CHYOBIOLOGY
14,
94-99
Xylem
( 1977)
Pressure Potential and Chlorophyll Fluorescence Indicators of Freezing Survival in Black Locust and Western Hemlock Seedlings 1
as
GREGORY N. BROWN, JAMES A. BIXBY, PETER K. MELCAREK, THOMAS M. HINCKLEY, AND ROBERT ROGERS School of Forestry,
Fisheries and Wildlife, UniocrsitrJ Columbia, Missouri 65201
and associated meter). \Vestern hemlock seedlings ( ?‘sugu heterophyZZa (Raf.) Sarg.), transplanted from a Weyerhaeuser Company research nursery near Centralia, Washington, at 6 months of age, were grown for an additional 16 weeks under the same conditions as described for black locust seedlings. All seedlings were grown in vermiculite maintained near field capacity. Freedug .surtiizjal cletemiaation. Black locust seedlings were used at various points throughout a hardening rc$me previously described by Brown and Bisby (1) to provide a range of hardiness. AYonhardened hemlock seedlings were used. Black locust seedlings at various sampling points were frozen in pots at 5”C/hr to various subfreezing temperatures. Following 30 min at 4”C, several stems were removed and placed in a Scholander pressure chamber (14, 16), and xylem pressure potential (P) was measured. The remaining stems were placed in a mist chamber for 7 davs i after 24 hr at 4°C. Long-term survival was easily determined since living black locust stems remained green with bark tightly bound as compared to dead seedlings with loose white or brown bark. Nonhardened hemlock seedlings either were maintained at 27”C, cooled rapidly
The rapid and accurate determination of the degree of cold hardiness, though fundamental in cold hardiness studies, remains a major problem. The generally accepted procedure is to freeze tissues to various temperatures, thaw them under controlled environmental conditions, and determine survival. Numerous methods have been employed which will be discussed later. This paper proposes two new methods survival; both of which for determining can be used immediately following freezing and thawing, can be done rapidly using a variable number of samples, are relatively repeatable, reduce subjective evaluations, and one of which can be used on chlorophyllous material. \Vhilc special equipment is required, this equipment is relatively inexpensive and can be used both in the laboratory and in the field. MATERIALS
of Missouri,
AND METHODS
Materials. Black locust seeds (Robinia pseudoacacia L. ) were germinated and grown for 4 weeks at 25°C day and 15°C night with a daylength of 15 hr and a light intensity of 90 pE/m”/sec PAR (measured using a Lambda LI 190s quantum sensor Received November 4, 1975. 1 Contrihtion from the Missouri Agricultural Experiment Station. Journal Series No. 7420. 94 Copyright All rights
0 1977 by Academic of reproduction in any
l’rcss, Inr. form r~servrd.
ISSN
0011
2240
to tither 0” or ---7.5 (:, and iriailrtaiiicd at these temperatures for 1 hr or were frozen to -12°C at G”C/hr. Needles from each treatment were thawed in the dark for 30 min at 27°C and used for fluorcsccncc determinations. The remaining foliated stems were placed for 7 days in the mist chamber. Living hemlock stems maintained green necdlcs as compared to dead seedlings with white to brown needles. Analyses of pressure chamber tleterminations. Two hundred and twenty-six paired readings of P and long-term survival were available for analysis. Each paired observation was subjected to both Spearman’s p and Kendall’s 7 tests in order to measure correlations between these two variables (2). I3oth tests indicated significant correlation (p < 0.05) exists bctnieen P and long-term survival. Inspection of plotted data (P vs longterm survival) prompted assigning longterm survival percentages to two classifications: those giving 50% or greater survival (survived) and those giving less than 5076 survival (dead). The probability of “surviving” based on immediate pressure chamber readings was estimated by fitting a sigmoid curve to all paired observations using regression techniques for a dichotomous dependent variable as applied by Hamilton (6). The X? goodness of fit test was employed to determine how well the observed probability distribution compared with the predicted probability distribution (2); i.e., it tested the adequacy of the logistic model. Fluorescence cleten,-Lirzations.Needles of ‘iVestern hemlock sampled at various freezing points were monitored for fluorescence at 25°C. The fluorescence-monitoring apparatus has been described by Melcarek and Brown (11). RESULTS
The magnitude of the X? test statistic differences indicated that no significant
F1c.1. Relationship between black locust seedling probability of survival-classification for longterm survival and xylem pressure potential (P) inmcdiately following freezing and thawing. Probability of survival-classification refers to the probability of fitting into a long-term “survival” classification defined as samples with seedling survival of 50% or freatcr.
existed between observed and predicted survival probability distributions of xylem pressure potential (P) readings (p < 0.05). Therefore, the logistic model was used to describe the functional relationship between the probabilities of “survival” and P readings in this experiment (Fig. 1). Vsing Fig. 1, a sample with an observed P of -10 bars would indicate a 0.75 probability of that sample fitting into the survived classification described under An&yses of pressure &umber determinations. In contrast, a P of -1 bar woulcl have a probability of only 0.2.5. Figures 2A and R illustrate on two different time scales the simultaneous measurement of fluorescence transients of hemlock needles subjtcted to four different temperature regimes. These complr~\ fluorescence chailges occur clue to the induction of photosynthetic activity after a long dark pretreatment ( 12). Fluorescence intensity is partially controlled by the osidationreduction state of the primary electron acceptor of photosystem II (3). Increases in electron transport generally quench fluercscence. Another controlling factor is the high-cnergv state of the thvlakoid mem-
BROWN ET AL.
9G
n
Ttme,sec.
occurs ( Fig. 211) . I’llis suggests that needles could csprcss a slightly survival than those -7.5”C even though a long-term basis.
diffcrcuI frozen higher frozen neither
rcspot Isc to -12°C capacity for rapidly to survived on ial
DISCUSSION
FIG. 2. Chlorophyll fluorescence transients of needles from western hemlock seedlings immediately following thawing from various freezing treatments in the dark. (A), Short-term fluorescence transients. (B), Long-term fluorescence transients.
brane (21). Hence any damage to the membrane, to photosystem II, or to elcctron transport will cause a modification of the fluorescence transients. The needles that survived showed the following fluorescence characteristics: (i) The initial fluorescence is considerably less than the maximum fluorescence which is achieved after approximately 1 set (Fig. 2-4); (ii) considerable fluorescence quenching to a steady state occurs following maximum fluorescence (Fig. 2B). The fluorescence transients of needles subjected to 0°C showed no qualitative differences from the control needle transients, and the needles also had long-term survival. The needles that did not have long-term survival showed (i) little difference between initial and maximum fluorescence (Fig. 2A) and (ii) little long-term fluorescence quenching (Fig. 2B). This is similar to the fluorescence from a chlorophyll solution which is invariant with time. Rapid freezing to -7.5”C appears to have a more adverse effect than slow freezing to -12°C. In the -75°C sample, initial fluorescence equals maximum fluorescence while in the -12°C sample a slight initial rise in fluorescence
The pressure chamber is a relatively inexpensive tool and is easily portable for field use. Xylem water is under varying tensions in plant stems and as xylem (symplastic) water content decreases, xylem pressure potential (P) becomes more negative. When cells are frozen intrncellularly, membrane fracture likely occurs, and a rapid loss of cellular (apoplastic) water should follow. Therefore, an increase iu P (less negative) should be expected with cell rupture and death of tissue. Grouping for survival above and below 50% is a stndard procedure for expressing survival (7). While the probabilities may not assure absolute prediction, any presently used method applied immediately following freezing and compared to longterm survival would likely yield predictive characteristics much less than 100%. Most methods have not been subjected to this type of statistical analysis in comparison with long-term survival. Since desiccation is known to increase as a result of the environmental growth conditions immediately prior to programmed freezing studies, during induction of hardiness ( 1, S), or during senescence, P determinations would be influenced by these variables, Lower P readings may be obtained, suggesting higher survival than is in fact true. Generally, visible observations of shoots for color and flexibility immediately prior to programmed freezing studies, plus good control of environmental moisture conditions, will reduce these problems. Also, since survival prediction is based on major differences in P, the slight
tlcsiculic~l~ IIiirclcr
d~lrillg imnwliatc:
lwdcliillg dctc,lnliiratiolts
sl~o~ilcl of
1101 SIX-
of ;I large: IJiagJiitJdc followin, (r short-term frccziiig and, hence, membrane damage, apoplastic loss of mater into symplast must have occurrcd. Se\wal detc,rmin~~tions should lx used. for nvcrages since individual deter~ninntions can be inconsistent. Readings of high P yielding high sur\iwl pose another problem. ,4 combination of relativdy high hydration before freezing and relatively high capacity for recovery from freezing damngc which occurs in semilrardenccl seedlings could account for these observations. Tlw vast majority of such observations did occur in scmihardcned sccdlings. Freezing treatments below supercooling tcmpcratures (10) would result in celhdar dehydration and esccssi\x cxtracellular water. This condition combined with relatively high survival as seen in semihardened and hardened seedlings also could result in high P values. To be useful, a distribution of P readings corresponding to a range of long-term survival percentages should be done at various dcgrces of cold hardiness for each species, tissue, and dc~~elopmental stage studied. %c then collld use P readings from any conrp~lral~le sample to compare with the estininted population P values plotted against long-term survival percentages to predict sample survival in excess of or be101~ SO>/;,. Otherwise, differences in freezing survival cnpacity, growth potential, aud internal water balance between species, tissues, and developmental stages would alter the relationship lx~t\\ww P values and survival perccwtagcs. The chlorophyll fluoresccncc indrlction phenomenon is a tool long used to demonstrate the functioning of the primary processes of photosynthesis related to chloroplast niembranc~s ( 12). -411~ changes iJ, meniln2ne charge separation, photosystem II, and elrctron transport arc easily identi-
\?\xi.
change
If oiic in
ol~ww
P
1)) ;L lc2f's ll~~orx~scc~~c~c cllaractcrislics t, I2 j. I m\x3 d;m~a(:(d 1,~ fwc~ziiig tculIwraliiiw show ;it~ alteration of tlicsc chnractclristics. Oftcii it is not possible to make :I quick \isllal deternlination of freezing damage to a coilif(>r nerdle as a deep green color c;iii persist loiig nftcr irreparable danlagc has occlirrcd. JIowc\~er~ monitoring of flunrescclw transients of damaged lcxvcss offers a quick n11c1con\vlimt dctcrnnination of IGbility. \C’hile fluoreswncc i~ieasurcnicnt may rq~~ire separate quipment and obviously are limited to chlorophyllous material, they do provide a rapid and efficient technifIue for d~~tc,niiiJiatior1 of leaf survival. The correlation with long-term tissue sur\-iv:11 is excellent, and detectable differences of sur\~ival can he found at very sharp Ailling points with a minimum of subjective c~\alllatioJl. The possibility of chloroplast niortality \ritli siibseqncnt ccl11siirvi\xl and chloroplast regeneration csists. Ilowevcr, all long-term survi\xl correlntions lvitli loss of fluorescence dccn!~ suggest that cellular rcco\wy is mllikcly when immediate damage to clioloroplast function occurs. Alany perennial plaits surjrive frcwing temperatures even though their lea\w arc not freezing rcsistailt. LYliilc wlliilar recover) is umlikcly in chlorophylious ~11~ of needles \vhen immediate damage to chloroplast function occurs, total plant survival might still occur e\wi with the loss of needles. 7’1~ loss of fluorcwcwcc decay iI,dicates inability of pliotos~5tcw I to rcoxiclime (2 ( 12), thus suggesting apparent irrc~versible damage to at least photosystem I Since photosystem I ill the cliloroplnst. provides rcduccd NADPH, the chloroplast loses its supply of rcductant to continue carbon fixation iir tlie Cal\-iii cycle. The instantaneous rise see11 in Fig. 24 for nonsurvi\al trcatmcuts indicates that no mechanism rcwiained in the quenching chloroplnsts. This loss of qiienching reflects destrllction of the photoclicmical apparatus. fial)l~~
9s
BROWN
Electrical in~pcdar~~~ (4, 5), second and third exothcrm ( lo), tissue discoloration (8, 9, 15), amino acid efflux (17), viability staining ( 7, 18), and long-term survival either under optimal growth conditions or in mist chambers (8, 13, 19) all have been employed to determine tissue survival following freezing tests. Long-term survival often introduces other survival variables with time, and theoretically a single parenchymatous cell could become activated and produce new tissue, while the vast majority of the tissue may have been killed. Nonetheless, long-term survival has been claimed to be the most accurate method of survival determination ( 7). Electrical impedance also appears to provide an accurate method for determination of hardiness with limited complications (4, 5). The remaining methods listed above are relatively nonrepeatable within a large population of frozen seedlings, are tedious to perform, or are limited in usage to tissues which can absorb viability stains rapidly and lack high concentrations of visible pigments. The pressure chamber and fluorescence methods reported in this paper provide rapid, immediate determination of survival with a relatively high degree of correlation with long-term survival. Both techniques also provide portability for field or greenhouse determinations, assuming facilities are available for control of freezing levels and rates (20). SUMMARY
Xylem pressure potential was determined using the Scholander pressure chamber on stems of cold hardened and nonhardened black locust (R&i& pseudoacucia L.) seedlings following freezing to various nonlethal and lethal temperatures and subsequent thawing. Correlation was found between immediate xylem pressure potential and long-term seedling survival. Chlorophyll fluorescence transients were monitored using needles of western hem-
ET At.
lock ( I~‘SU~CLlwteropkylh (Iiaf.) Sarg.) seedlings following freezing to various nonlethal and lethal temperatures and subsequent thawing. Immediate and repeatable differences in fluorescence transients correlated with long-term seedling survival. Methodology is described and correlations discussed relative to using either chlorophyll fluorescence or xylem pressure potential as an immediate indicator of Iongterm freezing survival in woody plant seedIings. REFERENCES 1. Brown, G. N., and Bixby, J. A. Soluble and insoluble protein patterns during induction of freezing tolerance in black locust seedlings. PhysioZ. Plant. 34, 187-191 (1975). 2. Conover, W. J. “Practical Nonparametric Statistics.” John Wiley, New York, 1971. 3. Duysens, I,. N. M., and Sweers, H. E. Mechanisms of two photochemical reactions in algae as studied by means of fluorescence. In “Studies on hficroalgae and Photosynthetic Bacteria” (S. h4iyachi, Ed.), pp. 353-372. University of Tokyo Press, Tokyo, 1963. 4. Glerum, C., and Krenciglowa, E. M. The dependence of electrical impedance of woody stems on various frequencies and tissues. Curd. J. Bot. 48, 2187-2192 ( 1970). 5. Greenham, C. G., and Daday, H. Electrical determination of cold hardiness in Trifolium repens L. and Medicago sativa L. Nature (LorIdon) 180, 541-543 (1957). 6. Hamilton, D. A., Jr. “Event Probabilities Estimated by Regression,” USDA Forest Service Research Paper INT-152. lntermountain Forest and Range Experiment Station, Ogden, Utah, 1974. 7. Levitt, J. “Responses of Plants to Environmental Stresses,” pp. 75-109. Academic Press, New York 1972. 8. Li, P. H., and Weiser, C. J. Increasing cold resistance of stem sections of Cornzis stoloniferu by artificial dehydration. Cryobiology 8, 108-111 (1971). 9. hlcKenzie, J. S., We&r, C. J., and Burke, M. J. Effects of red and far red light on the initiation of cold acclimation in Cornus stolonifera Michz. Plant Physiol. 53, 783789 (1974).
INDICATORS 10. McLeester,
R. C., Weiser,
OF FREEZING C. J., and Hall,
T.
SURVIVAT,
IN
SEEDLINGS
09
sen, E. A., and Bradstrect,
E. D.
Ilydro-
C. Multiple freezing points as a test for
static pressure and osmotic potential in
viability of plant stems in the determination of frost hardiness. Plotlt PhrJsiol. 44, 37-
lenves of mnngroves and some other plants.
44 (1969).
(19B3).
11. hlelcnrek, I’. K., ant1 Brown, G. S. In \-iv0 chlorophyll fluorescence monitoring with solid state photosensors. Physiol. Plant. 35, 147-151 (1975). 12. Papageorgious, G. Chloropl~yll fluorescence: An intrinsic probe of photosynthesis. 111. “Bioenergetics of l’hotosyntliesis” ( Covindjce, Ed.), p. 319. Acndrmic Press, New York, 1975. 13. Pellett, N. E., and m’hite, D. B. Soil-air temperature relationships and cold acclimation of container grown Jnliilxrus chiiW~si.s “Hetzi.” J. Amer. Sot. Hart. Kci. 94, 453456 (19G9). 14. Ritchie, G. A., and Hinckley, T. ?\I. The pressure chamber as an instrument for ecological research. 1~ “Advances in Ecological Research” (A. 1IncFayden, Ed.), \‘ol. 9, p. 165. Academic Press, Sew York, 1975. 15. Snkai, A. Characteristics of winter hardiness in extremely hardy twigs of woody plants. Plant Cell Physiol. 14, l-9 (1973). 16. Scholandrr, P. F., Iimnmcl, H. T., Ilemming-
Proc. Nat.
Acrid.
Sci.
USA
52,
119-125
D., Therricn, II., Gfcller, F., 17. Siminovitch, and Rhearnne, 1~. The qnantitative cstimntion of frost injury and resistance in black locust, alfalfa, and wheat tissnes By determination of amino acitls and other ninhydrinreacting sulxtanccs released after thnwinq. Catlad. J. Bat. 42, ($37~649 ( 1964). 18. Steponkus, I’. L., and Lnnphenr, F. 0. Refinement of the triphcnyl tetrazolium chloride method of determining cold injury. Plant Ph~siol. 42, 1423-1426 ( 1967). 19. Stergios, B. G., and IIowell, G. S., Jr. Evaluation of \-iability tests for cold stressed plants. J. Amer. Sec. Hart. Sci. 98, 325-330 (1973). 20. Wiltbank, \\‘. J,, and Rouae, R. E. A portable freezing unit for determining leaf freezing points of citrus 1~~s. Ilort. Sci. 8, 510511 ( 1973). 21. Wraight, C. i4., and Crofts, A. R. Energydependent quenching of chlorophyll a Auorescence in isolated chloroplasts. J. Rio&em. 17, 319-327 (1970).
14,
94-99
Xylem
( 1977)
Pressure Potential and Chlorophyll Fluorescence Indicators of Freezing Survival in Black Locust and Western Hemlock Seedlings 1
as
GREGORY N. BROWN, JAMES A. BIXBY, PETER K. MELCAREK, THOMAS M. HINCKLEY, AND ROBERT ROGERS School of Forestry,
Fisheries and Wildlife, UniocrsitrJ Columbia, Missouri 65201
and associated meter). \Vestern hemlock seedlings ( ?‘sugu heterophyZZa (Raf.) Sarg.), transplanted from a Weyerhaeuser Company research nursery near Centralia, Washington, at 6 months of age, were grown for an additional 16 weeks under the same conditions as described for black locust seedlings. All seedlings were grown in vermiculite maintained near field capacity. Freedug .surtiizjal cletemiaation. Black locust seedlings were used at various points throughout a hardening rc$me previously described by Brown and Bisby (1) to provide a range of hardiness. AYonhardened hemlock seedlings were used. Black locust seedlings at various sampling points were frozen in pots at 5”C/hr to various subfreezing temperatures. Following 30 min at 4”C, several stems were removed and placed in a Scholander pressure chamber (14, 16), and xylem pressure potential (P) was measured. The remaining stems were placed in a mist chamber for 7 davs i after 24 hr at 4°C. Long-term survival was easily determined since living black locust stems remained green with bark tightly bound as compared to dead seedlings with loose white or brown bark. Nonhardened hemlock seedlings either were maintained at 27”C, cooled rapidly
The rapid and accurate determination of the degree of cold hardiness, though fundamental in cold hardiness studies, remains a major problem. The generally accepted procedure is to freeze tissues to various temperatures, thaw them under controlled environmental conditions, and determine survival. Numerous methods have been employed which will be discussed later. This paper proposes two new methods survival; both of which for determining can be used immediately following freezing and thawing, can be done rapidly using a variable number of samples, are relatively repeatable, reduce subjective evaluations, and one of which can be used on chlorophyllous material. \Vhilc special equipment is required, this equipment is relatively inexpensive and can be used both in the laboratory and in the field. MATERIALS
of Missouri,
AND METHODS
Materials. Black locust seeds (Robinia pseudoacacia L. ) were germinated and grown for 4 weeks at 25°C day and 15°C night with a daylength of 15 hr and a light intensity of 90 pE/m”/sec PAR (measured using a Lambda LI 190s quantum sensor Received November 4, 1975. 1 Contrihtion from the Missouri Agricultural Experiment Station. Journal Series No. 7420. 94 Copyright All rights
0 1977 by Academic of reproduction in any
l’rcss, Inr. form r~servrd.
ISSN
0011
2240
to tither 0” or ---7.5 (:, and iriailrtaiiicd at these temperatures for 1 hr or were frozen to -12°C at G”C/hr. Needles from each treatment were thawed in the dark for 30 min at 27°C and used for fluorcsccncc determinations. The remaining foliated stems were placed for 7 days in the mist chamber. Living hemlock stems maintained green necdlcs as compared to dead seedlings with white to brown needles. Analyses of pressure chamber tleterminations. Two hundred and twenty-six paired readings of P and long-term survival were available for analysis. Each paired observation was subjected to both Spearman’s p and Kendall’s 7 tests in order to measure correlations between these two variables (2). I3oth tests indicated significant correlation (p < 0.05) exists bctnieen P and long-term survival. Inspection of plotted data (P vs longterm survival) prompted assigning longterm survival percentages to two classifications: those giving 50% or greater survival (survived) and those giving less than 5076 survival (dead). The probability of “surviving” based on immediate pressure chamber readings was estimated by fitting a sigmoid curve to all paired observations using regression techniques for a dichotomous dependent variable as applied by Hamilton (6). The X? goodness of fit test was employed to determine how well the observed probability distribution compared with the predicted probability distribution (2); i.e., it tested the adequacy of the logistic model. Fluorescence cleten,-Lirzations.Needles of ‘iVestern hemlock sampled at various freezing points were monitored for fluorescence at 25°C. The fluorescence-monitoring apparatus has been described by Melcarek and Brown (11). RESULTS
The magnitude of the X? test statistic differences indicated that no significant
F1c.1. Relationship between black locust seedling probability of survival-classification for longterm survival and xylem pressure potential (P) inmcdiately following freezing and thawing. Probability of survival-classification refers to the probability of fitting into a long-term “survival” classification defined as samples with seedling survival of 50% or freatcr.
existed between observed and predicted survival probability distributions of xylem pressure potential (P) readings (p < 0.05). Therefore, the logistic model was used to describe the functional relationship between the probabilities of “survival” and P readings in this experiment (Fig. 1). Vsing Fig. 1, a sample with an observed P of -10 bars would indicate a 0.75 probability of that sample fitting into the survived classification described under An&yses of pressure &umber determinations. In contrast, a P of -1 bar woulcl have a probability of only 0.2.5. Figures 2A and R illustrate on two different time scales the simultaneous measurement of fluorescence transients of hemlock needles subjtcted to four different temperature regimes. These complr~\ fluorescence chailges occur clue to the induction of photosynthetic activity after a long dark pretreatment ( 12). Fluorescence intensity is partially controlled by the osidationreduction state of the primary electron acceptor of photosystem II (3). Increases in electron transport generally quench fluercscence. Another controlling factor is the high-cnergv state of the thvlakoid mem-
BROWN ET AL.
9G
n
Ttme,sec.
occurs ( Fig. 211) . I’llis suggests that needles could csprcss a slightly survival than those -7.5”C even though a long-term basis.
diffcrcuI frozen higher frozen neither
rcspot Isc to -12°C capacity for rapidly to survived on ial
DISCUSSION
FIG. 2. Chlorophyll fluorescence transients of needles from western hemlock seedlings immediately following thawing from various freezing treatments in the dark. (A), Short-term fluorescence transients. (B), Long-term fluorescence transients.
brane (21). Hence any damage to the membrane, to photosystem II, or to elcctron transport will cause a modification of the fluorescence transients. The needles that survived showed the following fluorescence characteristics: (i) The initial fluorescence is considerably less than the maximum fluorescence which is achieved after approximately 1 set (Fig. 2-4); (ii) considerable fluorescence quenching to a steady state occurs following maximum fluorescence (Fig. 2B). The fluorescence transients of needles subjected to 0°C showed no qualitative differences from the control needle transients, and the needles also had long-term survival. The needles that did not have long-term survival showed (i) little difference between initial and maximum fluorescence (Fig. 2A) and (ii) little long-term fluorescence quenching (Fig. 2B). This is similar to the fluorescence from a chlorophyll solution which is invariant with time. Rapid freezing to -7.5”C appears to have a more adverse effect than slow freezing to -12°C. In the -75°C sample, initial fluorescence equals maximum fluorescence while in the -12°C sample a slight initial rise in fluorescence
The pressure chamber is a relatively inexpensive tool and is easily portable for field use. Xylem water is under varying tensions in plant stems and as xylem (symplastic) water content decreases, xylem pressure potential (P) becomes more negative. When cells are frozen intrncellularly, membrane fracture likely occurs, and a rapid loss of cellular (apoplastic) water should follow. Therefore, an increase iu P (less negative) should be expected with cell rupture and death of tissue. Grouping for survival above and below 50% is a stndard procedure for expressing survival (7). While the probabilities may not assure absolute prediction, any presently used method applied immediately following freezing and compared to longterm survival would likely yield predictive characteristics much less than 100%. Most methods have not been subjected to this type of statistical analysis in comparison with long-term survival. Since desiccation is known to increase as a result of the environmental growth conditions immediately prior to programmed freezing studies, during induction of hardiness ( 1, S), or during senescence, P determinations would be influenced by these variables, Lower P readings may be obtained, suggesting higher survival than is in fact true. Generally, visible observations of shoots for color and flexibility immediately prior to programmed freezing studies, plus good control of environmental moisture conditions, will reduce these problems. Also, since survival prediction is based on major differences in P, the slight
tlcsiculic~l~ IIiirclcr
d~lrillg imnwliatc:
lwdcliillg dctc,lnliiratiolts
sl~o~ilcl of
1101 SIX-
of ;I large: IJiagJiitJdc followin, (r short-term frccziiig and, hence, membrane damage, apoplastic loss of mater into symplast must have occurrcd. Se\wal detc,rmin~~tions should lx used. for nvcrages since individual deter~ninntions can be inconsistent. Readings of high P yielding high sur\iwl pose another problem. ,4 combination of relativdy high hydration before freezing and relatively high capacity for recovery from freezing damngc which occurs in semilrardenccl seedlings could account for these observations. Tlw vast majority of such observations did occur in scmihardcned sccdlings. Freezing treatments below supercooling tcmpcratures (10) would result in celhdar dehydration and esccssi\x cxtracellular water. This condition combined with relatively high survival as seen in semihardened and hardened seedlings also could result in high P values. To be useful, a distribution of P readings corresponding to a range of long-term survival percentages should be done at various dcgrces of cold hardiness for each species, tissue, and dc~~elopmental stage studied. %c then collld use P readings from any conrp~lral~le sample to compare with the estininted population P values plotted against long-term survival percentages to predict sample survival in excess of or be101~ SO>/;,. Otherwise, differences in freezing survival cnpacity, growth potential, aud internal water balance between species, tissues, and developmental stages would alter the relationship lx~t\\ww P values and survival perccwtagcs. The chlorophyll fluoresccncc indrlction phenomenon is a tool long used to demonstrate the functioning of the primary processes of photosynthesis related to chloroplast niembranc~s ( 12). -411~ changes iJ, meniln2ne charge separation, photosystem II, and elrctron transport arc easily identi-
\?\xi.
change
If oiic in
ol~ww
P
1)) ;L lc2f's ll~~orx~scc~~c~c cllaractcrislics t, I2 j. I m\x3 d;m~a(:(d 1,~ fwc~ziiig tculIwraliiiw show ;it~ alteration of tlicsc chnractclristics. Oftcii it is not possible to make :I quick \isllal deternlination of freezing damage to a coilif(>r nerdle as a deep green color c;iii persist loiig nftcr irreparable danlagc has occlirrcd. JIowc\~er~ monitoring of flunrescclw transients of damaged lcxvcss offers a quick n11c1con\vlimt dctcrnnination of IGbility. \C’hile fluoreswncc i~ieasurcnicnt may rq~~ire separate quipment and obviously are limited to chlorophyllous material, they do provide a rapid and efficient technifIue for d~~tc,niiiJiatior1 of leaf survival. The correlation with long-term tissue sur\-iv:11 is excellent, and detectable differences of sur\~ival can he found at very sharp Ailling points with a minimum of subjective c~\alllatioJl. The possibility of chloroplast niortality \ritli siibseqncnt ccl11siirvi\xl and chloroplast regeneration csists. Ilowevcr, all long-term survi\xl correlntions lvitli loss of fluorescence dccn!~ suggest that cellular rcco\wy is mllikcly when immediate damage to clioloroplast function occurs. Alany perennial plaits surjrive frcwing temperatures even though their lea\w arc not freezing rcsistailt. LYliilc wlliilar recover) is umlikcly in chlorophylious ~11~ of needles \vhen immediate damage to chloroplast function occurs, total plant survival might still occur e\wi with the loss of needles. 7’1~ loss of fluorcwcwcc decay iI,dicates inability of pliotos~5tcw I to rcoxiclime (2 ( 12), thus suggesting apparent irrc~versible damage to at least photosystem I Since photosystem I ill the cliloroplnst. provides rcduccd NADPH, the chloroplast loses its supply of rcductant to continue carbon fixation iir tlie Cal\-iii cycle. The instantaneous rise see11 in Fig. 24 for nonsurvi\al trcatmcuts indicates that no mechanism rcwiained in the quenching chloroplnsts. This loss of qiienching reflects destrllction of the photoclicmical apparatus. fial)l~~
9s
BROWN
Electrical in~pcdar~~~ (4, 5), second and third exothcrm ( lo), tissue discoloration (8, 9, 15), amino acid efflux (17), viability staining ( 7, 18), and long-term survival either under optimal growth conditions or in mist chambers (8, 13, 19) all have been employed to determine tissue survival following freezing tests. Long-term survival often introduces other survival variables with time, and theoretically a single parenchymatous cell could become activated and produce new tissue, while the vast majority of the tissue may have been killed. Nonetheless, long-term survival has been claimed to be the most accurate method of survival determination ( 7). Electrical impedance also appears to provide an accurate method for determination of hardiness with limited complications (4, 5). The remaining methods listed above are relatively nonrepeatable within a large population of frozen seedlings, are tedious to perform, or are limited in usage to tissues which can absorb viability stains rapidly and lack high concentrations of visible pigments. The pressure chamber and fluorescence methods reported in this paper provide rapid, immediate determination of survival with a relatively high degree of correlation with long-term survival. Both techniques also provide portability for field or greenhouse determinations, assuming facilities are available for control of freezing levels and rates (20). SUMMARY
Xylem pressure potential was determined using the Scholander pressure chamber on stems of cold hardened and nonhardened black locust (R&i& pseudoacucia L.) seedlings following freezing to various nonlethal and lethal temperatures and subsequent thawing. Correlation was found between immediate xylem pressure potential and long-term seedling survival. Chlorophyll fluorescence transients were monitored using needles of western hem-
ET At.
lock ( I~‘SU~CLlwteropkylh (Iiaf.) Sarg.) seedlings following freezing to various nonlethal and lethal temperatures and subsequent thawing. Immediate and repeatable differences in fluorescence transients correlated with long-term seedling survival. Methodology is described and correlations discussed relative to using either chlorophyll fluorescence or xylem pressure potential as an immediate indicator of Iongterm freezing survival in woody plant seedIings. REFERENCES 1. Brown, G. N., and Bixby, J. A. Soluble and insoluble protein patterns during induction of freezing tolerance in black locust seedlings. PhysioZ. Plant. 34, 187-191 (1975). 2. Conover, W. J. “Practical Nonparametric Statistics.” John Wiley, New York, 1971. 3. Duysens, I,. N. M., and Sweers, H. E. Mechanisms of two photochemical reactions in algae as studied by means of fluorescence. In “Studies on hficroalgae and Photosynthetic Bacteria” (S. h4iyachi, Ed.), pp. 353-372. University of Tokyo Press, Tokyo, 1963. 4. Glerum, C., and Krenciglowa, E. M. The dependence of electrical impedance of woody stems on various frequencies and tissues. Curd. J. Bot. 48, 2187-2192 ( 1970). 5. Greenham, C. G., and Daday, H. Electrical determination of cold hardiness in Trifolium repens L. and Medicago sativa L. Nature (LorIdon) 180, 541-543 (1957). 6. Hamilton, D. A., Jr. “Event Probabilities Estimated by Regression,” USDA Forest Service Research Paper INT-152. lntermountain Forest and Range Experiment Station, Ogden, Utah, 1974. 7. Levitt, J. “Responses of Plants to Environmental Stresses,” pp. 75-109. Academic Press, New York 1972. 8. Li, P. H., and Weiser, C. J. Increasing cold resistance of stem sections of Cornzis stoloniferu by artificial dehydration. Cryobiology 8, 108-111 (1971). 9. hlcKenzie, J. S., We&r, C. J., and Burke, M. J. Effects of red and far red light on the initiation of cold acclimation in Cornus stolonifera Michz. Plant Physiol. 53, 783789 (1974).
INDICATORS 10. McLeester,
R. C., Weiser,
OF FREEZING C. J., and Hall,
T.
SURVIVAT,
IN
SEEDLINGS
09
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11. hlelcnrek, I’. K., ant1 Brown, G. S. In \-iv0 chlorophyll fluorescence monitoring with solid state photosensors. Physiol. Plant. 35, 147-151 (1975). 12. Papageorgious, G. Chloropl~yll fluorescence: An intrinsic probe of photosynthesis. 111. “Bioenergetics of l’hotosyntliesis” ( Covindjce, Ed.), p. 319. Acndrmic Press, New York, 1975. 13. Pellett, N. E., and m’hite, D. B. Soil-air temperature relationships and cold acclimation of container grown Jnliilxrus chiiW~si.s “Hetzi.” J. Amer. Sot. Hart. Kci. 94, 453456 (19G9). 14. Ritchie, G. A., and Hinckley, T. ?\I. The pressure chamber as an instrument for ecological research. 1~ “Advances in Ecological Research” (A. 1IncFayden, Ed.), \‘ol. 9, p. 165. Academic Press, Sew York, 1975. 15. Snkai, A. Characteristics of winter hardiness in extremely hardy twigs of woody plants. Plant Cell Physiol. 14, l-9 (1973). 16. Scholandrr, P. F., Iimnmcl, H. T., Ilemming-
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D., Therricn, II., Gfcller, F., 17. Siminovitch, and Rhearnne, 1~. The qnantitative cstimntion of frost injury and resistance in black locust, alfalfa, and wheat tissnes By determination of amino acitls and other ninhydrinreacting sulxtanccs released after thnwinq. Catlad. J. Bat. 42, ($37~649 ( 1964). 18. Steponkus, I’. L., and Lnnphenr, F. 0. Refinement of the triphcnyl tetrazolium chloride method of determining cold injury. Plant Ph~siol. 42, 1423-1426 ( 1967). 19. Stergios, B. G., and IIowell, G. S., Jr. Evaluation of \-iability tests for cold stressed plants. J. Amer. Sec. Hart. Sci. 98, 325-330 (1973). 20. Wiltbank, \\‘. J,, and Rouae, R. E. A portable freezing unit for determining leaf freezing points of citrus 1~~s. Ilort. Sci. 8, 510511 ( 1973). 21. Wraight, C. i4., and Crofts, A. R. Energydependent quenching of chlorophyll a Auorescence in isolated chloroplasts. J. Rio&em. 17, 319-327 (1970).
