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Seasonal trends in energy contents and storage substances of the Mediterranean species Dittrichia viscosa and Thymelaea tartonraira
Environmental and Experimental Botany 39 (1998) 21 – 32
Seasonal trends in energy contents and storage substances of the Mediterranean species Dittrichia 6iscosa and Thymelaea tartonraira M.S. Meletiou-Christou *, G. P. Banilas, S. Diamantoglou Institute of General Botany, Uni6ersity of Athens, Panepistimiopolis, Athens 157 84, Greece Received 1 June 1997; accepted 2 September 1997
Abstract The annual fluctuations of soluble sugars, lipids, starch and nitrogen content were determined in leaves, stems and roots of Dittrichia 6iscosa and Thymelaea tartonraira. The energy content of storage substances was calculated. Soluble sugars increased in all parts of both species during the summer while starch and total lipids decreased. The annual variations of lipids, total and protein-nitrogen and the energy content of storage substances differed between the leaves of the two species. In contrast, starch and soluble sugars showed similar annual fluctuations in the leaves of both species. During the periods of growth foliar nitrogen concentration was correlated with foliar soluble sugar and starch concentrations, but relationships varied with time and between species. The differences in leaf lifespan between species affected only the leaf storage contents and not the stem and root contents. A sink storage system may be functioning between stems and roots of both examined species. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Carbohydrates; Dittrichia 6iscosa; Lipids; Nitrogen; Thymelaea tartonraira
1. Introduction The energy content of storage substances has been studied in Mediterranean evergreen (Diamantoglou and Kull, 1982; Larcher and Thomaser-Thin, 1988; Meletiou-Christou et al., 1994) and deciduous species (Diamantoglou et al., 1989), as well as in characteristic small dimorphic dwarf shrubs of the Greek scrub vegetation phry* Corresponding author. Tel.: + 30 1 7284503; fax: + 30 1 7234136; e-mail: [email protected]
gana (Meletiou-Christou et al., 1992). Although the plants investigated in most of the above studies grow under similar environmental conditions of the east Mediterranean ecosystem, different storage patterns have been reported among the different life forms and species (Diamantoglou and Kull, 1982; Larcher and Thomaser-Thin, 1988; Diamantoglou et al., 1989; Meletiou-Christou et al., 1992). Starch content, which is easily converted to soluble carbohydrates under stress conditions (Larcher and Thomaser-Thin, 1988; Amundson et al., 1993), appears to be much more
S0098-8472/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 9 8 - 8 4 7 2 ( 9 7 ) 0 0 0 2 2 - 1
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influenced by climatic conditions than lipid content; the annual storage cycle of lipids appears to be primarily subject to endogenous control, and only reacts to drastic environmental stressors such as drought and frost (Larcher and Thomaser-Thin, 1988). Furthermore, the role of lipids as true storage compounds in leaves and bark has been a subject of controversy (Hetherington et al., 1984; Diehl et al., 1993). Seasonally, phenological and developmental processes are of paramount importance to understand the cumulative partitioning of carbon into the various plant parts (Peoples and Gifford, 1990). In many cases, storage compounds can act as a buffer between asynchrony of resource supply and demand for resource use in growth (Bloom et al., 1985). As a result, different plant parts show different strategies in storage of such compounds (Diamantoglou and Meletiou-Christou, 1980; Meletiou-Christou et al., 1992). The two species that were investigated in the present study grow in the east Mediterranean region, experience the same environmental conditions, but exhibit different growth activities. Moreover, they seem to be more tolerant to the Mediterranean hot and dry summer than other species of the same area. Thymelaea tartonraira is an evergreen shrub (phanerophyte) that flowers in March, dominates phryganic ecosystems (Adamandiadou et al., 1978), but does not exhibit leaf dimorphism: i.e. this plant species, in contrast to other phrygana species, survives with the same leaves throughout the year. Dittrichia 6iscosa is a hemicryptophyte that flowers in August, during the drought period and produces epicuticular material that reduces cuticular transpiration (Stefanou and Manetas, 1995). The annual fluctuations of soluble sugars, starch, total lipids and nitrogen along the root – stem–leaf continuum of T. tartonraira and D. 6iscosa were investigated in the present study. The energy content of storage substances was calculated from the amounts measured. Results were compared and discussed in relation to storage patterns reported for Mediterranean evergreen and deciduous species as well as for phrygana species. The main objective of the present study
was to assess whether species with different growth activities but growing under the same environmental conditions exhibit differences in their strategies of storing compounds. A secondary objective was to estimate the importance of the fluctuations of storage compounds as factors contributing to the adaptive mechanisms produced under stress conditions. The hypothesis tested in the present study was the suggestion made by Chapin (1991) that starch accumulates in leaves of nitrogen-limited plants because there is sufficient nitrogen for continued carbon fixation but insufficient nitrogen to maintain normal growth. Since the growth activities differ in the two species, the source availabilities and sink demands might differ between the two species throughout the year by changing the positive relationship between foliar nitrogen and foliar carbohydrate reserves (Amundson et al., 1995), the annual fluctuations of storage compounds and the relationships between nitrogen and carbohydrate concentrations are expected to be related to growth periods.
2. Materials and methods
2.1. Plant material Plants of Dittrichia 6iscosa (L.) W. Greuter (syn. Inula 6iscosa (L.) Aiton) (Asteraceae) and Thymelaea tartonraira (L.) All. (Thymelaeaceae) were sampled between March 1991 and February 1992 from an open field, at the base of Mt. Hymettus, near Athens (latitude 38°57.5%, longitude 23°48.0%, altitude : 250 m a.s.l.). One whole plant of each species was sampled every sampling date. The collection was made at random. The plants were grown in calcareous and shallow rocky soil (Fouseki and Margaris, 1981). The height of the plants was 0.4–1.3 m for D. 6iscosa and 0.2–0.5 m for T. tartonraira. The root system of both species did penetrate the soil up to 0.4 m. In both species, sprouting started at the end of March 1991 and lasted until May. D. 6iscosa lost its leaves in December 1991 and new leaves appeared in April 1992.
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T. tartonraira is evergreen and new leaves were produced in March. Flowering occurred between August and October in D. 6iscosa, and between March and May in T. tartonraira. Plant material was collected at monthly intervals, early in the morning (07:00 to 08:00 h) to avoid diurnal variations (Kimura, 1969). Leaves, stems and roots, packed into plastic bags, were brought to the laboratory in a very short time (approx. 5 min), where (still in the bags) they were dipped into boiling water for 10 min to achieve stabilization (Diamantoglou and Kull, 1988; Diamantoglou et al., 1989) and dried at 60°C in an oven to a constant weight. The dried material was then powdered, using an MFC mill (Janke and Kunkel, Germany). Climatic data of the experimental period were obtained from a standard meteorological enclosure 5.0 km from the test area (Meletiou-Christou et al., 1994).
2.2. Methods Soluble sugars were extracted from 1 g of dry powdered material with acetone and were determined colorimetrically according to the phenol– sulphuric acid method of Dubois et al. (1956). Quantitative determination of starch content was accomplished with the residue after the extraction of sugars, using the anthrone method (McCready et al., 1950). Total nitrogen (N) content was measured by the method of Kjeldahl (1883), which was also used for quantitation of the protein-N after precipitation of total proteins with trichloroacetic acid (Diamantoglou and Kull, 1988). The difference between total-N and protein-N is indicated as soluble-N (Hollwarth, 1976). Total lipids were extracted from the dried powdered material with a chloroform – methanol solution (2:1, v/v) according to Bligh and Dyer (1959). The energy contents of the storage substances (ECS) were calculated and added (Diamantoglou and Kull, 1982; Merino et al., 1984). Determinations were repeated twice (n = 3). Values reported in the present study are given as proportion of dry weight (DW)9S.E.M. In Fig. 5, S.E.M. are not indicated, because the data were derived from calculations and not directly from measurements.
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2.3. Data analysis Soluble sugars, starch, lipid and nitrogen contents were subjected to analyses of variance (twoway analysis of variance (ANOVA) between species and among months and one-way ANOVA among months for the leaves within a species). Duncan’s multiple range test was used for comparison of means at 5% probability. The relationships between foliar nitrogen and carbohydrates in both species and the relationship between the variation of soluble sugars and total lipids in the leaves of T. tartonraira were estimated using linear regression analysis at pB 0.05.
3. Results The seasonal trends of soluble sugars in the leaves, stems and roots of D. 6iscosa and T. tartonraira are shown in Fig. 1. In all cases, soluble sugars increased during the growth period and the following drought period and declined thereafter, even if a secondary growth occurred as in T. tartonraira. The decline of soluble sugars in the leaves of D. 6iscosa was probably due to leaf senescing which occurred during this period. In the leaves of both species, the maximum values were reached in August, when the hemicryptophyte D. 6iscosa accumulated more soluble sugars than the evergreen T. tartonraira (Duncan’s test, p= 0.003). During the summer drought period, higher values of soluble sugar content were recorded in D. 6iscosa stems than in T. tartonraira (Duncan’s test, p B0.02), while in the roots the differences were generally not statistically significantly different (p\ 0.05). Starch content (Fig. 2) increased in the leaves of both species during their growth period. The maximum value was reached in June. During the following months, a continuous decrease of starch was observed in D. 6iscosa, until the dehiscence of the leaves, while in T. tartonraira starch decreased up to August and remained at a level of 30–35 g g − 1 DW during autumn and winter. The seasonal variations of the starch content were similar in stems and roots of both species. Positive linear relationships were found between starch concen-
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Fig. 1. Soluble sugar content in leaves, stems and roots of D. 6iscosa (closed symbols) and T. tartonraira (open symbols) during the course of a year. The order of months is from March up to February. S.E.M. values ranged from 0.0 to 0.2%, n = 3. Means followed by the same letters, within a plant part (capital letter for T. tartonraira, small letter for D. 6iscosa), are not statistically significantly different (p \0.05). *Statistically significant difference between species at p B0.05 within a given month and plant part.
tration in the stems (R 2 =0.63, p =0.000) and in the roots (R 2 =0.62, p =0.000) within and between the two species (R 2 =0.71, p = 0.000 for T. tartonraira and R 2 =0.74, p = 0.000 for D. 6iscosa). The maximum value was recorded in June. Thereafter, a decrease was observed until Septem-
ber; during autumn and winter the values of the starch content fluctuated between 25–30 g g − 1 DW in stems and 20–25 g g − 1 DW in roots. The total lipid content (Fig. 3) of T. tartonraira leaves showed its maximum values (65 mg g − 1 DW) from January to March. Thereafter, during
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Fig. 2. Starch accumulation in leaves, stems and roots of D. 6iscosa (closed symbols) and T. tartonraira (open symbols) during the course of a year. S.E.M. values ranged from 0.1 to 0.3%, n =3. Means followed by the same letters, within a plant part (capital letter for T. tartonraira, small letter for D. 6iscosa), are not statistically significantly different (p \0.05). *Significant difference between species at p B 0.05 within a given month and plant part.
the growing period and the following dry summer season, in contrast to soluble sugars, the total lipid content showed a continuous decrease to reach a minimum value (20 mg g − 1 DW) in August. From March to August a negative linear relationship (R 2 =0.80, p =0.015) was found be-
tween soluble sugar and lipid content in the leaves of T. tartonraira. During autumn and winter, a large accumulation of lipids was observed in T. tartonraira leaves (from September until February the values of total lipid concentrations were statistically significantly different at pB 0.05; see Fig.
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Fig. 3. Total lipid content in leaves, stems and roots of D. 6iscosa (closed symbols) and T. tartonraira (open symbols) during the course of a year. S.E.M. values ranged from 0.0 to 2.0 mg g − 1, n =3. Means followed by the same letters, within a plant part (capital letter for T. tartonraira, small letter for D. 6iscosa), are not statistically significantly different (p \ 0.05). *Significant difference between species at pB0.05 within a given month and plant part.
3). In the leaves of D. 6iscosa the seasonal fluctuations of total lipids were completely different from those of T. tartonraira. The total lipid concentrations in the leaves of D. 6iscosa were not correlated with those of T. tartonraira leaves (R 2 =0.054, p = 0.27). From April onwards,
young expanding leaves of D. 6iscosa accumulated lipids; the maximum value (45 mg g − 1 DW) was reached in June. Thereafter, during the summer drought period, total lipids decreased to spring values; the decrease was continued in senescing leaves until their dehiscence in November. The
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Fig. 4. Total nitrogen content in leaves, stems and roots of D. 6iscosa (closed symbols) and T. tartonraira (open symbols) during the course of a year. S.E.M. values ranged from 0.0 to 1.2 mg g − 1, n =3. Means followed by the same letters, within a plant part (capital letter for T. tartonraira, small letter for D. 6iscosa), are not statistically significantly different (p \0.05). *Statistically significant difference between species at pB 0.05 within a given month and plant part.
annual fluctuations of total lipids were similar in the stems and roots of both examined species both with a minimum during the summer. Positive linear relationships were found between lipid concentration in the stems (R 2 =0.70, p =0.000) and the roots (R 2 =0.28, p =0.001) within and be-
tween the two species (R 2 = 0.88, p= 0.000 for T. tartonraira and R 2 = 0.47, p= 0.000 for D. 6iscosa). Total-N and protein-N concentrations (Fig. 4) declined in young expanding leaves of D. 6iscosa. From March until June the relationships between
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Table 1 Relationships between foliar nitrogen (N) concentration (mg g−1) and foliar carbohydrate concentration (mg g−1) during the different periods of the year Plant species
Time period
Equation
R2
p
D. 6iscosa
March–June
Starch = 6.1112−0.1175N Sugars = 5.7639−0.0794N Starch = 4.1611−0.0357N Sugars = 2.5667+0.15714N Starch = 5.8332−0.1688N Sugars = 23.196−0.8201N
0.65 0.74 0.01 0.02 0.34 0.58
0.008* 0.003* 0.805 0.743 0.046* 0.004*
Starch = 0.94906+0.14897N Sugars =0.48176+0.17844N Starch=−0.5726+0.20806N Sugars =13.433−0.3093N Starch =2.4624+0.04626N Sugars =0.29521+0.30648N Starch =2.7669+0.02862N Sugars =4.9474−0.0621N Starch= 1.4828+0.09371N Sugars =−2.307+0.25695N
0.91 0.82 0.42 0.38 0.19 0.63 0.06 0.22 0.29 0.71
0.000* 0.000* 0.060 0.076 0.277 0.010* 0.551 0.192 0.130 0.004*
June–August August–November
T. tartonraira
March–June June–August August–October October–December December–February
*Relationships with pB0.05.
total-N and soluble sugars and between total-N and starch were negative (Table 1). On the other hand, nitrogen concentration increased in the leaves of T. tartonraira during the growth period and was positively correlated with foliar starch and soluble sugar concentrations (Table 1). Thereafter, from June until August, it remained unchanged in both species and was not correlated either with foliar starch or with soluble sugar concentration (Table 1). Total-N storage after leaf drop of D. 6iscosa (November through February) was different in the two species for stem and root tissues (Fig. 4). In early spring the concentration of soluble-N increased in all parts of both species. New leaves of D. 6iscosa attained higher proportions of soluble-N than the leaves of the evergreen T. tartonraira, which they maintained throughout their lifespan. An increase of soluble-N was observed in young expanding leaves of D. 6iscosa and a decrease in senescing leaves before their dehiscence. In the roots soluble-N increased in autumn in parallel with leaf senescence and decreased during the growth of new leaves. The energy content of storage substances (Fig. 5) mainly reflected variations of total lipids. In the hemicryptophyte D. 6iscosa the values ranged
from 1.1 to 3.1 kJ g − 1 DW, an increase was observed in young expanding leaves and the maximum value was recorded in June. During the following months, the energy content of storage substances decreased continuously until leaf senescence and defoliation. In T. tartonraira, which is evergreen, the energy content of storage substances ranged from 2.5 to 3.5 kJ g − 1 DW, the minimum values were recorded at the end of the summer drought period and the maximum ones during winter and early spring. Stems and roots, of both species, showed similar annual fluctuations of the energy content of storage substances. The minimum values were recorded in autumn and the maximum ones during winter and spring.
4. Discussion The increase of soluble sugars during the growth and the following drought period in the leaves of both examined species (Fig. 1) is in agreement with previous publications on Mediterranean evergreen and deciduous species (Diamantoglou and Meletiou-Christou, 1980, 1981; Diamantoglou et al., 1989). In contrast to dimor-
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Fig. 5. The energy content of storage substances (ECS) in D. 6iscosa (shaded bars) and T. tartonraira (open bars) during the year.
phic phrygana species (Meletiou-Christou et al., 1992), the leaves of T. tartonraira and D. 6iscosa exhibited only one summer peak of soluble sugars. During the summer, leaves and stems of the hemicryptophyte D. 6iscosa reached higher proportions of soluble sugars than leaves and stems of T. tartonraira. This difference was also found, in leaves and bark, between Mediterranean ever-
greens, which are considered to be drought tolerators, and deciduous species, which have intermediary characteristics between evergreen drought tolerators and deciduous drought avoiders (Diamantoglou and Meletiou-Christou, 1980; Diamantoglou et al., 1989). A uniform pattern of starch storage was exhibited by stems and roots of both species (Fig. 2).
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The same phenomenon was also reported for phrygana dimorphic species (Meletiou-Christou et al., 1992). Presumably, as in phrygana species, the shallow root systems of T. tartonraira and D. 6iscosa were exposed to the same environmental changes as the above-ground parts of the plants. In contrast the annual fluctuations of starch differed between shoots and roots of the deep-rooted Mediterranean evergreen sclerophylls (Mooney and Chu, 1974; Larcher and Thomaser-Thin, 1988). Starch was accumulated in all parts from March to June (Fig. 2), in agreement with the postulate that in Mediterranean areas a maximum in carbon input occurs during spring (Tenhunen et al., 1984) and suggesting that, during this main growth period (Rhizopoulou et al., 1991), sugar was produced in excess (source \ sink) and condensed to starch in order to overcome the stress of summer that follows. During the summer drought period, starch was depleted in all parts, as was also found in the leaves of Mediterranean evergreen (Diamantoglou and Meletiou-Christou, 1978, 1980; Larcher and Thomaser-Thin, 1988; Meletiou-Christou et al., 1994) and in the leaves, stems and roots of phrygana species (MeletiouChristou et al., 1992). The decrease of starch during summer was accompanied by a depletion of total lipids. In contrast, during the same period, osmotically active soluble sugars increased. Thus, water might be withheld during the period of water stress at the expense of reserves stored in the leaves as well as the roots (Amundson et al., 1993). Furthermore, it has been suggested that species with substantial reserves withstand periods of reduced carbon fixation, such as during the summer in the Mediterranean area (Tenhunen et al., 1984), by utilizing reserves for maintenance (Amundson et al., 1993). The difference in the seasonal change of the total lipid content between the leaves of T. tartonraira and D. 6iscosa (Fig. 3) support our hypothesis that the variation of storage compounds was positively related to growth periods. Our results were also found in Mediterranean evergreen and deciduous species (Diamantoglou and MeletiouChristou, 1979; Diamantoglou et al., 1989). T. tartonraira, the evergreen species, accumulated lipids in the autumn and winter and broke them
down during the growth and the following drought period, in contrast to soluble sugar fluctuation. D. 6iscosa accumulated lipids during the growth period and depleted them during summer drought. The nearly parallel fluctuations of total lipids in roots and stems of both examined species (Fig. 3) were also found in roots and stems of phrygana dimorphic species (Diamantoglou and Rhizopoulou, 1990; Meletiou-Christou et al., 1992). According to our hypothesis, the relationships between nitrogen and starch and between nitrogen and soluble sugars varied with species and time of year and were related to growth periods. The negative relationships that were found between nitrogen and starch and between nitrogen and soluble sugars (Table 1), during the growth of the leaves of D. 6iscosa, resulted from the decrease of nitrogen concentration and the increase of starch and soluble sugar concentrations. The decrease of nitrogen concentration during the growth period of the leaves of Mediterranean shrubs has been considered as a dilution effect (Field and Mooney, 1983). The positive relationships between foliar nitrogen and starch and between foliar nitrogen and soluble sugars from March up to June in the leaves of T. tartonraira (Table 1) resulted from the increase of nitrogen, starch and soluble sugar concentrations. Chapin (1991) suggests that starch accumulates in leaves of nitrogen-limited plants because there is sufficient nitrogen for continued carbon fixation but insufficient nitrogen to maintain normal growth. The results of Amundson et al. (1995) also support that hypothesis. During the period of drought stress neither starch nor sugar concentration was correlated with foliar nitrogen of both plants (Table 1) as was also found in the needles of Picea rubens during the cold hardening period (Amundson et al., 1995). From August until November, during leaf senescence of D. 6iscosa, foliage is exporting carbon and the negative relationships between nitrogen and starch and between nitrogen and soluble sugars (Table 1) resulted primarily from the decrease of carbohydrate concentrations. The energy content of storage substances (Fig. 5) in the leaves of D. 6iscosa and T. tartonraira was within the range of energy values reported for
M.S. Meletiou-Christou et al. / En6ironmental and Experimental Botany 39 (1998) 21–32
Mediterranean evergreen (Diamantoglou and Kull, 1982; Meletiou-Christou et al., 1994) and deciduous species (Diamantoglou et al., 1989) and lower than the values found in phrygana species (Meletiou-Christou et al., 1992). The different fluctuations of the energy content of storage substances between the leaves of the two examined species support the argument of Larcher and Thomaser-Thin (1988), that the seasonal changes in energy content are in intimate connection with the seasonal periodicity of growth activity, rather than that they directly respond to weather conditions. It is interesting that stems and roots of both species showed similar annual fluctuations of energy content of storage substances, in contrast to our hypothesis as stated in the Section 1. It seems that the differences in leaf lifespan did not affect the stem and root contents but only the leaf contents.
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Peoples, M.B., Gifford, R.M., 1990. Long-distance transport of carbon and nitrogen from sources to sinks in higher plants. In: Dennis, D.T., Turpin, D.H. (Eds.), Plant Physiology, Biochemistry and Molecular Biology. Longman, London, pp. 434 – 447. Rhizopoulou, S., Meletiou-Christou, M.S., Diamantoglou, S., 1991. Water relations for sun and shade leaves of four Mediterranean evergreen sclerophylls. J. Exp. Bot. 238, 627 –635. Stefanou, M., Manetas, Y., 1995. Allelopathic and water conserving functions of leaf epicuticular exudates in the Mediterranean shrub Dittrichia 6iscosa. Aust. J. Plant Physiol. 22, 755 – 759. Tenhunen, J.D., Meister, H.P., Caldwell, M.M., Lange, O.L., 1984. Environmental constraints on productivity of the Mediterranean shrub Quercus coccifera. Options Mediterr. 1, 33 – 53.
Seasonal trends in energy contents and storage substances of the Mediterranean species Dittrichia 6iscosa and Thymelaea tartonraira M.S. Meletiou-Christou *, G. P. Banilas, S. Diamantoglou Institute of General Botany, Uni6ersity of Athens, Panepistimiopolis, Athens 157 84, Greece Received 1 June 1997; accepted 2 September 1997
Abstract The annual fluctuations of soluble sugars, lipids, starch and nitrogen content were determined in leaves, stems and roots of Dittrichia 6iscosa and Thymelaea tartonraira. The energy content of storage substances was calculated. Soluble sugars increased in all parts of both species during the summer while starch and total lipids decreased. The annual variations of lipids, total and protein-nitrogen and the energy content of storage substances differed between the leaves of the two species. In contrast, starch and soluble sugars showed similar annual fluctuations in the leaves of both species. During the periods of growth foliar nitrogen concentration was correlated with foliar soluble sugar and starch concentrations, but relationships varied with time and between species. The differences in leaf lifespan between species affected only the leaf storage contents and not the stem and root contents. A sink storage system may be functioning between stems and roots of both examined species. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Carbohydrates; Dittrichia 6iscosa; Lipids; Nitrogen; Thymelaea tartonraira
1. Introduction The energy content of storage substances has been studied in Mediterranean evergreen (Diamantoglou and Kull, 1982; Larcher and Thomaser-Thin, 1988; Meletiou-Christou et al., 1994) and deciduous species (Diamantoglou et al., 1989), as well as in characteristic small dimorphic dwarf shrubs of the Greek scrub vegetation phry* Corresponding author. Tel.: + 30 1 7284503; fax: + 30 1 7234136; e-mail: [email protected]
gana (Meletiou-Christou et al., 1992). Although the plants investigated in most of the above studies grow under similar environmental conditions of the east Mediterranean ecosystem, different storage patterns have been reported among the different life forms and species (Diamantoglou and Kull, 1982; Larcher and Thomaser-Thin, 1988; Diamantoglou et al., 1989; Meletiou-Christou et al., 1992). Starch content, which is easily converted to soluble carbohydrates under stress conditions (Larcher and Thomaser-Thin, 1988; Amundson et al., 1993), appears to be much more
S0098-8472/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 9 8 - 8 4 7 2 ( 9 7 ) 0 0 0 2 2 - 1
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influenced by climatic conditions than lipid content; the annual storage cycle of lipids appears to be primarily subject to endogenous control, and only reacts to drastic environmental stressors such as drought and frost (Larcher and Thomaser-Thin, 1988). Furthermore, the role of lipids as true storage compounds in leaves and bark has been a subject of controversy (Hetherington et al., 1984; Diehl et al., 1993). Seasonally, phenological and developmental processes are of paramount importance to understand the cumulative partitioning of carbon into the various plant parts (Peoples and Gifford, 1990). In many cases, storage compounds can act as a buffer between asynchrony of resource supply and demand for resource use in growth (Bloom et al., 1985). As a result, different plant parts show different strategies in storage of such compounds (Diamantoglou and Meletiou-Christou, 1980; Meletiou-Christou et al., 1992). The two species that were investigated in the present study grow in the east Mediterranean region, experience the same environmental conditions, but exhibit different growth activities. Moreover, they seem to be more tolerant to the Mediterranean hot and dry summer than other species of the same area. Thymelaea tartonraira is an evergreen shrub (phanerophyte) that flowers in March, dominates phryganic ecosystems (Adamandiadou et al., 1978), but does not exhibit leaf dimorphism: i.e. this plant species, in contrast to other phrygana species, survives with the same leaves throughout the year. Dittrichia 6iscosa is a hemicryptophyte that flowers in August, during the drought period and produces epicuticular material that reduces cuticular transpiration (Stefanou and Manetas, 1995). The annual fluctuations of soluble sugars, starch, total lipids and nitrogen along the root – stem–leaf continuum of T. tartonraira and D. 6iscosa were investigated in the present study. The energy content of storage substances was calculated from the amounts measured. Results were compared and discussed in relation to storage patterns reported for Mediterranean evergreen and deciduous species as well as for phrygana species. The main objective of the present study
was to assess whether species with different growth activities but growing under the same environmental conditions exhibit differences in their strategies of storing compounds. A secondary objective was to estimate the importance of the fluctuations of storage compounds as factors contributing to the adaptive mechanisms produced under stress conditions. The hypothesis tested in the present study was the suggestion made by Chapin (1991) that starch accumulates in leaves of nitrogen-limited plants because there is sufficient nitrogen for continued carbon fixation but insufficient nitrogen to maintain normal growth. Since the growth activities differ in the two species, the source availabilities and sink demands might differ between the two species throughout the year by changing the positive relationship between foliar nitrogen and foliar carbohydrate reserves (Amundson et al., 1995), the annual fluctuations of storage compounds and the relationships between nitrogen and carbohydrate concentrations are expected to be related to growth periods.
2. Materials and methods
2.1. Plant material Plants of Dittrichia 6iscosa (L.) W. Greuter (syn. Inula 6iscosa (L.) Aiton) (Asteraceae) and Thymelaea tartonraira (L.) All. (Thymelaeaceae) were sampled between March 1991 and February 1992 from an open field, at the base of Mt. Hymettus, near Athens (latitude 38°57.5%, longitude 23°48.0%, altitude : 250 m a.s.l.). One whole plant of each species was sampled every sampling date. The collection was made at random. The plants were grown in calcareous and shallow rocky soil (Fouseki and Margaris, 1981). The height of the plants was 0.4–1.3 m for D. 6iscosa and 0.2–0.5 m for T. tartonraira. The root system of both species did penetrate the soil up to 0.4 m. In both species, sprouting started at the end of March 1991 and lasted until May. D. 6iscosa lost its leaves in December 1991 and new leaves appeared in April 1992.
M.S. Meletiou-Christou et al. / En6ironmental and Experimental Botany 39 (1998) 21–32
T. tartonraira is evergreen and new leaves were produced in March. Flowering occurred between August and October in D. 6iscosa, and between March and May in T. tartonraira. Plant material was collected at monthly intervals, early in the morning (07:00 to 08:00 h) to avoid diurnal variations (Kimura, 1969). Leaves, stems and roots, packed into plastic bags, were brought to the laboratory in a very short time (approx. 5 min), where (still in the bags) they were dipped into boiling water for 10 min to achieve stabilization (Diamantoglou and Kull, 1988; Diamantoglou et al., 1989) and dried at 60°C in an oven to a constant weight. The dried material was then powdered, using an MFC mill (Janke and Kunkel, Germany). Climatic data of the experimental period were obtained from a standard meteorological enclosure 5.0 km from the test area (Meletiou-Christou et al., 1994).
2.2. Methods Soluble sugars were extracted from 1 g of dry powdered material with acetone and were determined colorimetrically according to the phenol– sulphuric acid method of Dubois et al. (1956). Quantitative determination of starch content was accomplished with the residue after the extraction of sugars, using the anthrone method (McCready et al., 1950). Total nitrogen (N) content was measured by the method of Kjeldahl (1883), which was also used for quantitation of the protein-N after precipitation of total proteins with trichloroacetic acid (Diamantoglou and Kull, 1988). The difference between total-N and protein-N is indicated as soluble-N (Hollwarth, 1976). Total lipids were extracted from the dried powdered material with a chloroform – methanol solution (2:1, v/v) according to Bligh and Dyer (1959). The energy contents of the storage substances (ECS) were calculated and added (Diamantoglou and Kull, 1982; Merino et al., 1984). Determinations were repeated twice (n = 3). Values reported in the present study are given as proportion of dry weight (DW)9S.E.M. In Fig. 5, S.E.M. are not indicated, because the data were derived from calculations and not directly from measurements.
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2.3. Data analysis Soluble sugars, starch, lipid and nitrogen contents were subjected to analyses of variance (twoway analysis of variance (ANOVA) between species and among months and one-way ANOVA among months for the leaves within a species). Duncan’s multiple range test was used for comparison of means at 5% probability. The relationships between foliar nitrogen and carbohydrates in both species and the relationship between the variation of soluble sugars and total lipids in the leaves of T. tartonraira were estimated using linear regression analysis at pB 0.05.
3. Results The seasonal trends of soluble sugars in the leaves, stems and roots of D. 6iscosa and T. tartonraira are shown in Fig. 1. In all cases, soluble sugars increased during the growth period and the following drought period and declined thereafter, even if a secondary growth occurred as in T. tartonraira. The decline of soluble sugars in the leaves of D. 6iscosa was probably due to leaf senescing which occurred during this period. In the leaves of both species, the maximum values were reached in August, when the hemicryptophyte D. 6iscosa accumulated more soluble sugars than the evergreen T. tartonraira (Duncan’s test, p= 0.003). During the summer drought period, higher values of soluble sugar content were recorded in D. 6iscosa stems than in T. tartonraira (Duncan’s test, p B0.02), while in the roots the differences were generally not statistically significantly different (p\ 0.05). Starch content (Fig. 2) increased in the leaves of both species during their growth period. The maximum value was reached in June. During the following months, a continuous decrease of starch was observed in D. 6iscosa, until the dehiscence of the leaves, while in T. tartonraira starch decreased up to August and remained at a level of 30–35 g g − 1 DW during autumn and winter. The seasonal variations of the starch content were similar in stems and roots of both species. Positive linear relationships were found between starch concen-
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Fig. 1. Soluble sugar content in leaves, stems and roots of D. 6iscosa (closed symbols) and T. tartonraira (open symbols) during the course of a year. The order of months is from March up to February. S.E.M. values ranged from 0.0 to 0.2%, n = 3. Means followed by the same letters, within a plant part (capital letter for T. tartonraira, small letter for D. 6iscosa), are not statistically significantly different (p \0.05). *Statistically significant difference between species at p B0.05 within a given month and plant part.
tration in the stems (R 2 =0.63, p =0.000) and in the roots (R 2 =0.62, p =0.000) within and between the two species (R 2 =0.71, p = 0.000 for T. tartonraira and R 2 =0.74, p = 0.000 for D. 6iscosa). The maximum value was recorded in June. Thereafter, a decrease was observed until Septem-
ber; during autumn and winter the values of the starch content fluctuated between 25–30 g g − 1 DW in stems and 20–25 g g − 1 DW in roots. The total lipid content (Fig. 3) of T. tartonraira leaves showed its maximum values (65 mg g − 1 DW) from January to March. Thereafter, during
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Fig. 2. Starch accumulation in leaves, stems and roots of D. 6iscosa (closed symbols) and T. tartonraira (open symbols) during the course of a year. S.E.M. values ranged from 0.1 to 0.3%, n =3. Means followed by the same letters, within a plant part (capital letter for T. tartonraira, small letter for D. 6iscosa), are not statistically significantly different (p \0.05). *Significant difference between species at p B 0.05 within a given month and plant part.
the growing period and the following dry summer season, in contrast to soluble sugars, the total lipid content showed a continuous decrease to reach a minimum value (20 mg g − 1 DW) in August. From March to August a negative linear relationship (R 2 =0.80, p =0.015) was found be-
tween soluble sugar and lipid content in the leaves of T. tartonraira. During autumn and winter, a large accumulation of lipids was observed in T. tartonraira leaves (from September until February the values of total lipid concentrations were statistically significantly different at pB 0.05; see Fig.
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Fig. 3. Total lipid content in leaves, stems and roots of D. 6iscosa (closed symbols) and T. tartonraira (open symbols) during the course of a year. S.E.M. values ranged from 0.0 to 2.0 mg g − 1, n =3. Means followed by the same letters, within a plant part (capital letter for T. tartonraira, small letter for D. 6iscosa), are not statistically significantly different (p \ 0.05). *Significant difference between species at pB0.05 within a given month and plant part.
3). In the leaves of D. 6iscosa the seasonal fluctuations of total lipids were completely different from those of T. tartonraira. The total lipid concentrations in the leaves of D. 6iscosa were not correlated with those of T. tartonraira leaves (R 2 =0.054, p = 0.27). From April onwards,
young expanding leaves of D. 6iscosa accumulated lipids; the maximum value (45 mg g − 1 DW) was reached in June. Thereafter, during the summer drought period, total lipids decreased to spring values; the decrease was continued in senescing leaves until their dehiscence in November. The
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Fig. 4. Total nitrogen content in leaves, stems and roots of D. 6iscosa (closed symbols) and T. tartonraira (open symbols) during the course of a year. S.E.M. values ranged from 0.0 to 1.2 mg g − 1, n =3. Means followed by the same letters, within a plant part (capital letter for T. tartonraira, small letter for D. 6iscosa), are not statistically significantly different (p \0.05). *Statistically significant difference between species at pB 0.05 within a given month and plant part.
annual fluctuations of total lipids were similar in the stems and roots of both examined species both with a minimum during the summer. Positive linear relationships were found between lipid concentration in the stems (R 2 =0.70, p =0.000) and the roots (R 2 =0.28, p =0.001) within and be-
tween the two species (R 2 = 0.88, p= 0.000 for T. tartonraira and R 2 = 0.47, p= 0.000 for D. 6iscosa). Total-N and protein-N concentrations (Fig. 4) declined in young expanding leaves of D. 6iscosa. From March until June the relationships between
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Table 1 Relationships between foliar nitrogen (N) concentration (mg g−1) and foliar carbohydrate concentration (mg g−1) during the different periods of the year Plant species
Time period
Equation
R2
p
D. 6iscosa
March–June
Starch = 6.1112−0.1175N Sugars = 5.7639−0.0794N Starch = 4.1611−0.0357N Sugars = 2.5667+0.15714N Starch = 5.8332−0.1688N Sugars = 23.196−0.8201N
0.65 0.74 0.01 0.02 0.34 0.58
0.008* 0.003* 0.805 0.743 0.046* 0.004*
Starch = 0.94906+0.14897N Sugars =0.48176+0.17844N Starch=−0.5726+0.20806N Sugars =13.433−0.3093N Starch =2.4624+0.04626N Sugars =0.29521+0.30648N Starch =2.7669+0.02862N Sugars =4.9474−0.0621N Starch= 1.4828+0.09371N Sugars =−2.307+0.25695N
0.91 0.82 0.42 0.38 0.19 0.63 0.06 0.22 0.29 0.71
0.000* 0.000* 0.060 0.076 0.277 0.010* 0.551 0.192 0.130 0.004*
June–August August–November
T. tartonraira
March–June June–August August–October October–December December–February
*Relationships with pB0.05.
total-N and soluble sugars and between total-N and starch were negative (Table 1). On the other hand, nitrogen concentration increased in the leaves of T. tartonraira during the growth period and was positively correlated with foliar starch and soluble sugar concentrations (Table 1). Thereafter, from June until August, it remained unchanged in both species and was not correlated either with foliar starch or with soluble sugar concentration (Table 1). Total-N storage after leaf drop of D. 6iscosa (November through February) was different in the two species for stem and root tissues (Fig. 4). In early spring the concentration of soluble-N increased in all parts of both species. New leaves of D. 6iscosa attained higher proportions of soluble-N than the leaves of the evergreen T. tartonraira, which they maintained throughout their lifespan. An increase of soluble-N was observed in young expanding leaves of D. 6iscosa and a decrease in senescing leaves before their dehiscence. In the roots soluble-N increased in autumn in parallel with leaf senescence and decreased during the growth of new leaves. The energy content of storage substances (Fig. 5) mainly reflected variations of total lipids. In the hemicryptophyte D. 6iscosa the values ranged
from 1.1 to 3.1 kJ g − 1 DW, an increase was observed in young expanding leaves and the maximum value was recorded in June. During the following months, the energy content of storage substances decreased continuously until leaf senescence and defoliation. In T. tartonraira, which is evergreen, the energy content of storage substances ranged from 2.5 to 3.5 kJ g − 1 DW, the minimum values were recorded at the end of the summer drought period and the maximum ones during winter and early spring. Stems and roots, of both species, showed similar annual fluctuations of the energy content of storage substances. The minimum values were recorded in autumn and the maximum ones during winter and spring.
4. Discussion The increase of soluble sugars during the growth and the following drought period in the leaves of both examined species (Fig. 1) is in agreement with previous publications on Mediterranean evergreen and deciduous species (Diamantoglou and Meletiou-Christou, 1980, 1981; Diamantoglou et al., 1989). In contrast to dimor-
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Fig. 5. The energy content of storage substances (ECS) in D. 6iscosa (shaded bars) and T. tartonraira (open bars) during the year.
phic phrygana species (Meletiou-Christou et al., 1992), the leaves of T. tartonraira and D. 6iscosa exhibited only one summer peak of soluble sugars. During the summer, leaves and stems of the hemicryptophyte D. 6iscosa reached higher proportions of soluble sugars than leaves and stems of T. tartonraira. This difference was also found, in leaves and bark, between Mediterranean ever-
greens, which are considered to be drought tolerators, and deciduous species, which have intermediary characteristics between evergreen drought tolerators and deciduous drought avoiders (Diamantoglou and Meletiou-Christou, 1980; Diamantoglou et al., 1989). A uniform pattern of starch storage was exhibited by stems and roots of both species (Fig. 2).
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The same phenomenon was also reported for phrygana dimorphic species (Meletiou-Christou et al., 1992). Presumably, as in phrygana species, the shallow root systems of T. tartonraira and D. 6iscosa were exposed to the same environmental changes as the above-ground parts of the plants. In contrast the annual fluctuations of starch differed between shoots and roots of the deep-rooted Mediterranean evergreen sclerophylls (Mooney and Chu, 1974; Larcher and Thomaser-Thin, 1988). Starch was accumulated in all parts from March to June (Fig. 2), in agreement with the postulate that in Mediterranean areas a maximum in carbon input occurs during spring (Tenhunen et al., 1984) and suggesting that, during this main growth period (Rhizopoulou et al., 1991), sugar was produced in excess (source \ sink) and condensed to starch in order to overcome the stress of summer that follows. During the summer drought period, starch was depleted in all parts, as was also found in the leaves of Mediterranean evergreen (Diamantoglou and Meletiou-Christou, 1978, 1980; Larcher and Thomaser-Thin, 1988; Meletiou-Christou et al., 1994) and in the leaves, stems and roots of phrygana species (MeletiouChristou et al., 1992). The decrease of starch during summer was accompanied by a depletion of total lipids. In contrast, during the same period, osmotically active soluble sugars increased. Thus, water might be withheld during the period of water stress at the expense of reserves stored in the leaves as well as the roots (Amundson et al., 1993). Furthermore, it has been suggested that species with substantial reserves withstand periods of reduced carbon fixation, such as during the summer in the Mediterranean area (Tenhunen et al., 1984), by utilizing reserves for maintenance (Amundson et al., 1993). The difference in the seasonal change of the total lipid content between the leaves of T. tartonraira and D. 6iscosa (Fig. 3) support our hypothesis that the variation of storage compounds was positively related to growth periods. Our results were also found in Mediterranean evergreen and deciduous species (Diamantoglou and MeletiouChristou, 1979; Diamantoglou et al., 1989). T. tartonraira, the evergreen species, accumulated lipids in the autumn and winter and broke them
down during the growth and the following drought period, in contrast to soluble sugar fluctuation. D. 6iscosa accumulated lipids during the growth period and depleted them during summer drought. The nearly parallel fluctuations of total lipids in roots and stems of both examined species (Fig. 3) were also found in roots and stems of phrygana dimorphic species (Diamantoglou and Rhizopoulou, 1990; Meletiou-Christou et al., 1992). According to our hypothesis, the relationships between nitrogen and starch and between nitrogen and soluble sugars varied with species and time of year and were related to growth periods. The negative relationships that were found between nitrogen and starch and between nitrogen and soluble sugars (Table 1), during the growth of the leaves of D. 6iscosa, resulted from the decrease of nitrogen concentration and the increase of starch and soluble sugar concentrations. The decrease of nitrogen concentration during the growth period of the leaves of Mediterranean shrubs has been considered as a dilution effect (Field and Mooney, 1983). The positive relationships between foliar nitrogen and starch and between foliar nitrogen and soluble sugars from March up to June in the leaves of T. tartonraira (Table 1) resulted from the increase of nitrogen, starch and soluble sugar concentrations. Chapin (1991) suggests that starch accumulates in leaves of nitrogen-limited plants because there is sufficient nitrogen for continued carbon fixation but insufficient nitrogen to maintain normal growth. The results of Amundson et al. (1995) also support that hypothesis. During the period of drought stress neither starch nor sugar concentration was correlated with foliar nitrogen of both plants (Table 1) as was also found in the needles of Picea rubens during the cold hardening period (Amundson et al., 1995). From August until November, during leaf senescence of D. 6iscosa, foliage is exporting carbon and the negative relationships between nitrogen and starch and between nitrogen and soluble sugars (Table 1) resulted primarily from the decrease of carbohydrate concentrations. The energy content of storage substances (Fig. 5) in the leaves of D. 6iscosa and T. tartonraira was within the range of energy values reported for
M.S. Meletiou-Christou et al. / En6ironmental and Experimental Botany 39 (1998) 21–32
Mediterranean evergreen (Diamantoglou and Kull, 1982; Meletiou-Christou et al., 1994) and deciduous species (Diamantoglou et al., 1989) and lower than the values found in phrygana species (Meletiou-Christou et al., 1992). The different fluctuations of the energy content of storage substances between the leaves of the two examined species support the argument of Larcher and Thomaser-Thin (1988), that the seasonal changes in energy content are in intimate connection with the seasonal periodicity of growth activity, rather than that they directly respond to weather conditions. It is interesting that stems and roots of both species showed similar annual fluctuations of energy content of storage substances, in contrast to our hypothesis as stated in the Section 1. It seems that the differences in leaf lifespan did not affect the stem and root contents but only the leaf contents.
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