Overexpression of RPOTmp Being Targeted to Either Mitochondria or Chloroplasts in Arabidopsis Leads to Overall Transcriptome Changes and Faster Growth.

The transcription of Arabidopsis organellar genes is performed by three nuclear-encoded RNA polymerases: RPOTm, RPOTmp, and RPOTp. The RPOTmp protein possesses ambiguous transit peptides, allowing participation in gene expression control in both mitochondria and chloroplasts, although its function in plastids is still under discussion. Here, we show that the overexpression of RPOTmp in Arabidopsis, targeted either to mitochondria or chloroplasts, disturbs the dormant seed state, and it causes the following effects: earlier germination, decreased ABA sensitivity, faster seedling growth, and earlier flowering. The germination of RPOTmp overexpressors is less sensitive to NaCl, while rpotmp knockout is highly vulnerable to salt stress. We found that mitochondrial dysfunction in the rpotmp mutant induces an unknown retrograde response pathway that bypasses AOX and ANAC017. Here, we show that RPOTmp transcribes the accD, clpP, and rpoB genes in plastids and up to 22 genes in mitochondria.

Plant biotechnology research with single-cell transcriptome: recent advancements and prospects.

KEY MESSAGE: Single-cell transcriptomic techniques have emerged as powerful tools in plant biology, offering high-resolution insights into gene expression at the individual cell level. This review highlights the rapid expansion of single-cell technologies in plants, their potential in understanding plant development, and their role in advancing plant biotechnology research. Single-cell techniques have emerged as powerful tools to enhance our understanding of biological systems, providing high-resolution transcriptomic analysis at the single-cell level. In plant biology, the adoption of single-cell transcriptomics has seen rapid expansion of available technologies and applications. This review article focuses on the latest advancements in the field of single-cell transcriptomic in plants and discusses the potential role of these approaches in plant development and expediting plant biotechnology research in the near future. Furthermore, inherent challenges and limitations of single-cell technology are critically examined to overcome them and enhance our knowledge and understanding.

A snapshot of the transcriptome of Medicago truncatula (Fabales: Fabaceae) shoots and roots in response to an arbuscular mycorrhizal fungus and the pea aphid (Acyrthosiphon pisum) (Hemiptera: Aphididae).

Plants simultaneously interact with belowground symbionts such as arbuscular mycorrhizal (AM) fungi and aboveground antagonists such as aphids. Generally, plants gain access to valuable resources including nutrients and water through the AM symbiosis and are more resistant to pests. Nevertheless, aphids' performance improves on mycorrhizal plants, and it remains unclear whether a more nutritious food source and/or attenuated defenses are the contributing factors. This study examined the shoot and root transcriptome of barrel medic (Medicago truncatula Gaertn.) plants highly colonized by the AM fungus Rhizophagus irregularis (Blaszk., Wubet, Renker, and Buscot) C. Walker and A. Schüßler (Glomerales: Glomeraceae) and exposed to 7 days of mixed age pea aphid (Acyrthosiphon pisum (Harris)) herbivory. The RNA-seq samples chosen for this study showed that aphids were heavier when fed mycorrhizal plants compared to nonmycorrhizal plants. We hypothesized that (i) insect-related plant defense pathways will be downregulated in shoots of mycorrhizal plants with aphids compared to nonmycorrhizal plants with aphids; (ii) pathways involved in nutrient acquisition, carbohydrate-related and amino acid transport will be upregulated in shoots of mycorrhizal plants with aphids compared to nonmycorrhizal plants with aphids; and (iii) roots of mycorrhizal plants with aphids will exhibit mycorrhiza-induced resistance. The transcriptome data revealed that the gene repertoire related to defenses, nutrient transport, and carbohydrates differs between nonmycorrhizal and mycorrhizal plants with aphids, which could explain the weight gain in aphids. We also identified novel candidate genes that are differentially expressed in nonmycorrhizal plants with aphids, thus setting the stage for future functional studies.

Establishment and Validation of a New Analysis Strategy for the Study of Plant Endophytic Microorganisms.

Amplicon sequencing of bacterial or fungal marker sequences is currently the main method for the study of endophytic microorganisms in plants. However, it cannot obtain all types of microorganisms, including bacteria, fungi, protozoa, etc., in samples, nor compare the relative content between endophytic microorganisms and plants and between different types of endophytes. Therefore, it is necessary to develop a better analysis strategy for endophytic microorganism investigation. In this study, a new analysis strategy was developed to obtain endophytic microbiome information from plant transcriptome data. Results showed that the new strategy can obtain the composition of microbial communities and the relative content between plants and endophytic microorganisms, and between different types of endophytic microorganisms from the plant transcriptome data. Compared with the amplicon sequencing method, more endophytic microorganisms and relative content information can be obtained with the new strategy, which can greatly broaden the research scope and save the experimental cost. Furthermore, the advantages and effectiveness of the new strategy were verified with different analysis of the microbial composition, correlation analysis, inoculant content test, and repeatability test.

A Comprehensive Guide to Potato Transcriptome Assembly.

We have witnessed a rapid advancement in high-throughput genome sequencing and the maturation of long-read technologies. However, an accurate assembly of polyploid potato genomes still remains challenging. Sequencing the double-monoploid genome of Solanum tuberosum Group Phureja (Xu et al., Nature 475:189-195, 2011) has enabled functional studies of polyploid potato cultivars using RNA sequencing (RNA-Seq) technologies, although with the limitation of not covering cultivar-specific gene expression. The accumulated RNA-Seq datasets from these cultivars can be leveraged to assemble tetraploid potato transcriptomes that enable the analysis of genes that are not limited to reference genome annotations. To increase transcriptomes' quality, short-read assemblies are nowadays complemented with full-length transcriptome sequencing using Pacific Biosciences or Oxford Nanopore platforms. In this chapter we give a detailed guide on a pipeline for de novo transcriptome assembly of polyploid potato genotypes and their integration into a pan-transcriptome.

Analysis and comprehensive comparison of PacBio and nanopore-based RNA sequencing of the Arabidopsis transcriptome.

BACKGROUND: The number of studies using third-generation sequencing utilising Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) is rapidly increasing in many different research areas. Among them, plant full-length single-molecule transcriptome studies have mostly used PacBio sequencing, whereas ONT is rarely used. Therefore, in this study, we examined ONT RNA sequencing methods in plants. We performed a detailed evaluation of reads from PacBio, Nanopore direct cDNA (ONT Dc), and Nanopore PCR cDNA (ONT Pc) sequencing including characteristics of raw data and identification of transcripts. In addition, matched Illumina data were generated for comparison. RESULTS: ONT Pc showed overall better raw data quality, whereas PacBio generated longer read lengths. In the transcriptome analysis, PacBio and ONT Pc performed similarly in transcript identification, simple sequence repeat analysis, and long non-coding RNA prediction. PacBio was superior in identifying alternative splicing events, whereas ONT Pc could estimate transcript expression levels. CONCLUSIONS: This paper made a comprehensive comparison of PacBio and nanopore-based RNA sequencing of the Arabidopsis transcriptome, the results indicate that ONT Pc is more cost-effective for generating extremely long reads and can characterise the transcriptome as well as quantify transcript expression. Therefore, ONT Pc is a new cost-effective and worthwhile method for full-length single-molecule transcriptome analysis in plants.

Predicting Antimicrobial and Other Cysteine-Rich Peptides in 1267 Plant Transcriptomes.

Antimicrobial peptides (AMPs) are a key component of innate immunity in various organisms including bacteria, insects, mammals, and plants. Their mode of action decreases the probability of developing resistance in pathogenic organisms, which makes them a promising object of study. However, molecular biology methods for searching for AMPs are laborious and expensive, especially for plants. Earlier, we developed a computational pipeline for identifying potential AMPs based on the cysteine motifs they usually possess. Since most motifs are too species-specific, a wide-scale screening of novel data is required to maintain the accuracy of searching algorithms. We have performed a search for potential AMPs in 1267 plant transcriptomes using our pipeline. On average, 50-150 peptides were revealed in each transcriptome. The data was verified by a BLASTp search in nr database to confirm peptide functions and by using random nucleotide sequences to estimate the fraction of erroneous predictions. The datasets obtained will be useful both for molecular biologists investigating AMPs in various organisms and for bioinformaticians developing novel algorithms of motif searching in transcriptomic and genomic sequences. The results obtained will represent a good reference point for future investigations in the fields mentioned above.

The transcriptome enables the identification of candidate genes behind medicinal value of Drumstick tree (Moringa oleifera).

Moringa oleifera is a plant well-known for its nutrition value, drought resistance and medicinal properties. cDNA libraries from five different tissues (leaf, root, stem, seed and flower) of M. oleifera cultivar Bhagya were generated and sequenced. We developed a bioinformatics pipeline to assemble transcriptome, along with the previously published M. oleifera genome, to predict 17,148 gene models. Few candidate genes related to biosynthesis of secondary metabolites, vitamins and ion transporters were identified. Expressions were further confirmed by real-time quantitative PCR experiments for few promising leads. Quantitative estimation of metabolites, as well as elemental analysis, was also carried out to support our observations. Enzymes in the biosynthesis of vitamins and metabolites like quercetin and kaempferol are highly expressed in leaves, flowers and seeds. The expression of iron transporters and calcium storage proteins were observed in root and leaves. In general, leaves retain the highest amount of small molecules of interest.

StarSeeker: an automated tool for mature duplex microRNA sequence identification based on secondary structure modeling of precursor molecule.

BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNA molecules that play a key role in gene regulation in both plants and animals. MicroRNA biogenesis involves the enzymatic processing of a primary RNA transcript. The final step is the production of a duplex molecule, often designated as miRNA:miRNA*, that will yield a functional miRNA by separation of the two strands. This miRNA will be incorporated into the RNA-induced silencing complex, which subsequently will bind to its target mRNA in order to suppress its expression. The analysis of miRNAs is still a developing area for computational biology with many open questions regarding the structure and function of this important class of molecules. Here, we present StarSeeker, a simple tool that outputs the putative miRNA* sequence given the precursor and the mature sequences. RESULTS: We evaluated StarSeeker using a dataset consisting of all plant sequences available in miRBase (6992 precursor sequences and 8496 mature sequences). The program returned a total of 15,468 predicted miRNA* sequences. Of these, 2650 sequences were matched to annotated miRNAs (~ 90% of the miRBase-annotated sequences). The remaining predictions could not be verified, mainly because they do not comply with the rule requiring the two overhanging nucleotides in the duplex molecule. CONCLUSIONS: The expression pattern of some miRNAs in plants can be altered under various abiotic stress conditions. Potential miRNA* molecules that do not degrade can thus be detected and also discovered in high-throughput sequencing data, helping us to understand their role in gene regulation.

Mitochondrial Biogenesis in Diverse Cauliflower Cultivars under Mild and Severe Drought. Impaired Coordination of Selected Transcript and Proteomic Responses, and Regulation of Various Multifunctional Proteins.

Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower (Brassica oleracea var. botrytis) cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly) in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Respiratory (e.g., complex II, IV (CII, CIV) and ATP synthase subunits), transporter (including diverse porin isoforms) and matrix multifunctional proteins (e.g., components of RNA editing machinery) were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots) immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX) regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation.

Plant Phenotypic and Transcriptional Changes Induced by Volatiles from the Fungal Root Pathogen Rhizoctonia solani.

Beneficial soil microorganisms can affect plant growth and resistance by the production of volatile organic compounds (VOCs). Yet, little is known on how VOCs from soil-borne plant pathogens affect plant growth and resistance. Here we show that VOCs released from mycelium and sclerotia of the fungal root pathogen Rhizoctonia solani enhance growth and accelerate development of Arabidopsis thaliana. Seedlings briefly exposed to the fungal VOCs showed similar phenotypes, suggesting that enhanced biomass and accelerated development are primed already at early developmental stages. Fungal VOCs did not affect plant resistance to infection by the VOC-producing pathogen itself but reduced aboveground resistance to the herbivore Mamestra brassicae. Transcriptomics of A. thaliana revealed that genes involved in auxin signaling were up-regulated, whereas ethylene and jasmonic acid signaling pathways were down-regulated by fungal VOCs. Mutants disrupted in these pathways showed similar VOC-mediated growth responses as the wild-type A. thaliana, suggesting that other yet unknown pathways play a more prominent role. We postulate that R. solani uses VOCs to predispose plants for infection from a distance by altering root architecture and enhancing root biomass. Alternatively, plants may use enhanced root growth upon fungal VOC perception to sacrifice part of the root biomass and accelerate development and reproduction to survive infection.

Catalyzing plant science research with RNA-seq.

Next generation DNA sequencing technologies are driving increasingly rapid, affordable and high resolution analyses of plant transcriptomes through sequencing of their associated cDNA (complementary DNA) populations; an analytical platform commonly referred to as RNA-sequencing (RNA-seq). Since entering the arena of whole genome profiling technologies only a few years ago, RNA-seq has proven itself to be a powerful tool with a remarkably diverse range of applications, from detailed studies of biological processes at the cell type-specific level, to providing insights into fundamental questions in plant biology on an evolutionary time scale. Applications include generating genomic data for heretofore unsequenced species, thus expanding the boundaries of what had been considered "model organisms," elucidating structural and regulatory gene networks, revealing how plants respond to developmental cues and their environment, allowing a better understanding of the relationships between genes and their products, and uniting the "omics" fields of transcriptomics, proteomics, and metabolomics into a now common systems biology paradigm. We provide an overview of the breadth of such studies and summarize the range of RNA-seq protocols that have been developed to address questions spanning cell type-specific-based transcriptomics, transcript secondary structure and gene mapping.