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The effects of transplant and drought stress on the water relations of western hemlock as measured with… Kandiko, Robert Alan 1978

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THE EFFECTS OF TRANSPLANT AND DROUGHT STRESS ON THE WATER RELATIONS OF WESTERN HEMLOCK AS MEASURED WITH A PRESSURE CHAMBER Robert A l a n Kandlko o B.A. , C o r n e l l U n i v e r s i t y , 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF » MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES F a c u l t y of F o r e s t r y We accept t h i s t h e s i s as conforming to the r e q u i r e d s tandard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1978 0 Robert A l a n K a n d i k o , 1978 In presenting th i s thes is in p a r t i a l fu l f i lment of the r e q u i r e m e n t s f o r an advanced degree at the Univers i ty of B r i t i s h C o l u m b i a , I a g r e e that the L ib rary sha l l make i t f ree l y ava i lab le for r e f e r e n c e and s t u d y . I fur ther agree that permission for extensive copying o f t h i s t h e s i s for scho la r l y purposes may be granted by the Head o f my Department or by h is representat ives . It is understood that c o p y i n g o r p u b l i c a t i o n o f th is thes is f o r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permission. Department of Forestry The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 25/4/78 6 - i i -ABSTRACT P r e l i m i n a r y exper iments were conducted u s i n g western hemlock p l u g s e e d l i n g s (Tsuga h e t e r o p h y l l a R a f . S a r g . ) to t e s t the p r e s s u r e chamber t e c h n i q u e . P ressu re - vo lume r e l a t i o n s h i p s were measured f o r the shoots and the r e l a t i v e importance of the m a t r i x p o t e n t i a l was a s s e s s e d . P ressure - vo lume r e l a t i o n s h i p s were a l s o measured f o r the r o o t s and d i f f e r e n c e s between the r o o t s and the shoots were i d e n t i f i e d . The e f f e c t s of t r a n s p l a n t and drought s t r e s s on these p r e s s u r e - v o l u m e r e l a t i o n s h i p s were t e s t e d and i t was found t h a t t r a n s p l a n t i n g has ve ry l i t t l e e f f e c t on the water r e l a t i o n s of the s e e d l i n g s . A two-seek drought s t r e s s of 10 atm. r e s u l t e d i n a l o w e r i n g of the osmot ic p o t e n t i a l of the shoot both at f u l l t u r g o r and a t i n c i p i e n t p l a s m o l y s i s . The r o o t s showed no s i g n i f i c a n t change. Shoot and roo t r e s i s t a n c e s to water f l o w were shown no t to change s i g n i f i c a n t l y w i t h e i t h e r t r e a t m e n t . The b u l k e l a s t i c modulus of the shoots was found to v a r y w i t h a change i n V , but was not shown to v a r y w i t h the drought s t r e s s . - i i i -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES v i VARIABLES DISCUSSED AND MEASURED . v i i i FOREWORD i x ACKNOWLEDGEMENTS x i PART I. TEST OF THE PRESSURE CHAMBER TECHNIQUE 1 Introduction 1 A. Measurement of P-V curves for Shoots 8 Methods and Materials 8 Results and Discussion 11 B. Measurement of Matrix Potential for Shoots 18 Introduction 18 Methods and Materials 18 Results and Discussion 19 C. Measurement of P-V curves for Roots 23 Introduction 23 Methods and Materials 23 Results and Discussion 24 Summary - Part I 25 - i v -PART I I . EFFECT OF TRANSPLANTING AND DROUGHT STRESS ON THE WATER RELATIONS OF HEMLOCK SEEDLINGS 26 Introduction 26 Methods and Materials 27 Results: ^ A. Comparison between Plug seedlings from Parts I and I I . 31 B. Comparison between Shoots and Roots 33 C. Comparison between Plug and Transplant Treatments. • • 36 D. Comparison between Transplant and Drought Stress Treatments 40 Discussion 41 Summary^. 53 BIBLIOGRAPHY 54 - v -LIST OF TABLES Table Page I Comparison of s t a t i s t i c s d e r i v e d from P-V curves f o r western and e a s t e r n hemlock 13 V I I R e l a t i v e importance of Y and 1^ i n the / ' m l i n e a r p o r t i o n of the P-V curve 22 I I I Comparison of s t a t i s t i c s f o r p l u g r o o t s and shoots from P a r t s I and I I 32 - v i -LIST OF FIGURES F i g u r e Page 1 P r e s s u r e chamber f o r measurement of sap p r e s s u r e i n the xylem of a shoot 3 2 T h e o r e t i c a l P-V curve 5 3 Diagrammatic r e p r e s e n t a t i o n of a t w i g i n a p ressure chamber 7 4 P-V curve f o r onehemlock shoot . . 12 5 R e l a t i o n s h i p between 1/VAT and percent water removed f o r 7 hemlock shoots 15 6 R e l a t i o n s h i p between VAT and percent water removed f o r 7 hemlock shoots 16 7 M a t r i x p o t e n t i a l f o r 4 hemlock shoots 20 8 Comparison of P-V curves f o r r o o t s and shoots of hemlock p l u g s e e d l i n g s 34 9 Comparison of osmotic p o t e n t i a l s f o r 3 t reatments 37 10 Stem r e s i s t a n c e ve rsus t r a n s p i r a t i o n r a t e s f o r 3 t reatments 38 11 Root r e s i s t a n c e versus uptake per u n i t root l e n g t h f o r 3 t reatments 39 12 Comparison of shoot r e s i s t a n c e s f o r western hemlock and D o u g l a s - f i r 43 13 Comparison of roo t r e s i s t a n c e s f o r western hemlock and D o u g l a s - f i r 44 14 Comparison of P-V curves f o r shoots from the t r a n s p l a n t and d r o u g h t - s t r e s s e d t reatments 45 15 Comparison of P-V curves f o r r o o t s from the t r a n s p l a n t and drought s t r e s s e d t reatments 46 - v i i -16 R e l a t i o n s h i p s of K f c ve rsus F and K ve rsus VAT f o r p l u g shoots 49 17 Comparison of K f o r p l u g shoots from P a r t s I and I I . 50 18 Comparison of VAT p ressures f o r p lug shoots from P a r t s I and I I . . . . ' 51 - v i i i -VARIABLES DISCUSSED AND MEASURED water p o t e n t i a l : the chemica l p o t e n t i a l of water and a measure of the energy a v a i l a b l e f o r s u c t i o n or movement osmotic p o t e n t i a l : the p o t e n t i a l w i t h which pure water w i l l d i f f u s e toward a s o l u t i o n osmotic p o t e n t i a l a t f u l l turgor osmotic p o t e n t i a l at i n c i p i e n t p l a s m o l y s i s or w i l t i n g p o i n t tu rgor p r e s s u r e : the p ressure exerted on the l i q u i d by the w a l l s of a t u r g i d c e l l volume-averaged tu rgor p r e s s u r e : the turgor p ressure f o r the c e l l s i n the shoot or root as measured u s i n g the pressure -vo lume curve p o t e n t i a l due to g r a v i t a t i o n a l f o r c e s m a t r i x p o t e n t i a l : the p o t e n t i a l due to a b s o r p t i v e f o r c e s r e l a t i v e volume of the symplast ; percent of V t o t a l volume of water (symplast and apop las t ) volume expressed and c o l l e c t e d i n tygon tub ing stem h y d r a u l i c r e s i s t a n c e roo t h y d r a u l i c r e s i s t a n c e t r a n s p i r a t i o n r a t e t measured f o r r o o t s w 0** measured f o r shoots •::w uptake per u n i t roo t l e n g t h b u l k e l a s t i c modulus of the shoot : f o r c e per u n i t area a s s o c i a t e d w i t h a change i n volume of t i s s u e per u n i t volume - i x -FOREWORD The experiments d i s c u s s e d i n t h i s t h e s i s were conducted d u r i n g my summer i n t e r n s h i p a t Weyerhaeuser Research Center i n C e n t r a l i a , Washington. The o b j e c t i v e s of my i n t e r n s h i p were (1) to adopt the procedures d e s c r i b e d by Farnum (1977) and by Tyree and Hammel (1972) f o r measuring the h y d r a u l i c and osmot ic p r o p e r t i e s of hemlock nurse ry s tock u s i n g a p ressure chamber; (2) to o b t a i n t y p i c a l mean v a l u e s and v a r i a n c e s of these p r o p e r t i e s both b e f o r e and a t v a r i o u s i n t e r v a l s a f t e r t r a n s p l a n t i n g to p o t s ; and i f t ime was a v a i l a b l e (3) to compare the h y d r a u l i c and osmotic r e l a t i o n s h i p s a f t e r a p e r i o d of reduced w a t e r i n g . The lengthy t ime r e q u i r e d to set up l a b o r a t o r y equipment and to measure the pressure -vo lume r e l a t i o n s h i p s l i m i t e d the number of s e e d l i n g s i n which the h y d r a u l i c p r o p e r t i e s were measured. I t was important to determine i f changes i n these p r o p e r t i e s o c c u r r e d . F u r t h e r exper imenta t ion i s now b e i n g conducted to determine q u a l i t a t i v e l y the changes repor ted i n t h i s t h e s i s . The t h e s i s i s d i v i d e d i n t o two s e c t i o n s each c o n t a i n i n g an i n t r o d u c t i o n . The f i r s t s e c t i o n d i s c u s s e s the use of the p ressure chamber techn ique f o r measuring xylem sap t e n s i o n as developed by D ixon (1914) , Scholander et a l . (1965) , Boyer (1967b), and Tyree and Hammel (1972) . T h i s techn ique was a p p l i e d to s e e d l i n g s of western hemlock (Tsuga h e t e r o p h y l l a ( R a f . ) S a r g . ) , and the r e s u l t s are compared w i t h those repor ted p r e v i o u s l y by Tyree et^ a l . (1973) f o r e a s t e r n hemlock (T_. canadensis (L . ) C a r r . ) , by H e l l k v i s t et: a l . (1974) f o r S i t k a spruce ( P i c e a s i t c h e n s i s B o n g a r d . ) , and by Boyer (1967b) f o r yew (Taxus c u s p i d a t a S i e b . and Z u c c ) . P ressure -vo lume curves were a l s o measured f o r r o o t s . T h i s i s an e x t e n s i o n of the p ressure chamber techn ique not repor ted p r e v i o u s l y i n the l i t e r a t u r e . The second s e c t i o n of the t h e s i s d e s c r i b e s an experiment that used the p r e s s u r e chamber t e c h n i q u e , combined w i t h measurements of p l a n t r e s i s t a n c e s (Farnum, 1977) , to i n v e s t i g a t e the e f f e c t s t h a t t r a n s p l a n t i n g and drought s t r e s s have on the water r e l a t i o n s of c o n t a i n e r nursery s t o c k . - x i -ACKNOWLEDGEMENTS I w i s h to express my a p p r e c i a t i o n to the personne l at Weyerhaeuser Research Center i n C e n t r a l i a , Washington whose a s s i s t a n c e made t h i s t h e s i s p o s s i b l e . S p e c i a l thanks a re extended to Roger Timmis and P e t e r Farnum f o r t h e i r e x c e l l e n t s u p e r v i s i o n , and c o n s u l t a t i o n and to S a l l y Johnson f o r her a d v i c e and encouragement. I a l s o w ish to thank Dan Dunham and Gary R i t c h i e f o r a s s i s t i n g i n the a n a l y s i s of my exper iments . I w i s h to acknowledge my g r a t i t u d e to my f a c u l t y s u p e r v i s o r Dr . John W o r r a l l f o r s h a r i n g h i s enthusiasm and wisdom and f o r ex tend ing h i s a d v i c e and c o n s t r u c t i v e c r i t i c i s m . - 1 -PART I. TEST OF THE PRESSURE CHAMBER TECHNIQUE. INTRODUCTION In recent y e a r s , the p ressure chamber has become the most e f f i c i e n t and r e l i a b l e inst rument to measure the water s t a t u s of p l a n t s (Boyer, 1967a; H e l l k v i s t et a l . , 1974) . The major development of the p ressure chamber took p l a c e over the l a s t 15 y e a r s . Scholander e_t a l . (1964, 1965) , f i r s t adapted D i x o n ' s " p r e s s u r e bomb" (Dixon, 1914) to measure the n e g a t i v e h y d r o s t a t i c p r e s s u r e i n mangroves (Rhizophora s p . ) . They a l s o observed v a r i a t i o n i n xylem sap p ressure at d i f f e r e n t h e i g h t s i n c o n i f e r o u s t r e e s and the e f f e c t t h a t h a b i t a t has on xylem sap p r e s s u r e . An e x c e l l e n t rev iew of the s t u d i e s conducted w i t h the p ressure chamber i s presented by R i t c h i e and H i n c k l e y (1975) . The d i s c u s s i o n tha t f o l l o w s i s a s i m p l i f i e d a n a l y s i s of the water s t a t u s w i t h i n a p l a n t . An i n - d e p t h a n a l y s i s of p l a n t water r e l a t i o n s has been w r i t t e n by S l a t y e r (1967) . The b a s i c p r i n c i p l e s of the pressure chamber techn ique are p r e s e n t e d . A q u a n t i t a t i v e examinat ion of the water r e l a t i o n s as measured by the p ressure chamber has been presented by Tyree and Hammel (1972) . When a p l a n t i s t r a n s p i r i n g there e x i s t s a f r e e energy g rad ien t between the r o o t s anchored i n the s o i l and the stomata which are exposed to the atmosphere. T h i s a c t s , i n e f f e c t , to p u l l water up through the p l a n t . I f the stem i s severed , the d r i v i n g f o r c e due to t r a n s p i r a t i o n no longer e x i s t s r e s u l t i n g i n a r e c e s s i o n of the water column u n t i l a - 2 -meniscus forms at bordered p i t s at some d i s t a n c e , X , from the cut s u r f a c e . T h i s d i s t a n c e i s p r o p o r t i o n a l to the water p o t e n t i a l d i f f e r e n c e between t h e atmosphere and the p l a n t (Scholander ej: a l . , 1965) . The e x t e r n a l p r e s s u r e needed to f o r c e the water column back to the cut s u r f a c e shou ld equa l the j f of the p l a n t when i t was severed . T h i s a p p l i c a t i o n of e x t e r n a l p ressure to measure the water s t a t u s of p l a n t s i s the p r i n c i p l e u n d e r l y i n g the p ressure chamber t e c h n i q u e . A f t e r the s e e d l i n g i s severed at the roo t c o l l a r , the shoot i s p l a c e d i n s i d e the p ressure chamber w i t h the severed end exposed (F igure 1 ) . N i t r o g e n gas i s pumped i n t o the sea led chamber r e s u l t i n g i n an i n c r e a s e of p r e s s u r e . The xy lem sap appears on the cut s u r f a c e when the p r e s s u r e exer ted by the a p p l i e d gas equals the of the w p l a n t when i t was severed . Two assumptions must be made be fo re the p ressure chamber technique i s accepted (Boyer, 1967a) . The water p o t e n t i a l s of the xylem sap and l e a f c e l l s must be i n e q u i l i b r i u m d u r i n g the t ime of measurement and the s p a t i a l arrangement of water w i t h i n the p l a n t when i t i s i n the p ressure chamber must be i d e n t i c a l w i t h tha t found i n the i n t a c t p l a n t . Boyer s t a t e s t h a t these assumptions can be e a s i l y accepted i n woody p l a n t s ( e . g . yew) tha t have s m a l l amounts of p i t h and t h a t are not prone to de fo rmat ion . The water p o t e n t i a l , ^ , of a p l a n t i s composed of four components which are represented i n the f o l l o w i n g e q u a t i o n : U> = /jf + f + p + f 1 w ' 1 m ' g Eq . 1 - 3 -gas pressure gas I I * release Figure 1. Pressure chamber for measurement of sap pressure i n the xylem of a shoot. - 4 -the osmotic p o t e n t i a l , / i s the m a t r i x p o t e n t i a l , P i s the t u r g o r p r e s s u r e , and jr* i s the g r a v i t a t i o n a l f o r c e . In l a b o r a t o r y experiments ^ i s n e g l i g i b l e so i t i s dropped from the e q u a t i o n . The osmotic p o t e n t i a l , tf, i s composed of f o r c e s due to f r e e s o l u t e s , whereas the m a t r i x p o t e n t i a l i s p r i m a r i l y a r e s u l t of a b s o r p t i v e f o r c e s of the c e l l w a l l s . Osmotic and m a t r i x p o t e n t i a l s are o f t e n so i n t e r r e l a t e d t h a t s e p a r a t i o n of t h e i r e f f e c t s becomes i m p o s s i b l e ( S l a t y e r , 1967) . Both of these f o r c e s a c t to lower the f and a re t h e r e f o r e n e g a t i v e i n s i g n . The p ressure p o t e n t i a l , P , can w e i t h e r be p o s i t i v e or n e g a t i v e depending on whether the p ressure a c t i n g on the molecu les i n q u e s t i o n i s below or above atmospher ic (Boyer, 1969) . In most cases P i s p o s i t i v e . Most of the e a r l y s t u d i e s u s i n g the p ressure chamber s imply measured one ^jf f o r a p a r t i c u l a r sample. Scholander et a l . (1965) , advanced the u s e f u l n e s s of the techn ique when they i n c r e a s e d the p ressure beyond the i n i t i a l ^ , and c o l l e c t e d and weighed the e x p e l l e d sap. The procedure was repeated s e v e r a l t imes f o r s m a l l increments of p r e s s u r e , and then a graph of the r e c i p r o c a l p ressures a g a i n s t the volumes c o l l e c t e d was p r e p a r e d , thus de te rmin ing a pressure -vo lume r e l a t i o n s h i p . Tyree and Hammel (1972) r e f i n e d t h i s technique and presented a q u a n t i t a t i v e a n a l y s i s of the P-V r e l a t i o n s h i p . A t h e o r e t i c a l P-V curve i s shown i n F i g u r e 2 . The volume of sap removed i s expressed as a percent of the t o t a l water conta ined i n the shoot which was c a l c u l a t e d a f t e r the dry weight of the shoot was measured. The curve has an i n i t i a l steep drop which i s a r e s u l t Percent water removed F i g u r e 2. T h e o r e t i c a l Pressure-volume Curve L i n e A-E i s P-V curve from d a t a ; B i s i n c i p i e n t p l a s m o l y s i s ; C i s osmotic p o t e n t i a l a t f u l l t u r -g or,"^; D i s r e l a t i v e volume of s y m p l a s t , V Q. - 6 -primarily of decreasing turgor pressure. When the curve becomes a straight l i n e , turgor potential i s no longer present (P = 0). The exact point at which P = 0 corresponds to incipient plasmolysis, or w i l t i n g point. The straight l i n e segment of the P-V curve i s composed of ffand j . The r e l a t i v e importance of these forces w i l l be discussed ' m i n Part I-B. If the straight l i n e segment of the P-V curve i s extrapolated back to the y-axis (Point C), a determination of at f u l l turgor, *ff » c a n be made. Simple subtraction of t h i s extrapolated l i n e from the curve segment (A) w i l l reveal the amount of turgor pressure at a pa r t i c u l a r water content, or the volume-average-turgor pressure, VAT (Tyree and Hammel, 1972). Extrapolation of the straight l i n e segment to the x-axis (Point D) determines the volume of water contained i n the protoplast (symplast) and the volume of water outside the protoplast (apoplast). Figure 3 i l l u s t r a t e s diagrammatically a shoot i n a pressure chamber. The apoplast i s the space of the interconnected c e l l walls including the xylem elements. The protoplasm of the leaf c e l l s i s connected by plasmodesmata which are not shown in Figure 3. This system of interconnected protoplasm i s the symplast (Salisbury and Ross, 1969). I t has been shown that within a given population of a particular species grown under similar environmental regimes, there i s l i t t l e v a r i a t i o n i n T T , the osmotic potential at incipient plasmolysis ( 1 Y ) , and the r e l a t i v e volume of the symplast (V ) (Tyree et a l . - 7 -Figure 3. Diagrammatic representation of a 'shoot i n the pressure chamber. Symplast i s composed of interconnected protoplasm. Apoplast i s composed of interconnected c e l l walls and xylem elements. (Adapted from Tyree and Hammel, 1972) - 8. -1973; H e l l k v i s t et al.,1974). These values however change with species (Scholander et a l . , 1965) , with different growing environments ( i b i d . ) , and with seasons (Hellkvist et a l . , 1974). The experiments described i n Part I were conducted to test the pressure chamber technique as described by Scholander et a l . (1965), Tyree and Hammel (1972), and Boyer (1967). The variations mentioned i n the previous paragraph w i l l be discussed i n Part I I . A. MEASUREMENT OF PRESSURE-VOLUME CURVES FOR SHOOTS OF HEMLOCK SEEDLINGS Methods and Materials The seedlings used i n this experiment were 2-year-old western hemlock plug seedlings, which were kept i n a research greenhouse and watered daily u n t i l they were used i n the experiment. The pressure chamber used i n this experiment (PMS Instrument Co., C o r v a l l i s , Ore.) i s similar i n design to the type developed by Scholander et a l . (1964). A hemlock plug seedling was watered to or near saturation, covered with a p l a s t i c bag, and placed i n a refrigerator overnight i n order to reduce transpiration and bring i t to f u l l turgor. The seedling was then removed from the refrigerator and severed at the root c o l l a r . The shoot was weighed on a Mettler a n a l y t i c a l balance before being positioned inside the pressure chamber. The pressure - 9 -chamber was lined with moist f i l t e r paper to prevent excessive loss of water due to evaporation (Cheung e_t a l . , 1975) . Nitrogen gas was pumped into the chamber increasing the pressure u n t i l f l u i d appeared on the cut surface of the protruding stem. This pressure corresponds to the water potential, j , of the " w shoot when i t was severed (Scholander e_t al. , 1965) . Elapsed time between the severing of the stem and the i n i t i a l pressure reading was less than two minutes. The i n i t i a l ^ w °f the shoots sampled was never larger than -2.5 atmospheres. The expected value of | = 0 r' w i s not achievable even with the pretreatment saturation conditions. This phenomenum has been reported i n other studies but has yet to be adequately explained (Ritchie and Hinckley, 1975). After the i n i t i a l \ was recorded, a pre-weighed 5 cm. ' w section of tygon tubing f i l l e d with tissue paper was placed over the cut surface (Cheung e_t a l . , 1975) . The pressure was then increased 5 atmospheres which resulted i n exudation of f l u i d from the cut surface; this f l u i d was absorbed by the tissue paper. After 10 minutes the tube was removed and weighed. I t was assumed that weight equaled volume for the collected f l u i d (Tyree and Hammel, 1972). The pressure was then released u n t i l the f l u i d was just v i s i b l e on the cut surface. This equilibrium pressure, or balance pressure, was located within 5 minutes of the removal of the tygon tubing. A fresh tube was then placed over the cut stem and the pressure was increased 5 atmospheres o above the balance pressure, and the procedure repeated. This continued u n t i l the volume collected at the end of successive repetitions became - 10 -comparatively small or the volume collected per unit pressure increase became constant which usually occurred between balance pressures of 25-35 atm. In this experiment 15 to 20 balance pressures recorded over 3.5 to 5 hours were s u f f i c i e n t to achieve both of these c r i t e r i a . I f a longer period than 5 minutes i s allowed for the balance pressure to be determined, a lower pressure w i l l be recorded since the water within the plant has more time to equilibrate throughout the tissues. No true equilibrium can be found, however, since the membrane and osmotic properties of the shoot change when stress i s applied (Tyree and Hammel, 1972). The time period for determination of balance pressure i s therefore arbitrary (Cheung, e_t a l . , 1975). After the f i n a l balance pressure was recorded the shoot was weighed and placed i n a drying oven for at least 48 hours to obtain the dry weight. In other studies (Tyree et a l . , 1973; Cheung et a l . , 1975), d i f f i c u l t i e s have arisen due to the long time periods over which the plant material i n the pressure chamber are exposed to pure nitrogen gas. This long exposure may result i n premature death of the c e l l s which was noted by the appearance of greenish sap on the cut surface and a sudden drop i n balance pressure. These studies indicated that by maintaining a p a r t i a l pressure of 2 to 3 atm. of compressed a i r within the chamber, the seedlings could survive for more than 10 hours. In this study, pure nitrogen gas was used since the design of the pressure chamber allowed for only one gas l i n e . However, none of the - 11 -diagnostic signs described above were observed during my experiment. Temperature control was also not possible due to the design of the apparatus. Fluctuating temperatures which result from the increase and decrease i n pressure have been measured by Puritch and Turner (1973), but i t i s not known how this fluctuation effects the as measured by the pressure chamber (Ritchie and Hinckley, 1975). Results and Discussion Figure 4 graphs the pressure-volume relationship for one of the seven shoots sampled i n this experiment. The curve closely resembles that reported for eastern hemlock (Tyree et a l . , 1973). Table I compares the values of TT , 1T, and V for eastern and western o p o hemlock. Tyree et al_. (1973) used a micro-catheter and an electronic balance to measure the percent water removed. Since the standard errors of the mean for the values of ft , n ., and V are similar for o 'p' o eastern and western hemlock as shown i n Table I, i t was concluded that the pressure chamber technique using tygon tubing as a co l l e c t i n g system i s as precise as the micro-catheter and the electronic balance. There are no available s t a t i s t i c a l methods for the determination of 1fp. The studies that have measured P-V curves (Tyree and Hammel, 1972; Tyree et a l . , 1973; Cheung et a l . , 1975), have simply used an ocular estimate to determine where the linear portion of the P-V curve sta r t s ; VAT=0 (M.T. Tyree, personal communication). I f the pressure chamber F i g u r e 4. P-V curve f o r one hemlock sho'ot P e r c e n t water removed Western hemlock Eastern hemlock Tyree et al.1973 : •. .v. • . mean -13.5 -16.5 standard error + 0.48 + 0.4 mean -17.1 not standard error + 0.59 reported relative amount of water in symplast;V . .me an 0.715 0. 767 standard error + 0.028 + 0.035 Table I. Comparison of statistics derived from P-V curves of shoots for western and eastern hemlock. - 14 -technique i s to be applied for diagnostic tests on nursery stock, a more accurate method for determining incipient plasmolysis w i l l have to be developed. There has been considerable controversy reported i n the l i t e r a t u r e concerning the measurement of the bulk e l a s t i c modulus (Hellkvist et a l . , 1974; Cheung et a l . , 1976). The e l a s t i c modulus i s the force per unit area associated with a change i n volume of tissue per unit volume (Hellkvist \et al_. , 1974) . The e l a s t i c modulus of c e l l walls within a shoot controls to a large extent the change i n VAT pressure (Tyree and Hammel, 1972). Tyree and Hammel found a linear relationship when log VAT was plotted against log c e l l volume. H e l l k v i s t et a l . (1974), however, found that i n Sitka spruce, a linear function was derived when log 1/VAT was plotted against volume expressed. As can be seen i n Figure 5 the data from the seven shoots i n this experiment supports the theory of H e l l k v i s t et a l . (1974). Bulk e l a s t i c modulus has been shown to have th i s same dependence with percent water removed (Hellkvist et a l . , 1974). The relationship between VAT, percent water removed, and the bulk e l a s t i c modulus i s discussed i n more depth when additional data i s presented i n Part I I . If the graph i n Figure 5 i s redrawn as log VAT against percent water removed as i s done i n Figure 6, i t i s possible to derive some useful parameters from the intercepts. When the regression l i n e i s extended to the y-axis the VAT pressure at f u l l turgor can be determined. The mean value for th i s VAT pressure for the sample of 7 shoots was 13.8 atm. - 15 -- 16 -- 17 -I f the r e g r e s s i o n l i n e i s extended to the x - a x i s (VAT=0.1 a t m . ) , the percent water removed a t approx imate ly i n c i p i e n t p l a s m o l y s i s can be determined. T h i s v a l u e i s near 1 5 . 5 percent f o r the 7 shoots sampled. Conf idence l i m i t s of these e x t r a p o l a t e d va lues are l a r g e which l i m i t s the p o s s i b l e d i a g n o s t i c u s e f u l n e s s of t h i s method to determine the percent water removed when VAT=0. \ - 18 -B. MEASUREMENT OF MATRIX POTENTIAL Introduction As discussed i n the previous s e c t i o n ^ ^ i n laboratory experiments i s composed of 3 components: 1 w " ' m It was stated e a r l i e r that the linear segment of the P-V curve contains only the.osmotic and matrix components of jf . In this experiment, these two components w i l l be separated and their r e l a t i v e importance assessed. The procedure follows that reported by Boyer (1967b) who measured the matrix potentials of rhododendron (Rhododendron roseum Rehd.), sunflower (Helianthus annus L.), and yew (Taxus cuspidata Sieb. and Z u c c ) . In his experiment, Boyer froze the shoot tissue at -20°C and thawed i t slowly. The freezing resulted i n the destruction of semi-permeable membranes. The pressure needed to bring the released c e l l sap to the cut surface of the shoot equals the osmotic potential of the sap at atmospheric pressure. A l l subsequent pressures are a function of matrix potential ( i b i d ) . Methods and Materials The 4 hemlock plug seedlings used i n this experiment were from the same seedlot and received the same c u l t u r a l treatment as those - 19 -used i n the p r e v i o u s exper iment . A f t e r a s e e d l i n g was removed from the r e f r i g e r a t o r , the shoot was submerged i n l i q u i d n i t r o g e n f o r 20 seconds and then a l lowed to thaw at room temperature . The shoot was severed a t the roo t c o l l a r , weighed on a M e t t l e r ba lance and p laced i n the p ressure chamber. -The procedure f o r r e c o r d i n g the P-V r e l a t i o n s h i p i s s i m i l a r to t h a t d e s c r i b e d p r e v i o u s l y except that i n i t i a l l y the p ressure a p p l i e d to expe l the c e l l sap (overpressure) was l i m i t e d to 3 atm. due to the l a r g e amounts of exuding c e l l sap. When the volume of c e l l sap decreased (between ba lance pressures of - 6 to - 9 atm.) the overpressure was i n c r e a s e d to 5 atm. At the end of the t e s t the shoot was weighed, p laced i n a d r y i n g oven f o r at l e a s t 48 hours , and then reweighed. R e s u l t s and D i s c u s s i o n F i g u r e 1 graphs the m a t r i x p o t e n t i a l a g a i n s t percent water removed. The curve i s drawn from a r e g r e s s i o n l i n e (r = - 0 . 9 0 6 ) c a l c u l a t e d from the m a t r i x p o t e n t i a l s of 4 shoots from 0 to 50 percent water removed. The l i n e i s e x t r a p o l a t e d towards the p o i n t J = - 1 8 . 9 atm. m (65.25% water removed) which i s the average v a l u e f o r the two s e e d l i n g s measured to t h i s percent water removed. The data corresponds c l o s e l y to B o y e r ' s (1967b) data f o r yew.which had a = - 2 . 0 atm. a t 50% water c o n t e n t , but i s l e s s , than the data f o r S i t k a spruce ( ^ = - 4 . 0 atm. a t 15% water removed) r e p o r t e d by H e l l k v i s t e_t al_. (1974) . Figure 7 . Matrix p e t e n t i a l f o r 4 hemlock shoots - 21 -Table I I compares t h e f f + l va lues de r i ved from the m 7 shoots i n the p rev ious experiment and the 7^ v a l u e s f o r the 4 shoots measured i n t h i s exper iment . ^ comprises a s m a l l but s i g n i f i c a n t p r o p o r t i o n of the ff + p o r t i o n of the P-V curve between 0 and 50 percent water removed. The p h y s i o l o g i c a l range of r e l a t i v e water contents f o r western hemlock has not been documented but i t c e r t a i n l y would not be l e s s than 50% water con ten t . The assumption tha t the l i n e a r p o r t i o n of the P-V curve i s composed p r i m a r i l y of Tf (Tyree and Hammel, 1972; H e l l k v i s t et a l . , 1974) , i s r e i n f o r c e d by the data presented h e r e . - 22 -Percent Water Removed (atm.) (atm.) X 100 0 -13.9 -0.67 4.8 10 -16.0 -0.82 5.1 20 -18.8 -1.01 5.4 30 -22.8 -1.31 5.7 40 -28.9 -1.87 6.5 50 -39,7 -3.28 8.3 (65.25) -91.5 -18.9 20. 7 Table I I . Relative importance of and j m i n the l i n e a r p o r t i o n of P-V curves f or Western hemlock. - 23 -MEASUREMENT OF P-V CURVES FOR ROOTS OF WESTERN HEMLOCK Introduction The pressure chamber technique has been applied only on a limited basis to measure the ^  of roots (DeRob, 1969; H e l l k v i s t et a l . , 1974; Gees et a l . , 1974). Comparison of the ^  for the shoot and root in shoot . . . portions of the same plant reveal that the j xn transpiring w plants i s more negative than the . r o o t (n eR 0o, 1969). In non-transpiring plants, however, the water potentials of the roots and shoots tend to be equal (Ibid.). Gees et a l . (1974) found that damaged roots had more negative than undisturbed roots and suggested w that the low water potential gradients reported by DeRoo for tobacco (Nicotiana tabacum L.) roots may have resulted from root damage during the experiment. To-date the pressure chamber has not been used to generate P-V curves for roots. Comparison of the P-V curves for roots and shoots would probably reveal some interesting differences i n their water status. Methods and Materials Western hemlock'plug seedlings used i n this experiment were from the same seedlot as those used i n the previous experiment and were brought to f u l l turgor i n the same manner. The seedlings were - 24 -removed from the refrigerator and the roots were soaked i n lukewarm water. The roots were massaged gently to remove the peat- vermiculite s o i l . Care was taken not to break or crush the roots. After removing the s o i l from the roots, they were covered with paper towels which absorbed the surface water. The seedlings were then severed at the root c o l l a r and the roots weighed on a Mettler balance. The roots were placed i n the pressure chamber and the i n i t i a l was recorded. The w procedure for c o l l e c t i n g the xylem sap follows that i n Part I-B with i n i t i a l overpressures of 3 atm. which were increased to 5 atm. between balance pressures of -6 to -9 atm. At the completion of the test the roots were weighed, dried for at least 48 hours, and then reweighed. Results and Discussion ^ The data from this experiment are not reported since i t was la t e r recognized that inaccuracies occurred due to incomplete s o i l removal from the dense plug root systems. I t i s important to note, however, that pressure-volume curves were generated from roots. This extension of the pressure chamber technique had not been reported previously i n the l i t e r a t u r e . Refinement of the pressure chamber technique for roots was achieved i n the experiment i n Part I I . - 25 -SUMMARY - PART I The preliminary tests conducted i n this section demonstrated that the pressure chamber i s a r e l i a b l e instrument to measure pressure-volume relationships for the shoots of hemlock seedlings. The data for western hemlock were found to be similar to that for eastern hemlock (Tyree et a l . , 1973). The standard errors of the means for the values of , TC , and V were similar for both species which o p o r demonstrated the r e l i a b i l i t y of the pressure chamber technique. VAT pressure was found to change as a logarithmic function of percent water removed, which reinforced the data presented by H e l l k v i s t et a l . (1974) for Sitka spruce. As reported i n previous studies (Tyree and Hammel, 1972; Boyer, 1967b), matrix potential comprises a small proportion of the linear segment of the P-V curve. The pressure chamber technique was found to generate pressure-volume curves for roots but unfortunately the data gathered i n this experiment were inaccurate due to incomplete s o i l removal. The preliminary results revealed, however, that the osmotic and turgor properties of root systems could be analyzed successfully using the pressure chamber technique. PART I I . EFFECT OF TRANSPLANTING AND DROUGHT STRESS ON THE WATER RELATIONS OF HEMLOCK SEEDLINGS I n t r o d u c t i o n In many n u r s e r i e s t r e e s e e d l i n g s are now being grown i n c o n t r o l l e d environment greenhouses which a l l o w f o r long growing seasons and optimum growing c o n d i t i o n s . I t i s u n c l e a r what advantages or d isadvantages t h i s p r o t e c t e d environment might confer on s e e d l i n g s which a re outp lanted on dry s i t e s . In a g r i c u l t u r a l s p e c i e s i t has been shown that p l a n t s grown i n the greenhouse are l e s s w e l l adapted to drought c o n d i t i o n s than a re those grown i n the f i e l d (McCree, 1974; M i l l a r et a l . , 1971; Thomas et a l . , 1976) . The most n o t a b l e d i f f e r e n c e i s s tomata l r e s i s t a n c e : the p l a n t s that were grown i n the f i e l d and thus had exper ienced a l t e r n a t e wet and dry p e r i o d s , were ab le to keep t h e i r stomates open at lower water p o t e n t i a l s than those grown i n the greenhouse and watered d a i l y . Kaufmann (1977a; 1977b) has observed the e f f e c t s of s o i l temperatures and d r y i n g c y c l e s on the water r e l a t i o n s and growth of P inus r a d i a t a D. Don, and has repor ted that a f t e r r e -w a t e r i n g , the p l a n t s sub jected to the d r y i n g c y c l e s had lower t r a n s p i r a t i o n r a t e s than s e e d l i n g s tha t were watered d a i l y . I t i s important to note tha t s e e d l i n g s exposed to d r y i n g c y c l e s have slower growth r a t e s immediately f o l l o w i n g r e - w a t e r i r i g , but at the end of the growing season o f t e n have more t o t a l growth ( M i l l e r , 1965; Kaufmann, 1977b) . - 27 -Since stomatal resistance i s a function of osmotic and turgor potentials, i t seemed l o g i c a l to measure the effects of drought stress using the pressure chamber technique as described i n Part I. In addition to the P-V curves which measure free energy gradients, plant resistances to water flow as measured by Farnum (1977) were calculated. Farnum divided the plant's resistances into 3 parts: stomatal, stem, and root resistance. By observing these resistances i t was thought that possible correlations between changes i n the osmotic and turgor properties and changes i n plant resistances might be i d e n t i f i e d . This information could possibly be incorporated into existing computer models to provide a better understanding of a seedling's reaction to drought stress (Farnum, personal communication). Methods and Materials 2-year-old plug hemlock seedlings were used i n this experiment. These seedlings were from a different seedlot than those used i n Part I. The seedlings were divided into three treatments. The f i r s t treatment (plug treatment) contained plug seedlings that were watered d a i l y . The second treatment (transplant treatment) contained plug seedlings that were transplanted into quart-sized p l a s t i c containers f i l l e d with sandy loam s o i l . These seedlings were watered daily for one month before their water properties were measured. The t h i r d treatment (drought stressed) was similar to the second treatment except following their transplanting, they were held at 10 atm. stress for two weeksJ Two - 28 -weeks are required to bring these seedlings to this stress l e v e l i n i t i a l l y so that the t o t a l time following transplanting was equal i n the trans-plant and the drought stressed treatments. The stress l e v e l i n the th i r d treatment was maintained by weighing the pots d a i l y , taking jr ' w measurements on a random selection of seedling's branches (this allowed for non-destructive sampling), and addition of measured amounts of water into the s o i l medium. A population of 100 seedlings was controlled i n t h i s manner, from which my sample of 20 seedlings was selected randomly. The procedure prior to measurement of the P-V curves corresponded to that described i n Part I, and again the roots and shoot from one seedling could not be measured since only one pressure chamber was available. The procedure for measurement of the stem and root resistances i s taken from Farnum (1977). The seedlings were watered u n t i l the potting medium was at or near saturation. The containers were then enclosed i n p l a s t i c bags which were sealed around the base of the stem. These p l a s t i c bags prevented soil-water evaporation during the transpiration measurements. The seedlings were placed i n a controlled environment growth chamber and were allowed to equilibrate for one hour. The pots were weighed at 30 minute intervals u n t i l a steady state transpiration rate was recorded. The seedling was then severed at the root c o l l a r and the f of f i r s t the shoot and then the root were w . measured using the pressure chamber. These measurements were recorded - 29 -within 2 minutes after the seedling was severed. Transpiration rates were altered by changing the humidity, wind speed, and l i g h t intensity. In order to prevent the v i s c o s i t y of water from influencing' the calculations of plant resistances, a l l measurements were corrected to 20°C. The hydraulic resistance from the root c o l l a r to the evaporation site s was computed by Farnum (1977) i n the following equation: R s Y shoot - root / w / w TR _3 where Rg i s the stem resistance (atm. sec. cm. ) and TR i s the trans-3 - 1 pi r a t i o n rate (cm. s. ). The basic assumption behind this calculation i s that the plant i s transpiring at a steady rate. This condition i s ensured by the experimental procedure. The root resistance i s computed as: roots TR R = : q = r r <-q L r _3 where R^  i s the root resistance (atm.cm.s.cm. ), q^ i s the uptake per 3 -1 unit root length (cm. s. ) and L i s the root length (cm.). The calculation of root length was made using the l i n e intercept method developed by Newman (1965). The equation used by Newman i s : NA L = 2H - 30 -where L i s the t o t a l l e n g t h of the roo t i n the f i e l d of area A and N i s the number of i n t e r s e c t i o n s between the r o o t s and random s t r a i g h t l i n e s of t o t a l l e n g t h H. The r o o t s were r i n s e d c l e a n of the s o i l medium and were cut i n t o 9 s e c t i o n s approx imate ly 1 cm. i n depth . The r o o t s from each s e c t i o n were spread out on a g r i d and the leng th was determined . The 9 lengths were summed to p rov ide the t o t a l roo t l e n g t h e s t i m a t e . As mentioned e a r l i e r , Farnum (1977) a l s o measured s tomata l r e s i s t a n c e . U n f o r t u n a t e l y , usab le s tomata l data were not c o l l e c t e d i n my experiment due to a c c i d e n t a l d e s t r u c t i o n of the shoots p r i o r to the measurement of l e a f a r e a . - 31 -Results II-A - Comparison of P-V curves from Parts I and I I Table I I I compares s t a t i s t i c s taken from P-V curves for plug shoots and roots from t h i s experiment with those sampled i n Part I. The shoots i n Part I I have larger f T both at f u l l turgor and at incipient plasmolysis. This difference could be a result of the v a r i a t i o n i n seedlot, different c u l t u r a l treatment prior to these experiments, or a change i n the basic water status i n the seedlings during the 8 weeks between the sampling dates (May to J u l y ) . The different values for the r e l a t i v e volume of symplast, V q , also are known to vary for the reasons just l i s t e d (Hellkvist et a l . , 1974). Comparison of the root s t a t i s t i c s i s not as straight forward as that for the shoots. As mentioned i n Part I there was an incomplete removal of s o i l p a r t i c l e s from the root systems before the P-V curves were measured. In Part I I more care was taken to remove the s o i l . I t i s f e l t that most of the change between the root s t a t i s t i c s seen i n Table I I I i s a result of the more complete removal of s o i l . Crushing or bending of the root systems were avoided, since this can result i n erroneous values of ^ t (Gees e_t a l . , 1975) . TT at f u l l turgor ;lto ^T*at i n c i p i e n t plasmo l y s i s ;7/p Relative volume of symplast;Vo Shoots -Part II -16.8 + 0.5 -20.3 + 0.7 0.610 + 0.03 Shoots -Part I -13.5 + 0.48 -17.1 + 0.59 0. 715 + 0.028 Roots -Part II -8.5 + 0.4 -14.8 +1.4 0.61 + 0.04 Roots -Part I -15.1 + 0.7 -18.2 + 01.53 0. 751 + 0.047 Table I I I . Comparison of s t a t i s t i c s for plug shoots and roots from Parts I and I I . - 33 -Results II-B - Comparison of P-V curves for Roots and Shoots Figure 8 compares the P-V curves for roots and shoots of plug seedlings. This graph can be compared with Figure 9 to show the difference between the roots measured i n Part I and those measured here. The shoots have much larger values of f£ and ^ than the roots. This could be a r e s u l t of greater concentrations of solutes i n the protoplasm of the c e l l s i n the shoots than i n those of the roots. Figure 8 shows that the roots lose a larger percentage of water while reaching inc i p i e n t plasmolysis at a lower stress l e v e l . The roots therefore appear to have a large amount of water that moves r e l a t i v e l y easily out of the surrounding tissues. The small osmotic potentials of the roots tend to reinforce t h i s assumption. I t i s interesting to note, however, that the shoot and the root have the same r e l a t i v e volume of symplast (61%) even though the tissue morphology i s vastly different between these two plant parts. As discussed previously i n Part I, the VAT can be calculated by subtracting the l i n e a r extrapolation of the P-V curve from the non-linear segment. This i s simply rearrangement of the equation V = ( V + T T ) + p I w • m to y - ( f + tr ) = p = VAT • w m Figure 8 . Comparison of P-V curves f o r shoots Percent water removed - 35 -The reciprocal nature of the ordinate values may deceive the reader since the absolute difference between the non-linear and linear segments i s much greater for the roots than for the shoots. If one looks, however, at the s t a t i s t i c s of *[fQ> i t i s seen that the shoots have the greater P; 16.8 versus 8.5 atm. respectively. This higher VAT pressure could be the resu l t of a higher proportion of dry matter i n the shoot as compared with the roots. The difference between *TT and "TT has been correlated with c e l l wall r i g i d i t y . O p o j Cheung et al_. (1975) stated that the difference between the osmotic potentials i s smaller i n c e l l s with more r i g i d walls. In this example, the shoots have a smaller difference (4.4 atm.) than the roots (6.7 atm.). This difference i n c e l l wall r i g i d i t y i s probably advantageous for each particular plant part: the shoot requires r i g i d c e l l walls for support and protection. The drop i n VAT and the loss of water i s controlled to a large extent by the c e l l wall e l a s t i c i t y . A discussion of this property i s presented separately below. - 36 -Results II-C - Comparison between Plug and Transplant Treatments Figure .9 compares the three treatments tested i n this experiment. The transplants' shoot and ff (-16.7 + 0.3 and -20.8 + 0.7, respectively) are not s i g n i f i c a n t l y different from the plug shoots' values (-16.8 + 0.5 and -20.3 + 0.7, respectively). The only s i g n i f i c a n t difference between the shoots of these two treatments i s the V q value (plugs = 0.61 + 0.03; transplant = 0.49 + 0.03). No s i g n i f i c a n t difference was found between the values fYQ> ^ J ^ , and V Q for the roots of the plugs and transplants. The V q value for the roots of these two treatments were 0.61 + 0.04 and 0.63 + 0.03, respectively. Figure 10 plots stem resistance against transpiration rate. A covariance analysis determined that the slopes of the transplant and plug regression l i n e s were not s i g n i f i c a n t l y different but the adjusted means were s i g n i f i c a n t l y different (ck = 0.05). Figure 11 graphs the root resistance as a function of uptake per unit root length. A covariance analysis determined that neither the slopes or the adjusted means were s i g n i f i c a n t l y different for the regression l i n e s of the plug and transplant treatments. •30.0 -25.0 -20.0 (atm.) -10.0 •5.0 Shoots Shoots Roots Roots X X >< X X X X X X X X X X X X A A / / / / / / / / / / / / / / / / / / / / / / X X X X X X X V. X \ X X -\ X \ X X •v. X >< X Plug Transplant Drought stressed Figure 9. Comparison of osmotic p o t e n t i a l s - 38 -e 10. Relationship between shoot hydraulic resistance and t r a n s p i r a t i o n rate f o r 3 treatments. <3 JL. I i—J.—Ui™4a^_^CT„.^^Jra...^,™Jra. A-—l__Jwi~JULi 3 A 5 6 7 8 9 1 0 2 3 4 5 6 7 « 9 10C 3 - 1 5 Transpiration rate (cm. sec. x 10 ) - 3 9 -Figure 11. Relationship between root hydraulic resistance and uptake per u n i t root length. i o o r q : uptake per u n i t root length (cm. sec. cm. x 10 ) - 40 -Results II-D - Comparison between Transplant and Drought Stressed Treatments In Figure 9 the differences i n fT and IT between transplant o p and stressed shoots can be seen c l e a r l y . The tf^ value for the stress and transplant shoots (-20.0 + 0.3 and -16.7 + 0.3, respectively) as well as the I t values (-25.6 + 0.6 and -20.8 + 0.5 respectively) are / s i g n i f i c a n t l y d ifferent ( = 0 . 0 5 ) . The r e l a t i v e volumes of the symplast were also s i g n i f i c a n t l y different ( 0 . 6 0 + 0.03 and 0.49 + 0.03, respectively). Figure 9 also shows the s t a t i s t i c s calculated from the P-V curves for the roots of these two treatments. Though no s i g n i f i c a n t differences exist i n the values of and IT* for the stress versus o p the transplant treatments (1^: - 1 0 . 9 + 0 . 8 1 and - 9 . 1 + 0.3; -17.7 + 3.2 and - 1 5 . 1 + 0.3, respectively) a trend i s apparent. The stressed samples tend to have greater values fortf^  and than the transplant samples. The V q for the stress and the transplant treatments were not s i g n i f i c a n t l y different ( 0 . 6 3 + 0.04 and 0.55 + 0.08, respectively). Figures 1 0 and 11' graph the stem and root resistances, respectively, for these two treatments. Analysis of covariance showed no si g n i f i c a n t differences i n either the slopes or the adjusted means of the regression l i n e s for these two treatments ( = 0 . 0 5 ) . - 41 -D i s c u s s i o n In t h i s experiment t r a n s p l a n t i n g western hemlock p lug s e e d l i n g s i n t o quart c o n t a i n e r s had very l i t t l e e f f e c t on the s e e d l i n g s ' water s t a t u s as measured one month a f t e r t r a n s p l a n t i n g . The on ly s i g n i f i c a n t change noted was the decrease i n V . The t r a n s p l a n t s e e d l i n g s had c o n s i d e r a b l e new roo t growth d u r i n g the 4 week p e r i o d betwen the a c t u a l t r a n s p l a n t i n g and the measurement of the h y d r a u l i c p r o p e r t i e s . The r e s u l t s i n d i c a t e that these new r o o t s have osmotic and tu rgor p r o p e r t i e s tha t are s i m i l a r to those of the suber i zed r o o t s found i n the p l u g s e e d l i n g s . The r e s u l t s do i n d i c a t e , however, that new root growth may e f f e c t root r e s i s t a n c e to water uptake . A c l o s e examinat ion of the 7 3 —I roo t r e s i s t a n c e s i n F i g u r e 11 r e v e a l s that f o r q^ ^ 10 x 10 cm. s e c . cm. the p lugs have the g r e a t e s t r e s i s t a n c e to water uptake w h i l e the t r a n s p l a n t s e e d l i n g s have the l e a s t . The s t r e s s e d s e e d l i n g s were observed J:o have l e s s new root growth than the t r a n s p l a n t e d s e e d l i n g s and t h e i r root r e s i s t a n c e s f o r the most par t f a l l between those of the t r a n s p l a n t s and p l u g s . The new root growth i n the t r a n s p l a n t and drought s t r e s s e d s e e d l i n g s would c o n t a i n l e s s s u b e r i n and c u t i n which p rov ide a b a r r i e r through which water passes s l o w l y (Cowan and M i l t h r o p e , 1968) . R e s i s t a n c e s of unsuber i zed r o o t s of l o b l o l l y p i n e (Pinus taeda L.) have been found to be almost one ten th those of suber i zed r o o t s (Kramer and B u l l o c k , 1966) . Va lues of q^ 10 x 10^ a r e i n d i c a t i v e of very low t r a n s p i r a t i o n r a t e s and the d i f f e r e n c e between the r e s i s t a n c e s - o f the - 42 -three treatments are smallest i n this range. The va r i a t i o n i n resistances between suberized and unsuberized roots becomes more evident at high transpiration rates where there i s a r e l a t i v e l y large f l u x of water through the roots. Figures 12 and 13 compare the shoot and root resistances, respectively, for western hemlock as measured i n my experiment with those for Douglas-fir (Pseudotsuga menziesii var. menziesii (Mirb.) Franco) as measured by Farnum (1977). The data for hemlock are considerably more scattered, which may be attributed to the va r i a t i o n i n size of the 2-0 plug seedlings. Farnum used 1-0 plug seedlings which have a more uniform growth habit. The data in ;Figure 13 indicate that hemlock seedlings have lower stem resistances than Douglas-fir seedlings. As mentioned previously, the calculation of root hydraulic resistance incorporates the uptake per unit root length. The differences i n magnitude for the abscissa values seen i n Figure 13 are due to root length and not to transpiration rate. The hemlock plug seedlings had a much larger t o t a l root length. This difference i n root length may be due to the different rooting characteristics as well as to the different age of the samples. The two week drought stress resulted i n a s i g n i f i c a n t change i n the water status of the seedlings. The changes i n TT , 'flf , and o p V Q described i n the results are integrated i n Figures 14 and 15 which compare the P-V curves for the transplant and stress treatments. - 43 -Figure 12 . Comparison between shoot hydraulic resistances f o r hemlock and Douglas-fir plug seedlings. ,.--1™-^™!., . . a — < . . . ' • . . . i . , , , , . - . , ,^ . . . , , . , . , - a — » X - . _ , ^ L ™ L , _ L . * L = . i » U 3 4 5 6 7 'C 9 10 2 3 4 5 . 6 7 8 9 , 3 - 1 , 5 , Transpiration rate (cm. sec. x 10 ) - 44 -1 3 . Comparison between root hydraulic resistances for hemlock and Douglas-fir plug seedlings. Figure 14. Comparison of P-V curves t o r shoots from transplant and drought stressed treatments. Figure 15. Comparison of P-V curves f o r roots from percent water removed The larger values of TT and ff associated with the drought stressed o 'p seedlings indicate an adaptation to drought conditions (Levitt, 1972). The increases i n fT* and *tV allow the seedlings to conserve water o p and continue growth at higher levels of stress. The dry weight fractions (dry weight/turgid weight) for both the shoots and the roots were s i g n i f i c a n t l y larger for the stress versus the transplant seedlings. The increase i n the dry weight f r a c t i o n i s also associated with drought hardiness (Petterson e_t a l . , 1957) . No test was conducted that d i r e c t l y measured the drought hardiness of the three treatments. L e v i t t (1972) points out the d i f f i c u l t y i n developing such an index of drought hardiness. The most useful test would have measured the percent survival after outplanting the seedlings i n a droughty environment. I t i s hypothesized that the drought stressed seedlings would have a higher s u r v i a l percentage due to the adaptations i n their osmotic, turgor, and dry weight properties. Values for TC, 7T > and V are known to be variable. These 'o p o properties change seasonally (Hellkvist et a l . , 1974), and with c u l t u r a l treatments as shown i n t h i s experiment. The e l a s t i c modulus of c e l l walls which controls the VAT pressure i s also thought to fluctuate though measurements of t h i s change have not been reported i n the l i t e r a t u r e (Tyree and Hammel, 1972). H e l l k v i s t e_t a_l. (1974) defined bulk e l a s t i c modulus of a tissue as the force per unit area associated with a change i n volume of tissue per unit volume, i . e . - 48 -where F i s the free water content defined as V - V F = ^ _§ E q > 4 V o where i s the volume expressed and V q i s the volume of symplast. Equation 3 can be rewritten to show small differences i n f i n i t e form as: P - P 1 2 K = ± ^ — Eq. 5 F - F 1 2 In t h i s study, the log VAT pressures for the shoots of one treatment were plotted against the percent water removed. A regression l i n e was calculated for this function for each of the treatments. These regression l i n e s could then be used to determine the K values for changes i n P of 1 arm. (Eq. 5). Figure 16 i s the graph of K versus F and K versus VAT for the plug shoots. This figure i s similar to that presented for Sitka spruce by H e l l k v i s t _et a_l. (1974) . The relationship between K and VAT was found to be constant for the shoots from Part I, the plug shoots from Part I I and the stress 1 shoots. The data for the transplant shoots did not form the lin e a r regression 2 .. , . function (r =0.58) that i s reported .in the l i t e r a t u r e (Hellkvist -et al.,1974). The relationship of K and F was very similar for the plug and stress shoots from Part I I , but i t was different for the shoots from Part I as i s shown i n Figure 17.. This difference i n Kfc values results i n a difference i n VAT values (Figure 18). V O Free water content Figure 16. Relationship between bulk e l a s t i c modulus and (a) free water content, and (b) Volume-averaged-turgor pressure for plug shoots. 800 600 Bulk E l a s t i c Modulus 400 K. t (atm.) 200 Figure 17. Comparison between bulk e l a s t i c modul from plug.shoots from Parts I and I I . Part I 1.00 0.95 Free water content 0.90 Figure 18. Comparison of VAT pressures f or plug shoots from Parts I and I I . - 52 -The p h y s i o l o g i c a l b a s i s f o r the changes i n K i s not known. From t h i s d a t a , i t can be concluded tha t the osmotic p o t e n t i a l of the symplast has no d i s c e r n i b l e e f f e c t s i n c e the r e l a t i o n s h i p between K f c and F i s s i m i l a r f o r the p lug and s t r e s s shoots form P a r t I I w h i l e the and ft' v a l u e s are s i g n i f i c a n t l y d i f f e r e n t (F igure 9 ) . k p The f u n c t i o n of K versus F may be s t r o n g l y i n f l u e n c e d by the V Q . The p l u g and s t r e s s s e e d l i n g s from P a r t I I had s i m i l a r V Q v a l u e s (0 .61 + 0 .03 and 0 .60 + 0 .03 r e s p e c t i v e l y ) w h i l e the p lug shoots from P a r t I had s i g n i f i c a n t l y d i f f e r e n t V Q v a l u e s (0 .72 + 0 . 0 3 ) . . Fu r ther exper imenta t ion w i l l have to show i f t h i s c o r r e l a t i o n between v a l u e s of K and V e x i s t s , t o - 53 -SUMMARY The experiment conducted i n P a r t I I showed that the water s t a t u s of hemlock s e e d l i n g s does not show s i g n i f i c a n t change one month a f t e r t r a n s p l a n t i n g . The two week drought s t r e s s of 10 atm. produced s i g n i f i c a n t changes i n "J^ and ff f o r the shoots . A s i m i l a r t rend was ev ident i n the data of the r o o t s but s i g n i f i c a n t changes were not r e c o r d e d . No s i g n i f i c a n t changes i n shoot or roo t h y d r a u l i c r e s i s t a n c e s were observed . The r e l a t i v e volume of the symplast appears to a f f e c t the b u l k e l a s t i c modu l i of the shoots but drought s t r e s s does not a l t e r t h i s p r o p e r t y . The r e s u l t s from these experiments i n d i c a t e tha t changes i n the osmotic and turgor p r o p e r t i e s of hemlock s e e d l i n g s r e s u l t i n g from drought s t r e s s can be s u c c e s s f u l l y monitored u s i n g the p ressure chamber. F u r t h e r exper imenta t ion should be conducted to determine what a d d i t i o n a l changes might occur a t more .severe s t r e s s l e v e l s ( e . g . 20 atm. and 30 a t m . ) . The r e l a t i o n between changes i n the osmotic and turgor p r o p e r t i e s and changes i n s tomata l r e s i s t a n c e and t r a n s p i r a t i o n r a t e should a l s o be i n v e s t i g a t e d . I n t e r - s p e c i f i c comparisons w i t h e c o l o g i c a l i m p l i c a t i o n s c o u l d a l s o be a s c e r t a i n e d u s i n g the p ressure chamber techn ique . The r e s u l t s from these experiments coupled w i t h the r e s u l t s from o u t p l a n t i n g t e s t s w i t h s t r e s s e d and uns t ressed s e e d l i n g s cou ld p rov ide the f o u n d a t i o n f o r s u p e r i o r t r e e p ropagat ion programs f o r droughty areas throughout the w o r l d . - 54 -BIBLIOGRAPHY Boyer, J.S. 1969. Measurement of the water status of plants. A. Rev. P l . Physiol. 19:351-354. , 1967a. Leaf water potentials measured with a pressure chamber. P l . Physiol. 42:133-137. . 1967b. Matric potentials of leaves. P l . Physiol. 42: 213-217. Cheung, Y.N.S., M.T. Tyree and J. Dainty. 1976. Some possible errors i n determining bulk e l a s t i c moduli and other parameters from pressure-volume curves of shoots and leaves. Can. J. Bot. 54:758-765. . 1975. Water r e l a t i o n parameters on single leaves obtained i n a pressure bomb and some ecological interpretations. Can. J. Bot. 53:1342-1346. Cowan, I.R. and F.L. Milthrope. 1968. Plant factors influencing the water status of plant tissues. In Water D e f i c i t s and Plant Growth. Vol. I. (ed. T.T. Kozlowski) pp. 137-193. DeRoo, H.C. 1969. Water stress gradients i n plants and soil - r o o t systems. Agron. J. 61:511-515. Dixon, H.H. 1914. Transpiration and the Ascent of Sap i n Plants. MacMillan, New York. Farnum, P. 1977. Post-planting water relations of container-grown seedlings. A mathematical model using f i n i t e elements. Ph.D. thesis. U. of Wash. College of Forest Resources. Gee, G.W., W. L i u , H. Oluang and B.E. Janes. 1974. Use of pressure bomb measurements to estimate root water potentials. Agron. J. 66:75-78. H e l l k v i s t , J . , G.P. Richards and P.G. Ja r v i s . 1974. V e r t i c a l gradients of water potential and tissue water relations i n Sitka spruce trees measured with the pressure chamber. J. Appl. Ecol. 11: 637-667. Kaufmann, M.R. 1977a. S o i l Temperature and Drought Effects on Growth of Monterey Pine. For. Sc i . 23; 3:317-325. . 1977b. S o i l temperature and drying cycle effects on water relations of Pinus radiata. Can.J. Bot. 55:2413-2418. - 55 -Kramer, P.J. and H.C. Bullock. 1966. Seasonal variations i n the proportions of suberized and unsuberized roots i n trees i n r e l a t i o n to the absorption of water. Am.J.Bot.53:200. L e v i t t , J. 1972. Responses of Plants to Environmental Stresses. Academic Press, New York and London. 697p. McCree, K.J. 1974. Changes i n the stomatal response characteristics of grain sorghum produced by water stress during growth. Crop S c i . 14:273-278. M i l l a r , A.A., W.R. Gardner and S.M. Goltz. 1971. Internal water stress and water transport i n seed onion plants. Agron. J . 63:779-784. M i l l e r , L.N. 1965. Changes i n r a d i o s e n s i t i v i t y of pine seedlings subjected to water stress during chronic gamma i r r a d i a t i o n . Health Physics 11:1653-1662. Newman, E.I. 1965. A method of estimating the t o t a l length of root i n a sample. J. Appl. Ecol. 2:139-145. Petterson, M.L.R., S.K.F. E l l i s , G.E.J. Gray and R.S.Smith. 1957. An inherent character of plant species of drier habitats. Nature, London. 180:698-699. Puritch, G.S. and Turner, J.A. 1973. Effects of pressure increase and release on temperature within a pressure chamber used to estimate plant water potential. J. Exp. Bot. 24:342-348. Ritchie, G..A. and T.M. Hinckley. 1975. The pressure chamber as an instrument for Ecological research. Adv. i n Ecol. Research 9:165-254. Salisbury, F.B. and C. Ross. Plant Physiology. 1969. Wadsworth Pub. Co. 747p. Scholander, P.F., H.T. Hammel, E.P. Bradstreet and E.A. Hemmingsen. 1965. Sap pressure i n vascular plants. Science, N.Y. 148: 339-46. Scholander, P.F., A.T. Hammel, E.A. Hemmingsen and E.D. Bradstreet. 1964. Hydrostatic pressure and osmotic potential i n leaves of mangroves and some other plants. Proc. Natn. Acad. S c i . USA, 52:119-25. Slatyer, R.O. 1967. Plant - Water Relationships. Academic Press. London and New York. 366p. Thomas, J.C, K.W. Brown and W.R. Jordan. 1976. Stomatal response to leaf water potential as affected by preconditioning water stress i n the f i e l d . Agron. J . 68:706-708. - 56 -Ty ree , M.T . , J . Dainty and M. Benn. 1973. The water r e l a t i o n s of hemlock (Tsuga c a n a d e n s i s ) . I. some e q u i l i b r i u m water r e l a t i o n s as measured by the pressure-bomb techn ique . Can. J . B o t . 51 :1471 -1480 . Ty ree , M.T. and Hammel, H.T. 1972. The measurement of the turgor p r e s s u r e and the water r e l a t i o n s of p l a n t s by the p r e s s u r e -bomb techn ique . J . Exp. B o t . 2 3 : 2 6 7 - 2 8 2 . 

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