The completion of sequencing of the genome of Arabidopsis thaliana (AGI, 2000) provides the first detailed description of the genetic blue print of a higher plant (Lin et al. 1999).The availability of genome sequence of Arabidopsis is likely to broaden and accelerate further research in plant sciences. The knowledge gained from research on Arabidopsis will certainly benefit research efforts in other crop plants like rice (Rensink and Buell, 2004). In order to completely understand all the growth and developmental processes of plants at molecular level and to intelligently use the information to engineer crop and other plants, it is imperative that we know the function of the DNA sequences present in Arabidopsis and other plants. Having identified a new sequence, the comparison with sequences in the databases is the simplest way to obtain functional information. Although efficient bioinformatics tools are becoming available for the annotation of genome sequences, in silico analysis by itself is only indicative and is generally not sufficient to define function of a gene. Even in those cases where some indications become available from in silico analyses, experimental evidences are required. One of the most challenging tasks before plant scientists is assigning functions to a large number of plant genes. The functions of ~69% of the genes were classified according to sequence similarity to proteins of known function in all organisms (Berardini et al. 2004). Definitive functions for individual genes have been thoroughly established for less than 10% of the genes. Out of the ~26000 genes identified in Arabidopsis, the functions of only a few thousand have been defined with great confidence (Bouche and Bouchez, 2001) and more than 30% of the predicted Arabidopsis genes could not be assigned any specific function (AGI, 2000; Berardini et al. 2004).

Several approaches are used to clone and gather information about the function(s) of gene(s). Among these, insertional mutagenesis has been extensively used for generation, isolation and characterization of mutants. This approach is based on the inactivation of a gene via insertion of a known DNA fragment. DNA elements that are able to insert at random within chromosomes such as transposons (Sundaresan et al. 1995) or the T-DNA of Agrobacterium tumefacians can be used as mutagens to create loss of function mutations in plants (Ostergaard and Yanofsky, 2004). The mutant population thus generated is then used for cloning genes, promoters, enhancers and other regulatory sequences. The present article describes and highlights the significance of one such strategy of insertional mutagenesis namely T-DNA tagging, for functional genomic studies in Arabidopsis. An attempt has also been made to compile recent developments on cloning, characterization and identification of genes and promoter elements in Arabidopsis, using the T-DNA tagging approach. We describe the various experimental approaches employed to clone genes and regulatory sequences from T-DNA tagged lines and point out some of the common difficulties and problems encountered in the process. In addition, we also provide a list of resources on the T-DNA tagged Arabidopsis lines available to the research community.

T-DNA tagging strategy, exploits the fact that Agrobacterium can be used to introduce a piece of DNA into plant genome and a transgenic plant carrying the transferred DNA (T-DNA) that is stably integrated into the genome is obtained. The T-DNA not only disrupts the expression of the gene into which it is inserted, but also acts as a marker for subsequent identification of the mutation. An advantage of using T-DNA as the insertional mutagen as compared to transposons is that the T-DNA segments do not transpose subsequent to insertion and are chemically and physically stable through multiple generations.

Simple insertional mutagenesis, like all gene disruption approaches, has some limitations. It is difficult to identify the function of redundant genes or of genes required in early embryogenesis or gametophytic development. To overcome these limitations, a variety of ingenious T-DNA based vectors called promoter trap vectors, enhancer trap vector, activation tag vectors, exon trap vectors and gene trap vectors etc. have been designed to clone and characterize genes, promoters, enhancers and other regulatory elements. During the last few years T-DNA tagged lines have proved to be invaluable for the isolation of regulatory sequences and eventually the isolation of specifically expressed genes. This approach is particularly relevant to identification and cloning of genes (and their regulatory sequences) expressed in tissues that are difficult to analyze, by traditional methods relying on RNA extraction. The general principle behind promoter or gene trapping approach is to integrate a reporter gene that either lacks a promoter (gene/promoter trap) or carries only a minimal promoter (enhancer trap), at random sites in the wild type genome. A reporter gene cassette containing a minimal promoter (enhancer trap) close to the end of the insertion element can be cis activated when inserted close to a transcriptional enhancer that will drive the expression of the reporter gene.

Large collections of the T-DNA insertion lines are available at the Arabidopsis stock centers at Ohio State University (USA) and Nottingham (UK). More than 175,000 T-DNA insertion lines of various types are already available from the Arabidopsis Biological Resource Center (ABRC). The Salk Institute, through the efforts of Ecker and coworkers has established a computer database that contains the precise genomic locations of over 94,000 T-DNA insertions (Alonso et al. 2003).

Activation tagging is a method, which complements conventional insertional mutagenesis. This system uses T-DNA or a transposable element containing multimerized Cauliflower Mosaic Virus (CaMV) 35S enhancer. Because enhancers can function in either orientation and at a considerable distance from the coding regions, they can cause transcriptional activation of nearby genes, resulting in dominant gain of functions mutations. Such gene activations may produce novel phenotypes that identify important genes that are either redundant members of a gene family or are essential for survival.

The isolation of the disrupted gene in a T-DNA tagged mutant can be achieved by a number of strategies. Since the sequence of the inserted element is known, the gene in which the insertion has occurred can be recovered, using various cloning or PCR-based strategies. Sequences flanking the insertion can be easily identified from single or low copy number lines using inverse PCR (IPCR), thermal asymmetric interlaced (TAIL) PCR, Adaptor PCR or anchored PCR, T-linker PCR, single oligonucleotide nested (SON)-PCR and plasmid rescue (Ostergaard and Yanofsky, 2004). It is also possible to construct a genomic library of the mutant and isolate clones containing T-DNA sequences and identify the clones containing flanking plant genomic sequences.

An important companion to gene identification are functional genomic investigations aimed at determining the pattern of the expression of genes in the whole organism. The use of activation elements, enhancer traps and promoter traps in gene tagging studies is complementary to loss of function studies, because it provides the opportunity to generate new types of gain of function mutants. Thus promoter traps and enhancer traps provides a means of identifying genes and characterizing in vivo, the expression patterns of the tagged genes. Activation tags containing enhancer elements provoke tissue specific up regulation of activation tagged genes which potentially reveals more about the function of the gene than constitutive over expression.

A variety of problems are encountered while analyzing the T-DNA tagged lines, in particular when attempts are made to clone flanking sequences from some T-DNA tagged mutant, because of multiple insertions, complex arrangement of T-DNA, insertion of vector backbone sequences, chromosomal duplication and rearrangements or a combination of these (Jorgensen et al. 1987). In spite of these problems, different populations of large number of T-DNA tagged lines are becoming available, which will provide a wealth of information on functional aspects of several Arabidopsis gene sequences. T-DNA mutational approach is now being successfully modified to tag genes in a number of economically important plant species, rice (Chen et al. 2003; Sha et al. 2004), tomato (Meissner et al. 2000), Brassica napus (Bade et al. 2003), Medicago truncatula (Trieu et al. 2000) and poplar (Groover et al. 2004). Thus T-DNA tagging in conjunction with other molecular biology techniques would not only continue to provide useful information in Arabidopsis but is likely to prove an efficient tool for functional genomics in other plants also.

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