Transcript No Slide Title

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
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
fresh 29.6
“dry” 19.9
100%
98%
1 y @ +4oC 1 y @ -2oC
88%
5%
“dry” = 2 days on the lab bench in Mississippi
97%
22%
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:
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!
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)
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
• “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%)
% M oisture
Moisture Content vs Day on Bench
40
30
20
10
0
year 1
year 2
0
2
4
Day
6
8
Germination vs Moisture Content
100
80
70
60
50
40
30
20
10
0
40
35
30
25
20
15
% Moisture
10
5
0
% Germination
90
year1
year2
0.355
28 50 .62
29 22 .29
29 20 .17
0.360
0.350
28 52 .54
0.345
0.340
0.335
A b so rba nce
0.330
0.325
0.320
Day 0
0.315
0.310
0.305
0.300
Day 4
0.295
0.290
0.285
0.280
3000
2900
W a ve n u m b e rs (c m -1 )
Membrane lipid -CH2- vibrations in cherrybark embryos
symmetric (2850 cm-1) and asymmetric (2920 cm-1)
0.355
Day 0
0.350
0.345
0.340
0.335
0.330
A b so rb a n ce
0.325
0.320
Day 9
0.315
0.310
0.305
0.300
Day 8
0.295
0.290
0.285
0.280
3000
2900
W avenum bers (c m -1)
Membrane lipid vibrations in cherrybark embryos
Peak frequencies at 2852.5, 2849.7, and 2850.3 cm-1
0.435
0.430
0.425
Day 0
0.420
0.415
0.410
A b so rb a n ce
0.405
0.400
0.395
0.390
Day 9
0.385
0.380
Day 8
0.375
0.370
0.365
0.360
3000
2900
W avenum bers (c m -1)
Membrane lipid vibrations in day 0, day 8, and day 9
cotyledons. Peak frequencies at 2851.9, 2847.2, and
2848.8 cm-1.
The isothermal gel point
Membrane Lipid Phase Transition
2853.7
2853.5
2853.4
2853.3
2853.2
2853.1
2853
2852.9
2852.8
2852.7
2852.6
35
30
25
20
Seed Moisture Content % H2O
15
10
Peak Fequency (cm-1)
2853.6
year 1
0.28
0.26
0.24
A b so rb a n ce
0.22
0.20
0.18
0.16
0.14
0.12
0.10
1800
1700
1600
W avenum bers (c m -1)
Storage lipid vibrations in day 0 cherrybark embryos
and cotyledons. Peak frequency at 1743 cm-1.
Lipid:protein ratio higher in cotyledons (oily seed).
0.34
Day 0
0.33
0.32
0.31
0.30
0.29
A b so rb a n ce
0.28
0.27
Day 8
0.26
0.25
0.24
0.23
0.22
0.21
0.20
0.19
0.18
1800
1700
1600
W avenum bers (c m -1)
Storage lipid vibrations in day 0 and day 8 cherrybark
cotyledons.
0.136
0.135
0.134
0.133
0.132
Day 4
embryos
cotyledons
0.131
A b so rb a n ce
0.130
0.129
0.128
0.127
0.126
0.125
0.124
0.123
0.122
0.121
3000
2900
W avenum bers (c m -1)
Membrane lipid vibrations in day 4 cherrybark embryos
and cotyledons. Peak frequencies at 2850.6 and
2848.4 cm-1 indicating differential drying.
0.285
0.280
0.275
A b so rb a n ce
0.270
0.265
0.260
0.255
0.250
0.245
0.240
1700
1650
1600
1550
W avenum bers (c m -1)
Protein (amide I and II) vibrations of day 0 cherrybark
embryos. Peak frequencies near 1640 and 1550 cm-1
0.285
Day 8
0.280
0.275
A b so rb a n ce
0.270
0.265
Day 9
Day 0
0.260
0.255
0.250
0.245
0.240
1700
1650
1600
1550
W avenum bers (c m -1)
Protein (amide) vibrations in cherrybark embryos.
Peak frequencies at 1638.5, 1635, and 1629.9 cm-1
0.325
0.320
Day 9
Day 0
0.315
0.310
A b so rb a n ce
0.305
Day 8
0.300
0.295
0.290
0.285
0.280
0.275
1700
1650
1600
1550
W avenum bers (c m -1)
Amide protein vibrations in day 0, day 8 and day 9
cherrybark cotyledons.
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 occur
• 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)
2
• 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 structure2 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
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
• 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
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