Functional analysis of maize RAD51 in meiosis and double-strand break repair.

In Saccharomyces cerevisiae, Rad51p plays a central role in homologous recombination and the repair of double-strand breaks (DSBs). Double mutants of the two Zea mays L. (maize) rad51 homologs are viable and develop well under normal conditions, but are male sterile and have substantially reduced seed set. Light microscopic analyses of male meiosis in these plants reveal reduced homologous pairing, synapsis of nonhomologous chromosomes, reduced bivalents at diakinesis, numerous chromosome breaks at anaphase I, and that >33% of quartets carry cells that either lack an organized nucleolus or have two nucleoli. This indicates that RAD51 is required for efficient chromosome pairing and its absence results in nonhomologous pairing and synapsis. These phenotypes differ from those of an Arabidopsis rad51 mutant that exhibits completely disrupted chromosome pairing and synapsis during meiosis. Unexpectedly, surviving female gametes produced by maize rad51 double mutants are euploid and exhibit near-normal rates of meiotic crossovers. The finding that maize rad51 double mutant embryos are extremely susceptible to radiation-induced DSBs demonstrates a conserved role for RAD51 in the repair of mitotic DSBs in plants, vertebrates, and yeast.

Identification of genes required for embryo development in Arabidopsis.

A long-term goal of Arabidopsis research is to define the minimal gene set needed to produce a viable plant with a normal phenotype under diverse conditions. This will require both forward and reverse genetics along with novel strategies to characterize multigene families and redundant biochemical pathways. Here we describe an initial dataset of 250 EMB genes required for normal embryo development in Arabidopsis. This represents the first large-scale dataset of essential genes in a flowering plant. When compared with 550 genes with other knockout phenotypes, EMB genes are enriched for basal cellular functions, deficient in transcription factors and signaling components, have fewer paralogs, and are more likely to have counterparts among essential genes of yeast (Saccharomyces cerevisiae) and worm (Caenorhabditis elegans). EMB genes also represent a valuable source of plant-specific proteins with unknown functions required for growth and development. Analyzing such unknowns is a central objective of genomics efforts worldwide. We focus here on 34 confirmed EMB genes with unknown functions, demonstrate that expression of these genes is not embryo-specific, validate a strategy for identifying interacting proteins through complementation with epitope-tagged proteins, and discuss the value of EMB genes in identifying novel proteins associated with important plant processes. Based on sequence comparison with essential genes in other model eukaryotes, we identify 244 candidate EMB genes without paralogs that represent promising targets for reverse genetics. These candidates should facilitate the recovery of additional genes required for seed development.

The Arabidopsis SeedGenes Project.

The SeedGenes database (http://www.seedgenes.org) presents molecular and phenotypic information on essential, non-redundant genes of Arabidopsis that give a seed phenotype when disrupted by mutation. Experimental details are synthesized for efficient use by the community and organized into two major sections in the database, one dealing with genes and the other with mutant alleles. The database can be queried for detailed information on a single gene to create a SeedGenes Profile. Queries can also generate lists of genes or mutants that fit specified criteria. The long-term goal is to establish a complete collection of Arabidopsis genes that give a knockout phenotype. This information is needed to focus attention on genes with important cellular functions in a model plant and to assess from a genetic perspective the extent of functional redundancy in the Arabidopsis genome.

A high-throughput Arabidopsis reverse genetics system.

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.

Condensin and cohesin knockouts in Arabidopsis exhibit a titan seed phenotype.

The titan (ttn) mutants of Arabidopsis exhibit striking alterations in chromosome dynamics and cell division during seed development. Endosperm defects include aberrant mitoses and giant polyploid nuclei. Mutant embryos differ in cell size, morphology and viability, depending on the locus involved. Here we demonstrate that three TTN genes encode chromosome scaffold proteins of the condensin (SMC2) and cohesin (SMC1 and SMC3) classes. These proteins have been studied extensively in yeast and animal systems, where they modulate chromosome condensation, chromatid separation, and dosage compensation. Arabidopsis contains single copies of SMC1 and SMC3 cohesins. We used forward genetics to identify duplicate T-DNA insertions in each gene. These mutants (ttn7 and ttn8) have similar titan phenotypes: giant endosperm nuclei and arrested embryos with a few small cells. A single SMC2 knockout (ttn3) was identified and confirmed by molecular complementation. The weak embryo phenotype observed in this mutant may result from expression of a related gene (AtSMC2) with overlapping functions. Further analysis of titan mutants and the SMC gene family in Arabidopsis should provide clues to chromosome mechanics in plants and insights into the regulation of nuclear activity during endosperm development.

Diversity of TITAN functions in Arabidopsis seed development.

The titan mutants of Arabidopsis exhibit striking defects in seed development. The defining feature is the presence of abnormal endosperm with giant polyploid nuclei. Several TTN genes encode structural maintenance of chromosome proteins (condensins and cohesins) required for chromosome function at mitosis. Another TTN gene product (TTN5) is related to the ARL2 class of GTP-binding proteins. Here, we identify four additional TTN genes and present a general model for the titan phenotype. TTN1 was cloned after two tagged alleles were identified through a large-scale screen of T-DNA insertion lines. The predicted gene product is related to tubulin-folding cofactor D, which interacts with ARL2 in fission yeast (Schizosaccharomyces pombe) and humans to regulate tubulin dynamics. We propose that TTN5 and TTN1 function in a similar manner to regulate microtubule function in seed development. The titan phenotype can therefore result from disruption of chromosome dynamics (ttn3, ttn7, and ttn8) or microtubule function (ttn1 and ttn5). Three other genes have been identified that affect endosperm nuclear morphology. TTN4 and TTN9 appear to encode plant-specific proteins of unknown function. TTN6 is related to the isopeptidase T class of deubiquitinating enzymes that recycle polyubiquitin chains following protein degradation. Disruption of this gene may reduce the stability of the structural maintenance of chromosome complex. Further analysis of the TITAN network should help to elucidate the regulation of microtubule function and chromosome dynamics in seed development.