Planta (Berl.) 91, 274--278 (1970)
Short Communication
Starch and Apparent Rate of Photosynthesis of Leaves of Lolium perenne Grown at Different Temperatures D. WILSON Grasslands Division, D.S.I.R., Palmerston North, New Zealand Received December 18, 1969 Summary. Leaves from Lolium perenne plants grown at day/night temperatures of 15~ ~ had slower rates of apparent photosynthesis at 15~ than leaves from plants grown at 250/20~ Electron microscopy showed a higher starch concentration in chloroplasts from low- compared with the higher temperature-grown plants. However, all differences in apparent photosynthesis were negatively associated with differences in size of mesophyll cells. It is suggested that the presence of starch per se had no effect on photosynthesis rate and that temperature during growth influences subsequent rates because leaves from the higher temperature have smaller cells and chloroplasts than those from the lower one.
Introduction I t is possible that starch accumulation in leaves m a y inhibit photosynthesis at saturating light levels (see review of Neales and Incoll, 1968), and mechanisms by which this could occur have been discussed (Warren Wilson, 1966; Neales and Incoll, 1968). However, although Warren Wilson (1.c.) found that slow rates of apparent photosynthesis in cold conditions were associated with high concentrations of sugar and starch, Haapala (1969) showed that build-up of starch in leaves of Stellaria media during prolonged illumination was without effect on photosynthesis. Clearly, changes in rates of photosynthesis with temperature during leaf growth may not be causally associated with starch accumulation but might result from parallel changes in some other rate-determining factor. I n L o l i u m perenne, small mesophyll cells seem to be advantageous for apparent light-saturated photosynthesis among leaves grown in similar environments (Wilson and Cooper, 1967, 1969a). Further, leaves grown in low temperatures (9 ~ or 15 ~ may have larger mesophyll cells than those grown in high (21 ~) and while this difference has not in general
Starch and Photosynthesis in Lolium
275
been f o u n d t o be followed b y t h e e x p e c t e d difference in p h o t o s y n t h e s i s , t h e r e a p p e a r s to be g e n o t y p e - e n v i r o n m e n t i n t e r a c t i o n s in this response (Wilson a n d Cooper, 1969 c). The p r e s e n t p a p e r i n v e s t i g a t e s the relationships b e t w e e n s t a r c h a c c u m u l a t i o n , a p p a r e n t p h o t o s y n t h e s i s , m e s o p h y l l cell a n d c h l o r o p l a s t size in L . perenne. Plants of Lolium perenne L. from Italy were grown singly in pots containing potting mixture, with six plants in each of two controlled environment cabinets. Day/night temperatures in one cabinet were 25~ ~ in the other, 15~ ~ Visible (400--700nm) light of 90W m -2 was supplied 12 hr daily by Philips 120W TLMF33 fluorescent tubes and Philips comptalux 150W tungsten lamps. The 6th young, fully expanded leaf on the main stem was detached from each plant 2 hr after beginning of the light period and rates of apparent lightsaturated photosynthesis of segments from the central part of a leaf determined manometrically (Wilson et al., 1969). Light intensity, from Philips 40OW HPLI~ mercury-vapour lamps, was 200 W m -2 (at 400--700 nm) and temperature 15~ After determination of rates of photosynthesis the leaf portions were sectioned and measurement made of mean mesophyll-cell size and mesophyll thickness (Wilson and Cooper, 1967). Leaf segments, proximal to those used above, were fixed at the time of first measurement of photosynthetic rate and embedded for subsequent thin sectioning, for examination of chloroplasts. The tissue was fixed for 2 hr in phosphate-buffered 3 % glutaraldehyde, pH 7.2, post fixed in 2 % buffered osmic acid, and dehydrated through a graded series of alcohols. Embedding in araldite followed passage through propylene oxide. Thin sections were cut of the same transverse leaf region as for cell-size and chloroplasts were measured and examined for starch in comparable mesophyll ceils. In thin sections, not all chloroplasts in any cell are cut medially; therefore, examinations were conducted on many chloroplasts and cells. For chloroplast-size estimates, the longest axis of 60--120 chloroplasts in each leaf was measured on photographs taken in a Philips EM-20O electron microscope. L a r g e differences in a m o u n t of s t a r c h in leaves e x i s t e d b e t w e e n t h e two t e m p e r a t u r e t r e a t m e n t s . M a n y s t a r c h grains were e v i d e n t in chlorop l a s t s of all m e s o p h y l l cells e x a m i n e d of leaves grown a t 15~ ~ whereas v e r y few could be d e t e c t e d in those grown a t 25~ ~ (Fig. 1). This effect of cold of increasing s t a r c h in leaves has been d o c u m e n t e d for o t h e r species ( W a r r e n Wilson, 1966 ; T a y l o r , 1968). L e a v e s p r o d u c e d in 15~ ~ also h a d slower r a t e s of a p p a r e n t photosynthesis a n d larger m e s o p h y l l cells t h a n those f r o m 25~ ~ (Fig. 2). This is similar to t h e r e a c t i o n of a n o t h e r L o l i u m g e n o t y p e o b s e r v e d p r e v i o u s l y (Wilson a n d Cooper, 1969e). F u r t h e r , the t e m p e r a t u r e effect on cell size a p p e a r e d to be p a r a l l e l e d b y a change in chloroplast diam e t e r ( 7 . 2 6 ~ 0 . 1 2 ~ a t 15~ ~ 6.55 =J=0.10 ~ a t 25~176 The d u a l effect of increasing s t a r c h a n d lowering a p p a r e n t p h o t o synthesis could, in t h e absence of other i n f o r m a t i o n , p r o v i d e a basis for suggesting t h a t a c c u m u l a t i o n of s t a r c h in t h e leaf m i g h t depress a s s i m i l a t i o n r a t e ( W a r r e n Wilson, 1966). ~ o w e v e r , this is clearly n o t
276
D. Wilson:
Fig. 1
Starch and Photosynthesis in Lolium 3 0 0 I-
_~ 2601. 2201" o ~ . ~ 180r
~, E 1001-
"~,
,
,'
,
,
x
,
277 ,
x, ",
',,
\
500 6;0 7;0 8;0 9~01d001100 Meen rnesophyllcell size (,~z) Fig. 2. Relationship between apparent rate of photosynthesis per unit volume of mesophyll and mean mesophyll-ccll cross-sectional area (cell size) among leaves of Lolium perenne grown at day/night temperatures of 15~ ~ (o) and 25o/20 ~ (o). Duncan's 5% LSR is shown (I). Data plotted on log log scale. Solid regression line: present data, log Y = -- 1.31 (~: 0.11) log X + 5.72. Broken regression line: calculated from data in Wilson and Cooper (1969b) where leaves grown at constant 15~ Log Y ~ - 1.64 ( • 0.09) log X + 6.71
a causal association since t h e relationship b e t w e e n a p p a r e n t photosynthesis a n d cell size is similar to t h a t f o u n d p r e v i o u s l y a m o n g L o l i u m leaves grown in a u n i f o r m e n v i r o n m e n t (Fig. 2). Reasons for t h e a p p a r e n t a d v a n t a g e of small cells for p h o t o s y n t h e s i s , where CO 2 is limtiing, h a v e been discussed elsewhere (Wilson a n d Cooper, 1969a), one simple e x p l a n a t i o n being t h e g r e a t e r cell-surface to v o l u m e r a t i o of small cells. Since chloroplast size also changes with t e m p e r a t u r e , it seems possible t h a t such an a d v a n t a g e m i g h t also exist w i t h small chloroplasts. Thus, t h e p r e s e n t results suggest t h a t h g h t - s a t u r a t e d p h o t o synthesis of L o l i u m perenne leaves is n o t affected b y t h e presence of starch, a t least a t t h e levels indicated. This agrees w i t h H a a p a l a ' s (1969) o b s e r v a t i o n t h a t there was no change in r a t e of p h o t o s y n t h e s i s of Stellaria media during both b u i l d u p a n d d e g r a d a t i o n of starch. Thanks are due to Mr. D. H. Hopcroft of the Applied Biochemistry Division, D.S.I.I~., Palmerston North for sectioning and photographing material for the chloroplast studies, Mr. K. I. Williamson of the same Division for critical discussions, Miss Elle Cornege, Grasslands Division, D.S.I.R. for technical assistance and Dr. K. J. Mitchell, Director, Plant Physiology Division, D.S.I.R., for controlled environment facilities.
Fig. 1. Mesophyll cells of a leaf grown at day/night temperatures of 15~ ~ (above) and 25o/20 ~ (below). Note presence of numberous starch grains (S) in chloroplasts from 15~ ~ regime. Stained with uranyl nitrate and lead citrate
278
D. Wilson: Starch and Photosynthesis in Lolium
References Haapala, H. : Photosynthesis and starch metabolism of chloroplasts during prolonged illumination. Planta (BEE.) 86, 259--266 (1969). Neales, T.F., Incoll, L. D. : The control of leaf photosynthetic rate by the level of assimilate concentration in the leaf: A review of the hypothesis. Bot. Rev. 24, 107--125 (1968). Taylor, A. O. : The influence of temperature and time of day on the level of readily fermentable carbohydrate in white clover leaves. N. Z. agric. Sci. 2, 172--173 (1968). Warren-Wilson, J.: An analysis of plant growth and its control in arctic environments. Ann. Bot., N.S. 30, 383--402 (1966). Wilson, D., Cooper, J . P . : Assimilation of Loloim in relation to leaf mesophyll. Nature (Lond.) 214, 989--992 (1967). - - - Effect of light intensity and CO2 on apparent photosynthesis and its relationship with leaf anatomy in genotypes of Lolium perenne L. New Phytol. 68, 627--644 (19699). - - - - Apparent photosynthesis and leaf characters in relation to leaf position and age, among contrasting Lolium genotypes. New Phytol. 68, 645--655 (1969b). - - - - Effect of temperature during growth on leaf anatomy and subsequent lightsaturated photosynthesis among contrasting Lolium genotypes. New Phytol. 68, 1115--1123 (1969c). - - T r e h a r n e , K . J . , Eagles, C.F., Jager, J. M. de: A manometric technique for determination of apparent photosynthesis of Lolium. J. exp. Bot. 20, 373--380 (1969). Dr. D. Wilson Grasslands Division D.S.I.R. Palmcrston North, New Zealand
Short Communication
Starch and Apparent Rate of Photosynthesis of Leaves of Lolium perenne Grown at Different Temperatures D. WILSON Grasslands Division, D.S.I.R., Palmerston North, New Zealand Received December 18, 1969 Summary. Leaves from Lolium perenne plants grown at day/night temperatures of 15~ ~ had slower rates of apparent photosynthesis at 15~ than leaves from plants grown at 250/20~ Electron microscopy showed a higher starch concentration in chloroplasts from low- compared with the higher temperature-grown plants. However, all differences in apparent photosynthesis were negatively associated with differences in size of mesophyll cells. It is suggested that the presence of starch per se had no effect on photosynthesis rate and that temperature during growth influences subsequent rates because leaves from the higher temperature have smaller cells and chloroplasts than those from the lower one.
Introduction I t is possible that starch accumulation in leaves m a y inhibit photosynthesis at saturating light levels (see review of Neales and Incoll, 1968), and mechanisms by which this could occur have been discussed (Warren Wilson, 1966; Neales and Incoll, 1968). However, although Warren Wilson (1.c.) found that slow rates of apparent photosynthesis in cold conditions were associated with high concentrations of sugar and starch, Haapala (1969) showed that build-up of starch in leaves of Stellaria media during prolonged illumination was without effect on photosynthesis. Clearly, changes in rates of photosynthesis with temperature during leaf growth may not be causally associated with starch accumulation but might result from parallel changes in some other rate-determining factor. I n L o l i u m perenne, small mesophyll cells seem to be advantageous for apparent light-saturated photosynthesis among leaves grown in similar environments (Wilson and Cooper, 1967, 1969a). Further, leaves grown in low temperatures (9 ~ or 15 ~ may have larger mesophyll cells than those grown in high (21 ~) and while this difference has not in general
Starch and Photosynthesis in Lolium
275
been f o u n d t o be followed b y t h e e x p e c t e d difference in p h o t o s y n t h e s i s , t h e r e a p p e a r s to be g e n o t y p e - e n v i r o n m e n t i n t e r a c t i o n s in this response (Wilson a n d Cooper, 1969 c). The p r e s e n t p a p e r i n v e s t i g a t e s the relationships b e t w e e n s t a r c h a c c u m u l a t i o n , a p p a r e n t p h o t o s y n t h e s i s , m e s o p h y l l cell a n d c h l o r o p l a s t size in L . perenne. Plants of Lolium perenne L. from Italy were grown singly in pots containing potting mixture, with six plants in each of two controlled environment cabinets. Day/night temperatures in one cabinet were 25~ ~ in the other, 15~ ~ Visible (400--700nm) light of 90W m -2 was supplied 12 hr daily by Philips 120W TLMF33 fluorescent tubes and Philips comptalux 150W tungsten lamps. The 6th young, fully expanded leaf on the main stem was detached from each plant 2 hr after beginning of the light period and rates of apparent lightsaturated photosynthesis of segments from the central part of a leaf determined manometrically (Wilson et al., 1969). Light intensity, from Philips 40OW HPLI~ mercury-vapour lamps, was 200 W m -2 (at 400--700 nm) and temperature 15~ After determination of rates of photosynthesis the leaf portions were sectioned and measurement made of mean mesophyll-cell size and mesophyll thickness (Wilson and Cooper, 1967). Leaf segments, proximal to those used above, were fixed at the time of first measurement of photosynthetic rate and embedded for subsequent thin sectioning, for examination of chloroplasts. The tissue was fixed for 2 hr in phosphate-buffered 3 % glutaraldehyde, pH 7.2, post fixed in 2 % buffered osmic acid, and dehydrated through a graded series of alcohols. Embedding in araldite followed passage through propylene oxide. Thin sections were cut of the same transverse leaf region as for cell-size and chloroplasts were measured and examined for starch in comparable mesophyll ceils. In thin sections, not all chloroplasts in any cell are cut medially; therefore, examinations were conducted on many chloroplasts and cells. For chloroplast-size estimates, the longest axis of 60--120 chloroplasts in each leaf was measured on photographs taken in a Philips EM-20O electron microscope. L a r g e differences in a m o u n t of s t a r c h in leaves e x i s t e d b e t w e e n t h e two t e m p e r a t u r e t r e a t m e n t s . M a n y s t a r c h grains were e v i d e n t in chlorop l a s t s of all m e s o p h y l l cells e x a m i n e d of leaves grown a t 15~ ~ whereas v e r y few could be d e t e c t e d in those grown a t 25~ ~ (Fig. 1). This effect of cold of increasing s t a r c h in leaves has been d o c u m e n t e d for o t h e r species ( W a r r e n Wilson, 1966 ; T a y l o r , 1968). L e a v e s p r o d u c e d in 15~ ~ also h a d slower r a t e s of a p p a r e n t photosynthesis a n d larger m e s o p h y l l cells t h a n those f r o m 25~ ~ (Fig. 2). This is similar to t h e r e a c t i o n of a n o t h e r L o l i u m g e n o t y p e o b s e r v e d p r e v i o u s l y (Wilson a n d Cooper, 1969e). F u r t h e r , the t e m p e r a t u r e effect on cell size a p p e a r e d to be p a r a l l e l e d b y a change in chloroplast diam e t e r ( 7 . 2 6 ~ 0 . 1 2 ~ a t 15~ ~ 6.55 =J=0.10 ~ a t 25~176 The d u a l effect of increasing s t a r c h a n d lowering a p p a r e n t p h o t o synthesis could, in t h e absence of other i n f o r m a t i o n , p r o v i d e a basis for suggesting t h a t a c c u m u l a t i o n of s t a r c h in t h e leaf m i g h t depress a s s i m i l a t i o n r a t e ( W a r r e n Wilson, 1966). ~ o w e v e r , this is clearly n o t
276
D. Wilson:
Fig. 1
Starch and Photosynthesis in Lolium 3 0 0 I-
_~ 2601. 2201" o ~ . ~ 180r
~, E 1001-
"~,
,
,'
,
,
x
,
277 ,
x, ",
',,
\
500 6;0 7;0 8;0 9~01d001100 Meen rnesophyllcell size (,~z) Fig. 2. Relationship between apparent rate of photosynthesis per unit volume of mesophyll and mean mesophyll-ccll cross-sectional area (cell size) among leaves of Lolium perenne grown at day/night temperatures of 15~ ~ (o) and 25o/20 ~ (o). Duncan's 5% LSR is shown (I). Data plotted on log log scale. Solid regression line: present data, log Y = -- 1.31 (~: 0.11) log X + 5.72. Broken regression line: calculated from data in Wilson and Cooper (1969b) where leaves grown at constant 15~ Log Y ~ - 1.64 ( • 0.09) log X + 6.71
a causal association since t h e relationship b e t w e e n a p p a r e n t photosynthesis a n d cell size is similar to t h a t f o u n d p r e v i o u s l y a m o n g L o l i u m leaves grown in a u n i f o r m e n v i r o n m e n t (Fig. 2). Reasons for t h e a p p a r e n t a d v a n t a g e of small cells for p h o t o s y n t h e s i s , where CO 2 is limtiing, h a v e been discussed elsewhere (Wilson a n d Cooper, 1969a), one simple e x p l a n a t i o n being t h e g r e a t e r cell-surface to v o l u m e r a t i o of small cells. Since chloroplast size also changes with t e m p e r a t u r e , it seems possible t h a t such an a d v a n t a g e m i g h t also exist w i t h small chloroplasts. Thus, t h e p r e s e n t results suggest t h a t h g h t - s a t u r a t e d p h o t o synthesis of L o l i u m perenne leaves is n o t affected b y t h e presence of starch, a t least a t t h e levels indicated. This agrees w i t h H a a p a l a ' s (1969) o b s e r v a t i o n t h a t there was no change in r a t e of p h o t o s y n t h e s i s of Stellaria media during both b u i l d u p a n d d e g r a d a t i o n of starch. Thanks are due to Mr. D. H. Hopcroft of the Applied Biochemistry Division, D.S.I.I~., Palmerston North for sectioning and photographing material for the chloroplast studies, Mr. K. I. Williamson of the same Division for critical discussions, Miss Elle Cornege, Grasslands Division, D.S.I.R. for technical assistance and Dr. K. J. Mitchell, Director, Plant Physiology Division, D.S.I.R., for controlled environment facilities.
Fig. 1. Mesophyll cells of a leaf grown at day/night temperatures of 15~ ~ (above) and 25o/20 ~ (below). Note presence of numberous starch grains (S) in chloroplasts from 15~ ~ regime. Stained with uranyl nitrate and lead citrate
278
D. Wilson: Starch and Photosynthesis in Lolium
References Haapala, H. : Photosynthesis and starch metabolism of chloroplasts during prolonged illumination. Planta (BEE.) 86, 259--266 (1969). Neales, T.F., Incoll, L. D. : The control of leaf photosynthetic rate by the level of assimilate concentration in the leaf: A review of the hypothesis. Bot. Rev. 24, 107--125 (1968). Taylor, A. O. : The influence of temperature and time of day on the level of readily fermentable carbohydrate in white clover leaves. N. Z. agric. Sci. 2, 172--173 (1968). Warren-Wilson, J.: An analysis of plant growth and its control in arctic environments. Ann. Bot., N.S. 30, 383--402 (1966). Wilson, D., Cooper, J . P . : Assimilation of Loloim in relation to leaf mesophyll. Nature (Lond.) 214, 989--992 (1967). - - - Effect of light intensity and CO2 on apparent photosynthesis and its relationship with leaf anatomy in genotypes of Lolium perenne L. New Phytol. 68, 627--644 (19699). - - - - Apparent photosynthesis and leaf characters in relation to leaf position and age, among contrasting Lolium genotypes. New Phytol. 68, 645--655 (1969b). - - - - Effect of temperature during growth on leaf anatomy and subsequent lightsaturated photosynthesis among contrasting Lolium genotypes. New Phytol. 68, 1115--1123 (1969c). - - T r e h a r n e , K . J . , Eagles, C.F., Jager, J. M. de: A manometric technique for determination of apparent photosynthesis of Lolium. J. exp. Bot. 20, 373--380 (1969). Dr. D. Wilson Grasslands Division D.S.I.R. Palmcrston North, New Zealand
