Mira Milisavljevic

Liming of anthropogenically acidified soil promotes phosphorus acquisition in the rhizosphere of wheat

We studied the effect of liming and P fertilization of extremely acid soil (accidently acidified by sulfidic mining waste) on P availability and the subsequent adaptive responses of wheat roots. The wheat plants were grown in rhizoboxes allowing precise sampling of rhizosphere and bulk soil for sequential extraction of P fractions and determination of exchangeable Al. Root exudates were collected by pieces of paper for electrophoresis and subjected to HPLC analysis. Expression of organic anions and Pi transporter genes was analyzed by a real-time quantitative PCR. The concomitant application of lime with P fertilization increased the concentrations of plant-available P fractions in both rhizosphere and bulk compartments. The applied soil amendments strongly affected plant growth, biomass partitioning and shoot P accumulation. Liming enhanced root exudation of citrate in P unfertilized plants, while the high malate efflux was maintained until both P deficiency and Al toxicity were eliminated by the amendments. We showed the importance of liming for recovering of P acquisition potential of wheat roots, which can be strongly impaired in acid soils. Our results clearly demonstrated that P-deficient roots not subjected to Al stress in the limed soil can maintain high efflux of malate and even increase efflux of citrate along with the enhanced expression of related anion transporters (TaMATE1 and TaALMT1).

The Metallothionein‐Like Gene from Buckwheat: Structural and Functional Analysis of the Promoter Region1

The buckwheat (Fagopyrum esculentum Moench) metallothionein‐like gene was cloned and its 5′ regulatory region was examined. Computer analysis of this region using overlapping data from three different databases predicted the existence of regulatory sequences that could be involved in responses to different hormonal and external stimuli (ERE, heat shock‐HSE, light and stress‐GT‐1, I‐box, GATA, G‐box, metal‐MRE) as well as the presence of putative binding sites for plant‐specific transcription factors (Dof1, NtBBF1, Athb‐1). Further investigation included analysis of the interactions of distal and proximal parts of the defined regulatory region and purified Dof1ΔC domain of maize Dof1 and the HD‐Zip‐1 domain of Arabidopsis thaliana Athb‐1 transcription factors as well as with buckwheat leaf nuclear extract. The identity of putative Dof1‐ and Athb‐1‐binding sites was confirmed in the proximal region. More important, we found that some proteins from buckwheat leaf nuclear extract compete for the Dof1‐binding sites, indicating the presence of a similar protein type in that extract. We also showed that buckwheat leaf nuclear extract itself interacted with both proximal and distal promoter regions. Interaction with the proximal region resulted in the formation of a single HMW complex, while five separate complexes with the distal region were detected, indicating interaction with predicted G‐ and I‐boxes, which are probably involved in light‐ and/or stress‐regulated MT3 gene expression. The promoter ability of the cloned buckwheat MT gene regulatory region was characterized by measuring the activity of the GUS reporter gene in the leaves of transgenic tobacco. Buckwheat MT3 promoter activity was also analyzed for its response to stress produced by either hydrogen peroxide or UV treatment. We found that both treatments increased GUS activity.

LAMMER kinase contributes to genome stability in Ustilago maydis

Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1(Q488*)) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1(Q488*)rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1(Q488*) mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1(Q488*) mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.

Bioavailability of Nutritional Resources From Cells Killed by Oxidation Supports Expansion of Survivors in Ustilago maydis Populations

After heavy exposure of Ustilago maydis cells to clastogens, a great increase in viability was observed if the treated cells were kept under starvation conditions. This restitution of viability is based on cell multiplication at the expense of the intracellular compounds freed from the damaged cells. Analysis of the effect of the leaked material on the growth of undamaged cells revealed opposing biological activity, indicating that U. maydis must possess cellular mechanisms involved not only in reabsorption of the released compounds from external environment but also in contending with their treatment-induced toxicity. From a screen for mutants defective in the restitution of viability, we identified four genes (adr1, did4, kel1, and tbp1) that contribute to the process. The mutants in did4, kel1, and tbp1 exhibited sensitivity to different genotoxic agents implying that the gene products are in some overlapping fashion involved in the protection of genome integrity. The genetic determinants identified by our analysis have already been known to play roles in growth regulation, protein turnover, cytoskeleton structure, and transcription. We discuss ecological and evolutionary implications of these results.

Collaboration in the actions of Brh2 with resolving functions during DNA repair and replication stress in Ustilago maydis

Cells maintain a small arsenal of resolving functions to process and eliminate complex DNA intermediates that result as a consequence of homologous recombination and distressed replication. Ordinarily the homologous recombination system serves as a high-fidelity mechanism to restore the integrity of a damaged genome, but in the absence of the appropriate resolving function it can turn DNA intermediates resulting from replication stress into pathological forms that are toxic to cells. Here we have investigated how the nucleases Mus81 and Gen1 and the helicase Blm contribute to survival after DNA damage or replication stress in Ustilago maydis cells with crippled yet homologous recombination-proficient forms of Brh2, the BRCA2 ortholog and primary Rad51 mediator. We found collaboration among the factors. Notable were three findings. First, the ability of Gen1 to rescue hydroxyurea sensitivity of dysfunctional Blm requires the absence of Mus81. Second, the response of mutants defective in Blm and Gen1 to hydroxyurea challenge is markedly similar suggesting cooperation of these factors in the same pathway. Third, the repair proficiency of Brh2 mutant variants deleted of its N-terminal DNA binding region requires not only Rad52 but also Gen1 and Mus81. We suggest these factors comprise a subpathway for channeling repair when Brh2 is compromised in its interplay with DNA.