in tolerant genotype. Enzymatic antioxidant (GR, APX, DHAR) activities were enhanced in tolerant genotype, while there were insignificant changes in these enzymes in sensitive genotype. The ratios of antioxidant molecules (ASC/DHA and GSH/GSSG) were higher in tolerant genotype as compared to sensitive genotype. In sensitive genotype, 6625 differentially expressed genes (DEGs) were upregulated and 5365 genes were downregulated. In tolerant genotype, 5206 genes were upregulated and 2793 genes were downregulated. In tolerant genotype, transketolase family protein, phosphoenolpyruvate carboxylase 3, temperature-induced lipocalin, and cytochrome oxidase were highly upregulated. Moreover, according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the drought tolerance may be attributed to upregulated starch and sucrose metabolism-related genes in tolerant genotype. Finally, quantitative real-time PCR confirmed the reproducibility of the RNA-seq data.

Transcriptomic and fatty acid analyses of Neochloris aquatica grown under different nitrogen concentration.

In this study, we characterized the fatty acid production in Neochloris aquatica at transcriptomics and biochemical levels under limiting, normal, and excess nitrate concentrations in different growth phases. At the stationary phase, N. aquatica mainly produced saturated fatty acids such as stearic acid under the limiting nitrate concentration, which is suitable for biodiesel production. However, it produced polyunsaturated fatty acids such as α-linolenic acid under the excess nitrate concentration, which has nutritional values as food supplements. In addition, RNA-seq was employed to identify genes and pathways that were being affected in N. aquatica for three growth phases in the presence of the different nitrate amounts. Genes that are responsible for the production of saturated fatty acids were upregulated in the cells grown under a limiting nitrogen amount while genes that are responsible for the production of polyunsaturated fatty acid were upregulated in the cells grown under excess nitrogen amount. Further analysis showed more genes differentially expressed (DEGs) at the logarithmic phase in all conditions while a relatively steady trend was observed during the transition from the logarithmic phase to the stationary phase under limiting and excess nitrogen. Our results provide a foundation for identifying developmentally important genes and understanding the biological processes in the different growth phases of the N. aquatica in terms of biomass and lipid production.

Gene expression profiling before and after internode culture for adventitious shoot formation in ipecac.

BACKGROUND: In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), adventitious shoots can be induced simply by placing internodal segments on phytohormone-free culture medium. The shoots form locally on the epidermis of the apical region of the segments, but not the basal region. Levels of endogenous auxin and cytokinin transiently increase in the segments after 1 week of culture. RESULTS: Here, we conducted RNA-seq analysis to compare gene expression patterns in apical and basal regions of segments before culture and after 1 week of culture for adventitious shoot formation. The results revealed 8987 differentially expressed genes in a de novo assembly of 76,684 genes. Among them, 276 genes were upregulated in the apical region after 1 week of culture relative to before culture and the basal region after 1 week of culture. These genes include 18 phytohormone-response genes and shoot-formation-related genes. Validation of the gene expression by quantitative real-time PCR assay confirmed that the expression patterns were similar to those of the RNA-seq data. CONCLUSIONS: The transcriptome data show that expression of cytokinin biosynthesis genes is induced along with the acquisition of cellular pluripotency and the initiation of cell division by wounding in the apical region of internodal segments, that trigger adventitious shoot formation without callusing.

Pathogen resistance in Sphagneticola trilobata (Singapore daisy): molecular associations and differentially expressed genes in response to disease from a widespread fungus.

Understanding the molecular associations underlying pathogen resistance in invasive plant species is likely to provide useful insights into the effective control of alien plants, thereby facilitating the conservation of native biodiversity. In the current study, we investigated pathogen resistance in an invasive clonal plant, Sphagneticola trilobata, at the molecular level. Sphagneticola trilobata (i.e., Singapore daisy) is a noxious weed that affects both terrestrial and aquatic ecosystems, and is less affected by pathogens in the wild than co-occurring native species. We used Illumina sequencing to investigate the transcriptome of S. trilobata following infection by a globally distributed generalist pathogen (Rhizoctonia solani). RNA was extracted from leaves of inoculated and un-inoculated control plants, and a draft transcriptome of S. trilobata was generated to examine the molecular response of this species following infection. We obtained a total of 49,961,014 (94.3%) clean reads for control (un-inoculated plants) and 54,182,844 (94.5%) for the infected treatment (inoculated with R. solani). Our analyses facilitated the discovery of 117,768 de novo assembled contigs and 78,916 unigenes. Of these, we identified 3506 differentially expressed genes and 60 hormones associated with pathogen resistance. Numerous genes, including candidate genes, were associated with plant-pathogen interactions and stress response in S. trilobata. Many recognitions, signaling, and defense genes were differentially regulated between treatments, which were confirmed by qRT-PCR. Overall, our findings improve our understanding of the genes and molecular associations involved in plant defense of a rapidly spreading invasive clonal weed, and serve as a valuable resource for further work on mechanism of disease resistance and managing invasive plants.

de novo transcriptome assembly for discovering gene expressed in Holothuria leucospilota with exposed to copper.

Sea cucumber is a bioremediator as it can composite organic matter and excrete inorganic matter. Sea cucumber has the potential to serve as a bioindicator in marine habitat as they provide an integrated insight into the status of their environment over long periods. Sea cucumbers are sensitive to the organic concentration in the marine environment and can effectively provide an early warning system for any organic contamination that can negatively impact the ecosystem. The availability of a reference transcriptome for sea cucumber would constitute an essential tool for identifying genes involved in crucial steps of the defence pathway. De novo assembly of RNA-seq data enables researchers to study the transcriptomes without needing a genome sequence. In this study, sea cucumbers fed with Kappaphycus alvarezii powder were treated with 0.20 mg/L copper concentration comprehensive transcriptome data containing 75,149 Unigenes, with a total length of 20,460,032 bp. A total of 8820 genes were predicted from the unigenes, annotated, and functionally categorized into 25 functional groups with approximately 20% cluster in signal transduction mechanism. The reference transcriptome presented and validated in this study is meaningful for identifying a wide range of gene(s) related to the bioindication of sea cucumber in a high copper environment.

De Novo Transcriptome Analysis of R. nigrum cv. Aldoniai in Response to Blackcurrant Reversion Virus Infection.

The most damaging pathogen in blackcurrant plantations is mite-transmitted blackcurrant reversion virus (BRV). Some Ribes species have an encoded genetic resistance to BRV. We performed RNA sequencing analysis of BRV-resistant blackcurrant cv. Aldoniai to evaluate the molecular mechanisms related to the BRV infection response. The RNA of virus-inoculated and mock-inoculated microshoots was sequenced, and the transcriptional changes at 2- and 4-days post inoculation (dpi) were analyzed. The accumulation and expression of BRV RNA1 were detected in infected plants. In total, 159,701 transcripts were obtained and 30.7% were unigenes, annotated in 7 databases. More than 25,000 differentially expressed genes (DEGs) according to FPKM were upregulated or downregulated. We observed 221 and 850 DEGs at 2 and 4 dpi, respectively, in BRV-infected microshoots related to the stress response. The proportion of upregulated DEGs at 4 dpi was about 3.5 times higher than at 2 dpi. Pathways of the virus defense response were activated, and key candidate genes were identified. The phenylpropanoid and the cutin, suberine, and wax biosynthesis pathways were activated in infected plants. Our comparative de novo analysis of the R. nigrum transcriptome provides clues not only for understanding the molecular BRV resistance mechanisms but also for breeding BRV-tolerant genotypes.

In Silico Characterisation of the Late Embryogenesis Abundant (LEA) Protein Families and Their Role in Desiccation Tolerance in Ramonda serbica Panc.

Ramonda serbica Panc. is an ancient resurrection plant able to survive a long desiccation period and recover metabolic functions upon watering. The accumulation of protective late embryogenesis abundant proteins (LEAPs) is a desiccation tolerance hallmark. To propose their role in R. serbica desiccation tolerance, we structurally characterised LEAPs and evaluated LEA gene expression levels in hydrated and desiccated leaves. By integrating de novo transcriptomics and homologues LEAP domains, 318 R. serbica LEAPs were identified and classified according to their conserved motifs and phylogeny. The in silico analysis revealed that hydrophilic LEA4 proteins exhibited an exceptionally high tendency to form amphipathic α-helices. The most abundant, atypical LEA2 group contained more hydrophobic proteins predicted to fold into the defined globular domains. Within the desiccation-upregulated LEA genes, the majority encoded highly disordered DEH1, LEA1, LEA4.2, and LEA4.3 proteins, while the greatest portion of downregulated genes encoded LEA2.3 and LEA2.5 proteins. While dehydrins might chelate metals and bind DNA under water deficit, other intrinsically disordered LEAPs might participate in forming intracellular proteinaceous condensates or adopt amphipathic α-helical conformation, enabling them to stabilise desiccation-sensitive proteins and membranes. This comprehensive LEAPs structural characterisation is essential to understanding their function and regulation during desiccation aiming at crop drought tolerance improvement.

Comparing de novo transcriptome assembly tools in di- and autotetraploid non-model plant species.

BACKGROUND: Polyploidy is very common in plants and can be seen as one of the key drivers in the domestication of crops and the establishment of important agronomic traits. It can be the main source of genomic repatterning and introduces gene duplications, affecting gene expression and alternative splicing. Since fully sequenced genomes are not yet available for many plant species including crops, de novo transcriptome assembly is the basis to understand molecular and functional mechanisms. However, in complex polyploid plants, de novo transcriptome assembly is challenging, leading to increased rates of fused or redundant transcripts. Since assemblers were developed mainly for diploid organisms, they may not well suited for polyploids. Also, comparative evaluations of these tools on higher polyploid plants are extremely rare. Thus, our aim was to fill this gap and to provide a basic guideline for choosing the optimal de novo assembly strategy focusing on autotetraploids, as the scientific interest in this type of polyploidy is steadily increasing. RESULTS: We present a comparison of two common (SOAPdenovo-Trans, Trinity) and one recently published transcriptome assembler (TransLiG) on diploid and autotetraploid species of the genera Acer and Vaccinium using Arabidopsis thaliana as a reference. The number of assembled transcripts was up to 11 and 14 times higher with an increased number of short transcripts for Acer and Vaccinium, respectively, compared to A. thaliana. In diploid samples, Trinity and TransLiG performed similarly good while in autotetraploids, TransLiG assembled most complete transcriptomes with an average of 1916 assembled BUSCOs vs. 1705 BUSCOs for Trinity. Of all three assemblers, SOAPdenovo-Trans performed worst (1133 complete BUSCOs). CONCLUSION: All three assembly tools produced complete assemblies when dealing with the model organism A. thaliana, independently of its ploidy level, but their performances differed extremely when it comes to non-model autotetraploids, where specifically TransLiG and Trinity produced a high number of redundant transcripts. The recently published assembler TransLiG has not been tested yet on any plant organism but showed highest completeness and full-length transcriptomes, especially in autotetraploids. Including such species during the development and testing of new assembly tools is highly appreciated and recommended as many important crops are polyploid.

Comparative phenotypic and transcriptomic analyses unravel conserved and distinct mechanisms underlying shade avoidance syndrome in Brassicaceae vegetables.

BACKGROUND: Plants grown under shade are exposed to low red/far-red ratio, thereby triggering an array of altered phenotypes called shade avoidance syndrome (SAS). Shade negatively influences plant growth, leading to a reduction in agricultural productivity. Understanding of SAS is crucial for sustainable agricultural practices, especially for high-density indoor farming. Brassicaceae vegetables are widely consumed around the world and are commonly cultivated in indoor farms. However, our understanding of SAS in Brassicaceae vegetables and their genome-wide transcriptional regulatory networks are still largely unexplored. RESULTS: Shade induced common signs of SAS, including hypocotyl elongation and reduced carotenoids/anthocyanins biosynthesis, in two different Brassicaceae species: Brassica rapa (Choy Sum and Pak Choy) and Brassica oleracea (Kai Lan). Phenotype-assisted transcriptome analysis identified a set of genes induced by shade in these species, many of which were related to auxin biosynthesis and signaling [e.g. YUCCA8 (YUC8), YUC9, and INDOLE-3-ACETIC ACID INDUCIBLE (IAAs)] and other phytohormones signaling pathways including brassinosteroids and ethylene. The genes functioning in plant defense (e.g. MYB29 and JASMONATE-ZIM-DOMAIN PROTEIN 9) as well as in biosynthesis of anthocyanins and glucosinolates were repressed upon shade. Besides, each species also exhibited distinct SAS phenotypes. Shade strongly reduced primary roots and elongated petioles of B. oleracea, Kai Lan. However, these SAS phenotypes were not clearly recognized in B. rapa, Choy Sum and Pak Choy. Some auxin signaling genes (e.g. AUXIN RESPONSE FACTOR 19, IAA10, and IAA20) were specifically induced in B. oleracea, while homologs in B. rapa were not up-regulated under shade. Contrastingly, shade-exposed B. rapa vegetables triggered the ethylene signaling pathway earlier than B. oleracea, Kai Lan. Interestingly, shade induced the transcript levels of LONG HYPOCOTYL IN FAR-RED 1 (HFR1) homolog in only Pak Choy as B. rapa. As HFR1 is a key negative regulator of SAS in Arabidopsis, our finding suggests that Pak Choy HFR1 homolog may also function in conferring higher shade tolerance in this variety. CONCLUSIONS: Our study shows that two Brassicaceae species not only share a conserved SAS mechanism but also exhibit distinct responses to shade, which will provide comprehensive information to develop new shade-tolerant cultivars that are suitable for high-density indoor farms.