Planta 9 by Springer-Verlag 1977

Planta i37, 145-151 (1977)

The Influence of Externally Supplied Sucrose on Phloem Transport in the Maize Leaf Strip* Wolfgang Heyser, Rosemarie Heyser, Walter Eschrich, and Eberhard Fritz Forstbotanisches Institut, UniversitS,t G6ttingen, Bfisgenweg2, D-3400 G6ttingen-Weende, Federal Republic of Germany

Abstract. Sucrose (2,5-1000mmol 1-1), labeled with [l#C]sucrose, was taken up by the xylem when supplied to one end of a 30-cm-long leaf strip of Z e a mays L. cv. Prior. The sugar was loaded into the phloem and transported to the opposite end, which was immersed in diluted H o a g l a n d ' s nutrient solution. When the H o a g l a n d ' s solution at the opposite end was replaced by unlabeled sucrose solution of the same molarity as the labeled one, the two solutions met near the middle of the leaf strip, as indicated by radioautographs. In the dark, translocation of ~4Clabeled assimilates was always directed away from the site of sucrose application, its distance depending on sugar concentration and translocation time. When sucrose was applied to both ends of the leaf strip, translocation of 14C-labeled assimilates was directed toward the lower sugar concentration. In the light, transport of ~4-C-labeled assimilates can be directed (1) toward the morphological base of the leaf strip only (light effect), (2) toward the base and away from the site of sucrose application (light and sucrose effect), or (3) away from the site of sucrose application independent of the (basipetal or acropetal) direction (sucrose effect). The strength of a sink, represented by the darkened half of a leaf strip, can be reduced by applying sucrose (at least 2 5 m m o l l -~) to the darkened end of the leaf strip. However, equimolar sucrose solutions applied to both ends do not affect the strength of the dark sink. Only above 75 m m o l lsucrose was the sink effect of the darkened part of the leaf strip reduced. Presumably, increasing the sucrose concentration replenishes the leaf tissue m o r e rapidly, and photosynthates from the illuminated part of the leaf strip are imported to a lesser extent by the dark sink. Key words: Phloem transport -

*

Sucrose - Zea.

Supported by Deutsche Forschungsgemeinschaft

Introduction

In a previous communication (Heyser et al., 1975), strips isolated from mature maize leaves were described as suitable objects for the study of phloem translocation. It was demonstrated that minute local changes of external conditions can trigger phloem transport and determine its direction. It also was shown that the natural polarity of phloem transport (basipetal export from mature leaves) is greatly repressed by starvation (plants kept in the dark for 48 h). In a further contribution (Heyser et al., 1976), the effects on translocation of several water-soluble substances, applied externally to one end of maize leaf strips, were studied. It was established that sucrose had by far the greatest influence on phloem transport, because it seemed to be loaded into the phloem much more easily than any of the other sugars investigated. This seems to relate to the widespread occurrence of sucrose in sieve-tube exudates (Zimmerm a n n and Ziegler, 1975). The present communication describes the extent to which externally supplied sucrose solutions can influence the transport of assimilates in a maize leaf strip.

Materials and Methods

Corn plants (Zea mays L. cv. Prior), 80 cm high and with 10 visible leaves, were either predarkened for 48 h or taken directly from the greenhouse at noontime. The 5th visible leaf, counted from above, was excised from each plant and 30-cmqong leaf strips were prepared as described previously (Heyser et al., 1975). For studying the effect of sucrose on phloem transport, leaf strips were placed with their median portions across a 1-cm-wide 14C2-Fixation compartment in a 3-compartment Plexiglaschamber (Fig. 1 in Heyser et al., 1975). The ends of the leaf strips were immersed either in sucrose solution (2,5mmoll 1 to 1000 mmol I 1 in 1/20 strength Hoagland's nutrient solution) or in the Hoagland's solution only (Eschrisch, 1976, p. 191). Both

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Fig. 1. Autoradiographs showing the distribution of sucrose in darkened maize leaf strips during different pretreatment periods, and its effect on the transport of tr assimilates. 1.2, 1.3, 1.5, and 1.7: [14C] sucrose distribution after 15 min, 2 h, 8 h and 16 h incubation at one end (P) of the leaf strip (B, basal; A, apical) with 75 m m o l 1 ~ sucrose containing 0.66 pCi/ml [~4C]sucrose. The other end of the leaf strip in 1/20 strength Hoagland solution. 1.2, 1.4, 1.6, and 1.8: 1+C-labeled assimilates, phloem loaded by 20 rain photosynthesis of 1+CO2 under 22,000 lx in a 1 cm section of the middle of the leaf strips. Subsequent translocation in the dark for 2.5 h. Leaf strips were taken from predarkened plants. Double-headed arrows (.I ~-) indicate front of l+C-label

basal and apical ends of the leaf strips were utilized for unilateral treatment with sucrose solution. For labeling assimilates with 14C, the narrow 14CO2-fixation c o m p a r t m e n t was made leak-proof with lanolin (Heyser et al., 1975). 30 pC| 14CO2 in 200 ml air (CO2 concentration ca. 0.031%) were passed through the compartment within 20 min+ Only this compartment, with l-cm-lengths of leaf strips, was exposed to mercury vapor light (Osram HQLS 400 W, 10-7 W m - 2 ~ 2 2 , 0 0 0 1 x ) . At the end of the labeling period, the lid of the compartment was removed. After a desired time of translocation (usually 2.5 h) the lanolin was wiped away and the leaf strips were frozen with crushed dry ice, freeze-dried and autoradiographed (Eschrich, 1966). Each experiment was repeated at least 3 times, using 8 parallel leaf strips. In Figs. 2, 5, 6 and 8, average distance and direction of phloem transport is indicated by solid-line arrows. Weak transport, which sometimes occurred in the major bundles, is indicated by dashed-line arrows. For checking the movement of sucrose, |4C-labeled sucrose (0.66 pCi/ml), purchased from Amersham-Buchler, Braunschweig (W. Germany), was added to the desired solution. Leaf strips then were rinsed carefully with water before processing for autoradiography.

Results

Sucrose solutions and other aqueous solutions are taken up primarily by the xylem vessels of a maize leaf strip, when applied externally to one end of the strip. When the leaf strip is provided with either water or aqueous solutions from both ends, transpiration

causes the water or solutions to meet somewhere near the middle of the strip. These were the observations of a study utilizing dye solutions and autoradiography of *4C-labeled solutes, which apparently were bound to apoplastic pathways (Heyser et al., 1976). During the same study, 14C-labeled sucrose was recorded by antoradiography at the end of the leaf strip opposite that of application within an hour of application, even though a stream of water was advancing against it in the xylem. The labeled sucrose had been taken up from the xylem into the phloem. By this method of phloem loading, sucrose exerts a force on phloem transport, which can be recorded with the use of labeled assimilates.

1. Influence of Sucrose Sohftions on Phloem Transport in the dark Sucrose concentrations as low as 2.5mmol1-1 proved sufficient to " p u s h " lgC-labeled assimilates from the middle of the leaf strip towards the opposite end. With sucrose concentrations of 25mmol 1 1 or higher, the label reached the opposite end within 2.5 h or less (Heyser et al., 1976).

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In the following experiment, a 75mmol 1-1 sucrose solution was used to pretreat one end of leaf strips from predarkened corn plants for 0, 15, 30 rain, and 1,2, 4, 8, and l 6 h in the dark. After the pretreatmerit, a 1-cm-wide section of the middle of each leaf strip was exposed to l+COz in light (22,000 lx) for 20 rain, and the labeled assimilates produced were allowed to proceed in the phloem for a further 2.5 h in darkness. Autoradiographs of leaf strips treated this way are shown in every second illustration of Figure 1 (1.2, 1.4, 1.6, 1.8). Independent of the natural polarity of the leaf, the 1+C-labeled assimilates loaded into the phloem in the middle of the leaf strip were driven away from the site of sucrose application (basal end, B, and apical end, A), except in the leaf strips pretreated with sucrose for 16 h. In addition, there was a gradual decrease in the extent of phloem transport in leaf strips pretreated from 15 rain to 16 h with sucrose solution. Every first illustration of Figure 1 (1.1, 1.3, 1.5, 1.7) shows autoradiographs of leaf strips pretreated with 75 mmol 1-1 sucrose to which 14C-labeled sucrose (0.66 gCi/ml) had been added. These autoradiographs show what happened during the pretreatment time in the dark. After 15 rain, the sucrose, which was restricted primarily to the main bundles, had moved to the middle of the leaf strip. After 2 h, the sucrose reached the opposite end and started to fill up the leaf tissue between the bundles. 8 h pretreatment shows the site of application of the leaf strip completely flooded with label. Within 16 h the whole length of the strip appears homogeneously provided with label. This experiment shows that prolonged loading of a leaf strip with sucrose in the dark progressively reduces phloem translocation. When the sucrose is evenly distributed throughout the darkened leaf strip, phloem transport presumably ceases completely. The results of these experiments are in agreement with the concept that phloem transport is regulated by the equilibration of supply and demand.

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Fig. 2. Influence on phloem transport of two different sucrose concentrations and three different translocation periods in light and darkness. Maize ieaf strips from predarkened plants, incubated at one end (~,) with 10 o1 1 2 5 m m o l l 1 sucrose (S), the other end in 1/20 strength Hoagland solution. 15 min pretreatment in darkness or light followed by 20 rain photosynthesis of ~4CO~ at 1 cm section of the middle of the leaf strip, and subsequent translocation periods of 0, 30 rain or 2.5 h either in darkness (shaded part of the figure) or in ]ight. Arrows indicate extent of label in autoradiograpbs. B, basal, A, apicaI ends of Jeaf strip

2. Influence of Sucrose Concentration and Translocation Time In the previous experiments (Fig. 1), 75 mmol 1-1 sucrose solution was used throughout for pretreatment, and the time for translocation of labeled assimilates was always 2.5 h. In this set of experiments the time of pretreatment was kept constant at 15 rain, but relatively low (10 mmol 1 1) and high (125 mmol. I-t) sucrose concentrations were applied for comparison. Figure 2 shows how far labeled assimilates moved during the 20 rain labeling period and 30 min and 2.5 h later. Whereas 15 rain pretreatment with 125mmol1-1 sucrose solution drove assimilates to the opposite end of the leaf strip within 30rain, pretreatment with 10mmolt a sucrose was much less effective. In the dark, no difference existed between basipetal and acropetal transport. When the same experiment was conducted in the light, photosynthetic phloem loading throughout the leaf strip contributed to the flooding with externally supplied sucrose. Nevertheless, the effect of unilateral sucrose application was nearly the same as in the darkness. The basipetal movement of labeled assimilates at 10 mmol 1- t sucrose pretreatment and 2.5 h transport time apparently is an expression of the natural polarity of transport (export in mature leaves) in the replenished transport system. The purpose of the next experiment was to test this assumption.

3. Compensation of Light and Sucrose Effects In an earlier publication (Heyser et al., 1975, Fig. 2.4), light was shown to direct translocation of ~+Clabeled assimilates always basipetally, i.e., towards the basal end of the maize leaf strip. This is equivalent to the natural transport polarity of a mature leaf. Figure 3.1 shows this "light effect". It cannot be counteracted by application of 2.5 mmoll- 1 sucrose

148

W. Heyser et al. : Influence of Sucrose on Phloem Transport

Fig, 3. Autoradiographs showing effect of different sucrose concentrations on transport of ~C-labeled assimilates in the light. Leaf strips from not predarkened plants, incubated with basal (B) or apical (A) end (--~) in sucrose and the other in diluted Hoagland solution. 15 rain pretreatment with sucrose, 20 rain photosynthesis with ~ C O z , and 2.5 h translocation time. Light of 22,000 lx. 3.1, "light effect": 2.5 mmot 1 ~ sucrose does not compensate photosynthetic phloem loading, 3.2, "light and sucrose effect": 10 mmol 1 ~ sucrose, basally applied, just compensates photosynthetic phloem loading. 3.3, "sucrose effect": 75 m m o i l - 1 sucrose completely suppresses effect of photosynthetic phloem loading

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solution to the basal end (B) of the leaf strip. A 10 mmol 1-~ sucrose solution, applied to the basal end, just compensates for the light effect (Fig. 3.2). Supplying the apical end (A) of the leaf strip with sucrose, will of course, lead to an increase of the light effect, because it supports export. With 75 mmol 1-1 sucrose, the light effect is completely negated (Fig. 3.3). The leaves used in these experiments were taken from plants that were not predarkened. In the following experiments, leaves of predarkened plants and plants that were not predarkened were compared. In addition, light intensity was reduced stepwise. Figure 4 shows the 2 factors light and sucrose in different combinations, and their effect on translocation. The dependency on each other is quite conspicuous. Increasing light intensity needs higher sucrose concentrations to compensate the light effect and vice versa. Leaf strips from predarkened plants show a slightly stronger response to sucrose than those from plants that were not predarkened. Referring to Figure 2 (right side, 10 mmol l - t sucrose applied to the basal end, and 2.5 h translocation time), it now becomes clear that the backward (basipetal) movement of the labeled assimilates must have been caused by the light effect. Photosynthesis had compensated for the sucrose effect. The values are in good agreement with those given in Figure 4.

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Fig. 4. Effect of different light intensities and sucrose concentrations on translocation of ~C-labeled assimilates. Shaded areas of the table are indicated as "light effect", "light and sucrose effect", and "sucrose effect", as recorded in that type of experiments shown in Figure 3. Time schedule and experimental conditions as in Figure 3. l l x = _ 2 2 x 1 0 ~ ~ -~

Darkening either the basal or the apical half of the leaf strip results in its becoming a strong sink (Heyser et al., 1975, Figs. 2.6, 2.7). The following experiment was designed to determine whether externally supplied sucrose solutions are able to counteract the dark sink. Results are recorded in Figure 5. Unilateral application of sucrose solutions of increasing concentration to the darkened end (S) of the leaf strip showed the first perceptible influence on basipetal phloem transport with 25 mmol 1- 1 Sucrose of higher concentrations " p u s h " the labeled assimilates to the opposite, illuminated ends, whether acropetal or basipetal. Regardless, the darkened area remained an effective sink. Sucrose solutions, applied to the illuminated end of the leaf strip (middle section of Fig. 5) strengthened the dark sink at every level of concentration, including 1000 mmol l - 1 sucrose. It should be possible to determine what concentration of sucrose solution compensates the dark sink-

W. Heyser et al. : Influence of Sucrose on Phloem Transport

149

solutions within the range of physiological concentrations. In the following experiment, equimolar sucrose solutions were applied to both ends of a leaf strip. Results showed two different effects occurring in leaf 10 mM AB strips taken from predarkened plants and kept dark except for the 20 rain labeling period (Fig. 6): (1) Natural polarity, i.e., basipetal transport of the labeled assimilates, was progressively reduced the longer the leaf strips were pretreated with sucrose solutions. Obviously, flooding the leaf strip with sucrose i!!!!!i~!!!!{!!!!!!~!~!::! { ~{