Plant Functional Genomics Research Articles

Sequencing and functional validation of the JGI Brachypodium distachyon T-DNA collection.

Due to a large and growing collection of genomic and experimental resources, Brachypodium distachyon has emerged as a powerful experimental model for the grasses. To add to these resources we sequenced 21165 T-DNA lines, 15569 of which were produced in this study. This increased the number of unique insertion sites in the T-DNA collection by 21078, bringing the overall total to 26112. Thirty-seven per cent (9754) of these insertion sites are within genes (including untranslated regions and introns) and 28% (7217) are within 500bp of a gene. Approximately 31% of the genes in the v.2.1 annotation have been tagged in this population. To demonstrate the utility of this collection, we phenotypically characterized six T-DNA lines with insertions in genes previously shown in other systems to be involved in cellulose biosynthesis, hemicellulose biosynthesis, secondary cell wall development, DNA damage repair, wax biosynthesis and chloroplast synthesis. In all cases, the phenotypes observed supported previous studies, demonstrating the utility of this collection for plant functional genomics. The Brachypodium T-DNA collection can be accessed at http://jgi.doe.gov/our-science/science-programs/plant-genomics/brachypodium/brachypodium-t-dna-collection/.

Identifying markers associated with yield traits in Nagina22 rice mutants grown in low phosphorus field or in alternate wet/dry conditions

Mutants are powerful genetic resources in plant breeding and functional genomics studies. Sixty seven stable ethyl methane sulphonate (EMS) induced rice mutants and the wild type parent Nagina 22 (N22) were characterized for plant height, tiller number, panicle number and grain yield under normal, low P field and alternate wet and dry (AWD) conditions in the same season. They were also genotyped with 44 SSR markers and four Pup1 (Phosphorus uptake1) gene specific markers. Genetic diversity was analysed by combining phenotype and marker data using Ward- MLM method. Single marker analysis showed significant association of four markers RM19696, RM263, RM3688 and RM1942 with grain yield in all three conditions. K-1, a Pup1 gene specific marker was significantly associated with tiller number only under low P conditions. The average dissimilarity between mutants was 0.86 and cophenetic correlation coefficient was 0.74. Six mutants were selected as gain-of-function mutants as they showed significantly higher grain yield in all three conditions, compared with N22. The selected mutants are an important resource for gene discovery for enhanced tolerance to low P and water stress conditions and associated markers can be useful in marker assisted selection.

Advances on molecular mechanisms of Rehmannia glutinosa consecutive monoculture problem formation in multi-omics era

Although consecutive monoculture problems have been studied for many years, no effective treatments are currently available. The complexity of systems triggered the formation of consecutive monoculture problems was one major cause. This paper elaborated the physiological and ecological mechanisms of consecutive monoculture problem formation based on the interaction relationship among multiple factors presented in the rhizosphere soil of consecutive monoculture plants. At same time, in this paper the multiple interactions among cultivated medicinal plants, autotoxic allelochemicals and rhizosphere microbial were proposed to be most important causes that derived the formation of consecutive monoculture problem. The paper also highlighted the advantage of 'omics' technologies integrating plant functional genomics and metabolomics as well as microbial macro-omics in understanding the multiple factor interaction under a particular ecological environment. Additionally, taking R. glutinosa as an example, the paper reviewed the molecular mechanism for the formation of R. glutinosa consecutive monoculture problem from the perspective of the accumulation of allelopathic autotoxins, the rhizosphere microecology catastrophe and theresponding of consecutive monoculture plants. Simultaneously, the roles of mutilple 'omics' technologies in comprehending these formation mechanism were described in detail. This paper provides finally a new insight to solve systematically the mechanism of consecutive monoculture problem formation on molecular level.

CRISPR/Cas9-Based Genome Editing in Plants.

Recently, genome editing technologies have shown great potential in plants. The newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a new generation of genome editing tool rapidly replacing the earlier zinc finger nucleases and transcription activator-like effector nucleases systems. Indeed, due to its advantages of simplicity and high efficiency, the CRISPR/Cas9-based genome editing system is becoming a powerful tool in plant science research. Here, we introduce the technical features of the plant CRISPR/Cas9-based genome editing system and its applications in plant functional genomics studies and genetic improvement.

Current Status of Targeting Induced Local Lesions IN Genomes (TILLING) and its Relevance to Plant Functional Genomics, Polymorphism Assessment and Plant Breeding with Relevant Case Studies on Different Crops

One of the most important breakthroughs in the history of genetics was the discovery that mutations can be induced. TILLING (Targeting induced local lesions in genomes) a newly developed general reverse genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It was first explored and introduced 17 years ago in the early 2000’s by the efforts of Claire McCallum and her collaborators. TILLING allows the rapid and inexpensive detection of induced point mutations in populations of physically/chemically mutagenized individuals. EcoTILLING is an extension of TILLING, which uncover natural alleles at a locus contrary to induced mutations. In This review the over view of history, techniques, of TILLING and EcoTILLING and their application in functional genomics, DNA polymorphism assessment and crop breeding with relevant case study in different crops illustrated. Furthermore, this review describes the potentials, challenges and future prospects of TILLING.

Current status of potential applications of repurposed Cas9 for structural and functional genomics of plants

Redesigned Cas9 has emerged as a tool with various applications like gene editing, gene regulation, epigenetic modification and chromosomal imaging. Target specific single guide RNA (sgRNA) can be used with Cas9 for precise gene editing with high efficiency than previously known methods. Further, nuclease-deactivated Cas9 (dCas9) can be fused with activator or repressor for activation (CRISPRa) and repression (CRISPRi) of gene expression, respectively. dCas9 fused with epigenetic modifier like methylase or acetylase further expand the scope of this technique. Fluorescent probes can be tagged to dCas9 to visualize the chromosome. Due to its wide-spread application, simplicity, accessibility, efficacy and universality, this technique is expanding the structural and functional genomic studies of plant and developing CRISPR crops. The present review focuses on current status of using repurposed Cas9 system in these various areas, with major focus on application in plants. Major challenges, concerns and future directions of using this technique are discussed in brief.

Reverse Genetics and High Throughput Sequencing Methodologies for Plant Functional Genomics.

In the post-genomic era, increasingly sophisticated genetic tools are being developed with the long-term goal of understanding how the coordinated activity of genes gives rise to a complex organism. With the advent of the next generation sequencing associated with effective computational approaches, wide variety of plant species have been fully sequenced giving a wealth of data sequence information on structure and organization of plant genomes. Since thousands of gene sequences are already known, recently developed functional genomics approaches provide powerful tools to analyze plant gene functions through various gene manipulation technologies. Integration of different omics platforms along with gene annotation and computational analysis may elucidate a complete view in a system biology level. Extensive investigations on reverse genetics methodologies were deployed for assigning biological function to a specific gene or gene product. We provide here an updated overview of these high throughout strategies highlighting recent advances in the knowledge of functional genomics in plants.

Editorial (Thematic Issue: Advances in Plant Functional Genomics: Challenges and Applications)

Editorial (Thematic Issue: Advances in Plant Functional Genomics: Challenges and Applications)

Comparative Transcriptome Reconstruction of Four Hypericum Species Focused on Hypericin Biosynthesis.

Next generation sequencing technology rapidly developed research applications in the field of plant functional genomics. Several Hypericum spp. with an aim to generate and enhance gene annotations especially for genes coding the enzymes supposedly included in biosynthesis of valuable bioactive compounds were analyzed. The first de novo transcriptome profiling of Hypericum annulatum Moris, H. tomentosum L., H. kalmianum L., and H. androsaemum L. leaves cultivated in vitro was accomplished. All four species with only limited genomic information were selected on the basis of differences in ability to synthesize hypericins and presence of dark nodules accumulating these metabolites with purpose to enrich genomic background of Hypericum spp. H. annulatum was chosen because of high number of the dark nodules and high content of hypericin. H. tomentosum leaves are typical for the presence of only 1–2 dark nodules localized in the apical part. Both H. kalmianum and H. androsaemum lack hypericin and have no dark nodules. Four separated datasets of the pair-end reads were gathered and used for de novo assembly by Trinity program. Assembled transcriptomes were annotated to the public databases Swiss-Prot and non-redundant protein database (NCBI-nr). Gene ontology analysis was performed. Differences of expression levels in the marginal tissues with dark nodules and inner part of leaves lacking these nodules indicate a potential genetic background for hypericin formation as the presumed site of hypericin biosynthesis is in the cells adjacent to these structures. Altogether 165 contigs in H. annulatum and 100 contigs in H. tomentosum were detected as significantly differentially expressed (P < 0.05) and upregulated in the leaf rim tissues containing the dark nodules. The new sequences homologous to octaketide synthase and enzymes catalyzing phenolic oxidative coupling reactions indispensable for hypericin biosynthesis were discovered. The presented transcriptomic sequence data will improve current knowledge about the selected Hypericum spp. with proposed relation to hypericin biosynthesis and will provide a useful resource of genomic information for consequential studies in the field of functional genomics, proteomics and metabolomics.

A viral satellite DNA vector-induced transcriptional gene silencing via DNA methylation of gene promoter in Nicotiana benthamiana

Virus-induced gene silencing (VIGS) has been widely used for plant functional genomics study at the post-transcriptional level using various DNA or RNA viral vectors. However, while virus-induced transcriptional gene silencing (VITGS) via DNA methylation of gene promoter was achieved using several plant RNA viral vectors, it has not yet been done using a satellite DNA viral vector. In this study, a viral satellite DNA associated with tomato yellow leaf curl China virus (TYLCCNV), which has been modified as a VIGS vector in previous research, was developed as a VITGS vector. Firstly, the viral satellite DNA VIGS vector was further optimized to a more convenient p1.7A+2mβ vector with high silencing efficiency of the phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. Secondly, the constructed VITGS vector (TYLCCNV:35S), which carried a portion of the cauliflower mosaic virus 35S promoter, could successfully induce heritable transcriptional gene silencing (TGS) of the green fluorescent protein (GFP) gene in the 35S-GFP transgenic N. benthamiana line 16c plants. Moreover, bisulfite sequencing results revealed higher methylated cytosine residues at CG, CHG and CHH sites of the 35S promoter sequence in TYLCCNV:35S-inoculated plants than in TYLCCNV-inoculated line 16c plants (control). Overall, these results demonstrated that the viral satellite DNA vector could be used as an effective VITGS vector to study DNA methylation in plant genomes.

AraPPISite: a database of fine-grained protein-protein interaction site annotations for Arabidopsis thaliana.

Knowledge about protein interaction sites provides detailed information of protein-protein interactions (PPIs). To date, nearly 20,000 of PPIs from Arabidopsis thaliana have been identified. Nevertheless, the interaction site information has been largely missed by previously published PPI databases. Here, AraPPISite, a database that presents fine-grained interaction details for A. thaliana PPIs is established. First, the experimentally determined 3D structures of 27 A. thaliana PPIs are collected from the Protein Data Bank database and the predicted 3D structures of 3023 A. thaliana PPIs are modeled by using two well-established template-based docking methods. For each experimental/predicted complex structure, AraPPISite not only provides an interactive user interface for browsing interaction sites, but also lists detailed evolutionary and physicochemical properties of these sites. Second, AraPPISite assigns domain-domain interactions or domain-motif interactions to 4286 PPIs whose 3D structures cannot be modeled. In this case, users can easily query protein interaction regions at the sequence level. AraPPISite is a free and user-friendly database, which does not require user registration or any configuration on local machines. We anticipate AraPPISite can serve as a helpful database resource for the users with less experience in structural biology or protein bioinformatics to probe the details of PPIs, and thus accelerate the studies of plant genetics and functional genomics. AraPPISite is available at http://systbio.cau.edu.cn/arappisite/index.html .

Applications and roles of the CRISPR system in genome editing of plants

Genome editing is a valuable tool to target specific DNA sequences for mutagenesis in the genomes of microbes, plants, and animals. Although different genome editing technologies are available, the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, which utilizes engineered endonucleases to generate a double-stranded DNA break (DSB) in the target DNA region and subsequently stimulates site-specific mutagenesis through DNA repair machineries, is emerging as a powerful genome editing tool for elucidating mechanisms of protection from plant viruses, plant disease resistance, and gene functions in basic and applied research. In this review, we provide an overview of recent advances in the CRISPR system associated genome editing in plants by focusing on application of this technology in model plants, crop plants, fruit plants, woody plants and grasses and discuss how genome editing associated with the CRISPR system can provide insights into genome modifications and functional genomics in plants.

Overviews and Applications of the CRISPR/Cas9 System in Plant Functional Genomics and Creation of New Plant Germplasm

Overviews and Applications of the CRISPR/Cas9 System in Plant Functional Genomics and Creation of New Plant Germplasm

Progress of targeted genome modification approaches in higher plants.

Transgene integration in plants is based on illegitimate recombination between non-homologous sequences. The low control of integration site and number of (trans/cis)gene copies might have negative consequences on the expression of transferred genes and their insertion within endogenous coding sequences. The first experiments conducted to use precise homologous recombination for gene integration commenced soon after the first demonstration that transgenic plants could be produced. Modern transgene targeting categories used in plant biology are: (a) homologous recombination-dependent gene targeting; (b) recombinase-mediated site-specific gene integration; (c) oligonucleotide-directed mutagenesis; (d) nuclease-mediated site-specific genome modifications. New tools enable precise gene replacement or stacking with exogenous sequences and targeted mutagenesis of endogeneous sequences. The possibility to engineer chimeric designer nucleases, which are able to target virtually any genomic site, and use them for inducing double-strand breaks in host DNA create new opportunities for both applied plant breeding and functional genomics. CRISPR is the most recent technology available for precise genome editing. Its rapid adoption in biological research is based on its inherent simplicity and efficacy. Its utilization, however, depends on available sequence information, especially for genome-wide analysis. We will review the approaches used for genome modification, specifically those for affecting gene integration and modification in higher plants. For each approach, the advantages and limitations will be noted. We also will speculate on how their actual commercial development and implementation in plant breeding will be affected by governmental regulations.

High throughput transformation of a Sorghum cDNA library for rice improvement

The improvement of rice productivity is an important subject due to great population pressure. As plant functional genomics rapidly develops, increasing numbers of genes from different organisms are documented for rice improvement. Sorghum, a relative of rice, has many novel phenotypic characteristics such as strong stature, large panicle, drought tolerance, and a tolerance for low soil fertility, making this species an excellent gene resource for rice improvement. A Sorghum cDNA library was transformed into rice generating an overexpression population with 1153 fertile transgenic lines. Over 900 of these lines displayed phenotypic changes including: plant height, leaf shape and size, effective panicles, and other vegetative characteristics. Importantly, there were 19 lines with higher grain yield than the wild type, which demonstrates that the high-throughput transformation of overexpressing transgenes from Sorghum into rice is a highly efficient method for rice improvement.

Plant Circular RNAs (circRNAs): Transcriptional Regulation Beyond miRNAs in Plants

Current plant functional genomics is converging on two aspects to provide sustainable solutions to cater to the demands of the growing population: 1) engineering crops for sustainable food security, where recently identified CRISPR/Cas is playing a detrimental role (Belhaj et al., 2014) and 2) identifying regulators of the post-transcriptional regulation, which can be functionally engineered. Canonical splicing has been widely seen and associated with functional protein diversity in plants (Min et al., 2015) (Figure 1).

System approaches to study root hairs as a single cell plant model: current status and future perspectives.

Our current understanding of plant functional genomics derives primarily from measurements of gene, protein and/or metabolite levels averaged over the whole plant or multicellular tissues. These approaches risk diluting the response of specific cells that might respond strongly to the treatment but whose signal is diluted by the larger proportion of non-responding cells. For example, if a gene is expressed at a low level, does this mean that it is indeed lowly expressed or is it highly expressed, but only in a few cells? In order to avoid these issues, we adopted the soybean root hair cell, derived from a single, differentiated root epidermal cell, as a single-cell model for functional genomics. Root hair cells are intrinsically interesting since they are major conduits for root water and nutrient uptake and are also the preferred site of infection by nitrogen-fixing rhizobium bacteria. Although a variety of other approaches have been used to study single plant cells or single cell types, the root hair system is perhaps unique in allowing application of the full repertoire of functional genomic and biochemical approaches. In this mini review, we summarize our published work and place this within the broader context of root biology, with a significant focus on understanding the initial events in the soybean-rhizobium interaction.

Plant Comparative and Functional Genomics.

Plant Comparative and Functional Genomics.

MR VIGS: microRNA-based virus-induced gene silencing in plants.

In plants, microRNA (miRNA)-based virus-induced gene silencing, dubbed MR VIGS, is a powerful technique to delineate the biological functions of genes. By targeting to a specific sequence, miRNAs can knock down expression of genes with fewer off-target effects. Here, using a modified Cabbage leaf curling virus (CaLCuV) and Tobacco rattle virus (TRV) as vectors, we describe two virus-based miRNA expression systems to perform MR VIGS for plant functional genomics assays.

Laser assisted microdissection, an efficient technique to understand tissue specific gene expression patterns and functional genomics in plants.

Laser assisted microdissection (LAM) is an advanced technology used to perform tissue or cell-specific expression profiling of genes and proteins, owing to its ability to isolate the desired tissue or cell type from a heterogeneous population. Due to the specificity and high efficiency acquired during its pioneering use in medical science, the LAM technique has quickly been adopted for use in many biological researches. Today, it has become a potent tool to address a wide range of questions in diverse field of plant biology. Beginning with comparative transcriptome analysis of different tissues such as reproductive parts, meristems, lateral organs, roots etc., LAM has also been extensively used in plant-pathogen interaction studies, proteomics, and metabolomics. In combination with next generation sequencing and proteomics analysis, LAM has opened up promising opportunities in the area of large scale functional studies in plants. Ever since the advent of this technique, significant improvements have been achieved in term of its instrumentation and method, which has made LAM a more efficient tool applicable in wider research areas. Here, we discuss the advancement of LAM technique with special emphasis on its methodology and highlight its scope in modern research areas of plant biology. Although we put emphasis on use of LAM in transcriptome studies, which is mostly used, we also discuss its recent application and scope in proteome and metabolome studies.