The estimation of sugar concentration in individual sieve-tube elements by negative staining

The presence of water-soluble compounds in sectioned plant tissue can be visualized in negative contrast by freeze-substitution in acetone, followed by embedment in Epon containing 6% Sudan B. The contents of mature sieve tubes and companion cells of bean (Phaseolus vulgaris L.) showed strong, and mostly uniform, negative staining. The degree of negative staining was measured by microspectrophotometry. Since, in sieve tubes, virtually all of the solute is the translocated sugar, the sugar concentration can be estimated by comparison with similar measurements made on sections from pith blocks which were infiltrated with sugar solutions and processed by the same procedures. Sieve tubes contained a solution of about 11.2% (w/v) sucrose; companion cells contained a similar concentration of sucrose. Negative staining, and therefore the sucrose concentration, in immature sieve-tube elements and companion cells was much less than in their mature counterparts.This work was presented in part at a joint U.S.-Australia Conference on Transport and Transfer Processes in Plants, Canberra, Australia, December 15–20, 1975; see Fisher (1976)     

The functional significance of phloem anastomoses in stems ofDahlia pinnata Cav.

The role of phloem anastomoses in translocation was studied experimentally in intact and wounded internodes ofDahlia pinnata Cav. Translocation was visualized with fluorescein, a fluorescent dye capable of movement in the phloem. Translocation was analyzed in large areas of living phloem tissue which were peeled from the xylem at the cambium region. Under normal conditions, fluorescein was observed in sieve tubes of the longitudinal phloem strands but very rarely in the sieve tubes of the anastomoses. However, when a few longitudinal strands were severed, fluorescein was translocated through the anastomoses located around the wound within 24 h. It is suggested that the phloem anastomoses in mature internodes ofDahlia serve mainly as an emergency system which enable a fast response to damage by providing alternative pathways for assimilates around the stem. A possible regulatory mechanism based on differences in resistance to flow in longitudinal versus lateral sieve tubes is discussed. This study was supported by an International Scientific Exchange award and an operating grant to C.A.P. from the Natural Sciences and Engineering Research Council of Canada.     

Phloem unloading in tobacco sink leaves: insensitivity to anoxia indicates a symplastic pathway

Phloem unloading in transition sink leaves of tobacco (Nicotiana tabacum L.) was analyzed by quantitative autoradiography. Detectable levels of labeled photoassimilates entered sink leaves approx. 1 h after source leaves were provided with ¹⁴CO₂. Samples of tissue were removed from sink leaves when label was first detected and further samples were taken at the end of an experimental phloem-unloading period. The amount of label in veins and in surrounding cells was determined by microdensitometry of autoradiographs using a microspectrophotometer. Photoassimilate unloaded from first-, second-and third-order veins but not from smaller veins. Import termination in individual veins was gradual. Import by the sink leaf was completely inhibited by exposing the sink leaf to anaerobic conditions, by placing the entire plant in the cold, or by steam-girdling the sink-leaf petiole. Phloem unloading was completely inhibited by cold; however, phloem unloading continued when the sink-leaf petiole was steam girdled or when the sink leaf was exposed to a N₂ atmosphere. Compartmental efflux-analysis indicated that only a small percentage of labeled nutrients was present in the free space after unloading from sink-leaf veins in a N₂ atmosphere. The results are consistent with passive symplastic transfer of photoassimilates from phloem to surrounding cells.Symbol VI radio of ¹⁴C in veins and interveinal tissue     

A morphometric analysis of the phloem-unloading pathway in developing tobacco leaves

A morphometric analysis of developing leaves of Nicotiana tabacum L. was conducted to determine whether imported photoassimilates could be unloaded by symplastic transport and whether interruption of symplastic transport could account for termination of import. Five classes of veins were recognized, based on numbers of cells in transverse section. Photoassimilate is unloaded primarily from Class III veins in tissue nearing the end of the sink phase of development. Smaller veins (Class IV and V) do not transport or unload photoassimilate in sink tissue because the sieve elements of these veins are immature until after the tissue stops importing. In Class III veins the sieve element-companion cell (SE-CC) complexes are surrounded by phloem parenchyma which abuts the bundle sheath. Along the most obvious unloading route, from SE-CC complex to phloem parenchyma to bundle sheath to mesophyll cells, the frequency of plasmodesmata at each interface increases. To determine whether this pattern of plasmodesmatal contact is consistent with symplastic unloading we first demonstrated, by derivation from Fick's law that the rate of diffusion from a compartment is proportional to a number N which is equal to the ratio of surface area to volume of the compartment multiplied by the frequency of pores (plasmodesmata) which connect it to the next compartment. N was calculated for each compartment within the vein which has the SE-CC complex as its center, and was shown to be statistically the same in all cases except one. These observations are consistent with a symplastic unloading route. As the leaf tissue matures and stops importing, plasmodesmatal frequency along the unloading route decreases and contact area between cells also decreases as intercellular spaces enlarge. As a result, the number of plasmodesmata between the SE-CC complex and the first layer of mesophyll cells declines in nonimporting tissue to 34% of the number found in importing tissue, indicating that loss of symplastic continuity between the phloem and surrounding cells plays a role in termination of photoassimilate unloading.Abbreviation SE-CC sieve element-companion cell     

Synchronous pressure-potential changes in the phloem of Fraxinus americana L.

Simultaneous measurement of the pressure potential of the phloem of F. americana made on two locations on the trunk over long periods of time showed synchronous oscillations of no fixed period during the day. The simultaneous changes in pressure in two different trees indicated environmental changes were responsible for the synchrony. The coincident changes of pressure 5 m apart on the same trunk implied that either transpiration had an immediate and direct effect upon the pressure potentials developed in the phloem because of the intimate relationship of the phloem water potential and the water potential of the adjacent transpiration stream, or factors affecting phloem loading resulted in pressure changes throughout the phloem.     

A guide to the use of the exuding-stylet technique in phloem physiology

The use of exuding stylets holds considerable promise for the investigation of sieve-tube physiology. However, largely because of difficulties in cutting insect stylets, the technique has been applied to only a few plant species. Based on our experience, a comparison is made of the available means of obtaining sieve-tube exudate from the exuding stylets of phloem-feeding insects, including aphids, scale and mealybugs. Forty-one plant species and approx. 35 insect species were tested for their ability to provide stylet exudate. Stylets on all but a few of the plant species tested yielded at least some exudate, but the success rate and duration of exudation on many species were unsatisfactory for detailed investigations of phloem transport. Plant species appears to be the most important factor for obtaining reliably exuding stylets, although the size of the insect species used and the physiological condition of the plant are also important variables. Armored scale provide a simple and reliable source of exuding stylets, but are impractical for most experimental purposes. Radio-frequency microcautery of aphid stylets was substantially the most effective means of cutting stylets. Instructions are provided for constructing a microcautery unit at minimal expense, using a citizen's band radio as the radio-frequency source.Abbreviations CB citizen's band - R.F. radio frequency     

Immunocytochemical localisation of phloem lectin from Cucurbita maxima using peroxidase and colloidal-gold labels

Antibodies were raised against lectin purified from the sieve-tube exudate of Cucurbita maxima. Immunocytochemistry, using peroxidase-labelled antibodies and Protein A-colloidal gold, was employed to determine the location of the lectin within the tissues and cells of C. maxima and other cucurbit species. The anti-lectin antibodies bound to P-protein aggregates in sieve elements and companion cells, predominantly in the extrafascicular phloem of C. maxima. This may reflect the low rate of translocation in these cells. Under the electron microscope, the lectin was shown to be a component of P-protein filaments and was also found in association with the sieve-tube reticulum which lines the plasmalemma. The anti-lectin antibodies reacted with sieve-tube proteins from other species of the genus Cucurbita but showed only limited reaction with other genera. We suggest that the lectin serves to anchor P-protein filaments and associated proteins to the parietal layer of sieve elements.Abbreviation SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Photoassimilate-transport characteristics of nonchlorophyllous and green tissue in variegated leaves of Coleus blumei Benth

The nonchlorophyllous (albino) tissue of mature C. blumei leaves is a sink for photoassimilate. Transport from the green to the albino region of the same leaf was inhibited by cold and anoxia. When the green tissue of mature leaves was removed, the remaining albino portion imported labeled translocate from other mature leaves in the phloem. Photoassimilate unloading in the albino region of mature leaves was studied by quantitative autoradiography. The unloading was inhibited by cold but not by anoxia. No labeled photoassimilate could be detected in the free space of mature albino tissue by compartmental efflux analysis as phloem unloading proceeded in a N₂ atmosphere, indicating that unloading, may occur by a symplastic pathway as it apparently does in sink leaves of other species. The minor veins of mature albino leaf tissue did not accumulate exogenous [¹⁴C]sucrose. Minor veins of green tissue in the same leaves accumulated [¹⁴C]sucrose but, in contrast to other species studied to date, this accumulation was insensitive to the inhibitor p-chloromercuribenzensulfonic acid (PCMBS).In its capacity to import and unload photoassimilate, and in the inability, of the minor veins to accumulate exogenous sucrose, the albino region of the mature C. blumei lamina differs from mature albino tobacco leaves and darkened mature leaves of other species. This, together with evidence indicating that phloem loading in C. blumei and other species may occur by different routes and with different sensitivity to PCMBS, indicates that the mechanism of transfer of photoassimilates between veins and surrounding tissues, and the mechanism of the sink-source transition, may not be the same in the leaves of all species. It is speculated that the unusual properties of the C. blumei leaf may be a consequence of the presence, in the minor veins, of intermediary cells, large companion cells connected to the bundle sheath by abundant plasmodesmata.Abbreviation PCMBS p-chloromercuribenzenesulfonic acid     

Sieve-element differentiation and fluoresceine translocation in wound-phloem of pea roots after complete severance of the stele

Experimental interruption of the root stele of Pisum sativum L. induces in the cortex tissue the development of wound-sieve tubes which bridge the wound and reconnect the vascular stumps. Outside the stele, sieve plates arise from primary pit fields. This origin is confirmed by the distribution of future sieve pores over the original parenchyma cell wall and by remnants of the pitfield cavity in developing sieve plates. Differentiation of wound-sieve elements is similar to that of bundle-sieve elements and includes the chromatolytic disintegration of nuclei as well as the development of typical sieve pores arising from pit-field plasmodesmata. The completion of first woundsieve tubes (indicated by a continuous chain of anilin-blue-positive sieve plates by-passing the wound) was observed 55–62 h after wounding. However, effective translocation, visualized with fluoresceine as a phloem-mobile marker, was not found until 10 h (on average) later. It is suggested that this time delay corresponds to the maturing of the last link within a chain of wound-sieve-tube members. Presumably, enucleate sieve elements with widened pores are a prerequisite for effective phloem translocation.Abbreviations DAPI 4,6-diamidino-2-phenylindole·2 H₂O - ER endoplasmic reticulum Preliminary results of this investigation have been presented at the International Phloem Transport Conference in Asilomar, Cal., USA 1985 (cf. Schulz 1986c)     

The microscopy of P-protein filaments in freeze-etched sieve pores

Intact vascular bundles from Nymphoides peltata (S.G. Gmel.) O. Kuntze, shown to have translocated carbon-14, were freeze-fractured and etched for electron microscopy. The interpretation of freezefractured and etched sieve pores and P-protein filaments seen in them is discussed. The entire widths of most of the sieve pores seen contained filaments separated by less than 100 nm. Their arrangement indicates too high a resistance to flow for pressure flow alone to drive translocation at known rates; pumps would be necessary at places along sieve tubes. However, calculations are presented to show that during the time taken to fix pores, by fast freezing or chemically, the filaments in them could rearrange and move further by Brownian and other motion than the distances between filaments which we need to measure. These calculations show that it is not possible, by microscopy alone, to answer the outstanding question “How are filaments arranged in translocating sieve pores?” with enough certainty to tell us whether pressure flow is adequate to explain translocation where filaments are present. The calculations are relevant also to microscopy of other cell structures which may move.     

Cytochemical localization of adenosine triphosphatase in the phloem of Pisum sativum and its relation to the function of transfer cells

The cytochemical localization of ATPase in differentiating and mature phloem cells of Pisum sativum L. has been studied using a lead precipitation technique. Phloem transfer cells at early stages of differentiation exhibit strong enzyme activity in the endoplasmic reticulum (ER) and some reaction product is deposited on the vacuolar and plasma membranes. As the phloem transfer cells mature and develop their characteristic wall structures, strong enzyme activity can be observed in association with the plasma membranes and nuclear envelopes. Mature phloem transfer cells with elaborate cell-wall ingrowths show ATPase activity evenly distributed on plasma-membrane surfaces. Differentiating sieve elements show little or no enzyme activity. When sieve elements are fully mature they have reaction product in the parietal and stacked cisternae of the ER. There is no ATPase activity associated with P-protein at any stage of sieve-element differentiation or with the sieve-element plasma membranes. It is suggested that the intensive ATPase activity on the plasma membranes of the transfer cells is evidence for a transport system involved in the active movement of photosynthetic products through these cells.Key to labeling in the figures ER endoplasmic reticulum - P parenchyma cell - PP P-protein - SE sieve element - SPP sieve-plate pore - TC transfer cell     

Intracellular redistribution of phytochrome in etiolated soybean (Glycine max L.) seedlings

The intracellular distribution of phytochrome in hypocotyl hooks of etiolated soybean (Glycine max L.) has been examined by immunofluorescence using a newly produced monoclonal antibody (Soy-1) directed to phytochrome purified from etiolated soybean shoots. Cortical cells in the hook region exhibit the strongest phytochrome-associated fluorescence, which is diffusely distributed throughout the cytosol in unirradiated, etiolated seedlings. A redistribution of immunocytochemically detectable hytochrome to discrete areas (sequestering) following irradiation with red light requires a few minutes at room temperature in soybean, whereas this redistribution is reversed rapidly following irradiation with far-red light. In contrast, sequestering in oat (Avena sativa L.) occurs within a few seconds (D. McCurdy and L. Pratt, 1986, Planta 167, 330–336) while its reversal by far-red light requires hours (J. M. Mackenzie Jr. et al., 1975, Proc. Natl. Acad. Sci. USA 72, 799–803). The time courses, however, of red-light-enhanced phytochrome pelletability and sequestering are similar for soybean as they are for oat. Thus, while these observations made with a dicotyledon are consistent with the previous conclusion derived from work with oat, namely that sequestering and enhanced pelletability are different manifestations of the same intracellular event, they are inconsistent with the hypothesis that either is a primary step in the mode of action of phytochrome.Abbreviations DIC differential interference contrast - FR far-red light - Ig immunoglobulin - Pfr, P far-red- and red-absorbing form of phytochrome, respectively - R red light This work was supported by National Science Foundation grant No. DCB-8703057.     

Phloem unloading following reactivation in predarkened mature maize leaves

Mature leaf blades of 48-h predarkened maize plants (Zea mays L. cv. Prior) were excised, and treated apically as the source (light, normal air) and basally as the sink (light or dark, air without CO₂). After providing the source portion with ¹⁴CO₂, the sink portions were harvested after 2, 7 or 14 h by freezing with liquid nitrogen, grinding, and freeze-drying. Extracts, fractionated by ionexchange resins into neutral, basic and acid fractions, were chromatographed on thin cellulose layers, and autoradiographed. Identification of labeled compounds was carried out by co-chromatography with authentic labeled substances. Activities of enzymes pertaining to the metabolism of sucrose were checked. Results show that the source supplies sucrose to the sink, where it is unloaded and metabolized by acid invertase (EC 3.2.1.26) in both the light and the dark. Starch appearing in the sink only in the light, after 7 h of re-illumination, yields labeled glucose upon hydrolysis. Although sucrose-phosphate synthetase (EC 2.4.1.14) is active in sinks and in isolated vascular-bundle fragments, it remains questionable whether sucrose unloaded from sieve tubes is metabolized by a method other than inversion. Sucrose synthetase (EC 2.4.1.13) was found to be inactive. Obviously, the main metabolite of unloaded sucrose is glucose-6-phosphate, giving access to the glycolytic pathway. The main difference between the sinks in the light and the dark is the lack of labeled glycine and serine in the dark. This indicates that in the light decarboxylation of glycine yields CO₂, which is recycled photosynthetically.Abbrevations Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - TLC thin-layer chromatography - UDPGlc uridine 5-diphosphate glucose     

Evidence against the involvement of phytochrome in UVB-induced inhibition of stem growth in green tomato plants

The effects of UVB on the kinetics of stem elongation of wild type (WT) and photomorphogenic mutants of tomato were studied by using linear voltage transducers connected to a computer. Twenty-one or twenty-six-day-old plants, grown in 12 h white light (150 μmol m⁻² s⁻¹ PAR)/12 h dark cycles, were first transferred to 200 μmol m⁻² s⁻¹ monochromatic yellow light for 12 h, then irradiated with 0.1 or 4.5 μmol m⁻² s⁻¹ UVB for 12 h and finally kept in darkness for another 24 h. The measurements of the kinetics of stem elongation started after 4 h under yellow light. Significant differences in stem growth during the irradiation with yellow light, as well as during the dark period, were found between the genotypes. In darkness, the magnitude of stem growth followed the order: tri > AC = fri > MMau > hp1. Two factors determined the large differences of growth in darkness: 1) the different stem elongation rate (SER) and 2) the different duration of the growing phase among the genotypes. In darkness the stem growth of au and hp1 mutants lasted for about 18 h, whereas it continued for the whole experimental period (36 h) in the other genotypes. UVB irradiation substantially reduced elongation growth of all genotypes (4.5 μmol m⁻² s⁻¹ being more effective than 0.1 μmol m⁻² s⁻¹). Both fluence rates of UVB induced a detectable reduction of SER already after 15 min of irradiation. Red light inhibited, while far red light promoted stem growth of all the genotypes tested. fri (phyA null), tri (phyB1 null), hp1 (exhibiting exaggerated phytochrome responses) mutants and WT tomato showed similar levels of UVB–induced inhibition of growth, while the aurea mutant showed the largest growth inhibition during the 12 h of irradiation. These results indicate that phytochrome is not directly involved in UVB control of stem elongation. The results of dichromatic irradiations UVB + red or UVB + far red indicate the presence of distinct and additive action of UVB photoreceptor and of the phytochrome system in the photoregulation of stem growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Immunoprecipitation of phytochrome from green Avena by rabbit antisera to phytochrome from etiolated Avena

Thirty-nine antiserum preparations from eight rabbits were screened for their ability to precipitate the immunochemically distinct phytochrome that is obtained from green oat (Avena sativa L.) shoots. The antisera were obtained from rabbits immunized with either proteolytically degraded, but still photoreversible, 60-kDa (kilodalton) phytochrome, or approx. 120-kDa phytochrome, both of which were purified from etiolated oat shoots. The ability of these antisera to precipitate phytochrome from green oats was independent of the size of phytochrome used for immunization. While crude antisera immunoprecipitated as much as 80% of the phytochrome isolated from green oat shoots, antibodies immunopurified from these sera with a column of highly purified, approx. 120-kDa phytochrome from etiolated oats precipitated no more than about 5–10%.Abbreviations kDa kilodalton - mU milliunit     

Transport of hexoses by the phloem of Ricinus communis L. seedlings

The sieve-tube sap of Ricinus communis L. seedlings has been analysed to determine whether or not hexoses can be taken up by the phloem. Under natural conditions, i.e. with the endosperm attached to the cotyledons, glucose and fructose occurred only in trace amounts in the sieve-tube sap. Incubation of the cotyledons with hexoses in the concentration range 25–200 mM caused a rapid and substantial uptake of hexoses into the phleom, where they appeared eventually in the sieve-tube sap at the same concentration as in the incubation medium. Phloem loading of glucose, 3-O-methyl-glucose and sorbitol occurred easily, whereas fructose was less well loaded. glucose and to a larger extent fructose were also transformed to sucrose, which was loaded into the phloem. The loading of hexoses into the sieve tubes as observed in the experimental exudation system also occurred in the intact seedling, but transloction in the latter soon came to a standstill, probably because of lack of consumption by the sink tissues. These results indicate that the virtual absence of hexoses in the sievetube sap under in-vivo conditions is not because of the inability of the phloem-loading system to transport the monosaccharides but because of the absence of sufficiently high concentrations in the apoplast.     

Detection of lectins in nodulated peanut and soybean plants

The direct double-antibody enzymelinked immunosorbent assay system was used in the detection and measurement of seed lectins from peanut (Arachis hypogaea L.) and soybean (Glycine max L.) plants (PSL and SBL, respectively) that had been inoculated with their respective rhizobia. Concentrations of PSL dropped to undetectable levels in peanut roots at 9 d and stems and leaves at 27 d after planting; SBL could no longer be detected in soybean roots at 9 d and in stems and leaves at 12 d. A lectin antigenically similar to PSL was first detected in root nodules of peanuts at 21 d reaching a maximum of 8 g/g at 29 d then decreasing to 2.5 g/g at 60 d. There was no evidence of a corresponding lectin in soybean nodules.Sugar haemagglutination inhibition tests with neuraminidase-treated human blood cells established that PSL and the peanut nodule lectin were both galactose/lactose-specific. Further tests with rabbit blood cells demonstrated a second mannosespecific lectin in peanut nodule extracts that was not detected in root extracts of four-week-old inoculated plants or six-week-old uninoculated plants, although six-week-old root extracts from inoculated plants showed weak lectin activity. The root extracts from both nodulated and uninoculated plants contained another peanut lectin that agglutinated rabbit but not human blood cells. Haemagglutination by this lectin was, however, not inhibited by simple sugars but a glycoprotein, asialothyroglobulin, was effective in this respect.Abbreviations DAS double antibody sandwich - ELISA enzyme-linked immunosorbent assay - PBS phosphate-buffered saline - PSL peanut seed lectin - SBL soybean lectin     

Transfer cells and solute uptake in minor veins of Pisum sativum leaves

Morphometric and physiological studies were conducted to determine whether the wall ingrowths of transfer cells in the minor-vein phloem of Pisum sativum L. leaves increase the capacity of the cells for solute influx. Size and number of wall ingrowths are positively correlated to the photon flux density (PFD) at which the plants are grown. An analysis of plasmodesmatal frequencies indicated that numerous plasmodesmata are present at all interfaces except those between the sieveelement-transfer-cell complex (SE-TCC) and surrounding cells where plasmodesmata are present but few in number. Flux of exogenous sucrose into the SE-TCC was estimated from kinetic profiles of net sucrose influx into leaf discs, quantitative autoradiography, and measurements of sucrose translocation. Flux based both on the saturable (carrier-mediated) and the linear components of influx was 47% greater in leaves of plants grown at high PFD (1000 mol·m^–2·s^–1) than those grown in low PFD (200 mol·m^–2·s^–1) and was paralleled by a 47% increase in SE-TCC plasmalemma surface area. Flux of endogenous photosynthate across the SE-TCC plasmalemma was calculated from carbon balance and morphometric data. The increase in flux in high-light leaves over that in low-light leaves can be explained on the basis of an increase in plasmalemma surface area. In intact leaves, a standing osmotic gradient may facilitate transport of solute into transfer cells with extensive wall elaborations.Abbreviations LPI leaf plastochron index - PCMBS p-chloromercuribenzenesulfonic acid - PFD(s) photon flux density (densities) - SE-TCC sieve-element-transfer-cell complex This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competitive Grant 90000854, and Hatch funds.     

The kinetics of sucrose concentration in the phloem of individual vascular bundles of the Ricinus communis seedling measured by nuclear magnetic resonance microimaging

The sucrose concentration was measured at 70-min intervals in the phloem of individual bundles of the hypocotyl of Ricinus seedlings by ¹H nuclear magnetic resonance (NMR) spectroscopic imaging. The sucrose concentration stayed fairly constant in all bundles for more than 7 h if the cotyledons were embedded in the endosperm or excised and incubated in 100 mM sucrose. If, however, the sucrose solution was replaced by sucrose-free buffer solution, the sucrose levels in the phloem decreased with a kinetic depending on the seedling: in some cases there was a smooth decline, in some a decline followed by a slight recovery and in some cases a clear-cut oscillation. The sucrose concentration was often not identical in the phloem of the individual bundles. The oscillations were larger in the phloem at the apex of the hypocotyl than in the phloem at the base of the hypocotyl. Cutting the petiole of one cotyledon led to a decrease in sucrose not only in the four bundles directly connected to the severed petiole but in all eight bundles of the hypocotyl. Cutting the petiole and dividing the vascular ring at the cotyledonary node and at the root crown did not prevent the decline of sucrose in all eight bundles. Therefore, a functional equilibration of translocated solutes between the eight bundles may occur within the 1-h measuring interval by radial diffusion through the parenchyma of the hypocotyl. Received 4 July 1997 / Accepted: 4 October 1997