Planta (1983)159:193-206
P l a n t a 9 Springer-Verlag 1983
Mieroautoradiographie studies of phloem loading and transport in the leaf of Zea mays L. Eberhard Fritz 1 *, Ray F. Evert 2 * and Wolfgang Heyser 3 Forstbotanisches Institut der Universit/it G6ttingen, Biisgenweg 2, D-3400 G6ttingen-Weende, Federal Republic of Germany, 2 Departments of Botany and Plant Pathology, University of Wisconsin, Madison, WI 53706, USA, and 3 Fachbereich 3 Biologie, Universit/it Bremen, D-2800 Bremen, Federal Republic of Germany
Abstract.
Microautoradiographs
showed
that
[14C]sucrose taken up in the xylem of small and
intermediate (longitudinal) vascular bundles of Zea mays leaf strips was quickly accumulated by vascular parenchyma cells abutting the vessels. The first sieve tubes to exhibit 14C-labeling during the [l~C]sucrose experiments were thick-walled sieve tubes contiguous to the more heavily labeled vascular parenchyma cells. (These two cell types typically have numerous plasmodesmatal connections.) With increasing [lr feeding periods, greater proportions of thick- and thin-walled sieve tubes became labeled, but few of the labeled thin-walled sieve tubes were associated with labeled companion cells. (Only the thin-walled sieve tubes are associated with companion cells.) When portions of leaf strips were exposed to ~4CO2 for 5 rain, the vascular parenchyma cells - regardless of their location in relation to the vessels or sieve tubes - were the most consistently labeled cells of small and intermediate bundles, and label ( ~ C photosynthate) appeared in a greater proportion of thin-walled sieve tubes than thick-walled sieve tubes. After a 5-rain chase with 12COa, the thinwalled sieve tubes were more heavily labeled than any other cell type of the leaf. After a 10-rain chase with 12CO> the thin-walled sieve tubes were even more heavily labeled. The companion cells generally were less heavily labeled than their associated thin-walled sieve tubes. Although all of the thickwalled sieve tubes were labeled in portions of leaf strips fed 14CO2 for 5 rain and given a 10-rain 12CO2 chase, only five of 72 vascular bundles below the *4CO2-exposed portions contained labeled thick-walled sieve tubes. Moreover, the few labeled thick-walled sieve tubes of the "transport region" always abutted ~4C-labeled vascular parenchyma * To whom correspondence should be addressed
cells. The results of this study indicate that (I) the vascular parenchyma cells are able to retrieve at least sucrose from the vessels and transfer it to the thick-walled sieve tubes, (2) the thick-walled sieve tubes are not involved in long-distance transport, and (3) the thin-walled sieve tubes are capable themselves of accumulating sucrose and photosynthates from the apoplast, without the companion cells serving as intermediary cells. Key words: Assimilate transport
- Leaf (14C transport) -- Phloem loading - Sieve tube - Vascular bundle - Zea (1~C transport).
Introduction
During their investigation of the vascular bundles in the leaves of Triticum aestivum and other festucoid grasses, Kuo and O'Brien (1974) noticed that the longitudinal bundles contained two types of sieve tube, one with thick, and the other with thin walls. Since then, similar types of sieve tubes have been found in longitudinal bundles of the leaves of Zea mays (Walsh 1974), Oryza sativa (Miyake and Maeda 1976), Themeda triandra (Botha et al. 1982), and Saccharum officinale (Colbert and Evert 1982). The thick-walled sieve tubes in the T. aestirum leaf are lignified, but those in the leaves of the other four species apparently are not. Further investigation of the small and intermediate bundles of the Z. mays leaf by Evert et al. (1978) showed that the thick-walled sieve tubes, which are the sieve tubes nearest the xylem and which commonly are in direct contact with the vessels, lack companion cells but have numerous poreplasmodesmata connections with vascular parenchyma cells. The thin-walled sieve tubes, which occur below the thick-walled sieve tubes and are asso-
194
E. Fritz et al. : Phloem loading and transport in Zea mays leaf
ciated with companion cells, have numerous poreplasmodesmata connections with their companion cells. Plasmodesmatal connections between the sieve tube-companion cell complexes and other cell types of the leaf, including vascular parenchyma cells and thick-walled sieve tubes, are rare, so that the sieve tube-companion cell complexes are virtually isolated symplastically from the rest of the leaf. The phloem of the longitudinal bundles of the maize leaf contains no fewer than two sieve tubes. When only two sieve tubes are present, they always differ from one another; that is, one is a thickwalled type and the other a thin-walled type. Moreover, all small and intermediate bundles contain both types of sieve tube (Evert et al. 1978; Evert 1980). (In the maize leaf, small and intermediate bundles intergrade with one another and at times those designated intermediate differ from small bundles primarily by the presence of a hypodermal sclerenchyma strand on one or both sides of the intermediate bundle between epidermis and bundle sheath; Evert et al. 1977a). The presence of two types of sieve tube in the grass leaves has led to speculation over their functions. Kuo and O'Brien (1974) have suggested that the thick-walled sieve tubes in the T. aestivum leaf may be specialized for long-distance transport or may serve as temporary storage reservoirs for sugar in excess of what can be transported by the thin-walled types. Results of a subsequent microautoradiographic study of 14C-photosynthate transport in T. aestivum leaves indicated, however, that the thick-walled sieve tubes in that species may be involved neither in storage nor directly in the transport of photosynthates (Cartwright et al. 1977). More recently, Evert et al. (1978) suggested that the thick-walled sieve tubes in Z. mays may play a role in the retrieval of solutes entering the leaf apoplast in the transpiration stream. This suggestion was based on the close spatial association of the thick-walled sieve tubes with the vessels, and on their possession of plasmalemma tubules, which apparently greatly increase the apoplast-symplast interface (Evert et al. 1977b). The present study was undertaken in an attempt to determine experimentally the function(s) of the two types of sieve tube in the Z. mays leaf. The experiments were carried out on maize leaf strips taken from mature leaves. This plant material has proved highly suitable for experimental study of the details of phloem loading and the phloem transport process (Heyser etal. 1975, 1976, 1977, 1978; Heyser
Material and methods
1980).
Plant material. The maize plants (Zea mays L., cv. Prior; Samen-Kr6bel, G6ttingen, FRG) were cultivated in soil, either in a greenhouse without artificial illumination for the experiments utilizing [a~C]sucrose, or in a growth chamber (25 ~ C; relative humidity 65% ; light 14 h, 540 lamol m - 2 s- x) with mercury-vapor light for experiments utilizing 14CO z. The plants utilized for [~4C]sucrose experiments were 1 m tall and kept in the dark for 25-29 h before removing leaf strips for the experiments. Those utilized for i4CO2-feeding experiments were 70 cm tall and kept in the dark for 41 h before removing leaf strips. The leaf strips were prepared under a green safelight by procedures described earlier by Heyser et al. (1975). The maize plants were predarkened in order to deplete the leaves of the large amounts of starch commonly found in the chloroplasts of the bundle-sheath cells. After 24 h of darkness, maize leaves are starch-free. In experiments with starchfree leaf strips, the direction and intensity of phloem transport can be switched on and off by simply changing the external conditions (Heyser et al. 1975, 1976, 1977). Moreover, in maize plants illuminated for several hours, ~4C-labeled assimilates are exported so rapidly after exposure to ~4CO2 that it is difficult to separate experimentally phloem-loading and phloem transport (Eschrich and Burchardt 1982).
Experiments utilizing [14C]sucrose. Strips 16 cm long and I cm wide were taken from the apical half of the blade of the fourth to seventh visible leaf counted from above. The basal end of each leaf strip was placed in 280 gl of a 25 mM sucrose solution, which contained 3.7.10 s Bq of uniformly labeled [a4C]sucrose (Amersham-Buchler, Braunschweig, FRG), and the apical end was immersed in tap water. Feeding with [14C]sucrose was allowed to continue for 2, 4, 6, or 8 min in the dark. After each of the feeding times, tissue samples I mm long and 1.5-4.5 mm from the fed ends of the leaf strips were removed for microautoradiographic examination of [14C]sucrose distribution. Microautoradiographs were made of 78-84 serial transverse sections from three to five leaf strips for each feeding time.
Experiments utilizing 14C02. Strips 25 cm long and 2 cm wide were taken from the apical half of the blade of the fourth visible leaf counted from above. The basal and apical ends of the strips were immersed in double-distilled water. The basal 15 cm of the strips were kept in the dark until the end of the experiment, in order to maintain a sink for the attraction of assimilates (Heyser et al. 1975). For this purpose, a blackened chamber with two compartments was used. The compartment containing the 15-cm-long portion of the leaf strip was covered with black plastic. The apical 10 cm of the strips were adapted to mercury-vapor light for 30 rain (400-W mercury-halide HQI lamp; Osram, Berlin; 155-180 lamol m -2 s-1) before application of 14CO2. Five cm of the light-adapted portions of three leaf strips were then allowed to assimilate 2.96-106 Bq of 1~CO2 for 5 rain. The ~4CO2 was absorbed in 0.8 ml I% K O H and injected with a syringe directly into a lucite chamber containing 10% HC10 4. After the 5-rain ~4CO z pulse, the lucite chamber was opened. One leaf strip was immediately removed and tissue samples I mm long were cut from the 14COz-fed portion for microautoradiographic analysis. The fed portion of a second leaf strip was given a 12CO2 chase for an additional 5 rain (5 rain 1"CO2+5 rain lzCO2), then tissue samples I mm long were cut from the fed portion for microautoradiographic analysis. The third leaf strip was given a 1zCO2 chase for 10 min
E. Fritz et al. : Phloem loading and transport in Zea mays leaf after the i4CO2-feeding (5 rain 14CO 2 + 10 min 12C02). By this time substantial transport of assimilates had taken place into the non-illuminated portion, or "transport region," of the strip, as determined with use of a Geiger tube. For microautoradiographic analysis, l-ram-long tissue samples were cut from both the fed portion and the transport region of the third leaf strip. Tissue samples from the transport region were taken 15-20 mm from the basal end of the fed portion of the strip and 3 5 4 5 mm from the tissue samples of the fed portion. Microautoradiographs were made of 52 serial transverse sections for the 5 rain ~4CO2 experiment, 51 for the 5 min 14COz+5 rain ~2CO2 experiment, 25 for the 14CO2-fed area of the 5 min ~4COz+ 10 min 12CO2 experiment, and 137 for the transport region of the latter experiment.
Microautoradiography. The method of the microautoradiography used is described in detail in Fritz (1980). In brief, after treatment with either the 14C02 or [l~C]sucrose, l-ram samples of tissue are immediately frozen in isopentane, which was cooled with liquid nitrogen, and then freeze-dried. The freezedried material is evacuated and infiltrated with diethyl ether and epon-araldite (DDSA: Serva, Heidelberg, F R G ; Epon 812Araldite M; Fluka A.G., Buchs, Switzerland) under high pressure. Sections 1 gm thick are cut with glass knives on an ultramicrotome, transferred to micro slides, coated with Ilford K5 or L4 emulsion (Ilford, Essex, UK), and developed in Amidol (Barkas 1963) or D19A/S (Sanderson 1981) developer, respectively. After exposure times of 3-60 d, the emulsion is developed and the sections stained with toluidine blue. The microautoradiographs were viewed and photographed with a Zeiss Ultraphot II photomicroscope (Zeiss, Oberkochen, FRG).
Analysis of the mieroautoradiographs. Inasmuch as the volume of material to be analyzed was very large and the measurement of radioactivity required for the purposes of this study needed to be at most semi-quantitative, it was decided that it was neither practical nor necessary to make visual grain counts. Initial analysis of the mieroautoradiographs was made simply to determine which cells were labeled (i.e. above background level) and which were not. During this analysis it became obvious that differences existed in the density of silver grains over the various cell types and tissues and that the density of silver grains could be rated roughly as light, moderate, or heavy. All of the microautoradiographs were then so analyzed. This included rating the density of silver grains over all of the vascular bundles and contiguous tissues in all of the serial sections. After the initial rating of densities was completed, a second one was made of selected sections from each experiment without reference to the original data, in order to check the reliability of the original ratings. The two sets of data were quite similar. The possible effects of scatter, background, and other factors affecting variability (see Rogers 1979) on the grain density over the various cell types were carefully monitored during analysis of all the microautoradiographs. Hereafter, when the densities of silver grains over different cell types are compared, "more (less) heavily labeled" will always mean higher (lower) density of silver grains.
Classification and identification of ceil types. The characters used in classification of the various cell types in the vascular bundles of the maize leaf are given in Evert et al. (1978). At the lightmicroscope level most companion cells and vascular parenchyma cells can be distinguished from one another in part by their size differences (the companion cells commonly being smaller than the vascular parenchyma cells) and partly by their spatial associations with the sieve tubes and bundle-sheath cells. In
195 addition, most thick-walled sieve tubes can be distinguished from thin-walled sieve tubes by their generally smaller size and spatial association with the xylem. Both types of parenchymatous element and both types of sieve tube intergrade in size with one another, however, so that it is not always easy to distinguish between them at the light-microscope level. During the present study it was sometimes necessary, therefore, to examine a given cell in several serial sections with oil-immersion optics in order to establish its identity. Under those circumstances, the thick-walled sieve tubes and vascular parenchyma cells were often identified by the characteristic type of plastid each contains (Evert et al. ~978).
Selection offigures. Approximately 540 photomicrographs were taken for possible use as figures to illustrate the results of this investigation. Most of these were eliminated for use when pertinent features, which were discernible by through-focusing on the emulsion and underlying section, were not captured in the single plane of the photomicrograph. During preparation of the manuscript, it soon became apparent that only one or two vascular bundles from a given experiment would not adequately illustrate the results of that experiment. Several vascular bundles of each experiment would have to be illustrated, but that would be prohibitive, and probably unnecessary, as long as the reader realizes that the figures were selected to illustrate and highlight only certain features from each of the experiments. It is pertinent to remember that the figures represent only a very small portion of the data accumulated during the analysis and interpretation of the microautoradiographs. In the following section an overview is presented of the results of each experiment followed by an explanation of the figures chosen to illustrate certain aspects of those results. The figures were selected so that when viewed collectively and sequentially (e.g., Figs. 1-8, 9-12, and 13-16) they would provide an overview of the salient results of the two groups of experiments (experiments utilizing [l~C]sucrose and 14CO2, respectively). Results
Experiments utilizing [14 C]sucrose. The microautoradiographs obtained of tissues from the 2-min feeding experiment included 51 small and 11 intermediate bundles. In all of these bundles the vessels were consistently labeled (i.e. above background level), as evidenced by the density of silver grains in the photographic emulsion above them. Moreover, in most of the bundles the vessels were the most heavily labeled components, although considTable l. Percentages of thick- and thin-walled sieve tubes labeled in small and intermediate vascular bundles of maize leaf strips fed [14C]sucrose solution for 2, 4, 6, or 8 rain in the dark Feeding time
% Thick-walled sieve tubes labeled
(n)
2 min 4 rain 6 rain 8 rain
37 58 81 88
(87) (50) (86) (64)
% Thin-walled sieve tubes labeled
P l a n t a 9 Springer-Verlag 1983
Mieroautoradiographie studies of phloem loading and transport in the leaf of Zea mays L. Eberhard Fritz 1 *, Ray F. Evert 2 * and Wolfgang Heyser 3 Forstbotanisches Institut der Universit/it G6ttingen, Biisgenweg 2, D-3400 G6ttingen-Weende, Federal Republic of Germany, 2 Departments of Botany and Plant Pathology, University of Wisconsin, Madison, WI 53706, USA, and 3 Fachbereich 3 Biologie, Universit/it Bremen, D-2800 Bremen, Federal Republic of Germany
Abstract.
Microautoradiographs
showed
that
[14C]sucrose taken up in the xylem of small and
intermediate (longitudinal) vascular bundles of Zea mays leaf strips was quickly accumulated by vascular parenchyma cells abutting the vessels. The first sieve tubes to exhibit 14C-labeling during the [l~C]sucrose experiments were thick-walled sieve tubes contiguous to the more heavily labeled vascular parenchyma cells. (These two cell types typically have numerous plasmodesmatal connections.) With increasing [lr feeding periods, greater proportions of thick- and thin-walled sieve tubes became labeled, but few of the labeled thin-walled sieve tubes were associated with labeled companion cells. (Only the thin-walled sieve tubes are associated with companion cells.) When portions of leaf strips were exposed to ~4CO2 for 5 rain, the vascular parenchyma cells - regardless of their location in relation to the vessels or sieve tubes - were the most consistently labeled cells of small and intermediate bundles, and label ( ~ C photosynthate) appeared in a greater proportion of thin-walled sieve tubes than thick-walled sieve tubes. After a 5-rain chase with 12COa, the thinwalled sieve tubes were more heavily labeled than any other cell type of the leaf. After a 10-rain chase with 12CO> the thin-walled sieve tubes were even more heavily labeled. The companion cells generally were less heavily labeled than their associated thin-walled sieve tubes. Although all of the thickwalled sieve tubes were labeled in portions of leaf strips fed 14CO2 for 5 rain and given a 10-rain 12CO2 chase, only five of 72 vascular bundles below the *4CO2-exposed portions contained labeled thick-walled sieve tubes. Moreover, the few labeled thick-walled sieve tubes of the "transport region" always abutted ~4C-labeled vascular parenchyma * To whom correspondence should be addressed
cells. The results of this study indicate that (I) the vascular parenchyma cells are able to retrieve at least sucrose from the vessels and transfer it to the thick-walled sieve tubes, (2) the thick-walled sieve tubes are not involved in long-distance transport, and (3) the thin-walled sieve tubes are capable themselves of accumulating sucrose and photosynthates from the apoplast, without the companion cells serving as intermediary cells. Key words: Assimilate transport
- Leaf (14C transport) -- Phloem loading - Sieve tube - Vascular bundle - Zea (1~C transport).
Introduction
During their investigation of the vascular bundles in the leaves of Triticum aestivum and other festucoid grasses, Kuo and O'Brien (1974) noticed that the longitudinal bundles contained two types of sieve tube, one with thick, and the other with thin walls. Since then, similar types of sieve tubes have been found in longitudinal bundles of the leaves of Zea mays (Walsh 1974), Oryza sativa (Miyake and Maeda 1976), Themeda triandra (Botha et al. 1982), and Saccharum officinale (Colbert and Evert 1982). The thick-walled sieve tubes in the T. aestirum leaf are lignified, but those in the leaves of the other four species apparently are not. Further investigation of the small and intermediate bundles of the Z. mays leaf by Evert et al. (1978) showed that the thick-walled sieve tubes, which are the sieve tubes nearest the xylem and which commonly are in direct contact with the vessels, lack companion cells but have numerous poreplasmodesmata connections with vascular parenchyma cells. The thin-walled sieve tubes, which occur below the thick-walled sieve tubes and are asso-
194
E. Fritz et al. : Phloem loading and transport in Zea mays leaf
ciated with companion cells, have numerous poreplasmodesmata connections with their companion cells. Plasmodesmatal connections between the sieve tube-companion cell complexes and other cell types of the leaf, including vascular parenchyma cells and thick-walled sieve tubes, are rare, so that the sieve tube-companion cell complexes are virtually isolated symplastically from the rest of the leaf. The phloem of the longitudinal bundles of the maize leaf contains no fewer than two sieve tubes. When only two sieve tubes are present, they always differ from one another; that is, one is a thickwalled type and the other a thin-walled type. Moreover, all small and intermediate bundles contain both types of sieve tube (Evert et al. 1978; Evert 1980). (In the maize leaf, small and intermediate bundles intergrade with one another and at times those designated intermediate differ from small bundles primarily by the presence of a hypodermal sclerenchyma strand on one or both sides of the intermediate bundle between epidermis and bundle sheath; Evert et al. 1977a). The presence of two types of sieve tube in the grass leaves has led to speculation over their functions. Kuo and O'Brien (1974) have suggested that the thick-walled sieve tubes in the T. aestivum leaf may be specialized for long-distance transport or may serve as temporary storage reservoirs for sugar in excess of what can be transported by the thin-walled types. Results of a subsequent microautoradiographic study of 14C-photosynthate transport in T. aestivum leaves indicated, however, that the thick-walled sieve tubes in that species may be involved neither in storage nor directly in the transport of photosynthates (Cartwright et al. 1977). More recently, Evert et al. (1978) suggested that the thick-walled sieve tubes in Z. mays may play a role in the retrieval of solutes entering the leaf apoplast in the transpiration stream. This suggestion was based on the close spatial association of the thick-walled sieve tubes with the vessels, and on their possession of plasmalemma tubules, which apparently greatly increase the apoplast-symplast interface (Evert et al. 1977b). The present study was undertaken in an attempt to determine experimentally the function(s) of the two types of sieve tube in the Z. mays leaf. The experiments were carried out on maize leaf strips taken from mature leaves. This plant material has proved highly suitable for experimental study of the details of phloem loading and the phloem transport process (Heyser etal. 1975, 1976, 1977, 1978; Heyser
Material and methods
1980).
Plant material. The maize plants (Zea mays L., cv. Prior; Samen-Kr6bel, G6ttingen, FRG) were cultivated in soil, either in a greenhouse without artificial illumination for the experiments utilizing [a~C]sucrose, or in a growth chamber (25 ~ C; relative humidity 65% ; light 14 h, 540 lamol m - 2 s- x) with mercury-vapor light for experiments utilizing 14CO z. The plants utilized for [~4C]sucrose experiments were 1 m tall and kept in the dark for 25-29 h before removing leaf strips for the experiments. Those utilized for i4CO2-feeding experiments were 70 cm tall and kept in the dark for 41 h before removing leaf strips. The leaf strips were prepared under a green safelight by procedures described earlier by Heyser et al. (1975). The maize plants were predarkened in order to deplete the leaves of the large amounts of starch commonly found in the chloroplasts of the bundle-sheath cells. After 24 h of darkness, maize leaves are starch-free. In experiments with starchfree leaf strips, the direction and intensity of phloem transport can be switched on and off by simply changing the external conditions (Heyser et al. 1975, 1976, 1977). Moreover, in maize plants illuminated for several hours, ~4C-labeled assimilates are exported so rapidly after exposure to ~4CO2 that it is difficult to separate experimentally phloem-loading and phloem transport (Eschrich and Burchardt 1982).
Experiments utilizing [14C]sucrose. Strips 16 cm long and I cm wide were taken from the apical half of the blade of the fourth to seventh visible leaf counted from above. The basal end of each leaf strip was placed in 280 gl of a 25 mM sucrose solution, which contained 3.7.10 s Bq of uniformly labeled [a4C]sucrose (Amersham-Buchler, Braunschweig, FRG), and the apical end was immersed in tap water. Feeding with [14C]sucrose was allowed to continue for 2, 4, 6, or 8 min in the dark. After each of the feeding times, tissue samples I mm long and 1.5-4.5 mm from the fed ends of the leaf strips were removed for microautoradiographic examination of [14C]sucrose distribution. Microautoradiographs were made of 78-84 serial transverse sections from three to five leaf strips for each feeding time.
Experiments utilizing 14C02. Strips 25 cm long and 2 cm wide were taken from the apical half of the blade of the fourth visible leaf counted from above. The basal and apical ends of the strips were immersed in double-distilled water. The basal 15 cm of the strips were kept in the dark until the end of the experiment, in order to maintain a sink for the attraction of assimilates (Heyser et al. 1975). For this purpose, a blackened chamber with two compartments was used. The compartment containing the 15-cm-long portion of the leaf strip was covered with black plastic. The apical 10 cm of the strips were adapted to mercury-vapor light for 30 rain (400-W mercury-halide HQI lamp; Osram, Berlin; 155-180 lamol m -2 s-1) before application of 14CO2. Five cm of the light-adapted portions of three leaf strips were then allowed to assimilate 2.96-106 Bq of 1~CO2 for 5 rain. The ~4CO2 was absorbed in 0.8 ml I% K O H and injected with a syringe directly into a lucite chamber containing 10% HC10 4. After the 5-rain ~4CO z pulse, the lucite chamber was opened. One leaf strip was immediately removed and tissue samples I mm long were cut from the 14COz-fed portion for microautoradiographic analysis. The fed portion of a second leaf strip was given a 12CO2 chase for an additional 5 rain (5 rain 1"CO2+5 rain lzCO2), then tissue samples I mm long were cut from the fed portion for microautoradiographic analysis. The third leaf strip was given a 1zCO2 chase for 10 min
E. Fritz et al. : Phloem loading and transport in Zea mays leaf after the i4CO2-feeding (5 rain 14CO 2 + 10 min 12C02). By this time substantial transport of assimilates had taken place into the non-illuminated portion, or "transport region," of the strip, as determined with use of a Geiger tube. For microautoradiographic analysis, l-ram-long tissue samples were cut from both the fed portion and the transport region of the third leaf strip. Tissue samples from the transport region were taken 15-20 mm from the basal end of the fed portion of the strip and 3 5 4 5 mm from the tissue samples of the fed portion. Microautoradiographs were made of 52 serial transverse sections for the 5 rain ~4CO2 experiment, 51 for the 5 min 14COz+5 rain ~2CO2 experiment, 25 for the 14CO2-fed area of the 5 min ~4COz+ 10 min 12CO2 experiment, and 137 for the transport region of the latter experiment.
Microautoradiography. The method of the microautoradiography used is described in detail in Fritz (1980). In brief, after treatment with either the 14C02 or [l~C]sucrose, l-ram samples of tissue are immediately frozen in isopentane, which was cooled with liquid nitrogen, and then freeze-dried. The freezedried material is evacuated and infiltrated with diethyl ether and epon-araldite (DDSA: Serva, Heidelberg, F R G ; Epon 812Araldite M; Fluka A.G., Buchs, Switzerland) under high pressure. Sections 1 gm thick are cut with glass knives on an ultramicrotome, transferred to micro slides, coated with Ilford K5 or L4 emulsion (Ilford, Essex, UK), and developed in Amidol (Barkas 1963) or D19A/S (Sanderson 1981) developer, respectively. After exposure times of 3-60 d, the emulsion is developed and the sections stained with toluidine blue. The microautoradiographs were viewed and photographed with a Zeiss Ultraphot II photomicroscope (Zeiss, Oberkochen, FRG).
Analysis of the mieroautoradiographs. Inasmuch as the volume of material to be analyzed was very large and the measurement of radioactivity required for the purposes of this study needed to be at most semi-quantitative, it was decided that it was neither practical nor necessary to make visual grain counts. Initial analysis of the mieroautoradiographs was made simply to determine which cells were labeled (i.e. above background level) and which were not. During this analysis it became obvious that differences existed in the density of silver grains over the various cell types and tissues and that the density of silver grains could be rated roughly as light, moderate, or heavy. All of the microautoradiographs were then so analyzed. This included rating the density of silver grains over all of the vascular bundles and contiguous tissues in all of the serial sections. After the initial rating of densities was completed, a second one was made of selected sections from each experiment without reference to the original data, in order to check the reliability of the original ratings. The two sets of data were quite similar. The possible effects of scatter, background, and other factors affecting variability (see Rogers 1979) on the grain density over the various cell types were carefully monitored during analysis of all the microautoradiographs. Hereafter, when the densities of silver grains over different cell types are compared, "more (less) heavily labeled" will always mean higher (lower) density of silver grains.
Classification and identification of ceil types. The characters used in classification of the various cell types in the vascular bundles of the maize leaf are given in Evert et al. (1978). At the lightmicroscope level most companion cells and vascular parenchyma cells can be distinguished from one another in part by their size differences (the companion cells commonly being smaller than the vascular parenchyma cells) and partly by their spatial associations with the sieve tubes and bundle-sheath cells. In
195 addition, most thick-walled sieve tubes can be distinguished from thin-walled sieve tubes by their generally smaller size and spatial association with the xylem. Both types of parenchymatous element and both types of sieve tube intergrade in size with one another, however, so that it is not always easy to distinguish between them at the light-microscope level. During the present study it was sometimes necessary, therefore, to examine a given cell in several serial sections with oil-immersion optics in order to establish its identity. Under those circumstances, the thick-walled sieve tubes and vascular parenchyma cells were often identified by the characteristic type of plastid each contains (Evert et al. ~978).
Selection offigures. Approximately 540 photomicrographs were taken for possible use as figures to illustrate the results of this investigation. Most of these were eliminated for use when pertinent features, which were discernible by through-focusing on the emulsion and underlying section, were not captured in the single plane of the photomicrograph. During preparation of the manuscript, it soon became apparent that only one or two vascular bundles from a given experiment would not adequately illustrate the results of that experiment. Several vascular bundles of each experiment would have to be illustrated, but that would be prohibitive, and probably unnecessary, as long as the reader realizes that the figures were selected to illustrate and highlight only certain features from each of the experiments. It is pertinent to remember that the figures represent only a very small portion of the data accumulated during the analysis and interpretation of the microautoradiographs. In the following section an overview is presented of the results of each experiment followed by an explanation of the figures chosen to illustrate certain aspects of those results. The figures were selected so that when viewed collectively and sequentially (e.g., Figs. 1-8, 9-12, and 13-16) they would provide an overview of the salient results of the two groups of experiments (experiments utilizing [l~C]sucrose and 14CO2, respectively). Results
Experiments utilizing [14 C]sucrose. The microautoradiographs obtained of tissues from the 2-min feeding experiment included 51 small and 11 intermediate bundles. In all of these bundles the vessels were consistently labeled (i.e. above background level), as evidenced by the density of silver grains in the photographic emulsion above them. Moreover, in most of the bundles the vessels were the most heavily labeled components, although considTable l. Percentages of thick- and thin-walled sieve tubes labeled in small and intermediate vascular bundles of maize leaf strips fed [14C]sucrose solution for 2, 4, 6, or 8 rain in the dark Feeding time
% Thick-walled sieve tubes labeled
(n)
2 min 4 rain 6 rain 8 rain
37 58 81 88
(87) (50) (86) (64)
% Thin-walled sieve tubes labeled
