Susan sigma xi 2013

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1. Coping with Extreme Stress-Resurrection! Susan John Advisor: Dr. Karl H. HasensteinDepartment of Biology University of Louisiana at Lafayette 2. In response to scarcity of water plants wilt, and eventually die. 3. Some plants however can tolerate water stressPolypodiumpolypodioides- Resurrection FernDehydrated frondsRehydrated (resurrected) Resurrection ferns have a remarkable ability to tolerate dehydration bycurling tightly and re-expand as soon as water (rain) becomes available. Their ability to adapt to changing environment makes them an ideal model to study the effects of stress 4. Early Studies on Stress Tolerance Various components listed below are known to be synthesized and suggested to be providing tolerance to desiccation tolerant plants Osmoprotectants e.g. proline, glycerol Hormones e.g. Abscisic acid, salicylic acid Sugars e.g. sucroseDrawback!There is no information on when different componentsare synthesized during stress! 5. A Challenging Question of our ResearchRehydrated frondDehydrated frond What enables Polypodium todry without dying? 6. HypothesisPolypodium fronds tolerate dehydrationwhen dried slowly e.g. at 30 C, but theycannot sustain exposure to hightemperatures e.g. 80 C 7. To test our hypothesis we will expose Polypodium to differentintensities of stress such as slow (30 C), intermediate (50 C)and fast (80 C) dehydration. The effects of dehydration will be determined by measuring-1.Water status (Relative Water Content)2.Glycerol content (an osmoprotectant known to besynthesized in response to stress) and3.Vitality (test whether dehydrated fronds are metabolicallyactive after rehydration) 8. Fresh fronds collected from Method oak treesFronds cut into half, floatedon water for 12 h, weighed (rehydrated weight, WT)Vitality test Resazurin Assay3Glycerol content measured rehydrated rehydrated 2according to Sturgeon et fronds dried atfronds placed inal., 197980 C for 96 h tooven for dryingmeasure dryat 30 C, 50weight (WD) C, and 80 CDrying fronds1 removed from ovenRelative Water Content (RWC) at specific timeRWC% = Fresh weight -Dry Weight 100intervals, andTurgid weight Dry weight weighed (WD) 9. 30 C ln RWC%RWC% 50 CTime (h) 80 C A Time (h) B 10. Glycerol mg g-1 DWTime (h)Figure 2: Glycerol content of 30 C, 50 C and 80 C dried fronds decreased at the rate of0.68, 0.59 and 1.57 mg h-1respectively during 12 h of dehydration. Between 24 - 72 h ofdehydration, glycerol accumulated in 30 C dried fronds at a slow rate 0.13 mg h-1 and in 50 C and80 C dried fronds the amount decreased at a rate of 0.18 and 0.09 mg h-1. There was nosignificant difference in glycerol content over the duration of dehydration (p>0.05). The data isrepresented as mean SE; N= 5 replicates per treatments 11. (Vitality) Fluorescence Intensity (a.u.) Drying Duration (h)Figure 3: Vitality of 30 C, 50 C and 80 C dried fronds were measured by incubating oven driedfronds with redox dye resazurin. The intensity of fluorescence corresponds to the metabolic stateof rehydrating fronds. 30 C dried fronds showed higher intensity (metabolic activity) compared to50 C and 80 C fronds. The intensities of 50 C and 80 C measured zero after 20 h ofdehydration, suggesting no metabolic activity. Fluorescence was measured ( 530 nm excitationand 590 nm emission)N= 3 replicates, mean SE 12. DiscussionMorphology In response to dehydration, fronds curled tightly and turned brittle. Such mechanisms is suggested to provide protection against extensive damage.Relative Water Content (RWC%) RWC is an ideal tool to measure water status of the fronds. In response to the intensity of stress, they adjusted their water status, a mechanism adopted to prevent permanent cell damage.Glycerol Content Slow drying (30 C) may induce synthesis / accumulation of glycerol (an osmoprotectant), enabling the fronds to withstand dehydration. However, temperatures 50 C affected glycerol content, suggesting sensitivity to heat.Vitality Slow dried (30 C) fronds were metabolically active (alive) even after 72 h of dehydration. However, intermediate (50 C) and fast (80 C) dried fronds were metabolically inactive, suggesting they were dead. 13. ConclusionSlow dried Polypodium can tolerate dehydration; nothigh temperature. Osmoprotectant glycerol is one ofthe component observed to be accumulated 12 h postdehydration, that allows fronds to withstand longerdurations of dehydration. 14. Future PlansUsing RNA seq (gene exprssion), we will determine the timeperiod when different components will be synthesized in adehydrating frond. Such information will provide deeperunderstanding on how Polypodiums remain dry for longerdurations without dying. 15. AcknowledgementsAdvisor: Dr. Karl H. HasensteinAssembling personal video : Robyn Noltingfor Sigma Xi showcase