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All Graduate Theses and Dissertations Graduate Studies

5-1977

The Influence of Certain Growth Hormones on Growth at Cold Temperatures

Michael D. Salvesen Utah State University

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ii

ACKNOWLEDGMENTS

I would like to thank Dr. Frank B. Salisbury for his assistance and continued encouragement and patience while serving as my major professor and advisor during the course of this study. Also special thanks to Dr. Herman H. Wiebe, and Dr. Schuyler D. Seeley for counsel and service rendered me while serving on my advisory committee.

For the financial support that made this study possible, I would like to thank the National Aeronautics and Space Administration.

I am grateful for the assistance and encouragement of my father­ in-law, Dr. George E. Stoddard and my wife, Karalee .

Michael D. Salvesen iii

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ii

LIST OF TABLES . iv

LIST OF FIGURES v

ABSTRACT . vi

INTRODUCTION 1

REVIEW OF LITERATURE 3

Geographic adaptation 3 Stage and rate of growth 4 moisture 6 Light 7 Externally applied hormones 8 Cellular influences 10 Cold injury and desiccation 14 Plant growth under the snow 15 Dehardening 15

MATERIALS AND METHODS 16

Growth under the snow 16

RESULTS AND DISCUSSION 23

Conditions, at the snow tunnel 23 Plant growth . 25 Soluble protein content 29

SUMMARY 31

LITERATURE CITED 32

APPENDIX 37

VITA 40 iv

LIST OF TABLES

Table Page

1. Average shoot length in em 38

2. Fresh weight per shoot in mg 38

3. Mg of soluble protein per ml of solution per gram of fresh weight • 39 v

LIST OF FIGURES

Figure Page

1. Tunnel uncovered 17

2. Tunnel partially covered .. 17

3. Tunnel covered 18

4. grove in Steep Hollow 18

5. Tray in tunnel 19

6. Central g r aph of soluble protein in mg/ml 21

7. Snow depth in Steep Hollow. Variation with time 23

8. Temperature in tunnel . Variation with time 25

9. Shoot length. Variation in time 26

10. Fresh weight. Variation in time 28

11. Soluble protein. Variation in time 30 vi

ABSTRACT

The Influence of Certain Growth Hormones

on Plant Growth at Cold Temperatures

by

Michael D. Salvesen, Master of Science

Utah State University, 1977

Major Professor: Dr. Frank B. Salisbury Department: Plant Science

Studies were conducted to determine the effect of externally applied growth hormones on rye (Secale cereale var. cougar).

The rye was grown under the snow in a specially prepared tunnel on a mountain side near Logan, Utah. These showed a remarkable ability to grow at 0°C. Three growth hormones, kinetin, gibberellic acid (GA), and (indole-3-acetic acid) were externally applied to rye seedlings growing in the tunnel. These seedlings were later weighed, measured and tested for soluble protein content. The three hormone treatments had no signficant effects on the fresh weights or shoot lengths of the rye. GA however, had a significant effect on the soluble protein content of the young shoots. Shoots treated with GA showed a marked decrease in soluble protein content. Kinetin and IAA had no significant effect on the soluble protein content of t he rye.

(45 pages) INTRODUCTION

Many environmental conditions influence the cold tolerance of plants . These influences are logically manifest through their effect on metabolic processes. Even though research studies attempt to isolate environmental fact ors t o determine their individual effects, in nature they interact in complex combinations of several factors.

Thus, some interpretive judgements are essential in reviewing published reports.

Natural adaptation to environmental conditions is evident in plants native to various climatic regions. The influences of micro­ climates as well as macro-climates are readily apparent as one observes plant distribution within and among climatic regions.

Cultured plants or those planted in nonnatural environments are often more susceptible to adverse c limatic conditions than plants in their natural environments . Hence, an understanding of the mechanisms of adaptation may have substantial economic as well as scientific value.

The mechanisms by which plants become cold hardy is not well known (Alden and Hermann, 1971) . In Lyons review (1973) it is suggested that all types of chilling injury can be the result of a change in membrane permeability, while other (Mazure, 1969), have suggested that injury involves a complex of several interacting events. The complex nature of the mechanisms probably accounts for the lack of more definitive information in published reports. 2

Hormone participation in plant responses have been cited in the following areas of plant development: The growth and development of stems, roots, and , formation of adventitious buds and roots, tubers, and in development and maturation, senescence, of buds and seeds, germination of seeds, and other phenomena (Salisbury & Ross, 1969).

A study as initiated to determine some of the influences of cold temperatures and of growth regulators on growth and chemical composition of winter rye (Secale cereale var. cougar) . 3

REVIEW OF LITERATURE

The natural environment includes several parameters: temperature, precipitation, soil fertility, soil moisture, light, and co-habitating plants and animals. Man's influences become important environmental factors when he enters a region. Each factor imposes its influences in a variable manner, sometimes periodic and sometimes quite sporatic.

Periodic influences may be diurnal, seasonal or yearly. All influences affect individual plants differently, depending on the combination of and sequence of their presence.

Low temperatures, more than any other environmental factor, influences the physiological and biochemical processes that impart cold hardiness to plants. Most plants begin to develop resistance to cold as temperature is gradually lowered below l0°C, if other environmental factors are also favorable for the development of cold hardiness. Duration of exposure to low temperatures and intensity of cold weather required for development of cold hardiness depend on the potential cold resistance of the plant as well as the· plant's past and current environment (Tumanov, 1964, 1967).

Research studies to identify the influences and their metabolic mechanisms have been varied but far too limited to identify more than a few of the associated factors.

Geographic adaptation

The ability of plants to survive sub-freezing temperatures is of interest in the study of distribution, succession, and migration of 4 plants, because climate is of paramount importance in controlling plant distribution. Seasonal sub-freezing temperatures as a single environmental factor, however, may not limit natural altitude and polar migration of plants in cold climates. Inadequate heat during the growing season appears more limiting to plant distribution

(Daubenmire, 1959). Large seasonal oscillations of other environmental factors, low productivity, and the youth of the ecosystems in polar climates are more limiting in the adaptation of plants than sub­ freezing temperatures (Dunbar, 1968). Seasonal sub-freezing tempera­ tures, however, force many plants to develop greater tolerance to cold than the minimum temperature of their ecological range.

Many plants have an inherent ability to develop high resistance to cold but lack correct biological timing in new climates (Weiser,

1968). Plants from warm climates introduced to a cold environmen t may be susceptible to cold injury because they fail to develop sufficient cold hardiness, do not harden rapidly enough for protection from early cold weather, deharden too rapidly (which makes them vulnerable to freezing temperatures in late winter), or because of a combination of these conditions.

Hardening cucumber seeds with 12 hours of exposur e daily to 0- 2°C for 10 to 15 days before germination is reported to enhance the cold hardiness of the young seedlings, particularly these of southern varieties (Velik, 1963).

Stage and rate of growth

Kappen (1964) found that sporophytes of and fern species were most sensitive to cold injury in the spring and that 5 cold resistance did not set in until the fronds were full-grown. Frost resistance of Mediterranian species was equal to the frost resistance of Central European plants in April.

Sakae (1962, 1966) reported that cold hardiness of growing twigs of woody -plants that are extremely sensitive to frost injury cannot be improved by chilling to O ~C. Frost resistance developed even without exposure to low temperatures after growth ceased, however. Maximum resistance to cold can be induced in red osier dogwood (Corus stolonifera Michx.) in seven weeks at any stage during its annual cycle of development except one or two months after growth resumes in the spring.

Clauson (1964) examined about fifty species of hepatics and found that freezing to -l0°C for four days destroyed undeveloped cells in young shoots, but fully developed cells survived the same treatment.

The androecium, particularly of spring wheat, was most sensitive to frost injury during development of the reproductive organs (Petrova and Drozdov, 1963).

That the development of cold hardiness in woody plants is inversely proportional to growth rate has been well established (Levitt, 1966,

1972). During the period of rapid growth in the spring, plants are exceptionally susceptible to frost injury. Environmental factors that depress growth, such as low temperature, insufficient moisture, short photoperiods in plants that accumulate starch, and low nitrogen levels, also enhanced that cold tolerance of most plants. Cox and Levitt (1969) reported, however, that l eaves of cabbage hardened when growth was rapid because proteins that promote both cold hardiness and growth were 6 synthesized at low temperatures. Spring ephemerals, (plants that com­ plete their growth in a brief period early in spring), exhibit their most rapid growth while temperatures are sometimes at freezing. Their cycle of growth is finished by the time other less hardy plants are just beginning to grow. (Salisbury et al., 1973; Salisbury, 1976).

Prolonged warm weather during the fall resulted in less dormancy and cold hardiness in citrus species and increased the severity of freezing injury (Cooper, et al., 1963). Whether dormancy and cold hardiness are separate and parallel processes or whether dormancy accounts for all the physiological changes that contribute to frost hardiness is not clear.

Winter annuals, on the other hand, have no permanent period of dormancy, and growth processes cease on exposure to low temperatures only. Late fall planting resulted in slower growth and greater winter hardiness (Young and Feltner, 1966). The relation of growth and development of frost hardiness in winter cereals is given complete coverage by Vasil'yev (1956).

Soil moisture

The ultimate tolerance of plants to cold temperatures and their ab ility to over-winter successfully in climates with seasonal sub­ freezing weather may depend on available moisture in the soil during the growing season. For example, abnormally low temperatures during the winter preceded by a fall drought is believed responsible for severe winter damage and the disappearance of Ceonothus velutinus Dougl. from the mountains of northern Utah (Holmgren, 1963). Given time for adaptation to a dry climate, frost-sensitive plants can tolerate more cold weather under dry conditions than under moist conditions. Low soil temperature also decreases uptake of water by thermophilic plants (Nezgovorov and Solovev, 1965).

Crops on dry and coarse textured are more likely to be injured by frost than crops on moist clay or loam soils. Water increases the conductivity of heat, stores heat during the day, and releases heat as it freezes (Hom, 1966).

The importance of light in plant processes is readily apparent.

Photosynthesis as a process of producing essential plant metabolites may be separate from the photoperiodism associated with seasonal variations, in adapting to cold temperatures.

Photosynthetic role. Early studies indicated that photosynthesis is important for the over-wintering ability of many plants. Dexter

(1933) found that plants such as winter wheat, alfalfa, cabbage, and tomatoes hardened more quickly at o•c when they were illuminated. More recently , McGuire and Flint (1962) showed that three-year-old seedlings of white spruce, Scotch pine, red pine, balsa fir and red pine increased the photosynthetic reserves of seedlings during the hardening process. The ability of plants to develop high tolerance to cold climates may depend on photosynthesis and the storage of photosynthates before dormancy and the hardening process.

Photoperiodic role. Light may affect the development of tolerance to cold through a photoperiodic response. In contrast to other reports, 8 short photoperiods throughout the summer are known to increase the cold tolerance of red clover to a greater degree than long photoperiods

(Umaerus, 1963).

Hardiness of alfalfa improved with increasing photoperiods to a maximum of two hours and with an increased light intensity of 12,000 lux. The low intensity of light and short photoperiod may indicate that the effect of light on development of resistance to cold is photoperiodic rather than photosynthetic (Calder, Macleod, and Hayden,

1966).

These findings indicate that photoperiod can influence the development of cold resistmlce in plants. The genetic potential for resistance is reached, however, only when plants are exposed to control­ led photoperiods (Hodgson, 1965). For this reason, the author suggested that the photoperiodic response may be mediated through a phytochrome system similar to those involved in dormancy and flowering.

Externally applied hormones

The gibberellins. Gibberellins generally cause an increase in the stem lengths of most growing plants. The elongation is caused by an increase in the length of cells, and to a lesser extent by an increased number of cells. Gibberellic acid may also overcome dormancy in s ome plants, and in some plants flowering can be enhanced by the application of gibberellin (Salisbury and Ross, 1969).

Exogenous applications of gibberellic acid have been shown to affect the cold hardiness of plants. Its mode of action is not known, however, even though many observations have been reported. Most reports 9 show that the gibberellins increase growth rate and decrease cold hardiness (Broks, 1963; Wunsche, 1966; Irving and Lanphear, 1968), while others report an opposite effect on cold hardiness. For example,

Proebsting and Mills (1965) found that solutions containing 80 to

1,000 ppm of gibberellin applied in August and September increased the winter hardiness of buds, but applications in November decreased bud hardiness. Gibberellin appeared to delay differentiation of flower primordia, which resulted in smaller buds and increased cold hardiness (Edgertan, 1966). Exogenous applications of gibberellin reduced the cold tolerance of some types of vegetables and increased the cold tolerance of others (Lao, Hwang and Tang, 1963).

The cytokinins. Cytokinins overcome apical dominances in some plants. Application of cytolinins helps leaves to remain green due to slower degradative processes; also, they may stimulate synthesis in the . Cytokinins stimulate the growth of dicot seedlings in the absence of light, largely due to increasing true size of the cells and not by stimulating cytokinesis as light does (Salisbury and Ross,

1969).

Spraying pea plants with an aqueous solution of benzyladenine

(a cytokinin) increased the plants' resistance to freezing, while cm1trols that were sprayed with water were almost dead within one week.

The cytokinin seemed to cause the pea plants to become frost hardy. In this study it was suggested that cytokinins improve hardiness by mobilizing cold-hardiness promoters (Kuraishi, et al., 1966). 10

Auxins. in general play a part in causing the elongation of stems in grass coleoptiles and of roots, leaves, and flowers of higher plants. This influence is caused mainly by the elongation of cells (Salisbury and Ross, 1969).

Cellular influences

Ultimately, the ability of plants to adapt to cold temperatures must be some change within the cells. The change may be associated with metabolic processes, components within the cell, or the physical structure of the cell. It may be some combination of these and may vary from one plant to another and from one set of environmental condi tiona to another.

Chloroplasts. Changes in as plants harden in response to low temperatures have been reported, but with two divergent explanations as to the effects. Some workers have observed that chloroplasts retain their integrity but migrate from a summer position near the ce l l wall to a crowded position in the cell interior, with some reports of a concomitant loss in content (Parker,

1963). Others (Salcheva and Samygin, 1963), believe that chloroplasts aggutinate, l ose their integrity, ar1d merge with each other to become a continuous mass from which chloroplasts re-form again as spring approaches.

Kimball and Salisbury (1973) reported a less pronounced change in the chloroplasts of the three species they studied; (Secale ce re ale L., ~ dactylon (L.) Pers., and Paspalum nootatum Fugge).

At reduced temperatures the plastids became grouped tc.gether within 11

the cells, changed cooformation, and became enlarged. This grouping

of chloroplasts seemed to correlate with the reduced growth activity

or death of the cells.

Enzyme activity. The changes in carbohydrates, amino acids,

proteins and other organic compounds when plants become acclimated to

cold must be brought about by changes in enzymes and enzyme activity.

Because biological reactions are less dependent on activation energy

or temperature when enzymes are highly active, much research has been

done on the effect of low temperature on enzymes. Hydolysis,

aminolysis, dehydration, oxydation, and decomposition of peroxide are

accelerated in ice despite limited diffusion and reduced kinetic

energy (Grant and Alburn, 1966). Virtually all work has confirmed that

frost-resistant plants have a higher enzymatic activity than non­

resistant and, therefore, the rates of enzymatically catalyzed reactions

do not decrease as rapidly when the temperature falls (Vasil'yev, 1956).

Alden and Hermann (1971) support the concept that increased

intramolecular formation of hydrogen bonds at low temperatures

results in increased folding of the enzyme and reduced contact between

the substrate and active site of the enzyme and this, in turn, reduces

catalytic activity.

Lipids and Lipoproteins. Levitt (1957) cited numerous studies that

reported an increase and a spring decline in intracellular oil and fat accumulations in plants. Recent studies show that plants accumulate lipids as part of their hardening process. Genkel' and

Oknina (1966) reported an increase in lipids and tannins in specific

strains of fruit as starch declined during the winter. An 12

accumulation of starch in the spring likewise accompanied a decrease

in lipids and tannins.

Some plants may not accumulate fats and lipids as they harden.

Chien and Wu (1956) found no evidence of these substances in wheat

during the winter .

Carbohydrates . That the reduction of carbohydrate reserves reduces

cold hardiness in wintering plants in well known. Starch decreases

to a minimum and sugar increases to a maximum as plants become

acclimated to cold temperatures . Levitt (1956) listed more than fifty

writers who have observed starch-to-sugar changes. Although starch-to­

sugar changes are fairly well understood, the conversions of sucrose

to other sugar complexes and the role of sugars in the frost hardening

mechanism have not been fully worked out (Parker, 1963).

Tumanov and Trunova (1963) and Trunova (1963, 1967) concluded

that the effectiveness of sugars in promoting cold hardiness depended

on the enzymatic ability of the cell to transform the sugars into

substances that provide maximum protection. The following sugars

afford decreasing protective action to winter annuals; sucrose,

glucose, rhamnose, maltose, lactose, and glactose.

Although numerous studies have been made on the relation of

changes in sugar content to frost resistance, all functions of sugars

in resistance to low temperatures have not been resolved. Substances

like sucrose are shown to retard growth of ice crystals and alter their

pattern without depressing the point of ice nucleation any more than the freezing point (Lusena, cited by Parker, 1963). Such action may 13 protect the proteins of membranes and enzymes from sudden loss of water during freezing. Sugars may also protect proteins directly by replacing some of the water of hydration, or they may serve to hold the water of hydration more firmly through hydrogen bonding in structures sensitive to dehydration as water is removed to form ice (Ullrich and Heber, 1957).

Large polymers in the cell wall interfere with the structure of ice crystals growing along cell walls, and by occupying sites in the crystal !attics, cause the crystals to become variable (Olien,

1965, 1967). Tissues highly resistant to injury develop more imperfect crystals upon free zing than do less resistant tissues. This interference with the ice lattice development results in an ice mass with small or imperfect crystals within the plant (Olien, 1967a, 1967b).

Soluble protein. Paulsen (1968) reported that decreasing tempera­ tures in winter wheat increased both cold tolerance and soluble protein content . All types of alfalfa that were hardened under short photo­ periods exhibited an increase in soluble protein (Hodgson, 1964) .

Hodgson and Bula (1956) suggested that water soluble protein is necessary for tolerance to cold in sweet clover (Melilotus spp).

Cell membranes. Several studies have related chilling injury at temperatures above o~c to physical and chemical changes in the lipid portion of cellular membranes that alter their permeability. It was suggested that chilling affects a lipid that is important to the structure of the cell membrane. The cell is disrupted when cell permeability is destroyed (Ibaniz, Casac and Redshaw, 1965). 14

In Lyons (1973) review of chilling injury in plants it was noted

that lipids controlled the physical state of the membrane . Phase

transitions of membrane or membrane-bound enzymes were observed in

chilling sensitive plant species but not in chilling resistant species.

Membrane lipids from chilling-sensitive plant species have a higher

ratio of saturated to unsaturated fatty acids than do chill resistant

plants.

Protoplasmic changes, Although it appears to be a potentially

important part of the cold adaptation process, little is known about

protoplasmic changes associated with this plant phenomenon. Proto­

plasmic streaming in some cold sensitive plants ceased at about 10•-12•c while streaming continued down to near o•c in cold hardy plants.

The role of streaming in normal metabolism is little understood, it

is likely that normal metabolism is upset by cessation of the process

(Lyons, 19 7 3).

Cold injury and desiccation

After hardy plants develop maximum resistance to cold, they can withstand extremely low temperatures without injury, provided that rapid

freezing is delayed until all readJ.ly freezable intercellular water

is removed to sites of extracellular ice formation (Tumanov and

Krasavtsev, 1966a, 1966b; Sakai, 1967; Sakai and Yoshida, 1967).

Rapid fluctuations in temperature above the critical freezing tempera­

ture that remove freezable intracellular water may cause injury, however, as a result of intracellular ice formation. This type of in­

jury is not always separated from that caused by frost drying. Injury 15 from frost drying supposedly occurs when the above-ground organs of the plant can not replace water lost by as air tempera­ tures rise to about 0°C, because the soil or vascular system remains frozen (Pisek, 1962; Weise, 1962).

Plant growth under the snow

Growth under the snow has been studied by Salisbury et al. (1973) in the mountainous vacinity of Logan, Utah, as well as some alpine snow banks in the Rockies. These studies show that some plants grow under the snow at a temperature of approximately 0°C.

Brevor (winter hardy) and Lemhi (nonhardy) wheat varieties can grow and develop at near freezing temperatures. Also observed was the growth of spring ephemerals demonstrating the ability to grow, develop, and in some cases, flower in and/or under the snow.

Dehardening

Maintenance of the hardened condition appears to depend on the same environmental conditions that inhibit growth, induce dormancy, and develop cold resistance. Growth impulses and gradual loss of hardiness brought by warm weather in the spring occurred simultaneous ly but independently of each other. Cold resistance in trees is lost not only during thaws, but also during periods when cold is less intense

(Tumanov and Krasavtsev, 1955). 16

MATERIALS AND ME'll!ODS

Growth under the snow

Field studies. The site chosen for this study was an aspen grove

(Figure 4) at an elevation of 2,350 meters (7700 feet) in Steep Hollow,

Cache Na tional in northern Utah. The location was selected as an area that is essentially undisturbed during the winter by wild animals or recreational vehicles, and where the snowpack is deep and lasts for an extended period of time .

An experimental tunnel was constructed with steel sides and top, and was buried parallel to the contour of the ground surface. (Figu res

1, 2, 3). It was 6.1 meters long, 1.8 meters high, and 1.2 meters wide. At one end, an entrance was provided by attaching a corregated steel culvert in a vertical position to a height of approximately 2 meters so as to be above the maximum outside snow level. The snow depth was measured at the tunnel entrance at each visit.

Along the upper portion of one side of the tunnel, a series of openings wit h detachable panel doors provided access to a balcony, an outside ground-level area where the seed trays were placed. A 2.5 em layer of gravel was placed in the tray holder to facilitate the removal of trays for sampling. The space was sufficient for 20 trays of 18 1 each in which the seeds were planted (Figure 5).

In October, 1971, a sandy loam soil was placed in each tray, and seeds of commercial winter rye (Secale cereale var. cougar) were planted at a depth of 1-2 em • About 150 seeds were planted per tray. 17

Figure 1. Tunnel uncovered

Figure 2. Tunnel partially covered. 18

Figure 3. Tunnel covered.

Figure 4. Aspen grove in Steep Hollow. ..

19

Figure 5. Tray in tunnel.

Prior to planting, the seed was divided into four groups and

each group soaked with a 10-3 M hormone solution as follows:

(1) indole-3-acetic acid, (2) Kinetin---6 furfurylaminepurine,

(3) Gibberellic acid- --GA . (4) Control treated with an alcohol 3 solution. To facilitate complete solution of each hormone, 20 ml of

95 percent methanol was added to each 230 ml of the hormone solution.

Soluble protein studies. Starting in December 1971, and period-

ically for the next six months, about 12 plants from each treatment

were taken for analysis. The plants were placed in plastic bags and

stored in an ice chest while being transported to the laboratory--

usually within 2 hours of their removal from the trays. 20

The shoots were removed from other portions of the seedlings and their lengths and weights determined. Soluble protein content of the shoots was determined according to the procedure of Lowry, et al.

(1951) as follows:

1. A weighed sample was ground for two minutes by hand, using a mortar and pestle. Ten ml of tris buffer were added before the grinding.

2. The ground sample was centrifuged at 10,000 rpm for ten minutes.

3. Of the plant extract, 0.25 ml was added to 0.25 ml of 2N NaOH.

4. The mixture (now 1 N NaOH) was allowed to stand at room temperature for one hour.

5. Five ml of the following solution was added: 2 percent Na co 2 3 and 0.5 percent cuso • 5 H 0 in 1 percent sodium nitrate . This 4 2 solution was mixed fresh daily.

6. The mixture was shaken well and kept at room temperature for

10 minutes, and then 0.5 ml of Folin reagent .. was added. The mixture was allowed to stand at room temperature for 30 minutes.

7. The solution was measured at 750 nm in a Beckman DB-G spectrophotometer against a blank (0.5 ml 1 H HaOH to which all reagents had been added), then compared with the control graph,

(Figure 6).

Statistical analysis. An analysis of variance was made on the growth of plants (length and weight of shoots) and the soluble protein content (Sokal and Rohlf, 1969). Mean values for each period were 21

360

320

280

240 § 0 ~ 200 '"',.,"' ....'"' (J) 160 Q) Cl" .... u ...."' 120 '"'0. 0

80

40

0 .8 .1 . 2 . 3 .4 . 5 .6 . 7

Figure 6. Contr ol graph of soluble protein in mg/ml. 22 treated as replicates for the analysis of each treatment group.

No interactions of treatment and time were tested. 23

RESULTS AND DISCUSSION

Conditions, at the snow tunnel

The snow depth as shown in Figure 7, at the tunnel site in

Steep Hollow demonstrates the heavy snow pack in this area in the late winter. During this particular winter the heavier snow fall carne in the months of March and April. The fast melt off of snow as shown in the graph is probably typical.

2.0

~ w~ ~w 5 I ~ ~ 1. 0 ~ ~

~ a0 ~

0.0 Dec Jan Feb Mar Apr May 1971 1972

Figure 7. Snow depth in Steep Hollow. Variation with time. 24

As shown in Figure 8, while there is ample snow cover, the temperature inside t he snow tunnel is essentially con stant at 0°C.

10.0 Q) "0 ,.."' ·~..."" Q) u"

"'Q) ,..Q) 0.0 ""Q) :::'- • •••• .. I I ,..Q) ...;:J ,.. ,."'Q) ~ f-<

-10.0 Dec Jan Feb Mar Apr May 1971 1972

Figure 8. Temperature in tunnel. Variation with time. Plant growth

Shoot length. Figure 9 shows a rapid growth during the month of

March in the shoot length of the groups treated with Gibberellic acid

(GA) and Indole acetic acid (IAA). This agrees with the information

summarized by Salisbury and Ross (1969). The rapid growth of these two

groups would be explained by the cell elongation caused by these growth

hormones.

The shoot length growth of the control group (OR) and the cytokinin

(K) group is more gradual, reaching a peak a few weeks later. According

to the data (Figure 9), the average shoot length of each group begins

to decline sometime in the last of March or the first of April. In

nature, as far as we know, shoots do not shrink in size so some other

possibilities must be considered for the indicated loss of shoot length.

Several possibilities for this occurrence are:

1. Random variations: The samples were too small to accurately

account for the random differences in the rye population. These small

samples were necessary because of the small number of plants that could

be grown in each treatment group.

2. Position effect in the tunnel: Plants taken later in the

year may have been growing in an area in the balcony of the snow

:unnel that was not as advantageous for their growth. Thus, the

shoots may have been shorter all the time. This alternative does

'ot really satisfy the problem, as all positions in the balcony

appeared to be equal in all aspects.

3. Death of longer shoots: The longer shoots may have died

out in the later months because of a greater stress on them due 26

9

8

GA OH

6 K 00 .... '-''" s •rl'" 5 '-' "u'" ~ I ..c: IAA '-' 4 bJl ,..,"'" 3

1

0~------L--L--4--L----~---L--~ ____J_ ____ , March April May

Figure 9 . Shoot length. Variation in time . 27

to their size. Some evidence of this was noted in the finding of a few

dead and shriveled shoots in the trays sampled in April and May. In my

opinion this is the most logical explanation for the shoot length

findings.

The apparant increase of growth in three of the four groups in

May is probably a response to higher temperatures in the snow tunnel.

This higher temperature is probably a result of lower snow depth and

higher outside May temperatures. The limited number of observations make it difficult to interpret these observations. There were no

statistically significant differences in shoot length among treatment

groups.

Fresh shoot weight. Rapid increases in the average shoot fresh weight of all four groups (Figure 10) coincides with the rapid growth

in shoot length (Figure 9). Each group peaked out in fresh weight at

approximately the same time. As the average shoot length decreased

during the winter, the average fresh weight did not. This trend is

probably explained by the fact that the shoots sampled were more bulky

than were the longer samples taken earlier.

The statistical analysis of the fresh weight data showed no

significant difference between the four experimental groups.

Interrelationships (length vs weight). By observation it appeared

that the rye had grown markedly by the time the first samples were

taken in March. This rapid growth continued through the month of March with plants gaining in both shoot length (Figure 9) and shoot weight

(Figure 10). Both cell division and cell elongation are responsible for this rapid growth. The GA group and the IAA group were the fastest 28

60

50 OH

GA

00 K s 40 btl""' IAA ...... _:!, I w 0 30 .c0 --...(/) w .c ....btl Q) :. .c (/) 20 Q) ""'"

10

Figure 10. Fresh weight. Variation in time. 29 growers in length and fresh weight, although that was not s t atistically significant.

Soluble protein content

As mentioned earlier, in past studies of the relationship between the gibberellins and cold hardiness in plants, the GA treatments have shown less cold hardiness. The mode of action is not known at this point. It appears that as the growth rate increases the cold hardiness of the plant goes down (Broke, 1963; Wunsche, 1966; Irving and Lanphear,

1968). In the present study with GA, it was demonstrated that plants treated with this hormone showed a significant decrease in soluble protein (Figure 11).

If as r eported by Paulsen (1968) increased cold t olerance is accompanied by an increase in soluble protein, and as reported by

Hodgson and Bula (1956) soluble protein is necessary for cold tolerance in come species, a decrease in soluble protein may be an indication of reduced cold tolerance. If so, the GA hormone may have a direct influence on the level of soluble protein found in some species of plants and thus also in their lower level of cold hardiness.

More experiments using GA and testing for soluble prctein level on different plant species needs to be run in order to explore the findings of this research project.

The other three groups, K, OH, and IAA showed no signficant difference in the amount of soluble protein found in their fresh weight. It would then appear that these hormones do not play an important roll in the level of soluble protein in the cold-hardiness mechanism in plants . 30

2 . 0

.; 1. 5 .c .... ""\l) :> .c

...; ..0"' .-< "0 C/)

O.QL------~--~--~-L------~--4-r-L-----~----~ March April May

Figure 11. Soluble protein. Variation in time. 31

SUMMARY

Cold hardiness in plants may be related to one single alteration of a complex of several interacting events. Plants that are able to withstand colder temperatures generally have more soluble protein.

Soluble protein has a definite but undeter mined connection with the cold-hardiness of a plant. The growt h hormones, gibberellic acid, indole-3-acetic acid, and kinetin, used in this experiment had no significant effect on shoot length or fresh weight. IAA and Kinetin had no significant effect on the soluble protein level, while the GA treatment showed a significant decrease. GA is imp licated in plants tha t are l ess cold-hardy with an accompaning lower level of soluble protein. 32

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APPENDIX 38

Table 1. Average shoot length in em.

DATE GA K IAA OH

March 17 5.0 5.1 6.8 5.2

March 24 8.4 4.7 7.3 5.4

March 31 8. 2 6.3 7 0 7 4.8

April 6 6.4 6.1 6.8 6.4

April 20 5.5 5.4 5.6 6.4

April 27 6.2 4.5 6.4 5.3

May 4 5.3 4.5 5.6 4.0

May 18 7.0 5.8 4.1 6.8

Table 2. Fresh weight per shoot in mg.

DATE GA K IAA OH

!1arch 17 23 23 33 27

March 24 36 30 49 35

March 31 49 40 48 41

April 6 41 45 43 48

April 20 38 38 44 50

April 27 46 33 48 43

May 4 37 47 36 43

May 18 45 40 38 49 39

Table 3. Mg of soluble protein per ml of solution per gram of fresh weight.

DATE GA K IAA OH

March 17 l. 420 2.000 l. 313 l. 453

March 24 0. 72 7 0.967 0.940 1.077

March 31 0 . 650 1.112 l. 097 0. 816

April 6 0. 831 1.080 1.110 1.234

April 20 0. 730 0. 645 0. 932 o. 739

April 2 7 0.667 l. 363 l. 414 l. 231

May 4 0.640 1.045 1.216 0.979

May 18 0. 682 1.094 0.787 0. 779 40

VITA

Michael D. Salvesen

Candidate for the Degree of

Master of Science

Thesis: The Influence of Certain Growth Hormones on Plant Growth at Cold Temperatures

Major Field: Plant Physiology

Biographical Information:

Personal Data: Born at Logan, Utah, February 5, 1946, son of Mervin N. and Jane W. Salvesen; married Karalee Stoddard, August 16, 1967; three children--Shalise, Chantel, and Michelle.

Education: Attended elementary school in Hyrum, Utah; graduated from South Cache High School in 1964; received the Bachelor of Science Degree in Biological Sciences with a Composite Major in Secondary Education; completed requirements for the Master of Science Degree, Majoring in Plant Physiology, at Utah State University in 1977.

Professional Experience: Worked for Utah State Fish and Game Department as a Biological assistant 1969. 1971, 1972, a graduate research assistant at Utah State University, working for Dr. Frank B. Salisbury. Fall of 1972 to present, teacher of Biological Sciences, South Cache Junior High School, Hyrum, Utah.