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RECALCITRANT BEHAVIOR OF CHERRYBARK (Quercus pagoda Raf) OAK
Sharon SowaChemistry Department and Biochemistry Program
Indiana University of PennsylvaniaIndiana, PA
Kristina F. ConnorUSDA- FS
Center for Bottomland Hardwood ResearchStarkville, MS
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RECALCITRANT “desiccation sensitive” and “homeohydrous”
e.g. many tropical species, also some temperates
JUST HOW “SENSITIVE” IS SENSITIVE??
Cherrybark Oak Seed Germination after Storage
% mc day 0 1 y @ +4oC 1 y @ -2oC
fresh 29.6 100% 88% 97%“dry” 19.9 98% 5% 22%
“dry” = 2 days on the lab bench in Mississippi
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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:
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WHY FTIR SPECTROSCOPY?• It identifies many functional groups in cells
• most dipoles are infrared absorbers• It can be quantitative
• Beer’s law applies• It’s fast
• all wavelengths collected simultaneously• It’s easy
• minimal sample preparation required• We have one!
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WHAT FUNCTIONAL GROUPS ARE IMPORTANT?• those found in membrane lipids
• -CH2- ; -CH3 ; -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
• CO2 production (that’s another story)
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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 103oC, 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
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• “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%)
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Moisture Content vs Day on Bench
010203040
0 2 4 6 8
Day
% M
oist
ure year 1
year 2
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Germination vs Moisture Content
0
10
20
30
40
50
60
70
80
90
100
0510152025303540
% Moisture
% G
erm
inat
ion
year1
year2
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2850.6
2
2920.1
7
2852.5
4
2922.2
9
0.280
0.285
0.290
0.295
0.300
0.305
0.310
0.315
0.320
0.325
0.330
0.335
0.340
0.345
0.350
0.355
0.360
Absorb
ance
2900 3000
Wavenumbers (cm-1)
Day 0
Day 4
Membrane lipid -CH2- vibrations in cherrybark embryossymmetric (2850 cm-1) and asymmetric (2920 cm-1)
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0.280
0.285
0.290
0.295
0.300
0.305
0.310
0.315
0.320
0.325
0.330
0.335
0.340
0.345
0.350
0.355
Abs
orba
nce
2900 3000
Wavenumbers (cm-1)
Membrane lipid vibrations in cherrybark embryosPeak frequencies at 2852.5, 2849.7, and 2850.3 cm-1
Day 0
Day 9
Day 8
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0.360
0.365
0.370
0.375
0.380
0.385
0.390
0.395
0.400
0.405
0.410
0.415
0.420
0.425
0.430
0.435
Abs
orba
nce
2900 3000
Wavenumbers (cm-1)
Membrane lipid vibrations in day 0, day 8, and day 9cotyledonscotyledons. Peak frequencies at 2851.9, 2847.2, and2848.8 cm-1.
Day 0
Day 8Day 9
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Membrane Lipid Phase Transition
2852.6
2852.7
2852.8
2852.9
2853
2853.1
2853.2
2853.3
2853.4
2853.5
2853.6
2853.7
101520253035
Seed Moisture Content % H2O
Pe
ak
Fe
qu
en
cy
(c
m-1
)
year 1
The isothermal gel point
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0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
Abs
orba
nce
1600 1700 1800
Wavenumbers (cm-1)
Storage lipid vibrations in day 0 cherrybark embryosand cotyledons. Peak frequency at 1743 cm-1.Lipid:protein ratio higher in cotyledonscotyledons (oily seed).
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0.18
0.19
0.20
0.21
0.22
0.23
0.24
0.25
0.26
0.27
0.28
0.29
0.30
0.31
0.32
0.33
0.34
Abs
orba
nce
1600 1700 1800
Wavenumbers (cm-1)
Storage lipid vibrations in day 0 and day 8 cherrybarkcotyledonscotyledons.
Day 0
Day 8
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0.121
0.122
0.123
0.124
0.125
0.126
0.127
0.128
0.129
0.130
0.131
0.132
0.133
0.134
0.135
0.136
Abs
orba
nce
2900 3000
Wavenumbers (cm-1)
Membrane lipid vibrations in day 4 cherrybark embryosembryosand cotyledonscotyledons. Peak frequencies at 2850.6 and 2848.4 cm-1 indicating differential drying.
Day 4embryoscotyledons
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0.240
0.245
0.250
0.255
0.260
0.265
0.270
0.275
0.280
0.285A
bsor
banc
e
1550 1600 1650 1700
Wavenumbers (cm-1)
Protein (amide I and II) vibrations of day 0 cherrybarkembryos. Peak frequencies near 1640 and 1550 cm-1
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0.240
0.245
0.250
0.255
0.260
0.265
0.270
0.275
0.280
0.285
Abs
orba
nce
1550 1600 1650 1700
Wavenumbers (cm-1)
Protein (amide) vibrations in cherrybark embryos.Peak frequencies at 1638.5, 1635, and 1629.9 cm-1
Day 0
Day 8
Day 9
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0.275
0.280
0.285
0.290
0.295
0.300
0.305
0.310
0.315
0.320
0.325
Abs
orba
nce
1550 1600 1650 1700
Wavenumbers (cm-1)
Amide protein vibrations in day 0, day 8 and day 9cherrybark cotyledonscotyledons.
Day 0Day 9
Day 8
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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 dryingand do NOT recover upon rehydration as viabilityis lost
• phase change or isothermal gel point occurs at moisture content where significant viability loss occurs
• phase change occurs first in cotyledons; water lossoccurs preferentially in cotyledons while embryosretain moisture as long as possible
1 H.L. Casal and H.H. Mantsch. Polymorphic phase behavior of phospholipidmembranes studied by infrared spectroscopy. Biochim. Biophys Acta. 779 (1984)
381-401.
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• cotyledon tissue has a higher lipid:protein ratio thanembryos
• no significant degree of lipid mobilization occurs during drying (we see sucrose mobilization inhigh-sugar seed such as white oak)
• changes in protein secondary structure2 occurred inboth 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
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• the most sensitive indicator of viability loss was achange in protein secondary structure to extendedbeta-sheet conformation (absorbance frequenciesless than 1630 cm-1)
• this is contrary to behavior observed in orthodoxseeds using infrared techniques: E.A. Golovina, W.F. Wolkers and F.A. Hoekstra. 86. Behavior ofmembranes and Proteins during Natural Seed Agingin: 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
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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