Plant Functional Genomics Research Articles

Targeting MAGO proteins with a peptide aptamer reinforces their essential roles in multiple rice developmental pathways.

Peptide aptamers are artificial short peptides that potentially interfere with the biological roles of their target proteins; however, this technology has not yet been applied to plant functional genomics. MAGO and Y14, the two core subunits of the exon junction complex (EJC), form obligate heterodimers in eukaryotes. In Oryza sativa L. (rice), each of the two genes has two homologs, designated OsMAGO1 and OsMAGO2, and OsY14a and OsY14b, respectively. Here, we characterized a 16-amino acida peptide aptamer (PAP) for the rice MAGO proteins. PAP and rice Y14 bound competitively to rice MAGO proteins. Specifically targeting the MAGO proteins by expressing the aptamer in transgenic rice plants did not affect the endogenous synthesis and accumulation of MAGO proteins; however, the phenotypic variations observed in multiple organs phenocopied those of transgenic rice plants harboring RNA interference (RNAi) constructs in which the accumulation of MAGO and/or OsY14a transcripts and MAGO proteins was downregulated severely. Morphologically, the aptamer transgenic plants were short with abnormally developed flowers, and the stamens exhibited reduced degradation and absorption of both the endothecium and tapetum, thus confirming that EJC core heterodimers play essential roles in rice development, growth and reproduction. This study reveals that as a complementary approach of RNAi, peptide aptamers are powerful tools for interfering with the function of proteins in higher plants.

Something new to explore: Plant viruses infecting and inducing gene silencing in filamentous fungi.

Functional genomics in plants has been facilitated greatly by the use of plant viruses to carry segments of host genes that can then promote the silencing of the RNAs expressed from the corresponding host genes; a process called virus-induced gene silencing (VIGS). The silencing of genes in filamentous fungi is either technically more problematic or labor-intensive, especially if transgenic plants need to be generated first. However, a recent paper from our team demonstrated that a plant virus could infect three related fungal species, as well as express a reporter gene ectopically, and also silence the correspondingly expressed reporter transgene. The gene expression and RNA silencing of the reporter gene was maintained for six passages in culture and also persisted in plants infected by the virus-infected fungus. Here, we consider how the virus can enter and migrate within the fungus, whether the virus can move back and forth between the fungus and the plant and the ramifications of this, the prospects for VIGS being used to silence fungal endogenes and possible biotechnological or therapeutic applications of using plant viruses for expressing foreign proteins in fungi or silencing fungal endogenes.

RNAi (RNA Interference) Vectors for Functional Genomics Study in Plants

The discovery of RNA interference (RNAi) has revolutionized gene silencing for basic as well as applied studies. RNAi is a homology-dependent gene silencing mechanism, triggered by the introduction of double-stranded RNA (dsRNA) molecule. Since it can specifically suppress function of the targeted gene, the technique has been very useful in functional genomic studies. For efficient knock-down of the targeted gene, Gateway cloning based RNAi Silencing (pRISI) vector was developed. When a target gene-specific trigger fragment is cloned as inverted repeats in the pRISI vector, it produces a hairpin containing RNA transcribed from a CaMV35S (in case of pRISI-Ca) or from an ubiquitin (in case of pRISI-Ub) promoter. Function of the pRISI-Ca vector was confirmed by RNAi-mediated gene silencing experiment in Arabidopsis thaliana for At2g30350 gene which was found to be involved in repair of methylated DNA. A similar vector, pRISI-Ub was also developed for gene silencing studies in monocotyledonous plants. Its function was successfully demonstrated by transient suppression of GUS (UidA) gene co-bombarded with GUS-RNAi construct in wheat leaf. The vectors would be useful in rapid and easy preparation of RNAi constructs for reverse genetic studies for functional genomics in plants.

The election process for the Board of Directors of the Metabolomics Society: call for nomination of new board directors

It’s election time! The International Metabolomics Society (www.metabolomicssociety.org) is a dynamic society, founded in 2004, and with a membership of almost one thousand scientists that is growing year on year. Historically, the primary activity of the Society has been to organize the premiere international conference in metabolomics each year, with alternating venues in North America, Europe and the greater Asia-Pacific region. While organizing this conference remains one of the most important events in the Society’s calendar, the Directors have recently put in place a range of other benefits and activities for its Members. The Society is led and managed by a Board of 13 Directors (http://www.metabolomicssociety.org/board_of_directors), consisting of leading metabolomics researchers from around the world, and which includes the three Officer positions of President, Treasurer and Secretary. The exiting Officers remain as part of the board for 2 further years to ensure a smooth transition of tasks and responsibilities. For operational efficiency, the Board devolves its daily activities across numerous committees and task groups. These include the Website & Communications Committee, the Publications Committee, a Membership Committee, a Conference & Training Committee and a Nominations & Election Committee. These committees typically consist of 3–6 board members. The task groups, such as the International Affiliation Task Group, the Industry Liaison Task Group, the Data Standards Task Group, the Metabolite Identification Task Group and the Society Strategy Task Group, each consist of a few board members and also have non-board participants from the community. Each Director commits to a term of 2 years to the board and the society. After that he/she has the option to stand for re-election for a second 2-year term. Around May/June each year the process for election of replacement Directors is initiated. In 2014, four current Directors will have their second term of duty finished and therefore will provide spaces for four new Directors to be elected. In addition, we have three Directors who will finish their first term and may stand for re-election. For the new positions available we are calling on our membership base to nominate outstanding and excited metabolomics research leaders who would be interested and have the time to be fully committed to serve on the Board of Directors of the Society. The Board is interested to have researchers nominated who are active in the metabolomics sciences and also who are keen and motivated to serve on the Board to develop and strengthen the visibility and viability of the Metabolomics Society further. The nominees also should be able to commit sufficient time to be actively involved in typically two of the Board’s tasks groups and committees, an average commitment of *2 h per week. The election process is driven and overseen by the Nominations & Election Committee, which consists of two Board members and two non-board Members who observe the process for transparency. Once the nomination process is finalized the Nominations & Election Committee will collect information from the nominees including a short biography and a statement of commitment which will be published on the Society’s website allowing the membership to select and vote for their preferred nominees. The voting process will finish in August each year. The Nominations & Election Committee then selects the nominees with the highest number of votes and invites them to become a Director. Beginning on October 1st, the newly elected Board members begin their term of office. U. Roessner Metabolomics Australia and Australian Centre for Plant Functional Genomics, School of Botany, The University of Melbourne, 3010 Parkville, VIC, Australia

TILLING for plant breeding.

TILLING is widely used in plant functional genomics. Mutagenesis and SNP detection is combined to allow for the isolation of mutations in genes of interest. It can also be used as a plant breeding tool, whereby variation in known or candidate genes of interest to breeding programs is generated. Here we describe a simple low-cost TILLING procedure.

A virus-induced gene silencing (VIGS) system for functional genomics in the parasitic plant Striga hermonthica.

BackgroundStriga hermonthica is a hemiparasitic weed that infects cereals in Sub Sahara Africa (SSA) resulting in up to 100% grain yield loss. This significant loss in grain yields is a major contributor to food insecurity and poverty in the region. Current strategies to control the parasite are costly, unavailable and remain unpracticed by small-scale farmers, underscoring the need for more economical and sustainable control strategies. Development of resistant germplasm is the most sustainable strategy in the control of S. hermonthica, but is constrained by paucity of resistance genes for introduction into crop germplasm. RNA interference (RNAi) has potential for developing host-derived resistance against S. hermonthica by transformation of host crops with RNAi sequences targeted at critical Striga genes. The application of RNAi in management of S. hermonthica is however constrained by lack of efficient high throughput screening protocols for the candidate genes for silencing, as well as sub optimal delivery of siRNAs into the parasite. In comparison to stable transformation, viral induced gene silencing (VIGS) is a rapid and powerful tool for plant functional genomics and provides an easy and effective strategy in screening for putative candidate genes to target through RNAi. In addition, VIGS allows for a secondary amplification of the RNAi signal increasing the siRNA threshold and facilitates siRNA transport through viral movement proteins. We tested the efficiency of the Tobacco rattle virus (TRV1 and TRV2) VIGS vectors in silencing S. hermonthica phytoene desaturase (PDS) gene through agrodrench and agro-infiltration.ResultsWe report the validation of VIGS in S. hermonthica using a silencing cassette generated from TRV with a PDS gene insert. Agro-infiltrated and agro-drenched S. hermonthica leaves showed photo-bleaching phenotypes typical for PDS silencing within 7 and 14 days post infection respectively. In both cases S. hermonthica plants recovered from photo-bleaching effects within 28 days post inoculation. The transformation efficiency of the VIGS protocol in S. hermonthica was (60 ± 2.9)%.ConclusionThese results demonstrate that the TRV-VIGS system work in S. hermonthica and can be used for candidate gene validation for their role in the parasite development and parasitism, with the ultimate goal of developing resistant transgenic maize.

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus-based gene-silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus-induced gene-silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene-silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N.benthamiana. GFP transgenic plants co-agro-inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat-shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV- but not BaMV-based vector could enhance gene-silencing efficiency in newly emerging leaves of N.benthamiana deficient in RNA-dependant RNA polymerase 6. The dual gene-silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.

English

Generation, screening and isolating mutants for any developmental and adaptive traits plays a major role in plant functional genomics research. Identification and exploitation of mutants possessing contrasting root growth behavior and salinity tolerance in rice will help us to identify key genes controlling these traits and in turn will be useful for manipulating abiotic stress tolerance through tilling and genetic engineering in rice. In this study, we have screened about 1500 mutants (M 2 generation) generated by treating an upland drought tolerant genotype Nagina 22 with Ethyl Methane Sulfonate (EMS), for their root growth behavior and salinity tolerance under hydroponic conditions. Six independent mutant lines possessing significantly shorter roots and three mutant lines exhibiting greater degree of salinity tolerance than the wild type plants were identified. The identified mutant lines were advanced to M 5 generation to allow the mutants to reach homozygosity, and the fixed mutants were confirmed for their phenotype. One mutant namely N22-C-241-5-6 was found to possess significantly shorter roots than wild type N22, and it was also noticed that the mutant was devoid of root cap. Among the three salinity tolerant mutant lines identified, N22-C-334-3 was found to possess a greater degree of tolerance upto 250 mM Nacl stress at germination stage. These identified mutant lines can be used for further physiological, biochemical and molecular biology experiments to identify candidate gene(s) controlling root growth behavior and salinity tolerance in rice. Keywords : Rice, mutation, EMS, altered rood growth and salinity tolerant mutant African Journal of Biotechnology Vol. 12(40), pp. 5852-5859

Progress on identification and analysis of DNase I hypersensitive sites in plant genomes

Eukaryotes's gene expression and regulation relies on the interaction of their cis-acting elements and trans-acting factors. The cis-elements are specific DNA sequences in the genome and frequently located in the untranslated regions. The trans-acting factors are usually considered to be proteins that bind to the cis-acting sequences to regulate gene expression. It is well known that the cis-elements are always associated with DNase I hypersensitive site, which is a signature of open chromatin. The identification of all the functional cis-element using high-throughput method in plant genome has not been initiated in plant genomes. With the rapid achievement of genomics studies, an increasing number of plant genomes have been sequenced. Genome-wide identification of DNase I hypersensitive sites will be a considerably efficient method to locate cis-element in plants, which will provide a vital potential for further plant functional genomics. The present review is to reveal the recent progresses on identification and analysis of DNase I hypersensitive site in plant genomes.

Epigenetic mechanisms of plant stress responses and adaptation

Epigenetics has become one of the hottest topics of research in plant functional genomics since it appears promising in deciphering and imparting stress-adaptive potential in crops and other plant species. Recently, numerous studies have provided new insights into the epigenetic control of stress adaptation. Epigenetic control of stress-induced phenotypic response of plants involves gene regulation. Growing evidence suggest that methylation of DNA in response to stress leads to the variation in phenotype. Transposon mobility, siRNA-mediated methylation and host methyltransferase activation have been implicated in this process. This review presents the current status of epigenetics of plant stress responses with a view to use this knowledge towards engineering plants for stress tolerance.

Novel genomes and genome constitutions identified by GISH and 5S rDNA and knotted1 genomic sequences in the genus Setaria

BackgroundThe Setaria genus is increasingly of interest to researchers, as its two species, S. viridis and S. italica, are being developed as models for understanding C4 photosynthesis and plant functional genomics. The genome constitution of Setaria species has been studied in the diploid species S. viridis, S. adhaerans and S. grisebachii, where three genomes A, B and C were identified respectively. Two allotetraploid species, S. verticillata and S. faberi, were found to have AABB genomes, and one autotetraploid species, S. queenslandica, with an AAAA genome, has also been identified. The genomes and genome constitutions of most other species remain unknown, even though it was thought there are approximately 125 species in the genus distributed world-wide.ResultsGISH was performed to detect the genome constitutions of Eurasia species of S. glauca, S. plicata, and S. arenaria, with the known A, B and C genomes as probes. No or very poor hybridization signal was detected indicating that their genomes are different from those already described. GISH was also performed reciprocally between S. glauca, S. plicata, and S. arenaria genomes, but no hybridization signals between each other were found. The two sets of chromosomes of S. lachnea both hybridized strong signals with only the known C genome of S. grisebachii. Chromosomes of Qing 9, an accession formerly considered as S. viridis, hybridized strong signal only to B genome of S. adherans. Phylogenetic trees constructed with 5S rDNA and knotted1 markers, clearly classify the samples in this study into six clusters, matching the GISH results, and suggesting that the F genome of S. arenaria is basal in the genus.ConclusionsThree novel genomes in the Setaria genus were identified and designated as genome D (S. glauca), E (S. plicata) and F (S. arenaria) respectively. The genome constitution of tetraploid S. lachnea is putatively CCC’C’. Qing 9 is a B genome species indigenous to China and is hypothesized to be a newly identified species. The difference in genome constitution and origin of S. verticillata and S. faberi is also discussed. The new genomes and the genome constitutions of Setaria species identified in this report provide useful information for Setaria germplasm management, foxtail millet breeding, grass evolution and the development of S. viridis and S. italica as a new model for functional genomics.

A series of TA-based and zero-background vectors for plant functional genomics.

With the sequencing of genomes from many organisms now complete and the development of high-throughput sequencing, life science research has entered the functional post-genome era. Therefore, deciphering the function of genes and how they interact is in greater demand. To study an unknown gene, the basic methods are either overexpression or gene knockout by creating transgenic plants, and gene construction is usually the first step. Although traditional cloning techniques using restriction enzymes or a site-specific recombination system (Gateway or Clontech cloning technology) are highly useful for efficiently transferring DNA fragments into destination plasmids, the process is time consuming and expensive. To facilitate the procedure of gene construction, we designed a TA-based cloning system in which only one step was needed to subclone a DNA fragment into vectors. Such a cloning system was developed from the pGreen binary vector, which has a minimal size and facilitates construction manipulation, combined with the negative selection marker gene ccdB, which has the advantages of eliminating the self-ligation background and directly enabling high-efficiency TA cloning technology. We previously developed a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, subcellular localization analysis and promoter activity detection. Our results show that such a system is highly efficient and serves as a high-throughput platform for transient or stable transformation in plants for functional genome research.

Induced Mutations Unleash the Potentials of Plant Genetic Resources for Food and Agriculture

The options for increasing food production by at least 70% over the next four decades so as to keep pace with a rapidly increasing human population are bedeviled by erratic climatic conditions, depleted arable lands, dwindling water resources and by the significant environmental and health costs for increasing the use of agrochemicals. Enhanced productivities through “smart” crop varieties that yield more with fewer inputs is a viable option. However, the genetic similarities amongst crop varieties—which render entire cropping systems vulnerable to the same stresses—coupled with unvarying parental materials limit the possibilities for uncovering novel alleles of genes and, hence, assembling new gene combinations to break yield plateaux and enhance resilience. Induced mutation unmasks novel alleles that are harnessed to breed superior crop varieties. The historical antecedents, theoretical and practical considerations, and the successes of induced mutations in crop improvement are reviewed along with how induced mutagenesis underpins plant functional genomics. The roles of cell and molecular biology techniques in enhancing the efficiencies for the induction, detection and deployment of mutation events are also reviewed. Also, the integration of phenomics into induced mutagenesis and the use of pre-breeding for facilitating the incorporation of mutants into crop improvement are advocated.

Recent Advances on Virus Induced Gene Silencing (VIGS): Plant Functional Genomics

Recent Advances on Virus Induced Gene Silencing (VIGS): Plant Functional Genomics

A simple plant gene delivery system using mesoporous silica nanoparticles as carriers.

A facile DNA delivery method would greatly facilitate studies of plant functional genomics. However, plant cell walls limit the utilization of nanoparticles on plant research. Here, we employed functionalized mesoporous silica nanoparticles (MSNs) to develop a MSN-mediated plant transient gene expression system. In this system, MSNs served as carriers to deliver foreign DNA into intact Arabidopsis thaliana roots without the aid of mechanical force. Gene expression was detected in the epidermal layer and in the more inner cortical and endodermal root tissues by both fluorescence and antibody labeling. This is a novel alternative to the conventional gene-gun or ultrasonic methods. In addition, the parameters that affect the MSN uptake and the mechanism and subcellular distribution of particles were also analyzed. The present study may provide valuable information on the manipulation of functional nanoparticles in plants and have significant impact on plant biotechnology.

Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening

Plant virus technology, in particular virus-induced gene silencing, is a widely used reverse- and forward-genetics tool in plant functional genomics. However the potential of virus technology to express genes to induce phenotypes or to complement mutants in order to understand the function of plant genes is not well documented. Here we exploit Potato virus X as a tool for virus-induced gene complementation (VIGC). Using VIGC in tomato, we demonstrated that ectopic viral expression of LeMADS-RIN, which encodes a MADS-box transcription factor (TF), resulted in functional complementation of the non-ripening rin mutant phenotype and caused fruits to ripen. Comparative gene expression analysis indicated that LeMADS-RIN up-regulated expression of the SBP-box (SQUAMOSA promoter binding protein-like) gene LeSPL-CNR, but down-regulated the expression of LeHB-1, an HD-Zip homeobox TF gene. Our data support the hypothesis that a transcriptional network may exist among key TFs in the modulation of fruit ripening in tomato.

High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants.

With the wide use of double-stranded RNA interference (RNAi) for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA) constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA) vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG) cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics.

High-throughput cryopreservation of plant cell cultures for functional genomics.

Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell–LSP solution mixtures was kept at −30°C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics.

HIGH EFFICIENCY TRANSFORMATION PLATFORMS FOR KIWIFRUIT (ACTINIDIA SPP.) FUNCTIONAL GENOMICS

ISHS XXVIII International Horticultural Congress on Science and Horticulture for People (IHC2010): International Symposium on Genomics and Genetic Transformation of Horticultural Crops HIGH EFFICIENCY TRANSFORMATION PLATFORMS FOR KIWIFRUIT (ACTINIDIA SPP.) FUNCTIONAL GENOMICS

Virus-induced gene silencing and its application in plant functional genomics

Virus-induced gene silencing is regarded as a powerful and efficient tool for the analysis of gene function in plants because it is simple, rapid and transformation-free. It has been used to perform both forward and reverse genetics to identify plant functional genes. Many viruses have been developed into virus-induced gene silencing vectors and gene functions involved in development, biotic and abiotic stresses, metabolism, and cellular signaling have been reported. In this review, we discuss the development and application of virus-induced gene silencing in plant functional genomics.