Differential regulation of five Pht1 phosphate transporters from maize (Zea mays L.).

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.

Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana.

The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.

Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning.

We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening approximately 38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

The TITAN5 gene of Arabidopsis encodes a protein related to the ADP ribosylation factor family of GTP binding proteins.

The titan (ttn) mutants of Arabidopsis exhibit dramatic alterations in mitosis and cell cycle control during seed development. Endosperm development in these mutants is characterized by the formation of giant polyploid nuclei with enlarged nucleoli. Embryo development is accompanied by significant cell enlargement in some mutants (ttn1 and ttn5) but not others (ttn2 and ttn3). We describe here the molecular cloning of TTN5 using a T-DNA-tagged allele. A second allele with a similar phenotype contains a nonsense mutation in the same coding region. The predicted protein is related to ADP ribosylation factors (ARFs), members of the RAS family of small GTP binding proteins that regulate various cellular functions in eukaryotes. TTN5 is most closely related in sequence to the ARL2 class of ARF-like proteins isolated from humans, rats, and mice. Although the cellular functions of ARL proteins remain unclear, the ttn5 phenotype is consistent with the known roles of ARFs in the regulation of intracellular vesicle transport.

Three-dimensional microscopy of the Rad51 recombination protein during meiotic prophase.

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 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 approximately 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.

A novel yeast expression/secretion system for the recombinant plant thiol endoprotease propapain.

A new high-yield yeast expression/secretion system has been adapted for the plant thiol endoprotease papain. The propapain gene, obtained from Carica papaya fruit, is expressed in the yeast Saccharomyces cerevisiae. The gene was cloned into a FLAG epitope-tagging expression vector downstream of the yeast alpha mating factor (alpha-factor) secretion signal sequence. Expression of the heterologous propapain in yeast is controlled by the glucose-repressible alcohol dehydrogenase isoenzyme II promoter (ADH2). Glycosylated FLAG-tagged propapain is secreted by a so-called 'super secretor' strain, pmr1 (ssc1), into the culture supernatant where it accumulates to approximately 1.7 mg/l. The proregion contains three consensus N-linked glycosylation sites, whereas there are only two such sites in previously reported cDNA sequences. Removal of this third N-linked glycosylation site results in a drastic reduction in the level of protease activity present in the culture supernatant. Two different types of affinity chromatography were used to purify either propapain or papain. The propapain precursor is autoproteolytically activated to mature papain (M(r) = 24 kDa) using conditions reported previously. The kinetic parameters obtained agree well with the literature values. The yields of active papain are 10-fold higher than those previously reported for propapain in other yeast or bacterial expression systems. This, together with the ease with which mutant proteins can be made, makes this yeast advantageous for a structure-function analysis of recombinant wild-type and mutant papain, and possibly for other related cysteine proteases as well.

Gene disruption with PCR products in Saccharomyces cerevisiae.

We describe here the generation of gene disruption constructs using PCR amplification of selectable markers with primers that provide homology to the target gene of interest. We find that regions of homology as short as 38 to 50 bp suffice to mediate homologous recombination in yeast. We describe applications of this technology to three specific yeast genes that would have been difficult to disrupt with current methods. By dispensing with the need to either clone the gene of interest or engineer a standard disruption construct, this method should facilitate analysis of sequenced genes of unknown function, which will soon include the entire yeast genome.