Seasonal changes of carbohydrates, lipids and nitrogen content in sun and shade leaves from four mediterranean evergreen sclerophylls

Environmental and Experimental Botany, Vol. 34, No. 2, pp. 12~140, 1994

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0098-8472(93)E0008-E

Pergamon

Copyright © 1994 Elsevier Sci. . . . . Ltd Printed in Great Britain. All rights reserved 0~9~?~8472]94 $6.00 + 0.00

S E A S O N A L C H A N G E S OF C A R B O H Y D R A T E S , LIPIDS A N D N I T R O G E N C O N T E N T IN S U N A N D SHADE LEAVES F R O M F O U R MEDITERRANEAN EVERGREEN SCLEROPHYLLS M. S. MELETIOU-CHRISTOU, S. RHIZOPOULOU and S. DIAMANTOGLOU Institute of General Botany, University of Athens, Panepistimiopolis, Athens 15784, Greece (Received 28 July 1993; accepted in revisedform 24 November 1993)

MELETIOU-CHRISTOUM. S., RHIZOPOULOU S. and DIAMANTOGLOUS. Seasonal changes of carbohydrates, lipids and nitrogen content in sun and shade leaves from four Mediterranean evergreen sclerophylls. ENVIRONMENTAL AND EXPERIMENTALBOTANY, 34, 129--140, 1994. Annual fluctuations of starch, soluble sugars, nitrogen, proteins, lipids and energy-content of storage substances were determined and compared in sun and shade leaves from four evergreen sclerophyll species: Arbutus unedo, Olea europaea, Pistacia lentiscus and Quercus coccifera. Sun leaves ofP. lentiscus and Q. coccifera contained higher amounts of starch compared to shade leaves; in A. unedo and O. europaea reciprocal results were obtained. The seasonal trends of soluble sugar content differed among species. Nitrogen content was high in young expanding leaves and decreased considerably during the growth period. Sun leaves contained higher amounts of proteins and lower amounts of soluble nitrogen when compared with shade leaves. The total lipid content was greater in sun leaves of P. lentiscus and Q. coccifera, whereas shade leaves of A. unedo and O. europaea contained higher amounts of total lipids at the beginning of the growing season. It is likely that these various accumulations in sun and shade leaves of these evergreen sclerophylls represent a species-specific response. Key words: Carbohydrates, lipids, nitrogen, sun and shade leaves.

INTRODUCTION

are in a different physiological state t h a n shade leaves, due to the c h a n g e d m e t a b o l i s m towards the a c c u m u l a t i o n of higher proportions o f lipids, IN n a t u r a l environments, most plants do not starch and soluble carbohydrates./a°! It has also experience continuous sunlight but, instead, experience frequent fluctuations in i r r a d i a n c e been r e p o r t e d that starch a c c u m u l a t i o n is a profrom full sun to w i t h i n - c a n o p y shading. (27! Thus, g r a m m e d process, inversely related to light intensity a n d possibly regulated by the same photoleaves exposed to full sunlight m a y exhibit a m o r p h o g e n e t i c controls that d e t e r m i n e leaf g r o w t h response which is different from that thickness a n d whole p l a n t morphology.(5i found in shade leaves that receive d a y l i g h t filtered t h r o u g h green leaves; a d a p t a t i o n s can be associ- However, the distribution of p h o t o s y n t h a t e s ated with morphological, physiological and ultrabetween starch a n d sugars varies in response to structural changes of the leaves./3°/However, sev- certain changes in e n v i r o n m e n t a l conditions. eral aspects of leaf shape a n d leaf position within Hence, when an e n v i r o n m e n t a l factor, such as the c a n o p y are likely to affect the w h o l e - p l a n t light or w a t e r becomes limiting, d e t r i m e n t a l energy c a p t u r e in sun versus shade. (2°/ Sun leaves effects of the less favorable condition m a y be mini129

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M . S . M E L E T I O U - C H R I S T O U et al.

mized by alterations in photosynthate partitioning./5'4~) It was reported that the a m o u n t of proteins found in sun leaves was higher than that in shade leaves, (2°) whereas shade leaves contained more soluble amino acids (~°! and chlorophyll. (37) Nevertheless, the utilization of nitrogen within a leaf canopy is a function of the competition between leaves as well as the altered partitioning within leaves. (~8) T h e Mediterranean climate is characterized by a prolonged dry summer and high solar irradiance. Mediterranean evergreen sclerophyll species represent the predominant life form with foliage longevity. T h e foliage of these species is non-randomly distributed with respect to azimuth angle; within each canopy layer, foliage azimuth and inclination angles are correlated. (4) These structures do not permit radiation to reach all leaves at the same intensity and quality. (23/ In regard to the species annual cycle, it is apparent that the accumulation and degradation of starch, soluble sugars, nitrogen and lipids are dependent on climatic conditions. (9 12) In a previous paper, water relations and chlorophyll and proline accumulation were investigated in sun and shade leaves of four Mediterranean evergreen sclerophylls: A'~butus unedo, Olea europaea, Pistacia lentiscus and Quercus coccifera. (37) The four species are major components of an East-Mediterranean ecosystem, grow under the same environmental conditions and exhibit large variation in leaf size, shape, structure and canopy form. (6'37'47)A step towards understanding the physiological behavior of sun and shade leaves of evergreen sclerophylls is to compare seasonal variations in starch, soluble sugars, nitrogen and lipid content between the two leaf types of the above-mentioned species. A secondary objective of the present study was to determine the energy content of storage substances in order to assess whether sun leaves accumulate more storage substances than shade leaves. MATERIALS AND METHODS

Plant material Sun and shade leaves of Arbutus unedo L., Olea europaea L. var. sylvestris (Miller) Lehr. or Olea europaea L. ssp. oleaster Hoffm. and Lind., Pistacia

lentiscus L. and Q uercus coccifera L. were used. Leaves were collected from shrubs that grow in a region near Athens (latitude 37057.5 ' , longitude 23048.0 ' , altitude ~ 4 0 0 m a.s.1.). Due to invariant values of plastochron ratio (about 1.04 + 0.07) for all species examined and throughout the year, (37) a section from the fifth up to the 10th leaf from branches of each species was selected for measurements. The leaves were collected during one year, on the 15th of each month, at the same hour of the day (0700-0800 hr) to avoid diurnal variations. (25) Sun leaves were collected from the top of the canopy where the photosynthetically active radiation is approximately 1800 #mol m -2 s l during spring (the main growth period). Shade leaves were collected from an average height of 0.5 m above the ground, where photosynthetically active radiation is approximately 400 pmol m 2 s-~ during spring. The plant material, packed into plastic bags, was brought to the laboratory in the bags, boiled (in water) for 10 min to achieve stabilization, and dried at 60°C to a constant weight; !9'~4)the dried material was then powdered, using a M F C mill [ J u n k e and Kunkel G M B H & Co (Germany)]. Values of precipitation and air temperature were obtained from a standard meteorological enclosure about 5.0 km distant from the research site (Fig. 1). Methods Soluble sugars were extracted from 1 g dry powdered material with acetone, and were determined colorimetrically according to the p h e n o l sulfuric acid method of Dubois et al. (~7) T h e absorbance was read at 490 nm using a Zeiss spectrophotometer. Reducing sugar content was determined colorimetrically using the method of Nelson as modified by Somogyi.(34'41) Glucose was used as a standard. The absorbance was read at 660 nm. Fructose was also determined colorimetrically. (38'49) Sucrose was calculated by the difference between total and reducing sugars, and glucose by the difference between reducing sugars and fructose. Quantitative determination of starch content was accomplished with the residue after the extraction of sugars, using the anthrone method.(31) T h e total nitrogen content was measured by a method (26) which was also used for the quantifi-

CARBOttYDRATES, LIPIDS AND N I T R O G E N IN SUN AND SHADE LEAVES

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Fro. 1. Climate diagram of the research site, referring to mean monthly temperature (closed squares) and mean monthly precipitation (closed cycles). The order of months is from March to February. Surface areas represent the arid (dotted) and the humid (shaded) periods.

cation of p r o t e i n - N after p r e c i p i t a t i o n of the proteins with trichloroacetic acid. !9) T h e difference between total-N a n d protein-N is indicated as soluble-N, c22iT h e protein fraction was calculated as described by D i a m a n t o g l o u et al. (I5) T h e ratio of protein to chlorophyll content was calculated from the values of protein content measured in the present study and the values of chlorophyll content published in a previous paper. ~37) T o t a l lipids were extracted from the dried p o w d e r e d m a t e r i a l with a chlorotbrm m e t h a n o l solution (2 : 1, v/v).is°i' T h e methylesters of the fatty acids were p r o d u c e d by saponification of the total lipids. (43/The qualitative a n d q u a n t i t a t i v e determ i n a t i o n of the tatty acid methylesters was performed by g a s - l i q u i d c h r o m a t o g r a p h y . '24'29! A Perkin E l m e r F11 gas c h r o m a t o g r a p h , e q u i p p e d with a flame ionization detector, was utilized. T h e stainless steel column h a d a length of 1.83 m, a d i a m e t e r of 0.63 cm and c o n t a i n e d 10% E G S S X on C h r o m o s o r b W . A W . D M C S 60 80 mesh. T h e column t e m p e r a t u r e was 180°C, and the injector and detector t e m p e r a t u r e 220°C. Nitrogen was the carrier gas, with a flow rate of 30 ml min 1. T h e methylester of p e n t a d e c a n o i c acid

131

(15:0) was used as an internal s t a n d a r d . T h e degree of u n s a t u r a t i o n is given as the ratio of the sum of the p o l y u n s a t u r a t e d fatty acids (18:2 + 18:3) to the s a t u r a t e d acids plus oleic acid. This ratio was selected because oleic acid shows the same a n n u a l fluctuations as the pred o m i n a t i n g s a t u r a t e d p a l m i t i c acid, in M e d i t e r r a n e a n p l a n t species.(12'13) T h e energy contents of the a b o v e - m e n t i o n e d storage substances (designated as ECS) were calculated and a d d e d . (~'3~) Values reported in the present study, given as m g per g of d r y weight, are the means of three replicates, ± S.E.M. In Figs 5, 6, 8 a n d 9 S.E.M.s are not i n d i c a t e d since the d a t a arise from calculations a n d not directly from measurements. S t u d e n t ' s t-tests were used ~35' to d e t e r m i n e differences in starch, soluble sugar, total nitrogen, and total lipid contents between sun a n d shade leaves. RESULTS

Starch content of the four evergreen sclerophyll species is given in Fig. 2. Sun leaves of P. lentiscus and Q. coccifera contained higher a m o u n t s of starch c o m p a r e d to shade leaves; the differences betwen sun and shade leaves were small in the case of Q. coccifera, b u t statistically significant (P < 0.001). I n A. unedo a n d O. europaea, reciprocal results were obtained. T h e highest values of starch a c c u m u l a t i o n were reached d u r i n g spring by shade leaves ofA. unedo and O. europaea, as well as by sun leaves of P. lentiscus. However, quite similar values of starch content ( ~ 2 0 mg g i d r y wt) were o b t a i n e d in sun leaves of A. unedo, O. europaea a n d Q. coccifera, whereas those of P. lentiscus were found to be two-fold higher. S h a d e leaves ofA. unedo and O. europaea exhibited similar values of starch content ( ~ 50 m g g i d r y wt). I n P. lentiscus, starch content was a r o u n d 30 mg g 1 d r y wt and the lowest values (10 mg g 1 d r y wt) were recorded in Q. coccifera. Soluble sugar content for the four species is given in Fig. 3. I n general, the highest a m o u n t s of sugars were found in A. unedo and the lowest in O. europaea a n d P. lentiscus. Seasonal variations of soluble sugars differed a m o n g species, b u t in c o m p a r i n g sun and shade leaves of each species, the differences were not substantial (P > 0.05 in

M . S . M E L E T I O U - C H R I S T O U et al.

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FIG. 2. Starch accumulation in sun (open squares) and shade (closed squares) leaves of four Mediterranean evergreen sclerophylls during the course of a year. S.E.M. values for A. unedo ranged from 0.2 to 4.2 mg g- 1, for O. europaea from 0.3 to 5.1 mg g- ~, for P. lentiscus from 0.2 to 5.0 mg g i and for Q. coccifera from 0.1 to 0.3 mg g-I, for n = 3.

most cases). Sucrose, glucose and fructose contents are given in Fig. 3 (insets). Sucrose predominated in almost all cases, i.e. between the two leaf types and a m o n g species. T h e highest amounts of fructose were found in A. unedo. The annual variations of total nitrogen (Fig. 4) in sun and shade leaves of all species were similar; young expanding leaves exhibited the highest values, and nitrogen content declined during the growth period. In general, sun leaves contained more nitrogen than shade leaves ( P < 0.001). T h e values of soluble nitrogen are given in Fig. 5. Shade leaves of O. europaea, P. lentiscus and Q. coccifera exhibited generally higher values of soluble nitrogen than sun leaves, whereas in A. unedo the differences between sun and shade leaves seemed negligible. Data for proteins (Fig. 6) showed a great similarity to those of total nitro-

gen. Sun and shade leaves differed with respect to the ratio of protein to chlorophyll content that was more pronounced in the case of sun leaves (Fig. 6, insets). Total lipid content is shown in Fig. 7. T h e amounts of total lipids were generally larger in sun leaves ofP. lentiscus and Q. coccifera (P < 0.05 in most cases). In contrast, shade leaves ofA. unedo compared to sun leaves showed higher values of total lipid content from March to M a y (P < 0.05) and those of O. europaea between M a r c h (P < 0.001) and April (P < 0.01), in coincidence with their growth period. Moreover, shade leaves of O. europaea exhibited the highest values of total lipids between October and December (P < 0.05). Sun and shade leaves of the four species contained the same fatty acids, namely lower fatty

CARBOHYDRATES, LIPIDS AND N I T R O G E N IN SUN AND SHADE LEAVES

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FIG. 3. Soluble sugar content in sun (open squares) and shade (closed squares) leaves of four Mediterranean evergreen sclerophylls during the course of a year. S.E.M. values for A. unedo ranged from 2.0 to 10.3 mg g - ' , for O. europaea from 0.3 to 2.5 mg g ', for P. lentiscus from 0.4 to 5.2 mg g ' and for Q. coccifera from 0.4 to 7.8 mg g - i , for n = 3. The sucrose (dotted bars), glucose (shaded bars) and fructose (closed bars) content is also shown in the insets. Due to the fact that soluble sugar concentrations differed substantially among the species, relative scales were used.

acids ( < 14:0), s a t u r a t e d fatty acids: myristic (14:0), p a l m i t i c (16:0) a n d stearic ( 18:0); unsatur a t e d fatty acids: palmitoleic ( 16:1 ), oleic (18:1), linoleic (18:2) a n d linolenic (18:3). I n spite of the same qualitative fatty acid composition, q u a n titative variations were observed which a p p e a r e d more related to climatic factors t h a n to leaf position in the canopy. T h e a n n u a l fluctuations of the degree o f u n s a t u r a t i o n (Fig. 8) were similar in sun a n d shade leaves of the four species examined; m i n i m a were recorded in s u m m e r a n d m a x i m a between N o v e m b e r a n d D e c e m b e r . T h e highest values of energy content of storage substances a m o n g all species e x a m i n e d were found in A. unedo (Fig. 9). I n t e r m e d i a t e values were o b t a i n e d in O. europaea, whereas P. lentiscus

and Q. coccifera exhibited the lowest values. G r e a t e r a m o u n t s of energy were a c c u m u l a t e d in sun leaves of P. lentiscus and Q. coccifera t h a n in shade leaves. I n the case of A. unedo a n d O. europaea, higher values were recorded in shade leaves d u r i n g spring and between O c t o b e r a n d December.

DISCUSSION T h e results are generally consistent with the hypothesis that sun leaves a c c u m u l a t e higher a m o u n t s of total nitrogen, proteins a n d lipids, when c o m p a r e d with shade leaves. I n contrast, the effect of light on starch a c c u m u l a t i o n at different c a n o p y levels varies a m o n g the four

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FIG. 4. Total nitrogen content in sun (open squares) and shade (closed squares) leaves of four Mediterranean evergreen sclerophylls, during the course of a year. S.E.M. values for A. unedoranged from 0.05 to 0.40 m g g ~, for O. europaeafrom 0.07 to 0.30 m g g - 1, for P. lentiscus from 0.07 to 0.20 mg g - i and for Q. coccifera from 0.07 to 0.20 mg g - l, for n = 3.

evergreen sclerophyll species (Fig. 2). It has been argued that decreasing light intensity leads to an increase in the a m o u n t of starch in the leaf. i4°/ Since starch synthesis seems to be a process possibly regulated by the same p h o t o m o r p h o g e n e t i c controls that d e t e r m i n e leaf thickness, (5! it seems likely that thick sun leaves would invest more in residual components than thin shade leaves. This is the case for A. unedo and O. europaea. However, sun leaves of P. lenliscus a n d Q. coccifera accumulated m o r e starch than shade leaves, in a g r e e m e n t with the a r g u m e n t of LICHWENTHALEI~.C3°/ I n M e d i t e r r a n e a n areas, a m a x i m u m in c a r b o n i n p u t occurs d u r i n g spring, i2'46/ when s t o m a t a l c o n d u c t a n c e of evergreen sclerophylls is high/a6'~7/ a n d whenever the highest values of starch a c c u m u l a t i o n were observed. T h e seasonal trends of soluble sugars differed a m o n g the four species. However, the highest

a m o u n t s of soluble sugars (Fig. 3) were found in A. unedo, which is the least deeply rooted species a m o n g the four species e x a m i n e d (3'39/ a n d which m a y possess the smallest mass of non-green respiring tissues. Also, A. unedo has the largest leaf surface (7) a m o n g these species, which m a y lead to a higher a c c u m u l a t i o n of soluble c a r b o h y d r a t e s in the leaves. In O. europaea and P. lentiscus, with the smallest leaf size, the lowest soluble sugar content was recorded. Nevertheless, c a r b o h y d r a t e s are a c c u m u l a t e d in the source leaves if the rate of photosynthesis exceeds the c a p a c i t y of the sinks to utilize the p h o t o s y n t h a t e for growth./42/ T o t a l nitrogen content in the two leaf types of the evergreen sclerophyll species decreased d u r ing the growth period (Fig. 4), a n d is c o m p a r a b l e to the values given for evergreen species in California. (19'2Ii Moreover, these species flower in late spring and are laden with fruit in a u t u m n (per-

CARBOHYDRATES, LIPIDS AND N I T R O G E N IN SUN AND SHADE LEAVES

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FIG. 5. Soluble nitrogen in sun (open squares) and shade (closed squares) leaves of A. unedo, O. europaea, P. lentiscus and Q. coccifera during the year.

sonal observation) indicating, therefore, nitrogen transport. High amounts of proteins, low amounts of soluble amino acids and low ratios of chlorophyll-to-protein content, reported as a trait of sun versus shade adapted plants, i2°'3°/ are in agreement with our results (Figs 5, 6 and Fig. 6 insets). However, in A. unedo, the differences in soluble nitrogen between sun and shade leaves were small despite the fact that proline was highly accumulated in shade leaves. '~7;' T h e results for total lipid content o f P . lentiscus and Q. coccifera sun leaves (Fig. 7) are similar to those of starch and in agreement with the finding that sun leaves accumulate more lipids than shade leaves.~3°i An increase in total lipids in O. europaea was observed in both leaf types during autumn, which coincides with fruit maturation. With regard to the fatty acid composition, the difference in light distribution within plant canopies

does not seem to affect the degree ofunsaturation (Fig. 8), although light stimulates the production of lipids rich in linolenic acid./~'3a'48/ It has been reported that low temperatures during winter were favorable for unsaturated fatty acid production, and that drought stress caused a change in the relative proportions of fatty acids, towards increased amounts of saturated fatty acids and oleic acid. (12'28'44'45)From the seasonal variation in the degree ofunsaturation (Fig. 8) it is clear that the adaptations cited above were found in both leaf types of Mediterranean evergreen sclerophylls investigated in the present study. T h e main portion of the energy content of storage substances (Fig. 9) is contributed by lipids/16/ and generally reflects differences in total lipids (Fig. 7). Sun leaves o f P . lentiscus and Q. coccifera invested more carbon in storage substances than shade leaves throughout the year, as stated in the

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Fro. 7. Total lipid content in sun (open squares) and shade (closed squares) leaves of A. unedo, O. europaea, P. lentiscus and Q. coccifera during the year. Vertical bars indicate S.E.M. values, for n = 3. 4

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FIG. 8. Degree of unsaturation (18:2+ 18:3/16:0+ 18:0+ 18:1) of sun (open squares) and shade (closed squares) leaves ofA. unedo, O. europaea, P. lentiscus and Q. coccifera during the year.

138

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FIG. 9. The energy content of storage substances (ECS) in sun (open bars) and shade (shaded bars) leaves of A. unedo, O. europaea, P. lentiscus and Q. coccifera during the year.

7.

8.

I n t r o d u c t i o n . However, shade leaves of A. unedo and O. europaea exhibited higher values of energy content of storage substances than sun leaves d u r ing spring a n d a u t u m n . It is presumed, therefore, that the response of M e d i t e r r a n e a n evergreen sclerophyll species to alterations in their environment, with respect to c a r b o n investment in storage substances (versus residual compounds) varies and that it is a species-specific response. This conclusion is similar to that of CHRISTODOULAKIS~6i who found that the structures p r o d u c e d in evergreen sclerophylls are far from being uniform although the plants growing in the same area experience much the same e n v i r o n m e n t a l conditions.

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