Plant Functional Genomics Research Articles

An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics.

The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is used extensively for efficient progress in plant functional genomics. Here we provide details about the establishment of the material, demonstrate the population's functionality and discuss results from quality control studies. T-DNA insertion mutants of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation. To allow selection of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed. DNA from leaves of T1 plants was extracted and used as a template for PCR-based amplification of DNA fragments spanning insertion site borders. After sequencing, the data were placed in a flanking sequence tag (FST) database describing which mutant allele was present in which line. Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions. Insertion sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes. Segregation analysis for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus. In quality control studies with gene-specific primers in combination with T-DNA primers, 76% of insertions could be confirmed. Finally, the functionality of the GABI-Kat population was demonstrated by exemplary confirmation of several new transparent testa alleles, as well as a number of other mutants, which were identified on the basis of the FST data.

Post-transcriptional gene silencing in plants by RNA.

RNA silencing, which is termed post-transcriptional gene silencing in plants, is an RNA degradation process through sequence-specific nucleotide interactions induced by double-stranded RNA. In plants, RNA silencing not only serves as a component of the defense mechanism, but also participates in the regulation of endogenous gene expression in a variety of developmental processes. This review elaborates the current progress on the understanding of the molecular basis of RNA silencing including a mechanistic link between the regulation of microRNA and RNA silencing. The practical use of RNA silencing as a reverse genetics approach in plant functional genomics is also discussed.

Single-nucleotide mutations for plant functional genomics.

In the present genomics era, powerful reverse-genetic strategies are needed to elucidate gene and protein function in the context of a whole organism. However, most current techniques lack the generality and high-throughput potential of descriptive genomic approaches, such as those that rely on microarray hybridization. For example, in plant research, effective insertional mutagenesis and transgenic methods are limited to relatively few species or are inefficient. Fortunately, single-nucleotide changes can be induced in any plant by using traditional chemical mutagens, and progress has been made in efficiently detecting changes. Because base substitutions in proteins provide allelic series, and not just knockouts, this strategy can yield refined insights into protein function. Here, we review recent progress that has been made in genome-wide screening for point mutations and natural variation in plants. Its general applicability leads to the expectation that traditional mutagenesis followed by high-throughput detection will become increasingly important for plant functional genomics.

Combinatorial modification of multiple lignin traits in trees through multigene cotransformation.

Lignin quantity and reactivity [which is associated with its syringyl/guaiacyl (S/G) constituent ratio] are two major barriers to wood-pulp production. To verify our contention that these traits are regulated by distinct monolignol biosynthesis genes, encoding 4-coumarate-CoA ligase (4CL) and coniferaldehyde 5-hydroxylase (CAld5H), we used Agrobacterium to cotransfer antisense 4CL and sense CAld5H genes into aspen (Populus tremuloides). Trees expressing each one and both of the transgenes were produced with high efficiency. Lignin reduction by as much as 40% with 14% cellulose augmentation was achieved in antisense 4CL plants; S/G-ratio increases as much as 3-fold were observed without lignin quantity change in sense CAld5H plants. Consistent with our contention, these effects were independent but additive, with plants expressing both transgenes having up to 52% less lignin, a 64% higher S/G ratio, and 30% more cellulose. An S/G-ratio increase also accelerated cell maturation in stem secondary xylem, pointing to a role for syringyl lignin moieties in coordinating xylem secondary wall biosynthesis. The results suggest that this multigene cotransfer system should be broadly useful for plant genetic engineering and functional genomics.

Correlated clustering and virtual display of gene expression patterns in the wheat life cycle by large-scale statistical analyses of expressed sequence tags.

Compared to rice, wheat exhibits characteristic growth habits and contains complex genome constituents. To assess global changes in gene expression patterns in the wheat life cycle, we conducted large-scale analysis of expressed sequence tags (ESTs) in common wheat. Ten wheat tissues were used to construct cDNA libraries: crown and root from 14-day-old seedlings; spikelet from early and late flowering stages; spike at the booting stage, heading date and flowering date; pistil at the heading date; and seeds at 10 and 30 days post-anthesis. Several thousand colonies were randomly selected from each of these 10 cDNA libraries and sequenced from both 5' and 3' ends. Consequently, a total of 116 232 sequences were accumulated and classified into 25 971 contigs based on sequence homology. By computing abundantly expressed ESTs, correlated expression patterns of genes across the tissues were identified. Furthermore, relationships of gene expression profiles among the 10 wheat tissues were inferred from global gene expression patterns. Genes with similar functions were grouped with one another by clustering gene expression profiles. This technique might enable estimation of the functions of anonymous genes. Multidimensional analysis of EST data that is analogous to the microarray experiments may offer new approaches to functional genomics of plants.

Unique Tissue‐Specific Cell Cycle in Physcomitrella

Abstract: The moss Physcomitrella patens (Hedw.) B.S.G. is a novel tool in plant functional genomics as it has an inimitable high gene targeting efficiency facilitating the establishment of gene/function relationships.Here we report, based on flow cytrometric (FCM) data, that the basic nuclear DNA content per cell of Physcomitrella is 0.53 pg, equating to a genome size of 1 C = 511 Mbp. Furthermore, we describe a unique tissue‐specific cell cycle change in this plant. Young plants consisting of only one cell type (chloronema) displayed one single peak of fluorescence in FCM analyses. As soon as the second cell type (caulonema) developed from chloronema, a second peak of fluorescence at half the intensity of the previous one became detectable, indicating that caulonema cells were predominantly at the G1/S transition, whereas chloronema cells were mainly accumulating at the G2/M transition. This conclusion was validated by further evidence: i) The addition of ammonium tartrate arrested Physcomitrella in the chloronema state and in G2/M. ii) Two different developmental mutants, known to be arrested in the chloronema/caulonema transition, remained in G2/M, regardless of age and treatment. iii) The addition of auxin or cytokinin induced the formation of caulonema, as well as decreasing the amount of cells in G2/M phase. Additionally, plant growth regulators promoted endopolyploidisation.Thus, cell cycle and cell differentiation are closely linked in Physcomitrella and effects of plant hormones and environmental factors on both processes can be analysed in a straight forward way. We speculate that this unique tissue‐specific cell cycle arrest may be the reason for the uniquely high rate of homologous recombination found in the Physcomitrella nuclear DNA.

Maintaining collections of mutants for plant functional genomics.

As the plant genomics era progresses and post-genomic functional research rapidly expands, varied genetic resources of unprecedented power and scope are being developed. Partially by the mandate of public funding, these resources are being shared via stock centers and private laboratories. The successful initiation of any new research requires that advantage be taken of these stocks. Information on most plant genomic resources can be obtained through simple yet powerful. Web searches, and ordering mechanisms are linked to the information. Hence, locating and obtaining materials is rapid and simple. Currently, available genomic resources are described, and references, links for Web data, and ordering information are also included.

Rice Gene Machine: A Vehicle for Finding Functions of Cereal Genes

Identifying functions for the predicted 25 000 to 40 000 plant genes is the new focus of genomic research following the sequencing of genomes from model plants like Arabidopsis and rice. It is important that researchers and plant breeders especially from Asian countries access the techniques and resources of plant functional genomics, not only to remain competitive globally in rice research and development activities, but also to increase yields by alleviating some of the rice production constraints. These newly identified genes and gene control sequences can be used in transformation breeding and as molecular markers in classical plant breeding. At CSIRO Plant Industry we are using an insertional mutagenesis approach in rice to relate genes to their functions. With national and international collaboration we hope to create a substantial "Rice Gene Machine" comprised of many thousands of rice insertion lines with their associated phenotypes and the gene sequences flanking insertion sites identified. This Rice Gene Machine will allow the identification of gene functions, which will benefit rice research and the rice industry for years to come.

Promoter subfragments of the sugar beet V-type H + -ATPase subunit c isoform drive the expression of transgenes in the moss Physcomitrella patens

Based on the relative ease of performing tar- geted nuclear gene knockout, the moss Physcomitrella patens has recently been developed as a model system for plant functional genomics. To address the need for new promoters that could drive expression of transgenes in this moss, we tested two fragments of the promoter re- gion of the gene for the sugar beet (Beta vulgaris) V-type H + -ATPase subunit isoform c. Four gene knockout con- structs were tested in which the neomycin phosphotrans- ferase II selection marker gene was put under the control of two distinct V-type H + -ATPase promoter fragments, the NOS promoter, or the CaMV 35S promoter. In each case the selection cassettes were flanked by moss FtsZ1 cDNA sequences to facilitate chromosomal targeting. From a total of more than 440 transformed plants, the number of plants generated per construct was monitored and found to be in the range of 5 to 11 stable transgenics per transformation. Both V-type H+-ATPase promoter fragments lead to NPTII expression levels that were suf- ficiently high to generate large numbers of stable trans- genic plants. The numbers of plants obtained with the two V-type H + -ATPase promoter fragments were compa- rable to those with constructs containing the standard NOS and 35S promoters. We propose that the higher plant V-type H+-ATPase promoter can be used for the expression of transgenes in the bryophyte P. patens.

Plant functional genomics.

Functional genome analysis of plants has entered the high-throughput stage. The complete genome information from key species such as Arabidopsis thaliana and rice is now available and will further boost the application of a range of new technologies to functional plant gene analysis. To broadly assign functions to unknown genes, different fast and multiparallel approaches are currently used and developed. These new technologies are based on known methods but are adapted and improved to accommodate for comprehensive, large-scale gene analysis, i.e. such techniques are novel in the sense that their design allows researchers to analyse many genes at the same time and at an unprecedented pace. Such methods allow analysis of the different constituents of the cell that help to deduce gene function, namely the transcripts, proteins and metabolites. Similarly the phenotypic variations of entire mutant collections can now be analysed in a much faster and more efficient way than before. The different methodologies have developed to form their own fields within the functional genomics technological platform and are termed transcriptomics, proteomics, metabolomics and phenomics. Gene function, however, cannot solely be inferred by using only one such approach. Rather, it is only by bringing together all the information collected by different functional genomic tools that one will be able to unequivocally assign functions to unknown plant genes. This review focuses on current technical developments and their impact on the field of plant functional genomics. The lower plant Physcomitrella is introduced as a new model system for gene function analysis, owing to its high rate of homologous recombination.

DNA microarrays for functional plant genomics.

DNA microarray technology is a key element in today's functional genomics toolbox. The power of the method lies in miniaturization, automation and parallelism permitting large-scale and genome-wide acquisition of quantitative biological information from multiple samples. DNA microarrays are currently fabricated and assayed by two main approaches involving either in situ synthesis of oligonucleotides ('oligonucleotide microarrays') or deposition of pre-synthesized DNA fragments ('cDNA microarrays') on solid surfaces. To date, the main applications of microarrays are in comprehensive, simultaneous gene expression monitoring and in DNA variation analyses for the identification and genotyping of mutations and polymorphisms. Already at a relatively early stage of its application in plant science, microarrays are being utilized to examine a range of biological issues including the circadian clock, plant defence, environmental stress responses, fruit ripening, phytochrome A signalling, seed development and nitrate assimilation. Novel insights are obtained into the molecular mechanisms co-ordinating metabolic pathways, regulatory and signalling networks. Exciting new information will be gained in the years to come not only from genome-wide expression analyses on a few model plant species, but also from extensive studies of less thoroughly studied species on a more limited scale. The value of microarray technology to our understanding of living processes will depend both on the amount of data to be generated and on its clever exploration and integration with other biological knowledge arising from complementary functional genomics tools for 'profiling' the genome, proteome, metabolome and phenome.

Media Law

External control of gene expression in plants is critical to both characterization of gene function and plant biotechnology. Ethanol-inducible gene expression system (‘alc switch’) is such a system suitable for regulated expression of transgene or native gene in plants temporally, spatially and quantitatively. The alc switch consists of two expression cassettes:p35S:alcR and palcA:target gene. The system has high specificity and efficiency. The induction exhibits a rapid, reversible and dose-dependent manner. Ethanol, the chemical inducer, is non-toxic, inexpensive, safe and easy to apply both in lab and field. The alc switch has been successfully used in Arabidopsis, tobacco, potato, oilseed rape and other plant species. It is believed that alc switch will have broad utility in plant functional genomics and molecular farming.

Growth Stage-Based Phenotypic Analysis of Arabidopsis: A Model for High Throughput Functional Genomics in Plants

Growth Stage-Based Phenotypic Analysis of Arabidopsis: A Model for High Throughput Functional Genomics in Plants

Growth Stage-Based Phenotypic Analysis of Arabidopsis: A Model for High Throughput Functional Genomics in Plants

Growth Stage-Based Phenotypic Analysis of Arabidopsis: A Model for High Throughput Functional Genomics in Plants

Metabolite profiling for plant functional genomics.

Multiparallel analyses of mRNA and proteins are central to today's functional genomics initiatives. We describe here the use of metabolite profiling as a new tool for a comparative display of gene function. It has the potential not only to provide deeper insight into complex regulatory processes but also to determine phenotype directly. Using gas chromatography/mass spectrometry (GC/MS), we automatically quantified 326 distinct compounds from Arabidopsis thaliana leaf extracts. It was possible to assign a chemical structure to approximately half of these compounds. Comparison of four Arabidopsis genotypes (two homozygous ecotypes and a mutant of each ecotype) showed that each genotype possesses a distinct metabolic profile. Data mining tools such as principal component analysis enabled the assignment of "metabolic phenotypes" using these large data sets. The metabolic phenotypes of the two ecotypes were more divergent than were the metabolic phenotypes of the single-loci mutant and their parental ecotypes. These results demonstrate the use of metabolite profiling as a tool to significantly extend and enhance the power of existing functional genomics approaches.

Plant genomics and functional genomics

Plant genomics and functional genomics

A transgenic perspective on plant functional genomics.

Transgenic crops are very much in the news due to the increasing public debate on their acceptance. In the scientific community though, transgenic plants are proving to be powerful tools to study various aspects of plant sciences. The emerging scientific revolution sparked by genomics based technologies is producing enormous amounts of DNA sequence information that, together with plant transformation methodology, is opening up new experimental opportunities for functional genomics analysis. An overview is provided here on the use of transgenic technology for the functional analysis of plant genes in model plants and a link made to their utilization in transgenic crops. In transgenic plants, insertional mutagenesis using heterologous maize transposons or Agrobacterium mediated T-DNA insertions, have been valuable tools for the identification and isolation of genes that display a mutant phenotype. To discover functions of genes that do not display phenotypes when mutated, insertion sequences have been engineered to monitor or change the expression pattern of adjacent genes. These gene detector insertions can detect adjacent promoters, enhancers or gene exons and precisely reflect the expression pattern of the tagged gene. Activation tag insertions can mis-express the adjacent gene and confer dominant phenotypes that help bridge the phenotype gap. Employment of various forms of gene silencing technology broadens the scope of recovering knockout phenotypes for genes with redundant function. All these transgenic strategies describing gene-phenotype relationships can be addressed by high throughput reverse genetics methods that will help provide functions to the genes discovered by genome sequencing. The gene functions discovered by insertional mutagenesis and silencing strategies along with expression pattern analysis will provide an integrated functional genomics perspective and offer unique applications in transgenic crops.

All We Are Saying Is Give Plants a Chance!

This article has no abstract.

A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations.

Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) flanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green fluorescent protein transgene that had been designed to contain a single base deletion. Recovery of fluorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.

Plant functional genomics.

Nucleotide sequencing of the Arabidopsis genome is nearing completion, sequencing of the rice genome has begun, and large amounts of expressed sequence tag information are being obtained for many other plants. There are many opportunities to use this wealth of sequence information to accelerate progress toward a comprehensive understanding of the genetic mechanisms that control plant growth and development and responses to the environment.