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RECALCITRANT BEHAVIOR OF CHERRYBARK (Quercus pagoda Raf) OAK Sharon Sowa Chemistry Department and Biochemistry Program Indiana University of Pennsylvania.

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Presentation on theme: "RECALCITRANT BEHAVIOR OF CHERRYBARK (Quercus pagoda Raf) OAK Sharon Sowa Chemistry Department and Biochemistry Program Indiana University of Pennsylvania."— Presentation transcript:

1 RECALCITRANT BEHAVIOR OF CHERRYBARK (Quercus pagoda Raf) OAK Sharon Sowa Chemistry Department and Biochemistry Program Indiana University of Pennsylvania Indiana, PA Kristina F. Connor USDA- FS Center for Bottomland Hardwood Research Starkville, MS

2 RECALCITRANT desiccation sensitive and homeohydrous e.g. many tropical species, also some temperates JUST HOW SENSITIVE IS SENSITIVE?? Cherrybark Oak Seed Germination after Storage % mcday 01 y @ +4 o C 1 y @ -2 o C fresh 29.6100% 88%97% dry 19.9 98% 5%22% dry = 2 days on the lab bench in Mississippi

3 WHAT MAKES SEED ORTHODOX ? hormone-triggered synthesis of LEA proteins, or dehydrins accumulation of sugars trehalose raffinose sucrose others thermodynamic events that result in stable dry states i.e. Nobody really knows for sure. WHAT MAKES SEED RECALCITRANT? Nobody really knows for sure, but we have learned some things:

4 WHY FTIR SPECTROSCOPY? It identifies many functional groups in cells most dipoles are infrared absorbers It can be quantitative Beers law applies Its fast all wavelengths collected simultaneously Its easy minimal sample preparation required We have one!

5 WHAT FUNCTIONAL GROUPS ARE IMPORTANT? those found in membrane lipids -CH 2 - ; -CH 3 ; -C=C- those found in storage lipids ester carbonyls those found in proteins amide carbonyls, C-N stretch, N-H bend those found in energy storage compounds phosphates those found in carbohydrates (like sucrose) -OH stretch those resulting from respiratory metabolism CO 2 production (thats another story)


7 WHAT WE DID: for two consecutive years presoak seed overnight to fully hydrate day 0 spread seed on blotter paper on lab bench randomly sample 175 seeds determine moisture content on 5 reps of 5 seed (chop up, weigh, dry overnight at 103 o C, reweigh, calculate mc on fw basis) germinate 100 seeds in greenhouse collect FTIR transmission spectra on at least 2 samples each of cotyledon and embryo tissue

8 day 2,4,6,8 determine mc collect FTIR spectra soak 150 seeds overnight for germination & FTIR day 1,3,5,7,9 germinate rehydrated seed collect FTIR spectra of same (Continue sampling until mc < 15%)



11 Day 0 Day 4 Membrane lipid -CH 2 - vibrations in cherrybark embryos symmetric (2850 cm -1 ) and asymmetric (2920 cm -1 )

12 Membrane lipid vibrations in cherrybark embryos Peak frequencies at 2852.5, 2849.7, and 2850.3 cm -1 Day 0 Day 9 Day 8

13 Membrane lipid vibrations in day 0, day 8, and day 9 cotyledons cotyledons. Peak frequencies at 2851.9, 2847.2, and 2848.8 cm -1. Day 0 Day 8 Day 9


15 The isothermal gel point

16 Storage lipid vibrations in day 0 cherrybark embryos and cotyledons. Peak frequency at 1743 cm -1. cotyledons Lipid:protein ratio higher in cotyledons (oily seed).

17 Storage lipid vibrations in day 0 and day 8 cherrybark cotyledons cotyledons. Day 0 Day 8

18 embryos Membrane lipid vibrations in day 4 cherrybark embryos cotyledons and cotyledons. Peak frequencies at 2850.6 and 2848.4 cm -1 indicating differential drying. Day 4 embryos cotyledons

19 Protein (amide I and II) vibrations of day 0 cherrybark embryos. Peak frequencies near 1640 and 1550 cm -1


21 Protein (amide) vibrations in cherrybark embryos. Peak frequencies at 1638.5, 1635, and 1629.9 cm -1 Day 0 Day 8 Day 9

22 Amide protein vibrations in day 0, day 8 and day 9 cotyledons cherrybark cotyledons. Day 0 Day 9 Day 8

23 WHAT WE LEARNED ABOUT CHERRYBARK: seed storage longevity is sensitive to mc and temp seed germination drops rapidly as moisture drops below a critical level (between 18 - 15%) membrane lipids in both embryos and cotyledons change phase (liquid crystalline to gel) 1 upon drying and do NOT recover upon rehydration as viability is lost phase change or isothermal gel point occurs at moisture content where significant viability loss occurs phase change occurs first in cotyledons; water loss occurs preferentially in cotyledons while embryos retain moisture as long as possible 1 H.L. Casal and H.H. Mantsch. Polymorphic phase behavior of phospholipid membranes studied by infrared spectroscopy. Biochim. Biophys Acta. 779 (1984) 381-401.

24 cotyledon tissue has a higher lipid:protein ratio than embryos no significant degree of lipid mobilization occurs during drying (we see sucrose mobilization in high-sugar seed such as white oak) changes in protein secondary structure 2 occurred in both embryos and cotyledons as moisture was lost in embryos, a significant shift in the amide I peak occurred upon dehydration, which did not recover upon rehydration in cotyledons, secondary structure was completely lost upon dehydration, and remained so upon rehydration of nonviable samples 2 S. Sowa, K.F. Connor and L.E. Towill. Temperature changes in lipid and protein structure measured by Fourier transform infrared spectroscopy in intact pollen grains. Plant Science 105 (1995) 23-30

25 the most sensitive indicator of viability loss was a change in protein secondary structure to extended beta-sheet conformation (absorbance frequencies less than 1630 cm -1 ) this is contrary to behavior observed in orthodox seeds using infrared techniques: E.A. Golovina, W. F. Wolkers and F.A. Hoekstra. 86. Behavior of membranes and Proteins during Natural Seed Aging in: Basic and Applied Aspects of Seed Biology, R.H. Ellis, M. Black, A.J. Murdoch. T.D. Hong (eds) (1997) Kluwer Academic Publishers, Dordrecht, pp. 787-796

26 THANKS TO THE TECHNICAL HELP: Terri Orkwiszewski Leroy Muya Jennifer Sloppy AND FOR THE SUPPORT OF The USDA Forest Service The Merck/AAAS Scholar Program

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