Planta (Berl.) 93, 171--174 (1970) 9 by Springer-Verlag 1970
Short Communication Phloem in Carrot Calluses A. D. H A ~ s o ~ a n d J. EDELMAI~* Queen Elizabeth College, London, W. 8 lZeceived May 4/26, 1970
Summary. Translocation of 14C-labelledphotosynthate across 2 cm long carrot calluses was not detectable even 6 hours after a 30 min 14CO2pulse. This is consistent with the discontinuous nature of the phloem as a whole, although small strands of contiguous, well differentiated sieve cells with companion cells were readily seen in electron micrographs. Classical a n a t o m i c a l investigations (Gautheret, 1959) on carrot a n d other callus cultures have shown t h a t phloem tissue is present i n the form of discrete islands, b u t the lack of detailed cytological a n d physiological d a t a i n this field has been stressed b y K a r s t e n s (1965). This c o m m u n i c a t i o n reports the use of the p h o t o s y n t h e t i c ~aCO~-fixation k n o w n to occur in carrot callus tissue (Roux a n d Tendille, 1954) followed b y a n i n c u b a t i o n period i n light a n d air to s t u d y translocation across callus pieces. 14C-translocation across the tissue, n o t expected because of the discontinuous phloem distribution, was n o t detected b u t the results of a p r e l i m i n a r y electron microscope i n v e s t i g a t i o n of the phloem show t h a t sieve elements with a typically " f u n c t i o n a l " appearance are present. This provides a striking example of the t y p e of difficulty e n c o u n t e r e d i n a t t e m p t i n g to correlate u l t r a s t r u c t u r e a n d f u n c t i o n i n the sieve tube, a subject recently reviewed b y W e a t h e r l y a n d J o h n s o n (1968). Carrot callus cultures were grown on the KS medium of Reinert and Backs (1968) modified by the addition of 15% (v/v) autoclaved coconut milk, under 1,300 lux of continuous fluorescent light at 26 • 2~ C. For translocation experiments callus pieces 20 x 7 • 7 mm weighing about 700 mg were cut from 4-week old calluses, and half of each piece was shaded with aluminium foil. Unshaded and totally shaded controls were also included. Following a 30 rain pre-illumination at 25,000 lux at 28~ C, the cMlus pieces were exposed to 50br Ci 14C02 (55 m Ci/mM) in air in 45 ml tubular vessels for 30 min. The callus pieces were then incubated on moist filter paper in petri dishes under the same conditions for up to 6 hours; the * With the technical collaboration of H. J. W. Edge. Botany Dept. Kings College, London. 12"
172
A. D. Hanson and J. Edelman:
Fig. 1. Two sieve ceils (SC) with fibrillar material and starch grains in the lumens. CC companion cell with m a n y mitochondria. P parenchyma cell. x 6,000 illuminated a n d shaded moieties were separated at intervals a n d extracted in ethanol. Aliquots of the ethanolic extracts were counted in a liquid scintillation
spectrometer, and insoluble residues were counted on planehets at infinite thickness using a thin end-window counter. Material for electron microscopy was fixed in glutaraldehyde and OsO4, embedded in Araidite and stained with uranyl acetate and lead citrate. Each partly shaded callus piece fixed about 0.5~Ci of laCOu during the 30 rain exposure; the major labelled products were sucrose and glutamine. No significant decline of total i~C was observed with increasing time up to 6 hours. The amount of label recovered in the insoluble fractions of both illuminated and shaded portions rose during this period from about 10% to about 25%.
Phloem in Carrot Calluses
173
Fig. 2. Single sieve plate pore showing pore fibrils extending into the lumen. Note the small amount of eallose formation, x 6,000 Table. Distribution o/ radioactivity between illuminated and shaded moieties after various times Hours after end of 14C0~ exposure
uC-aetivity in / shaded moiety / laC-aetivity in shaded and unshaded moieties (%)
14C activity in shaded moiety a corrected for dark fixation (%)
0
21.5
9.2
1
19.8
7.5
2 4 6
18.2 17.7 14.0
5.9 5.4 1.7
a Calculated from the observation that a totally shaded callus piece fixed 12.3% of the amount of 14C02incorporated by a fully illuminated piece of the same volume. This Table indicates t h a t t r a n s l o c a t i o n from the i l l u m i n a t e d to the shaded m o i e t y did n o t occur as (a) during translocation the level of r a d i o a c t i v i t y i n the d a r k e n e d half would be expected to rise. This did n o t happen. The low levels a c t u a l l y f o u n d are ascribed to light scatter d u r i n g the 30 rain exposure to 1 4 C 0 2 ; (b) i n fact, the r a d i o a c t i v i t y in the d a r k e n e d half fell with time (table, second column) i n d i c a t i n g t h a t the fl-earboxylation products of dark 14CO2 fixation were respired; this was supported
174
A. D. Hanson and J. Edelman: Phloem in Carrot Calluses
b y the ionic n a t u r e of the radioactive c o m p o u n d s f o u n d i n the d a r k e n e d half. W e have also f o u n d t h a t the very low level of 14C i n sucrose in the shaded tissue did n o t rise even after 6 hours, a time period t h a t allows extensive t r a n s l o c a t i o n to occur i n whole p l a n t s (for example: Hofstra a n d Nelson, 1969). E l e c t r o n micrographs (Figs. l, 2) show small s t r a n d s of u p to 5 contiguous sieve cells, some with c o m p a n i o n cells (Fig. 1). The fine s t r u c t u r e of the c y t o p l a s m a n d of the sieve plate pores, which b o t h c o n t a i n filamentous m a t e r i a l (Fig. 2) appears to be typical of n o r m a l l y developed " f u n c t i o n a l " sieve elements (Weatherly a n d J o h n s o n , 1968). This work was supported in part by a grant from Tare and Lyle Ltd.
Re~erences Gautheret, R. J. : La culture des Tissus V6g~taux, Paris: Masson & Cie. 1959. Hofstra, G., Nelson, C. D. : A comparative study of transloeation of assimilated 14C from leaves of different species. Planta (Berl.) 88, 103--112 (1969). Karstens, W. K. H. : Phloem formation in explants and tissue cultures, from an anatomical point of view. Proceedings of an Intcrnat. Conference on Plant Tissue Culture, p. 309. California: MeCutchan Publishing Corp. 1965. Reinert, J., Backs, D.: Control of totipoteney in plant cells growing in vitro. Nature (Loud.) 220, 1340 (1968). Roux, E., Tendille, C.: Pigments des ehloroplastes et photosynth~se. C. R. Acad. Sci. (Paris) 288, 1261--1263 (1954). Weatherly, P. E., Johnson, R. P. C. : The form and function of the sieve tube: a problem in reconciliation. Int. Rev. Cytol. 24, 149--192 (1968). Prof. Dr. J. Edelman Department of Biology Atkins Building Campden Hill, London, W. 8, U. K.
Short Communication Phloem in Carrot Calluses A. D. H A ~ s o ~ a n d J. EDELMAI~* Queen Elizabeth College, London, W. 8 lZeceived May 4/26, 1970
Summary. Translocation of 14C-labelledphotosynthate across 2 cm long carrot calluses was not detectable even 6 hours after a 30 min 14CO2pulse. This is consistent with the discontinuous nature of the phloem as a whole, although small strands of contiguous, well differentiated sieve cells with companion cells were readily seen in electron micrographs. Classical a n a t o m i c a l investigations (Gautheret, 1959) on carrot a n d other callus cultures have shown t h a t phloem tissue is present i n the form of discrete islands, b u t the lack of detailed cytological a n d physiological d a t a i n this field has been stressed b y K a r s t e n s (1965). This c o m m u n i c a t i o n reports the use of the p h o t o s y n t h e t i c ~aCO~-fixation k n o w n to occur in carrot callus tissue (Roux a n d Tendille, 1954) followed b y a n i n c u b a t i o n period i n light a n d air to s t u d y translocation across callus pieces. 14C-translocation across the tissue, n o t expected because of the discontinuous phloem distribution, was n o t detected b u t the results of a p r e l i m i n a r y electron microscope i n v e s t i g a t i o n of the phloem show t h a t sieve elements with a typically " f u n c t i o n a l " appearance are present. This provides a striking example of the t y p e of difficulty e n c o u n t e r e d i n a t t e m p t i n g to correlate u l t r a s t r u c t u r e a n d f u n c t i o n i n the sieve tube, a subject recently reviewed b y W e a t h e r l y a n d J o h n s o n (1968). Carrot callus cultures were grown on the KS medium of Reinert and Backs (1968) modified by the addition of 15% (v/v) autoclaved coconut milk, under 1,300 lux of continuous fluorescent light at 26 • 2~ C. For translocation experiments callus pieces 20 x 7 • 7 mm weighing about 700 mg were cut from 4-week old calluses, and half of each piece was shaded with aluminium foil. Unshaded and totally shaded controls were also included. Following a 30 rain pre-illumination at 25,000 lux at 28~ C, the cMlus pieces were exposed to 50br Ci 14C02 (55 m Ci/mM) in air in 45 ml tubular vessels for 30 min. The callus pieces were then incubated on moist filter paper in petri dishes under the same conditions for up to 6 hours; the * With the technical collaboration of H. J. W. Edge. Botany Dept. Kings College, London. 12"
172
A. D. Hanson and J. Edelman:
Fig. 1. Two sieve ceils (SC) with fibrillar material and starch grains in the lumens. CC companion cell with m a n y mitochondria. P parenchyma cell. x 6,000 illuminated a n d shaded moieties were separated at intervals a n d extracted in ethanol. Aliquots of the ethanolic extracts were counted in a liquid scintillation
spectrometer, and insoluble residues were counted on planehets at infinite thickness using a thin end-window counter. Material for electron microscopy was fixed in glutaraldehyde and OsO4, embedded in Araidite and stained with uranyl acetate and lead citrate. Each partly shaded callus piece fixed about 0.5~Ci of laCOu during the 30 rain exposure; the major labelled products were sucrose and glutamine. No significant decline of total i~C was observed with increasing time up to 6 hours. The amount of label recovered in the insoluble fractions of both illuminated and shaded portions rose during this period from about 10% to about 25%.
Phloem in Carrot Calluses
173
Fig. 2. Single sieve plate pore showing pore fibrils extending into the lumen. Note the small amount of eallose formation, x 6,000 Table. Distribution o/ radioactivity between illuminated and shaded moieties after various times Hours after end of 14C0~ exposure
uC-aetivity in / shaded moiety / laC-aetivity in shaded and unshaded moieties (%)
14C activity in shaded moiety a corrected for dark fixation (%)
0
21.5
9.2
1
19.8
7.5
2 4 6
18.2 17.7 14.0
5.9 5.4 1.7
a Calculated from the observation that a totally shaded callus piece fixed 12.3% of the amount of 14C02incorporated by a fully illuminated piece of the same volume. This Table indicates t h a t t r a n s l o c a t i o n from the i l l u m i n a t e d to the shaded m o i e t y did n o t occur as (a) during translocation the level of r a d i o a c t i v i t y i n the d a r k e n e d half would be expected to rise. This did n o t happen. The low levels a c t u a l l y f o u n d are ascribed to light scatter d u r i n g the 30 rain exposure to 1 4 C 0 2 ; (b) i n fact, the r a d i o a c t i v i t y in the d a r k e n e d half fell with time (table, second column) i n d i c a t i n g t h a t the fl-earboxylation products of dark 14CO2 fixation were respired; this was supported
174
A. D. Hanson and J. Edelman: Phloem in Carrot Calluses
b y the ionic n a t u r e of the radioactive c o m p o u n d s f o u n d i n the d a r k e n e d half. W e have also f o u n d t h a t the very low level of 14C i n sucrose in the shaded tissue did n o t rise even after 6 hours, a time period t h a t allows extensive t r a n s l o c a t i o n to occur i n whole p l a n t s (for example: Hofstra a n d Nelson, 1969). E l e c t r o n micrographs (Figs. l, 2) show small s t r a n d s of u p to 5 contiguous sieve cells, some with c o m p a n i o n cells (Fig. 1). The fine s t r u c t u r e of the c y t o p l a s m a n d of the sieve plate pores, which b o t h c o n t a i n filamentous m a t e r i a l (Fig. 2) appears to be typical of n o r m a l l y developed " f u n c t i o n a l " sieve elements (Weatherly a n d J o h n s o n , 1968). This work was supported in part by a grant from Tare and Lyle Ltd.
Re~erences Gautheret, R. J. : La culture des Tissus V6g~taux, Paris: Masson & Cie. 1959. Hofstra, G., Nelson, C. D. : A comparative study of transloeation of assimilated 14C from leaves of different species. Planta (Berl.) 88, 103--112 (1969). Karstens, W. K. H. : Phloem formation in explants and tissue cultures, from an anatomical point of view. Proceedings of an Intcrnat. Conference on Plant Tissue Culture, p. 309. California: MeCutchan Publishing Corp. 1965. Reinert, J., Backs, D.: Control of totipoteney in plant cells growing in vitro. Nature (Loud.) 220, 1340 (1968). Roux, E., Tendille, C.: Pigments des ehloroplastes et photosynth~se. C. R. Acad. Sci. (Paris) 288, 1261--1263 (1954). Weatherly, P. E., Johnson, R. P. C. : The form and function of the sieve tube: a problem in reconciliation. Int. Rev. Cytol. 24, 149--192 (1968). Prof. Dr. J. Edelman Department of Biology Atkins Building Campden Hill, London, W. 8, U. K.
