Phytoremediation of copper-contaminated soils by rapeseed (Brassica napus L.) and underlying molecular mechanisms for copper absorption and sequestration.

High levels of copper released in the soil, mainly from anthropogenic activity, can be hazardous to plants, animals, and humans. The present research aimed to estimate the suitability and effectiveness of rapeseed (Brassica napus L.) as a possible soil remediation option and to uncover underlying adaptive mechanisms A pot experiment was conducted to explore the effect of copper stress on agronomic and yield traits for 32 rapeseed genotypes. The copper-tolerant genotype H2009 and copper-sensitive genotype ZYZ16 were selected for further physiological, metabolomic, and transcriptomic analyses. The results exhibited a significant genotypic variation in copper stress tolerance in rapeseed. Specifically, the ratio of seed yield under copper stress to control ranged from 0.29 to 0.74. Furthermore, the proline content and antioxidant enzymatic activities in the roots were greater than those in the shoots. The accumulated copper in the roots accounted for about 50% of the total amount absorbed by plants; thus, the genotypes possessing high root volumes can be used for rhizofiltration to uptake and sequester copper. Additionally, the pectin and hemicellulose contents were significantly increased by 15.6% and 162%, respectively, under copper stress for the copper-tolerant genotype, allowing for greater sequestration of copper ions in the cell wall and lower oxidative stress. Comparative analysis of transcriptomes and metabolomes revealed that excessive copper enhanced the up-regulation of functional genes or metabolites related to cell wall binding, copper transportation, and chelation in the copper-tolerant genotype. Our results suggest that copper-tolerant rapeseed can thrive in heavily copper-polluted soils with a 5.85% remediation efficiency as well as produce seed and vegetable oil without exceeding food quality standards for the industry. This multi-omics comparison study provides insights into breeding copper-tolerant genotypes that can be used for the phytoremediation of heavy metal-polluted soils.

Structural parameters of palindromic repeats determine the specificity of nuclease attack of secondary structures.

Palindromic sequences are a potent source of chromosomal instability in many organisms and are implicated in the pathogenesis of human diseases. In this study, we investigate which nucleases are responsible for cleavage of the hairpin and cruciform structures and generation of double-strand breaks at inverted repeats in Saccharomyces cerevisiae. We demonstrate that the involvement of structure-specific nucleases in palindrome fragility depends on the distance between inverted repeats and their transcriptional status. The attack by the Mre11 complex is constrained to hairpins with loops <9 nucleotides. This restriction is alleviated upon RPA depletion, indicating that RPA controls the stability and/or formation of secondary structures otherwise responsible for replication fork stalling and DSB formation. Mus81-Mms4 cleavage of cruciforms occurs at divergently but not convergently transcribed or nontranscribed repeats. Our study also reveals the third pathway for fragility at perfect and quasi-palindromes, which involves cruciform resolution during the G2 phase of the cell cycle.

GC content elevates mutation and recombination rates in the yeast Saccharomyces cerevisiae.

The chromosomes of many eukaryotes have regions of high GC content interspersed with regions of low GC content. In the yeast Saccharomyces cerevisiae, high-GC regions are often associated with high levels of meiotic recombination. In this study, we constructed URA3 genes that differ substantially in their base composition [URA3-AT (31% GC), URA3-WT (43% GC), and URA3-GC (63% GC)] but encode proteins with the same amino acid sequence. The strain with URA3-GC had an approximately sevenfold elevated rate of ura3 mutations compared with the strains with URA3-WT or URA3-AT About half of these mutations were single-base substitutions and were dependent on the error-prone DNA polymerase ζ. About 30% were deletions or duplications between short (5-10 base) direct repeats resulting from DNA polymerase slippage. The URA3-GC gene also had elevated rates of meiotic and mitotic recombination relative to the URA3-AT or URA3-WT genes. Thus, base composition has a substantial effect on the basic parameters of genome stability and evolution.

Break-induced replication promotes formation of lethal joint molecules dissolved by Srs2.

Break-induced replication (BIR) is a DNA double-strand break repair pathway that leads to genomic instabilities similar to those observed in cancer. BIR proceeds by a migrating bubble where asynchrony between leading and lagging strand synthesis leads to accumulation of long single-stranded DNA (ssDNA). It remains unknown how this ssDNA is prevented from unscheduled pairing with the template, which can lead to genomic instability. Here, we propose that uncontrolled Rad51 binding to this ssDNA promotes formation of toxic joint molecules that are counteracted by Srs2. First, Srs2 dislodges Rad51 from ssDNA preventing promiscuous strand invasions. Second, it dismantles toxic intermediates that have already formed. Rare survivors in the absence of Srs2 rely on structure-specific endonucleases, Mus81 and Yen1, that resolve toxic joint-molecules. Overall, we uncover a new feature of BIR and propose that tight control of ssDNA accumulated during this process is essential to prevent its channeling into toxic structures threatening cell viability.

Rtt107 Is a Multi-functional Scaffold Supporting Replication Progression with Partner SUMO and Ubiquitin Ligases.

Elucidating the individual and collaborative functions of genome maintenance factors is critical for understanding how genome duplication is achieved. Here, we investigate a conserved scaffold in budding yeast, Rtt107, and its three partners: a SUMO E3 complex, a ubiquitin E3 complex, and Slx4. Biochemical and genetic findings show that Rtt107 interacts separately with these partners and contributes to their individual functions, including a role in replisome sumoylation. We also provide evidence that Rtt107 associates with replisome components, and both itself and its associated E3s are important for replicating regions far from initiation sites. Corroborating these results, replication defects due to Rtt107 loss and genotoxic sensitivities in mutants of Rtt107 and its associated E3s are rescued by increasing replication initiation events through mutating two master repressors of late origins, Mrc1 and Mec1. These findings suggest that Rtt107 functions as a multi-functional platform to support replication progression with its partner E3 enzymes.

Genome-wide screen reveals replication pathway for quasi-palindrome fragility dependent on homologous recombination.

Inverted repeats capable of forming hairpin and cruciform structures present a threat to chromosomal integrity. They induce double strand breaks, which lead to gross chromosomal rearrangements, the hallmarks of cancers and hereditary diseases. Secondary structure formation at this motif has been proposed to be the driving force for the instability, albeit the mechanisms leading to the fragility are not well-understood. We carried out a genome-wide screen to uncover the genetic players that govern fragility of homologous and homeologous Alu quasi-palindromes in the yeast Saccharomyces cerevisiae. We found that depletion or lack of components of the DNA replication machinery, proteins involved in Fe-S cluster biogenesis, the replication-pausing checkpoint pathway, the telomere maintenance complex or the Sgs1-Top3-Rmi1 dissolvasome augment fragility at Alu-IRs. Rad51, a component of the homologous recombination pathway, was found to be required for replication arrest and breakage at the repeats specifically in replication-deficient strains. These data demonstrate that Rad51 is required for the formation of breakage-prone secondary structures in situations when replication is compromised while another mechanism operates in DSB formation in replication-proficient strains.

A reversible histone H3 acetylation cooperates with mismatch repair and replicative polymerases in maintaining genome stability.

Mutations are a major driving force of evolution and genetic disease. In eukaryotes, mutations are produced in the chromatin environment, but the impact of chromatin on mutagenesis is poorly understood. Previous studies have determined that in yeast Saccharomyces cerevisiae, Rtt109-dependent acetylation of histone H3 on K56 is an abundant modification that is introduced in chromatin in S phase and removed by Hst3 and Hst4 in G2/M. We show here that the chromatin deacetylation on histone H3 K56 by Hst3 and Hst4 is required for the suppression of spontaneous gross chromosomal rearrangements, base substitutions, 1-bp insertions/deletions, and complex mutations. The rate of base substitutions in hst3Δ hst4Δ is similar to that in isogenic mismatch repair-deficient msh2Δ mutant. We also provide evidence that H3 K56 acetylation by Rtt109 is important for safeguarding DNA from small insertions/deletions and complex mutations. Furthermore, we reveal that both the deacetylation and acetylation on histone H3 K56 are involved in mutation avoidance mechanisms that cooperate with mismatch repair and the proofreading activities of replicative DNA polymerases in suppressing spontaneous mutagenesis. Our results suggest that cyclic acetylation and deacetylation of chromatin contribute to replication fidelity and play important roles in the protection of nuclear DNA from diverse spontaneous mutations.

Fragile DNA motifs trigger mutagenesis at distant chromosomal loci in saccharomyces cerevisiae.

DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes.

Modulation of anti-oxidation ability by proanthocyanidins during germination of Arabidopsis thaliana seeds.

Proanthocyanidins (PAs) as the end products of flavonoid biosynthetic pathway mainly accumulate in seed coat but their biological function is largely unknown. We studied the anti-oxidation ability in seed coat and germination changes under externally applied oxidative stresses in PAs-deficient mutants of Arabidopsis. Germination of PAs-deficient mutant seeds was faster than that of wild-type under low or no oxidative stress, suggesting a PAs-induced inhibition of germination. When the applied oxidative stress was high, germination of PAs-deficient mutants was lower than that of wild-type, suggesting a loss of PAs-related anti-oxidation ability in the mutants. Using ABA signaling mutants, our studies demonstrated that both ABA signaling pathway and PAs were important for the response to serve oxidative stress during seed germination. However, the discrepancy of the response between abi mutants and PAs mutants to oxidative stress suggests that ABA signaling pathway may not play a major role in PAs' action in alleviating oxidative stress. Under low or no oxidative stress, germination was mainly determined by the ABA content in seed and the PAs-deficient mutant seeds germinated faster due to their lower ABA content than wild-type. However, oxidative injury inhibited germination when PAs-deficient seeds germinated under high oxidative stress. Wild-type exhibited higher germination under the high oxidative stress due to the PAs' anti-oxidation ability. Oxidative stress applied externally led to changes in endogenous PAs contents that coincided with the expression changes of PAs biogenesis genes. PAs modulated the activities of some key enzymes that controlled the levels of reactive oxygen species and the anti-oxidation capacity during the seed germination. This work suggests that PAs contribute to the adaptive mechanism that helps germination under environmental stresses by playing dual roles in both germination control and anti-oxidation reaction.