WNWWWNIHWIWHIHHIHIIIHIIWHIIW Date MM 29/ /?595 0-7 639 LIBRARY I Michigan State l University This is to certify that the thesis entitled A Comparison of Freeze-Induced Carbohydrate Changes in Winter Barley Crowns presented by David Palmer Livingston III has been accepted towards fulfillment of the requirements for MasLeLS——degree in Crop and Soil Science fiflflo @2ch Major professor Russel D. Freed MS U is an Affirmative Action/Equal Opportunity Institution MSU BEURN1N_§_J1£\_T_§£L§L§I Place in book drop to LIBRAfiJES remove this checkout from .3“. your record. FINES will be charged if book is returned after the date stamped below. A COMPARISON OF FREEZE-INDUCED CARBOHYDRATE CHANGES IN WINTER BARLEY CROWNS By David Palmer Livingston III A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Science 1985 ABSTRACT A COMPARISON OF FREEZE-INDUCED CARBOHYDRATE CHANGES 4 IN WINTER BARLEY CROWNS BY David Palmer Livingston 111 Past studies have shown that conversion of fructan to fructose and sucrose occurs in winter cereal crowns when they are frozen for 24 hours at ~30. Simple sugars reportedly increase in extracellular spaces and seem to provide resistance to adhesive stress due to freezing. Differences between rye and barley for freeze-induced conversion were reported. Differences within species may provide a basis for improvement of freezing resistance in that species. Four winter barley cultivars were grown in controlled conditions and frozen for 24 hours at -5C. Frozen crowns and an unfrozen control were extracted with ethanol and water. Carbohydrates were separated and quantified using HPLC, refractive index detection, and electronic peak area determination. Experimental variation was too high to show a significant interaction between cultivars and treatments but patterns in the variation indicate that under other conditions differences may be shown. Combining this component of winter hardiness with other freezing resistance mechanisms could provide a basis for improvement of overall hardiness in winter barley. TO MARIA and JESSE ii ACKNOWLEDGEMENTS I would like to express sincere thanks to Dr. Freed, my major professor for his valuable advice and moral support throughout this study. 1 also appreciate the help of Dr. Howel l in finding a reasonable procedure for the analyses of the plant material. I am indebted to Dr. Kindel for the use of his lab and guidance in developing final procedures. To my friend and "scientific father", Dr Olien, who made this study possible, I wish to remain indentured for at least 3 more years. I thank Bernie Marchetti for his help in keeping the various chambers operating and his advice in growing plants. I thank Dr. Cress for invaluable statistical guidance. Thanks to my wife, Maria, whose career as a mother and wife made coming home throughout this study a truly pleasurable experience. iii TABLE OF CONTENTS PAGE L181. OE‘ TABLESOOOOOOOOOOOOOOOOO ....... OOOOOOOOOOOOOOOCOOV LIST OF FIGURES........................................ vi INTRODUCTION............................................ 1 CEREAL CROWNS........................................... FREEZING STRESS..................... ...... .............. Nonequilibrium Freezing............................ Intracellular Freezing........................ Extracellular Ice............................. Equilibrium freezing............................... Adhesion...................................... Protoplast Desiccation........................ FRUCTANOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO...OOOOOOOOOOOOOOO Types.............................................. Biosynthesis....................................... Storage........................................... Utilization....................................... Fructan and Adhesion Inhibition................... 11 _s._s -‘O\O\O CO \lOONU'IAKN \N N MATERIALS and METHODSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 15 Plant Material.................................... 15 Harvest.............. ....................... ...... 15 Sugar Extraction.................................. 14 Chromatography...... ........ ...................... 15 RESULTS AND DISCUSSION................................. 17 Confirmation of Past Findings...... ..... .......... 17 Cultivar Differences.............................. 19 Screening.................................... 19 Four cultivars............................... 19 Patterns in Experimental Variability.............. 23 CONCLUSIONOOOOOOOOOOOOI0.000000000IOOOOOOOOOOOOOOIOOOOO 27 APPENDIXOOOOOOOOOOO0.....0...0.0.0.0....OOOOOOOOOOOOOOO 28 LITERATURE CITEDOOOOOO...OOOOOOOOOOOOOOOOO0.0.0.0000... 35 iv LIST OF TABLES TABLE PAGE 1. Mean squares from analysis of variance for percentage carbohydrates in rye and barley... 17 2. Mean percentages of total carbohydrates for Rosen rye and Hudson barley and differences between treatments.uu.u.uu.n. 18 3. Mean percentages of total carbohydrates for 4 barley cultivars and differences between treatmentSOOOOO0.0.00.0...OOOOOIOOOOOOOOOOOOO 21 4 Mean squares from analysis of variance for percentage carbohydrates in 4 barley CUltivarS...0.0.0....OOOOOOOOOOOIOOOOOOOOO0.0 22 LIST OF FIGURES FIGURE PAGE 1. Nonequilibrium freezing. Liquid water in g/g dry matter as a function of temperature.OOOOOOOOOOOOOOOOOOOOOO0.00...... 4 2. Mg. fructan and fructose per gram dry weight of extract and their ratio for 14 winter cereals.......................... 20 5. Percentage fructan before and after freezing and percentage converted, showing partitioning into available and unavailable amounts.................... 24 vi INTRODUCTION Winter cereals, where adapted, are generally preferred over spring types (11) because winter cereals produce a higher yielding and better quality crop. A fall planting utilizes winter moisture and stored reserves by beginning spring regrowth when soil temperatures are warm enough. This promotes maturation before many summer diseases and insects reach damaging levels. By contrast, spring varieties cannot be planted until soil moisture levels allow entry into the field. Freezing injury limits winter cereal production to areas where conditions are favorable; it also restricts production of less hardy winter cereals, such as barley and oats. For this reason, although winter wheat in 1985 accounted for 78% of the total wheat acreage in the U.S. (51), less than 1% of the barley acreage was winter habit in the state of Michigan. Improving freezing resistance of winter cereals is, therefore, an important aspect of crop improvement. But, increasing winterhardiness as a whole is a broad, complex problem. Identifying individual freezing stresses and resistance mechanisms, then combining favorable characteristics through plant breeding would be a more manageable approach. CEREAL CROWNS After germination in the fall, winter cereals establish a vegetative system of several tillers and secondary roots. Roots and leaves do not usually survive low winter temperatures, but spring recovery is not dependent on these organs. Provided crown meristems are not seriously damaged, and are capable of differentiating a functional root system, complete regeneration is possible. Cultivar hardiness will therefore, depend on the crown's withstanding individual freezing stresses as it overwinters. A cereal crown consists of a densely packed transitional zone of differentiated intertwining vascular elements, surrounded by "a mantle of parenchymatous promeristem". This promeristem does not continuously differentiate, but has continuous meristematic potential (29). The promeristem includes axial meristems which differentiate into roots or leaves, and apical meristems which give rise to floral primordia after a vernalization period. Freezing barley crowns causes vascular disruption in the transitional zone of the inner and lower crown resulting in tissue death (29). The extent of injury depends on cultivar hardiness, extent of hardening before freezing, and type of freezing stress. Uninjured, upper promeristems can generate leaves and roots which will be the basis for spring recovery (29). FREEZING STRESS Stresses resulting in tissue death involve water transitions and redistribution as crowns freeze. Two basic transistion patterns are followed as freezing progresses. First, there is a nonequilibrium pattern which involves "large displacements of temperature from the balanced state". Second, there is an equilibrium pattern caused by smaller temperature displacements which allow equilibration after each incremental decrease in temperature (15). Nonequilibrium freezing: Placing normally growing plants in freezing temperatures causes a disruption of the balanced state between plants and their environment. Plant liquid usually cools without freezing. As ice formation begins in extracellular spaces latent;heat.is released providing energy for ice crystal growth (15). The following diagram illustrates nonequilibrium freezing. 33 :ELOP 3:.9. D ES.8~ 3 .7L *5 .e. 5 .5- »“3 .4» Z 8 .3L gg.2- E J- .J m 0 1 1 1 1 1 1 1 1 1 1 1 (I I O'l '2 “3'4 ‘5'6 “7 ‘8 ‘9 “IO TEMPERATURE(C) (15) Figure 1. Nonequilibrium freezing. Liquid water in gm/gm dry matter as a function of temperature. See text for explanation. Nonequilibrium freezing can be further subdivided into two kinds of stress: 1J intracellular freezing and 2J extracellular ice. Intracellular freezing: The solid line is the equilibrium transition pattern while the broken line represents nonequilibrium freezing. The distance between points a and d depict the amount of supercooling caused by initial temperature displacement from equilibrium. The temperature rise in stage I as freezing progresses from a to b is due to latent heat release. This provides crystalization energy "that can cause ice crystals to grow from the outer free space into the protoplastfl (15) usually resulting in cell deatmn The amount of energy available for crystal growth depends on the extent of supercooling below the protoplast freezing point (16). Intracellular freezing resistance is provided by plasmalemma stabilization during hardening (mechanical resistance) and, freezing point depression through water loss, solute accumulation, and matric interactions (2,15). Stage I of nonequilibrium freezing is an instance of high freezing intensity (high degree of supercooling) with low freezing capacity (small amount of liquid water). By contrast, stage II (b to 0), consists of low freezing intensity (a small displacement from equilibrium) and high capacity (large amounts of liquid water). This causes a different kind of stress. Extracellular Ice: As extracellular freezing progresses from b to c (fig.1) it results in large extracellular ice crystals. This can cause extensive disruption of intertwining vascular elements and meristems in crowns (17). Protection involves the interaction of polymers, normally cell wall constituents, with growing ice crystals. Certain mucilaginous polymers inhibit ice crystal growth by forming films on crystal surfaces (17,21). Other polymers divert crystal growth to non-critical regions by inhibiting initiation of freezing; this protects vital meristematic tissue (17,21). These polymers can be extracted and their inhibiting activity rated invitro. Rye (Secale cereale) polymers have greater freeze inhibiting activity than those of barley (Hordeum vulgare)(17,21). Both types of nonequilibrium freezing stresses usually occur at temperatures around -5C. Equilibrium freezing stresses, however, take place at temperatures between -1OC and -20C (15). Equilibrium freezing: While nonequilibrium stresses depend on large displacements from equilibrium and result in high crystalization energies, equilibrium stresses involve relatively small displacements and therefore smaller crystalization energies. As ice crystals grow during equilibrium freezing two other types of stress occur: 1.) Adhesion and 2.) desiccation. Adhesion: Equilibrium freezing begins with a nonequilibrium freeze to provide crystalization energy fortextracellular liquid. After initial crystalization, freezing progresses such that very small displacements from equilibrium occur. The advancing ice lattice reaching the cell wall competes with it for the intervening liquid. This causes adhesion between ice and the wall or wall and plasmalemma. It can be a significant contribution to overall freezing stress (18,19,20). As freezing progresses and intracellular desiccation removes water from the protoplast, adhesions can cause irreversible distortions of the plasmamembrane when it shrinks. This kind of damage is histologically different from other types of injury (18). Resistance to adhesive damage is seemingly through adhesion inhibition by solutes which maintain a fluid barrier between ice and the plasmalemma. A dilute solute concentration in the extracellular spaces in winter cereals helps prevent the growth of disease-causing microorgansisms. This allows ice crystals to grow up to hydrophylic cell walls“ When frozen, the cell reversively releases sugars outside the protoplast and maintains a fluid barrier neccessary for prevention of adhesions (14). Minimization of adhesion allows freezing to progress to temperatures where intracellular desiccation occurs. Protoplast Desiccation: Freeze-induced desiccation ensues when ice acts as a water accumulator. As freezing progresses, the vapor pressure of extracellular ice becomes lower than that of the protOplast and a gradient is established (8). The crystal grows at the expense of intracellular water and the protoplasm shrinks (8L.When water is withdrawn to some critial level intracellular injury results. Burke (2) Steponkus (50) and Levitt (8) discuss probable causes of desiccation injury such as, concentration of salts or ions, pH changes, disruption of protein function, and mechanical injury to the plasmallema. Resistance to desiccation stress is probably through alterations of membrane proteins (9) and elevation of intracellular solutes. But, precise mechanisms are not well understood (9,54% These four types of stress provide a basis for overall winter-hardiness improvement. The hardiness of winter cereals deficient in one or more resistance mechanisms may be increased if genetic variation for those mechanisms exist within that species and optimum combinations are made through breeding. Adhesive stress resistance through solute release from the protoplast is a mechanism for which variation exists between species (15). The storage polymer fructan is involved in this solute release. FRUCTAN Unlike tropical and subtropical grasses which produce starch (a glucose polymer) as the main storage polysaccharide, temperate and cool zone grasses accumulate fructose polymers called fructans or fructosans (52). They are produced not only in monocots but dicots as wel l and are distributed in stems, leaves, inflorescences, and seeds (12). In grass stem bases, at certain growth stages, fructan may constitute up to 45% of the dry weight (7). Types: Three kinds of fructan, depending on glycosidic linkages, have been described. They are: inulin, levan (or phlein), and branched. Inulin occurs mainly in dicots and has 2-1, beta linkages between its fructofuranose residues. Levan is the principle monocot fructan and consists of 2-6, beta linkages. Branched types have been found in many monocots and have either a levan or inulin backbone with one or more short fructofuran branches (12). The structure of levan, the major fructan of winter cereals, is shown below. lunuO my ”8}?” OIEOH QIQOH (}hOH Biosynthesis: Fructan (inulin) synthesis in Compositae (12) and Liliaceae reportedly occurs through the concerted action of two enzymes. The first, sucrose-sucrose fructosyltransferase, forms a trisaccharide (glu-fru-fru) from two sucrose molecules (with a glucose released). The second, fructan-fructan fructosyltransferase, transfers a single terminal fructose residue from an oligosaccharide to the same carbon position on another molecule (5,5,24). One 10 oligosaccharide, therefore, grows at the expense of another. Fructan (levan) synthesis in grasses is less well understood but in Dactylis glomerata where trisaccharide intermediates do not accumulate, the polymer apparently grows by direct enzymatic transfer of fructose residues from sucrose (22,24). The remaining glucose is reportedly converted back to sucrose (22,24). Other grasses (Lolium temulentum, wheat, and barley) show a trisaccharide accumulation; fructan synthesis here is probably similar to inulin synthesis in Compositae (25,52). Pollock.suggests that alternate biosynthetic mechanisms may be present in grasses depending on species and growth stage (25). The average number of fructofuranose units attached to the terminal glucopyranose vary considerably depending on species and growing conditions but are from 260 in timothy (7) down to 10 in brome grass (12). Storage: Environmental factors, affecting growth rates, have a significant influence on fructan accumulation. Grasses can store large quantities during slow growth periods, when assimilate production exceeds demand. This typically occurs in the fall with low temperatures and continuing photosynthesis (1,4,26). Fructan accumulation for four different grasses was highest between November and January'(25). Nutrient availability also affects fructan storage. 11 Archibold showed an inverse relationship of nitrogen and phosphorus levels to fructan amounts in barley and a positive relationship of potassium to fructan (1). Besides confirming the inverse nitrogen relationship, Westhafer (55) showed that fructan was the most responsive carbohydrate to nitrogen levels in Kentucky bluegrass stems. Utilization: Mobilization of fructan reserves in grasses usually begins when active growth starts. For example, fructan decreases most rapidly in grasses when tillers begin spring growth (25). In addition, large fructan (inulin) decreases (with corresponding fructose and sucrose increases) occur in chicory (Cichorium intybus) within 5 days when normally growing plants are subjected to 2-6C temperatures (27). Fructan conversion to fructose and sucrose is through hydrolysis by beta-fructofuranosidases. The molecule is reportedly hydrolysed stepwise from the fructose end (exo- action) until a sucrose remains (28). There is evidence for fructofuranosidase specificity since levan hydrolase will not hydrolize inulin (28). Fructan and Adhesion Inhibition: Rye and barley crowns undergo fructan conversion when hardened seedlings are frozen for 24 hours at -5C. Fructan decreases (15) while intercellular fructose and sucrose increase. Furthermore, this release to intercellular spaces 12 is reversible and recovery (to prefrozen levels) takes less than one hour (14). Rye plants frozen directly to ~1ZC do not convert fructan significantly and have a lower survival rate than those first incubated at -5C for 24 hours (15). In addition, Hudson barley converts fructan to a lesser extent and has a lower survival rate. Olien proposes that the protection seemingly offered by the intercellular solutes is due to adhesion inhibition (14). The purpose of this research was to investigate fructan conversion in barley. If genetic differences can be found between cultivars for fructan conversion and solute release, a basis for improving the hardiness of winter barley may be established. MATERIALS and METHODS Plant material: The following winter cereals were used: C.I. Number TRITICALE BARLEY OAC Wintry *Hudson Breeder Selected Winter Tennessee 4655 WHEAT Kearney 7580 Genesee Breeder Selected *Dictoo Breeder Selected Wong Breeder Selected RYE Reno 6561 *Rosen Breeder Selected *Khayyam 1117 8-6 *Durani 6516 Asterook Merkator *before and after freeze comparison. Eight plants per 1255 cm pot were grown from seed in washed, steam sterilized sand. They were raised in a growth chamber at 150 with 18 hour/day of light at 300 Em‘23’1 (80% white flourescent, 20% incandescent). After five weeks, they were transferred to a 10 hardening chamber with continuous light at 200 Em‘zs‘1 for 5 weeks. They were watered daily and fertilized 5 times weekly with a Hoaglands solution for the entire growth period. Harvest: After 8 weeks, plants were at the three leaf stage with 5-4 tillers each. They were removed from pots, washed free of sand in ice water, and trimmed to four cm shoots and two cm roots. Plantlets were placed in slots cut in damp, circular, cellulose sponges with a pipe flange 15 14 assembly through the center to promote thermostabilization. Eight plants, from eight different pots, were used for each sample. In the comparison experiments plants were paired by size providing 2 identical sets, one for frozen and one for unfrozen (control) treatments. A randomized complete block design with 14 cultivars and four replications (separated over time, one week/rep) was used in the initial screening. A split plot design with four replications (separated according to crown size), two treatments (frozen and unfrozen),tand four cultivars was used for the before and after freeze comparison. Sponges containing plants were inoculated with ice, covered with plastic to help prevent desiccation, and placed in a freezer at -5C (-5C in the comparison experiment with 4 barley cultivars) for 24 hours. Thermocouples placed near crowns in the sponges were used to monitor tissue temperature. Actual time at equilibruim with the freezer was between 18 and 22 hours. Sugar Extraction: After the treatments, plantlet roots were trimmed with a razor in a cold room at 6C. The transition zone was grated with a fine toothed hand grater and 1/2 cm of the stem was trimmed with a razor. The combined tissue was placed in a mortar containing 10 mls of 80% (vol./vol.) EtOH and mascerated for aproximately 2 min. with a motor driven mortar and pestle. The ground tissue was transfered 15 to a 100ml beaker and the mortar and pestle rinsed with 20mls 80% EtOH which was added to the beaker. The tissue was placed on a 700 water bath for 10 min. to inactivate enzymes, and shaken for 15 min on a gyratory shaker. The supernatant was transferred to an Erlenmeyer flask and the tissue extracted an additional two times with double distilled water (50 mls each) at 21C. A fourth water extraction revealed less than 5% additional carbohydrate. The combined supernatants were swirled by hand and a 25ml alaquat was transferred to a 50 ml graduated centrifuge tube. 400mg of mixed bed resin was added and the mixture was shaken for 10 min (to eliminate sulfate ions damaging to the separation column). Samples were centrifuged at 6800 for 10 min. and 10mls of the supernatant decanted to a preweighed round bottom flask. They were taken to dryness under vacuum at 550 (the screening experiment was taken to dryness on a 500 water bath under forced air) and placed in a vacuum desiccator over P205 for approximately 12 hours. Samples were reweighed, brought to a concentration of 10 mg dry extract per ml double distilled water and passed through a .45 micron Millipore filter. Chromatography: Cabohydrates were separated by high pressure liquid chromatography (HPLC) using a Bio-Rad Aminex HPX-87P Column heated to 85C.‘The mobile phase consisted of degassed HPLC grade water (Baker Chemical (kn) with a flow rate of .4 16 ml/min. A Micromeritics 771 Refractometer was used to detect carbohydrates and they were quantitated by co-chromatography with external standards. Rosen rye fructan, collected off the column and freeze-dried, was used as the fructan standard for all cultivars in the initial screening and fructan from Hudson barley for barley cultivars in the comparative study. Peak areas were determined by a Hewlett Packard 5590A integrator. Retention times in minutes were: fructan 9.2, sucrose 14.8, glucose 18.4, and fructose 25.8. Data is presented as a percentage of total water , soluable carbohydrate because using dry weight as a base caused erroneous results. This was because complete recovery of dried crown tissue and removal of sand was difficult. There was no significant difference in the total extractable carbohydrate per dry weight for treatments. The average total cabohydrate for each sample was approximately 353 mg/g dry weight.1mus varied slightly between cultivars and experiments. Percentages used in this study represent a proportion of this amount. RESULTS and DISCUSSION Confirmation 9f past findings: The first part of this study was done to confirm differences in fructan conversion between species. Treatment effects and species by treatment interaction were both highly significant (table 1) for all four sugars. Fructan decrease with sucrose and fructose increase was larger in Rosen rye than in Hudson barley (table 2).<31ucose change in Rosen was not significant but was in Hudson. The glucose increase could be a result of sucrose hydrolysis from decompartmentation caused by freezing injury (15). Table 1. Mean squares from analyses of variance for percentage carbohydrates in rye and barley. Mean squares Source d.f. Fructan Sucrose Glucose Fructose RepIication 5 0.19 5.75 1.79 1.62 Species (S) 1 100.50** 155.14** 11.22* 25.00** Error (a) 5 0.24 5.54 0.74 0.25 Treatment (T) 1 118.26** 45.25** 1.00** 10.89** T x S 1 10.72** 11.06* 1.56** 1.56** Error (b) 6 0.91 1.16 0.07 0.11 *,** = Significant at 2% and 1% level of probability, respectively. 17 18 Table 2. Mean percentages of total carbohydrates for Rosen rye and Hudson barley and differences between treatmentsa. Species Sugars Rye Barley LSDb Fructan H 8004 75'? HF 73.5 69.9 2.50% D -701 ‘308 Sucrose H 11.5 18.8 HF 16.5 20.4 2.80% D +5.0 +1.6 Glucose H 4.5 5.5 HF 4.5 6.6 .68% D -0.2 +1.1 Fructose H 5.8 2.0 HF 6.1 5.0 .88% D +2.5 +1.0 H = Hardened plants (controI7 HF = Hardened and then frozen. 0 = Difference between H and HF. a = Frozen 24 hours at -50. b = LSD at 1% probability for treatments. Changes in fructan, sucrose, and fructose confirm the findings of Olien and are the basis for the remainder of the study. If differences exist for the magnitude of sugar change between species then differences may also exist within the same species. 19 Cultivar differences: Screening: Fourteen winter cereals were screened by measuring carbohydrate levels after a freeze treatment. A fructan:fructose weight ratio was used to separate high and low converting lines. The results (figure 2) suggest ratio differences between some cultivars of different species, but provide no evidence for differences between barley cultivars. Four cultivars: Two pairs of barley cultivars with the largest ratio differences (Durani and Khayyam-high, Hudson and Dictoo-low) were used in this experiment (Dictoo was selected instead of Winter Tennessee because of its hardiness). These were subjected to the same comparison test used previously for rye and barley. A significant treatment effect (table 5) was found for all four sugars but a non-significant interaction for fructan, sucrose and glucose. While this confirms freeze- induced fructan conversion generally, it provides no evidence for a difference in the magnitude of fructan change between these four cultivars. Jeiutm SIEGJSQ 2() Mg. Carbohydrate/g. extract dry wt. ‘§i§;, ,4 E: g: 23 \\\\\\\\\\\\\\\\\\\V a»): ‘3’ 4 <0 \V) 3:95 ‘1 “’ ‘- ii :>>\\‘\§J.\\\\\\.\\V ”-" i /z a g g ’ h: d P. :———le—-'E:§i$§§ i i“’jj 3w '2 aJnfita 0:12; staui pus 'steaaao Jaiutm ”I 10; ioeaixa go iufitam flap mes? Jed asoionig pue ueionag 21 Table 5. Mean squares from analyses of variance for percentage carbohydrates in 4 barley cultivars. Mean squares Source d.f. Fructan Sucrose Glucose Fructose Replication 5 8.65 4.21 0.41 0.12 Cultivar (C) 5 247.25** 159.52** 8.45** O.99** Error (a) 9 1.69 1.79 0.06 0.02 Treatment (T) 1 55.65** 0.45ns 8.61** 26.64** T x C 5 1.10ns 0.11ns 0.05ns 0.51** Error (b) 12 1.18 0.54 0.15 0.04 ** US Significant at 1% level of probability. not significantly different at 5% level. The significant cultivar by treatment interaction for fructose may have been a function of sucrose hydrolysis. Fructan hydrolysis results in sucrose (as well as fructose) increase (15g14,27,28). Hydrolysis of sucrose (possibly injury induced, since these cultivars were frozen at a lower temperature) from converted fructan could explain the lack of evidence for sucrose change and the significant increase in glucose. Highly significant cultivar differences were found for all four carbohydrates. Fructan differences may be related to partitioning as discussed in the next section. Winter hardiness has been related to accumulation of simple sugars during hardening; with a few exceptions, hardier varieties of a species accumulate more sugars than non-hardy ones (8). In this study, the hardier cultivar, Dictoo, shows about 10% 22 more simple sugars than the less hardy cultivars, Khayyam and Durani. Rosen rye's lower sugar accumulation compared to Hudson barley‘s at first seems to contradict this observation. But, Rosen's greater ability to convert fructan and release sugars when incubated probably provides more protection from freezing stress than Hudson's higher sugar content. Table 4. Mean percentages of total carbohydrates for 4 barley cultivars and differences between treatmentsa. Cultivars Sugars Hudson Dictoo Khayyam Durani Fructan H 75.6 71.5 81.8 82.8 HF 72.1 68.9 78.9 81.8 D -305 -204 “.209 ”107 Sucrose H 19.0 25.8 15.2 15.9 HF 19.0 25.5 15.0 15.4 D 0.0 -03 -02 -05 Glucose H 4.4 5.7 2.2 2.5 HF 5.5 4.8 5.4 5.2 D +1.1 +1.1 +1.2 +.7 Fructose H 1.1 1.3 0.9 0.7 HF 5.5 2.8 2.7 2.5 D +2.4 +1.5 +1.8 +1.6 Hardened plants (control) Difference between H and HF. H HF = Hardened and then frozen. 0 a Frozen 24 hours at -5C. 25 Patterns ig variability: Experimental variability was too high in this study to find genetic differences in fructan conversion between the four barley cultivars. However, patterns in the variability suggest that under other experimental conditions differences might be shown. Freeze-induced fructan hydrolysis is probably complete within 24 hours (Olien unpublished). With relatively high and consistent starting amounts the same proportion of the total fructan is always converted. This suggests a genetically specified equilibrium point for total fructan conversion and/or fructan partioning into available and unavailable amounts. This study suggests that partitioning may be occurring. In figure 5 the vertical line for Dictoo (% fructan converted) could be viewed as an extension of the abscissa (the scale is the same for both axes). So the percentage converted (ordinant) plus the percentage post-freeze levels (abscissa) equals the initial starting amount. For example, Dictoo's highest intial fructan level (a) was approximately 74% (4.8% + 68.9%) and the lowest was 69.5% (.6% + 68.9%). With or without partitioning one would expect different fructan levels depending on environmental conditions. Without partitioning, however, all the fructan would be available for conversion and similar proportions would probably be converted regardless of initial amounts. The 211 Percent Available for Conversion (Actual percent converted). mwmcnm_w.wmwom3dmmm wncoams dmwonm mun madmw Hammuwsm mum vmnomsdem ood