Cell Biological Analysis Of FB1-induced Programmed Cell Death In Arabidopsis Thaliana

Fumonisin B1 is an important mycotoxin with a sphingoid-like long chain base structure, produced by the necrotrophic fungal pathogen Fusarium verticillioides. It infects plants including corn, rice, sorghum, wheat, soybeans, peanuts, tomatoes, green peppers, bananas and other agricultural products and feed. It is a potential carcinogen for the mammal. As its wide infection range involving plants, animals and human health and food safety, it is an urgent need to study and explore its mechanism of toxicity in cells. Although it has been shown that FB1 can induce programmed cell death （PCD） and cause diseases in plants, signals and events mediating PCD and targets of FB1 are still unclear.In this work, we applied Arabidopsis wild-type, Arabidopsis lines expression green fluorescent protein （GFP） targeted to various organelles and acd5 mutant plants, and used cell biology approach, combined with inhibitor treatments to analyze FB1. We illustrated effects of FB1 on signal transduction, cellular physiology, dynamic movement of organelles and ultrastructural changes in the process of PCD.Combining fluorescence microscopy, laser scanning confocal microscopy and flow cytometry, we found that mitochondrial oxidative burst happened in the early period, accompanied by mitochondrial transmembrane potential loss. N-acetyl-L-cysteine, a free radical scavenger, can partially prevent FB1-induced cell death. We determined intracellular calcium concentration with the calcium-specific fluorescent dye Fluo-4 staining and YC-FRET method, and nitric oxide （NO） with specific fluorescent dye DAF staining respectively. We found that FB1 does not cause significant changes in the level of intracellular calcium and NO.By large sample analysis of three （xyz） and four （xyzt） dimensional confocal micrographs of mitochondria, the quantity, volume and distribution of mitochondria in three-dimension space, and track and velocity of mitochondria in four-dimension space were measured for the first time. We found irregularly clumped distribution of mitochondria in the early period after FB1 treatment, and observed a dramatic increase in the size of individual mitochondrion and a concomitant decrease in the number of mitochondria per cell. At the same time, significant decrease of the velocity and complexity of mitochondrial movement was also observed.We found that the transition of mitochondrial distribution and dynamic morphological alteration were dependent on actin filaments, but independent on microtubules and intracellular ATP. Actin-depolymerizing agents facilitate FB1-induced cell death in Arabidopsis protoplasts and leaves. After Arabidopsis GFP-FABD2 line treated with FB1, we found that actin cytoskeleton has suffered a serious depolymerization and degradation before the significant changes of mitochondrial morphology and dynamic movement.We examined the effects of FB1 on subcellular morphological changes and the subcellular localization of H₂O₂ with CeCl3 staining during cell death in Arabidopsis leaves. Mitochondrion-derived H₂O₂ was mainly localized on the outer membrane. Under transmission electron microscope, H₂O₂ were firstly observed on cell wall at the early period, and production of H₂O₂ increased as time went on. Later, H₂O₂ appeared on mitochondrial outer membrane, which gradually became the main source of H₂O₂. H₂O₂ signal disappeared during execution of cell death.In the aspects of subcellular morphology, we observed condensed chromatin in the perinuclear membrane, which is typical feature of programmed cell death. In the late period of cell death, cell contents disordered with a lot of lipid drops in chloroplasts and cytoplasm. We observed the papilla structure induced by FB1. The same feature of signal and structural changes was also observed in non-inoculated leaves adjacent to the injected ones, suggesting that FB1 may transit through the vascular tissues to other leaves and then expand the scope of disease.After both FB1 treatment and Fusarium verticillioides infection, acd5 mutant plants appeared to be more sensitive than wild type plants. More dead cells were observed in acd5 mutant than wild-type plants at early stage of FB1 treatment. The initiation and intensity of H₂O₂ signals and callose apposition were much later and weaker in acd5 than the wild type. We speculate that the reduced resistance in acd5 mutants may result from the delay or weakened synthesis of defensive signals and structures due to metabolic disorders of sphingolipids.