Planta (Berl.)128, 185-193 (1976)
Pl~.OH~ 9 by Springer-Verlag 1976
Translocation and Incorporation of 14C into the Petiole from Different Regions within Developing Cottonwood Leaves J.G. Isebrands*, Richard E. Dickson**, and Philip R. Larson** North Central Forest Experiment Station, U.S. Department of Agriculture, Forest Service, Rhinelander, Wisconsin 54501, USA
Summary. The ability of a developing cottonwood (Populus deltoides Bartr.) leaf to export 14C-labeled assimilates begins at the lamina tip and progresses basipetally with increasing LPI. This progression indicates that portions of leaves function quasi-independently in their ability to export 14C-photosynthate. Although most of the exported radioactivity was recovered in the petiole as water-80% alcohol-soluble compounds, there was also substantial incorporation into the chloroform and insoluble fractions. This observation indicates that assimilates translocated from the lamina are used in structural development of the petiole. Freeze substitution and epoxy embedding were used to prepare microautoradiographs for localization of water-soluble compounds. Radioactivity was found in all cell types within specific subsidiary bundles of the petiole. However, radioactive assimilates appeared to move from the translocation pathway in the phloem toward active sinks in the walls of the expanding metaxylem cells. Translocation in the mature xylem vessels was not observed.
Introduction
Different parts within a leaf import and export photosynthate quasi-independently, each according to its stage of development (Biddulph and Cory, 1965; Wardlaw, 1968). We have demonstrated in previous studies that different portions of cottonwood leaves vary in their ability to incorporate 14C when individual leaves were photosynthetically fed ~4CO2 (Larson etaI., 1972). For example, the percentage of total 14C incorporated decreased in the lamina tip and increased in the base with increasing leaf age. These results indicate that additional information is needed on the ability of various portions of a developing leaf to export pho* Wood Anatomist. ** Plant Physiologist.
tosynthate. Therefore, in this study we have augmented our previous work by labeling selected portions of leaves. To elucidate the translocation pathway to and from different portions of the leaf, it is first necessary to examine the anatomical organization of the petiole in relation to the lamina. The most useful sampling position in the petiole is where the individual bundles are discrete and easily discernible (Schmitz, 1970). However, within the petiole, the vasculature varies with position depending upon the frequency of anastomoses (Howard, 1974). In cottonwood the petiolar bundles are relatively discrete 1 cm below the juncture of the lamina and petiole. The petiole itself is thought to play a passive role in the translocation process (Mortimer, 1965), and the vascular pathways of import and export through the petiole are not well established. Investigators agree that export from a developing leaf into the petiole occurs in the phloem, but there is much less agreement concerning the high levels of labeled metabolites that occur in the xylem (Jones et al., 1959; Biddulph and Cory, 1965; Bieleski, 1966; Kipps and Boulter, 1974). Decisions as to whether photosynthates are moving in the phloem or the xylem along the translocation pathway are usually based on the interpretation of autoradiographs; these autoradiographs often suffer from poor resolution because of inadequate techniques. Conclusions about translocation pathways have often been drawn from suface autoradiographs when in fact microautoradiography of the vascular bundles was necessary for proper interpretation. Furthermore, discrepancies also exist among techniques for the application of labeled compounds to leaves as well as for the localization of water-soluble compounds (Eschrich and Fritz, 1972). We employed the improved methods of Fisher (1972) and Fisher and Housley (1972) for freeze substitution and microautoradiography to localize 14C_radioactivity within the developing vascular bundles of the cottonwood petiole. The objectives of this study were (1) to investigate
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J.G. Isebrands et af. : Translocation and 14C Incorporation in Populus Leaves
the export of photosynthetically fixed 14C from specific regions within young cottonwood leaves by labeling individual veins, (2) to determine what major chemical fractions were labeled in the petiole, and (3) to determine the vascular pathway of the 14C-radioactivity as it is exported into and through the petiole. Material and Methods Plant Material. Eastern cottonwood (Populus deltoides Bartr.) plants averaging 26.2 cm in height were grown from seed in sand culture with nutrient solution in a controlled-environment room (Dickson and Larson, 1975). Plants were selected at comparable morphological and anatomical stages of development when they reached a plastochron index (PI) of 16.0 (Larson and Isebrands, 1971). At PI 16.0, the 16th leaf from the base (index leaf) is 2 cm in length and has a leaf plastochron index (LPI) of 0. At LPI 4, the leaf lamina is approximately one-half expanded, with a mature lamina tip and immature base and petiole; at LPI 6, it is almost fully expanded and photosynthetically mature; and at LPt 7, the lamina is fully expanded and mature. Such a vertical aging series from LPI 0 to 7 allows one to study the ontogenetic development of the cottonwood leaf (Dickmann, 1971).
Experiment I." Translocation by Individual. Veins To determine the export pathways from portions of leaves at various stages of development, three lamina positions- tip, middle, b a s e and three developmental stages- LPI 4, 5, and 6 - w e r e labeled with 1"CO2 during photosynthesis (Fig. l). Labeling for each developmental stage and each lamina position within the leaf was replicated 3 times, each on a different plant for a total of 27 plants. The
~4CO2 was released by slowly adding 1 ml of 10% lactic acid from a syringe into the side arm of a standard Warburg flask which contained 30 gCi of dry Na214CO3. The flask opening was constricted to 2 cm 2 with a plastic insert, and was attached with lanolin to the abaxial surface of the leaf beneath the vein to be treated. The plants were preconditioned for 1 h, and each leaf was then treated for 1 h in a chamber at a light intensity of 5 x 105 ergs cm -2 sec -1 provided by four 150 w photofloods suspended in a waterbath. The plants were returned to the controlled environment room for an additional translocation period of 1 h. At harvest, a l-cm sample was collected about 1 cm below the juncture of the petiole andlamina. The petiole samples were digested with NCS Solubilizer (Amersham-Searle)x for liquid scintillation counting as described previously (Larson et al., 1972) to determine the amount of activity translocated. The treated laminae were quick-frozen at - 8 5 C in a FTS Multi-Cool ~ and freeze-dried for the preparation of whole-leaf autoradiographs.
Experiment H: Chemical Fractionation of Exported Products in the Petiole In this experiment, leaves at LPI 4 and 7 were supplied with 60 ~Ci of 14CO2 for 30 rain by treatment of the whole leaf (Larson et al., 1972). Entire petioles from two replicates of treated leaves were quick-frozen, freeze-dried, ground to a powder, and fractionated 2, 6, 12, 24 and 48h after 14C-labeling (Dickson and Larson, 1975). The fractionation procedure involved the separation into [1] water-80% alcohol-soluble compounds, [2] CHC13-soluble constituents, followed by the enzymatic removal of proteins with pronase, and non-structural carbohydrates with Clarase; and [3] the remaining residue, comprised of insoluble constituents such as hemicelluloses, cellulose and other structural compounds. All fractions were solubilized in NCS as in Expt. I.
Experiment Ill: Localization of Translocation Products ( Microautoradiography ) In this experiment the midvein at the lamina tip was labeled and the location of 14C within the vascular system of the petiole was examined by microautoradiography. To insure adequate radioactivity in the petiole, a 2 cm 2 spot located on the abaxial side of the midvein at the tip of each of 10 leaves at LPI 6 was treated with 60 gCi laCO2 for 2 h as described in Expt. I. After a 2 h translocation period (i.e., 4 h after initial release of ik4CO2), a 1 mm cross section of the petiole was collected at the same sampling position as in Expt. I. The samples were quick-frozen, freeze-substituted in acetone, embedded in Spurr's (1969) low-viscosity resin, and sectioned on the ultramicrotome (2 p.m) for microautoradiography (Fisher, 1972; Fisher and Housley, 1972).
Results
Experiment I: Translocation into Petiole from Different Portions of the Lamina The ability of the lamina to export photosynthate into the petiole increased basipetally as the young cottonwood leaf developed. At LPI 4, when the lamina had attained approximately one-half its mature size, only Fig. 1. Mature leaf showing the three treatment positions: tip, middle and base. Arrow = sampling position in the petiole
1 Reference to a trade name is for the convenience of the reader and should not be interpreted as endorsement by the U.S. Forest Service.
J.G. Isebrands et al. : Translocation and '4C Incorporation in Populus Leaves
187
Table 1. Radioactivity in treatment spots and 14C-activity recovered from petioles after labeling different lamina positions within different-aged cottonwood leaves a Leaf plastochron index
Labeling position in lamina
Radioactivity in treatment spot
Radioactivity in petiole c
(dpmmg - I 10 3)
(dpmmg-1)
4
tip middle base
1,471 b 2,014 1,778
2,070 4 29
5
tip middle base
1,587 2,100 1,559
8,267 1,548 280
6
tip middle base
1,345 1,274 1,495
7,447 8,807 8,435
(LPI)
Treatment time = 2 h (30 IxCi applied). b Average of 5 replicates per position. Differences between positions not significantly different (1% probability level). ~ Average of 3 replicates per position.
the lamina tip exported *4C-radioactivity into the petiole. Although some '4C was fixed in the immature middle and basal portions of the lamina, almost no radioactivity was exported from them (Table 1). Movement of a4C from the precociously mature lamina tip of a leaf at LP! 4 is illustrated by the wholeleaf autoradiograph in Fig. 2a. One plastochron later (LPI 5), not only did the amount of radioactivity exported from the lamina tip increase, but the ability to export assimilates also progressed basipetally into the middle portion of the leaf (Fig. 2 b). Radioactivity determined in the petiole after treatment of the basal portion of LPI 5 remained low, but it was nonetheless higher than in the immature portions of a leaf at LPI 4 (Table 1). No radioactivity was evident moving from the treatment spot in Fig. 2 c. By LPI 6, the tip, middle and basal portions of the leaf were exporting high amounts of labeled assimilates (Table 1, Fig. 2 d-f), a fact which indicated that the leaf was approaching functional maturity. Our method of treating selected positions within the lamina resulted in very little detectable diffusion of ~4C in the mesophyll, in contrast to the procedure of Penny and Nelson (1970). Some inter-veinal tran sfer did occur, however, even in restricted spot labeling (e.g., Fig. 2 d). On the average, only 25% (1.66 x 107 dpm) of the total radioactivity presented to a leaf was incorporated (Table 1). The remaining ~ C O 2 passed directly through the leaf during treatment; this loss has been minimized in subsequent studies by sealing a glass cover-slip over the treated area. Differences in 14C-
Fig. 2 a-f. Autoradiographs of leaves after individual vein treatment at various stages of leaf development. Leaf ages and treatment positions were LPI 4, tip (a); LPI 5, middle (b); LPI 5, base (c); LPI 6, tip (d); LPI 6, middle (e); LPI 6, base (f)
incorporation between spots at various treatment positions within leaves at LPI 4 to 6 were found to be non-significant at the 1% probability level by analysis of variance (Table 1).
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J.G. Isebrands et al. : Translocation and t4C Incorporation in Populus Leaves
Table 2. Amoun.t and percent of total radioactivity in various chemical fractions within the petioles of LPI 4 and LPI 7 in cottonwood. Values=dpmmg l;in()=%a LPI 4 Fraction
LPI 7
Translocation time
Water-80% ethanol soluble u CHC13-soluble+protein Structural carbohydrates Starch Total
2h
6h
2h
6h
708 (51.3) 442 (32.0) 97 (7.0) 134 (9.7)
600 (46.9) 429 (33.6) 143 (11.2) 106 (8.3)
21,117 (93.3) 1,097 (4.9) 272 (1.2) 136 (0.6)
41,300 (77.7) 6,082 (11.4) 3,221 (6.1) 2,524 (4.8)
22,622
53,127
1,381
1,278
a Percent based on total counts in petiole. Average 2 replicates per treatment. b Combined activity from 8 extractions.
Experiment H: Chemical Fractions Recovered in Petiole At LPI 4, ca. 50% of the total radioactivity found in the petiole after treatment of the whole leaf was in the form of water-80% ethanol soluble compounds (Table 2). The amount of soluble 14C-activity declined slightly with time: this fraction consists of sugars, amino acids and organic acids (Dickson and Larson, 1975). Over 30% of the activity in the petiole at LPI 4 was in the CHC13 +protein fraction (Table 2) at both the 2 and 6 h sampling times. 14C recovery from the structural carbohydrate fraction, which included cellulose, hemicelluloses and lignin, increased from 7% after 2 h to 11.2% at the end of 6 h. This increase indicated that developing vascular tissue of the young petiole continued to incorporate photosynthate exported from its own lamina over time. Preliminary work for this study showed that there was no detectable recycling of ~4C-labeled compounds from the roots or stem into the treated leaf during these treatment times. Of the total radioactivity, 8-10% was incorporated into starch. Starch may be temporarily stored in parenchyma cells adjacent to the vascular bundles >-
F
40
Iu
30
LPI 7 i
o
5
B.
20
/
/
LPI
4
.-o
within the developing petiole for incorporation into cellular components at a later time. Two h after treatment of the whole leaf at LPI 7, 93 % of the radioactivity in the petiole was in the water80% ethanol-soluble fraction (Table 2). Percentage recoveries from the remaining fractions were relatively low, but the total recovery in dpm/mg was higher than from LPI 4. Even though the percentages were low, substantial 14C-incorporation occurred in both the protein and structural-carbohydrate fractions. Total radioactivity in the mature petiole (LPI 7) increased substantially after 6 h (Table 2). The percentage of radioactivity in the water-ethanol-soluble fraction declined while that in the other fractions increased between 2 and 6 h. The increase in both the CHC13 + protein and the structural-carbohydrate fractions indicate that cellular differentiation and vascularization continue in the petiole even after expansion of the lamina ceases. Considerable photosynthate for this cellular activity is translocated from the lamina to the petiole rather than from other leaves. Activity incorporated into the starch fraction of mature petioles also increased, as has been reported for other species by Crafts and Crisp (1971). The percentages of total activity in the petiole associated with structural carbohydrates for both LPI 4 and 7 are shown in Fig. 3 for the five translocation times. At LPI 4, the percentage of radioactivity incorporated into structural carbohydrates in the petiole peaked at 18% in 12 h and then leveled off. By contrast, incorporation at LPI 7 leveled off at around 25% after 24 h.
b- z__ z IO [aJ we, laJ o_
o/
6
i?
a'4
TRANSLOCATION
4'8 TIME
(HR)
Fig. 3. Percentage of total 14C radioactivity incorporated into structural carbohydrates in the petioles of LPI 4 and 7 with increasing translocation times. Each point represents the average of 2 replicates
Experiment III: Location of Activity within l/ascular System (Petiole Anatomy) At the node, the cottonwood vascular system consists of three leaf traces--a median or central trace (C)
J.G. Isebrands et al. : Translocation and 14C Incorporation in Populus Leaves
189
and two laterals (R and L). As these traces enter the petiole from the node, they anastomose to form the petiolar bundles. These bundles are arranged in various patterns depending upon the point of observation within the petiole (Howard, 1974). Petiolar bundles in cottonwood are subdivided into components called "subsidiary bundles" that originate in the nodal region (Larson, in press). Each subsidiary bundle is associated with a specific portion of the vascular system of the leaf (Watari, 1936; Schmitz, 1970) and is a functional subunit within the petiolar bundle. A knowledge of petiole vasculature is necessary if one is to follow the pathway of exported a4C-assimilates from portions of the leaf into the petiole. The developmental aspects of the cottonwood petiole with respect to structure and function will be the subject of a subsequent paper. The sampling position located 1 cm below the juncture of the leaf and petiole of a leaf at LPI 6 was chosen both for reproduciblity of vascular arrangement as shown in other dicotyledons (Watari, 1936; Howard, 1974) and for independence of the subsidiary bundles. At this position, each of the four petiolar bundles contain both xylem and phloem (Fig. 4). The dorsal petiolar bundle [1] consists of an upper (UD) and a lower (LD) arc. The lower arc leads directly to the lamina tip; it is composed of subsidiary bundles originating primarily from the C-trace but with lesser contributions from each of the lateral traces. The ventral petiolar bundle [2] consists of an upper (UV) and a lower (LV) arc. It is composed of subsidiary bundles originating from both the C-trace and the two lateral (R and L) leaf traces; these subsidiaries are the main contributors to the lateral veins of the leaf. Similarly, the right (RS) and left (LS) supra-ventral petiolar bundles [3 and 4] originate from the lateral leaf traces and are associated with the lowermost lateral veins of the leaf.
Microautoradiography of the Petiole
The leaf at LPI 6 was chosen for microautoradiography because at this stage of development it consistently exported 14C-radioactivity into the petiole from all portions of its lamina (Table 1). 14C-labeled photosynthates were exported from each of the three treated leaf positions--tip, middle, and base--via subsidiary bundles within different petiolar bundles. However, regardless of treatment position, exported radioactivity was located in both the phloem and xylem of the subsidiary bundles within 4 h. When the midvein of the lamina tip was treated, radioactivity was localized in the subsidiary bundles of the upper and lower arcs of the dorsal petiolar bundle (Fig. 4). The highest con-
Fig. 4. Cross-sectionof an LPI 6 petioleat samplingposition shown in Fig. 1. Each of the four petiolar bundles is associated with a specificportion of the leaf lamina. UD and LD = upper and lower arcs of the dorsal petiolar bundle (1); UV and LV=upper and lower arcs of the ventral petiolar bundle (2); RS and LS=right and left supra-ventral bundles (3 and 4), respectively. Rectangle outlines central subsidiary bundle of the lower arc of the dorsal petiole bundle. Dark field microscopy, x 90
centration of radioactivity was in the central subsidiary bundle of the lower arc connected directly to the lamina tip (Rectangle, Fig. 4). It was apparent that not only was radioactivity concentrated in specific sieve-element groups of the
190
J.G. Isebrands et al. : Translocation and 1~C Incorporation in Populus Leaves
Fig. 5. Microautoradiograph of the central subsidiary bundle of the lower arc of the dorsal bundle (rectangle, Fig. 4) after labeling of tip (LPI 6). ~4C-activity occurs in the phloem (P) and xylem (X) as well as the developing cells between them. Note concentric incorporation into the developing metaxylem vessel cell walls. Nomarski, x 352
Fig. 6. Higher magnification of Fig. 5 showing the radioactivity coincident with the secondary cell wall thickenings of the pitted metaxylem vessel elements. Phase microscopy, x 880
J.G. Isebrandset al. : Translocationand 1~C Incorporationin PopulusLeaves phloem but also in the developing cambial cells between the phloem and xylem (Fig. 5). Furthermore, the cell walls of immature metaxylem vessels were also heavily labeled. Fig. 5 shows concentric incorporation of 14C over the cell walls of developing vessels. Some radioactivity was also observed in the lumen of these cells. At a higher magnification, location of the label appeared to coincide with the secondary cell wall thickenings of the pitted metaxylem vessels (Fig. 6). However, the cell walls of mature vessel elements in the same subsidiary bundle were not labeled, indicating that most 14C-labeled assimilates in the developing xylem were being incorporated rather than being translocated.
Discussion
The results of this study indicate that the ability to export 14C-labeled assimilates from a young cottonwood leaf begins at the lamina tip and progresses basipetally with increasing leaf maturity. Therefore, portions of leaves function quasi-independently in terms of their ability to export 14C-photosynthate as shown in Pelargoniurn by Schmitz (1970). Basipetal maturation in terms of the lamina's ability to export radioactivity also confirms the synchrony of structure and function associated with leaf development (Isebrands and Larson, 1973). Maturity begins with the precocious development of the leaf tip (LPI 4) and progresses basipetally until the leaf is fully expanded and all portions are exporting (LPI 6 or 7). The cottonwood leaf apparently does not reach a plateau in export ability when the leaf is 50% expanded, as does the leaf of Beta vulgaris (Fellows and Geiger, 1974), but rather at a later stage of development (Fig. 5 0f Larson and Dickson, 1973). Furthermore, as the leaf matures, the tip gradually declines in relative export ability when compared to other portions of the lamina, as evidenced by the slightly lower export from the tip at LPI 6 than at LPI 5 (Table 1). When the entire lamina of LPI 4 was treated with a4COz, its petiole had radioactivity in both the waterethanol and the CHC13-soluble and the insoluble fractions after 2 h. Of this radioactivity, 50% was in waterethanol-soluble compounds such as sugars, amino acids and organic acids (Dickson, 1974). This agrees with the work on Phaseolus by K6cher and Leonard (1971). Wardlaw (1974) suggests that the developing petiole is an important sink for exported photosynthate from its own lamina. Our data not only confirm this idea, but also indicate that the cottonwood petiole is a major sink for imported photosynthates (Dickson and Larson, 1975). Apparently the developing petiole
191
siphons off the nearest available photosynthate from either the import or export pathways during development (Canny, 1973). Moore et al. (1974) also found a similar accumulation of activity in the petioles of Sinapis cotyledons from photosynthate produced by either the attached leaf or imported from another source. Most of the radioactive water-alcohol-soluble compounds recovered from the petioles of LPI 4 and 7 were undoubtedly in transit through the phloem. However, the high percentage of activity recovered in the CHC13 +protein fraction from the petiole of LPI 4 after 2 h (Table 2) indicated either [1] movement out of the phloem and incorporation into components necessary for vascular development, or [2] metabolism in the phloem. The increasing radioactivity recovered in the structural-carbohydrate fraction with time indicated significant incorporation into cell-wall constituents. The substantial quantities of radioactivity incorporated into membranes and cell walls in the petiole of LPI 7 are evidence that the cottonwood petiole is the last portion of the leaf to mature, as also found in Pisum (Wardlaw and Mortimer, 1970). The pathways of bidirectional translocation in the petiole and midvein of developing leaves are not well understood. Although some authors have suggested separate import-export roles for different portions of the vascular system (Biddulph and Cory, 1965; Fritz, 1973), most investigators agree that export from leaves occurs mainly in the phloem (Crafts and Crisp, 1971). Nevertheless, numerous studies have indicated the presence of labeled assimilates in the xylem of developing leaves while many investigators have interpreted xylem movement from surface autoradiographs of mature leaves (Biddulph and Cory, 1965; Bieleski, 1966; Fritz, 1973; Larson and Dickson, 1973). Various interpretations are offered in the literature for the occurrence of 14C-labeled photosynthates in the xylem. These include [1] lateral leakage from the phloem to the xylem, an artifact caused by techniques; [2] active cross transfer from the phloem to the xylem; [3] xylem translocation (e.g., translocation from the stem or roots), and [4] xylem incorporation. Unfortunately, interpretation has been complicated by discrepancies betwen various labeling techniques. For example, externally applied labeled compounds and assimilated label can move by different pathways (Trip and Gorham, 1968). Furthermore, the amount of wounding during application of radioisotopes may influence the translocation mechanism (Hoddinott and Gorham, 1974). Photosynthetic labeling should help to avoid some of these artifacts. On the other hand, vein labeling on whole-leaf autoradiographs has often been interpreted as evidence for xylem transfer. We believe that surface autoradiographs are
192
J.G. Isebrands et al. : Translocation and ~4C Incorporation in Populus Leaves
not adequate for determining the precise pathway of assimilate movement even though they are useful for determining the presence of label. Further discrepancies in the interpretation of translocation literature result from the methods employed for localization and retention of water-soluble compounds. Until recently, microautoradiographic results were highly variable because of inadvertent movement of radioactivity during various stages of histological processing. The newer techniques of freeze substitution have proven much more reliable (Fisher, 1972; Fisher and Housley, 1972). Although labeled photosynthates were found in the phloem, they were also present in the xylem and the cambial cells between the phloem and xylem. We interpret the radioactivity found in all cell types of the subsidiary bundles of cottonwood petioles as movement from the phloem translocation pathway toward an active sink for incorporation. One of the sinks is created by the secondary cell walls of the rapidly expanding metaxylem vessels. This interpretation is in contrast to the point of view of Jones et al. (1959), who implied that large amounts of permanent incorporation in the xylem elements indicated that radioactive sugars were carried in them, and to our previous work, suggesting xylem transport in mature cottonwood leaves (Larson and Dickson, 1973). Our observations indicate that the developing metaxylem is the most active sink of attached cottonwood petioles rather than the parenchyma. Such parenchyma sinks are common in detached leaves of Apium and Phaseolus as reported by Bieleski (1966), and K6cher and Leonard (1971), respectively. Furthermore, our microautoradiographs show that the incorporated label coincided perfectly with the discontinuous pitted secondary cell walls of the expanding metaxylem vessels (Fig. 6). However, this label was not present in the mature metaxylem. This difference suggests that most of the activity was being incorporated, because xylem translocation would have no doubt involved some movement in the mature vessels. Spot feeding the tips of leaves with 14CO2 is a useful technique for studying translocation in developing leaves and stems. For example, feeding of the leaf tip makes it possible to restrict most of the ~4C-radioactivity exported from a labeled leaf to the central trace, because the tips of leaves translocate primarily via subsidiary bundles of the petiole that are in continuity with those of the central leaf trace. This procedure facilitates the localization of translocation products from a source leaf because import-export patterns normally conform to vascular phyllotaxy (Larson and Dickson, 1973). Furthermore, this technique allows one to follow the ~4C-activity from a specific portion of the leaf into a specific part of the vascular
system of the stem; this specificity of labeling would no doubt aid in the study of vascular development. The authors gratefully acknowledge the technical assistance of Mr. Gary Buchschacher and Mr. Gary Garton.
References Biddulph, O., Cory, R. : Translocation of C 14 metabolites in the phloem of the bean plant. Plant Physiol. 40, 119-129 (1965) Bieleski, R.L. : Sites of accumulation in excised phloem and vascular tissues. Plant Physiol. 41,455~466 (1966) Canny, M.J.: Phloem translocation. London: Cambridge Univ. Press 1973 Crafts, A.A., Crisp, C.E. : Phloem transport in plants. San Francisco: Freeman 1971 Dickmann, D.I.: Photosynthesis and respiration by developing leaves of cottonwood (Populus deltoides Bartr.). Bot. Gaz. 132, 253-259 (1971) Dickson, R,E.: Source-sink relationships in 14C-labelling of structural carbohydrates in cottonwood. (Abstr.) Plant Physiol. 53, Suppl., 68 (1974) Dickson, R.E., Larson, P.R. : Incorporation of i4C-photosynthate into major chemical fractions of source and sink leaves of cottonwood. Plant Physiol. 56, 185-193 (1975) Eschrich, W., Fritz, E.: Microautoradiography of water soluble organic compounds. In: Microautoradiography and electron probe analysis, pp. 99-122. Ed.: Luttge, U. Berlin-HeidelbergNew York:Springer 1972 Fellows, R.J., Geiger, D.R. : Structural and physiological changes in sugar beet leaves during sink to source conversion. Plant Physiol. 54, 877-885 (1974) Fisher, D.B. : Artifacts in the embedment of water soluble compounds for light microscopy. Plant Physiol. 49, 161 165 (1972) Fisher, D.B., Housley, T.L. : The retention of water soluble compounds during freeze substitution and microautoradiography. Plant Physiol. 49, 166-171 (1972) Fritz, E. : Microautoradiographic investigations in bidirectional translocation in the phloem of Vicia faba. Planta (Bed.) 112, 169-179 (1973) Hoddinott, J., Gorham, P.R. : Translocation of 14C-labelled assimilates in petioles and phloem loops of Heracleurn lanatum. Canad. J. Bot 52, 349-353 (1974) Howard, R.A. : The stem-node-leaf continuum of the Dicotyledoneae. J. Arnold Arb. 55, 125-170 (1974) Isebrands, J.G., Larson, P.R. : Anatomical changes during leaf ontogeny in Populus deltoides. Amer. J. Bot. 60, 199-208 (1973) Jones, H. Martin, R.V., Porter, H.K.: Translocation of ~%arbon in tobacco following assimilation of 1%arbon dioxide by a single leaf. Ann. Bot 23, 494-508 (1959) Kipps, A.E., Boulter, D. : Origins of the amino acids in pods and seeds of Viciafaba L. New Phytol. 73, 675-684 (1974) K6cher, H., Leonard, O.A. : Translocation and metabolic conversion of ~4C-labelled assimilates in detached and attached leaves of Phaseolus vulgaris L. in different phases of leaf expansion. Plant Physiol. 47, 212-216 (1971) Larson, P.R. : Organization and development of the primary vascular system in Populus deltoides according to phyllotaxy. Amer. J. Bot. (in press) Larson, P.R., Dickson, R.E. : Distribution of imported t~C in developing leaves of eastern cottonwood according to phyllotaxy. Planta (Bed.) 111, 95-112 (1973) Larson, P.R., Isebrands, J.G. : The plastochron index as applied to developmental studies of cottonwood. Canad. J. For. Res. 1, 1-11 (1971)
J.G. Isebrands et al. : Translocation and ~ C Incorporation in Populus Leaves Larson, P.R., Isebrands, J.G., Dickson, R.E.: Fixation patterns of 1~C within developing leaves of eastern cottonwood. Planta 107, (Berl.) 301-314 (1972) Moore, K.G., Illsley, A., Lovell, P.H. : Effects of sucrose on petiolar carbohydrate accumulation and photosynthesis in excised Sinapis cotyledons. J. exp. Bot. 25, 887-898 (1974) Mortimer, D.C. : Translocation of the products of photosynthesis in sugar beet petioles. Canad. J. Bot. 43, 26%280 (1965) Penny, P., Nelson, C.D.: Movement within leaves and plants of 14C applied as 14CO2. Canad. J. Bot. 48, 1033-1037 (1970) Schmitz, K. : Untersuchungen zur funktionellen Anatomie des Leitgewebesystems im Blatt von Pelargonium zonale. Planta (Berl.) 92, 208-221 (1970) Spurr, A.R. : A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31M3 (1969)
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Trip, P., Gorham, P.R. : Translocation of radioactive sugars in the vascular tissues of soybean plants. Canad. J. Bot. 46, 1129 1133 (1968) Wardlaw, I.F. : The control and pattern of movement of carbohydrates in plants. Bot. Rev. 34, 79 105 (1968) Wardlaw, I.F. : Phloem transport : Physical, chemical, or impossible. Ann. Rev. Plant Physiol. 25, 515-539 (1974) Wardlaw, I.F., Mortimer, D.C.: Carbohydrate movement in pea plants in relation to axillary bud growth and vascular development. Canad. J. Bot. 48, 229-237 (1970) Watari, S. : Anatomical studies on the vascular system in the petioles of some species of Acer with notes on external morphological features. J. Fac. Sci. Univ. Tokyo, Sec. III, 5, 1-73 (1936) Received 17 June; accepted 27 June 1975
Pl~.OH~ 9 by Springer-Verlag 1976
Translocation and Incorporation of 14C into the Petiole from Different Regions within Developing Cottonwood Leaves J.G. Isebrands*, Richard E. Dickson**, and Philip R. Larson** North Central Forest Experiment Station, U.S. Department of Agriculture, Forest Service, Rhinelander, Wisconsin 54501, USA
Summary. The ability of a developing cottonwood (Populus deltoides Bartr.) leaf to export 14C-labeled assimilates begins at the lamina tip and progresses basipetally with increasing LPI. This progression indicates that portions of leaves function quasi-independently in their ability to export 14C-photosynthate. Although most of the exported radioactivity was recovered in the petiole as water-80% alcohol-soluble compounds, there was also substantial incorporation into the chloroform and insoluble fractions. This observation indicates that assimilates translocated from the lamina are used in structural development of the petiole. Freeze substitution and epoxy embedding were used to prepare microautoradiographs for localization of water-soluble compounds. Radioactivity was found in all cell types within specific subsidiary bundles of the petiole. However, radioactive assimilates appeared to move from the translocation pathway in the phloem toward active sinks in the walls of the expanding metaxylem cells. Translocation in the mature xylem vessels was not observed.
Introduction
Different parts within a leaf import and export photosynthate quasi-independently, each according to its stage of development (Biddulph and Cory, 1965; Wardlaw, 1968). We have demonstrated in previous studies that different portions of cottonwood leaves vary in their ability to incorporate 14C when individual leaves were photosynthetically fed ~4CO2 (Larson etaI., 1972). For example, the percentage of total 14C incorporated decreased in the lamina tip and increased in the base with increasing leaf age. These results indicate that additional information is needed on the ability of various portions of a developing leaf to export pho* Wood Anatomist. ** Plant Physiologist.
tosynthate. Therefore, in this study we have augmented our previous work by labeling selected portions of leaves. To elucidate the translocation pathway to and from different portions of the leaf, it is first necessary to examine the anatomical organization of the petiole in relation to the lamina. The most useful sampling position in the petiole is where the individual bundles are discrete and easily discernible (Schmitz, 1970). However, within the petiole, the vasculature varies with position depending upon the frequency of anastomoses (Howard, 1974). In cottonwood the petiolar bundles are relatively discrete 1 cm below the juncture of the lamina and petiole. The petiole itself is thought to play a passive role in the translocation process (Mortimer, 1965), and the vascular pathways of import and export through the petiole are not well established. Investigators agree that export from a developing leaf into the petiole occurs in the phloem, but there is much less agreement concerning the high levels of labeled metabolites that occur in the xylem (Jones et al., 1959; Biddulph and Cory, 1965; Bieleski, 1966; Kipps and Boulter, 1974). Decisions as to whether photosynthates are moving in the phloem or the xylem along the translocation pathway are usually based on the interpretation of autoradiographs; these autoradiographs often suffer from poor resolution because of inadequate techniques. Conclusions about translocation pathways have often been drawn from suface autoradiographs when in fact microautoradiography of the vascular bundles was necessary for proper interpretation. Furthermore, discrepancies also exist among techniques for the application of labeled compounds to leaves as well as for the localization of water-soluble compounds (Eschrich and Fritz, 1972). We employed the improved methods of Fisher (1972) and Fisher and Housley (1972) for freeze substitution and microautoradiography to localize 14C_radioactivity within the developing vascular bundles of the cottonwood petiole. The objectives of this study were (1) to investigate
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J.G. Isebrands et af. : Translocation and 14C Incorporation in Populus Leaves
the export of photosynthetically fixed 14C from specific regions within young cottonwood leaves by labeling individual veins, (2) to determine what major chemical fractions were labeled in the petiole, and (3) to determine the vascular pathway of the 14C-radioactivity as it is exported into and through the petiole. Material and Methods Plant Material. Eastern cottonwood (Populus deltoides Bartr.) plants averaging 26.2 cm in height were grown from seed in sand culture with nutrient solution in a controlled-environment room (Dickson and Larson, 1975). Plants were selected at comparable morphological and anatomical stages of development when they reached a plastochron index (PI) of 16.0 (Larson and Isebrands, 1971). At PI 16.0, the 16th leaf from the base (index leaf) is 2 cm in length and has a leaf plastochron index (LPI) of 0. At LPI 4, the leaf lamina is approximately one-half expanded, with a mature lamina tip and immature base and petiole; at LPI 6, it is almost fully expanded and photosynthetically mature; and at LPt 7, the lamina is fully expanded and mature. Such a vertical aging series from LPI 0 to 7 allows one to study the ontogenetic development of the cottonwood leaf (Dickmann, 1971).
Experiment I." Translocation by Individual. Veins To determine the export pathways from portions of leaves at various stages of development, three lamina positions- tip, middle, b a s e and three developmental stages- LPI 4, 5, and 6 - w e r e labeled with 1"CO2 during photosynthesis (Fig. l). Labeling for each developmental stage and each lamina position within the leaf was replicated 3 times, each on a different plant for a total of 27 plants. The
~4CO2 was released by slowly adding 1 ml of 10% lactic acid from a syringe into the side arm of a standard Warburg flask which contained 30 gCi of dry Na214CO3. The flask opening was constricted to 2 cm 2 with a plastic insert, and was attached with lanolin to the abaxial surface of the leaf beneath the vein to be treated. The plants were preconditioned for 1 h, and each leaf was then treated for 1 h in a chamber at a light intensity of 5 x 105 ergs cm -2 sec -1 provided by four 150 w photofloods suspended in a waterbath. The plants were returned to the controlled environment room for an additional translocation period of 1 h. At harvest, a l-cm sample was collected about 1 cm below the juncture of the petiole andlamina. The petiole samples were digested with NCS Solubilizer (Amersham-Searle)x for liquid scintillation counting as described previously (Larson et al., 1972) to determine the amount of activity translocated. The treated laminae were quick-frozen at - 8 5 C in a FTS Multi-Cool ~ and freeze-dried for the preparation of whole-leaf autoradiographs.
Experiment H: Chemical Fractionation of Exported Products in the Petiole In this experiment, leaves at LPI 4 and 7 were supplied with 60 ~Ci of 14CO2 for 30 rain by treatment of the whole leaf (Larson et al., 1972). Entire petioles from two replicates of treated leaves were quick-frozen, freeze-dried, ground to a powder, and fractionated 2, 6, 12, 24 and 48h after 14C-labeling (Dickson and Larson, 1975). The fractionation procedure involved the separation into [1] water-80% alcohol-soluble compounds, [2] CHC13-soluble constituents, followed by the enzymatic removal of proteins with pronase, and non-structural carbohydrates with Clarase; and [3] the remaining residue, comprised of insoluble constituents such as hemicelluloses, cellulose and other structural compounds. All fractions were solubilized in NCS as in Expt. I.
Experiment Ill: Localization of Translocation Products ( Microautoradiography ) In this experiment the midvein at the lamina tip was labeled and the location of 14C within the vascular system of the petiole was examined by microautoradiography. To insure adequate radioactivity in the petiole, a 2 cm 2 spot located on the abaxial side of the midvein at the tip of each of 10 leaves at LPI 6 was treated with 60 gCi laCO2 for 2 h as described in Expt. I. After a 2 h translocation period (i.e., 4 h after initial release of ik4CO2), a 1 mm cross section of the petiole was collected at the same sampling position as in Expt. I. The samples were quick-frozen, freeze-substituted in acetone, embedded in Spurr's (1969) low-viscosity resin, and sectioned on the ultramicrotome (2 p.m) for microautoradiography (Fisher, 1972; Fisher and Housley, 1972).
Results
Experiment I: Translocation into Petiole from Different Portions of the Lamina The ability of the lamina to export photosynthate into the petiole increased basipetally as the young cottonwood leaf developed. At LPI 4, when the lamina had attained approximately one-half its mature size, only Fig. 1. Mature leaf showing the three treatment positions: tip, middle and base. Arrow = sampling position in the petiole
1 Reference to a trade name is for the convenience of the reader and should not be interpreted as endorsement by the U.S. Forest Service.
J.G. Isebrands et al. : Translocation and '4C Incorporation in Populus Leaves
187
Table 1. Radioactivity in treatment spots and 14C-activity recovered from petioles after labeling different lamina positions within different-aged cottonwood leaves a Leaf plastochron index
Labeling position in lamina
Radioactivity in treatment spot
Radioactivity in petiole c
(dpmmg - I 10 3)
(dpmmg-1)
4
tip middle base
1,471 b 2,014 1,778
2,070 4 29
5
tip middle base
1,587 2,100 1,559
8,267 1,548 280
6
tip middle base
1,345 1,274 1,495
7,447 8,807 8,435
(LPI)
Treatment time = 2 h (30 IxCi applied). b Average of 5 replicates per position. Differences between positions not significantly different (1% probability level). ~ Average of 3 replicates per position.
the lamina tip exported *4C-radioactivity into the petiole. Although some '4C was fixed in the immature middle and basal portions of the lamina, almost no radioactivity was exported from them (Table 1). Movement of a4C from the precociously mature lamina tip of a leaf at LP! 4 is illustrated by the wholeleaf autoradiograph in Fig. 2a. One plastochron later (LPI 5), not only did the amount of radioactivity exported from the lamina tip increase, but the ability to export assimilates also progressed basipetally into the middle portion of the leaf (Fig. 2 b). Radioactivity determined in the petiole after treatment of the basal portion of LPI 5 remained low, but it was nonetheless higher than in the immature portions of a leaf at LPI 4 (Table 1). No radioactivity was evident moving from the treatment spot in Fig. 2 c. By LPI 6, the tip, middle and basal portions of the leaf were exporting high amounts of labeled assimilates (Table 1, Fig. 2 d-f), a fact which indicated that the leaf was approaching functional maturity. Our method of treating selected positions within the lamina resulted in very little detectable diffusion of ~4C in the mesophyll, in contrast to the procedure of Penny and Nelson (1970). Some inter-veinal tran sfer did occur, however, even in restricted spot labeling (e.g., Fig. 2 d). On the average, only 25% (1.66 x 107 dpm) of the total radioactivity presented to a leaf was incorporated (Table 1). The remaining ~ C O 2 passed directly through the leaf during treatment; this loss has been minimized in subsequent studies by sealing a glass cover-slip over the treated area. Differences in 14C-
Fig. 2 a-f. Autoradiographs of leaves after individual vein treatment at various stages of leaf development. Leaf ages and treatment positions were LPI 4, tip (a); LPI 5, middle (b); LPI 5, base (c); LPI 6, tip (d); LPI 6, middle (e); LPI 6, base (f)
incorporation between spots at various treatment positions within leaves at LPI 4 to 6 were found to be non-significant at the 1% probability level by analysis of variance (Table 1).
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J.G. Isebrands et al. : Translocation and t4C Incorporation in Populus Leaves
Table 2. Amoun.t and percent of total radioactivity in various chemical fractions within the petioles of LPI 4 and LPI 7 in cottonwood. Values=dpmmg l;in()=%a LPI 4 Fraction
LPI 7
Translocation time
Water-80% ethanol soluble u CHC13-soluble+protein Structural carbohydrates Starch Total
2h
6h
2h
6h
708 (51.3) 442 (32.0) 97 (7.0) 134 (9.7)
600 (46.9) 429 (33.6) 143 (11.2) 106 (8.3)
21,117 (93.3) 1,097 (4.9) 272 (1.2) 136 (0.6)
41,300 (77.7) 6,082 (11.4) 3,221 (6.1) 2,524 (4.8)
22,622
53,127
1,381
1,278
a Percent based on total counts in petiole. Average 2 replicates per treatment. b Combined activity from 8 extractions.
Experiment H: Chemical Fractions Recovered in Petiole At LPI 4, ca. 50% of the total radioactivity found in the petiole after treatment of the whole leaf was in the form of water-80% ethanol soluble compounds (Table 2). The amount of soluble 14C-activity declined slightly with time: this fraction consists of sugars, amino acids and organic acids (Dickson and Larson, 1975). Over 30% of the activity in the petiole at LPI 4 was in the CHC13 +protein fraction (Table 2) at both the 2 and 6 h sampling times. 14C recovery from the structural carbohydrate fraction, which included cellulose, hemicelluloses and lignin, increased from 7% after 2 h to 11.2% at the end of 6 h. This increase indicated that developing vascular tissue of the young petiole continued to incorporate photosynthate exported from its own lamina over time. Preliminary work for this study showed that there was no detectable recycling of ~4C-labeled compounds from the roots or stem into the treated leaf during these treatment times. Of the total radioactivity, 8-10% was incorporated into starch. Starch may be temporarily stored in parenchyma cells adjacent to the vascular bundles >-
F
40
Iu
30
LPI 7 i
o
5
B.
20
/
/
LPI
4
.-o
within the developing petiole for incorporation into cellular components at a later time. Two h after treatment of the whole leaf at LPI 7, 93 % of the radioactivity in the petiole was in the water80% ethanol-soluble fraction (Table 2). Percentage recoveries from the remaining fractions were relatively low, but the total recovery in dpm/mg was higher than from LPI 4. Even though the percentages were low, substantial 14C-incorporation occurred in both the protein and structural-carbohydrate fractions. Total radioactivity in the mature petiole (LPI 7) increased substantially after 6 h (Table 2). The percentage of radioactivity in the water-ethanol-soluble fraction declined while that in the other fractions increased between 2 and 6 h. The increase in both the CHC13 + protein and the structural-carbohydrate fractions indicate that cellular differentiation and vascularization continue in the petiole even after expansion of the lamina ceases. Considerable photosynthate for this cellular activity is translocated from the lamina to the petiole rather than from other leaves. Activity incorporated into the starch fraction of mature petioles also increased, as has been reported for other species by Crafts and Crisp (1971). The percentages of total activity in the petiole associated with structural carbohydrates for both LPI 4 and 7 are shown in Fig. 3 for the five translocation times. At LPI 4, the percentage of radioactivity incorporated into structural carbohydrates in the petiole peaked at 18% in 12 h and then leveled off. By contrast, incorporation at LPI 7 leveled off at around 25% after 24 h.
b- z__ z IO [aJ we, laJ o_
o/
6
i?
a'4
TRANSLOCATION
4'8 TIME
(HR)
Fig. 3. Percentage of total 14C radioactivity incorporated into structural carbohydrates in the petioles of LPI 4 and 7 with increasing translocation times. Each point represents the average of 2 replicates
Experiment III: Location of Activity within l/ascular System (Petiole Anatomy) At the node, the cottonwood vascular system consists of three leaf traces--a median or central trace (C)
J.G. Isebrands et al. : Translocation and 14C Incorporation in Populus Leaves
189
and two laterals (R and L). As these traces enter the petiole from the node, they anastomose to form the petiolar bundles. These bundles are arranged in various patterns depending upon the point of observation within the petiole (Howard, 1974). Petiolar bundles in cottonwood are subdivided into components called "subsidiary bundles" that originate in the nodal region (Larson, in press). Each subsidiary bundle is associated with a specific portion of the vascular system of the leaf (Watari, 1936; Schmitz, 1970) and is a functional subunit within the petiolar bundle. A knowledge of petiole vasculature is necessary if one is to follow the pathway of exported a4C-assimilates from portions of the leaf into the petiole. The developmental aspects of the cottonwood petiole with respect to structure and function will be the subject of a subsequent paper. The sampling position located 1 cm below the juncture of the leaf and petiole of a leaf at LPI 6 was chosen both for reproduciblity of vascular arrangement as shown in other dicotyledons (Watari, 1936; Howard, 1974) and for independence of the subsidiary bundles. At this position, each of the four petiolar bundles contain both xylem and phloem (Fig. 4). The dorsal petiolar bundle [1] consists of an upper (UD) and a lower (LD) arc. The lower arc leads directly to the lamina tip; it is composed of subsidiary bundles originating primarily from the C-trace but with lesser contributions from each of the lateral traces. The ventral petiolar bundle [2] consists of an upper (UV) and a lower (LV) arc. It is composed of subsidiary bundles originating from both the C-trace and the two lateral (R and L) leaf traces; these subsidiaries are the main contributors to the lateral veins of the leaf. Similarly, the right (RS) and left (LS) supra-ventral petiolar bundles [3 and 4] originate from the lateral leaf traces and are associated with the lowermost lateral veins of the leaf.
Microautoradiography of the Petiole
The leaf at LPI 6 was chosen for microautoradiography because at this stage of development it consistently exported 14C-radioactivity into the petiole from all portions of its lamina (Table 1). 14C-labeled photosynthates were exported from each of the three treated leaf positions--tip, middle, and base--via subsidiary bundles within different petiolar bundles. However, regardless of treatment position, exported radioactivity was located in both the phloem and xylem of the subsidiary bundles within 4 h. When the midvein of the lamina tip was treated, radioactivity was localized in the subsidiary bundles of the upper and lower arcs of the dorsal petiolar bundle (Fig. 4). The highest con-
Fig. 4. Cross-sectionof an LPI 6 petioleat samplingposition shown in Fig. 1. Each of the four petiolar bundles is associated with a specificportion of the leaf lamina. UD and LD = upper and lower arcs of the dorsal petiolar bundle (1); UV and LV=upper and lower arcs of the ventral petiolar bundle (2); RS and LS=right and left supra-ventral bundles (3 and 4), respectively. Rectangle outlines central subsidiary bundle of the lower arc of the dorsal petiole bundle. Dark field microscopy, x 90
centration of radioactivity was in the central subsidiary bundle of the lower arc connected directly to the lamina tip (Rectangle, Fig. 4). It was apparent that not only was radioactivity concentrated in specific sieve-element groups of the
190
J.G. Isebrands et al. : Translocation and 1~C Incorporation in Populus Leaves
Fig. 5. Microautoradiograph of the central subsidiary bundle of the lower arc of the dorsal bundle (rectangle, Fig. 4) after labeling of tip (LPI 6). ~4C-activity occurs in the phloem (P) and xylem (X) as well as the developing cells between them. Note concentric incorporation into the developing metaxylem vessel cell walls. Nomarski, x 352
Fig. 6. Higher magnification of Fig. 5 showing the radioactivity coincident with the secondary cell wall thickenings of the pitted metaxylem vessel elements. Phase microscopy, x 880
J.G. Isebrandset al. : Translocationand 1~C Incorporationin PopulusLeaves phloem but also in the developing cambial cells between the phloem and xylem (Fig. 5). Furthermore, the cell walls of immature metaxylem vessels were also heavily labeled. Fig. 5 shows concentric incorporation of 14C over the cell walls of developing vessels. Some radioactivity was also observed in the lumen of these cells. At a higher magnification, location of the label appeared to coincide with the secondary cell wall thickenings of the pitted metaxylem vessels (Fig. 6). However, the cell walls of mature vessel elements in the same subsidiary bundle were not labeled, indicating that most 14C-labeled assimilates in the developing xylem were being incorporated rather than being translocated.
Discussion
The results of this study indicate that the ability to export 14C-labeled assimilates from a young cottonwood leaf begins at the lamina tip and progresses basipetally with increasing leaf maturity. Therefore, portions of leaves function quasi-independently in terms of their ability to export 14C-photosynthate as shown in Pelargoniurn by Schmitz (1970). Basipetal maturation in terms of the lamina's ability to export radioactivity also confirms the synchrony of structure and function associated with leaf development (Isebrands and Larson, 1973). Maturity begins with the precocious development of the leaf tip (LPI 4) and progresses basipetally until the leaf is fully expanded and all portions are exporting (LPI 6 or 7). The cottonwood leaf apparently does not reach a plateau in export ability when the leaf is 50% expanded, as does the leaf of Beta vulgaris (Fellows and Geiger, 1974), but rather at a later stage of development (Fig. 5 0f Larson and Dickson, 1973). Furthermore, as the leaf matures, the tip gradually declines in relative export ability when compared to other portions of the lamina, as evidenced by the slightly lower export from the tip at LPI 6 than at LPI 5 (Table 1). When the entire lamina of LPI 4 was treated with a4COz, its petiole had radioactivity in both the waterethanol and the CHC13-soluble and the insoluble fractions after 2 h. Of this radioactivity, 50% was in waterethanol-soluble compounds such as sugars, amino acids and organic acids (Dickson, 1974). This agrees with the work on Phaseolus by K6cher and Leonard (1971). Wardlaw (1974) suggests that the developing petiole is an important sink for exported photosynthate from its own lamina. Our data not only confirm this idea, but also indicate that the cottonwood petiole is a major sink for imported photosynthates (Dickson and Larson, 1975). Apparently the developing petiole
191
siphons off the nearest available photosynthate from either the import or export pathways during development (Canny, 1973). Moore et al. (1974) also found a similar accumulation of activity in the petioles of Sinapis cotyledons from photosynthate produced by either the attached leaf or imported from another source. Most of the radioactive water-alcohol-soluble compounds recovered from the petioles of LPI 4 and 7 were undoubtedly in transit through the phloem. However, the high percentage of activity recovered in the CHC13 +protein fraction from the petiole of LPI 4 after 2 h (Table 2) indicated either [1] movement out of the phloem and incorporation into components necessary for vascular development, or [2] metabolism in the phloem. The increasing radioactivity recovered in the structural-carbohydrate fraction with time indicated significant incorporation into cell-wall constituents. The substantial quantities of radioactivity incorporated into membranes and cell walls in the petiole of LPI 7 are evidence that the cottonwood petiole is the last portion of the leaf to mature, as also found in Pisum (Wardlaw and Mortimer, 1970). The pathways of bidirectional translocation in the petiole and midvein of developing leaves are not well understood. Although some authors have suggested separate import-export roles for different portions of the vascular system (Biddulph and Cory, 1965; Fritz, 1973), most investigators agree that export from leaves occurs mainly in the phloem (Crafts and Crisp, 1971). Nevertheless, numerous studies have indicated the presence of labeled assimilates in the xylem of developing leaves while many investigators have interpreted xylem movement from surface autoradiographs of mature leaves (Biddulph and Cory, 1965; Bieleski, 1966; Fritz, 1973; Larson and Dickson, 1973). Various interpretations are offered in the literature for the occurrence of 14C-labeled photosynthates in the xylem. These include [1] lateral leakage from the phloem to the xylem, an artifact caused by techniques; [2] active cross transfer from the phloem to the xylem; [3] xylem translocation (e.g., translocation from the stem or roots), and [4] xylem incorporation. Unfortunately, interpretation has been complicated by discrepancies betwen various labeling techniques. For example, externally applied labeled compounds and assimilated label can move by different pathways (Trip and Gorham, 1968). Furthermore, the amount of wounding during application of radioisotopes may influence the translocation mechanism (Hoddinott and Gorham, 1974). Photosynthetic labeling should help to avoid some of these artifacts. On the other hand, vein labeling on whole-leaf autoradiographs has often been interpreted as evidence for xylem transfer. We believe that surface autoradiographs are
192
J.G. Isebrands et al. : Translocation and ~4C Incorporation in Populus Leaves
not adequate for determining the precise pathway of assimilate movement even though they are useful for determining the presence of label. Further discrepancies in the interpretation of translocation literature result from the methods employed for localization and retention of water-soluble compounds. Until recently, microautoradiographic results were highly variable because of inadvertent movement of radioactivity during various stages of histological processing. The newer techniques of freeze substitution have proven much more reliable (Fisher, 1972; Fisher and Housley, 1972). Although labeled photosynthates were found in the phloem, they were also present in the xylem and the cambial cells between the phloem and xylem. We interpret the radioactivity found in all cell types of the subsidiary bundles of cottonwood petioles as movement from the phloem translocation pathway toward an active sink for incorporation. One of the sinks is created by the secondary cell walls of the rapidly expanding metaxylem vessels. This interpretation is in contrast to the point of view of Jones et al. (1959), who implied that large amounts of permanent incorporation in the xylem elements indicated that radioactive sugars were carried in them, and to our previous work, suggesting xylem transport in mature cottonwood leaves (Larson and Dickson, 1973). Our observations indicate that the developing metaxylem is the most active sink of attached cottonwood petioles rather than the parenchyma. Such parenchyma sinks are common in detached leaves of Apium and Phaseolus as reported by Bieleski (1966), and K6cher and Leonard (1971), respectively. Furthermore, our microautoradiographs show that the incorporated label coincided perfectly with the discontinuous pitted secondary cell walls of the expanding metaxylem vessels (Fig. 6). However, this label was not present in the mature metaxylem. This difference suggests that most of the activity was being incorporated, because xylem translocation would have no doubt involved some movement in the mature vessels. Spot feeding the tips of leaves with 14CO2 is a useful technique for studying translocation in developing leaves and stems. For example, feeding of the leaf tip makes it possible to restrict most of the ~4C-radioactivity exported from a labeled leaf to the central trace, because the tips of leaves translocate primarily via subsidiary bundles of the petiole that are in continuity with those of the central leaf trace. This procedure facilitates the localization of translocation products from a source leaf because import-export patterns normally conform to vascular phyllotaxy (Larson and Dickson, 1973). Furthermore, this technique allows one to follow the ~4C-activity from a specific portion of the leaf into a specific part of the vascular
system of the stem; this specificity of labeling would no doubt aid in the study of vascular development. The authors gratefully acknowledge the technical assistance of Mr. Gary Buchschacher and Mr. Gary Garton.
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