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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.

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Maize is one of the most important crops in the developing world, where adverse soil conditions and low fertilizer input are the two main constraints for stable food supply. Understanding the molecular and biochemical mechanisms involved in nutrient uptake is expected to support the development of future breeding strategies aimed at improving maize productivity on infertile soils. Phosphorus is the least mobile macronutrient in the soils and it is often limiting plant growth. In this work, five genes encoding Pht1 phosphate transporters which contribute to phosphate uptake and allocation in maize were identified. In phosphate-starved plants, transcripts of most of the five transporters were present in roots and leaves. Independent of the phosphate supply, expression of two genes was predominant in pollen or in roots colonized by symbiotic mycorrhizal fungi, respectively. Interestingly, high transcript levels of the mycorrhiza-inducible gene were also detectable in leaves of phosphate-starved plants. Thus, differential expression of Pht1 phosphate transporters in maize suggests involvement of the encoded proteins in diverse processes, including phosphate uptake from soil and transport at the symbiotic interface in mycorrhizas, phosphate (re)translocation in the shoot, and phosphate uptake during pollen tube growth.

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.

In the present study, we determined the amount of homology required for targeted integration of DNA fragments into the yeast genome. The procedure described here facilitates the manipulation of the yeast genome and eliminates the need to clone sequences homologous to a target site. In addition, this method is useful for applications in which only limited sequence information of the target is available. The procedure comprises of: (i) production of PCR primers to amplify a selectable marker containing flanking homology to the target of choice; (ii) transformation of yeast cells and (iii) selection of integrants.Since the first development of this technique (1) different groups reported versions of this technique (2-4). However, the minimum length of homology required for targeted integration and the exact efficiencies of targeting as a function of homology length has not to date been determined.To determine the minimum amount of homology needed for integration of DNA fragments into the genome of yeast, DNA fragments were created by PCR amplification that contained the URA3 gene flanked by different amounts of homology (5-90 bp) to the LYS2 or the ADE2 gene (Fig. 1). These fragments were produced by using primers that contained 5, 15, 25, 30, 45, 60, 75 or 90 bp of homology to the LYS2 gene and 20 bp of sequence homologous to pBR322 flanking the URA3 sequence to amplify URA3. For integration into the ADE2 gene, the primers contained 25 or 30 bp homology toADE2 and 12 bp ofhomology to pBR322. Strains RSY12 containing a complete deletion of the URA3 fragment (ura3A; 5), RSY6 (ura3-52) and W303 (ura3-1) were transformed with the PCR products described above via a high efficiency transfonnation method (6,7). Integration can occur into LYS2 by homologous integration or into sequences other than LYS2 by illegitimate integration (5). These transformations yielded 2-14 transformants/jg of DNA. No net increase in the overall yield of transformants with increasing homology was found. Homologous integration into the LYS2 gene results in lysine deficiency whereas illegitimate integration does not. To determine the fraction of homologous integration events we replica-plated -200 colonies obtained by transformation with each of the different amplification products (modules) onto medium lacking lysine (Table 1). To our surprise, when we transformed LYS2-B which contains 15 bp of homology on each side of the fragment we obtained 3.6% lysine-deficient colonies (Table 1). When we used 25 bp of homology on each side (LYS2-C) we obtained 4.1% lysinedeficient colonies. This frequency jumped to 54% with 30 bp of homology on each side (LYS2-D, Table 1). With longer homology (LYS2-E to LYS2-H, Table 1) -80% of lysine deficient colonies were obtained. To compare the above results with integration events in the presence of extended homology we used a URA3 fragment flanked by 672 bp of LYS2 sequences on one side and 1200 bp on the other side. Similar to the experiments with limited homology starting at 45 bp 80% of the colonies obtained we...

An open question in meiosis is whether the Rad51 recombination protein functions solely in meiotic recombination or whether it is also involved in the chromosome homology search. To address this question, we have performed three-dimensional high-resolution immunofluorescence microscopy to visualize native Rad51 structures in maize male meio-cytes. Maize has two closely related RAD51 genes that are expressed at low levels in differentiated tissues and at higher levels in mitotic and meiotic tissues. Cells and nuclei were specially fixed and embedded in polyacrylamide to maintain both native chromosome structure and the three dimensionality of the specimens. Analysis of Rad51 in maize meiocytes revealed that when chromosomes condense during leptotene, Rad51 is diffuse within the nucleus. Rad51 foci form on the chromosomes at the beginning of zygotene and rise to 500 per nucleus by mid-zygotene when chromosomes are pairing and synapsing. During chromosome pairing, we consistently found two contiguous Rad51 foci on paired chromosomes. These paired foci may identify the sites where DNA sequence homology is being compared. During pachytene, the number of Rad51 foci drops to seven to 22 per nucleus. This higher number corresponds approximately to the number of chiasmata in maize meiosis. These observations are consistent with a role for Rad51 in the homology search phase of chromosome pairing in addition to its known role in meiotic recombination.

An open question in meiosis is whether the Rad51 recombination protein functions solely in meiotic recombination or whether it is also involved in the chromosome homology search. To address this question, we have performed threedimensional high-resolution immunofluorescence microscopy to visualize native Rad51 structures in maize male meiocytes. Maize has two closely related RAD51 genes that are expressed at low levels in differentiated tissues and at higher levels in mitotic and meiotic tissues. Cells and nuclei were specially fixed and embedded in polyacrylamide to maintain both native chromosome structure and the three dimensionality of the specimens. Analysis of Rad51 in maize meiocytes revealed that when chromosomes condense during leptotene, Rad51 is diffuse within the nucleus. Rad51 foci form on the chromosomes at the beginning of zygotene and rise to ‫ف‬ 500 per nucleus by mid-zygotene when chromosomes are pairing and synapsing. During chromosome pairing, we consistently found two contiguous Rad51 foci on paired chromosomes. These paired foci may identify the sites where DNA sequence homology is being compared. During pachytene, the number of Rad51 foci drops to seven to 22 per nucleus. This higher number corresponds approximately to the number of chiasmata in maize meiosis. These observations are consistent with a role for Rad51 in the homology search phase of chromosome pairing in addition to its known role in meiotic recombination. INTRODUCTIONMeiosis results in halving the chromosome complement, a process that is required for the formation of haploid gametes or cells. This involves a complex orchestration of events, including large-scale chromosome remodeling and movement within the nucleus. Cytological analysis of meiosis has revealed that the distinctive stages of meiotic prophase (leptotene, zygotene, pachytene, diplotene, and diakinesis) can be found in the plant, animal, and fungal kingdoms (John, 1990;Roeder, 1995). During leptotene, chromosomes condense, and the axial elements of the synaptonemal complex (SC) are assembled onto the chromosomes. During zygotene, homologous chromosomes synapse as defined by the installation of the central element of the SC (Moses, 1956). The beginning of zygotene is distinguished by the de novo formation of the telomere bouquet (Scherthan et al., 1996;Bass et al., 1997). The telomere bouquet is a conspicuous nuclear arrangement in which all of the telomeres are clustered together on the nuclear envelope (Dernburg et al., 1995). This arrangement may aid in pairing by coorienting the chromosomes (Gillies, 1975;Bass et al., 1997). By pachytene, chromosomes are synapsed along their entire length, and meiotic recombination is completed (Padmore et al., 1991). Finally, in diplotene, the SC disassembles, and the chiasmata responsible for holding the homologs together become cytologically evident (Creighton and McClintock, 1931; Stern, 1931).Proteins involved in homologous recombination are expressed during meiosis. The Rad51 recombination protein and its meiotic-specif...