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

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

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

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%)

Moisture Content vs Day on Bench

010203040

0 2 4 6 8

Day

% M

oist

ure year 1

year 2

Germination vs Moisture Content

0

10

20

30

40

50

60

70

80

90

100

0510152025303540

% Moisture

% G

erm

inat

ion

year1

year2

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)

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

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

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

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).

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

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

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

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

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

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.

• 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

• 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

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