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University Microfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106 8315522 Wolpert, James Alan COLD ACCLIMATION OF ‘CONCORD’ GRAPEVINES PH.D. Michigan State University University Microfilms International 300 N. Zeeb Road, Ann Arbor, M I 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V 1. 2. . Glossy photographs or pages______ Colored illustrations, paper or print_____ 3. Photographs with dark background 4. Illustrations are poor copy______ 5. Pages with black marks, not original 6. Print shows through as there is text on both sides of page______ 7. Indistinct, broken or small print on several pages 8. Print exceeds margin requirements______ 9. Tightly bound copy with print lost in spine______ 10. Computer printout pages with indistinct print______ 11. Page(s)___________ lacking when material received, and not available from school or author. 12. Page(s)___________ seem to be missing in numbering only as text follows. 13. Two pages numbered____________. Text follows. 14. Curling and wrinkled pages______ 15. Other_____________________________________________________________________ copy__ University Microfilms International COLD ACCLIMATION OF 'CONCORD' GRAPEVINES By James A. Wolpert A DISSERTATION Submitted to Mi c h i g a n .State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1983 ABSTRACT COLD ACCLIMATION OF ’CONCORD' GRAPEVINES By James A. Wolpert Cold acclimation of 'Concord' grapevines (Vitis labruscana Bailey), investigated in Michigan during late summer and fall of three years, began in early August and continued to increase gradually through early November. Hardening occurred first in basal bud and shoot tissues (nodes 2-4), followed in order by middle (nodes 6-8) and apical (nodes 10-12) tissues. Increased hardiness to about -13°C in the early portion of the acclimation period (through late Septem­ ber) was related to loss of tissue water, but later hardiness increases were unrelated to water content. Increased hardiness was also positively related to shoot exposure to sunlight and advanced tissue maturation, the latter evidenced by a change in shoot color from green to brown. The relationship of cold hardiness, shoot maturation, and tissue water content is discussed. Potted vines exposed to night-interrupted (NI) photoperiods had a greater percentage of shoots with active growing points and a greater number of nodes per shoot, when compared to plants exposed to naturally decreasing daylength in late summer and fall. Apical tissues of plants under NI photoperiods showed slightly inhibited James A. Wolpert cold acclimation but NI treatment did not prevent full acclimation. Root resistance, measured as suction-induced water flow through detopped root systems, increased throughout the acclimation period, but was not influenced by NI photoperiod. Root resistance, measured at 500-590 torr, was significantly affected by premeasurement handling such as soil removal or low pressure pretreatment (5 min at 150 torr). Differential thermal analysis (DTA) of freezing in acclimating shoot segments indicated that changes in tissue maturation were accompanied by changes in freezing pattern. During acclimation DTA profiles changed from that of a single bulk water exotherm to one showing 3 distinct exotherms. This dissertation is dedicated to my parents Louis and Vera Wolpert for their love and unfailing support ACKNOWLEDGMENTS I would like to express my appreciation to the members of my guidance committee, Drs. Robert Olien, Norman Good, James Flore, and David Dilley, for their suggestions during the conduct of the research and for thoughtful criticisms of the dissertation. I wish to thank my friends, fellow graduate students, and faculty for their advice and encouragement; thanks especially to Keith Striegler, Tim Mansfield, and Hector Escamilla for their efforts in the viticultural research program which allowed me the extra time I needed to complete the dissertation. A special thanks is extended to the National Grape Coopera­ tive and Mr. William Grevelding for financial support and to grape growers, Bob Rogers and Bill and Brian Cronenwett, for their fine cooperation in the field aspects of this research. Finally, I wish to express a deep sense of gratitude to my advisor and friend, Stan Howell, for his guidance, counsel, and inspiration during my development as a scientist. About him I can say what Jacques Monod said of his mentor, Andre Lwoff— he is a man "... whose example I have done no more than try to follow, from whom I have learned that the sharpest criticism can also be generous and that the severest scientific exactitude, rather than forbidding actually authorizes and encourages enthusiasm for the boldest specu­ lations." iii TABLE OF CONTENTS Page LIST OF T A B L E S ................ vii LIST OF F I G U R E S .............................................. INTRODUCTION . . ix .......................................... 1 ........................................... 3 Acclimation of Plants under Field Conditions . . . Acclimation under Artificial Conditions ............. Photoperiod ....................................... Temperature ....................................... Water . Changes in Tissue Accompanying Acclimation . . . . Ecological Aspects of Acclimation .................... Logic of the Dissertation R e s e a r c h ................ 3 3 4 6 7 8 13 14 LITERATURE REVIEW LITERATURE C I T E D .......................................... SECTION I: 17 NATURAL ACCLIMATION OF 'CONCORD'BUD AND CANE TISSUES INTRODUCTION ................................................. 30 MATERIALS AND M E T H O D S ....................................... 33 Cold Hardiness A s s e s s m e n t .......................... Tissue Water Measurement ............................. 34 35 R E S U L T S .................................................... 36 Study 1 Study 2 .............................................. .............................................. 36 38 D I S C U S S I O N ................................ 40 LITERATURE CITED 58 .......................................... iv Page SECTION II: RELATIONSHIP OF COLD HARDINESS, WATER CONTENT, AND TISSUE MATURATION IN 'CONCORD' GRAPEVINES INTRODUCTION ................................................. 63 MATERIALS AND M E T H O D S ....................................... 65 R E S U L T S .................................................... 66 D I S C U S S I O N .................................... 68 LITERATURE C I T E D .......................................... 76 SECTION III: EFFECT OF NATURAL AND NIGHT-INTERRUPTED PHOTOPERIODS ON ACCLIMATION OF POTTED 'CONCORD' GRAPEVINES INTRODUCTION ................................................. 79 MATERIALS AND METHODS ....................................... 81 T e c h n i q u e s ........................................... Hardiness Assessments ............................. Tissue Water Content Measurements .................. Root Resistance ....................... 83 83 83 83 R E S U L T S .................................................... 85 DISCUSSION 87 LITERATURE C I T E D ...................... 98 SECTION IV: PRELIMINARY STUDIES ON DIFFERENTIAL THERMAL ANALYSIS OF FREEZING IN CANES OF ACCLITING 'CONCORD' GRAPEVINES INTRODUCTION ................................................. 101 Studies on Higher Plants . Stem S t u d i e s ....................................... Bud S t u d i e s ....................................... 104 104 105 MATERIALS AND M E T H O D S ................ ^..................... 109 Differential Thermal Analysis ....................... Tissue Water Content.... ............................... v 109 Ill Page RESULTS AND DISCUSSION LITERATURE CITED SECTION V: .................................... .......................................... 112 122 STUDIES ON ROOT RESISTANCE OF POTTED 'CONCORD' GRAPEVINES INTRODUCTION ................................................. 127 MATERIALS AND M E T H O D S ....................................... 128 Study 1 Study 2 Study 3 .............................................. .............................................. .............................................. RESULTS AND DISCUSSION LITERATURE CITED .................................... .......................................... vi 128 128 129 130 139 LIST OF TABLES Table Page Section I 1. 2. 3. 4. 5. 6. Potential factors affecting cold acclimation of 'Concord* grape buds and c a n e s ....................... 43 Effect of vine size on hardiness (T50) and water content of primary buds and canes of 'Concord' grapevines, September 16, 1979 44 Effect of cluster number per shoot on hardiness (T50) and water content of primary buds and canes of 'Concord' grapevines ........................... 45 Effect of shoot exposure to sunlight on hardiness (T50) and water content of primary buds and canes of 'Concord' grapevines, Lawton, Michigan, September 30, 1979 .................................................... 46 Effect of node position on cold hardiness (T50) and water content of primary buds of 'Concord' garpevines, 1979 .................................................... 47 Effect of node position on cold hardiness (T50) and water content of canes of 'Concord' grapevines, 1979 .................................... 48 Section II 1. 2. 3. Relationship of cold hardiness (T50) » water content, and color of acclimating shoots of 'Concord' grape­ vines, 1980 71 Effect of cane color change on cold hardiness (T50) and water content of primary buds and canes of 'Concord' grapevines, October 7, 1979 .................. 72 Effect cane color changes on cold hardiness (T50) and water content of primary buds and canes of 'Concord' grapevines, October 13, 1979 73 vii Table 4. 5. Page Effect of cane color change on cold hardiness (T^q ) and water content of primary buds and canes of 'Concord* grapevines, September 25, 1980 74 Hardiness (T5 0 ) , water content, and water saturation deficit of canes of 'Concord' grapevines during fall, 1 9 8 1 ..................................................... 75 Section III 1. 2. 3. Growth and maturation of shoots of potted 'Concord' grapevines under natural and night-interrupted photo­ periods. Observations taken on September 2, 1981 . . 92 Effect of night interruption on cold acclimation of bud and cane tissues of potted 'Concord' grapeviness and the response of plants split between the two photoperiods ........................................... 93 Water content of bud and cane tissues of potted 'Con­ cord' grapevines in response to natural daylength and night-interrupted photoperiods and response of plants split between two photoperiods .................... 95 Section IV 1. Water content of stem tissues of 'Concord' grapevines Oil T..1 OUXJf It 4.-S » 1O Q 1 JL • • « • • viii • • • • • • » • 115 - - LIST OF FIGURES Page Figure Section I 1. 2. 3. 4. 5. Hardiness (T50) and water content of primary buds of 'Concord' grapevines during cold acclimation, August ................ to November, 1980, Lawton, Michigan 49 Hardiness (T50) and water content of canes of 'Concord' grapevines during cold acclimation, August to November, 1980, Lawton, Michigan ................ 51 Hardiness (T50) and water content of primary buds of 'Concord' grapevines during cold acclimation, August to November, 1981, Lawton, Michigan ................ 53 Hardiness (T50) and water content of primary buds of 'Concord' grapevines during cold acclimation, August to November, 1981, Lawton, Michigan ................ 55 Maximum and minimum air temperatures (°C) during late summer and fall, 1981, at Lawton, Michigan . 57 Section III 1. 2. Schematic diagram of apparatus used to measure root resistance by suction-induced water flow through detopped root systems ................................. 96 Root resistance of detopped potted 'Concord' grape­ vines in response to natural and night-interrupted photoperiods and the response of plants split between the two photoperiods (split plants) ................. 97 Section IV 1. Schematic diagram of apparatus used in differential thermal analysis (A) and close-up of aluminum block heat sink and thermocouple placement (B) ix 116 Page Figure 2. 3. 4. Differential thermal profiles of stem cross-section of various internode positions from a single shoot of 'Concord1 grapevine, July 27, 1981 ..................... 118 Differential thermal profiles of stem cross-sections of various internode positions from a single shoot of 'Concord' grapevine, September 4, 1982 .. 120 Differential thermal profile of a stem cross-section of a seven-node shoot segment of 'Concord' grapevines . 121 Section V 1. 2. 3. 4. 5. Schematic diagram of apparatus used to measure root resistance by suction-induced water flow through detopped root systems . . . . .................... 134 Schematic diagram apparatus used to measure root resistance by suction-induced water flow, modified to prevent interference by air bubbles . . . 135 Relationship of suetion-induced water flow to the total pressure applied to the stem end of detopped root systems of potted 'Concord' grapevines ................ 136 Effect of soil removal and low pressure pretreatment on suction-induced water flow through detopped root sys­ tems of potted 'Concord' grapevines . . ............. 137 Suction-induced water flow through detopped root sys­ tems of potted 'Concord' grapevines, comparing living root systems with root systems killed by freezing and boiling ................................................. 138 x INTRODUCTION In temperate climates, perennial plants must be adapted to winter temperatures if they are to survive and flourish. Inadequate cold hardiness results in cold injury and leads to the destruction of plant tissues which are important to crop production. The importance of winter survival can be seen from the fact that nearly 14% of all crop losses in the United States over the past 40 years were due to cold temperatures (6). Of interest to agriculturists is how survival is accomplished and how injury can be prevented. The experiments comprising this dissertation constitute an effort to understand how plants become adapted to low temperature stress. Experiments are arranged in sections, each of which addresses an aspect of cold acclimation of grapevines. Each sec­ tion has its own introduction, description of experiments, and discussion of results and it is intended that each will be sub­ mitted later to a journal for publication, after condensation and style modification. The purpose of the literature review, which follows, is to present a detailed background in several areas of cold acclimation of woody plants. Not all of the topics discussed in the review will be addressed in this dissertation. Rather, it is hoped that the review will provide a general frame of reference against which the experiments and conclusions can be judged. 2 Reviewing literature and drawing general principles about cold acclimation from diverse studies is complicated by several considerations. 1. Plant species used in various studies differ dramatically in their abilities to acclimate to cold; herbaceous plants gain very limited resistance, while some woody plant tissues can accli­ mate to survive immersion in liquid nitrogen (-196°C). The mechan­ isms by which these plants avoid or tolerate freezing stresses may be quite different and unrelated. 2. The method of exposure of plants to low temperatures (either field or laboratory conditions, at rates which vary by 2 orders of magnitude) may influence the amount of injury. 3. Evaluating injury by different means, such as regrowth, tissue browning, solute leakage, etc., may preclude meaningful comparison of reuslts from study to study. Recognition of the above difference is crucial in avoiding erroneous conclusions. LITERATURE REVIEW Woody plants actively growing in summer have little innate cold hardiness. Current season's succulent growth may survive a freeze of only a few degrees below 0°C. In late summer and early fall these tissues undergo a dramatic increase in cold resistance so that by early winter, tissues are able to tolerate temperatures of -40°C and below. This increase in hardiness, termed cold acclimation, has been studied in detail and will be discussed in several sections: (1) acclimation of plants under field conditions, of plants under artificial conditions, (2) acclimation (3) changes in tissues accompanying acclimation, and (4) ecological aspects of acclima­ tion. Acclimation of Plants under Field Conditions Increases in cold hardiness which occur under natural field conditions have been monitored in a number of plants by use of frequent, artificial freezing tests. Hardiness begins to increase in late summer or early fall, in most cases well in advance of freezing temperatures. In one-year-old shoots of Cornus (127) and apple (42), the increase in hardiness occurs in two stages, separated by a plateau of several weeks. Hardiness in the first stage reaches 3 -15° to - 2 5 °C while in the second stage it is from -50° to below -90°. Increases in hardiness in the second stage occur very rapidly as much as 60 to 70°C in a 2- or 3-week period (127), and coincide with the occurrence of the first freezing temperatures in the fall (42). Acclimation appears to occur in only one stage in living bark of black locust (108) and stems of Pinus contorta (97) and Hedera helix (112). Differences in the pattern of cold acclimation can be seen in ecotypes and races of several species and these will be discussed in a later section. Acclimation under Artificial Conditions Research to determine the physiological mechanisms under­ lying cold acclimation has concentrated on manipulating regimes of light, temperature, and water under controlled environment condi­ tions. These factors will be discussed separately. Photoperiod The results of many experiments with altered photoperiod is a generally accepted hypothesis that the first stage of cold acclimation is induced by short days (SD) (28). The SD effect is best expressed if accompanied by warm temperatures (ca. 20°C) implying that important metabolic changes are taking place. (28), When plants growing under long days (LD) (16 hr) and warm temperatures are transferred to a SD regime, 4 to 5 weeks are needed to induce the SD effect (28, 132). Leaves are the site of reception (28, 48) Although the leaves do function as a simple trigger (48) , there is also a required photosynthetic function (114). periods (less than 6 hr) Very short photo­ (75) or low light intensities (122, 125) inhibit full hardiness development. However, in Hedera h e l i x , light-enhanced acclimation is not due to accumulated photosynthetic products (112). Early speculations that cold acclimation was mediated by photochrome (48, 126) were subsequently borne out. In plants which respond to SD, acclimation is inhibited by interruption of of the long night by red light (76, 122, 125, 129). Indications that the amount of inhibition is related to the intensity of the interruption (75) suggests that the effect may be more complex than the more common "on-off" photochrome response. Further information about mechanisms of hardening has been gained from studies involving plants modified by the following treat ments: defoliation, girdling, grafting together genotypes which differ in their acclimation response, and by exposing portions of plants to different environments (split plants). Results from these studies indicate that the SD-leaf produces a hardiness promoter (27, 42, 49) which moves in the phloem (27) and which can induce hardiness in portions of a split plant under noninductive conditions (42). The LD-leaf is also the source of a hardiness inhibitor, as evidenced from experiments in which defoliation of the LD side of a SD/LD split plant gave a hardiness increase (42, 49) Despite these demonstrations of SD promotion and LD inhibi­ tion, LD conditions have failed in several studies to prevent 6 eventual acclimation under field (76) and controlled environment conditions (42, 50, 124). The critical daylength which triggers the SD response differs among species and ecotypes. In red-osier dogwood, a 12-hr photoperiod is SD for the clone from Dickenson, North Dakota, but is LD for the clone from Seattle, Washington (28). Growth cessa­ tion in ecotypes of Norway spruce from high latitudes is triggered when plants are shifted from continuous light (24 hr) tp "short days" of 16-20 hr (35). Temperature Low temperatures (LT) are also effective in inducing cold hardiness (99). In plants which respond to SD, LT (15°/5°C, day/ night) regimes confer an additional increase in cold hardiness, but only after completion of the SD response (127). The addition of 1 hr of freezing temperatures (-2 to -5°C) results in maximum hardi­ ness (34). In those plants, LT during LD can give hardiness increases, but only to about -10° (127). gradually However, in some plants, decreasing temperatures can alone result in maximum hardiness (33, 113). The LT effect is oxygen-requiring (34, 37) and is best elicited if it occurs in the dark rather than the light portion of the photoperiodic cycle (132). However, light per se is not required, provided that plants have sufficient carbohydrates available (49, 112). Unlike the photoperiodic response, the LT 7 effect cannot be translocated from the cold half to the warm half of a split plant (42). Neither SD nor LT can induce cold hardiness when plants are undergoing active growth (127) and one aspect of the SD response is to cause shoot growth cessation by terminal bud set (129). Although it is often suggested that growth cessation is a prerequisite to cold acclimation (81, 128), there is no evidence to support the notion that the two are necessarily at odds at all times. In fact, Levitt (61) cites a French reference as concluding that the base of an actively growing shoot can, by lignification, become mated while the tips remain cold tender. cold accli­ Prior to natural acclimation, buds enter physiological rest (110, 127), but entry into rest is not a prerequisite to acclimation (50, 122). Water Subjecting potted plants of red-osier dogwood to water shortage increases hardiness from 6° to 10°C over controls, regard­ less of photoperiod (19, 10), but after plants are returned to wellwatered conditions for 7 days, the LD plants lose stress-induced hardiness while SD plants retain it (85). Although SD treatments do alter water relations of dogwood (77, 84) (discussed in a later section), the effects of SD and water stress are apparently inde­ pendent and additive (18). Maximum hardiness can be achieved without soil water stress (28), and. in fact, the wet, marshy natural habi­ tat of dogwood does not apparently inhibit its hardening. On the other hand, in Douglas-fir, even though water stress enhances growth cessation, it actually inhibits both photoperiodic- and temperatureinduced hardiness increases (121). This suggests that a severe water stress may inhibit metabolic changes required for acclimation. Changes in Tissue Accompanying Acclimation During cold acclimation, many changes take place in plants and these have been investigated to determine which, if any, are causally related to cold hardiness increases. which have been examined include: Cellular constituents sugar (68, 83); starch (68) (including starch-sugar interconversions [25]); proteins (8, 33, 65, 68); free amino acids (68); organic phosphate (69); nucleic acids (9, 33, 34, 65, 66, 105), phospholipids (109, 131); fatty acyl chains (56); and glycolipids (130). Although it is largely agreed that sugars do not offer much cold protection by colligative action, hardiness increases from infiltration of millimolal quantities of sugar (112) suggests a special protective role, which may include hydrogen bonding to sensitive proteins in membranes (103). Demonstration of this pro­ tection is lacking in woody plants which develop resistance to very low temperatures. Studies on the living bark of black locust have led to con­ clusions that increases in soluble proteins are most closely related to acclimation (107). The source of the protein precursors may be senescing leaves (111), but demonstration of soluble protein 9 increases in leaves of evergreen boxwood reduces the likelihood that the changes are due to leaf senescence, unrelated to acclimation (33). Also seen are very high rates of protein synthesis, as evi­ denced by incorporation of *^C-glycine (105) and the entire process is preceded by a sharp rise in several types of RNA (105). The net result of this protein synthesis is "augmentation" of cell proto­ plasm (105), Lack of any increase in DNA (33, 105) suggests that cell division is not responsible for the observed changes. However, it has not yet been determined that the agumentation of protoplasm is causally related to acclimation. Membrane phospholipids have been the subject of investiga­ tion in acclimation, in part because of the attention paid to their role in chilling injury. Alterations in classes of phospholipids or in the relative unsaturation of their fatty acid moieties have been monitored because they may contribute to observed increases in membrane permeability (63, 64, 78). changes is contradictory. Evidence concerning these Full hardiness can be attained in black locust bark without changes in fatty acid unsaturation (109), but this is not a prima facie case against the theory because membrane permeability has not been shown to be a limiting factor in locust acclimation. Increases in fatty acid unsaturation may be due to increased activity of desaturase enzyme at low temperatures. Changes in enzymes accompany acclimation (3, 24, 74, 98). a very detailed comparison of ribulose bisphosphate carboxylaseoxygenase (RuBPCase) from hardened and unhardened wheat and rye, In 10 Huner and Macdowall observed that RuBPCase from hardened plants was more stable in low temperature (-20°C) storage and had a lower Km for CO2 at temperatures below 10°C (and a higher Km above 10°C) In the case of potato (43, 46). (47), RuBPCase from hardy and nonhardy species did not differ in KM for CO2 but differed dramatically in Vmax of CO2 fixa­ tion; at low temperature (5°C), the hardy plant had a higher Vmax than the nonhardy plant, but at a higher temperature (25°C) the former had a much lower Vmax. In both cases, differences in catalytic properties were related to conformational changes in enzyme subunits, as evidenced by differences in net charge and number of exposed SH groups (44, 45, 47). Increases in the permeability of cells to water and salts during cold acclimation were one of the first well-documented changes (63). Recently, in dogwood, membrane permeability as measured by the efflux of tritiated water, increased during the SD phase of cold acclimation but did not change in response to low temperature treat­ ment (78). Although Stout et al. (120) agree that permeability of the plasmalemma increases during acclimation, they report that it is a very small part (0.5%) of the total resistance to water movement, even when fast (31°C/min) cooling rates are used (119). Rather, membrane injury may occur as a result of cell contraction during freezing (117, 118). Also, injury to spruce needles may result from tonoplast damage, leading :to loss of compartmentalization and cellular disruption (133). With the advent of electron microscopy, researchers have attempted to relate ultrastructural changes to increases in cold 11 hardiness. One feature common to 2 studies is an increase in plasma- lemma material, leading to membrane invaginations and a "pinching off" of membrane-bound vesicles (80, 90). This is consistent with reported increases in total membrane phospholipids (109) and may account for changes in membrane permeability, discussed previously. During acclimation of pine needles, changes in ultrastructure of chloroplasts includes a breakdown of the stroma thylakoids (72), and this may explain observed decreases in the efficiency of photosyn­ thetic electron transport (73, 82, 104). More studies are needed before confidence can be placed on the relationship between physio­ logical and ultrastructural changes. During early acclimation, hardiness increases can, in part, be explained by tissue water loss (7, 33, 77, 78, 87). Plant water loss may occur as a result of decreased water uptake, increased transpiration, or tissue maturation. Root suberization may account for increased root resistance (77, 84), but fully suberized root systems are capable of absorbing large quantities of water (22). However, since high soil water levels do not affect tissue water content or cold acclimation (86), the role of roots in influencing cold acclimation, by regulating water uptake, remains a question. Decreasing stomatal resistance seen in leaves of dogwood when exposed to SD conditions in growth chambers (77, 84) also may be responsible for declining tissue water. However, under field conditions, no difference in stomatal resistance was found among dogwood clones which differed in stem water content and cold hardi­ ness (7). Nor were decreases in stomatal resistance in autumn found 12 in leaves of other hardwoods (29, 123) nor in hardened spruce and pine seedlings (21). The evidence seems to indicate that differ­ ences seen under growth chamber conditions may not reflect those in the field. Tissue water content, when expressed on a fresh or dry wt basis, may change by virtue of a change in dry wt alone (59). When these changes are corrected for by use of water saturation deficit (WSD) (20, 59), a reduction in tissue water content is still seen. This decline is closely related to a SD-induced senescence and dehydration of pith cells (77). Examination of stem water by nuclear magnetic resonance (NMR) spectroscopy indicates that acclimation occurs from base to apex of a shoot (11, 76). SD and LD light treat­ ments, which yielded the previously reported hardiness differences had NMR spectra which were identical at the third node from the apex, but different at the fifth node from the apex (76). This invites the suggestion that daylength may influence hardiness by lowering water content at different rates. Although water content can be credited with a great deal of importance in cold hardiness (14, 15, 51), hardiness fluctuations cannot always be equated with changes in water content (33). In spite of the importance placed by Li and Weiser (67) on their experiments which demonstrate that artificially decreasing water content of dogwood stem segments pro­ portionately increased cold hardiness, they overlook instances in their data in which field water content decreased substantially with no increase in hardiness and a 50°C hardiness increase was not accompanied by any change in water content. 13 Even though it was thought for many years that supercooling of water provided only limited protection from freeze injury (62), recent evidence has shown that supercooling is crucial to survival of many fruit crops, such as peach (1, 92), grape (89), apple (94, 95), blueberry (4), and cranberry (23), and a variety of other woody plants (31, 32, 38, 101, 102). This may be responsible for differ­ ences in ecological distribution of woody plants (2, 30). Ability of tissues to supercool changes during acclimation (53, 54, 79, 8 8), and during tissue maturation (53, 54) in response to changes in water content (53, 55) and low temperature (37). Ecological Aspects of Acclimation Studying ecotypes of a single species has provided a con­ venient tool for examining cold hardiness, in general, and acclima­ tion specifically. The most common procedure, called a provenance test, involves the collecting at one site individuals which represent the extreme conditions of a population’s geographical distribution, including factors such as: latitude (and with it photoperiod), longitude, altitude, average annual minimum temperature, and length of growing season. In this way one can identify factors which may be related to the adaptation of an ecotype to its environment. In general, accessions from northern latitudes acclimate first (16, 26, 60, 71, 96, 110) and those from high elevations before those from low elevations (60, 97). However, in dogwood, clones from a similar latitude (Dickenson, North Dakota; and Seattle, Washington) accli­ mate quite differently. Their acclimation pattern (77) correlates better with the length of growing season of their native range (110). Differences in hardiness among these populations are not necessarily present throughout acclimation; oak accessions differ most in hardi­ ness in October (26), while pines differ more in September (97). Growth cessation and bud set correlate well with resistance to injury from the first fall frost (60, 96), but neither factor correlates well with resistance to freezing injury in early winter (96). Redfeldt (96) speculates that resistance to frost damage can be achieved if bud set is sufficiently early to complete shoot lignification and that both this process and resistance to later freezing injury are under genetic control, but are not necessarily related. Climate of origin may not be the deciding factor in poten­ tial hardiness. Willow species, collected from tropical and sub­ tropical climates and planted on 1 site in southwest Japan, were able do survive -15° and could be hardened (by storage at -3°C for 14 days) to survive -50°C (100). This may indicate that some species introduced to a warm climate may still develop a hardiness level which exceeds that required for survival (10). Logic of the Dissertation Research Research into mechanisms of perennial plant cold acclimation has been primarily done on noncrop plants, such as dogwood and black locust (12, 106, 128). In mature tree fruits the ripening crop may present an additional facet of complexity in the acclimation picture; ripening fruit are sources of hormones and sinks for 15 photosynthates and minerals. Indeed, in some plants acclimation and fruit ripening are occurring simultaneously and interactions of the two processes are poorly understood. Although cold acclimation has been studied in a number of important horticultural fruit crops, such as blueberry (5), rasberry (52), cranberry (23), apple (57, 58), cherry (40), and peach (93), interpretation of the results is hindered by 2 aspects of the methods. Firstly, most studies did not include artificial freezing tests often enough to determine when and at what rate acclimation occurred. Differences in hardiness among treatments may vary during the course of acclimation and may influence subsequent mid-winter survival. Secondly, studies did not indicate from what portion of the plant sampling material was taken. Differences in cold resistance due to position-related tissue maturation (39) and due to buds (17, 39) or shoot segments (70) on the growing shoot,suggest that caution must be exercised in order to collect the position of uniform samples over the duration of a long study. Grapevines are an interesting choice for cold hardiness research. Some cultivars have the capacity to develop high resistance to cold injury and are gro"wn near the northern limit of perennial plant culture in many countries. When compared to other woody plants used in hardiness research, grapevines differ in growth habit, most notably in the production of very long shoots which show indetermin­ ate growth (91). The lack of growth cessation by terminal bud set provides an interesting contrast to other plants. 16 In addition, it is known that mid-winter cold hardiness of grapevines can be affected by cultural practices in the previous growing season which influence vine growth and fruit yield and matura­ tion; these include: growing site, method of training, cropping stress, exposure of leaves to sunlight and presence of adequate leaf area, and weed control (13, 39, 41, 115, 116). effects are manifested is not known. How these It may be enlightening to know more about the acclimation period in order to understand how summer practices affect winter hardiness. Because very little is known about acclimation of grapevines, the objectives of this dissertation were to provide a sound back­ ground for further study of acclimation and, specifically, to answer the following questions: 1. What constitutes a rational set of sampling criteria for hardiness tests in acclimating grapevines? 2. 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Soc. Hort. Sci. 89:706-713. 133. Ziegler, P. and 0. Handler. 1980. Tonoplast stability as a critical factors in frost injury and hardening of spruce (Picea abies L. Karst.) needles. Z. Pflanzenphysiol. 99: 393-410. SECTION I NATURAL ACCLIMATION OF 'CONCORD1 BUD AND CANE TISSUES 29 INTRODUCTION Cold injury to grapevines is an important problem, especially at the northern limits of culture where extensive damage to buds can result in severe economic loss. Efforts to understand how injury occurs in grapevines have concentrated on the relationship between cultural practices during the growing season and subsequent mid­ winter hardiness. Cold hardiness differences have been related to growing site (34), cropping stress (14), defoliation (21, 35), weed control (6 ), and training-induced exposure of shoots to sunlight during the growing season (12, 35). How these practices exert an effect on hardiness is not well understood. In an effort to understand how hardiness is gained, much research attention has focused on cold acclimation, a period in late summer or early fall during which plant tissues undergo a dramatic increase in cold resistance. Increases in cold hardiness have been monitored in a number of plants by use of frequent artificial freez­ ing tests. Hardiness begins to increase in late summer or early fall, in most cases well in advance of freezing temperatures. Cold acclimation occurs in 2 stages in stems of dogwood (36) and apple (15), separated by a plateau at about -15° to -20°C, but it appears to occur in only 1 stage in tissues of other plants, such as living bark of black locust (30) and stems of pine (29) and English ivy (32). 30 31 During early portions of the cold acclimation period, hardi­ ness increases can be explained, in part, by tissue water loss (3, 10, 23, 24, 26). Research on dogwood has indicated that SD- induced acclimation involves an increase in root resistance and a decrease in stomatal resistance (23, 26) which may lead to stem water loss. Decline in stem water content occurs from base to apex of a shoot (4, 24) and apparently involves senescence and dehydration of pith cells (24). Whether death of pith cells alone can account for hardiness increases in the whole stem is not known. However, most of the research into mechanisms of perennial plant cold acclimation has been done primarily on noncrop plants (5, 31, 37). In mature fruit trees the ripening crop may present an additional facet of complexity in understanding acclimation since ripening fruits are sources of hormones and sinks for photosynthates and minerals. In some plants the two processes of fruit maturation and cold acclimation are occurring simultaneously and their inter­ actions are poorly understood. Cold acclimation has been studied in a number of important horticultural fruit crops, such as blueberry (2), raspberry (16), cranberry (8 ), apple (18, 19), cherry (13), and peach (28), but interpretation of the results is hindered by 2 aspects of the • methods. Firstly, most studies did not perform artificial freezing tests often enough to determine when and at what rate acclimation occurred. Differences in hardiness among treatments may vary during the course of acclimation and may influence subsequent mid-winter 32 survival. Secondly, studies did not indicate from what portion of the plant sampling material was taken. Differences in cold resis­ tance due to position-related tissue maturation (12) and due to the position of buds (7, 12) or shoot segments (20) on the growing shoot, suggests that caution must be exercised in order to collect uniform samples over the duration of a long study. Grapevines also provide an interesting contrast to other woody plants in that their growth habit is indeterminate (27); they do not produce a terminal bud. Since growth cessation is considered a prerequisite for acclimation (37), a test for the hypothesis may be possible. With these considerations in mind, a study was undertaken with two objectives: (1) to identify sampling criteria which would minimize extraneous variability, and (2 ) to determine the onset and rate of acclimation of 'Concord* grapevines. MATERIALS AND METHODS Studies were conducted over a 3-year period in mature 'Concord' vineyards (Vitis labruscana Bailey) at 2 locations in Michigan. Vines growing in the Wm Cronenwett vineyard in Lawton were of medium vine size (0.4 - 1.4 kg of cane prunings per vine), yielding 5 - 7 tons/acre, and vines growing at the Horticulture Research Center of Michigan State University in East Lansing were of large vine size (1 - 3 kg of cane prunings per vine) yielding 5 - 6 tons/acre. Vines in both vineyards were trained to an upper-wire (1.7m) bi-lateral cordon (also called Hudson River Umbrella), and throughout the experiment were balance-pruned by means of a 30 + 10 formula (i.e., 30 buds retained for the first 0.4 kg of cane prunings and 10 buds for each additional 0.4 kg). Sampling criteria varied within an individual study (as detailed in the Results), but in all cases, sampling material con­ sisting of 1-year-old, 8 - 12 cm node-intemode pieces. Samples gathered in the field were sealed in plastic, stored at ambient temperature for less than 24 hr before being randomly divided into 2 lots for separate determinations of cold hardiness and water content of buds and canes. 33 34 Cold Hardiness Assessment The freezing technique was slightly modified from that of Howell and Weiser (15) and Stergios and Howell (34). Samples were inserted into vacuum flasks in contact with layered aluminum foil to facilitate heat removal and were placed into a Revco chest freezer. Moist cheesecloth in contact with the base of cane pieces served to inoculate the samples with ice, preventing supercooling (22). Freezer temperature was manually lowered to provide a freezing rate of 3 - 5°C/hr for samples; tissue temperature was monitored via a 24 gauge copper-constantan thermocouple inserted into the pith of a representative cane piece in each flask. Flasks were removed at each of several test temperatures and allowed to warm slowly over­ night in a 2°C cooler. A temperature range was chosen such that the warmest temperature produced no injury and the coldest was lethal for all tissues. If this was not achieved, then it was assumed for the purposes of calculations that the next warmest or coldest tempera­ ture would have, respectively, produced complete survival or complete death. Thawed samples were placed into a humid chamber for 7 - 1 0 days, after which tissues were sectioned and rated as alive or dead by the method of tissue browning (33).. Buds were rated as dead when primordia were brown and water-soaked; canes were rated as dead when the phloem-cambium layer was brown-colored. Hardiness was expressed at T ^ (the temperature at which 50% of tissues are theoretically killed), which was calculated by means of the Spearman-Karber equation (1). The formula used was as follows: where: t>n is the percent of dead tissues at one test temperature is the percent dead at the next lowest test temperature t is the value of one test temperature in -°C and t ,, is that for the next lowest test n+1 temperature 2 T 50 values were statistically separated by a Chi method (17) in which the numbers of live and dead tissues for each treatment, com­ bined across selected test temperatures, were fitted into an R x 2 contingency table (25). Chi Chi 2 2 The calculations were that of a standard test and results were compared to the tabular value of a 1-tailed distribution, p = 0.05. When the overall comparison of treat­ ments indicated significance, individual treatment T^ q values were separated in a pair-wise manner. Tissue Water Measurement Tissue water content of treatments was determined by placing 2 - 4 bud or cane pieces each into air-tight glass weighing vials fitted with ground-glass stoppers. Tissues were oven-dried for 36 hr at 70°C (vials open) and reweighed. Water content was calcu­ lated by difference after correction for vial wt and expressed as g water/g tissue dry wt. RESULTS Results are divided into 2 studies: Study 1 was designed to more clearly define a set of sampling criteria which would permit hardiness measurements over time with a minimum of extraneous vari­ ability and Study 2 traced the natural acclimation of bud and cane tissues for two years employing the sampling criteria from Study 1. Study 1 Potential sources of variation in a grapevine canopy were identified along with levels of each factor to be investigated (Figure 1). Factors were studied individually and asterisks indi­ cate at which level other factors were held constant when not being tested. For example, when 3 levels of vine size were being tested, all sampled material was from middle node positions, on exposed shoots, with 3 clusters. Vine size (Table 2) had no significant effect on hardiness or water content of either primary bud or cane tissues. An increas­ ing number of clusters per shoot from 0 to 3 (Table 3) decreased cane hardiness by 2°C in 1979, but had no effect on bud hardiness or on water content for either tissue. When the effect was investi­ gated again in 1981 in a different vineyard, the presence of clusters had no effect on hardiness at any of 3 node positions (Table 3). 36 37 To test the effect of shoot exposure to sunlight on hardiness, material was sampled from the exterior (exposed) and interior (shaded) portions of a vine canopy. After collection, it was noticed that the material from shaded positions could be divided into 2 groups, based on whether or not the shoots had begun to develop brown coloration. These distinctions were retained throughout the hardiness evaluation and calculations. Bud and cane tissues from exposed positions (Table 4) were 2° to 6 °C hardier than both groups of shaded tissues. In the shaded category, if tissues had begun to develop brown colora­ tions, they were about 4°C hardier than shaded green shoots. Tissue water content was lowest in the hardiest treatment and greatest for the least hardy. On 5 dates throughout the acclimation period, node position on the shoot was investigated for its influence on hardiness content of primary buds (Table 5) and canes (Table 6). and canes are similar and will be discussed together. and water Data for buds All tissues increased in hardiness and decreased in water content from August 25 through November 4. Water content increased on the last sampling date (November 9) but in comparison to other years, these data appear anomalous. The greatest hardiness effect due to node position was in August when basal tissues were about 5°C hardier tissues. than apical Differences in hardiness of 3°C were also present in September in buds and hardiness ranking was inversely related to water content. By October 26, hardiness and water content differ­ ences in both buds and canes were not found. In November, basal 38 bud and cane tissues were 2 - 3°C hardier than middle or apical tissues, but these differences were not reflected in changes in water content. Study 2 Natural acclimation pattern was determined for bud and cane tissues for 1980 and 1981. Based on data from Tables 1 through 6 , the sampling method was to take whole canes from positions well exposed to sunlight, but without respect to vine size or number of clusters per shoot. This would allow the monitoring of effects of node position (Tables 5 and 6 ). Although cane hardiness was affected by cluster number, that factor was not considered because of the difficulty of obtaining sufficient material of one category for the duration of the study. Primary bud and cane hardiness and water content data for 1980 and 1981 are presented in Figures 1 through 4. In all cases increasing hardiness (decreasing T,-q ) was accompanied by decreasing water content through about the end of September. Water content decline was most rapid from early August (2 - 3 g l^O/g dry wt) to mid-September (1 g/g dry w t ) . Acclimation increases seemed to be linear, except for primary buds in 1981, when a distinct plateau was seen between mid-September and mid-October. Hardiness increases after the end of September were not accompanied by changes in water content. In both years, base bud and cane tissues acclimated before apical tissues and this was accompanied by earlier water loss in those tissues (Figures 1 through A). With a few exceptions, differ ences in early acclimation were related to water loss; when these water content differences disappeared in mid-September so did hardi ness differences. However, further increases in hardiness not related to water loss may have been due in 1981 to low temperature in late October (Figure 5). DISCUSSION Results presented here support the conclusion that a study of acclimation requires specification of sampling criteria. Dif­ ferences due to exposure of shoots to sunlight (Table 4) are in agreement with those of Howell and Shaulis. (12) who reported that exposed shoots were 6 °C hardier than shaded shoots in September and 8 °C hardier in November. Position of the sampled material on cur­ rent season’s growth influence hardiness (Tables 5 and 6) and in this and other studies it is basal tissues which are hardier than apical tissues (4, 7, 12, 20, 38). Reasons for these positional effects are not known but Section II provides further data and pre­ sents an hypothesis to explain the effect. Effect of fruit load on acclimation was minor (Table 2), but a more detailed study is needed. Vine cold hardiness is reduced when vines are stressed either by retaining excess fruit (12, 14) or by defoliation (35). In this study, use of vigorous vines pruned to provide a recommended crop load may have precluded the importance of different fruit loads per shoot; soluble solids translocation within a vine occurs quite readily in response to separation of fruit sinks from source leaves (21). Acclimation of buds and canes (Figures 1 through 4) begins in early August and increases in a smooth, uninterrupted manner through 40 mid-November. Only primary bud tissues in 1981 (Figure 3) show a plateau (mid-September to mid-October) similar to that seen in acclimation of dogwood (36) and apple (15) stems. These latter studies have been interpreted in light of evidence that 2-stage acclimation is controlled first by short day photoperiod (9, 15) and then by low temperatures (11, 15). Presumably, observed plateaus are caused by a temporal separation of the environmental signals. One can speculate that if low temperatures were to come immediately after completion of the short day response, no plateau would be seen and acclimation would appear as a single, uninterrupted hardi­ ness increase. If this is true, seasonal differences in temperature decline might account for differences in bud acclimation in 1980 (Figure 1) and 1981 (Figure 3). Experiments in controlled environment conditions are needed to determine if both photoperiod and tempera­ ture are required for grapevine acclimation. Decline in water content in both buds and canes over 3 seasons (Tables 5 and 6 , Figures 1 through A) accompanies increase in hardiness suggesting that the two may be related early in the acclimation period. These results agree with those for dogwood stems (23) and leaves of evergreen boxwood (10) where tissue water decline correlates with early stages of hardiness increased, but not later stages. Water content may also explain differences in exposure (Table 4) and node position (Tables 5 and 6 ) in which lower hardiness values were usually accompanied by high water content. These factors are addressed in Section II which discusses interactions of hardiness, water content, and tissue maturation. 42 These studies can be used as a basis for further research into the mechanisms of cold acclimation of grapevines. More research will be needed to understand how cultural practices influence winter hardiness and to what extent these processes can be elucidated by studying cold acclimation. Sound management decisions can then be made in order to ameloriate winter injury and reduce economic loss. 43 Table 1. Potential factors affecting cold acclimation of 'Concord' grape buds and canes.2 Source of Variation Factor Level Within vineyard Vine size (kg of cane prunings) 0.4 ± 0.1 0.9 ± 0.1 1.4 ± 0.1* Within vine Exposure of shoot to sunlight Exposed* Shaded Clusters per shoot Within shoot Node position 0 1 3* Base (#2-4) Middle (#6-8 )* Apex (#10-12) zFactors were investigated individually. Asterisks indicate the level of each factor held constant when other factors were being tested. 44 Table 2. Effect of vine size on hardiness (Tso)2 and water contenty of primary buds and canes of 'Concord' grapevines, September 16, 1979.x Canes Primary Buds Vine size (kg of cane prunings) T50 h 2o T50 h 2o .4 ± .1 - 11.1a 1.47 a - 11.1a 1.23a .9 ± .1 - 9.4a 1.37 a -10.9a 1.32a 1.3 ± .1 - 9.9a 1.38a - 9.9a 1.26a z T5Q calculated by means of Spearman-Karber equation, separations within columns by test, p = 0.05. H^O/g dry wt, differences were not significant at p = 0.05 when separated within columns by Duncan's multiple range test. x Sampled material was from middle node positions (#6-8 ) on shoots well-exposed to sunlight, each shoot possessing 3 clusters. 45 Table 3. Clusters per shoot Effect of cluster number per shoot on hardiness (T50)z and water contenty of primary buds and canes of ’Concord' grapevines. Node positions Cane Primary Bud T X50 h 2o T50 September 15, 1979,, Lawton, Michigan h 2o X 0 6-8 - 9.8a 1.22a -13.1a 1.02a 1 6-8 - 9.4a 1.16a - 12.2ab 1 .10a 3 6-8 - 9.5a 1.24a - 11.1b 1 .10a October 22, 1981, East Lansing, Michiganw 0 2-3 z T5Q 2-4 -14.0a — -14.0a — 6-8 -14.0a — -14.0a — 10-12 -13.7a — -14.0a — 2-4 -13.7a — -14.0a — 6-8 -13.7a — -14.0a — 10-12 -13.7a — -14.0a — calculated by means of Spearman-Karber equation, separa- tions within columns by test, p = 0.05. I^O/g dry wt, value separated within columns by means of Duncan’s multiple range test, p = 0.05. M a t e r i a l sampled from shoots well-exposed to sunlight on vines with 1.4 ± 0.1 kg of cane prunings. wMaterial sampled from shoots well-exposed to sunlight without respect to vine size. 46 Table 4. Effect of shoot exposure to sunlight on hardiness (T^q ) and water contentY of primary buds and canes of 'Concord' grapevines, Lawton, Michigan, September 30, 1979. Treatment w Primary Bud Cane Color T 50 h 2o Cane T 50 H 2° Exposed Brown -13.2a 0.90c -13.5a 0.91c Shaded Lt. Brown -10.5b 1 .11b -11.5b 1.24b Shaded Green - 6.4c 1.69a - 7.5c 2.71a 2 T n calculated by means of Spearman-Karber equation, separations within columns by x 2 test, p = 0.05. I^O/g dry wt, mean separation within columns by Duncan's multiple range test, p = 0,05. x Sampled material was from middle node positions (#6-8 ) on shoots with 3 clusters. Vines had 1.4 ± 0.1 kg cane prunings. w Exposure status was visually determined. Table 5. Effect of node position on cold hardiness (T^q)Z and water content^ of primary buds of 'Concord1 grapevines, 1979.x Sampling dates Node position August 25 T50 H 2° September 13 T 50 h 2o October 26 T 50 h 2o November 4 h T 50 2o November 9 T 50 h 2o Base (#2-4) -8.3a -10.7a 1 .20c -16.5a •81b -18.6a .67a - 2 0 .0a .97a Middle (#6-8 ) -6 .8b - 9.7a 1.32b -16.6a .87a -17.0b .71a -19.0b .95a Apex (#10- 12) -3.0c - 7.2b 1 .58a -15.1a •88a -15.9b .72a -18.5b .95a z T50 calculated by means of Spearman-Karber equation, separations by x 2 test, p = 0.05. I^O/g dry wt, mean separations by Duncan's multiple range test, p = 0.05. x Sampled material was from shoots well-exposed to sunlight, each shoot possessing 3 clusters. Vines had 1.4 ± 0.1kg of cane prunings. Table 6 - Effect of node position on cold hardiness (T^q) 'Concord' grapevines, 1979.X Z Y an^ water content-7 of canes of Sampling dates Node position August 25 T 50 H 2° September 13 T 50 h 2o October 26 November 4 November 9 T T T 50 h 2o 50 h 2o 50 H 2° Base (#2-4) -8.4a - 11.2a 1.09b -16.8 a •86b -20.9a .70 a -20.5a .95a Middle (#6-8 ) -7.3a - 12.2a 1 .20a -16.5a .92a -18.3b .78a -17.8b .94a Apex (#10- 12) -3.9b -11.5a 1 .22a -15.2b .85b -17.1b •76a -16.7c .90a z z T 50 calculated by means of Spearman-Karber equation, separation within columns by x test, p - 0.05. ^g H 20/g dry wt, mean separations within columns by Duncan's multiple range test, p = 0.05.. x Sampled material was from shoots well-exposed to sunlight, each shoot possessiong 3 clusters. Vines had 1.4 ± 0.1 kg of cane prunings. r 49 Figure 1 Hardiness (T5 0 ) and water content of primary buds of 'Concord* grapevines during cold acclimation, August to November, 1980, Lawton, Michigan. Qa PRIMARY BUD 1980 -o- S' 'A. \ Basal nodes (2 -4) a-»a- Middle nodes (6-8) o-,«- Apical nodes (10-12) “A. '□« 1 AUG 1 SEPT 1 OCT NOV 51 Figure 2 Hardiness (T5 0 ) and water content of canes of 'Concord' grapevines during cold acclimation, August to November, 1980, Lawton, Michigan. CANE 1980 o-»«" Basal nodes (2 -4) a - ,* - Middle nodes (6-8) o-,*~ Apical nodes (10-12) 3.0 2.5 >• ■o a., 2.0 1.0 £ 0.5 I AUG SEPT OCT 53 Figure 3 Hardiness (T5 0 ) and water content of primary buds of 'Concord' grapevines during cold acclimation, August to November, 1981, Lawton, Michigan. 0 PRIMARY BUD 1981 ■"Basal nodes (2-41 A—Middle nodes (6-81 (OoOSj.) SS3NIQUVH •-A pical nodes (10-12) “ N \ \ 1 AUG SEPT OCT NOV 55 Figure 4 Hardiness (T5 0 ) and water content of primary buds of 1Concord 1 grapevines during cold acclimation, August to November, 1981, Lawton, Michigan. HARDINESS (T50 °C) (o— ,A-, O-) to 01 to A o A 01 i o 01 DO AUG a> o SEPT DO 08 OCT as M NOV o o to to WATER CONTENT (g/g dry wt.) (■-, A - , •-) 9S o Air Temperature (C o Maximum Minimum 30 20 11 l« AUG Figure 5. SEPT OCT NOV Maximum and minimum air temperatures (°C) during late summer and fall, 1981, at Lawton, Michigan. LITERATURE CITED 1. Bittenbender, H.C. and G.S. Howell. 1974. Adaptation of the Spearman-Karber method for estimating the T 50 of coldstressed flower buds. J. Amer. Soc. Hort. Sci. 99:187-190. 2. Bittenbender, H.C. and G.S. Howell. 1976. Cold hardiness of flower buds from selected highbush blueberry cultivars (Vaccinum australe Small). J. Amer. Soc. Hort. Sci. 101: 135-139. 3. Bray, E.A. and L.R. Parsons. 1981. Clonal variations in the water relations of red-osier dogwood during cold acclima­ tion. Can. J. Plant Sci. 61:351-363. 4. Burke, M.J., R.G. Bryant, and C.J. Weiser. 1974. Nuclear magnetic resonance of water in cold acclimating red-osier dogwood stem. Plant Physiol. 54:392-398. 5. Burke, M.J., L.V. Gusta, H.A. Quamme, C.J. Weiser, and P.H. Li. 1976. Freezing and injury in plants. Ann. Rev. Plant. Physiol. 27:507-528. 6 . Byrne, M.R. and G.S. Howell. 1978. Initial response of Baco noir grapevines to pruning severity, sucker removal, and weed control. Amer. J. Enol. Vitic. 29:192-198. 7. Chaplin, C.E. and G.W. Schneider. 1974. Peach rootstock/scion hardiness effects. J. Amer. Soc. Hort. Sci. 99:231-234. 8 . Eaton, G.W. and B.J. Mahrt. cranberry flower buds. 9. 10. 1977. Cold hardiness testing of Can. J. Plant Sci. 57:461-465. Fuchigami, L.H., C.J. Weiser, and D.R. Evert. 1971. of cold acclimation in Cornus stolonifera Michx. Physiol. 47:98-103. Induction Plant Gusta, L.V. and C.J. Weiser. 1972. Nucleic acid and protein changes in relation to cold acclimation and freezing injury of Korean boxwood leaves. Plant Physiol. 49:91-96. 58 59 11. Harrison, L.C., C.J. Weiser, and M.J. Burke. 1978. Environ­ mental and seasonal factors affecting the frost-induced stage of: cold acclimation in Cornus stolonifera Michx. Plant Physiol. 62:849-898. 12. Howell, G.S. and N. Shaulis. 1980. Factors influencing withinvine variation in the cold resistance of cane and primary bud tissues. Amer. J. Enol. Vitic. 31:158-161. 13. Howell, G.S. and S.S. Stackhouse. 1973. The effect of defolia­ tion time on acclimation and dehardening in tart cherry (Prunus cerasus L.). J. Amer. Soc. Hort. Sci. 98:132-136. 14. Howell, G.S., B.G. Stergios, and S.S. Stackhouse. 1978. Inter­ relation of productivity and cold hardiness of 'Concord' grapevines. Amer. J. Enol. Vitic. 29: 187-191. 15. Howell, G.S. and C. J. Weiser. 1970. The environmental control of cold acclimation in apple. Plant Physiol. 45:390-394. 16. Jennings, D.L. and E. Carmichael. 1972. Variation in the time of acclimation of raspberry canes in Scotland and Ireland and its significance for hardiness. Hort. Res. 12:187-200. 17. Johnson, D.E. and G.S. Howell. 1981. The effect of cane morhology and cultivar on the phenological development and critical temperatures of primary buds on grape canes. J. Amer. Soc. Hort. Sci. 106:545-549. 18. Ketchie, D.O. and C.H. Beeman. 1973. Cold acclimation in 'Red Delicious' apple trees under natural conditions during 4 winters. J. Amer. Soc. Hort. Sci. 98:257-261. 19. Ketchie, D.O., C.H. Beeman, and A.L. Ballard. 1972. Relation­ ship of electrolytic conductance to cold injury and acclima­ tion in fruit trees. J. Amer. Soc. Hort. Sci. 97:403-406. 20. Mair, B. 1968. sequences. summary.) 21. Mansfield, T.K. and G.S. Howell. 1980. Response of soluble solids accumulation, fruitfulness, cold resistance, and onset of bud growth to differential defoliation stress at veraison in 'Concord' grapevines. Amer. J. Enol. Vitic. 32:200-205. 22. McKenzie, J.S., and C.J. Weiser. 1975. Technique to inoculate woody plant stem sections with ice during artifical freez­ ing. Can. J. Plant Sci. 55:651-653. A gradient in cold-resistance of ash bud Planta 82:164-169. (In German with English 60 23. McKenzie, J.S., C.J. Weiser, and P.H. Li. 1974. Changes in water relations of Cornus stolonifera during cold acclima­ tion. J. Amer. Soc. Hort. Sci. 99:223-228. 24. McKenzie, J.S., C.J. Weiser, and M.U. Burke. 1974. Effects of red and far-red light on the initiation of cold acclima­ tion in Cornus stolonifera Michx. Plant Physiol. 53: 783-789. 25. Meddis, R. 1975. Statistical handbook for non-statisticians. McGraw-Hill C o . , London. 26. Parsons, L.R. 1978. Water relations, stomatal behavior and root conductivity of red-osier dogwood during acclimation to freezing temperatures. Plant Physiol. 62:64-70. 27. Pratt, C. 1974. Vegetative anatomy of cultivated grapes— a review. Amer. J. Enol. Vitic. 25:131-150. 28. Proebsting, E .L., Jr. 1963. The role of air temperatures and bud development in determining hardiness of dormant Elberta peach fruit buds. Proc. Amer. Soc. Hort. Sci. 83:259-269. 29. Rehfeldt, G.E. 1980. Cold acclimation of populations of Pinus contorta from the Northern Rocky Mountains. Bot. Gaz. 141:458-463. 30. Siminovitch, D., B. Rheaume, R. Sachar. 1967. Seasonal increase in protoplasm and metabolic capacity in tree cells during adaptation to freezing. In Molecular mechanisms of temperature adaptation. (C.L. Prosser, ed.), pp. 3-40. Publ. No. 84. Amer. Soc. Adv. Sci., Wash. D.C. 31. Siminovitch, D. 1981. Common and disparate elements in the processes of adaptation of harbaceous and woody plants to freezing— a perspective. Cryobiology 18:166-185. 32. Steponkus, P.L. 1971. Cold acclimation of Hedera h e l i x : evidence for a two-phase process. Plant Physiol. 47:175180. 33. Stergios, B.G. and G.S. Howell. 1973. Evaluation of viability tests for cold stressed plants. J. Amer. Soc. Hort. Sci. 98:325-330. 34. Stergios, B.G. and G.S. Howell. 1977. Effect of site on cold acclimation and deacclimation of 'Concord' grapevines. Amer. J. Enol. Vitic. 18:43-48. 61 35. Stergios, B.G. and G.S. Howell. 1977. Effects of defoliation, trellis height, and cropping stress on cold hardiness of 'Concord' grapevines. Amer. J. Enol. Vitic. 28:34-42. 36. van Huystee, R.B., C.J. Weiser, and P.H. Li. 1967. Cold accli­ mation in Cornus stolonifera under natural and controlled photoperiod and temperatures. Bot. Gaz. 128:200-205. 37. Weiser, C.J. 1970. Cold resistance and injury in wood plants. Science 169:1269-1278. 38. Williams, G.J. Ill and C. McMillan. 1971. Frost tolerance of Liquidambar styraciflua native to the United States, Mexico, and Central America. Can. J. Bot. 49:1551-1558. SECTION II RELATIONSHIP OF COLD HARDINESS, WATER CONTENT, AND TISSUE MATURATION IN 'CONCORD1 GRAPEVINES 62 INTRODUCTION Cold resistance of a large plant often varies as a function of the position from which tissue samples are taken (9). One of the most widely reported hardiness differences is that found between the basal and apical bud and twig tissues. Greater levels of hardiness in basal tissues have been reported for ash buds (12), peach twigs (3, 4), raspberry canes (10), and sweetgum twigs (18). In dogwood stems, nuclear magnatic resonance (NMR) spectros­ copy has shown that line broadening from about 20 Hz to 250 Hz is associated with a dramatic increase in hardiness (from -5° to -196°C) and the change begins at the base of a stem and develops acropetally (2, 13). Burke et al. (2) report that basal stem tissues of accli­ mating and nonacclimating dogwood plants have similar wide NMR line widths, but authors do not comment on the relative hardiness of the basal tissues. If NMR line widths are indicative of hardiness, one can speculate that cold hardening may be occurring in actively growing "nonacclimating" plants, perhaps, as a result of advanced physiological age of base tissues. Unfortunately, when positional effects were studied in dogwood, numbering was done from the apex without any indication of relative distance from the base (13, 14). In a detailed study, variation in cold hardiness of tissues within a grapevine canopy was found to be dependent on leaf exposure 63 64 to the sky, cane diameter, persistent lateral status, and presence and color of periderm tissue (9). Specifically, an intact 6-node cane segment was found to have hardier tissues at the basal end (-12° hardier canes and -15°C hardier buds) and was related closely to development of periderm. In Section I differences due to node position on the shoot were also seen and these were associated with water content differences. In this section, additional data are presented which relate cold hardiness, water content, and tissue maturation. The decision to present these data in a one separate section was made after experimentation was finished. The data do not represent an attempt to thoroughly investigate the relationship among the 3 factors. Rather, these data will serve to raise questions about the interpre­ tation of data from previous studies and the conduct of future studies in woody plant cold hardiness. MATERIALS AND METHODS Vines and sampling method used in this study were identical to those used in Section I. Hardiness and water content was measured as detailed in Section I. In addition, water saturation deficit (WSD) (11) was measured on canes in one case, using a formula and method as follows: saturation wt - fresh wt ,nr> WSD = — --- — ---3---- — x 100 saturation wt - dry wt Saturation weight was determined, after fresh weight was taken, by placing cane internode segments (2 - 3 cm long) into glass weighing vials (24 x 48 mm) with ground-glass stoppers, the cut ends in contact with ca. 2 ml water. In closed vials, cane pieces were allowed to take up water for 48 hr. Preliminary data indicated that consistent weight was achieved by this time. Pieces were removed, blotted dry of surface water, placed into a dry weighing vial, and handled as detailed in Section I. Shoot color changes brought on primarily by the development of periderm (16) were assessed subjectively by eye. Relationship of shoot color to the presence or absence of periderm (9) is also sub­ jective, but a gross relationship is known to exist 65 (15, 16). RESULTS Data in Table 1 were taken on samples collected with respect to node position only; color assessments reflected the status of a representative twig. Increased hardiness was accompanied by a decrease in water content and a change in shoot color (Table 1). When shoots were green (August 9), water content was high (2.0 2.6 g/g dry wt) and hardiness was low (-2.0°C). Three weeks later (August 30), color had begun to develop at base nodes while apical nodes were still green. and lowest water content. These brown nodes had the greatest hardiness With all nodes showing brown color (September 13), base nodes were still the hardiest and lowest in water content. On the last date (October 4), a hardiness difference of almost 4°C between base and apical shoot segments was not asso­ ciated with differences in water content or color. Later in the acclimation period, when the color change was further toward the apex, samples were collected with respect to the node position where the color change was most pronounced green to brown (Tables 2 and 3). That node was counted as #4 on the 6-node segment and counting proceeded from that point. On both October 7 (Table 2) and October 13 (Table 3), the 6-node segment had dramatic hardiness differences (7 - 10°C for buds and 6 - 7°C for canes) and brown base tissues in all cases were greater in hardiness and lower in water content than green apical tissues. 66 67 A color change, yellow-green to brown was also seen on persistent lateral shoots (Table 4) and 3-node segments were collected in a manner which maximized the color difference along the segment. Again, brown nodes were hardier and lower in water content than yellow-green apical tissues. Also, hardiness and water content values were comparable to node positions of similar color on the main shoot (Table 2, nodes 3 - 5 ) . Hardiness and water content data presented in Table 5 are the same as in Section I, Figure 5, but here are compared to WSD values. WSD showed a general increase throughout the acclimation period except for a temporary decline on September 26. WSD values showed little relationship to hardiness; little change occurred in WSD when hardiness increased (August 30 to September 5), and a large change occurred when hardiness was constant (September 26 to October 3). WSD was greatest in apical tissues even when no hardi­ ness or water content differences were apparent. DISCUSSION The stem color change which takes place in August and September (Table 1) is due to the development of periderm (16). Periderm in Vitis species forms in nonconducting primary phloem, by a redifferentiation of parenchymatic cells (5, 6 , 7). The formation of suberized cells (phellerm) to the outside of the phellogen isolates the epidermis and cortex, resulting in the death of these tissues. Anatomical aspects of periderm formation have been studied in grapevines (5, 6 ), but little is known about factors which influ­ ence its formation or development. Dark cane color, favored by exposure of shoots to sunlight, is associated with periderm formation (and increased hardiness) 7. ._ (9) but a detailed study of this has not _ J ~ u c e u lilauc • The progression of periderm development along a shoot in grapevines has not been followed, but in other woody plants the development is acropetal (1), similar to the progression of stem color change (Tables 1-4). Tissue maturation in grapevines may be much more complex than just periderm formation; it may include pith cell senescence (14) and changes in cell walls (7). In an intensive series of experiments on several woody species, Borger and Kozlowski determined that periderm formation is influenced 68 69 by photoperiod, light intensity, temperature, water stress, defolia­ tion and growth regulators (see 1). influence acclimation. Many of these factors also If a close relationship exists between shoot maturation and cold acclimation as suggested by data here (Tables 1 to 4), a much more detailed study of periderm and factors which influence its formation, would be very useful. These data raise questions about how to compare treatments in which tissues differ in shoot maturation if that maturation results in hardiness differences. For example, if stems from two plants are different in hardiness at the tips, but of equal hardi­ ness at bases, is the difference important? question one must know the plant species. Clearly, to answer the For grapevines in which relatively short basal portions of canes are retained for fruiting, the two plants should be considered equal in terms of potential to bear a crop of fruit. However, for forest species used for lumber in which maintenance of uninjured terminal buds is crucial to develop­ ment of a straight tree (and hence a saleable product), the hardiness difference at the tips may be of paramount importance. Clearly, then the sampling method which is used should reflect the type of question one wishes to answer. Often, studies do not indicate from what part of a current season's growth samples are taken (8 , 17) or do so by numbering nodes from the apex (13, 14), a method which cannot take into account the relative status of matura­ tion which begins at the base. These studies are not unique and they point to the fact that full interpretation of the physiological 70 importance of studies cannot be done until sampling criteria are more clearly defined. Section III provides further data on the relationship of cold hardiness and tissue maturation as affected by photoperiod and presents additional speculation about the interpretation of other studies. 71 Table 1. Twig Section Relationship of cold hardiness (T5o)> water content, and color of acclimating shoots of 'Concord' grapevines, 1980. Sampling Date Aug 9 Aug 30 Sept 13 Oct 4 10.7a -13.7a rp 2 50 Base -2 .0a -9.8a Middle -2 .0a -7.0b 9 .Oab -13.3a Apex -2 .0a -5.3b 7.8b - 1 0 .0b Water Content (g/g dry wt)y Base 2 .02a 1.28a 1 .01a 0.96a Middle 2.44b 1.48a 1.13a 1 .00a Apex 2.63b 1.95b 1.47b 1 .00a Shoot Color Base Green Brown Brown Brown Middle Green Yellow/Brown Brown Brown Apex Green Green Brown Brown 2 T c q calculated by means of Spearman-Karber equation, means separated within columns by x 2 test, p = 0.05. ^Mean separations of water content within columns by Duncan's multiple range test, p = 0.05. Base = indes #2-4, middle = nodes #6-8 , and apex = nodes #10 - 1 2 . 72 Table 2. Effect of cane color change on cold hardiness (T5 q)z and water contentY of primary buds and canes of 'Concord* grapevines, October 7, 1979. Node position on cane segment Cane color Primary Bud T 50 h 2o Cane T50 h 2o 1 (Basal) Brown -13.5a 1 .22a -13.7a .94a 2 Brown -13.4a 1.42bc -13.5a .99ab 3 Lt. brown -11.5b 1.37b - 1 2 .3ab 1.06b 4 Yellow - 9.7c 1.62d -11.5b 1 .20c 5 Green - 6 .Id 1.60d - 8 .1c 1.41d 6 (Apical) Green - 3.1e 1.55cd - 6.5d 1.403 2 T50 calculated by means of Spearman-Karber equation, separa­ tions by x 2 test, p = 0.05. ^gl^O/g dry wt, mean separations by Duncan's multiple range test, p = 0.05. 73 Table 3. Effect cane color change on cold hardiness (T5 q)z and water contenty of primary buds and canes of ’Concord’ grapevines, October 13, 1979. Node position on cane segment Cane color Cane Primary bud T 50 h 2o h T 50 2o 1 (Basal) Brown - 1 1 .4a l.Olab -11.7b .86a 2 Brown - 8.7b 1.07bc - 9 .2bc .90a 3 L t . brown - 6.7c 1.15cd - 8 .2c .94ab 4 Yellow - 4. Id 1.18cd - 6.5c 1 .00b 5 Green - 4.8d 1.28de - 6 .1c 1.18c 6 (Apical) Green - 4.3d 1.33e - 5.7c 1 .20c Dk. brown -13.0a .90a -13.9a .86a Well exposed ZTrn calculated by means of Spearman-Karber equation; values 2 separated within columns by x test, p = 0.05. y g I^O/g dry wt, means separated within columns by Duncan's multiple range test, p ® 0.05. 74 2 Table 4. Effect of cane color change on cold hardiness and water contentY of primary buds and canes of 'Concord' grapevines, September 25, 1980. Node position on lateral cane segment Cane color 1 (Basal) Brown 2 Lt. brown/ yellow 3 (Apical) Yellow/ green Cane Primary bud T 50 h 2o T 50 h 2o 1.56a - 12.0a -8 .6ab 1 .66b - 9.2 1 .10b -6 .4b 1.93c - 7.0b 1.38c -10.5a .95a ZT,-n calculated by means of Spearman-Karber equation, separations by x 2 test. ^g H 20/g dry wt, values separated by means of Duncan's multiple range test, p = .05. Table 5. Twig Section Hardiness (T50) , water content, and water saturation deficit of canes of 'Concord' grapevines during fall, 1981. Sampling Date Aug 30 Sept 5 Sept 12 Sept 19 rri Sept 26 Oct 3 Oct 31 -14.8a -14.5a -14.5a -2 1 .6a -2 1 .Oab -2 0 .6b Z 50 Base Middle Apex -9.8a -8.5a -5.8b -12.7a - 11.0b - 9.3c -12.3a - 12.0a - 12.0a -13.0a -11.7b -11.5b -14.5a -14.5a - 1 4 .0a Water Content (g/g dry wt)y Base Middle Apex 1.17a 1.33ab 1.59b 0.99a 1.04ab 1.25b 0.93a 0.95a 0.99a 0.94a 0.96a 0.91a 0.97ab 0.99a 0.94b 0 .86a 0.90a 0 .86a 0.89a 0 .88a 0.84a Water !Saturation Deficit51 Base Middle Apex 11.0a 10.1a 11.6a 12.6a 1 2 .1a 12.9a 13.2a 13.6a 17.0b 13.9a 14.0a 16.1b 12.3a 11.1a 14.9b 16.6a 18.1a 2 0 .2b 19.9a 2 1 .8b 22.5b z X T calculated by means of Spearman-Karber equation, means separated with columns by test, p = 0.05. ^Mean separations of water content within columns by Duncan's multiple range test, p = 0.05. xWater saturation deficit calculated as follows: WSD = sa^ura^ on_Y t--------- V A x saturation wt - dry wt Mean separations by Duncan's multiple range test, p = 0.05. iqO LITERATURE CITED 1. Borger, G.A. 1973. Development and shedding of bark. In Shedding of plant parts. (T.T. Kozlowski, ed.), pp. 205236. Academic Press, New York. 2. Burke, M.J., R.G. Bryant, and C.J. Weiser. 1974. Nuclear magnetic resonance of water in cold acclimating redosier dogwood stem. Plant Physiol. 54:392-398. 3. Cain, D.W. and R.L. Andersen. 1976. Sampling procedures for minimizing non-genetic wood hardiness variation in peach. J. Amer. Soc. Hort. Sci. 101:668-671. 4. Chaplin, C.E. and G.W. Schneider. 1974. Peach rootstock/scion hardiness effects. J. Amer. Soc. Hort. Sci. 99:231-234. 5. Davis, J.D. and R.F. Evert. 1970. Seasonal cycle of phloem development in woody vines. Bot. Gaz. 131:128-138. 6 . Esau, K. 1948. Phloem structure in the grapevine, and its seasonal changes. Hilgardia 18:217-296. 7. Esau, K. 1977. New York. Anatomy of seed plants. John Wiley and Sons, 8 . Fuchigami, L = H . S C.J- Weiser. and D.E. Evert. 1971. of cold acclimation in Cornus stolonifera Michx. Physiol. 47:98-103. 9. Induction Plant Howell, G.S. and N. Shaulis. 1980. Factors influencing withinvine variation in the cold resistance of cane and primary bud tissues. Amer. J. Enol. Vitic. 31:158-161. 10. Jennings, D.L. and E. Carmichael. 1972. Variation in the time of acclimation of raspberry canes in Scotland and Ireland and its significance for hardiness. Hort. Res. 12:187-200. 11. Kramer, P.J. 1969. Plant and soil water relationships— a modern synthesis. McGraw-Hill, New York. 12. Mair, B. 1968. sequences. summary.) A gradient of cold-resistance of ash bud Planta 82:164-169. (In German with English 76 77 13. McKenzie, J.S., C.J. Weiser, and M.J. Burke. 1974. Effects of red and far-red light on the initiation of cold acclimation in Cornus stolonifera Michx. Plant Physiol. 53:783-789. 14. McKenzie, J.S., C.J. Weiser, and P.H. Li. 1974. Changes in water relations of Cornus stolonifera during cold acclimation. J. Amer. Soc. Hort. Sci. 99:223-228. 15. Perold, A.I. 1927. A treatise on viticulture. and Co., London. 16. Pratt, C. 1974. Vegetative anatomy of cultivated grapes— a review. Amer. J. Enol. Vitic. 25:131-150. 17. van Huystee, R.B., C.J. Weiser, and P.H. Li. 1967. Cold acclimation in Cornus stolonifera under natural and con­ trolled photoperiod and temperature. Bot. Gaz. 128:200-205. 18. Williams, G.J. Ill and C. McMillan. 1971. Frost tolerance of Liquidambar styraciflua native to the United States, Mexico, and Central America. Can. J. Bot. 49:1551-1558. MacMillian SECTION III EFFECT OF NATURAL AND NIGHT-INTERRUPTED PHOTOPERIODS ON ACCLIMATION OF POTTED 'CONCORD' GRAPEVINES 78 INTRODUCTION Acclimation in grapevines is not well understood. Although we have dealt with the natural acclimation pattern and within-canopy variation (see Sections I and II), little is known about how acclima­ tion in grapevines is initiated. Reports on dogwood and other woody plants have strongly sug­ gested that the first stage of acclimation is initiated by short days (SD) (6 , 20) and mediated by phytochrome (12, 23). Leaves are the site of reception (9) and must be present to facilitate full harden­ ing (6 ). SD leaves produce a hardiness promoter (5) and long day (LD) leaves a hardiness inhibitor (11). Plants split between the inductive (SD) and noninductive (LD) photoperiods are intermediate in hardiness (8 ), suggesting an interaction of the two regulators rather than a single overriding control mechanisms. An important aspect of cold acclimation appears to be the SDinduced decline in tissue water content (13), a portion of which results from pith senescence and dehydration (2, 13). et al. McKenzie (13) and Parsons (14) claim overall plant water decline may be facilitated, or perhaps controlled, by increased root resistance and decreased stomatal resistance (13, 14). If root suberization is the cause of increased resistance to water flow, it may account for observations that plants acclimate regardless of the amount of water 79 present in the root environment (15, 22). This is likely too simplis­ tic an explanation, in view of reports that water stress can promote (3) and inhibit acclimation (19). The purpose of this study was to answer the following ques­ tions: (1) Is short-day photoperiod a requirement for cold acclima­ tion in grapevines? acclimation? That is, will night interruption prevent (2) If so, is it associated with growth cessation, tissue maturation, and/or decline in tissue water content? (3) Is an increase in root resistance related to acclimation of grapevines and is it related to photoperiod? (4) Will split plants simultane­ ously subjected to two photoperiods be intermediate in hardiness or will they be influenced more by one light regime than the other? MATERIALS AND METHODS Plants used in this study were purchased in 1980 from a commercial nursery as 1-year-old rooted cuttings and were potted into 3-gallon plastic pots, containing a sterilized medium of loam soil, sand, and peat (1:1:1 by volume). Plants were thinned to 2 shoots per pot, tied to bamboo stakes, and grown throughout the year without treatment. After a killing fall frost, plants were transferred to a protected lathhouse and mulched over winter. In spring of 1981, 110 uniform vines were randomly assigned to 2 blocks and equally spaced on a 5m x 15m flat concrete area. shoots, Plants were trained to 2 1 on each of the 2 trunks which grew the previous year, tied to bamboo stakes, and trained when growth permitted to an overhead trellis (1.7m) constructed of a grid of wire and twine. Lateral shoots were removed on a regular basis. Plants were watered as needed by means of a drip irrigation system. High pH water (7.8 - 8.0) necessitated the occasional adjustment of soil pH back to 6.0 - 6.5 optimum by use of iron sul­ fate. Fertilization consisted of 2 ca. 1-liter applications of water soluble (12-12-12) fertilizer (300 mg actual N per plot per applica­ tion), the first in early June and the second in early July. In the middle of each block a light barrier was erected (in a N-S orientation) which consisted of a black plastic wall (2m high) 81 82 on a wooden frame. Light treatments were the following: natural daylength (ND), in which plants were exposed to naturally decreasing daylengths (after June 22) and no artificial lighting; nightinterrupted (NI) photoperiod, in which plants were exposed to natural daylength plus % hr of white incandescent light (2.4 ± .8 yEcm _2 ) in the middle of the night period; and split plants (SP), which had 1 of the 2 shoots trained through the light barrier and exposed to natural daylengths (ND-SP) and the other shoot exposed to nightinterrupted (NI-SP). Night interruption was begun on July 27 when daylengths were 14.5 hr and continued until October 30, even after leaves were killed by frost. Measurements of shoot growth (total nodes), shoot maturation (extent of change in cane color, green to brown), and percent actively growing apices were taken at the beginning of night interruption and after 5 wks (September 3) to determine the influence of photoperiod on these parameters. Actively growing tips were those which had young leaves with a fresh green appearance. On 4 sampling dates throughout the acclimation period, plants randomly selected from the three light treatments were assessed for hardiness, tissue water content (first 2 sampling dates only) and root resistance. 83 Techniques Hardiness Assessments Because of the importance of node position in studies in Section 1, nodes were separated into 3 groups: base, #2-4; middle, #6- 8 ; and apex, # 10-12 (numbered from the base). Tissue Water Content Measurements Water content of buds and canes was measured (as described in Section 1) on nodes from positions 1, 5, 9, and 13, numbering from the base. Root Resistance Root resistance was measured in the same manner as that described by McKenzie et al. (13) and Parsons (14). After collection of shoot growth for.hardiness and water content measurements, vines were removed from pots and soil washed off by gentle agitation in a basin of water. Root systems were transferred to 7-liter buckets of fresh, room temperature tap water. Just prior to being attached to the root resistance apparatus, the stem was cut about 8 - 10 cm above soil line, loose bark was removed, and the outer surface of the stem was coated with vacuum grease to prevent entry of water or air. The apparatus used in measuring root resistance is presented as a schematic diagram in Figure 1. upright 2 ml (2 x 0.01) glass pipet The apparatus consisted of an (held in place by small V-jaw clamps), vacuum tubing, and a glass "Y" tube. The bottom end of the glass "Y" was connected to the cut stem by vacuum tubing and secured 84 by a tightened hose clamp. One arm of the "Y" was connected to the pipet by vacuum tubing while the other arm was fitted with a short piece of tubing and a serum stopper. This formed a single unit which was connected to 5 other units by means of a manifold con­ structed of vacuum tubing and glass "T" tubes. The manifold was connected to a manometer at one end and to a vacuum pump at the other end. All connections were secured by hose clamps or were wrapped with parafilm and checked regularly for leaks. Measurement of root resistance was done as follows. With roots in water, the vacuum tubing was attached, secured, and filled partially with water. The pressure was reduced to 150 torr for 5 min to clear the root system of air bubbles. The source of bubbles was not known, but the treatment was necessary to prevent air bubbles from lodging in the pipet leater in measurement. Water in the glass "Y" tube could be observed to ensure that bubbling had stopped. The vacuum was then released and a treatment at reduced pressure of 500 torr was applied. The water level at each position was individu­ ally adjusted to the lower end of the pipets by introducing water from a 50 ml syringe through the serum cap on the arm of the glass "Y". An initial reading of each pipet was taken followed by a final reading after 15 - 30 min. After measurement of 3 - 6 replicates, root systems were oven dried and weighted (only the fibrous portion was weighed, excluding the stem portion of the original cutting). as water flow per hr per 100 g of dry roots. Water flow was expressed RESULTS The influence of light treatments on growth cessation, total shoot growth, and extent of shoot maturation is shown in Table 1. Plants (NI) and shoots (NI-SP) exposed to night-interrupted photo­ periods had a greater percentage of actively growing shoots than plants exposed to natural daylengths. Data taken on total nodes per shoot, mature nodes per shoot, and percent mature nodes per shoot are presented as overall averages of plants in the treatment and as averages for actively growing shoots for comparison purposes (Table 1). Since not all shoots were actively growing, this will allow a compari­ son of these shoots versus the average shoot in the treatment. NI plants had the greatest average number of nodes, about 2 or 3 more than other treatments. Those shoots still actively growing on September 3 ahd about 3 or 4 nodes more than the overall average. However, there were no differences among light treatments in numbers of mature nodes (i.e., nodes with brown coloration, indicating devel­ opment of periderm). Actively growing shoots also had mature nodes, although perhaps an average of 1 node less per plant. This resulted in differences in % mature nodes per shoot, ND-treated plants, and shoots having the most, and actively growing shoots having the least. Hardiness assessments were made on 4 dates throughout accli­ mation (Table 2). By the first sampling date, September 15, primary 85 86 bud and cane tissues had attained significant hardiness. In general, there were only small (2 to 4°C) hardiness increases for most tissues on the following 3 sampling dates. Hardiness differences due to light treatments or node posi­ tions on the cane were sporadic .(Table 2), but generalities can be made. Where differences exist as a function of light treatment, NI-treated plants were least hardy and ND-treated plants most hardy. Shoots of split plants showed no clear relationship to one light treatment or the other, although on several occasions, NI-SP shoots had the lower hardiness level of Nl-treated plants. Differences in hardiness due to node position were most prominent on the first and last sampling dates when apical nodes were less hardy than basal n o d e s . Water content measurements (Table 3) were taken only on the first 2 sampling dates, but no consistent differences were seen due to light treatment. Differences due to node position were only seen on the second sampling date when node 13 was included in the study. Light treatment had no effect on vine root resistance, as measured by suction-induced water flow (Table 4). However, root resistance increased over the course of the experiment as indicated by a decrease in water flow from 2.5 to 0.9 ml per hr per 100 g of dry roots. DISCUSSION Night interruption significantly delays cessation of shoot growth as evidenced by the greater number of nodes per shoot and the percent of shoots with actively growing tips (Table 1). Although cessation is not accompanied by the formation of a terminal bud in grapevines, as it is in other woody plants (18), the process does seem to be under photoperiodic control. Why only 17% of the NI- treated plants had actively growing tips, and not more, is unknown, but one might speculate that the night interruption was insufficiently intense. Although the intensity used here is comparable to that used in other studies (6 , 8 ), the light intensity threshold for photoperiodic response in grapevines is not known. Cool night tem­ peratures also may have played a role; several nights in August tem­ peratures (Figure 3) were below 10°C and low temperature may be abie to override a photoperiodic signal (8 ). Shoot maturation (Table 1) was monitored as nodes developing brown cane color (7), which is associated with periderm development and death of epidermal and cortical tissues exterior to its origin (16). Periderm development in young seedlings of woody plants is affected by many environmental parameters (1), including photoperiod, but factors affecting periderm development in grapevines are unknown. 87 88 Neither night-interrupted photoperiod nor presence of an actively growing shoot tip affected shoot maturation (Table 1). Although light treatments yielded minor (1 - 3°C) differences in hardiness, night-interrupted photoperiods did not prevent acclima­ tion in this study (Table 2) and these results agree with those reported for Cornus (12), apple (8 ), and Viburnum (10). Consequently, without a clear distinction between hardiness of ND and NI plants, it is impossible to draw conclusions about split v i n e s . Since the data implicating short-day control and phytochrome involvement (12) are so conclusive in growth chamber studies, field results are puzzling. 1. Several explanations may be proposed: Plants are responding to a signal from an endogenous rhythm, which may bring about acclimation even under "noninductive" conditions (8 , 21). It is well known that certain tropical tree species exhibit discontinuous shoot growth without any apparent environmental signal (24). Acclimation may be brought about by internally controlled shoot growth cessation without the SD trigger. 2. The most noticeable difference between the two sets of conflicting reports is the total use of artificial light in growth chamber studies (6 , 20) versus the use of natural light in outdoor or greenhouse studies (8 , 12). During the course of the studies using natural light, the sun changes in elevation. This causes changes in spectral distribution especially the ratio of red to farred light at twilight (17) and these changes may have influenced or overriden night break or red light treatments. 89 3. Another possible explanation may lie in the reinterpreta­ tion of growth chamber studies. In these studies, plants are grown as rooted cuttings under warm temperatures and 16 hr photoperiods for 8 - 1 0 weeks (12) after which they are placed into short-day conditions or retained under long-day photoperiods. After a few weeks, SD plants set a terminal bud while LD plants presumably con­ tinue to grow. assessed. It is generally at this point where hardiness is Reinterpretation is required because no indication is given as to where the stem segment for hardiness was taken (6 , 13). If the sample were taken from the tip, a hardiness difference would be expected because actively growing tissue on LD plants would be much more succulent than tissues from shoots which had set a terminal bud (13). Evidence indicates that both shoot maturation and acclima­ tion proceed from the base of a shoot to the apex.(2, Section I ) , but a cause and effect relationship has not been shown. One can speculate that the SD induction of terminal bud set allows acropetal maturation and acclimation to catch up with shoot growth, while in LD plants shoot growth continues. One of the unknowns in this specu­ lation is the maturation status of long- and short-day shoots and whether SD photoperiod actually promotes maturation or if it is related to tissue age. The results might be to place shoot matura­ tion between photoperiodic growth cessation and cold acclimation as an intermediate process. In view of the lack of photoperiodic effect on acclimation, it is not surprising that root resistance (Figure 2) was not affected 90 by light treatment. The increase in root resistance over the course of the experiment (Figure 2) may reflect increased root suberization (13). McKenzie et al. (13) found increased root resistance in SD plants and used this observation as an explanation of water loss in dogwood stems. They imply that root suberization may restrict water absorption enough to cause the tissue water decline, but this argument has several inconsistencies: (1) suberized roots still have the capacity to absorb large quantities of water (4); (2) root-induced stem water stress should have increased stomatal resistance not decreased it; and (3) authors point to large increases in water flow (decreases in root resistance) when roots were killed by boiling. If suberization, a physical barrier, was the cause for increased resistance, killing protoplasts should have had no effect. It seems more likely that the decrease in root resistance may have been due to the death of a physiological barrier, perhaps the root cambium as proposed by Chung and Kramer (4) or due to a physical alteration of the suberin by boiling, i.e., an artifact. In summary, plants under night-interrupted photoperiods had a greater number of nodes and a greater percentage of shoots with actively growing tips, but number of nodes with periderm was not affected. Cold acclimation was not prevented by night-interruption and in all treatments hardiness early in the acclimation period was greater at the basal node positions than at apical node positions. Root resistance increased throughout the acclimation period, but was not different among treatments. 91 Further research is needed to determine how environmental parameters affect shoot maturation and whether these affect hardi­ ness. If photoperiod is not critical to acclimation, as suggested in this study, temperature would be an interesting parameter to investigate next. A study of low temperature or of temperature- photoperiod interactions may provide clues as to how cold acclimation in grapevine is controlled. Table 1. Growth and maturation of shoots of potted 'Concord' grapevines under natural and night-interrupted photoperiods. Observations taken on September 3, 1981. Total nodes per shoot Actively growing shoots Light Treatment2 No. of actively growing shoots Total shoots observed ND 0 40 ND-SP 1 15 NI-SP 3 15 11 40 NI Per­ cent^ 0a 0verallx average 20.1b Total mature nodes per shoot Avg. of shoots actively growing 0verallx average Avg. of shoots actively growing — 14.8a — Percent mature nodes per shoot OverallX average A v g . of shoots actively growing — 73.9a 19.6b 23.0 14.3a 13.0 73.2a 56.5 20.0b 20.7b 24.3 13.6a 12.6 66.7b 50.8 27.5b 22.8a 25.5 13.8a 13.0 61.8b 51.0 6. 7ab zLight treatment abbreviations: ND = natural daylength; NI = night interruption (h hr incandescent light, 2.4 ± .8 yEcm-^) in the middle of the dark period; SP = designation of split plant; 1 shoot trained into natural daylength (ND-SP) and 1 shoot trained into night-interrupted photoperiod (NI-SP). ^Mean separation within columm by x test, p = 0.05. ^ e a n separation within column by Duncan's multiple range test, p = 0.05. 93 Table 2. Date Sept 15 Effect of night interruption on cold acclimation of bud and cane tissues of potted ’Concord' grapevines and the response of plants split between the two photoperiods.2 Tissue 1° bud Cane Sept 24 1° bud Cane Twig Section Light Treatment57 ND ND-SP NI-SP NI Base -12.4a a -13.0a a -14.2a a -13.6a a Middle -13.0a -13.0a -13.0a -14.5a Apex -11.2b a -10.7b ab - 8.3b b - 9.5b b Base -11.8a -13.6a -13.6a -13.6a Middle -11.8a a -13.0ab a -11.3a a -13.0a a Apex -11.2a a -10.1b ab - 8.3b b - 8.3b b Base -15.5a a -14.5a be -15.0a ab -13.9a c Middle -15.0a -15.5a -13.2ab -13.2a Apex -14.5a a -14.0a ab -12.5b b -12.5b b Base -15.3a -15.5a -15.0a -14.0a Middle -15.5a -15.5a -15.0a -13.0ab Apex -13.0b -12.5b -12.0b -12.0b 94 Table 2. Continued Date Tissue Oct 10 1° bud Cane Oct 29 1° bud Cane Twig Section Light TreatmentY ND ND-SP NI-SP NI Base -15.5a a -15.2a a -15.8a a -15.4a a Middle -14.9b a -15.3a a -14.5a a -14.7a a Apex -14.8b a -15.3a a -13.2b b -13.2b b Base -16.2a a -16.6a a -15.8a a -15.8a a Middle -15.8a a -15.8a a -15.8a a -15.8a a Apex -15.8a a -15.8a a -15.3a a -14.9b a Base -19.5a a -19.5a a -17.0a a -17.4a a Middle -18.5a a -19.0a a -17.5a a -18.5a a Apex -16.2b a -17.9a a 16.0a a -15.7b a Base -21.3a a -21.5a a -20.0a ab -19.3a b Middle -21.3a a -20.5ab a -10.5a a -19.8a a Apex -19.0b a -19.0b a -17.5b a -17.5b a zT5q calculated by means of Spearman-Karber equation, mean sepa­ ration by x2 test, p <= 0.05. Letters following values indicate sig­ nificance within columns for an individual date and tissue and letters below values indicate significance within rows. The same letter within a column or row indicates that respective values are not sig­ nificantly different. ^Light treatment, abbreviations: ND = natural daylength, NI = night interruption, ND-SP = shoot of split-plant exposed to natural daylength, NI-SP = shoot of split-plant exposed to interrupted nights. Table 3. Sampling Date Water content of bud and cane tissues of potted 'Concord' grapevines in response to natural daylength arid nightinterrupted photoperiods and response of plants split between two photoperiods. Tissue Light Treatment2 Nodex Number ND ND-SP NI-SP NX Water content (g l^O/g dry wt)^ Sept 10 Sept 24 1° bud 1 5 9 0.83 0.81 1.02 0.95 0.97 1.09 0.85 0.88 1.06 0.85 0.95 0.99 Cane 1 5 9 0.86 0.91 0.91 0.85 0.97 0.95 0.87 0.98 1.00 0.88 0.91 0.91 1° bud 1 0.82a a 0.84a a 0.90a a 1.17b a 0.90a a 0.90a a 0.99a a 1.24b a 0.80a a 0.89a a 0.93b a 1.44c b 0.78a a 0.88ab a 0.96b a 1.25c a 0.89a b 0.86ab a 0.85ab a 0.81b a 0.79a a 0.84a a 0.85a a 0.83a a 0.83a ab 0.84a a 0.85a a 0.92b b 0.84a ab 0.87a a 0.85a a 0.90a b 5 9 13 Cane 1 5 9 13 zLight treatment abbreviations: ND == natural daylength, NI = night interruption, ND-SP = shoot of split plant exposed to natural daylength, NI-SP = shoot of split plant exposed to interrupted night. ^Mean separattion by Duncan' multiple range test, p = 0,05. values for sampling date, September 10, are not significant. xNodes numbered from the base of the shoot. All 96 to vacuum pump replicate set-up to manometer 2ml pipet replicate set-up vacuum tubing 50 cc syringe Figure 1. glass ‘Y* tube serum cap water or soil ~ detopped — root system Schematic diagram of apparatus used to measure root resistance by suction-induced water flow through detopped root systems. 97 o -o Natural daylength Interrupted night Split plant !* > la O) o o ; 1.0 i ■o o o 3 ■o Pressure = 500±12 torr I c o o 3 C/) 10 24 SEPT Figure 2. 30 10 OCT Root resistance of detopped potted 'Concord' grapevines in response to natural and night-interrupted photoperiods and the response of plants split between the two photo­ periods (split plants). Root resistance measured as suction-induced water flow (pressure = 500 ± 12 torr) . LITERATURE CITED 1. Borger, G.A. 1973. Development and shedding of bark. In Shedding of plant parts (T.T. Kozlowski, ed.), pp. 205-236. Academic Press, New York. 2. Burke, M.J., R.G. Bryant, and C.J. Weiser. 1974. Nuclear magnetic resonance of water in cold acclimating red-osier dogwood stem. Plant Physiol. 3. Chen, P., P.H. Li, and C.J. Weiser. 1975. Induction of frost hardiness in red-osier dogwood stems by water stress. HortScience 10:372-374. 4. Chung, H.-H. and P.J. Kramer. 1975. Absorption of water and 32p through suberized and unsuberized roots of loblolly pine. Can. J. For. Res. 5:229-235. 5. Fuchigami, L . H . , D.R. Evert, and C.J. Weiser. 1971. A translocatable cold hardiness promoter. Plant Physiol. 47:164167. 6. Fuchigami, L.H., C.J. Weiser, and D.R. Evert. 1971. Induction of cold acclimation in Cornus stolonifera Michx. Plant Physiol. 47:98-103. 7. Howell, G.S. and N. Shauiis. 1980. Factors influencing within­ vine variation in the cold resistance of cane and primary bud tissues. Amer. J. Enol. Vitic. 31:158-161. 8. Howell, G.S. and C.J. Weiser. 1970. The environmental control of cold acclimation in apple. Plant Physiol. 45:390-394. 9. Hurst, C., T.C. Hall, and C.J. Weiser. 1967. Reception of the light stimulus for cold acclimation in Cornus stolonifera Michx. HortScience 2:164-66. 10. Irving, R.M. and F.O. Lanphear. 1967. Environmental control of cold acclimation in woody plants. Plant Physiol. 42: 1191-1196. 11. Irving, R.M. and F.O. Lanphear. 1967. The long day leaf as a source of cold hardiness inhibitors. Plant Physiol. 42: 1384-1388. 98 99 12. McKenzie, J.S., C.J. Weiser, and M.U. Burke. 1974. Effects of red and far-red light on the initiation of cold acclimation in Cornus stolonifera Michx. Plant Physiol. 53:783-789. 13. McKenzie, J.S., C.J. Weiser, and P.H. Li. 1974. Changes in water relations of Cornus stolonifera during cold acclima­ tion. J. Amer. Soc. Hort Sci. 99:223-228. 14. Parsons, L.R. 1978. Water relations, stomatal behavior and root conductivity of red-osier dogwood during acclimation to freezing temperatures. Plant Physiol. 62:64-70. 15. Pellett, N.E. and D.B. White. 1969. Effect of soil nitrogen and soil moisture levels on the cold acclimation of container grown Juniperus chinensis 'Hetzii'. J. Amer. Soc. Hort. Sci. 94:457-459. 16. Pratt, C. 1974. Vegetative anatomy of cultivated grapes— a review. Amer. J. Enol. Vitic. 25:131-150. 17. Robertson, G.W. 1966. The light composition of solar and sky spectra available to plants. Ecology 47:640-643. 18. Smithberg, M.H. and C.J. Weiser. 1968. Patterns of variation among races of red-osier dogwood. Ecology 49:495-505. 19. Timmis, R. and Y. Tanaka. 1976. Effects of container density and plant water stress on growth and cold hardiness of Douglas-fir seedlings. For. Sci. 22:167-172. 20. van Huystee, R . B . , C.J. Weiser, and P.H. Li. 1967. Cold acclimation in Cornus tolnnifera under natural and controlled photoperiod and temperature. Bot. Gaz. 128:200-205. 21. Weiser, C.J. 1970. Cold resistance and injury in woody plants. Science 169:1269-1278. 22. Wildung, D . K . , C.J. Weiser, and H.M. Pellett. 1973. and moisture effects on hardening of apple roots. Science 8:53-55. 23. Williams, B.J., Jr., N.E. Pellett, and R.M. Klien. 1972. Phytochrome control of growth cessation and initiation of cold acclimation in selected woody plants. Plant Physiol. 50:262-265. 24. Zimmerman, M.H. and C.L. Brown. 1971. Tress— structure and function. Springer-Verlag, New York. Temperature Hort- SECTION IV PRELIMINARY STUDIES ON DIFFERENTIAL THERMAL ANALYSIS OF FREEZING IN CANES OF ACCLIMATING 'CONCORD' GRAPEVINES 100 INTRODUCTION The freezing of water in plant tissues has attracted the attention of researchers for many years. The early reports by Weigand (41) that ice crystals were observed only in intercellular spaces of hardy woody plants and not within the cells, concentrated attention on extracellular freezing and resultant cellular dehydration. Analy­ sis of the freezing process by Luyet and Gehenio (23) confirmed the presence of a double freezing point in living plant tissue and they proposed that the first point was due to freezing of extracellular water and the second to freezing of cellular water. Whether cellular water froze inside or outside the cell was not determined. This dis­ pelled the then current notion that one of the two freezing points was an artifact of technique (see 23). Later suggestion that the second freezing point resulted from melting and then refreezing of extracellular water induced by outflow of cellular solutes (15) was not further confirmed. In the above studies and in others (22, 25, 26, 38) initia­ tion of the second freezing point rarely occurred below a temperature of -8 to — 10°C. So, in spite of suggestions by Tumanov and coworkers (39, 40) that supercooled water occurred at much lower temperatures and was important in survival of hardy plants, it was still a commonly 101 102 held belief in the early 1970s that supercooling played no major role in freezing resistance of higher plants (21). Since that time numerous reports have demonstrated that freezing avoidance by deep supercooling plays a crucial role in winter survival of plants or plant parts (5). These advances have been possible in large measure by development and refinement of 3 techniques: differential thermal analysis (DTA), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR) spectroscopy. Emphasis here will be placed on the DTA studies. Along with technological advances, development of the theoretical foundation for the properties of supercooled water molecules (34, 35, 36) has provided a framework for interpretation of plant data. Supercooling (also known as undercooling or subcooling) occurs when water is cooled below its equilibrium melting point (34). The water is said to be in a "metastable" state, that is, stable until either a more stable, solid (ice) phase is introduced or until the lower temperature limit is reached (see 6). This lower tempera­ ture limit, termed the homogeneous nucleation temperature (HNT), is the point at which ice forms spontaneously in water (34). In practice, the HNT is determined by cooling, to very low temperatures, an emulsion composed of ultra-pure water finely dis­ persed in an organic solvent. This method effectively prevents the water from contacting foreign surfaces or suspended insoluble parti­ cles which catalyze ice nucleation. determined to be -38 ± 1 ° The HNT of pure water has been and is in agreement with theoretical 103 calculations (34). An endotherm near 0°C during a melting cycle of pure water provides evidence that supercooling is taking place and that freezing was not an artifact of the technique. Solute such as glucose, NaCl, or urea added to water lowers the HNT more than it lowers the melting point. This suggests that the effects of solutes at very low temperatures are more complex than the simple reduction in water activity which depresses the melting point. Why water freezes at the HNT may be hypothesized as follows: supercooled water shows a tendency towards "icelikeness" through formation of clusters of water molecules; these clusters are subcritical in size, but favor a structure from which ice can easily nucleate (35). The authors proposed that this metastable, subcriti- cal state becomes unstable at the HNT: (a) cluster distribution increases in mole fraction and in average and maximum cluster size with supercooling, such that some clusters reach critical size at the HNT, or (b) the critical cluster size capable of growth to macro­ scopic size is an exponential, function of supercooling, decreasing as supercooling increases, so that below the HNT clusters smaller than those that would allow nuleation at -40°C assume critical char­ acter. More detailed discussion of behavior of supercooled water can be found (34, 35). Application of this theoretical information to living cells (36) indicates that yeast cells do not survive indefinitely at temperatures above the HNT but, in fact, die by first order kinetics. The rate constant of death is a function of storage temperature. 104 Rasmussen et al. suggest that yeast (and perhaps higher plants) gen­ erate substrates on which ice can form, but corroboration has not yet been provided. Studies on Higher Plants Stem Studies Early experiments on stems of deciduons agalea performed in 1967 by Graham and Mullin, published must later (10), demonstrate that an exotherm seen at -40.5°C is consistently associated with xylem injury. Later Quamme and coworkers (31, 32) reported that apple twigs have 4 exotherms— the first 3 occurring at relatively high temperatures, unassociated with injury of any tissue, while the 4th coincides with killing temperature of xylem ray parenchyma. This lowest exotherm is independent of bulk water freezing, cooling rate, and tissue viability. Presence of a melting endotherm at -5°C pro­ vides further evidence that the exotherm is, in fact, due to freezing of supercooled water. Studies on infiltration of apple wood with various solutes led Krasavtsev (20) to conclude that supercooled water is prevented from flowing out of cells by a complex barrier composed of the cell wall, the plasmalemma, and the outer layer of protoplasm. The sug­ gestion of a role for plasmalemma and protoplasm contradicts con­ clusions of Quamme et al. (32) by DTA and Burke et al., (4) by NMR that the living state of tissue is not responsible for the freezing pattern in xylem parenchyma. The potential for artifacts caused by 105 solute infiltration seems quite high and so might place a question on Krasavtsev1s data (20). In several woody plants species, xylem freezing occurs over a span of 20°C when single cells or groups of cells freeze in inde­ pendent events; water or ice migration between frozen and unfrozen cells is slow, if present at all (11). Death of ray cells as assessed by staining and browning increases over the 20°C span in proportion to the % frozen water (13). Low temperature exotherms in apply can be lowered or raised by pretreatment with cold (-3°C) or warm (+5°C) temperatures respectively (12) even if treated for a very short period of time. Bud Studies Early experiments of Graham and Mullin (10) suggest that bud primordia of deciduous azalea survive low temperatures by super­ cooling as evidenced by the one-to-one association of the number of exotherms (between -17° and -27°C) and number of dead florets. A large exotherm seen at -8°C was attributed to freezing (without injury) in floral axes and bud scales. Later evidence that primorida supercooling is indeed taking place was provided by DTA and NMR studies (9) and further corroboration by DSC indicated that all of the the heat released was due to freezing water (7). That dead primordia also yield exotherms suggested to the authors that super­ cooling is due to structural features of primordia not to living state of protoplasm. 106 Studies on water relations of Rhododendron flower buds indi­ cate that water in florets migrates to frozen bud scales in response to a low temperature (-5°C) treatment, resulting in increased floret supercooling (16). Time required for water migration from primordia to bud scales may explain the dependence of exotherm temperatures on cooling rate during floret supercooling (9, 19); at faster freezing rates, water cannot migrate fast enough and primordia with greater water content supercool to a lesser extent. Natural dehydra­ tion of florets during cold acclimation (18) coincides with a shift in DTA pattern from 1 large exotherm in autumn to 3 smaller sharp exotherms in winter (19). After studying dye movement in Prunus buds, Quamme (30) pro­ posed that, when bud scales freeze, water migrates from the bud axis to scales creating a dry zone which acts as a physical barrier to ice inoculation from stem xylem. Recently, Ashworth (1) provided evidence that it is viability of the bud axis, not the primordium, on which supercooling depends;if the axis does not freeze in a "normal" fashion, then supercooling does not take place. The absence of xylem connections would also facilitate supercooling by isolating primordia from stem freezing. Subsequent xylem development in spring might serve as a mechanism for irre­ versible dehardening. In a study of Prunus buds (2), 11 of 14 species which had no xylem connections to bud primordia (as indicated by dye movement) demonstrated supercooling. The remaining 3 species had xylem connections and did not supercool, but authors did not specify how this related to primordia cold hardiness. 107 The two types of enhancement of bud supercooling, water migration from a floret (16) and physical isolation of a primordium by creation of a dry zone due to water movement in the bud axis (30) are not mutually exclusive and may to some extent be occurring simultaneously. This remains to be elucidated. Water migration may also be important in shoot primordia in conifer buds. Primordia show exotherms at -30°C when left intact in an excised terminal bud, but exotherm at only -12°C when excised from the bud (37). Since artificially decreasing water content of excised shoot primordia decreases the exotherm and since bud scale removal has no effect, Sakai (37) suggested that "crown" tissue of the shoot primordium is the recipient of migrating water. It is clear from these studies that while supercooling in both stems and reproductive primordia depends on structural integrity, the 2 processes differ in that the latter is dependent on cooling rates and presence of viable collateral tissues while the former is not. The presence of low temperature exotherms has ecological and horticultural ramifications. Plants cannot be grown in climates which have a likelihood of frequently achieving the temperature at which exotherms occur and this can limit the natural distribution of woody plants (8, 3) and the location of temperate fruit tree produc­ tion areas (29). Since presence of exotherms or the temperature at which they occur is not uniform for all species (33), introgressing desirable supercooling characteristics may improve hardiness of perennial crops. 108 Studies on freezing pattern of buds and stems of grapevines are very limited. Cane material of Vitis riparia (27) have 5 exo­ therms, 2 nonlethal, and 3 lethal. Of the lethal exotherms, one each is associated with injury to phloem ray parenchyma, to nonray phloem and to xylem tissue. Third and fourth exotherms cannot be seen if freezing rate is very slow, but authors did not indicate whether this affected the presence or absence of injury. The stem low tem­ perature exotherm is about -45°C and rate dependent (28). Unlike tissues previously cited, V_. riparia buds have no bulk water exotherm. Exotherms are not present in fully acclimated buds (27) indicating that freezable water is either absent or in low, undectable amounts. If buds are thawed, 1 exotherm appears for each of the 3 bud primorida (28) leading authors to speculate that the degree of hydration is responsible for changes in seasonal pattern of bud hardiness. This section of the dissertation began with 2 objectives in mind: (a) to assemble the equipment necessary to measure freezing events by DTA and (b) to study acclimation by DTA in hope that changes in the freezing pattern would shed light on important changes accom­ panying cold hardening. Because these efforts were only partially successful, the results presented here should be viewed as a prelim­ inary investigation. Not all points raised in the introduction will be addressed by these studies, but they will provide a frame of reference for interpreting the results. MATERIALS AND METHODS Plant material used in these studies were collected from shoots of mature 'Concord' grapevines growing at the Horticulture Research Center of Michigan State University in 1980-82. Vines were trained to an upper-wire (1.8m) bilateral cordon (Hudson River Umbrella) and were large (1.2 - 2.8 kg of cane prunings) and yielded 5 - 6 tons/acre. Shoots were selected from canopy positions well exposed to sunlight, but were chosen without respect to vine size or number of clusters per shoot. The node position on the shoot from which a sample was taken is indicated in each figure and nodes were numbered from the base of the shoot. Material consisted of stem cross- sections, 0.8 - 1.0 cm in length, taken from the middle portion of an internode. Differential Thermal Analysis The apparatus used for measuring freezing events by differ­ ential thermal analysis (DTA) was similar to that described by Quamme (30) and is shown in a schematic diagram (Figures 1A and IB). The differential thermocouple was made in the laboratory by connect­ ing 2 copper-constantan thermocouples (26 gauge) in series. wire junctions were twisted together, soldered, and sharpened 109 The 110 slightly. One thermocouple was inserted into the pith of a freshly cut stem section and the other was inserted into an oven-dried sample, the latter being used as a reference. Dry biological or nonbiological material may be used as a reference, the main criterion being that the reference not exhibit a phase transition within the experimental range of temperatures. The heat sink (Figure IB) was made of a cylindrical aluminum block (2" diameter x 3" high) into which 2 holes had been bored (0.5" x 2.5" deep). One stem tissue was placed into each hole and held in place by a cork stopper. Mineral oil was introduced into both holes to provide thermal contact between the heat sink and the tissues, and it showed no phase transition to at least -50°C. A single copper-constantan thermocouple was also placed into the hole with the reference tissue in order to record the temperature at which freezing events were taking place. Sample temperature and differential temperature were recorded simultaneously on a Leeds and Northrup Speedomax 250, 2-pen poten­ tiometer. The differential signal was amplified through a Leeds and Northrup linear amplifier while the temperature signal was recorded directly. Temperature drop was achieved by placing the heat sink into a Revco chest freezer which was held at -65° to -85°C. The differential thermocouple generated a signal when the cooling rate of the sample differed from that of the reference. Data recorded on a time x temperature scale were transformed to a temperature x exothermic temperature scale. Ill Tissue Water Content Tissue water content was measured and calculated as described in Section I. RESULTS AND DISCUSSION The shape of DTA patterns presented here (Figures 2 - 4 ) is a function of chart speed, rate of temperature drop, scale of differential response and sample dimensions. The rate of tempera­ ture drop is, in turn, dependent on freezer temperature and size of the heat sink. These factors were not consistent throughout the study as the technique was evolving and this complicates comparisons of groups of DTA patterns. Two studies were made of the influence of node position in DTA pattern, one in mid-summer (July 25) and one in late summer (September 4). In the mid-summer study (Figure 2), 1 to 2 exoterms were seen depending on whether the shoot section was from basal or apical positions. The change from 2 to 1 exotherm occurred between the iith and 14th node position (the 14th having a small shoulder) and corresponded closely to an increase in water content (Table 1), especially in the pith. Exotherm differences may be due to a lower­ ing of the freezing point of water in a certain tissue or to struc­ tural inhibition of ice crystal growth (17, 38). Data were not taken on whether the freezing pattern had any relationship to killing temperature of these nodes. Hardiness data presented in Section 1 indicate that differences between basal and apical tissues are seen as early as the first week in August, but differ slightly from year to year. 112 113 Decline in stem water content due to death and dehydration of pith cells has been correlated with the first stage of cold acclima­ tion in dogwood (24). However, whether the changes in freezing pattern (Figure 2) are causally related to pith water changes and the extent to which these are important in cold resistance needs further investigation. In Section II (Tables 1 - 3 ) were associated with increase in water content. significant hardiness increases tissue maturation and decline in Figure 4 shows DTA patterns from portions of a 7-node shoot section which included mature (brown-colored) nodes at the basal end and less mature (green-colored) nodes at the apical end. Basal shoot sections (Figure 4a) showed 4 distinct and separate exo­ therms, while apical tissues (Figure 4c) showed 3 exotherms, poorly separated and at warmer temperatures (except for the bulk water exo­ therm) . This strongly suggests that maturation-associated increases in hardiness (Section II, Tables 1 - 3 ) may be mediated through changes in the freezing process. The DTA system used in this study (Figure 1) needs refine­ ment, especially in control of the rate of temperature drop, in order to provide better information. bud exotherm measurements. Further sophistication would allow This would be advantageous because primary bud hardiness in 'Concord1 grapevines is usually less than that of critical stem tissues (14). Further research is needed to determine if a close relation­ ship exists between hardiness and DTA profile. This has not been 114 done in grape stem tissues and is especially important because the critical tissue differs. In apple (31), Pyrus and Prunus species (38) and other woody species (1, 8), the most cold sensitive tissue in mid-winter is the xylem ray parenchyma, which yields a large, easily detectable exotherm at the killing temperature. In grape stems, the critical tissue is the phloem-cambium layer, which is usually killed at a warmer temperature than the xylem, precluding the importance of the LTE (28). Phloem injury in wild grape (Vitis riparia) is associated with 2 exotherms, 1 coinciding with death of phloem ray parenchyma and 1 with nonray phloem tissue (27). These exotherms are smaller than the xylem exotherm and can even disappear during very slow rates of freezing. Although the authors do not indicate whether a change in injury was associated with exotherm disappearance, this does point up the need for further investigation into the relationship between DTA patterns and hardiness of critical tissues. 115 Table 1. Water content of stem tissues of 'Concord' grapevines on July 25, 1981.z Tissue Node number Phloem + cortex Xylem + pith Xylem only Pith only 2 3.05a 2.Old 1.56b 5.34a 5 2.94a 2.02d 1.60b 4.53b 8 2.78b 1.96d 1.58b 5.68b 11 2.48c 2.18c 1.50b 4.95b 14 2.33d 2.47b 1.58b 7.10a 17 2.49c 3.07a 1.88a 7.52a zWater content expressed as g l^O/g dry wt. Mean separations within columns by Duncan's Multiple Range test, p = 0.05. xNodes numbered from the base of the shoot to the apex. 116 reference thermocouple differential thermocouple chest freezer linear amplifier 2-pen potentiometer heat sink + to amplifier I to potentiometer (reference signal) ^-cork stopper fresh twig sample dry reference mineral oil aluminum block heat sink Figure 1 Schematic diagram of apparatus used in differential thermal analysis (A) and close-up of aluminum block heat sink and thermocouple placement (B). Exotheirmic i O 117 Temperature I (°C) 118 -15 >. -15 Exothermic -10 -15 / , Tem perature (°C) Figure 2. Differential thermal profiles of stem cross-sections of various internode positions from a single shoot of 'Concord' grapevine, July 27, 1981. Sections were collected from the middle of an internode apical to a numbered node and number­ ing commenced from the base. Profile a = node 2, b = node 5, c = node 8, d = node 11, e = node 14, and f = node 17. Exothermic 119 1 1 I -3 0 Temperature °C -20 i -40 120 Exothermic yk ■20 -30 Temperature °C Figure 3. 40 Differential thermal profiles of stem cross-sections of various internode positions from a single shoot of 'Concord1 grapevine, September 4, 1982. Sections were collected from the middle of an internode apical to a numbered node and numbering commenced from the base. Profile a = node 2, b = node 5, c = node 8, d = note 11, e = node 14, f = node 17, and g = node 20. TEMPERATURE (°C) Figure 4. Differential thermal profile of a stem cross-sections of a seven-node shoot segment of 'Concord' grapevines. The segment was selected so that it was mature and browncolored at the base (profile a), less mature and greencolored at the tip (profile c), and light brown-yellow in the intervening area between the tip and base (pro­ file b). LITERATURE CITED Ashworth, E.N. 1982. Properties of peach flower buds which facilitate supercooling. Plant physiol. 70:1475-1479. Ashworth, E.N. and D.J. Rowse. 1982. Vascular development in dormant Prunus flower buds and its relationship to super­ cooling. HortScience 17:790-791. Becwar, M . R . , C. Rajashekar, K.J.H. Bristow, and M.J. Burke. 1981. Deep supercooling of tissue water and winter hardi­ ness limitations in timberline flora. Plant Physiol. 68: 111-114. Burke, M.J., R.G. Bryant, and C.J. Weiser. 1974. Nuclear magnetic resonance of water in cold acclimating red osier dogwood stem. Plant Physiol. 54:392-398. Burke, M.J., L. V. Gusta, H.A. Quamme, C.J. Weiser, and P.H. Li. 1976. Freezing and injury in plants. Ann. Rev. Plant Physiol. 27:507-528. George, M.F. and M.J. Burke. 1976. The occurrence of deep supercooling in cold hardy plants. Curr. Adv. Plant Sci. 22 349-360. Georges M.F. and M.J. Burke. 1977. Supercooling in overwinter­ ing azalea flower buds; additional freezing parameters. Plant Physiol. 59:326-328. George, M.F., M.J. Burke, H.M. Pellett, and A.G. Johnson. 1974. Low temperature exotherms and woody plant distribution. HortScience 9:519-522. George, M.F., M.J. Burke, and C.J. Weiser. 1974. Supercooling in overwintering azalea flower buds. Plant Physiol. 54: 29-35. Graham, P.R. and R. Mullin. 1976. The determination of lethal freezing temperatures in buds and stems of deciduous azalea by a freezing curve method. J. Amer. Soc. Hort. Sci. 101:3-7. 123 11. 12 . Hong, S.-G. and E. Sucoff. 1980. Units of freezing of deep supercooled water in woody xylem. Plant Physiol. 66:40-45. Hong, S.-G. and E. Sucoff. 1982. Rapid increase in deep supercooling of xylem parenchyma. Plant Physiol. 69:698700. 13. Hong, S.-G., E. Sucoff, and O.K.Y. Lee-Stadelmann. 1980. Effect of freezing deep supercooled water on the viability of ray cells. Bot. Gaz. 141:464-468. 14. Howell, G.S., B.G. Stergios, and S.S. Stackhouse. 1978. Inter­ relation of productivity and cold hardiness of 'Concord' grapevines. Amer. J. Enol. Vitic. 29:187-191. 15. Hudson, M.A. and D.B. Idle. 1962. The formation of ice in plant tissues. Planta 57:718-730. 16. Ishikawa, M. and A. Sakai. 1981. Freezing avoidance mechan­ isms by supercooling in some Rhododendron flower buds with reference to water relations. Plant Cell Physiol. 22:953962. 17. Kaku, S. 1971. A possible role of the endodermis as a barrier for ice propagation in the freezing of pine needles. Plant Cell Physiol. 12:941-948. 18. Kaku, S., M. Iwaya, and K.B. Joen. 1981. Supercooling ability, water content and hardiness of Rhododendron flower buds during cold acclimation and deacclimation. Plant Cell Physiol. 22:1561-1569. 19. Kaku. S., M. Iwaya, and M. Kunishige. 1980. Supercooling ability of Rhododendron flower buds in relation to cooling rate and cold hardiness. Plant Cell Physiol. 21:1205-1216. 20. Krasavtsev, O.A. 1979. Delay of supercooled water outflow from parenchyma cells of apple wood. Sov. Plant Physiol. 26:330-335. 21 . Levitt, J. 1972. Responses of plants to environmental stress. Academic Press, New York. 22 . Lucas, J.W. 1954. Subcooling and ice nucleation in lemons. Plant Physiol. 29:245-251. 23. Luyet, J. and P.M. Gehenio. 1937. The double freezing point of living tissues. Biodynamica 1 (30):1-23. 124 24. McKenzie, J.S., C.J. Weiser, and P.H. Li. 1974. Changes in water relations of Cornus stolonifera during cold acclima­ tion. J. Amer. Soc. Hort. Sci. 99:223-228. 25. McLeester, R.C., C.J. Weiser, and T.C. Hall. 1968. Seasonal variations in freezing curves of stem sections of Cornus stolonifera Michx. Plant Cell Physiol. 9:807-817. 26. McLeester, R.C., C.J. Weiser, and T.C. Hall. 1969. Multiple freezing points as a test for viability of plant stems in the determination of frost hardiness. Plant Physiol. 44: 37-44. 27. Pierquet, P. and C. Stushnoff. 1980. Relationship of low temperature exotherms to cold injury in Vitis riparia Michx. Amer. J. Enol. Vitic. 31:1-6. 28. Pierquet, P., C. Stushnoff, and M.J. Burke. 1977. Low tempera­ ture exotherms in stem and bud tissues of Vitis riparia Michx. J. Amer. Soc. Hort. Sci. 102:54-55. 29. Quamme, H. 1976. Relationship of the low temperature exotherm to apple and pear production in North America. Can. J. Plant Sci. 56:493-500. 30. Quamme, H. 1978. Mechanism of supercooling in overwintering peach flower buds. J. Amer. Soc. Hort. Sci. 103:57-61. 31. Quamme, H . , C. Stushnoff, and C.J. Weiser. 1972. The relation­ ship of exotherm to cold injury in apple stem tissues. J. Amer. Soc. Hort. Sci. 97:608-613. 32. Quamme, K., C.J. Weiser, and C. Stushnoff, 1973The mechanism of freezing in xylem of winter apple twigs. Plant Physiol. 51:273-277. 33. Rajaskekar, C. and M.J. Burke. 1978. The occurrence of deep supercooling in the genera Pyrus, Prunus and R o s a : a pre­ liminary report. In Plant cold hardiness and freezing stress, (ed. P.H. Li and A. Sakai), pp. 213-225. Academic Press, New York. 34. Rasmussen, D.H. and A.P. Mackenzie. 1972. Effect of solute on ice-solution interfacial free energy; calculation from measured homogeneous nucleation temperatures. In Water structure at the water-polymer interface (ed. H.H.G. Jellinek), pp. 126-145. Plenum Press, New York. 35. Rasmussen, D.H. and A.P. Mackenzie. 1973. Clustering in super­ cooled water. J. Chem. Phys. 59:5003-5013. 125 36. Rasmussen, D.H., M.N. Macaulay and A.P. MacKenzie. 1975. Supercooling and nucleation of ice in single cel l s . Cryobiology 12:328-339. 37. Sakai, A. 1979. Freezing avoidance mechanism of primordial shoots of conifer buds. Plant Cell Physiol. 20:1381-1390. 38. Salt, R.W. and S. Kaku. 1967. Ice nucleation and propagation in spruce needles. Can. J. Bot. 45:1335-1346. 39. Tumanov, I.I. and O.A. Krasavtsev. 1959. Hardening of northern woody plants by temperatures below zero. Sov. Plant Physiol. 6:663-673. 40. Tumanov, I.I., O.A. Krasavtsev, and T.I. Trunova. 1969. Investigation of the ice formation process in plants by measuring heat evolution. Sov. Plant Physiol. 17:754-760. 41. Wiegand, K.M. 1906. Some studies regarding the biology of buds and twigs in winter. Bot. Gaz. 41:373-424. SECTION V STUDIES ON ROOT RESISTANCE OF POTTED ’CONCORD’ GRAPEVINES 126 INTRODUCTION Water flow through detopped root systems in response to reduced pressure has been used as a measure of root resistance to water uptake (7, 8, 10, 1975). Water flow in response to different levels of applied suction was found to be linear or log-linear, depending on whether the suction was increasing or decreasing (6, 10). Root resistance of dogwood increased as a result of exposure to short-day (SD) photoperiods (7, 8) and was correlated with increases in cold hardiness, leading the authors to claim that the SD influence on cold acclimation may be mediated in part by changes in root resistance via root suberization (7). However, when roots were killed by boiling, water flow increased 8 - lOx (7) which sug­ gests that the resistance was due to cell protoplasm or membranes or that boiling disrupted the periderm layer. Grapevines under natural day and night-interrupted photoperiods had equivalent root resistance values (Section III, Figure 2). Since this technique has not been previously reported to have been used on grapevines, additional studies were conducted to characterize water flow. Also, the technique used in Section III employed 2 pretreatments of soil removal and low pressure (to remove air bubbles) prior to flow measurement and the effect of these is not known. In order to address these areas and again background on root resistance in grapevines, several studies were conducted. 127 MATERIALS AND METHODS Potted 'Concord' grapevines used in the following studies were grown identically to those used in Section II. Root resistance was measured as suction-induced water flow, employing either the system used in Section II (and shown again here in Figure 1) or a slightly modified system (Figure 2), detailed below. Handling of plants prior to resistance measurements differed among the studies. All root systems (except those in Study 2) were prepared as follows: soil was removed by a light pressure water spray; roots were placed into buckets of water and subjected to low total pres­ sure (150 ramHg) which removed air bubbles, preventing them from lodging in the pipet and interfering with subsequent flow measure­ ments. In Study 2, these 2 factors-r-soil removal and low pressure pretreatment— were investigated for their effect on water flow as explained below. Study 1 Root systems were subjected to different levels of pressure, either in an increasing or decreasing manner (Figure 3a and 3b, respectively). Study 2 Pretreatment conditions, soil removal and low pressure removal of air bubbles, were investigated separately for their effects on 128 129 root resistance measurements. Soil was removed by a light pressure water spray, but not subjected to low pressure pretreatment. Low pressure (150 mmHg) was investigated on an apparatus modified (Figure 2) to trap air bubbles away from the pipet and nullify interference. Low pressure was applied either once or twice to see if it effected flow and if the effect was cumulative. Study 3 Flow through dead root systems was studied by killing roots either by submersion in boiling water for 2 min or by wrapping roots in plastic bags and placing them in a -70°C chest freezer for 12 hr. RESULTS AND DISCUSSION Water flow through root systems of 'Concord* vines varies with pressure, but the exact relationship differs, depending on whether the pressure is increasing or decreasing. When pressure is decreasing (i.e., increasing suction) water flow increases in a linear fashion (Figure 3a) while when pressure is increasing (i.e., decreasing suction) flow decreases in a log-linear fashion (Figure 3b). Shirazi et al. (10) reported log-linear pressure/flow relation­ ships in tomato bean and cotton, but the response was seen for both increasing and decreasing pressure. On the other hand, Kuiper (6) showed results opposite to that found in this study, i.e., linear response during increasing pressure and log-linear response during decreasing pressure. The reason for differences from linearity are not known. Removing air bubbles from root systems by pretreatment expo­ sure to low pressure (150 mmHg) (Figure 4) affected the subsequent flow measurement at higher pressure (590 mmHg). This may be due to the unexplained effect of log-linear flow reduction when pressure is increased (Figure 3); part of the flow difference is an artifact of measurement. Because of the design of the modified system (Fig­ ure 2), air bubbles collect in a part of the tubing where they do not interfere with pipet readings. However, the volume of water they 130 131 displace at the collection site will nevertheless be erroneously reflected as water flow in the p i p e t . the difference. This may account for some of An additional treatment of low pressure for 10 min (Figure 4) did not significantly decrease flow. Air bubbles were a problem in measuring root resistance of dogwood, but unreported design features of the system negated the artifact (L. Parsons, personal communication). Although soil removal from roots of dogwood did not affect root resistance (7), removal of soil from grapevines (Figure 4) greatly decreased water flow. Reason for this is not known, but may reflect loss of root surface by breakage of fine roots during handling. Flow through root systems killed by boiling and freezing (Figure 5) presents differing results. In the first trial, boiling roots yielded a large decrease in root resistance, in agreement with dogwood results (7), but no differences were found in the second trial. The only known difference between the two trials is that in the first case the explant was pretreated with low pressure while in the second it was not. The relationship between boiling and low pressure treatment may be coincidental, but requires more study to be understood. If killing the roots alone is sufficient to decrease root resistance, then freeze-killing should be effective, but it was not (Figure 5). Rather, freezing at -70°C for 12 hr had no effect in the first trial and slightly increased root resistance (decreased flow) 132 in the second. McKenzie et al. (7) suggest that root resistance is due at least in part to living root protoplasm which can be removed by killing it; however, at the same time they hypothesize that it is which root suberization plays the crucial role. ments are contradictory. These 2 state­ If suberization imposes a physical barrier due to development of an impermeable waxy substance, then boiling should have no effect on it except as an artifact. It is well known that suberized root surfaces are quite permeable (2) by virtue, of lenticels, branch roots, and cracks in the periderm (1) and account for a large percentage of water and ion uptake in woody perennials (4, 5). Suberization may account for an increase in root resistance of 2x (2) but not the 8 - lOx seen in dogwood (McKenzie et al., 1974). This indicates that protoplasmic resistance is involved and may occur at the root cambium (2, 3). results here (Figure 4) indicate that handling may influence these conclusions, but living protoplasm cannot account for the bulk of root resistance in grapevines (Figure 5). Root resistance also increases when root temperature is lowered (3, 10). This occurs in dead root systems and may be caused by increased water viscosity (3). The resistance increase is greater in live systems than dead again pointing to root protoplasm as a component of resistance. Root resistance also changes diurnally (9, 10) and detopped roots maintain the cycling for up to 2 - 3 days (9). The regulation of the diurnal resistance flux seems to be due to the light 133 environment of shoots because when the shoot light/dark cycle was changed, root resistance changed with it (9). However, the mechanism for this continued cycling in resistance of detopped plants is unknown. In this study neither root temperature nor time of day were standardized and these may have influenced variability in water flow from study to study or from plant to plant. Further research is needed to determine how these differences in techniques influence suction-induced water flow in order to elucidate the role of roots in regulating water uptake during cold acclimation. 134 to vacuum pump to m anom eter -> replicate set-up replicate set-up 2m l pipet vacuum tubing 5 0 cc syringe glass ‘Y* tube water or soil ~ Figure 1. serum cap detopped — root system Schematic diagram of apparatus used to measure root resistance by suction-induced water flow through detopped root systems. to vacuum pump to manometer replicate set-up replicate set-up 2 ml pipet vacuum tubing > 50 cc syringe serum cap glass ‘Y’ tube water or soil "n Figure 2. detopped „ root systen Schematic diagram of apparatus used to measure root resistance by suetion-induced water flow, modified to prevent interference by air bubbles. Suction-induced water flow (ml-hr-1) 136 1.5 1.0 Y = 2 .8 9 -0 .0 0 4 6 X 0.5 Log (Suction-induced water flow (ml-hr'1)) 300 400 500 400 500 Pressure (torr) 1.0 Y* 2.59-0.0071 X 1.0 ■ 300 Pressure (torr) Figure 3. Relationship of suction-induced water flow to the total pressure applied to the stem end of detopped root systems of potted 'Concord' grapevines. Numbers near each point indicate the order in which measurements were taken: A = decreasing pressure (increasing suction) B = increasing pressure (decreasing suction). Suet ion-induced water flow (m l-h r-1) 137 NO YES SOIL REMOVAL LOW PRESSURE PRETREATMENT (10 MIN AT 150 TORR) Figure 4. Effect of soil removal and low pressure pretreatment on suetion-induced water flow through detopped root systems of potted ’Concord* grapevines. 138 2.5 Suction-induced water flow (ml?hr"1) 2.0 1.5 1.0 a a 0.5 NO YES TRIAL 1 NO YES TRIAL 2 KILLED BY FREEZING Figure 5. NO YES TRIAL 1 NO YES TRIAL 2 KILLED BY BOILING Suetion-induced water flow through detopped root systems of potted 'Concord' grapevines, comparing living root systems with root systems killed by freezing and boiling. LITERATURE CITED 1. Addoms, R.M. 1946. Entrance of water into suberized roots of trees. Plant Physiol. 21:109-111. 2. Chung,H.-H. and P.J. Kramer. 1975. Absorption of water and through suberized and unsuberized roots of loblolly pine. Can. J. For. Res. 5:229-235. 3. Kramer, P.J. 1940. Root resistance as a cause of decreased water absorption at low temperatures. Plant Physiol. 15: 63-80. 4. Kramer, P.J. 1946. Absorption of water through suberized roots of trees. Plant Physiol. 21:37-41. 5. Kramer, P.J. and H.C. Bullock. 1966. Seasonal variations in the proportions of suberized and unsuberized roots of trees in relation to the absorption of water. Amer. J. Bot. 53:200-204. 6. Kuiper, P.J.C. 1963. Some considerations on water transport across living cell membranes. Li Stomata and water rela­ tions in plants. (I. Zelitch, ed.) Conn. Agric. Expt. Sta. Bull. 664:59-68. /• McKenzie, J.b. , C . j . weiser , and P.U. Li. 1974. Changes in water relations of Cornus stolonifera during cold acclima­ tion. J. Amer. Soc. Hort. Sci. 99:223-228. 8. Parsons, L.R. 1978. Water relations., stomatal behavior, and root conductivity of red-osier dogwood during acclimation to freezing temperatures. Plant Physiol. 62:64-70. 9. Parsons, L.R. and P.J. Kramer. 1974. Diurnal cycling in root resistance to water movement. Plant Physiol. 30:19-23. 10. 32 P Shirazi, G.A., J.F. Stone, L.I. Croy, and G.W. Todd. 1975. Changes in root resistance as a function of applied suction, time of day and root temperature. Physiol. Plant. 33:214218. 139