Planta (Berl.) 123, 53--62 (1975) 9 Springer-Verlag 1!}75
Leaf Development and Phloem Transport in Cucurbita pepo: Carbon Economy Robert Turgeon * and J.A. Webb ** Department o:~Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada Received 14 October; accepted 10 December, 1974 S u m m a r y . Net photosynthesis, dark respiration and growth for leaf 5 of Cueurbita pepo L. plants grown under controlled conditions were measured and the data used for an assessment of the changes in carbon balance during growth of the leaf through expansion to maturity. The blade is first capable of net COs fixation when ca. 8 % expanded but the initial rapid growth during this period is sustained almost entirely through imported nutrients. When the growth rate starts to decline rapidly the net photosynthetic capacity of the blade begins to increase. This increase is accompanied by an expansion of the intercellular spaces and by decreasing dark respiration measured at night and in dark periods during the day. The blade becomes completely independent of phloem imported nutrients and begins to export excess photosynthate when the phase of rapid decrease in relative growth rate is almost complete at about 45% expansion. Maximum net photosynthesis of ca. 11 mg CO~.h-1 dm-2 is achieved at 70% expansion. The first detectable synthesis of the transport sugars stachyose and raffinose in the blade coincides with the beginning of intralaminar phloem transport fl'om the tip to the base of the leaf. The synthesis of sucrose, the other major transport sugar, is detectable at all stages of leaf development.
Introduction
Normal growth of a developing and expanding leaf initially depends on phloem-imported nutrients. With further expansion of the leaf its capacity for assimilating atmospheric CO 2 gradually increases and the direction of transport in the phloem is eventually reversed as carbohydrates accumulate, import stops and the export of soluble sugar begins (Milthorpe and 1Vfoorby, 1969). I n an earlier paper we presented a detailed analysis of this import-export transition in leaf 5 of C u c u r b i t a p e p o L. using the leaf plastochron index (LPI) as a measure of leaf age (Turgeon and Webb, 1973). The transition from import to export does not occur simultaneously throughout the blade but develops in a basipetal direction. I m p o r t into the blade tip terminates when the leaf is 10% expanded (LPI 0.3) and into the blade base when the leaf is about 45% expanded (LPI 1.3). Export from each region of the blade commences soon after import has ceased. The carbohydrates exported from the blade tip are initially imported into the less mature basal regions so that export out of the blade cannot be detected until the leaf is about 35% expanded (LPI 1.1). The transition from import to export is a function of the development of the photosynthetic system with the achievement of a net positive carbon balance. I n this paper we report an analysis of net photosynthesis, dark respiration * Present address: The Rockefeller University, New York, N.Y. 10021, USA. **
To whom reprint requests should be addressed.
54
R. Turgeon and J. A. Webb
a n d g r o w t h d u r i n g t h e d e v e l o p m e n t of leaf 5 of C. ~oepo g r o w n u n d e r c o n t r o l l e d c o n d i t i o n s . F r o m t h e s e m e a s u r e m e n t s c h a n g e s in t h e c a r b o n b a l a n c e of t h e dev e l o p i n g leaf h a v e b e e n assessed.
Materials and Methods
Culture o/ Plants. Seeds of the Early Prolific Straight-Neck squash, Cucurbita pepo L. var. melopepo f. torticollis Bailey 1 were germinated in perlite under controlled conditions as previously described (Turgeon and Webb, 1973). When the plants were 7 days old they were transferred to individual pots in perlite and placed in a controlled environment cabinet. They were illuminated for i8 h each day from incandescent lights at a constant intensity at the level of leaf 5 of 110 ~E m -2 s-1 between 400 and 700 nm. Once a day the plants were watered with a modified Hoagland's solution (Webb and Gorham, 1964). The temperature remained constant at 26 ~. These conditions were chosen because plants raised in this environment most closely resembled the morphological development and rate of growth of those which were grown in the greenhouse for our previous studies (Turgeon and Webb, 1973). The light intensity in the controlled-environment cabinets permitted mature leaves to achieve approximately twothirds of the maximum net photosynthetic rate attained by similar leaves grown under greenhouse conditions. The environment of the growth cabinet with its constant daily 18 h periods of illumination therefore represented a compromise with the natural but fluctuating light conditions experienced by greenhouse-grown plants. Lea/Age. The age of leaf 5 was determined by the leaf plastochron index (LPI), as previously described (Turgeon and Webb, 1973). In order to correlate these studies with our previous experiments using greenhouse-grown plants (Turgeon and Webb, 1973), it was necessary to repeat the earlier l~C-displacement studies with similar plants raised in the growth cabinet. In either case when leaf age was determined by the L P I the transition from import to export took place during the same period, i.e., from L P I 0.3 to L P I 1.3. The LPI is linearly related to time between LPI 0.0 and 7.0 and plants raised in the controlled environment cabinet grew at a mean rate of one plastochron unit per 1.62 days. At maturity lamina 5 attained a mean surface area of 130 cm 2. Photosynthesis and Respiration. Experimental operations on leaf 5 were performed in the same controlled-environment cabinet in which the plants were grown. Analyses of COz flux were made with a Beckman Infrared Analyzer (model 215A). Lucite leaf chambers were designed with removable lids so that the blade could be sealed into the chamber while remaining attached to the plant. A pump pulled ambient air from the growth cabinet through a flow meter, the Lucite leaf chamber, a drying tube, and the sample cell of the infra-red analyzer. Connections were made with Tygon tubing and during operation the reference cell of the infrared analyzer was filled with C02-free N2 at atmospheric pressure. Large leaves were enclosed in a 1225-cm 3 Lucite chamber and air was passed through at a rate of 750 cm 3 min-L Smaller leaves were enclosed in a Lucite chamber of 475 cm a volume and an air flow rate of 160 em 3 rain -1 was maintained. Blade temperature was measured with a fine-gauge (0.0124 SWG) ehromel-constantan thermoeouple (Science Products Corp., Dover, N J , USA) inserted into a large vein. Leaf temperature ranged between 2 and 4 ~ above ambient during an analysis. The rates of net photosynthesis and dark respiration were calculated from the difference in CO2 concentration between ambient air in the growth cabinet and air emerging from the leaf chamber, t~espiration rates during the 18-h light period ("daytime dark respiration") were determined by turning off the lights of the growth cabinet and measuring CO~ flux when a constant reading was obtained, usually within 5 rain. Sugar Synthesis. Plants taken from the growth chamber were equilibrated for 30 min in a laboratory fumehood ill uminated with light from water-filtered incandescent lamps at a constant intensity at the level of leaf 5 of 100 ~E m -2 s-1 between 400 and 700 nm. The blade of leaf 5 was excised and quickly inserted into a 2-1 Erlenmeyer flask in which 0.1 ml of 1 m C i m l -1 Na214CO3 (95% 14C) had previously been mixed with 5N I-I~SO4. After 5 min continuous ex1 W.A. Burpee, Seed Growers, Philadelphia, Pa, USA.
Leaf Development and Phloem Transport in Cucurbita LIGHTS
OFF
LIGHTS ON
MiDr~IGHT
6 P,M
55
lO-
?
L~
E O0
O
0
-3~ NOON
6 PM
NOON
Fig. 1. Exchange of CO2 by a single leaf blade of Cucurbita pepo over a 24-h period (,). Values for respiration in the dark during the day are also included (o)
posure to 14CO, the blade was frozen in liquid nitrogen, ground to a fine powder, and extracted twice for a total of I h with 80% ethanol at 70~ The extract was filtered, reduced in volume by flash evaporation at 70~ and passed through weak anion (Rexyn 203-O~t-Fisher research grade, 16-50 mesh) and strong cation (Rexyn 101-H-Fisher research grade, 40-100 mesh) exchange resins. The neutral fraction was collected, reduced to dryness, and redissolved in sufficient water to give a final isotope concentration of ca. i00000 dpm mt -1. Samples of 50 [• were chromatographed on Whatman 3MM paper with n-propanol: ethyl acetate:water (7:1:2, v/v) as solvent, Chromatograms were analyzed with a Packard radiochromatogram strip scanner. Known standard sugars were run as alternate samples on the chromatogram sheets and their positions located with an ammoniacal silver-nitrate reagent (Partridge, 1948). Measurement o/Intercellular Spaces. Pieces of blade tissue were fixed in 6 % glutaraldehyde in 0.05 M sodium-cacodylatebuffer, pH 6.8, dehydrated in a graded ethanol series, and embedded in Epon-Araldite resin (N[ollenhauer, 1964). Transections were cut 1 ~zmthick with glass knives, placed on slides, and stained with methylene blue in sodium borate, ca. pH 10. Photographs were taken of mesophyll regions free from all vascular tissue including minor veins. Intercellular spaces and cells, including the epidermis, were carefully cut from enlarged photographic prints, and weighed. Dividing the weight of the pat3er representing intercellular spaces by the total weight gave a measure of the percent volume of the blade occupied by the air spaces.
Results
1. Photosynthesis and Respiration I n p r e l i m i n a r y e x p e r i m e n t s w i t h p l a n t s in which leaf 5 was fully m a t u r e ( L P I 4.0-7.0) n e t p h o t o s y n t h e s i s , d a y t i m e d a r k r e s p i r a t i o n a n d n i g h t r e s p i r a t i o n were m e a s u r e d a t 3-h i n t e r v a l s over a 24-h period. T h e results of a r e p r e s e n t a t i v e e x p e r i m e n t a r e shown in Fig. 1. N e t p h o t o s y n t h e s i s r e m a i n e d c o n s t a n t t h r o u g h o u t t h e d a y . D u r i n g ~he night, r e s p i r a t i o n was i n i t i a l l y e q u a l ~o d a r k r e s p i r a t i o n d u r i n g t h e d a y , b u t in ma~ure leaves i~ d r o p p e d to a Iower level a n d r e m a i n e d c o n s t a n t for t h e rest of t h e n i g h t period. F r o m t h e results of these e x p e r i m e n t s i t was concluded ~hat reliable m e a s u r e m e n t s of p h o t o s y n t h e s i s could be m a d e a t a n y p e r i o d of t h e d a y , a n d t h a t n i g h t r e s p i r a t i o n was b e s t m e a s u r e d a f t e r 2 a.m. (14:00).
56
R. Turgeon and J. A. Webb ---{ I0-
?
5-
Eo
-5LEAF R.ASTOCHRON INDEX
Fig. 2. Net photosynthesis (.), night respiration (*), and dark daytime respiration (o) plotted against leaf age. Vertical bars = 2 • standard error of the mean Measurements of net photosynthesis and dark respiration during the day were made on blade 5 of 50 plants. For night respiration measurements, 20 plants were used. Upon completion of each experiment determinations were made of the leaf plastoehron index (LPI) and blade dry weight. At L P I 0.0 blade 5 is incapable of net photosynthesis (Fig. 2) and approximately 1 mg CO S h -1 dm -2 is lost from these leaves. As the blade expands photosynthetic capacity increases rapidly. Net photosynthesis begins at ca. L P I 0.1 (8% leaf expansion) and reaches a maximum rate at the prevailing light intensity of ca. 11 mg COs h -1 dm -2 at L P I 2.0 (70% leaf expansion) or shortly thereafter. With increasing age photosynthetic capacity slowly declines to ca. 8.5 mg CO s h -1 dm -2 at L P I 7.0. Young leaves (LPI 0.0) lose ca. 5 mg CO2 h -1 dm -~ by dark respiration during the day and approximately the same amount at night. As the leaf grows respiration declines and levels off between L P I 1,0 and 2.0. Mature leaves respire 0.75 mg COS h -1 dm -2 at night and about 3 times this amount in the dark during the day. 2. C a r b o n B a l a n c e
The carbon balance over a 24-h period in a developing leaf or a mature leaf blade may be defined by the following equation, G r o w t h = N e t Photosynthesis--Night Respiration• where growth is the increase in weight of C in the blade over a 24-h period, net photosynthesis is the weight of C assimilated during the light period, night respiration is the weight of C respired during the dark period, and translocation is the weight of C either imported (-k) or exported (--) in 24 h. Note that growth is defined simply as an accumulation of C without regard for distribution. Night "dark" respiration should not be confused with "dark" respiration during the day. Values for net photosynthesis and night respiration were calculated from the
Leaf Development and Phloem Transport in Cucurbita
57
0.5"
)r~
o
0.1
9
-I
,
0
,
I
LEAF
2
PLASTOCHRONINDEX
,
~
3
4
Fig. 3. Dry weight increase plotted against leaf age. Vertical bars = 2 • standard error of the mean
~ N T H E S r S
40
30 20
/
',,/
~
GROWTH
INTOLEAF
3 o
~'
NIGHTI - - \
-I0 ~
.~PIRATION \
O
U
-20
+
T
OFLEAF
~TION
-30 -4C
I
2
LEAFPLA$TROCHRONINDEX
3
Fig. 4. Carbon balance plotted against leaf age. Values are calculated as instantaneous rates
and expressed as the weight of carbon exchanged over a 24-h period. Values for net photosynthesis and nigh~ respiration were calculated for 18-h and 6-h periods, respectively
data given in Fig. 2 and expressed as the total weight of C exchanged by the blade during the 18-h day and the 6-h night, respectively. Calculations of instantaneous growth rate were made graphically from the curve of dry-weight increase of leaf 5 with time (Fig. 3). I n order to convert this into C accumulation the dry-weight was assumed to be 45% C (Stout, 1961). The weight of C either imported or exported was calculated by difference using the above equation.
58
R. Turgeon and J. A. Webb
1.5.~'~C.~ OWTH z t.0, i ~~OSYNTHESI$ 0.5' -0.5' o
TRANSLOCATION i
0
~
LEAKPLAST0CHRONtNDEX
Fig. 5. Relative carbon balance plotted against leaf age. A value of 1.0 on t~heordinate indicates an increase or exchange of carbon, over a 24-h period, equivalent to ~he weight of carbon in the lamina
The balance sheet for C flux in leaf 5 obtained in this fashion is presented graphically in Fig. 4. At L P I 0.0 the quantity of C imported is equal to the amount required for growth. As the blade expands net photosynthetic capacity increases. The quantity of C translocated into the leaf increases until the growth rate peaks and then falls sharply. At approximately L P I 0.8 the contributions of net photosynthesis and transloca~ion to ~he influx of C into the blade are equal. When net photosynthesis supplies sufficient C to meet the demands of growth and night respiration, import ceases and export from the blade begins (LPI 1.1). Although these measurements provide an accounting of C flux, they do not properly reflect the efficiency of the C exchange processes since the weight of the blade is not taken into account. To obtain a measure of efficiency per unit weight as the leaf grows it is necessary to divide each factor in the above equation by the weight of the C in the blade. If this is done the weight units may be cancelled out and the values for net photosynthesis, dark respiration, growth and translocatiou become percentage or relative figures, each expressed as dw/dt 1/W. These relative values are a true measure of the efficiency of C flux, i.e., the ability of a unit weight of leaf material to add or remove C. I t should be noted that the relative growth factor in the above equation (dw/dt 1/W) is Blackman's Efficiency Index (Blackman, 1919) a well-known index of relative growth efficiency. Relative C flux is plotted against leaf age in Fig. 5. As in Fig. 4 the growth and import curves are almost identical for young leaves. Relative growth after unfolding reaches a maximum of 150 % increase in weight over a 24-h period at L P I 0.3. This coincides in time with the cessation of import by the leaf tip (Turgeon and Webb, 1973). The reIative growth rate falls sharply and then levels out to a slowly decreasing value. Shortly before the sharp decline in relative growth ends the leaf base ceases to import (LPI 1.3 ; Turgeon and Webb, t973). Therefore, cessation of import and the decline in relative growth rate after unfolding occur during the same period, L P I 0.3-1.3.
Leaf Development and Phloem Transport in Cucurbita
59
25. w
~ 20, "J 15
~) IO,
oJ0
3
4
LEAF PLASTOCHRON INDEX
Fig. 6. Intercellular-space enlargement in the distal (,) and proximal (o) regions of the expanding lamina expressed as a percentage of the tmtal volume of cells and air spaces excluding vascular tissue. Vertical bars = 2 • standard error of the mean
3. Intercellular Spaces Air spaces greatly enhance the distribution of CO z throughout the mesophyll and measurements were made of the enlargement of intercellular spaces during the growth of leaf 5. Sixteen plants were chosen in which the age of the leaf was between L P I 0.0 and 4.0. Two pieces of the blade, one near the apex and one near the base, were removed and the percentage of the cross-sectional area of the blade occupied b y air spaces was determined as described in Material and Methods. Care was taken to sample comparable regions in each leaf. Air spaces develop in the distal region of the blade in advance of the proximal region (Fig. 6). Air space enlargement and the development of net photosynthetic capacity are closely correlated (compare Fig. 2 and 3). However, air space enlargement is complete at the leaf apex b y L P I 1.3 and in the leaf base b y L P I 1.7, whereas photosynthesis reaches its m a x i m u m value at L P I 2.0 or slightly later. I n the mature leaf we calculated air spaces to occupy ca. 23 % of the blade volume (excluding vascular tissue).
4. Sugar Synthesis Carbon is transloeated in C. pepo primarily as sucrose, stachyose and raffinose. Earlier studies (Webb and Gotham, 1964) indicated that staehyose and raffinose are synthesized from 1~C0~ at approximately the same time as export from the blade begins. I t was of interest therefore to determine more precisely the time of initial synthesis of these transport sugars. Leaf 5 was excised from 20 plants and supplied 14C0~ continuously for 5 rain. []aC] Sucrose is synthesized in leaves which have just unfolded. [14C] Staehyose and [14C]raffinose are first synthesized at approximately L P I 0.6 (Fig. 7). The incorporation of 14C into stachyose and raffinose increases rapidly as the leaf matures and under the experimental conditions exceeds the incorporation into sucrose by L P I 1.0.
60
R. Turgeon and J. A. Webb
~0
z o
b 20' J
z
0
o
i
~
~,
4
LEAF PLASTOCHRON INDEX
Fig. 7. Incorporation of 14C,by expanding leaves, into the transport sugars sucrose (o), stachyrose (o) and raffinose (A)during exposure to 14C02for 5 min. Vertical bars = 2 • standard error of the mean
Discussion Our data on photosynthesis and respiration in growing leaves of Cucurbita are similar to those published for a variety of dicotyledonous species (Dickman, 1971 ; Hopkinson, 1964; Kriedemann, 1968; Larson and Gordon, 1969; Larson et al., 1972; Richardson, 1957; Ryle, 1972). Young leaves are initially incapable of net CO~ uptake in the light. The coincidence of the curves for growth and import of newly unfolded leaves show their high dependence on a sustained supply of imported nutrients. As the blade expands photosynthetic potential rapidly increases. At L P I 1.1 import ceases and thereafter the net amount of atmospheric carbon fixed by the lamina is sufficient to sustain growth, night respiration and to supply soluble carbohydrates for export. Maximum net photosynthesis is attained when leaves are between threequarters to fully expanded and thereafter slowly declines. In immature leaves the rates of respiration at night and in dark periods during the day are almost identical. A decrease in the respiratory capacity at night first becomes evident during the import-export transition at approximately L P I 1.0. This suggests that young growing leaves continue to import carbon and to respire at an undiminished rate during the night while the respiration of non-importing leaves becomes limited either through a shortage of or an inaccessibility to suitable substrates or through a partial inhibition of the respiratory system. At the transition point between import and export, L P I 1.1, the relative growth rate of the lamina has decreased considerably. However, the transition from import to export does not take place instantaneously throughout the leaf but occurs over a single plastochron interval, beginning at the leaf tip at L P I 0.3 when the relative growth rate of the whole blade is still very high (Turgeon and Webb, 1973).
Leaf Development and Phloem Transport in Cucurbita
61
In a consideration of leaf development it is therefore important to distinguish between the apparent properties of the blade summed as a whole and those properties of its component parts, presumably down to the level of individual mesophyll cells. Since the growth of leaves proceeds in a basipetal direction (Avery, 11933) and the capacity for photosynthesis also develops basipetally (Larson and Gordon, 1969) it is reasonable to assume that the data obtained from whole leaves may be applied to smaller regions of the lamina. I t is clearly an advantage to expanding tissue to have the import-export transition occur only after the growth rate has considerably declined. Rapid growth requires a constant supply of nutrients. If, during this critical period, reliance were placed entirely on photosynthetically assimilated carbon, growth would be unduly affected at night or during lbimes of inclement weather. During these periods, adequate substrates are assured through their continued import from the mature parts of the plant. The increase in relative growth rate between leaf unfolding and the stage when the leaf tip stops importing indicates an improvement in growth conditions perhaps from enhanced nutrient supply or release from internal inhibition. This has been reported for wheat leaves in which the rate of relative growth increases after leaf initiation to a maximum shortly before emergence (Williams, 1960). However, in the dicotyledonous species Tri/olium repens (Denne, 1966) and Fragaria vesca (Arney, 1953) the relative growth declines steadily after initiation. The pattern of relative growth before unfolding in Leaf 5 of Cucurbita pepo, the cause of the increase in growth rate after unfolding, and the relationship of this increase to the supply of imported substrates remain to be explored. Increased photosynthetic capacity is accompanied by the basipetal development of the intercellular spaces in the leaf mesophyll. A similar result was obtained by Isebrands and Larson (1973) using Populus deltoides leaves. Although air spaces cease expanding by L P I 1.6 the net photosynthesis rate continues to increase until L P I 2.0. While air spaces enhance the efficiency of gas exchange it would appear that at least in Cucurbita pepo their development does not limit photosynthesis in the developing leaf as suggested by Isebrands and Larson for their material (1973). Carbon is exported from mature Cucurbita leaves primarily as sucrose, stachyose and raffinose (Webb and Gorham, 1964). Sucrose synthesis occurs at the earliest stages of leaf development whereas synthesis of stachyose and raffinose is first detectable at L P I 0.6. Intralaminar transport between the leaf tip and the leaf base was observed at L P I 0.7 (Turgeon and Webb, 1973). At this stage the distal 30% of the blade no longer imports but the leaf blade does not export. Perhaps ~he inability to detect intralaminar transport between L P I 0.3, when import at the leaf tip ceases, and L P I 0.7 is in some way connected to the lack of available stachyose and raffinose. Sucrose is synthesized between L P I 0.3 and 0.6 but it appears not to be available for transport during this developmental stage. I t is intriguing that the syntheses of stachyose and raffinose coincides with the initiation of translocation. 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.
62
R. Turgeon and J. A. Webb
References Arney, S. E. : The initiation, growth, and emergence of leaf primorida in Fragaria. Ann. Bot. 1~.S. 17,477-492 (1953) Avery, G. S., Jr. : Structure and development of the tobacco leaf. Amer. J. Bot. 20, 565-592 (1933) Btaekman, V. It.: The compound interest law and plant growth. Ann. Bot. 13, 353-360 (1919) Denne, P. M. : Leaf development in Tri/olium repens. Bot. Gaz. 127, 202-210 {1966) Dickmann, D. I. : Photosynthesis and respiration by developing leaves of cottonwood (Populus deltoides Bartr.). Bot. Gaz. 132, 253-259 (1971) Hopkinson, J. M.: Studies on the expansion of the leaf surface. IV. The carbon and phosphorus economy of a leaf. J. exp. Bot. 15, 125-137 (1964) Isebrands, J. G., Larson, P. R. : Anatomical changes during leaf ontogeny in Populus deltoides. Amer. J. Bot. 60, 199-208 (1973) Kriedemann, P. E. : Photosynthesis in vine leaves as a function of light intensity, temperature and leaf age. Vitis 7, 213-220 (1968) Larson, P. R., Gordon, J.C.: Leaf development, photosynthesis, and 14C distribution in Populus deltoides seedlings. Amer. J. Bot. 56, 1058-1066 (1969) Larson, P. R., Isebrands, J. C., Dickson, R. E. : Fixation patterns of 14C within developing leaves of eastern cottonwood. Planta (Berl.) 107, 301-314 (1972) Milthorpe, F. L., Moorby, J. : Vascular transport and its significance in plant growth. Ann. Rev. Plant Physiol. 20, 117-138 (1969) Mollenhauer, tI. H. : Plastic embedding mixtures for use in electron microscopy. Stain Teehn. 39, 111-114 (1964) Partridge, S. M. : Filter-paper partition chromatography of sugar. L General description and application to the qualitative analysis of sugars in apple juice, egg white and foetal blood of sheep. Biochem. J. 42, 938-953 (1948) Richardson, S. D.: The effect of leaf age on the rate of photosynthesis in detached leaves of tree seedlings. Acta bot. neerl. 6, 445-457 (1957) RyIe, G. J. A.: A quantitative analysis of the uptake of carbon and of the supply of 14C-labelled assimilates to areas of meristematic growth in Lolium temutentum. Ann. Bot. 36, 497-512 (1972) Stout, P. R. L.: Micronutrient needs for plant growth. Calif. Fertilizer Conf. Proc. (California Fertilizer Assoc.) 9, 21-23 (1961) Turgeon, R., Webb, J. A. : Leaf development and phloem transport in Cucurbita pepo: transition from import to export. Planta (Berl.) 113, 179-191 (1973) Webb, J. A., Gorham, P. R. : Translocation of photosynthetically assimilated 14C in straightnecked squash. Plant Physiol. 39, 663-672 (1964) Williams, R. F. : The physiology of growth in the wheat plant. I. Seedling growth and the pattern of growth at the shoot apex. Austral. J. Biol. Sci. 13, 401-428 (1960)
Leaf Development and Phloem Transport in Cucurbita pepo: Carbon Economy Robert Turgeon * and J.A. Webb ** Department o:~Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada Received 14 October; accepted 10 December, 1974 S u m m a r y . Net photosynthesis, dark respiration and growth for leaf 5 of Cueurbita pepo L. plants grown under controlled conditions were measured and the data used for an assessment of the changes in carbon balance during growth of the leaf through expansion to maturity. The blade is first capable of net COs fixation when ca. 8 % expanded but the initial rapid growth during this period is sustained almost entirely through imported nutrients. When the growth rate starts to decline rapidly the net photosynthetic capacity of the blade begins to increase. This increase is accompanied by an expansion of the intercellular spaces and by decreasing dark respiration measured at night and in dark periods during the day. The blade becomes completely independent of phloem imported nutrients and begins to export excess photosynthate when the phase of rapid decrease in relative growth rate is almost complete at about 45% expansion. Maximum net photosynthesis of ca. 11 mg CO~.h-1 dm-2 is achieved at 70% expansion. The first detectable synthesis of the transport sugars stachyose and raffinose in the blade coincides with the beginning of intralaminar phloem transport fl'om the tip to the base of the leaf. The synthesis of sucrose, the other major transport sugar, is detectable at all stages of leaf development.
Introduction
Normal growth of a developing and expanding leaf initially depends on phloem-imported nutrients. With further expansion of the leaf its capacity for assimilating atmospheric CO 2 gradually increases and the direction of transport in the phloem is eventually reversed as carbohydrates accumulate, import stops and the export of soluble sugar begins (Milthorpe and 1Vfoorby, 1969). I n an earlier paper we presented a detailed analysis of this import-export transition in leaf 5 of C u c u r b i t a p e p o L. using the leaf plastochron index (LPI) as a measure of leaf age (Turgeon and Webb, 1973). The transition from import to export does not occur simultaneously throughout the blade but develops in a basipetal direction. I m p o r t into the blade tip terminates when the leaf is 10% expanded (LPI 0.3) and into the blade base when the leaf is about 45% expanded (LPI 1.3). Export from each region of the blade commences soon after import has ceased. The carbohydrates exported from the blade tip are initially imported into the less mature basal regions so that export out of the blade cannot be detected until the leaf is about 35% expanded (LPI 1.1). The transition from import to export is a function of the development of the photosynthetic system with the achievement of a net positive carbon balance. I n this paper we report an analysis of net photosynthesis, dark respiration * Present address: The Rockefeller University, New York, N.Y. 10021, USA. **
To whom reprint requests should be addressed.
54
R. Turgeon and J. A. Webb
a n d g r o w t h d u r i n g t h e d e v e l o p m e n t of leaf 5 of C. ~oepo g r o w n u n d e r c o n t r o l l e d c o n d i t i o n s . F r o m t h e s e m e a s u r e m e n t s c h a n g e s in t h e c a r b o n b a l a n c e of t h e dev e l o p i n g leaf h a v e b e e n assessed.
Materials and Methods
Culture o/ Plants. Seeds of the Early Prolific Straight-Neck squash, Cucurbita pepo L. var. melopepo f. torticollis Bailey 1 were germinated in perlite under controlled conditions as previously described (Turgeon and Webb, 1973). When the plants were 7 days old they were transferred to individual pots in perlite and placed in a controlled environment cabinet. They were illuminated for i8 h each day from incandescent lights at a constant intensity at the level of leaf 5 of 110 ~E m -2 s-1 between 400 and 700 nm. Once a day the plants were watered with a modified Hoagland's solution (Webb and Gorham, 1964). The temperature remained constant at 26 ~. These conditions were chosen because plants raised in this environment most closely resembled the morphological development and rate of growth of those which were grown in the greenhouse for our previous studies (Turgeon and Webb, 1973). The light intensity in the controlled-environment cabinets permitted mature leaves to achieve approximately twothirds of the maximum net photosynthetic rate attained by similar leaves grown under greenhouse conditions. The environment of the growth cabinet with its constant daily 18 h periods of illumination therefore represented a compromise with the natural but fluctuating light conditions experienced by greenhouse-grown plants. Lea/Age. The age of leaf 5 was determined by the leaf plastochron index (LPI), as previously described (Turgeon and Webb, 1973). In order to correlate these studies with our previous experiments using greenhouse-grown plants (Turgeon and Webb, 1973), it was necessary to repeat the earlier l~C-displacement studies with similar plants raised in the growth cabinet. In either case when leaf age was determined by the L P I the transition from import to export took place during the same period, i.e., from L P I 0.3 to L P I 1.3. The LPI is linearly related to time between LPI 0.0 and 7.0 and plants raised in the controlled environment cabinet grew at a mean rate of one plastochron unit per 1.62 days. At maturity lamina 5 attained a mean surface area of 130 cm 2. Photosynthesis and Respiration. Experimental operations on leaf 5 were performed in the same controlled-environment cabinet in which the plants were grown. Analyses of COz flux were made with a Beckman Infrared Analyzer (model 215A). Lucite leaf chambers were designed with removable lids so that the blade could be sealed into the chamber while remaining attached to the plant. A pump pulled ambient air from the growth cabinet through a flow meter, the Lucite leaf chamber, a drying tube, and the sample cell of the infra-red analyzer. Connections were made with Tygon tubing and during operation the reference cell of the infrared analyzer was filled with C02-free N2 at atmospheric pressure. Large leaves were enclosed in a 1225-cm 3 Lucite chamber and air was passed through at a rate of 750 cm 3 min-L Smaller leaves were enclosed in a Lucite chamber of 475 cm a volume and an air flow rate of 160 em 3 rain -1 was maintained. Blade temperature was measured with a fine-gauge (0.0124 SWG) ehromel-constantan thermoeouple (Science Products Corp., Dover, N J , USA) inserted into a large vein. Leaf temperature ranged between 2 and 4 ~ above ambient during an analysis. The rates of net photosynthesis and dark respiration were calculated from the difference in CO2 concentration between ambient air in the growth cabinet and air emerging from the leaf chamber, t~espiration rates during the 18-h light period ("daytime dark respiration") were determined by turning off the lights of the growth cabinet and measuring CO~ flux when a constant reading was obtained, usually within 5 rain. Sugar Synthesis. Plants taken from the growth chamber were equilibrated for 30 min in a laboratory fumehood ill uminated with light from water-filtered incandescent lamps at a constant intensity at the level of leaf 5 of 100 ~E m -2 s-1 between 400 and 700 nm. The blade of leaf 5 was excised and quickly inserted into a 2-1 Erlenmeyer flask in which 0.1 ml of 1 m C i m l -1 Na214CO3 (95% 14C) had previously been mixed with 5N I-I~SO4. After 5 min continuous ex1 W.A. Burpee, Seed Growers, Philadelphia, Pa, USA.
Leaf Development and Phloem Transport in Cucurbita LIGHTS
OFF
LIGHTS ON
MiDr~IGHT
6 P,M
55
lO-
?
L~
E O0
O
0
-3~ NOON
6 PM
NOON
Fig. 1. Exchange of CO2 by a single leaf blade of Cucurbita pepo over a 24-h period (,). Values for respiration in the dark during the day are also included (o)
posure to 14CO, the blade was frozen in liquid nitrogen, ground to a fine powder, and extracted twice for a total of I h with 80% ethanol at 70~ The extract was filtered, reduced in volume by flash evaporation at 70~ and passed through weak anion (Rexyn 203-O~t-Fisher research grade, 16-50 mesh) and strong cation (Rexyn 101-H-Fisher research grade, 40-100 mesh) exchange resins. The neutral fraction was collected, reduced to dryness, and redissolved in sufficient water to give a final isotope concentration of ca. i00000 dpm mt -1. Samples of 50 [• were chromatographed on Whatman 3MM paper with n-propanol: ethyl acetate:water (7:1:2, v/v) as solvent, Chromatograms were analyzed with a Packard radiochromatogram strip scanner. Known standard sugars were run as alternate samples on the chromatogram sheets and their positions located with an ammoniacal silver-nitrate reagent (Partridge, 1948). Measurement o/Intercellular Spaces. Pieces of blade tissue were fixed in 6 % glutaraldehyde in 0.05 M sodium-cacodylatebuffer, pH 6.8, dehydrated in a graded ethanol series, and embedded in Epon-Araldite resin (N[ollenhauer, 1964). Transections were cut 1 ~zmthick with glass knives, placed on slides, and stained with methylene blue in sodium borate, ca. pH 10. Photographs were taken of mesophyll regions free from all vascular tissue including minor veins. Intercellular spaces and cells, including the epidermis, were carefully cut from enlarged photographic prints, and weighed. Dividing the weight of the pat3er representing intercellular spaces by the total weight gave a measure of the percent volume of the blade occupied by the air spaces.
Results
1. Photosynthesis and Respiration I n p r e l i m i n a r y e x p e r i m e n t s w i t h p l a n t s in which leaf 5 was fully m a t u r e ( L P I 4.0-7.0) n e t p h o t o s y n t h e s i s , d a y t i m e d a r k r e s p i r a t i o n a n d n i g h t r e s p i r a t i o n were m e a s u r e d a t 3-h i n t e r v a l s over a 24-h period. T h e results of a r e p r e s e n t a t i v e e x p e r i m e n t a r e shown in Fig. 1. N e t p h o t o s y n t h e s i s r e m a i n e d c o n s t a n t t h r o u g h o u t t h e d a y . D u r i n g ~he night, r e s p i r a t i o n was i n i t i a l l y e q u a l ~o d a r k r e s p i r a t i o n d u r i n g t h e d a y , b u t in ma~ure leaves i~ d r o p p e d to a Iower level a n d r e m a i n e d c o n s t a n t for t h e rest of t h e n i g h t period. F r o m t h e results of these e x p e r i m e n t s i t was concluded ~hat reliable m e a s u r e m e n t s of p h o t o s y n t h e s i s could be m a d e a t a n y p e r i o d of t h e d a y , a n d t h a t n i g h t r e s p i r a t i o n was b e s t m e a s u r e d a f t e r 2 a.m. (14:00).
56
R. Turgeon and J. A. Webb ---{ I0-
?
5-
Eo
-5LEAF R.ASTOCHRON INDEX
Fig. 2. Net photosynthesis (.), night respiration (*), and dark daytime respiration (o) plotted against leaf age. Vertical bars = 2 • standard error of the mean Measurements of net photosynthesis and dark respiration during the day were made on blade 5 of 50 plants. For night respiration measurements, 20 plants were used. Upon completion of each experiment determinations were made of the leaf plastoehron index (LPI) and blade dry weight. At L P I 0.0 blade 5 is incapable of net photosynthesis (Fig. 2) and approximately 1 mg CO S h -1 dm -2 is lost from these leaves. As the blade expands photosynthetic capacity increases rapidly. Net photosynthesis begins at ca. L P I 0.1 (8% leaf expansion) and reaches a maximum rate at the prevailing light intensity of ca. 11 mg COs h -1 dm -2 at L P I 2.0 (70% leaf expansion) or shortly thereafter. With increasing age photosynthetic capacity slowly declines to ca. 8.5 mg CO s h -1 dm -2 at L P I 7.0. Young leaves (LPI 0.0) lose ca. 5 mg CO2 h -1 dm -~ by dark respiration during the day and approximately the same amount at night. As the leaf grows respiration declines and levels off between L P I 1,0 and 2.0. Mature leaves respire 0.75 mg COS h -1 dm -2 at night and about 3 times this amount in the dark during the day. 2. C a r b o n B a l a n c e
The carbon balance over a 24-h period in a developing leaf or a mature leaf blade may be defined by the following equation, G r o w t h = N e t Photosynthesis--Night Respiration• where growth is the increase in weight of C in the blade over a 24-h period, net photosynthesis is the weight of C assimilated during the light period, night respiration is the weight of C respired during the dark period, and translocation is the weight of C either imported (-k) or exported (--) in 24 h. Note that growth is defined simply as an accumulation of C without regard for distribution. Night "dark" respiration should not be confused with "dark" respiration during the day. Values for net photosynthesis and night respiration were calculated from the
Leaf Development and Phloem Transport in Cucurbita
57
0.5"
)r~
o
0.1
9
-I
,
0
,
I
LEAF
2
PLASTOCHRONINDEX
,
~
3
4
Fig. 3. Dry weight increase plotted against leaf age. Vertical bars = 2 • standard error of the mean
~ N T H E S r S
40
30 20
/
',,/
~
GROWTH
INTOLEAF
3 o
~'
NIGHTI - - \
-I0 ~
.~PIRATION \
O
U
-20
+
T
OFLEAF
~TION
-30 -4C
I
2
LEAFPLA$TROCHRONINDEX
3
Fig. 4. Carbon balance plotted against leaf age. Values are calculated as instantaneous rates
and expressed as the weight of carbon exchanged over a 24-h period. Values for net photosynthesis and nigh~ respiration were calculated for 18-h and 6-h periods, respectively
data given in Fig. 2 and expressed as the total weight of C exchanged by the blade during the 18-h day and the 6-h night, respectively. Calculations of instantaneous growth rate were made graphically from the curve of dry-weight increase of leaf 5 with time (Fig. 3). I n order to convert this into C accumulation the dry-weight was assumed to be 45% C (Stout, 1961). The weight of C either imported or exported was calculated by difference using the above equation.
58
R. Turgeon and J. A. Webb
1.5.~'~C.~ OWTH z t.0, i ~~OSYNTHESI$ 0.5' -0.5' o
TRANSLOCATION i
0
~
LEAKPLAST0CHRONtNDEX
Fig. 5. Relative carbon balance plotted against leaf age. A value of 1.0 on t~heordinate indicates an increase or exchange of carbon, over a 24-h period, equivalent to ~he weight of carbon in the lamina
The balance sheet for C flux in leaf 5 obtained in this fashion is presented graphically in Fig. 4. At L P I 0.0 the quantity of C imported is equal to the amount required for growth. As the blade expands net photosynthetic capacity increases. The quantity of C translocated into the leaf increases until the growth rate peaks and then falls sharply. At approximately L P I 0.8 the contributions of net photosynthesis and transloca~ion to ~he influx of C into the blade are equal. When net photosynthesis supplies sufficient C to meet the demands of growth and night respiration, import ceases and export from the blade begins (LPI 1.1). Although these measurements provide an accounting of C flux, they do not properly reflect the efficiency of the C exchange processes since the weight of the blade is not taken into account. To obtain a measure of efficiency per unit weight as the leaf grows it is necessary to divide each factor in the above equation by the weight of the C in the blade. If this is done the weight units may be cancelled out and the values for net photosynthesis, dark respiration, growth and translocatiou become percentage or relative figures, each expressed as dw/dt 1/W. These relative values are a true measure of the efficiency of C flux, i.e., the ability of a unit weight of leaf material to add or remove C. I t should be noted that the relative growth factor in the above equation (dw/dt 1/W) is Blackman's Efficiency Index (Blackman, 1919) a well-known index of relative growth efficiency. Relative C flux is plotted against leaf age in Fig. 5. As in Fig. 4 the growth and import curves are almost identical for young leaves. Relative growth after unfolding reaches a maximum of 150 % increase in weight over a 24-h period at L P I 0.3. This coincides in time with the cessation of import by the leaf tip (Turgeon and Webb, 1973). The reIative growth rate falls sharply and then levels out to a slowly decreasing value. Shortly before the sharp decline in relative growth ends the leaf base ceases to import (LPI 1.3 ; Turgeon and Webb, t973). Therefore, cessation of import and the decline in relative growth rate after unfolding occur during the same period, L P I 0.3-1.3.
Leaf Development and Phloem Transport in Cucurbita
59
25. w
~ 20, "J 15
~) IO,
oJ0
3
4
LEAF PLASTOCHRON INDEX
Fig. 6. Intercellular-space enlargement in the distal (,) and proximal (o) regions of the expanding lamina expressed as a percentage of the tmtal volume of cells and air spaces excluding vascular tissue. Vertical bars = 2 • standard error of the mean
3. Intercellular Spaces Air spaces greatly enhance the distribution of CO z throughout the mesophyll and measurements were made of the enlargement of intercellular spaces during the growth of leaf 5. Sixteen plants were chosen in which the age of the leaf was between L P I 0.0 and 4.0. Two pieces of the blade, one near the apex and one near the base, were removed and the percentage of the cross-sectional area of the blade occupied b y air spaces was determined as described in Material and Methods. Care was taken to sample comparable regions in each leaf. Air spaces develop in the distal region of the blade in advance of the proximal region (Fig. 6). Air space enlargement and the development of net photosynthetic capacity are closely correlated (compare Fig. 2 and 3). However, air space enlargement is complete at the leaf apex b y L P I 1.3 and in the leaf base b y L P I 1.7, whereas photosynthesis reaches its m a x i m u m value at L P I 2.0 or slightly later. I n the mature leaf we calculated air spaces to occupy ca. 23 % of the blade volume (excluding vascular tissue).
4. Sugar Synthesis Carbon is transloeated in C. pepo primarily as sucrose, stachyose and raffinose. Earlier studies (Webb and Gotham, 1964) indicated that staehyose and raffinose are synthesized from 1~C0~ at approximately the same time as export from the blade begins. I t was of interest therefore to determine more precisely the time of initial synthesis of these transport sugars. Leaf 5 was excised from 20 plants and supplied 14C0~ continuously for 5 rain. []aC] Sucrose is synthesized in leaves which have just unfolded. [14C] Staehyose and [14C]raffinose are first synthesized at approximately L P I 0.6 (Fig. 7). The incorporation of 14C into stachyose and raffinose increases rapidly as the leaf matures and under the experimental conditions exceeds the incorporation into sucrose by L P I 1.0.
60
R. Turgeon and J. A. Webb
~0
z o
b 20' J
z
0
o
i
~
~,
4
LEAF PLASTOCHRON INDEX
Fig. 7. Incorporation of 14C,by expanding leaves, into the transport sugars sucrose (o), stachyrose (o) and raffinose (A)during exposure to 14C02for 5 min. Vertical bars = 2 • standard error of the mean
Discussion Our data on photosynthesis and respiration in growing leaves of Cucurbita are similar to those published for a variety of dicotyledonous species (Dickman, 1971 ; Hopkinson, 1964; Kriedemann, 1968; Larson and Gordon, 1969; Larson et al., 1972; Richardson, 1957; Ryle, 1972). Young leaves are initially incapable of net CO~ uptake in the light. The coincidence of the curves for growth and import of newly unfolded leaves show their high dependence on a sustained supply of imported nutrients. As the blade expands photosynthetic potential rapidly increases. At L P I 1.1 import ceases and thereafter the net amount of atmospheric carbon fixed by the lamina is sufficient to sustain growth, night respiration and to supply soluble carbohydrates for export. Maximum net photosynthesis is attained when leaves are between threequarters to fully expanded and thereafter slowly declines. In immature leaves the rates of respiration at night and in dark periods during the day are almost identical. A decrease in the respiratory capacity at night first becomes evident during the import-export transition at approximately L P I 1.0. This suggests that young growing leaves continue to import carbon and to respire at an undiminished rate during the night while the respiration of non-importing leaves becomes limited either through a shortage of or an inaccessibility to suitable substrates or through a partial inhibition of the respiratory system. At the transition point between import and export, L P I 1.1, the relative growth rate of the lamina has decreased considerably. However, the transition from import to export does not take place instantaneously throughout the leaf but occurs over a single plastochron interval, beginning at the leaf tip at L P I 0.3 when the relative growth rate of the whole blade is still very high (Turgeon and Webb, 1973).
Leaf Development and Phloem Transport in Cucurbita
61
In a consideration of leaf development it is therefore important to distinguish between the apparent properties of the blade summed as a whole and those properties of its component parts, presumably down to the level of individual mesophyll cells. Since the growth of leaves proceeds in a basipetal direction (Avery, 11933) and the capacity for photosynthesis also develops basipetally (Larson and Gordon, 1969) it is reasonable to assume that the data obtained from whole leaves may be applied to smaller regions of the lamina. I t is clearly an advantage to expanding tissue to have the import-export transition occur only after the growth rate has considerably declined. Rapid growth requires a constant supply of nutrients. If, during this critical period, reliance were placed entirely on photosynthetically assimilated carbon, growth would be unduly affected at night or during lbimes of inclement weather. During these periods, adequate substrates are assured through their continued import from the mature parts of the plant. The increase in relative growth rate between leaf unfolding and the stage when the leaf tip stops importing indicates an improvement in growth conditions perhaps from enhanced nutrient supply or release from internal inhibition. This has been reported for wheat leaves in which the rate of relative growth increases after leaf initiation to a maximum shortly before emergence (Williams, 1960). However, in the dicotyledonous species Tri/olium repens (Denne, 1966) and Fragaria vesca (Arney, 1953) the relative growth declines steadily after initiation. The pattern of relative growth before unfolding in Leaf 5 of Cucurbita pepo, the cause of the increase in growth rate after unfolding, and the relationship of this increase to the supply of imported substrates remain to be explored. Increased photosynthetic capacity is accompanied by the basipetal development of the intercellular spaces in the leaf mesophyll. A similar result was obtained by Isebrands and Larson (1973) using Populus deltoides leaves. Although air spaces cease expanding by L P I 1.6 the net photosynthesis rate continues to increase until L P I 2.0. While air spaces enhance the efficiency of gas exchange it would appear that at least in Cucurbita pepo their development does not limit photosynthesis in the developing leaf as suggested by Isebrands and Larson for their material (1973). Carbon is exported from mature Cucurbita leaves primarily as sucrose, stachyose and raffinose (Webb and Gorham, 1964). Sucrose synthesis occurs at the earliest stages of leaf development whereas synthesis of stachyose and raffinose is first detectable at L P I 0.6. Intralaminar transport between the leaf tip and the leaf base was observed at L P I 0.7 (Turgeon and Webb, 1973). At this stage the distal 30% of the blade no longer imports but the leaf blade does not export. Perhaps ~he inability to detect intralaminar transport between L P I 0.3, when import at the leaf tip ceases, and L P I 0.7 is in some way connected to the lack of available stachyose and raffinose. Sucrose is synthesized between L P I 0.3 and 0.6 but it appears not to be available for transport during this developmental stage. I t is intriguing that the syntheses of stachyose and raffinose coincides with the initiation of translocation. 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.
62
R. Turgeon and J. A. Webb
References Arney, S. E. : The initiation, growth, and emergence of leaf primorida in Fragaria. Ann. Bot. 1~.S. 17,477-492 (1953) Avery, G. S., Jr. : Structure and development of the tobacco leaf. Amer. J. Bot. 20, 565-592 (1933) Btaekman, V. It.: The compound interest law and plant growth. Ann. Bot. 13, 353-360 (1919) Denne, P. M. : Leaf development in Tri/olium repens. Bot. Gaz. 127, 202-210 {1966) Dickmann, D. I. : Photosynthesis and respiration by developing leaves of cottonwood (Populus deltoides Bartr.). Bot. Gaz. 132, 253-259 (1971) Hopkinson, J. M.: Studies on the expansion of the leaf surface. IV. The carbon and phosphorus economy of a leaf. J. exp. Bot. 15, 125-137 (1964) Isebrands, J. G., Larson, P. R. : Anatomical changes during leaf ontogeny in Populus deltoides. Amer. J. Bot. 60, 199-208 (1973) Kriedemann, P. E. : Photosynthesis in vine leaves as a function of light intensity, temperature and leaf age. Vitis 7, 213-220 (1968) Larson, P. R., Gordon, J.C.: Leaf development, photosynthesis, and 14C distribution in Populus deltoides seedlings. Amer. J. Bot. 56, 1058-1066 (1969) Larson, P. R., Isebrands, J. C., Dickson, R. E. : Fixation patterns of 14C within developing leaves of eastern cottonwood. Planta (Berl.) 107, 301-314 (1972) Milthorpe, F. L., Moorby, J. : Vascular transport and its significance in plant growth. Ann. Rev. Plant Physiol. 20, 117-138 (1969) Mollenhauer, tI. H. : Plastic embedding mixtures for use in electron microscopy. Stain Teehn. 39, 111-114 (1964) Partridge, S. M. : Filter-paper partition chromatography of sugar. L General description and application to the qualitative analysis of sugars in apple juice, egg white and foetal blood of sheep. Biochem. J. 42, 938-953 (1948) Richardson, S. D.: The effect of leaf age on the rate of photosynthesis in detached leaves of tree seedlings. Acta bot. neerl. 6, 445-457 (1957) RyIe, G. J. A.: A quantitative analysis of the uptake of carbon and of the supply of 14C-labelled assimilates to areas of meristematic growth in Lolium temutentum. Ann. Bot. 36, 497-512 (1972) Stout, P. R. L.: Micronutrient needs for plant growth. Calif. Fertilizer Conf. Proc. (California Fertilizer Assoc.) 9, 21-23 (1961) Turgeon, R., Webb, J. A. : Leaf development and phloem transport in Cucurbita pepo: transition from import to export. Planta (Berl.) 113, 179-191 (1973) Webb, J. A., Gorham, P. R. : Translocation of photosynthetically assimilated 14C in straightnecked squash. Plant Physiol. 39, 663-672 (1964) Williams, R. F. : The physiology of growth in the wheat plant. I. Seedling growth and the pattern of growth at the shoot apex. Austral. J. Biol. Sci. 13, 401-428 (1960)
