Planta
Planta (Berl.)129, 265-269 (1975)
9 by Springer-Verlag 1976
Leaf Development and Phloem Transport in C.cm'bita pepo" Maturation of the Minor Veins Robert Turgeon* and J.A. Webb Biology Department, Carleton University Ottawa, Ontario, K1S 5B6, Canada
Summary. Young leaves of Cucurbita pepo L. were examined by whole-leaf autoradiography and serial paradermal sections were examined by light microscopy to determine whether commencement of sugar export depends upon the minor vein phloem achieving structural maturity. Maturation of these veins develops progressively from the largest toward the smallest elements with the minor veins in the distal region of the leaf maturing before those in the proximal region. Commencement of sugar export is coincident with maturation of the abaxial phloem of the minor veins delimiting the areoles. The abaxial phloem elements of the larger minor veins, which are probably capable of vein loading too but border only relatively few areoles, mature before export starts. The adaxial phloem surrounding the areoles and the xylem elements, mature in advance of the abaxial phloem and well before the beginning of sugar export. It is therefore considered unlikely that structural development alone directly governs the initiation of export. The results suggest that some other rate controlling step is involved.
Introduction
During initial development the normal growth rate of a Cucurbit leaf is maintained by importing soluble carbohydrates in the phloem from mature leaves. As leaf development progresses the growth rate declines but the photosynthetic capacity of the young leaf continues to increase thereby diminishing its dependence on the older leaves for additional carbohydrate supplies. When the leaf has grown to approximately half its maximal size this dependence is finally severed and flow in the phloem begins to reverse direction *
Present address." The Rockefeller University New York, N.Y.,
10021, U.S.A.
as the leaf now commences to export soluble sugars (Turgeon and Webb, 1975). This transition of the leaf from an importing to an exporting organ has been studied in a number of dicotyledonous species (e.g., Larson et al., 1972; Turgeon and Webb, 1973; Fellows and Geiger, 1974) and in all cases the transition has been observed to commence at the leaf tip and to pass quickly through the blade in a basipetal direction. The export of soluble carbohydrates from the lamina is the result of a multi-step transport process (Webb, 1970) commencing with the movement of photosynthesized molecules from within the chloroplasts and ending with a flow of soluble sugars into the minor vein phloem, a step generally termed "vein loading". The minor vein system permeates the mesophyll tissue and it is the smallest veins in this size class (seventh order in C. pepo according to Fischer, 1885) that delimit the areoles and are therefore considered to be primarily responsible for the vein loading. Larger veins occasionally border areoles and may also be capable of loading, but they constitute a relatively small proportion of the total minor vein length. It was of interest to discover whether the initiation of sugar export depends upon the development of certain structural features in the minor vein phloem thus permitting them to become functionally active in vein loading and transport. We, therefore, undertook a detailed microscopic examination of the structural development of the minor veins delimiting single areoles in Cucurbita pepo L. leaves and attempted to correlate the observed structures with the ability of these veins to export soluble sugar. Materials and Methods Plants of Cucurbita pepo L. var. rnelopepo f. torticollis Bailey were grown in a controlled environment chamber (Turgeon and Webb, 1975). Microscopic observations and l~C-autoradiographs were
266
R. Turgeon and J.A. Webb: Leaf Development and Phloem Transport Fig. 1-4. Light micrographs of mature minor veins Fig. 1. Transection of a minor vein and surrounding cells. Two tracheids are present, probably due to sectioning through the overlapping ends of a single file of cells. Scale = 15 gm Fig. 2. Paradermal section of adaxial sieve elements. Scale= 15 p,m Fig. 3. Paradermal section of an adaxiat companion celt with underlying sieve elements. Scale=20 Ixm Fig. 4. Paradermal section of abaxial phloem. Scale =25 p,m. CC companion cell, SE sieve element, P parenchyma cell, T tracheid, I intermediary cell, BS bundle sheath cell
made on leaf no. 5 at stages of development around the transition stage. Leaf no. 3 of appropriately aged plants was supplied 14CO2 for 5 minutes. Following a 2 h period to allow transport of 14Clabelled sugars from leaf 3 to leaf 5 (Turgeon and Webb, 1973), the lamina of leaf 5 was excised. Two small pieces of tissue, from the distal and proximal ends of the lamina respectively, were immediately excised for microscopy and the rest of the lamina was rapidly frozen and autoradiographed. Procedures for 14CO2 labelling and whole leaf autoradiography have been fully described previously (Turgeon and Webb, 1973). The excised tissue samples were fixed in glutaraldehyde followed by osmic acid and embedded in Epon-Araldite resin (Turgeon et al., 1975). Serial paradermal 1 ~t sections were cut with glass knives, and stained on glass slides with methylene blue in sodium borate, pH ca. 10.0. When required, thin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate and examined with a Siemens Elmiskop 101 electron microscope at 80 KV.
1885) a n d recently with the e l e c t r o n m i c r o s c o p e (Turg e o n et al., 1975). In s u m m a r y , the m i n o r veins are b i c o l l a t e r a l a n d in t r a n s e c t i o n consist o f a highly o r d e r e d series o f cells (Fig. 1). T h e a d a x i a l p h l o e m is c o m p r i s e d o f a single sieve e l e m e n t (Fig. 2) a n d c o m p a n i o n cell (Fig. 3). T h e c o m p a n i o n cell occupies the a d a x i a l position. A single p a r e n c h y m a cell lies between the a d a x i a l sieve element a n d the x y l e m w h i c h in the smallest veins (seventh o r d e r ) consists o f only one file o f t r a c h e a r y elements. T h e r e m a y be f r o m one to three c o m p a n i o n cells a n d a s s o c i a t e d sieve elements in the a b a x i a l p h l o e m (Fig. 4). T h e c o m p a n i o n cells, w h i c h a r e large a n d d w a r f the d i m i n u t i v e sieve elements, were t e r m e d intermediary cells ( U b e r g a n g s z e l l e n ) by F i s c h e r (1885).
Results 1. Structure o f Minor Veins T h e structure o f the m i n o r veins o f Cucurbita pepo h a s been investigated by light m i c r o s c o p y (Fischer,
2. Maturation o f the Minor Veins T h e structure o f the m i n o r vein n e t w o r k o f d e v e l o p i n g leaves was r e c o n s t r u c t e d f r o m serial p a r a d e r m a l sec-
R. Turgeon and J.A. Webb: Leaf Development and Phloem Transport
tions. In Figure 5 schematic diagrams of representative areoles from four leaves are drawn in the paradermal plane at levels of the adaxial sieve elements, xylem, and abaxial sieve elements. Adaxial sieve elements were considered structurally mature when the pores of the sieve plates had been perforated (Fig. 2). Since the pores of abaxial sieve elements are not visible in the light microscope (Fig. 4) the criterion used for structural maturity was the absence of stainable protoplasm. In certain cases thin sections were examined in the electron microscope to determine if the pores between these apparently mature abaxial sieve elements had, in fact, been perforated. The vascular network is delineated long before the import-export transition, but the elements of the minor veins are undifferentiated and the cell types are recognizable only by their position within the vein. As the leaf ages the minor veins mature progressively from the largest toward the smallest veins (Fig. 5). The terminal elements which form the blind endings are the last to mature (Figs. 5 and 6). Maturation of both the adaxial and abaxial sieve elements is continuous but the xylem occasionally matures discontinuously, i.e., structurally mature elements are flanked at both ends by immature cells. As expected the minor veins in the distal region of the lamina mature and cease importing before those in the proximal region (Fig. 5, leaf 1). In the adaxial phloem the companion cells undergo a marked structural differentiation. The cytoplasm becomes highly condensed and the vacuoles enlarge (Fig. 3). These changes occur simultaneously with the loss of cell content and the perforation of the sieve plate pores of contiguous sieve elements. 3. Maturation o f Minor Veins and the Direction o f Phloem Transport
The direction of phloem transport in the tissue samples at the time of fixation was determined by examining the corresponding whole leaf autoradiographs (Fig. 5). Darkening of the autoradiographs indicates that the leaf tissue had been importing at the time of excision (Turgeon and Webb, 1973). There is no apparent correlation between structural maturation of the adaxial phloem and the transition from import to export. Sieve elements of the adaxial phloem initially mature more rapidly than those in the abaxial phloem and many areoles in importing tissue have mature adaxial sieve elements in the surrounding minor veins (Fig. 5, leaf 1, distal sample; leaf 3, proximal sample). Therefore, before export begins the adaxial sieve elements have formed an open conduit beginning at the minor veins surrounding the areoles and leading out of the leaf. Elements of the
267 ADAXIAL SIEVE ELEMENTS
XYLEM
ABAXIAL SIEVE ELEMENTS
..:..-I..-J..~-'"
. X s ~ NX ( ~.
: ~...I...i.."
~, ~ .~~. ]'..
9..N"
.~X./. . ." "~ .
"./..~"~ .~." " "5"+"L"
2 -_
"+.."
.~..'~..i.../."-
3 '
'
:1..I.. i.- L "
.v;.
"-....4..?" "
Fig. 5. Schematic outline drawings of maturing areoles in paradermal view. Autoradiographs of leaves from which the samples were taken are drawn at the left. Arrows point from the position in the leaf where the sample originated to a series of drawings of a representative areote from that sample. The areoles are drawn at three levels: adaxial sieve elements, xylem, and abaxiaI sieve elements. Lines normal to the Iongitudinal axes of the cells indicate the positions of end walls. Scale beneath autoradiographs=4 cm, beneath areoles= 100 ~tm. A light micrograph of the areole from the proximal position of leaf sample 3 at the level of the adaxial sieve elements is shown in Figure 6. Legend: A d a x i a l sieve elements with perforated sieve plate pores ( m a t u r e ) - - without sieve plate pores (immature) . . . . X y l e m tracheids without c y t o p l a s m - - tracheids with cytoplasm, secondary walls deposited . . . . tracheid precursors, without secondary walls . . . . A b a x i a l sieve elements with no cytoplasm visible in the light microscope (mature) filled with cytoplasm (immature) . . . .
xylem mature at about the same rate as adaxial sieve elements forming an effective channel for water conduction before sugar export begins. The structural maturation of abaxial sieve elements, unlike that of adaxial sieve elements and xylem, is closely correlated with the initiation of assimilate export. Compare, for example, the abaxial sieve elements in the proximal regions of leaves 3 and 4 in Figure 5. The coincidence of initial export and the appearance of structurally mature sieve elements in the abaxial phloem is most clearly seen in
268
R. Turgeon and J.A. Webb: Leaf Development and Phloem Transport
Fig. 6. Paradermal section of a maturing areole in the plane of the adaxial sieve elements. Toward the terminal ending the sieve plates have not yet been perforated (arrows). Pores are open in the rest of the areole (arrowheads). This areoie is drawn schematically in Figure 5 (proximal position, teaf sample 3), Scale=30 gm
the proximal region of the lamina where the cessation of import is immediately followed by assimilate export (Turgeon and Webb, 1973). In the distal region of the lamina mature abaxial sieve elements do not appear immediately following the cessation of import (Fig. 5, leaf 2) and neither does export begin as soon as import stops (Turgeon and Webb, 1973). Selected samples from tissue which had just begun to export were examined in the electron microscope to determine if the sieve plate pores of cells which appeared mature in the light microscope were, in fact, perforated. It was difficult to determine the exact position of cells within the areole because the sections normally pass in and out of the plane of any single cell type. However, it appeared that in most, but not all, of the areoles some of the sieve plate pores in the abaxial phloem had been perforated. Minor veins (fifth and sixth order) slightly larger than those which normally border the areoles all contained structurally mature abaxial sieve elements with perforated pores prior to the onset of export. Discussion
Structural maturation of abaxial sieve elements of minor veins surrounding the areoles in C. pepo leaves
coincides with initial export of soluble carbohydrate. This evidence suggests that the abaxial phloem of the minor veins is responsible for the transport of assimilate from the mesophyll toward the major phloem pathways. Previous observations on the structure (Fischer, 1885) and the ultrastructure (Turgeon et al., 1975) of mature minor veins of this species led to the same conclusion. The role of the adaxial phloem in the minor veins of the Cucurbitaceae is still in doubt. However the evidence presented here on the timing of structural maturation in relation to the direction of assimilate flow suggests that the adaxial phloem constitutes a preferential pathway for the import of assimilate to the expanding mesophyll tissue. This suggestion has some experimental support from the work of Peterson and Currier (1969) who found that in bidirectionally transporting leaves of Ecbaltium elaterium (Cucurbitaceae) fluorescein dye travels toward the leaf in the internal (adaxial) phloem and away from the leaf in the external (abaxial) phloem. Similarly Bonnemain (1969), using the technique of tissue autoradiography, found that import to and export from developing leaves of tomato occur in the internal and external phloem, respectively. In the petiole of C. pepo, the phloem of the internal and external systems transports
R. Turgeon and J.A. Webb: Leaf Deveiopment and Phloem Transport
sugars equally and in either direction depending on the developmental stage of the leaf (Webb and Gorham, 1965). Collectively these results suggest that while the internal phloem is specialized for assimilate import and the external phloem for export, these systems are capable of transport in either direction and supplement each other when transport occurs in one direction only. The central question this paper addresses is whether structural maturation limits initial export. Although the presence of mature abaxial sieve elements in the seventh order veins surrounding the areoles undoubtedly facilitates the initial export of photoassimilate it remains problematical whether export by still younger tissue is limited by the availability of functionally mature sieve tubes. A close examination of available evidence suggests that this is not the case. The abaxial phloem of the larger (fifth and sixth order) minor veins matures before export begins. Although these larger veins constitute a relatively small percentage of the total minor vein length and border a limited number of areoles they are probably capable of vein loading since they are embedded directly in the mesophyll and contain large intermediary cells (Turgeon et al., 1975). The fact that these veins are not utilized for export before the smaller veins mature suggests that export is limited by another step in the vein loading-transport system. Fellows and Geiger (1974) suggested that this limiting step is the attainment of a threshold concentration of solute in the phloem of the minor veins. In their study, Fellows and Geiger (1974) examined minor vein structure in Beta leaves. They could find no correlation between structural maturation and initial export and concluded that the vascular network matures before export begins. However, Isebrands and Larson (1973) concluded that structural maturation of minor veins limits initial export in cottonwood leaves. These discrepancies may represent real differences between plant species. From the above considerations it would appear that the structural maturation of phloem in the minor veins of C. pepo does not limit transport. Although there is a clear correlation between the initiation of
269
export and the maturation of the phloem elements primarily responsible for vein loading, the adaptive advantage of this coordinated development is not immediately obvious. It may be that delayed maturation of the abaxial sieve elements maintains cellular plasticity during continued expansion growth. This, in turn, may avoid undue stretching and possible collapse of the very narrow phloem elements which will constitute the major export channel. This study was supported by an Operating Grant (No. A2827) from the National Research Council of Canada. R.T. was the recipient of a NRCC Postgraduate Scholarship.
References Bonnemain, J.-L.: Transport du ~4C assimil6 ~t partir des feuilles de Tomate en voie de croissance et vers celles-ci. C. R. Acad. Sci. (Paris) 269 D, 1660 1663 (1969) 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) Fischer, A. : Studien fiber die SiebrShren der Dicotylenbl/itter. Bet. Verh. Kon. Sachs. Ges. Wiss. Leipzig, Math-Phys. C1.37, 245290 (1885) Isebrands, J.G., Larson, P.R.: Anatomical changes during leaf ontogeny in Populus dehoides. Amer. J. Bot. 60, 199-208 (1973) Larson, P.R., Isebrands, J.G., Dickson, R.E.: Fixation patterns of a~C within developing leaves of eastern cottonwood. Planta (Berl.) 107, 301-314 (1972) Peterson, C.A., Currier, H.B.: An investigation of bidirectional translocation in the phloem. Physiologia Plantar. 22, 12381250 (1969) Turgeon, R., Webb, J.A, : Leaf development and phloem transport in Cucurbita pepo: transition from import to export. Planta (Berl.) 113, 179-191 (1973) Turgeon, R., Webb, J.A. : Leaf development and phloem transport in Cucurbita pepo: carbon economy. Planta (Berl.) 123, 53-62 (1975) Turgeon, R., Webb, J.A., Evert, R.F.: Ultrastructure of minor veins in Cucurbitapepo leaves. Protoplasma 83, 217-232 (1975) Webb, J.A.: The translocation of sugars in Cucurbita melopepo. V. The effect of leaf blade temperature on assimilation and transport. Canad. J. Bot. 48, 935-942 (1970) Webb, J.A., Gorham, P,R. : Radial movement of t4C-translocates from Squash phloem. Can. J. Botany 43, 97-103 (1965)
Received 7 October; accepted 8 December 1975
Planta (Berl.)129, 265-269 (1975)
9 by Springer-Verlag 1976
Leaf Development and Phloem Transport in C.cm'bita pepo" Maturation of the Minor Veins Robert Turgeon* and J.A. Webb Biology Department, Carleton University Ottawa, Ontario, K1S 5B6, Canada
Summary. Young leaves of Cucurbita pepo L. were examined by whole-leaf autoradiography and serial paradermal sections were examined by light microscopy to determine whether commencement of sugar export depends upon the minor vein phloem achieving structural maturity. Maturation of these veins develops progressively from the largest toward the smallest elements with the minor veins in the distal region of the leaf maturing before those in the proximal region. Commencement of sugar export is coincident with maturation of the abaxial phloem of the minor veins delimiting the areoles. The abaxial phloem elements of the larger minor veins, which are probably capable of vein loading too but border only relatively few areoles, mature before export starts. The adaxial phloem surrounding the areoles and the xylem elements, mature in advance of the abaxial phloem and well before the beginning of sugar export. It is therefore considered unlikely that structural development alone directly governs the initiation of export. The results suggest that some other rate controlling step is involved.
Introduction
During initial development the normal growth rate of a Cucurbit leaf is maintained by importing soluble carbohydrates in the phloem from mature leaves. As leaf development progresses the growth rate declines but the photosynthetic capacity of the young leaf continues to increase thereby diminishing its dependence on the older leaves for additional carbohydrate supplies. When the leaf has grown to approximately half its maximal size this dependence is finally severed and flow in the phloem begins to reverse direction *
Present address." The Rockefeller University New York, N.Y.,
10021, U.S.A.
as the leaf now commences to export soluble sugars (Turgeon and Webb, 1975). This transition of the leaf from an importing to an exporting organ has been studied in a number of dicotyledonous species (e.g., Larson et al., 1972; Turgeon and Webb, 1973; Fellows and Geiger, 1974) and in all cases the transition has been observed to commence at the leaf tip and to pass quickly through the blade in a basipetal direction. The export of soluble carbohydrates from the lamina is the result of a multi-step transport process (Webb, 1970) commencing with the movement of photosynthesized molecules from within the chloroplasts and ending with a flow of soluble sugars into the minor vein phloem, a step generally termed "vein loading". The minor vein system permeates the mesophyll tissue and it is the smallest veins in this size class (seventh order in C. pepo according to Fischer, 1885) that delimit the areoles and are therefore considered to be primarily responsible for the vein loading. Larger veins occasionally border areoles and may also be capable of loading, but they constitute a relatively small proportion of the total minor vein length. It was of interest to discover whether the initiation of sugar export depends upon the development of certain structural features in the minor vein phloem thus permitting them to become functionally active in vein loading and transport. We, therefore, undertook a detailed microscopic examination of the structural development of the minor veins delimiting single areoles in Cucurbita pepo L. leaves and attempted to correlate the observed structures with the ability of these veins to export soluble sugar. Materials and Methods Plants of Cucurbita pepo L. var. rnelopepo f. torticollis Bailey were grown in a controlled environment chamber (Turgeon and Webb, 1975). Microscopic observations and l~C-autoradiographs were
266
R. Turgeon and J.A. Webb: Leaf Development and Phloem Transport Fig. 1-4. Light micrographs of mature minor veins Fig. 1. Transection of a minor vein and surrounding cells. Two tracheids are present, probably due to sectioning through the overlapping ends of a single file of cells. Scale = 15 gm Fig. 2. Paradermal section of adaxial sieve elements. Scale= 15 p,m Fig. 3. Paradermal section of an adaxiat companion celt with underlying sieve elements. Scale=20 Ixm Fig. 4. Paradermal section of abaxial phloem. Scale =25 p,m. CC companion cell, SE sieve element, P parenchyma cell, T tracheid, I intermediary cell, BS bundle sheath cell
made on leaf no. 5 at stages of development around the transition stage. Leaf no. 3 of appropriately aged plants was supplied 14CO2 for 5 minutes. Following a 2 h period to allow transport of 14Clabelled sugars from leaf 3 to leaf 5 (Turgeon and Webb, 1973), the lamina of leaf 5 was excised. Two small pieces of tissue, from the distal and proximal ends of the lamina respectively, were immediately excised for microscopy and the rest of the lamina was rapidly frozen and autoradiographed. Procedures for 14CO2 labelling and whole leaf autoradiography have been fully described previously (Turgeon and Webb, 1973). The excised tissue samples were fixed in glutaraldehyde followed by osmic acid and embedded in Epon-Araldite resin (Turgeon et al., 1975). Serial paradermal 1 ~t sections were cut with glass knives, and stained on glass slides with methylene blue in sodium borate, pH ca. 10.0. When required, thin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate and examined with a Siemens Elmiskop 101 electron microscope at 80 KV.
1885) a n d recently with the e l e c t r o n m i c r o s c o p e (Turg e o n et al., 1975). In s u m m a r y , the m i n o r veins are b i c o l l a t e r a l a n d in t r a n s e c t i o n consist o f a highly o r d e r e d series o f cells (Fig. 1). T h e a d a x i a l p h l o e m is c o m p r i s e d o f a single sieve e l e m e n t (Fig. 2) a n d c o m p a n i o n cell (Fig. 3). T h e c o m p a n i o n cell occupies the a d a x i a l position. A single p a r e n c h y m a cell lies between the a d a x i a l sieve element a n d the x y l e m w h i c h in the smallest veins (seventh o r d e r ) consists o f only one file o f t r a c h e a r y elements. T h e r e m a y be f r o m one to three c o m p a n i o n cells a n d a s s o c i a t e d sieve elements in the a b a x i a l p h l o e m (Fig. 4). T h e c o m p a n i o n cells, w h i c h a r e large a n d d w a r f the d i m i n u t i v e sieve elements, were t e r m e d intermediary cells ( U b e r g a n g s z e l l e n ) by F i s c h e r (1885).
Results 1. Structure o f Minor Veins T h e structure o f the m i n o r veins o f Cucurbita pepo h a s been investigated by light m i c r o s c o p y (Fischer,
2. Maturation o f the Minor Veins T h e structure o f the m i n o r vein n e t w o r k o f d e v e l o p i n g leaves was r e c o n s t r u c t e d f r o m serial p a r a d e r m a l sec-
R. Turgeon and J.A. Webb: Leaf Development and Phloem Transport
tions. In Figure 5 schematic diagrams of representative areoles from four leaves are drawn in the paradermal plane at levels of the adaxial sieve elements, xylem, and abaxial sieve elements. Adaxial sieve elements were considered structurally mature when the pores of the sieve plates had been perforated (Fig. 2). Since the pores of abaxial sieve elements are not visible in the light microscope (Fig. 4) the criterion used for structural maturity was the absence of stainable protoplasm. In certain cases thin sections were examined in the electron microscope to determine if the pores between these apparently mature abaxial sieve elements had, in fact, been perforated. The vascular network is delineated long before the import-export transition, but the elements of the minor veins are undifferentiated and the cell types are recognizable only by their position within the vein. As the leaf ages the minor veins mature progressively from the largest toward the smallest veins (Fig. 5). The terminal elements which form the blind endings are the last to mature (Figs. 5 and 6). Maturation of both the adaxial and abaxial sieve elements is continuous but the xylem occasionally matures discontinuously, i.e., structurally mature elements are flanked at both ends by immature cells. As expected the minor veins in the distal region of the lamina mature and cease importing before those in the proximal region (Fig. 5, leaf 1). In the adaxial phloem the companion cells undergo a marked structural differentiation. The cytoplasm becomes highly condensed and the vacuoles enlarge (Fig. 3). These changes occur simultaneously with the loss of cell content and the perforation of the sieve plate pores of contiguous sieve elements. 3. Maturation o f Minor Veins and the Direction o f Phloem Transport
The direction of phloem transport in the tissue samples at the time of fixation was determined by examining the corresponding whole leaf autoradiographs (Fig. 5). Darkening of the autoradiographs indicates that the leaf tissue had been importing at the time of excision (Turgeon and Webb, 1973). There is no apparent correlation between structural maturation of the adaxial phloem and the transition from import to export. Sieve elements of the adaxial phloem initially mature more rapidly than those in the abaxial phloem and many areoles in importing tissue have mature adaxial sieve elements in the surrounding minor veins (Fig. 5, leaf 1, distal sample; leaf 3, proximal sample). Therefore, before export begins the adaxial sieve elements have formed an open conduit beginning at the minor veins surrounding the areoles and leading out of the leaf. Elements of the
267 ADAXIAL SIEVE ELEMENTS
XYLEM
ABAXIAL SIEVE ELEMENTS
..:..-I..-J..~-'"
. X s ~ NX ( ~.
: ~...I...i.."
~, ~ .~~. ]'..
9..N"
.~X./. . ." "~ .
"./..~"~ .~." " "5"+"L"
2 -_
"+.."
.~..'~..i.../."-
3 '
'
:1..I.. i.- L "
.v;.
"-....4..?" "
Fig. 5. Schematic outline drawings of maturing areoles in paradermal view. Autoradiographs of leaves from which the samples were taken are drawn at the left. Arrows point from the position in the leaf where the sample originated to a series of drawings of a representative areote from that sample. The areoles are drawn at three levels: adaxial sieve elements, xylem, and abaxiaI sieve elements. Lines normal to the Iongitudinal axes of the cells indicate the positions of end walls. Scale beneath autoradiographs=4 cm, beneath areoles= 100 ~tm. A light micrograph of the areole from the proximal position of leaf sample 3 at the level of the adaxial sieve elements is shown in Figure 6. Legend: A d a x i a l sieve elements with perforated sieve plate pores ( m a t u r e ) - - without sieve plate pores (immature) . . . . X y l e m tracheids without c y t o p l a s m - - tracheids with cytoplasm, secondary walls deposited . . . . tracheid precursors, without secondary walls . . . . A b a x i a l sieve elements with no cytoplasm visible in the light microscope (mature) filled with cytoplasm (immature) . . . .
xylem mature at about the same rate as adaxial sieve elements forming an effective channel for water conduction before sugar export begins. The structural maturation of abaxial sieve elements, unlike that of adaxial sieve elements and xylem, is closely correlated with the initiation of assimilate export. Compare, for example, the abaxial sieve elements in the proximal regions of leaves 3 and 4 in Figure 5. The coincidence of initial export and the appearance of structurally mature sieve elements in the abaxial phloem is most clearly seen in
268
R. Turgeon and J.A. Webb: Leaf Development and Phloem Transport
Fig. 6. Paradermal section of a maturing areole in the plane of the adaxial sieve elements. Toward the terminal ending the sieve plates have not yet been perforated (arrows). Pores are open in the rest of the areole (arrowheads). This areoie is drawn schematically in Figure 5 (proximal position, teaf sample 3), Scale=30 gm
the proximal region of the lamina where the cessation of import is immediately followed by assimilate export (Turgeon and Webb, 1973). In the distal region of the lamina mature abaxial sieve elements do not appear immediately following the cessation of import (Fig. 5, leaf 2) and neither does export begin as soon as import stops (Turgeon and Webb, 1973). Selected samples from tissue which had just begun to export were examined in the electron microscope to determine if the sieve plate pores of cells which appeared mature in the light microscope were, in fact, perforated. It was difficult to determine the exact position of cells within the areole because the sections normally pass in and out of the plane of any single cell type. However, it appeared that in most, but not all, of the areoles some of the sieve plate pores in the abaxial phloem had been perforated. Minor veins (fifth and sixth order) slightly larger than those which normally border the areoles all contained structurally mature abaxial sieve elements with perforated pores prior to the onset of export. Discussion
Structural maturation of abaxial sieve elements of minor veins surrounding the areoles in C. pepo leaves
coincides with initial export of soluble carbohydrate. This evidence suggests that the abaxial phloem of the minor veins is responsible for the transport of assimilate from the mesophyll toward the major phloem pathways. Previous observations on the structure (Fischer, 1885) and the ultrastructure (Turgeon et al., 1975) of mature minor veins of this species led to the same conclusion. The role of the adaxial phloem in the minor veins of the Cucurbitaceae is still in doubt. However the evidence presented here on the timing of structural maturation in relation to the direction of assimilate flow suggests that the adaxial phloem constitutes a preferential pathway for the import of assimilate to the expanding mesophyll tissue. This suggestion has some experimental support from the work of Peterson and Currier (1969) who found that in bidirectionally transporting leaves of Ecbaltium elaterium (Cucurbitaceae) fluorescein dye travels toward the leaf in the internal (adaxial) phloem and away from the leaf in the external (abaxial) phloem. Similarly Bonnemain (1969), using the technique of tissue autoradiography, found that import to and export from developing leaves of tomato occur in the internal and external phloem, respectively. In the petiole of C. pepo, the phloem of the internal and external systems transports
R. Turgeon and J.A. Webb: Leaf Deveiopment and Phloem Transport
sugars equally and in either direction depending on the developmental stage of the leaf (Webb and Gorham, 1965). Collectively these results suggest that while the internal phloem is specialized for assimilate import and the external phloem for export, these systems are capable of transport in either direction and supplement each other when transport occurs in one direction only. The central question this paper addresses is whether structural maturation limits initial export. Although the presence of mature abaxial sieve elements in the seventh order veins surrounding the areoles undoubtedly facilitates the initial export of photoassimilate it remains problematical whether export by still younger tissue is limited by the availability of functionally mature sieve tubes. A close examination of available evidence suggests that this is not the case. The abaxial phloem of the larger (fifth and sixth order) minor veins matures before export begins. Although these larger veins constitute a relatively small percentage of the total minor vein length and border a limited number of areoles they are probably capable of vein loading since they are embedded directly in the mesophyll and contain large intermediary cells (Turgeon et al., 1975). The fact that these veins are not utilized for export before the smaller veins mature suggests that export is limited by another step in the vein loading-transport system. Fellows and Geiger (1974) suggested that this limiting step is the attainment of a threshold concentration of solute in the phloem of the minor veins. In their study, Fellows and Geiger (1974) examined minor vein structure in Beta leaves. They could find no correlation between structural maturation and initial export and concluded that the vascular network matures before export begins. However, Isebrands and Larson (1973) concluded that structural maturation of minor veins limits initial export in cottonwood leaves. These discrepancies may represent real differences between plant species. From the above considerations it would appear that the structural maturation of phloem in the minor veins of C. pepo does not limit transport. Although there is a clear correlation between the initiation of
269
export and the maturation of the phloem elements primarily responsible for vein loading, the adaptive advantage of this coordinated development is not immediately obvious. It may be that delayed maturation of the abaxial sieve elements maintains cellular plasticity during continued expansion growth. This, in turn, may avoid undue stretching and possible collapse of the very narrow phloem elements which will constitute the major export channel. This study was supported by an Operating Grant (No. A2827) from the National Research Council of Canada. R.T. was the recipient of a NRCC Postgraduate Scholarship.
References Bonnemain, J.-L.: Transport du ~4C assimil6 ~t partir des feuilles de Tomate en voie de croissance et vers celles-ci. C. R. Acad. Sci. (Paris) 269 D, 1660 1663 (1969) 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) Fischer, A. : Studien fiber die SiebrShren der Dicotylenbl/itter. Bet. Verh. Kon. Sachs. Ges. Wiss. Leipzig, Math-Phys. C1.37, 245290 (1885) Isebrands, J.G., Larson, P.R.: Anatomical changes during leaf ontogeny in Populus dehoides. Amer. J. Bot. 60, 199-208 (1973) Larson, P.R., Isebrands, J.G., Dickson, R.E.: Fixation patterns of a~C within developing leaves of eastern cottonwood. Planta (Berl.) 107, 301-314 (1972) Peterson, C.A., Currier, H.B.: An investigation of bidirectional translocation in the phloem. Physiologia Plantar. 22, 12381250 (1969) Turgeon, R., Webb, J.A, : Leaf development and phloem transport in Cucurbita pepo: transition from import to export. Planta (Berl.) 113, 179-191 (1973) Turgeon, R., Webb, J.A. : Leaf development and phloem transport in Cucurbita pepo: carbon economy. Planta (Berl.) 123, 53-62 (1975) Turgeon, R., Webb, J.A., Evert, R.F.: Ultrastructure of minor veins in Cucurbitapepo leaves. Protoplasma 83, 217-232 (1975) Webb, J.A.: The translocation of sugars in Cucurbita melopepo. V. The effect of leaf blade temperature on assimilation and transport. Canad. J. Bot. 48, 935-942 (1970) Webb, J.A., Gorham, P,R. : Radial movement of t4C-translocates from Squash phloem. Can. J. Botany 43, 97-103 (1965)
Received 7 October; accepted 8 December 1975
