Project description:ABSTRACT: Sphingolipid synthesis is initiated by condensation of serine with palmitoyl-CoA to produce 3-ketodihydrosphinganine (3-KDS), which is subsequently reduced by a 3-KDS reductase to dihydrosphinganine (DHS). Serine palmitoyltransferase was recently shown to be essential for plant viability, but the 3-KDS reductase step of sphingolipid synthesis has not been investigated in plants. Arabidopsis thaliana contains two genes (At3g06060/TSC10A and At5g19200/TSC10B) that encode proteins with significant homology to the yeast 3-KDS reductase, Tsc10p. Heterologous expression, in yeast, of either A. thaliana gene restored 3-KDS reductase activity to the yeast tsc10Δ mutant, thereby identifying both as bona fide 3-KDS reductase genes. Consistent with previous evidence that sphingolipids have essential functions in plants, double mutant progeny lacking both genes were not recovered. Although the 3-KDS reductase genes are functionally redundant and ubiquitously expressed in A. thaliana, 3-KDS reductase activity was reduced to approximately 10% of wild-type levels in the loss-of-function tsc10a mutant, leading to an altered sphingolipid profile compared to wild-type plants. Interestingly, this perturbation of sphingolipid biosynthesis in the A. thaliana tsc10a mutant leads to significant alterations in the leaf ionome, including increases in Na, K and Rb (a K analogue), and decreases in Mg, Ca, Fe, and Mo. Reciprocal grafting revealed that these changes in the leaf ionome are driven by the root, and are associated with both increases in root suberin, and elevation of expression in the root of genes involved in Fe homoeostasis, including the Fe-transporter IRT1. Keywords: genomic hybridization bulked segregant analysis Hybridizations from a set of Bulk Segregant analysis. An F2 population from 7113 in the col-0 background crossed to Ler-0 was analyzed. This series contains the 3 hybs from Ler used to identify Single Feature Polymorphisms, the 3 hybs of Col-0 they were compared to, and 1 hyb for each pool from the BSA mapping(low Ca/Mg Pool and wild type pools).

Project description:Lipid intermediates derived from sphingolipid metabolism are crucial regulators of mitochondrial function from yeast to humans. Among these intermediates, trans-2-hexadecenal (t-2hex) within the sphingolipid degradation pathway exhibits remarkable efficiency in inducing mitochondria-mediated cell death. In yeast cell cultures, the addition of t-2-hex triggers complete disintegration of the mitochondrial network, leading to subsequent cell death. This effect is particularly pronounced in yeast cells lacking the activity of the t-2-hex degrading enzyme, Hfd1. However, the molecular mechanisms of t-2-hex induction of mitochondrial dysfunction are completely unknown. In this project, we want to exploit the unprecedented power of yeast genetics to unveil novel genetic determinants involved in t-2-hex's pro-apoptotic function. To accomplish this, we employed the SATAY method, which combines saturated transposon mutagenesis with high-throughput sequencing to functionally explore the yeast genome. In our screening approach, hfd1 mutant cells harboring a plasmid-encoded inducible MiniDs transposon were induced by galactose, resulting in extensive integration of the transposon throughout the yeast genome. Cells with the plasmid excised and the transposon genomically integrated were pooled together, creating a high-density transposon library comprising approximately 2.3E+06 independent insertion mutants. Subsequently, the pooled mutant library was subjected to treatment with the mitochondria-mediated death inducer, t-2-hexadecenal. As a control, cells were also incubated with the solvent dimethyl sulfoxide (DMSO), in which hexadecenal is dissolved. Following the treatments, cells were collected for genomic DNA extraction and digestion, using restriction enzymes with frequent four-base pair recognition sites. The resulting library fragments were circularized using T4 DNA ligase, and the transposon-genome junctions were selectively amplified through PCR with outward-facing primers specific to the transposon. Finally, the pooled and purified amplicons were subjected to massive sequencing on an Illumina MiSeq platform. The obtained sequences were then aligned to the reference genome of Saccharomyces cerevisiae, allowing for the mapping of transposon insertions and the calculation of transposon counts per gene. This project enabled the identification of genes required for the resistance and toxicity to t-2hex.

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Wickerhamomyces ciferrii

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