Global climate change has a detrimental effect on plant growth and health, causing serious losses in agriculture. Investigation of the molecular mechanisms of plant responses to various environmental pressures and the generation of plants tolerant to abiotic stress are imperative to modern plant science. In this paper, we focus on the application of the well-established technology CRISPR/Cas9 genome editing to better understand the functioning of the intrinsically disordered protein DSS1 in plant response to oxidative stress. The Arabidopsis genome contains two highly homologous DSS1 genes, AtDSS1(I) and AtDSS1(V). This study was designed to identify the functional differences between AtDSS1s, focusing on their potential roles in oxidative stress. We generated single dss1(I) and dss1(V) mutant lines of both Arabidopsis DSS1 genes using CRISPR/Cas9 technology. The homozygous mutant lines with large indels (dss1(I)del25 and dss1(V)ins18) were phenotypically characterized during plant development and their sensitivity to oxidative stress was analyzed. The characterization of mutant lines revealed differences in root and stem lengths, and rosette area size. Plants with a disrupted AtDSS1(V) gene exhibited lower survival rates and increased levels of oxidized proteins in comparison to WT plants exposed to oxidative stress induced by hydrogen peroxide. In this work, the dss1 double mutant was not obtained due to embryonic lethality. These results suggest that the DSS1(V) protein could be an important molecular component in plant abiotic stress response.

Ustilago maydis and Saccharomyces cerevisiae differ considerably in their response to water-transfer treatments. When stationary phase cells were transferred to pure water and incubated under limited supply of oxygen, the U. maydis cells suffered a catastrophic loss of viability while the S. cerevisiae population was virtually unaffected by the treatment. The major factor underlying the death of the U. maydis cells under those conditions was an oxygen-consuming cellular activity that generated a hypoxic environment, thereby inducing oxidative stress and accumulation of reactive oxygen species, which resulted in lethality. Importantly, a small residue of U. maydis cells that did survive was able to resume growth and repopulate up to the initial culture density when sufficient aeration was restored. The regrowth was dependent on the cellular factors (Adr1, Did4, Kel1, and Tbp1), previously identified as required for repopulation, after killing with hydrogen peroxide. Surprisingly, the survivors were also able to resume growth under apparently hypoxic conditions, indicating that these remnant cells likely switched to a fermentative mode of growth. We discuss the findings in terms of their possible relevance to the eco-evolutionary adaptation of U. maydis to risky environments.

Much headway has been made in understanding the numerous strategies that enable microorganisms to counteract various types of environmental stress, but little is known about how microbial populations recover after a massive death caused by exposure to extreme conditions. Using the yeast-like fungus Ustilago maydis as a model, our recent post-stress regrowth under starvation (RUS) studies have demonstrated that this organism reconstitutes devastated populations with remarkable efficiency. Subsequently, we have identified four RUS-gene products. Two of these, Did4 and Tbp1, play parallel roles in protecting the genome. To identify additional molecular components, we took a molecular-genetic and a transcriptomic approach. By employing a simple and novel screening method, we identified five RUS-deficient mutants (snf8, slm1, vrg4, snf5, hsf1), three of which (snf8, slm1, and hsf1) displayed sensitivity to different genotoxic agents, indicating that the corresponding gene products have roles in genome protection. The global transcriptomic changes of cells grown in supernatants derived from peroxide-treated cell suspensions revealed sets of uniquely expressed genes. Importantly, among the genes induced by the substrates was Chk1, which encodes a protein kinase required for checkpoint-mediated cell cycle arrest in response to DNA damage. Mutants of U. maydis deleted of Chk1 are severely incapacitated in RUS.

The recombinant aspartic proteinase-like protein (FeAPL1-His6) was overexpressed in the tobacco BY-2 cell line and the expected pepstatin A-sensitive enzymatic activity was confirmed at pH 3.0. Immunocytochemistry and protein gel blot analysis of the transformed BY-2 cells and their protoplasts showed extracellular localization of rFeAPL1-His6 in the cell wall. Based on the obtained results, potential functions of FeAPL1 are discussed.

Oxidative stress represents a critical facet of the array of abiotic stresses affecting crop growth and yield. In this paper, we investigated the potential differences in the functions of two highly homologous Arabidopsis DSS1 proteins in terms of maintaining genome integrity and response to oxidative stress. In the context of homologous recombination (HR), it was shown that overexpressing AtDSS1(I) using a functional complementation test increases the resistance of the Δdss1 mutant of Ustilago maydis to genotoxic agents. This indicates its conserved role in DNA repair via HR. To investigate the global transcriptome changes occurring in dss1 plant mutant lines, gene expression analysis was conducted using Illumina RNA sequencing technology. Individual RNA libraries were constructed from three total RNA samples isolated from dss1(I), dss1(V), and wild-type (WT) plants under hydrogen peroxide-induced stress. RNA-Seq data analysis and real-time PCR identification revealed major changes in gene expression between mutant lines and WT, while the dss1(I) and dss1(V) mutant lines exhibited analogous transcription profiles. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed significantly enriched metabolic pathways. Notably, genes associated with HR were upregulated in dss1 mutants compared to the WT. Otherwise, genes of the metabolic pathway responsible for the synthesis of secondary metabolites were downregulated in both dss1 mutant lines. These findings highlight the importance of understanding the molecular mechanisms of plant responses to oxidative stress.