Search for: All records

Abstract DNA inverted repeats (IRs) are widespread across many eukaryotic genomes. Their ability to form stable hairpin/cruciform secondary structures is causative in triggering chromosome instability leading to several human diseases. Distance and sequence divergence between IRs are inversely correlated with their ability to induce gross chromosomal rearrangements (GCRs) because of a lesser probability of secondary structure formation and chromosomal breakage. In this study, we demonstrate that structural parameters that normally constrain the instability of IRs are overcome when the repeats interact in single-stranded DNA (ssDNA). We established a system in budding yeast whereby >73 kb of ssDNA can be formed in cdc13-707fs mutants. We found that in ssDNA, 12 bp or 30 kb spaced Alu-IRs show similarly high levels of GCRs, while heterology only beyond 25% suppresses IR-induced instability. Mechanistically, rearrangements arise after cis-interaction of IRs leading to a DNA fold-back and the formation of a dicentric chromosome, which requires Rad52/Rad59 for IR annealing as well as Rad1-Rad10, Slx4, Msh2/Msh3 and Saw1 proteins for nonhomologous tail removal. Importantly, using structural characteristics rendering IRs permissive to DNA fold-back in yeast, we found that ssDNA regions mapped in cancer genomes contain a substantial number of potentially interacting and unstable IRs. 

Stomatal pores close rapidly in response to low-air-humidity-induced leaf-to-air vapor pressure difference (VPD) increases, thereby reducing excessive water loss. The hydroactive signal-transduction mechanisms mediating high VPD–induced stomatal closure remain largely unknown. The kinetics of stomatal high-VPD responses were investigated by using time-resolved gas-exchange analyses of higher-order mutants in guard-cell signal-transduction branches. We show that the slow-type anion channel SLAC1 plays a relatively more substantial role than the rapid-type anion channel ALMT12/QUAC1 in stomatal VPD signaling. VPD-induced stomatal closure is not affected in mpk12 / mpk4GC double mutants that completely disrupt stomatal CO 2 signaling, indicating that VPD signaling is independent of the early CO 2 signal-transduction pathway. Calcium imaging shows that osmotic stress causes cytoplasmic Ca 2+ transients in guard cells. Nevertheless, osca1-2 / 1.3 / 2.2 / 2.3 / 3.1 Ca 2+ -permeable channel quintuple, osca1.3 / 1.7 -channel double, cngc5 / 6 -channel double, cngc20 -channel single, cngc19 / 20crispr -channel double, glr3.2 / 3.3 -channel double, cpk- kinase quintuple, cbl1 / 4 / 5 / 8 / 9 quintuple, and cbl2 / 3rf double mutants showed wild-type-like stomatal VPD responses. A B3-family Raf-like mitogen-activated protein (MAP)-kinase kinase kinase, M3Kδ5/RAF6, activates the OST1/SnRK2.6 kinase in plant cells. Interestingly, B3 Raf-kinase m3kδ5 and m3kδ1 / δ5 / δ6 / δ7 ( raf3 / 6 / 5 / 4 ) quadruple mutants, but not a 14-gene raf-kinase mutant including osmotic stress-linked B4-family Raf-kinases, exhibited slowed high-VPD responses, suggesting that B3-family Raf-kinases play an important role in stomatal VPD signaling. Moreover, high VPD–induced stomatal closure was impaired in receptor-like pseudokinase GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) mutant alleles. Notably, the classical transient “wrong-way” VPD response was absent in ghr1 mutant alleles. These findings reveal genes and signaling mechanisms in the elusive high VPD–induced stomatal closing response pathway. 

Xerophyllum asphodeloides (Xerophyllaceae), known as eastern turkeybeard, is an herbaceous perennial found in eastern North America. Due to decline and destruction of its habitat, several states rank X. asphodeloides as “Imperiled” to “Critically Imperiled”. Protocols for seed cryopreservation, in vitro germination, sustainable shoot micropropagation, shoot establishment in soil, and seed germination are presented. Seeds from two tested sources were viable after 20 months of cryopreservation. Germination of isolated embryos in vitro was necessary to overcome strong seed dormancy. Shoot multiplication and elongation occurred on ½ MS medium without PGRs. Shoots rooted in vitro without PGRs or with 0.5 mg/L NAA or after NAA rooting powder treatment and placement in potting mix. When planted in wet, peaty soil mixes, shoots grew for two months and then declined. When planted in a drier planting mix containing aged bark, most plants continued growth. In the field, plant survival was 73% after three growing seasons. Safeguarding this species both ex situ and in situ is possible and offers a successful approach to conservation. Whole seeds germinated after double dormancy was overcome by incubation under warm moist conditions for 12 weeks followed by 12 weeks cold at 4 °C and then warm.