Journal o f Chemical Ecology, Vol. 9, No. 8, 1983
CAFFEINE HAZARDS AND THEIR PREVENTION IN G E R M I N A T I N G S E E D S OF C O F F E E (Coffea arabica L.) 1
JACOB
FRIEDMAN
2 and GEORGE
R. W A L L E R
Department o/" Biochemistry Oklahoma Agricultural Experiment Station Oklahoma State University Stillwater, Oklahoma 74078 (Received November 22, 1982; revised March 14, 1983) Abstract--The inhibition of growth of seedlings of coffee (CqlTfea arabica L.) exposed to 10 mM caffeine was found to occur in the rootlet: mitosis and cell plate formation were also inhibited. Since concentrations of endogenous caffeine in the imbibed seed are 40-60 mM, 4-6 times as high as in the seedlings, we conclude that coffee embryos have specific means of avoiding caffeine autotoxicity. Observations indicate that cell divisions in root tips start only after the latter are pushed away from the caffeine-rich endosperm by elongation of the hypocotyl and maintained through cell elongation. Caffeine is introduced into the embryonic cotyledons mostly after cell division is completed there. Thus, coffee seedlings may avoid autotoxic effects of endogenous caffeine by separation between sites where mitosis is occurring and those where caffeine is stored. This is achieved in root tips by separation is space but in the cotyledons by separation in time. Caffeine is liberated from the tree litter in coffee plantations and eventually will produce autotoxic effects, resulting in some degeneration.
Key Words--Colfea arabica, coffee, caffeine, theophylline, germination inhibitors, avoidance of autotoxicity.
INTRODUCTION Some secondary metabolites stored in s e e d p r e d a t o r s a n d t h u s f u n c t i o n as e m b r y o (Bell, 1978). A n u m b e r o f t h e s e a c t i v i t y ( R i c e , 1974), i.e., i n h i b i t g r o w t h
seeds exhibit toxicity against various natural protectants of the quiescent compounds may also have phytotoxic or germination of various species, but
~Published as Journal Article No. J-4112 of the Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74078. 2On sabbatical leave of absence from the Department of Botany, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 69978, Israel. 1099 0098-0331,'83/0800-1099503.00/0 9 1983 Plenum Publishing Corporation
1 I00
FRIEDMAN AND WALLER
may also adversely affect their own species. Phytotoxins with autotoxic activity are often present in the outer parts of seeds or diaspores. If these toxins are not sufficiently leached out by rainfall or metabolized by soil microflora, they hold germination in check and ensure that this will occur only after the amounts of rainfall are sufficient for the establishment of seedlings (Evenari, 1949). Other toxins, however, are stored within the seeds and are hardly leachable; these are natural protectants, e.g., caffeine in Coffea arabica and strychnine in Strychnos nux vomica (Evenari, 1949). When either of these compounds is applied exogenously to the seed that stores it, germination is inhibited, even when the concentration of the exogenous inhibitor is much lower than that found endogenously in the imbibed seed (Evenari, 1949). This suggests that such seeds have the means to avoid the effects of their own phytotoxins. Fowden and Lea (1979) describe mechanisms by which plants avoid autotoxicity by their phytotoxic secondary metabolites, especially by nonprotein amino acids. This work was undertaken to quantify the effect of exogenous caffeine and one of its degradation products, theophylline, on germination and growth of seedlings of coffee, to identify in the coffee embryo sites either susceptible or resistant to caffeine, and to speculate on the strategies by which the coffee embryo avoids autotoxic hazard during germination (Scheme 1).
METHODS AND MATERIALS
Seeds of Coffea arabica cv. Bourbon, 3-6 months after collecting, containing 32 + 3% water (on dry weight basis), were obtained from Conafruit, Jalapa, Mexico. As described by Valio (1976), endocarp-free seeds
0 H3G.N
GIH3 N-
0 H.G.
I
I
OH3
GH3 THEOPHYLLINE
CAFFEINE SCHEME I.
H
CAFFEINE IN G E R M I N A T I N G COFFEE SEEDS
1101
were allowed to germinate at 27 _+ 0. I~ in darkness. Seeds were initially soaked and shaken in 0-20 mM aqueous solutions of caffeine, pH 5.2-6.0, for 48 hr at 27~ and later washed and set in the middle of four layers of strips (10 X 50 cm) of Whatman chromatographic paper, 0.3 mm thick. These were rolled and placed in glass tanks (30 X 30 X l0 cm) each with a different concentration of caffeine. In each tank, 150 seeds, divided into three groups (replicas) of 50 each, were set. To avoid gross changes in caffeine concentration by either uptake or evaporation of water, 300 ml of solution per tank were used and tanks were sealed. The effect of exogenous theophylline was also similarly tested. Lengths of rootlets and hypocotyls were periodically measured. Extraction and quantitative determination of caffeine were conducted by a method of Chou and Waller (1980b), slightly modified. Caffeine content was determined for each treatment in each of the five seedlings weekly and was measured separately for the rootlet, hypocotyl, cotyledons, endosperm, and the germination medium. The effect of caffeine on cell division was studied in cells of root tips of coffee seedlings. The root tips were allowed to develop on filter paper in distilled water for 4 weeks, at 27 ~C in the dark, and then placed on filter paper immersed in 10 m M caffeine in a tightly covered glass tank (30 X 30 X 9 cm). Root tips were removed after 24 hr, fixed, and prepared for microscopic examination, using a modification of Warmake's method (Warmake, 1935).
RESULTS
When seeds of coffee were allowed to germinate in aqueous solutions of caffeine of various concentrations, elongation of hypocotyls was reduced in all cases and growth of rootlets was almost completely inhibited by 10 mM caffeine (Figure 1, Table 1). Root tips darkened and 4-5 days later deteriorated. A similar although milder inhibition was observed in response to theophylline. Suppression of growth occurred, although concentration of the endogenous caffeine in the imbibed coffee seeds was 40-60 mM. This suggested that embryos of germinating coffee seeds must be able to avoid autotoxic hazards from their endogenous caffeine. Following the level of caffeine in germinating coffee seeds showed that in the quiescent seeds, the embryos were nearly caffeine-free, containing only about 0.6% (on dry weight basis), whereas the adjacent endosperm contained 1.4% caffeine. However, during germination (ca. 4 weeks after seed wetting), substantial amounts of caffeine (ca. 75% of the total amount) were found in the developing embryo (Figure 2). Other portions of the caffeine were distributed as follows; 5.6 _+ 3.2% of the total amount was found in the leachate, and 6.4 _+ 2.2% was left in the residual, unutilized endosperm. The rest, 13.0 + 4.1%, could not be located, but the appearance of some metabolic products, including theo-
1102
FRIEDMAN AND WALLER
FIG. 1. Seedlings o f coffee g e r m i n a t e d in distilled w a t e r (left), in 2.5 (center) and 25 m M (right) caffeine, t h r e e weeks after wetting, in dark. In all c a f f e i n e - t r e a t e d seeds, e m e r g e n c e o f t h e h y p o c o t y l did o c c u r but r o o t l e t g r o w t h was a r r e s t e d (• natural size).
TABLE 1. GROWTH OF ROOTLET AND HYPOCOTYL OF 4uWEEK-OLD COFFEE SEEDLINGS GERMINATED IN VARIOUS CONCENTRATIONS OF CAFFEINE OR THEOPHYLLINE a
Concentration (mM)
Caffeine Rootlet Hypocotyl Theophylline Rootlet Hypocotyl
5
10
20
72 _+ 8 52 + 7
3 -+ 2 61 _ 19
3 -+ 3 39 • 12
59 -+ 7 58 -+ 4
27 _+ 4 48 -+ 11
14 • 7 54 -+_ 6
aData presented as percentage of growth (length) of the control (distilled water), average of 150 seedlings (_+ SE)
1103
CAFFEINE IN G E R M I N A T I N G COFFEE SEEDS
100
!
~
"
~
.
=_. 80
. \
~
Total in seed ~---4
,
,
o
"O ' "~~
60
~m
.=_ ~g
40 o 20
0
1
2 3 4 Germination Time (weeks)
5
6
FIG. 2. Changes in localization of endogenous caffeine during germination of coffee. Calculated as percentage of the initial amount (100%) found in the quiescent seed.
phylline and theobromine, in the hypocotyl and in the cotyledons, during the third week of germination (Figure 2), suggested that this portion was catabolized by the embryo. Of caffeine within the embryo, most (83.8 -+ 8.7%) was found in the cotyledons, lesser amounts (14.2 • 4.3%) in the hypocotyl, and the remaining 2.0 • I. 1% in the rootlet. This pattern of distribution of caffeine was nearly constant during seed germination, starting at the third week after seed wetting. From either of the seedling organs caffeine could be easily extracted by water and was therefore believed to be stored in a soluble form. The rootlet of a coffee embryo may be more susceptible to caffeine damage than the cotyledons, as caffeine is known to interfere with mitosis in root tips of several higher plants (Kihlman, 1977). We therefore tested effects of exogenous caffeine on mitosis in root tips of seedlings of coffee and followed the relation between accumulation of endogenous caffeine and mitosis in the cotyledons. Cell division was inhibited in root tips that were exposed to 10 mM caffeine for 24 hr. Also cell plate formation was blocked, yielding some binucleate cells (Figure 3). Similar results have been obtained on onion root tips (Kihlman, 1949). Tetranucleate cells could not be detected in our study, even after exposure f o r 4 8 or 72 hr or to higher caffeine concentration.
1104
FRIEDMAN AND WALLER
FIG. 3. Binucleate cells in root tips of coffee seedlings after exposure to l0 rnM caffeine
for 24 hr (X1200).
DISCUSSION
The embryo in the quiescent coffee seed is about 3 m m long, including two cotyledons and a long hypocotyl terminating with a minute radicle (ca. 0.2-0.5 m m long). Initial stages of germination (1-4 days after seed wetting) were characterized by elongation of the hypoeotyl and were observed under all treatments, but at this stage no cell division could be detected in either the hypocotyl or the radicle. In the root tips grown in distilled water, cell division started only after the rootlet was pushed by elongation of the hypocotyl 1-3 m m out of the endosperm, which had occurred 5 or 6 days after seed wetting. This growth of the hypocotyl is achieved by cell elongation only after the tip of the rootlet has become separated from the caffeine-rich endosperm before cell division starts in this part. The zone where mitosis occurs is thus separated in space from that where caffeine is localized. The means by which caffeine is sequestered in the endosperm apart from the embryo are not clear. In the cotyledons of the germinating embryo, such a separation cannot occur. In the quiescent seed,these small organs (1.1-1.5 m m in diameter) are
CAFFEINE IN G E R M I N A T I N G COFFEE SEEDS
1105
embedded in the caffeine-rich endosperm and serve as the embryo's haustoria, expanding remarkably (16-20 mm) until germination is completed (5-6 weeks after seed wetting). We therefore supposed that mitosis in the cotyledons was completed at stages of embryonic differentiation while the seed was still on the mother plant. However, observations during germination disproved this view. Cell division in the embryonic cotyledons started 9-11 days after seed wetting; as translocation of endogenous caffeine progressed (Figure 2), mitosis was much reduced, and almost stopped completely 3 weeks after seed wetting. Therefore in the very young cotyledons, the process of mitosis and accumulation of caffeine appear to be separated in time, and thus caffeine autotoxicity is avoided. Whether caffeine is the signal to stop mitosis, or whether each process is separately regulated, is unknown. Separations between caffeine and mitosis either by space, as in the root tips, or by time, as in the cotyledons, are the plant's means of avoiding autotoxicity. Catabolism of caffeine in germinating coffee embryos was indirectly measured, both by disappearance of about 13% of the amount of caffeine initially present within the quiescent seed, as well as by the presence of some products of biodegraded caffeine (theobromine and theophylline) (Suzuki and Waller, 1981) in the cotyledons and hypocotyl, as already noted. The small amount of catabolism suggests that it plays a minor role in the prevention of autotoxic effects. Conversely, about 75% of the caffeine stored in the endosperm is later found in the embryo, most of it in the cotyledons. This amount did not decrease even in seedlings kept in continuous dark and starved for 84 days at 27~ The fact that caffeine is preserved despite starvation suggests that the alkaloid is not easily utilized either for energy or as a nitrogen source. Nevertheless, uptake of the alkaloid into the cotyledons, despite its potential hazard, suggests that caffeine is an important agent for the plant. Its wide spectrum of biological activity, e.g., inhibition of fungi (Rizvi et al. 1980a) and bacteria (Kihlman, 1977), and its chemosterilant effect on the seed beetle (Callosobruchus chinensis) (Rizvi et al., 1981), as well as its possible allelopathic effects on weeds (Anaya-Lang et al., 1978; Chou and Waller, 1980a; Rizvi et al., 1980b), all support the view that it is a natural plant protectant. Although the toxic effects induced by caffeine on coffee seedlings in the laboratory were not extremely dramatic, autotoxicity may occur in nature. Around old coffee trees, considerable amounts of caffeine may be released from the tree's own litter and accumulate in the vicinity of roots over the years. The annual amount of litter leaves produced, plus about 10% of lost fruits, in old coffee trees is 150-200 g dry matter/m2/year (Epifanio, 1981). We reason that this litter may release 1-2 g caffeine/m2/year, with some additional amounts of caffeine derivatives. The antimicrobial activity of caffeine may reduce catabolism of the alkaloid in the soil and thus prolong its retention and increase caffeine accumulation. Since most roots of coffee develop in the
1106
FRIEDMAN AND WALLER
u p p e r soil l a y e r i m m e d i a t e l y u n d e r t h e t r e e ' s o w n litter, a u t o t o x i c i t y c a n b e m a n i f e s t e d . T h u s it is p o s s i b l e t h a t t h e w o r l d w i d e p h e n o m e n o n o f e a r l y d e g e n e r a t i o n o f c o f f e e p l a n t a t i o n s a t 1 0 - 2 5 y e a r s o f a g e ( W e l l m a n , 1961) is due in part to autotoxicity, and caffeine, the factor which allows for survival o f c o f f e e p l a n t s i n a h o s t i l e e n v i r o n m e n t , is a l s o r e s p o n s i b l e f o r s h o r t e n i n g t h e i r lives. Acknowledgments--We acknowledge the help of Mrs. Nurit Friedman for the cytological observations in coffee embryos and that of Dr. R. Panciera and Dr. P. Richardson in giving us access to their laboratories for microscopical work. This work was supported in part by NSF grant PCM-78-23160, and the Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74078.
REFERENCES
ANAYA-LANG,A.L., RoY-OCOTLA,G., and ORTIZ-ORTEGA,L. 1978. Allelopathic potential of coffee plantation. 2rid Int. Cong. Ecol. Jerusalem, Israel, September 10-16, p. 8 (abstract). BELL, E.A. 1978. Toxins in seeds, pp. 143-161, in J.B. Harborne (ed.). Biochemical Aspects of Plant and Animal Co-evolution. Academic Press, New York. CHOU, C.-H., and WALLER, G.R. 1980a. Possible allelopathic constituents of Coffea arabiea. J. Chem. Ecol. 6:643-659. CHOU, C.-H., and WALLER,G.R. 1980b. Isolation and identification by mass spectrometry of phytotoxins in Coffea arabica. Bot. Bull. Acad. Sinica 21:25-34. EPIFANIO, J.A. 1981. Ecologia del Agroecosistema Cafetalero. PhD thesis. Universidad Nacional Autonoma de Mexico, Mexico City, Mexico, p. 166. EVENARI, M. 1949. Germination inhibitors, Bot. Rev. 15(3): 153-194. FOWDEN, L., and LEA, P.J. 1979. Mechanisms of plant avoidance of autotoxicity by secondary metabolites, especially by nonprotein amino acids, pp. 135-160, in G.A. Rosenthal and D.H. Janzen (eds.). Herbivores, Their Interaction with Secondary Plant Metabolites. Academic Press, New York. KIHLMAN, B.A. 1949. The cytological effect of caffeine. Hereditas 35:109-111. KIHLMAN, B.A. 1977. Caffeine and Chromosomes. Elsevier, Amsterdam, pp. 297-301. RICE, E.L. 1974. Allelopathy. Academic Press, New York, p. 232. RlzvI, S.J.H., JAISWAL, V., MUKERJI, D., and MATHUR, S.N. 1980a. Antifungal properties of 1,3,7-trimethylxanthine isolated from Coffea arabica. Naturwissenschaften 67:459-460. RIzvI, S.J.H., MUKERJI, D., and MATHUR, S.N.J. 1980b. A possible new source of a natural herbicide. ,L Exp. Biol. 18:77-778. Rlzvi, S.J.H., PADNEY, S.K., MUKERJI, D., and MATHUS, S.N. 1981. 1,3,7-Trimethylxanthine, a new chemosterilant for stored grain pest Callosobruchus chinensis. Z. Angew. Entomol. 90:378-380. SUZUKI, T., and WALLER, G.R. 1981. Biodegradation of caffeine by excised fruits of Coffea arabica L. Fed. Proc. 41:608 (abstract 2394). VALIO, I.F.M. 1976. Germination of coffee seeds (Coffea arabica L. cv. Mundo Novo). J. Exp. Bot. 27:983-991. WARMAKE~H.E. 1935. A permanent root tip smear method, Stain Teehnol. 10:101-103. WELLMAN,F.L. 1961. Coffee: Botany, Cultivation and Utilization. Interscience Publishers, New York, pp. 345-347.
CAFFEINE HAZARDS AND THEIR PREVENTION IN G E R M I N A T I N G S E E D S OF C O F F E E (Coffea arabica L.) 1
JACOB
FRIEDMAN
2 and GEORGE
R. W A L L E R
Department o/" Biochemistry Oklahoma Agricultural Experiment Station Oklahoma State University Stillwater, Oklahoma 74078 (Received November 22, 1982; revised March 14, 1983) Abstract--The inhibition of growth of seedlings of coffee (CqlTfea arabica L.) exposed to 10 mM caffeine was found to occur in the rootlet: mitosis and cell plate formation were also inhibited. Since concentrations of endogenous caffeine in the imbibed seed are 40-60 mM, 4-6 times as high as in the seedlings, we conclude that coffee embryos have specific means of avoiding caffeine autotoxicity. Observations indicate that cell divisions in root tips start only after the latter are pushed away from the caffeine-rich endosperm by elongation of the hypocotyl and maintained through cell elongation. Caffeine is introduced into the embryonic cotyledons mostly after cell division is completed there. Thus, coffee seedlings may avoid autotoxic effects of endogenous caffeine by separation between sites where mitosis is occurring and those where caffeine is stored. This is achieved in root tips by separation is space but in the cotyledons by separation in time. Caffeine is liberated from the tree litter in coffee plantations and eventually will produce autotoxic effects, resulting in some degeneration.
Key Words--Colfea arabica, coffee, caffeine, theophylline, germination inhibitors, avoidance of autotoxicity.
INTRODUCTION Some secondary metabolites stored in s e e d p r e d a t o r s a n d t h u s f u n c t i o n as e m b r y o (Bell, 1978). A n u m b e r o f t h e s e a c t i v i t y ( R i c e , 1974), i.e., i n h i b i t g r o w t h
seeds exhibit toxicity against various natural protectants of the quiescent compounds may also have phytotoxic or germination of various species, but
~Published as Journal Article No. J-4112 of the Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74078. 2On sabbatical leave of absence from the Department of Botany, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 69978, Israel. 1099 0098-0331,'83/0800-1099503.00/0 9 1983 Plenum Publishing Corporation
1 I00
FRIEDMAN AND WALLER
may also adversely affect their own species. Phytotoxins with autotoxic activity are often present in the outer parts of seeds or diaspores. If these toxins are not sufficiently leached out by rainfall or metabolized by soil microflora, they hold germination in check and ensure that this will occur only after the amounts of rainfall are sufficient for the establishment of seedlings (Evenari, 1949). Other toxins, however, are stored within the seeds and are hardly leachable; these are natural protectants, e.g., caffeine in Coffea arabica and strychnine in Strychnos nux vomica (Evenari, 1949). When either of these compounds is applied exogenously to the seed that stores it, germination is inhibited, even when the concentration of the exogenous inhibitor is much lower than that found endogenously in the imbibed seed (Evenari, 1949). This suggests that such seeds have the means to avoid the effects of their own phytotoxins. Fowden and Lea (1979) describe mechanisms by which plants avoid autotoxicity by their phytotoxic secondary metabolites, especially by nonprotein amino acids. This work was undertaken to quantify the effect of exogenous caffeine and one of its degradation products, theophylline, on germination and growth of seedlings of coffee, to identify in the coffee embryo sites either susceptible or resistant to caffeine, and to speculate on the strategies by which the coffee embryo avoids autotoxic hazard during germination (Scheme 1).
METHODS AND MATERIALS
Seeds of Coffea arabica cv. Bourbon, 3-6 months after collecting, containing 32 + 3% water (on dry weight basis), were obtained from Conafruit, Jalapa, Mexico. As described by Valio (1976), endocarp-free seeds
0 H3G.N
GIH3 N-
0 H.G.
I
I
OH3
GH3 THEOPHYLLINE
CAFFEINE SCHEME I.
H
CAFFEINE IN G E R M I N A T I N G COFFEE SEEDS
1101
were allowed to germinate at 27 _+ 0. I~ in darkness. Seeds were initially soaked and shaken in 0-20 mM aqueous solutions of caffeine, pH 5.2-6.0, for 48 hr at 27~ and later washed and set in the middle of four layers of strips (10 X 50 cm) of Whatman chromatographic paper, 0.3 mm thick. These were rolled and placed in glass tanks (30 X 30 X l0 cm) each with a different concentration of caffeine. In each tank, 150 seeds, divided into three groups (replicas) of 50 each, were set. To avoid gross changes in caffeine concentration by either uptake or evaporation of water, 300 ml of solution per tank were used and tanks were sealed. The effect of exogenous theophylline was also similarly tested. Lengths of rootlets and hypocotyls were periodically measured. Extraction and quantitative determination of caffeine were conducted by a method of Chou and Waller (1980b), slightly modified. Caffeine content was determined for each treatment in each of the five seedlings weekly and was measured separately for the rootlet, hypocotyl, cotyledons, endosperm, and the germination medium. The effect of caffeine on cell division was studied in cells of root tips of coffee seedlings. The root tips were allowed to develop on filter paper in distilled water for 4 weeks, at 27 ~C in the dark, and then placed on filter paper immersed in 10 m M caffeine in a tightly covered glass tank (30 X 30 X 9 cm). Root tips were removed after 24 hr, fixed, and prepared for microscopic examination, using a modification of Warmake's method (Warmake, 1935).
RESULTS
When seeds of coffee were allowed to germinate in aqueous solutions of caffeine of various concentrations, elongation of hypocotyls was reduced in all cases and growth of rootlets was almost completely inhibited by 10 mM caffeine (Figure 1, Table 1). Root tips darkened and 4-5 days later deteriorated. A similar although milder inhibition was observed in response to theophylline. Suppression of growth occurred, although concentration of the endogenous caffeine in the imbibed coffee seeds was 40-60 mM. This suggested that embryos of germinating coffee seeds must be able to avoid autotoxic hazards from their endogenous caffeine. Following the level of caffeine in germinating coffee seeds showed that in the quiescent seeds, the embryos were nearly caffeine-free, containing only about 0.6% (on dry weight basis), whereas the adjacent endosperm contained 1.4% caffeine. However, during germination (ca. 4 weeks after seed wetting), substantial amounts of caffeine (ca. 75% of the total amount) were found in the developing embryo (Figure 2). Other portions of the caffeine were distributed as follows; 5.6 _+ 3.2% of the total amount was found in the leachate, and 6.4 _+ 2.2% was left in the residual, unutilized endosperm. The rest, 13.0 + 4.1%, could not be located, but the appearance of some metabolic products, including theo-
1102
FRIEDMAN AND WALLER
FIG. 1. Seedlings o f coffee g e r m i n a t e d in distilled w a t e r (left), in 2.5 (center) and 25 m M (right) caffeine, t h r e e weeks after wetting, in dark. In all c a f f e i n e - t r e a t e d seeds, e m e r g e n c e o f t h e h y p o c o t y l did o c c u r but r o o t l e t g r o w t h was a r r e s t e d (• natural size).
TABLE 1. GROWTH OF ROOTLET AND HYPOCOTYL OF 4uWEEK-OLD COFFEE SEEDLINGS GERMINATED IN VARIOUS CONCENTRATIONS OF CAFFEINE OR THEOPHYLLINE a
Concentration (mM)
Caffeine Rootlet Hypocotyl Theophylline Rootlet Hypocotyl
5
10
20
72 _+ 8 52 + 7
3 -+ 2 61 _ 19
3 -+ 3 39 • 12
59 -+ 7 58 -+ 4
27 _+ 4 48 -+ 11
14 • 7 54 -+_ 6
aData presented as percentage of growth (length) of the control (distilled water), average of 150 seedlings (_+ SE)
1103
CAFFEINE IN G E R M I N A T I N G COFFEE SEEDS
100
!
~
"
~
.
=_. 80
. \
~
Total in seed ~---4
,
,
o
"O ' "~~
60
~m
.=_ ~g
40 o 20
0
1
2 3 4 Germination Time (weeks)
5
6
FIG. 2. Changes in localization of endogenous caffeine during germination of coffee. Calculated as percentage of the initial amount (100%) found in the quiescent seed.
phylline and theobromine, in the hypocotyl and in the cotyledons, during the third week of germination (Figure 2), suggested that this portion was catabolized by the embryo. Of caffeine within the embryo, most (83.8 -+ 8.7%) was found in the cotyledons, lesser amounts (14.2 • 4.3%) in the hypocotyl, and the remaining 2.0 • I. 1% in the rootlet. This pattern of distribution of caffeine was nearly constant during seed germination, starting at the third week after seed wetting. From either of the seedling organs caffeine could be easily extracted by water and was therefore believed to be stored in a soluble form. The rootlet of a coffee embryo may be more susceptible to caffeine damage than the cotyledons, as caffeine is known to interfere with mitosis in root tips of several higher plants (Kihlman, 1977). We therefore tested effects of exogenous caffeine on mitosis in root tips of seedlings of coffee and followed the relation between accumulation of endogenous caffeine and mitosis in the cotyledons. Cell division was inhibited in root tips that were exposed to 10 mM caffeine for 24 hr. Also cell plate formation was blocked, yielding some binucleate cells (Figure 3). Similar results have been obtained on onion root tips (Kihlman, 1949). Tetranucleate cells could not be detected in our study, even after exposure f o r 4 8 or 72 hr or to higher caffeine concentration.
1104
FRIEDMAN AND WALLER
FIG. 3. Binucleate cells in root tips of coffee seedlings after exposure to l0 rnM caffeine
for 24 hr (X1200).
DISCUSSION
The embryo in the quiescent coffee seed is about 3 m m long, including two cotyledons and a long hypocotyl terminating with a minute radicle (ca. 0.2-0.5 m m long). Initial stages of germination (1-4 days after seed wetting) were characterized by elongation of the hypoeotyl and were observed under all treatments, but at this stage no cell division could be detected in either the hypocotyl or the radicle. In the root tips grown in distilled water, cell division started only after the rootlet was pushed by elongation of the hypocotyl 1-3 m m out of the endosperm, which had occurred 5 or 6 days after seed wetting. This growth of the hypocotyl is achieved by cell elongation only after the tip of the rootlet has become separated from the caffeine-rich endosperm before cell division starts in this part. The zone where mitosis occurs is thus separated in space from that where caffeine is localized. The means by which caffeine is sequestered in the endosperm apart from the embryo are not clear. In the cotyledons of the germinating embryo, such a separation cannot occur. In the quiescent seed,these small organs (1.1-1.5 m m in diameter) are
CAFFEINE IN G E R M I N A T I N G COFFEE SEEDS
1105
embedded in the caffeine-rich endosperm and serve as the embryo's haustoria, expanding remarkably (16-20 mm) until germination is completed (5-6 weeks after seed wetting). We therefore supposed that mitosis in the cotyledons was completed at stages of embryonic differentiation while the seed was still on the mother plant. However, observations during germination disproved this view. Cell division in the embryonic cotyledons started 9-11 days after seed wetting; as translocation of endogenous caffeine progressed (Figure 2), mitosis was much reduced, and almost stopped completely 3 weeks after seed wetting. Therefore in the very young cotyledons, the process of mitosis and accumulation of caffeine appear to be separated in time, and thus caffeine autotoxicity is avoided. Whether caffeine is the signal to stop mitosis, or whether each process is separately regulated, is unknown. Separations between caffeine and mitosis either by space, as in the root tips, or by time, as in the cotyledons, are the plant's means of avoiding autotoxicity. Catabolism of caffeine in germinating coffee embryos was indirectly measured, both by disappearance of about 13% of the amount of caffeine initially present within the quiescent seed, as well as by the presence of some products of biodegraded caffeine (theobromine and theophylline) (Suzuki and Waller, 1981) in the cotyledons and hypocotyl, as already noted. The small amount of catabolism suggests that it plays a minor role in the prevention of autotoxic effects. Conversely, about 75% of the caffeine stored in the endosperm is later found in the embryo, most of it in the cotyledons. This amount did not decrease even in seedlings kept in continuous dark and starved for 84 days at 27~ The fact that caffeine is preserved despite starvation suggests that the alkaloid is not easily utilized either for energy or as a nitrogen source. Nevertheless, uptake of the alkaloid into the cotyledons, despite its potential hazard, suggests that caffeine is an important agent for the plant. Its wide spectrum of biological activity, e.g., inhibition of fungi (Rizvi et al. 1980a) and bacteria (Kihlman, 1977), and its chemosterilant effect on the seed beetle (Callosobruchus chinensis) (Rizvi et al., 1981), as well as its possible allelopathic effects on weeds (Anaya-Lang et al., 1978; Chou and Waller, 1980a; Rizvi et al., 1980b), all support the view that it is a natural plant protectant. Although the toxic effects induced by caffeine on coffee seedlings in the laboratory were not extremely dramatic, autotoxicity may occur in nature. Around old coffee trees, considerable amounts of caffeine may be released from the tree's own litter and accumulate in the vicinity of roots over the years. The annual amount of litter leaves produced, plus about 10% of lost fruits, in old coffee trees is 150-200 g dry matter/m2/year (Epifanio, 1981). We reason that this litter may release 1-2 g caffeine/m2/year, with some additional amounts of caffeine derivatives. The antimicrobial activity of caffeine may reduce catabolism of the alkaloid in the soil and thus prolong its retention and increase caffeine accumulation. Since most roots of coffee develop in the
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FRIEDMAN AND WALLER
u p p e r soil l a y e r i m m e d i a t e l y u n d e r t h e t r e e ' s o w n litter, a u t o t o x i c i t y c a n b e m a n i f e s t e d . T h u s it is p o s s i b l e t h a t t h e w o r l d w i d e p h e n o m e n o n o f e a r l y d e g e n e r a t i o n o f c o f f e e p l a n t a t i o n s a t 1 0 - 2 5 y e a r s o f a g e ( W e l l m a n , 1961) is due in part to autotoxicity, and caffeine, the factor which allows for survival o f c o f f e e p l a n t s i n a h o s t i l e e n v i r o n m e n t , is a l s o r e s p o n s i b l e f o r s h o r t e n i n g t h e i r lives. Acknowledgments--We acknowledge the help of Mrs. Nurit Friedman for the cytological observations in coffee embryos and that of Dr. R. Panciera and Dr. P. Richardson in giving us access to their laboratories for microscopical work. This work was supported in part by NSF grant PCM-78-23160, and the Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74078.
REFERENCES
ANAYA-LANG,A.L., RoY-OCOTLA,G., and ORTIZ-ORTEGA,L. 1978. Allelopathic potential of coffee plantation. 2rid Int. Cong. Ecol. Jerusalem, Israel, September 10-16, p. 8 (abstract). BELL, E.A. 1978. Toxins in seeds, pp. 143-161, in J.B. Harborne (ed.). Biochemical Aspects of Plant and Animal Co-evolution. Academic Press, New York. CHOU, C.-H., and WALLER, G.R. 1980a. Possible allelopathic constituents of Coffea arabiea. J. Chem. Ecol. 6:643-659. CHOU, C.-H., and WALLER,G.R. 1980b. Isolation and identification by mass spectrometry of phytotoxins in Coffea arabica. Bot. Bull. Acad. Sinica 21:25-34. EPIFANIO, J.A. 1981. Ecologia del Agroecosistema Cafetalero. PhD thesis. Universidad Nacional Autonoma de Mexico, Mexico City, Mexico, p. 166. EVENARI, M. 1949. Germination inhibitors, Bot. Rev. 15(3): 153-194. FOWDEN, L., and LEA, P.J. 1979. Mechanisms of plant avoidance of autotoxicity by secondary metabolites, especially by nonprotein amino acids, pp. 135-160, in G.A. Rosenthal and D.H. Janzen (eds.). Herbivores, Their Interaction with Secondary Plant Metabolites. Academic Press, New York. KIHLMAN, B.A. 1949. The cytological effect of caffeine. Hereditas 35:109-111. KIHLMAN, B.A. 1977. Caffeine and Chromosomes. Elsevier, Amsterdam, pp. 297-301. RICE, E.L. 1974. Allelopathy. Academic Press, New York, p. 232. RlzvI, S.J.H., JAISWAL, V., MUKERJI, D., and MATHUR, S.N. 1980a. Antifungal properties of 1,3,7-trimethylxanthine isolated from Coffea arabica. Naturwissenschaften 67:459-460. RIzvI, S.J.H., MUKERJI, D., and MATHUR, S.N.J. 1980b. A possible new source of a natural herbicide. ,L Exp. Biol. 18:77-778. Rlzvi, S.J.H., PADNEY, S.K., MUKERJI, D., and MATHUS, S.N. 1981. 1,3,7-Trimethylxanthine, a new chemosterilant for stored grain pest Callosobruchus chinensis. Z. Angew. Entomol. 90:378-380. SUZUKI, T., and WALLER, G.R. 1981. Biodegradation of caffeine by excised fruits of Coffea arabica L. Fed. Proc. 41:608 (abstract 2394). VALIO, I.F.M. 1976. Germination of coffee seeds (Coffea arabica L. cv. Mundo Novo). J. Exp. Bot. 27:983-991. WARMAKE~H.E. 1935. A permanent root tip smear method, Stain Teehnol. 10:101-103. WELLMAN,F.L. 1961. Coffee: Botany, Cultivation and Utilization. Interscience Publishers, New York, pp. 345-347.
