Planta (Berl.) 111, 95--112 (1973) 9 by Springer-Verlag (1973)

Distribution of Imported 14C in Developing Leaves of Eastern Cottonwood According to Phyllotaxy Philip R. Larson* a n d R i c h a r d E. Diekson* North Central Forest Experiment Station, U.S. Department of Agriculture, Forest Service, Star Route No. 2, Rhinelander, Wisconsin 5450i, U.S.A. Received October 27 / December 6, 1972

Summary. Individual leaves of eastern cottonwood (Populus deltoides Bartr.), representing an ontogenetic series from leaf plastochron index (LPI) 3.0 to 8.0, were fed 1~C02 and harvested after 2-24 h. Importing leaves from LPI --1.0 through 8.0 on each plant were sectioned into 9 parts, and each part was quantitatively assayed for 14C activity. The highest level of 14C import was by leaves from LPI 1.0 to 3.0, irrespective of source-leaf age. 14C was translocated preferentially to either the right or left lamina-half depending on the position of the importing leaf in the phytlotactic sequence and its stage of development. For example, import was high when the importing leaf and the source leaf had two vascular bundles in common, moderately high with one bundle in common, and low with no bundles in common. The distribution of 14C within young importing leaves was highest in the lamina tip and decreased toward the base. With increasing leaf age, incorporation declined in the lamina tip and increased in the base. It may be concluded that each cottonwood leaf progresses through a continuum of importing and exporting stages as its lamina expands. The photosynthate imported by a given leaf is compartmentalized, with different exporting leaves supplying photosynthate to rather restricted regions of the lamina. Such localizatio3 within the importing leaf depends on its vascular connections with each of the exporting leaves, and these are predictable from a knowledge of the phyllotaxy. Introduction D u r i n g t h e m o s t r a p i d phase of l a m i n a expansion, a y o u n g leaf s i m u l t a n e o u s l y i m p o r t s a n d e x p o r t s p h o t o s y n t h a t e s (Hale a n d W e a v e r , 1962; H a n s e n , 1967; L a r s o n a n d Gordon, 1969). F u r t h e r i n v e s t i g a t i o n of this d u a l role suggests t h a t t h e p h o t o s y n t h e t i c a n d t h e i m p o r t - e x p o r t functions are segregated w i t h i n t h e leaf. A leaf is n o t homogeneous with r e g a r d to p h o t o s y n t h e t i c a c t i v i t y either during e x p a n s i o n ( B a l d y a n d L e B u h a n , 1971) or when a p p r o a c h i n g senescence ( H a r d w i c k et al., 1968). W a d a a n d K u r o d a (1968) d e m o n s t r a t e d , for example, t h a t p h o t o s y n t h e t i c a c t i v i t y in fully e x p a n d e d t o b a c c o leaves was low in t h e apex, interm e d i a t e in t h e middle, a n d high in t h e base. Similar results h a v e been r e p o r t e d for laC02 f i x a t i o n p a t t e r n s in developing c o t t o n w o o d leaves * Plant Physiologists. 7 Planta (Berl.),Bd. i l l

96

P. I~. Larson and 1~. E. Diekson: Table 1. Treatment schedule and experimental design

Treatment time a (h)

Presentation time b (rain)

14C0e presented (~C)

LPI of source leafc

2 6 6 12 24

60 60 60 60 60

2.2 2.2 8.0 8.0 8.0

3.2 3.3 3.1 3.0 3.0

4.0 4.1 4.0 4.0 4.3

5.0 5.1 5.2 5.0 5.1

6.0 6.0 6.2 6.4 6.0

7.0 7.4 7.1 7.4 7.0

8.0 8.0 8.1 8.3 8.4

a Elapsed time between introduction of 14C02into leaf chamber and harvest. b Aebual time leaf was exposed to 14CO2in chamber. c Leal plastochron index of leaf at time of 14C0e feeding; each source leaf represents a separate plant. (Larson et al., 1972). I n the latter study, photosynthate produced by the precociously mature tip was exported out of the leaf and none was redistributed to the still-expanding base. Failure of one region of the leaf to supply another with photosynthates also appears to be a prevalent characteristic of soybean (Aronoff, 1955; Thaine et al., 1959; Penny and Nelson, 1970) and tobacco (Jones and Eagles, 1962; Yamamoto et al., 1969). The relative level of import into young, expanding leaves depends on their stage o5 development and vascular connection with the exporting ]eaves (Shiroya et al., 1961 ; Wardlaw, 1968). Young leaves in the most rapid phase of expansion are generally the most active importers, but the relative level of import depends on the position of the leaf on the plant. A number of authors have suggested a causal relation between import patterns and organization of the vascular system (Jones et al., 1959 ; l~inne and Langston, 1960 ; Joy, 1964; Quinlan, 1965). Although a relation between the vascular system and leaf phyllotaxy is implied in most of these studies, it has been specifically studied in only a few cases (Shiroya et al., 1961; Brown, 1968; Ho and Peel, 1969; Vasilevskaya and Ermolaeva, 1970). For example, when the 12th leaf of a tobacco plant with a 2/5 phyllotaxy was fed 14CO~, the 17 th leaf beeame heavily labeled; the 12th and 17th leaves were on the same orthostichy and had direct vascular connections (Yamamoto, 1967). With the exception of Vasilevskaya and Ermolaeva (1970), all the Iorementioned authors relied either on external morphology or presumed vascular organization for evaluating phyllotaxy. The present study was designed to examine the 14C-import patterns of developing leaves of eastern cottonwood (Populus deltoides Bartr.) plants, to quantify import by these leaves on a relative basis, and to relate the import patterns of the leaves to the phyllotactic arrangement of the vascular system as determined by anatomical investigation.

Distribution of Imported ~4C in Leaves

97

Fig. 1. Sampling scheme for determining the distribution of 1~C within importing leaves of cottonwood. T tip, U M upper middle, L M lower middle, B base. Prefix letters R and L refer to the right and left lamina sectors, respectively, when the adaxial surface is viewed from the petiole. M V midvein, P petiole

Materials and Methods A previous communication (Larson et al., 1972) described the 14Cfixation patterns within source leaves of eastern cottonwood (Populu~ deltoides ]3artr.) fed 14C02. In the present study, the 14C distribution patterns within the importing leaves were examined. The same plants were used in both investigations. 7*

98

P . R . Larson and R. E. Dickson: 0 O x

~o 'O

o

x 4'

TOTAL

E Q_

RECOVERED o

/

.J I--

O

I--

. , TOTAL

/,t o

........

/ . / ' - - .

-I ~ -

EXPORTED

/ /

_//

/

/

/

-

LP{ OF SOURCE LEAF

:Fig. 2. Recovery alld export of 14C in d p m from source leaves located at different LPIs in the 8-~C series..---, Total 14C recovered a t harvest from all plant parts combined; each point is the m e a n of 3 t r e a t m e n t times. Variation around means: 9 6 h; • 12 h; A 24 h. ~ 1 7 6 Total 14C exported b y the source leaf; each point is the mean of 3 t r e a t m e n t times The plants were raised from seed in nutrient-sand culture under controlled environmental conditions as previously described (Larson and Gordon, 1969), and were selected for t r e a t m e n t when they reached a plastochron index (PI) of 16.0 to 16.4 (Erickson and ~ichelini, 1957; Larson and Isebrands, 1971). A t P I 16.0, the 16th leaf from the base was exactly 2.0 cm long, whereas at P I 16.4 the 16th leaf h a d advanced 0.4 of a plastochron beyond the 2.0-cm length. Leaves in the ontogenetic series down the plant were numbered according to leaf plastochron index (LPI). For example, the 16th leaf on a p l a n t of P I 16.0 would have a n L P I of 0.0, the next-older leaf L P I 1.0, and so on down the stem. Two unreplicated series of plants were used (Table 1). I n the first series, plants were fed 2.2 [xC of 14C02 per leaf; total t r e a t m e n t times from introduction of 1~C02 until harvest were 2 a n d 6 h. I n the second series, plants were fed 8.0 t~Ci of 14C02 per leaf; total t r e a t m e n t times were 6, 12 and 24 h. For each t r e a t m e n t time a single leaf on each of 6 different plants was fed ; the fed leaves rangedfrom L P I 3.0 through 8.0. Therefore, a t r e a t m e n t time consisted of 6 plants, each with a fed leaf a t a different LPI. A t harvest, the importing leaves were severed from the p l a n t below the pulvinus, immediately quick-frozen, and then freeze-dried. Each leaf was autoradiographed according to the procedure of Crafts a n d Yamaguchi (1964). The dried leaves were next sectioned as shown in Fig. 1 to provide data on 14C incorporation in the right and left halves of the lamina, in longitudinal sectors of the lamina, and in the petiole and midvein. Nine importing leaves on each p l a n t were sectioned, ranging from L P I -- 1.0, the smallest leaf possible to dissect, to L P I 8.0, the t h i r d fully-expanded leaf below the apex; these leaves will be referred to as the assayed leaves. The remaining leaves on each plant were freeze-dried and sampled for total

Distribution of Imported 14C in Leaves

99

I00"

>.

80'

,~'J 60

/ /

X~x

40-

Z ILl L) E ~. 20-

x /

/ o /

o

~ / ~ _1~ . . .7. - - -6- - - - - ~ A J

EXPORTEDuPwARD TOTAL RECOVERED TOTAL

EXPORTED

LPI OF SOURCE LEAF

Fig. 3. Gross patterns of 1~C recovery and export. 9 Total 14C recovered at harvest from all plant parts combined expressed as percentage of the 1~C0~presented to the source leaf. A Total 14C exported by the source leaf expressed as percentage of total 14C recovered. • Quantity of 1~C exported upward to all plant parts above the source leaf expressed as a percentage of the total 1'C exported by the source leaf. Each point is the mean of 5 treatment times 14C activity. Bark, which included the phloem and all tissues external to it, and wood were separately analyzed for each internode of the stem. The roots were sampled as a whole. Each leaf section and plant part was weighed, ground to a small particle size, and sub-sampled. The 3- to 5-rag sub-samples were prepared for liquid scintillation spectrometry as previously described (Larson et al., 1972). Differences in ~4C recovery and export were observed among the time treatments, but were not consistent from one source leaf to another. Therefore, in calculating gross patterns of ~4Crecovery and export, the data for the five time treatments were averaged. Fig. 2 shows the range of variation of 14C in dpm recovered and exported for the 8.0-,ae series. Gas exchange data were presented in a previous paper.

Results

Gross Patterns o/laC Recovery and Export The total laC recovered a t h a r v e s t from all i m p o r t i n g p l a n t parts combined varied with the age of the source leaf. I t ranged from 6.5% of the 1~CO~ presented i n leaves one-third e x p a n d e d at L P I 3.0, to 34% i n leaves fully e x p a n d e d at L P I 6.0 to 8.0 (Fig. 3). The low recoveries from m a t u r e leaves was due p r i m a r i l y to the 1-h p r e s e n t a t i o n time. U n d e r our conditions, 14C02 u p t a k e generally continues for a b o u t 2 h. The t o t a l ~4C exported b y the source leaf expressed as a percentage of the total x4C recovered also increased with age of the source leaf, r a n g i n g from 2.5 % i n leaves of L P I 3.0 to a b o u t 26 % i n leaves of L P I 6.0 a n d older (Fig. 3).

100

P . R . Larson and R. E. Dickson: I00-

BOt.U j....

(~ 0. X I,,IJ

60-

"~""~'"'~

~ "

'40 ~

APICAL UNIT

%%%. 9 A S S A Y E D L~'AVFS

I-Z hi U 20 n" Ixl 0-

/

x

/

X

~

x BARK

I0-

r

I

:5

i

I

i

i

4 5 6 7 LPI OF SOURCE LEAF

i

8

Fig. 4. Partitioning of the total 14C exported by a source leaf into major plant parts. Solid lines: 9 Apical unit includes all leaves on the plant plus the apex and immature internodes above LPI 4.0. x Bark includes all hand-peeled material external to the woody cylinder below LPI 4.0. A Woody cylinder below LPI 4.0. 9 Roots. Dashed line: The percentage of total 14C exported by a source leaf to the assayed leaves (LPI --1.0 through LPI 8.0). Each point is the mean of 5 time treatments. (Note change of scale on ordinate)

T h e q u a n t i t y of 14C e x p o r t e d u p w a r d to all p l a n t p a r t s a b o v e t h e source leaf expressed as percentage of t h e t o t a l 14C e x p o r t e d b y t h e source leaf increased from L P I 3.0 to L P I 5.0, a n d t h e n decreased to L P I 8.0 (Fig. 3). A source leaf a t L P I 3.0 n o t o n l y e x p o r t e d v e r y little of t h e 14C fixed, b u t m o s t of t h e e x p o r t was downward. T h e p a r t i t i o n i n g of t h e 14C e x p o r t e d b y t h e source leaf into t h e m a j o r p l a n t p a r t s is shown in Fig. 4. B y far t h e largest p r o p o r t i o n of 14C occurred in t h e leaves a n d i m m a t u r e internodes a b o v e L P I 4.0. Over 90 % was recovered from this apical u n i t when t h e source leaf was L P I 3.0, a n d 53 % when it was L P I 8.0. As t h e p e r c e n t a g e of 14C i n c o r p o r a t e d b y t h e apical u n i t decreased w i t h age of t h e source leaf, t h e p e r c e n t a g e i n c o r p o r a t e d into b a r k , wood a n d roots increased. No a c t i v i t y was recovered from t h e roots when t h e source leaf was L P I 3.0 or 4.0, regardless of t r e a t m e n t time. H o w e v e r , as t r e a t m e n t t i m e increased, 14C i n c o r p o r a t i o n into b a r k a n d wood a n d t r a n s l o c a t i o n to t h e roots increased from t h e older source leaves. These l a t t e r t r e n d s are obscured b y t h e gross averaging.

Distribution of Imported 14C in Leaves

101

14C Export to Leaves within a Growth Unit

The 9 leaves intensively assayed (LPI --1.0 through L P I 8.0) are the principal leaves in the zone of leaf development of a cottonwood shoot. Since cottonwood has a 3/8 phyllotaxy, 8 leaves would occur within the stem section corresponding to a unit of an orthostichy. These 8 leaves will be referred to as a growth unit, and the 9 assayed leaves would comprise one complete growth unit plus the first leaf of another. E x p o r t to the 9 leaves, expressed as percentage of the total laC exported b y the source leaf, declined from 85% at L P I 3.0 to 34% at L P I 8.0 (Fig. 4). When interpreted on the basis of the recovery and export curves (Fig. 3), this means t h a t a source leaf at L P I 3.0 fixed about 6.5 % of the 14C0~ presented. Of this quantity, 2.5 % was exported from the source leaf and, of the latter, 85 % was imported by the assayed leaves. I n contrast, a source leaf at L P I 8.0 fixed about 34% of the 14C02 presented. Of this quantity, 26% was exported, and 34% of the latter was imported by the assayed leaves. This export pattern is summarized in Fig. 5 where the total quantity of laC exported to the assayed leaves is expressed as a percentage of the total 1~C0~ presented to the source leaf. The values increased from a low of 0.13 % for leaves at L P I 3.0 to 4.60% at L P I 6.0, and declined thereafter. The total 14C exported to the assayed leaves (Fig. 4) was next analyzed for the percentage of the 14C incorporated into each of these leaves (Table 2). The purpose of this analysis was to determine the relation between the amount of ~ac imported by a leaf and its position in the phyllotactic sequence. To interpret these data, it is first necessary to briefly describe the relation of the vascular system to phyllotaxy, hereafter briefly referred to as "vascular phyllotaxy". 16-leaf cottonwood plants have a 3/8 phyllotaxy (unpublished data). Each leaf contributes 3 traces to the vascular cylinder, and a complete 8-leaf growth unit therefore consists of 24 stem bundles. The arrangement of bundles within one growth unit of a stem with left phyllotaxy is presented in Fig. 6. Since the figure was drawn to aid interpretation of the tabular data, lea~ No. 1 was designated the source leaf and subsequent leaves within the growth unit were numbered in the acropetal direction. In a similar manner, leaves above the source leaf were numbered upward on the right of Table 2 to conform with Fig. 6. These numbers should not be confused with the basipetal, ontogenetie L P I numbering sequence appearing on the left of the table. The percentage of 14C imported by a given leaf above the source leaf (No. 1)is found by reading diagonally upward from right to left. Four time series have been included in Table 2 to illustrate the relative consistency of the data.

102

P. 1~. Larson a n d 1%. E. Dickson: 5-

=4

/\

,,,

I--

\ x

nO I1. X 3Ld

x

z u r~ tlJ n

-//

.

.

.

3

4LPI

.

.

5

. 6

7

8

OF SOURCE L E A F

Fig. 5. The total quantity of 14C imported by the assayed leaves (LPI 1.0 through L P I 8.0) expressed as percentage of the 14C0~ presented to the source leaf. Each point is the mean of 5 time treatments and a n assumed presentation of 8 ~C t4G02 - -

COTTONWOOD VASCULAR SYSTEM 3/8

LEFT PHYLLOTAXY IC 4R 5L\ ~

I

/6L

/7R

"

:

t

3C

--7C

5L 8C"

4L

"SR

3R'~L 2L

~ T L ] ~)R

2C

5C Fig. 6. Orientation of the vascular bundles within one 8-leaf growth unit of a cottonwood plant with 3/8 phyllotaxy. Each leaf contributes 3 bundles (Central, and Right and Left laterals) to the vascular cylinder. Reconstruction of the vascular system was based on serial microsections from the stem of a 16-leaf cottonwood plant. The source leaf is No. 1 irrespective of its position on the stem; importing leaves are numbered acropetally

0.1 0.6 4.5 38.7 * 40.4 14.2 0.2 0.8 0.5

--1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

2.8 0.4 31.1 14.7 31.2 * 8.9 4.7 4.2 2.0

1.0 0.3 38.9 24,2 31.2 * 3.2 0.2 0.4 0.6

0.4 15.5 0.8 40.0 18.8 15.6 * 1.6 7.3 0.0

1.3 38.1 1.1 44.6 13.3 0.7 * 0.5 0.2 0.2

1.7 32.2 30.1 10.1 17.9 1.2 3.1 3.1 0.6 *

0.4 21.l 15.9 10.1 42.9 1.8 5.9 1.0 0,9 *

6-h series 3.6 11.5 0.6 11.4 36.6 2.7 0.7 53.8 48.6 1.8 9.4 16.7 0.3 0.7 * 0,8 0.1 * 0,1 0.6

8.0

8.9 7.1 9.1 45.7 1.2 25.4 1.3 1.0 * 0.3

7.9 2.9 45.7 1.0 34.9 5.5 1.2 * 0.5 0.4

24-h series

7.0

0.0 0.6 26.0 23.7 * 30.5 7,4 5.4 5.3 1.1

0.6 1,7 2.0 17.4 * 41.1 22.6 7.0 4.6 3.0

1.5 0.1 3.4 0.7 9,0 * 11.9 18,2 7.2 48.0

0,7 0.6 4.9 11.4 11.7 * 29.5 39.8 0.5 0.9

4.0

0.5 12.5 0.8 32.6 32.0 7.4 * 1.7 11.9 0.6

0.6 3.2 0.6 48.4 23.4 21.9 * 1.0 0.6 0.3

5.0

2-h series 2,3 1.7 29.8 2.5 38.4 13.6 3.0 * 3.1 5.6

4.3 1.7 40.1 1.9 45.5 4.7 1.4 * 0.2 0.2

12-h series

6.0

6.8 7.7 4.6 37.1 3.3 36.6 3.6 0.1 * 0.2

32.5 13.2 20.7 26.5 1.2 3.3 1.5 0.5 * 0.6

7,0

0.3 30.7 30.0 4.8 25.4 0,5 2.8 1.9 3.6 *

0.5 27.0 20.9 10.8 34.2 1.4 3.2 1.1 0.9 *

8.0

10 9 8 7 6 5 4 3 2 f

10 9 8 7 6 5 4 3 2 1

Leaf No b

T h e figures are t h e p e r c e n t a g e s c o m p u t e d on basis of i m p o r t b y a s s a y e d l e a v e s t o t a l i n g 1 0 0 % ; t o t a l d p m p e r leaf. L e a f d a t a i n c l u d e m i d v e i n a n d petiole. b L e a f n u m b e r s a b o v e t h e s o u r c e leaf c o n f o r m to Fig. 6. T h e p o s i t i o n of e a c h s o u r c e leaf (No. 1) is m a r k e d b y a n a s t e r i s k (*), a n d t h e n u m b e r of t h e leaf a b o v e t h e s o u r c e leaf is f o u n d b y r e a d i n g d i a g o n a l l y u p w a r d f r o m r i g h t to left.

0.9 1.8 16.0 16.6 * 21.8 18.5 14,8 9.6 0.0

6.0

3.0

5.0

3.0

4,0

L P I s o u r c e leaf

L P I source leaf

--1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

LPI importing leaf

T a b l e 2. I n c o r p o r a t i o n of 14C in a s s a y e d l e a v e s as a p e r c e n t a g e of t h e to~al 1~C e x p o r t e d b y t h e s o u r c e leaf ~

9~

~3

z

9

104

P.R. Larson and R. E. Dickson:

Acropetal translocation of 14C ~from a source leaf to an importing leaf varied according to the position of the importing leaf in the phyllotactic sequence and to the stage of development of both the source leaf and the importing leaf. For example, from Table 2 it is evident t h a t a large percentage of ltC was exported to leaves No. 4 and 6 (italics) above source leaf No. 1 in each time series. By referring to Fig. 6, it can be seen that in both cases the central and one lateral bundle of an importing leaf were in contact with one lateral and the central bundle, respectively, of the source leaf; i.e., 4C and 1L and 4 R and 1C. A high level of import occurred, however, only when the importing leaf was in a rapid stage of expansion, generally between L P I 1.0 and 3.0. A moderately high percentage of 14C was imported by a leaf when one of its laterals was in contact with one of the lateral bundles of the source leaf, such as 2 R and 8L (Fig. 6). Because leaves No. 2 and 8 are well separated longitudinally on the stem, they also illustrate the importance of stage of development in determining a leaf's abihty to import ~4C from a particular source leaf. For example, in the 24-h series (Table 2), when the source leaf was L P I 8.0, import by leaf No. 2, a mature leaf, was only 0.6%, whereas import by leaf No. 8, an expanding leaf, was 30.1%. As the L P I of the source leaf decreased, the age of leaf No. 2 directly above it decreased and 14C import increased correspondingly. I n contrast, as the L P I of the source leaf decreased, 14C import by leaf No. 8 also decreased. I n the latter case, the age of leaf No. 8 was less, but the leaves became successively smaller near the apex and their import capacity declined sharply. Regardless of source-leaf age and treatment time, import by leaves No. 2 and 8 appeared to be greatest during the most rapid stages of leaf expansion at an L P I of 1.0 to 3.0. Within the range of data (Table 2), the percentage of 140 import tended to increase from L P I 7.0 to 2.0 when the importing leaf was No. 2 above the source leaf, and to decrease from L P I 1.0 to -- 1.0 when the importing leaf was No. 8 above the source leaf. A moderately low percentage of 14C was imported by a leaf when one of its laterals lay between, but not in direct contact with, the central and lateral bundles of the source leaf; i.e., 3 L and 7 R (Fig. 6). I m p o r t was again greatest when the importing leaf was at a position between L P I 1.0 and 3.0. Leaf No. 5, located on the opposite side of the stem from the source leaf, imported a very low percentage of laC regardless of stage of development. Leaf No. 9 was the first leaf in the next higher growth unit and its vascular bundles should theoretically be located immediately adjacent to those of the source leaf. Within the limits of our data, leaf No. 9 incorporated high levels of 14C activity when the source leaf was either L P I 7.0 or 8.0.

Distribution of Imported 1~C in Leaves

105

Basipetal translocation of ~4C into leaves below the source leaf did not always conform to the phyllotactic sequence. Although considerable downward export occurred from source leaves at L P I 3.0 and 4.0 (Table 2, Fig. 2), overall export was erratic. I t should also be recalled t h a t both total ~4C recovery and export from these leaves was extremely low (Fig. 3).

14C Import by Right and Left Lamina Halves The assayed leaves were next examined for distribution of laC activity in the right and left lamina halves. The investigated cottonwoods possessed right and left phyllotaxy in approximately a 1 : 1 ratio ; therefore all data were arithmetically transposed to left phyllotaxy. The data for the right and left lamina halves are presented as the ratio R / L in Table 3. When interpreted with the aid of Fig. 6, the data of Table 3 indicate a close association between incorporation within an importing leaf and the phyllotaxy of its vascular bundles relative to those of the source leaf. For example, leaf No. 4 (italics) had a high R/L ratio because its right bundle (4R) was in contact with the central trace (1C) of the source leaf. Similarly, leaf No. 7 (italics) had a high I~/L ratio and leaf No. 8 a low one because their right and left bundles, respectively, lay adjacent to bundles from the source leaf. Leaf No. 7 also illustrates the importance of the developmental stage on distribution of imported 1~C. Leaf No. 7 was not among the highest importers of laC (Table 2) and its right bundle (7R) was not in direct contact with those of the source leaf (Fig. 6); nonetheless it incorporated a high proportion of 14C in the right half of the lamina because its lamina was expanding rapidly.

Longitudinal Distribution o/14C within Importing Leaves Longitudinal distribution within each importing leaf was analyzed by examining 14C activity in the lamina sectors described in Fig. 7. The data represent the means for the 12- and 24-h series for source leaves L P I 3.0, 5.0, and 8.0. Distribution of 14C activity is based on dpm/mg of dry leaf tissue to adjust for differences in size and weight of the ]amina sectors, and it is expressed as a percentage of the total 14C imported by each leaf. Recovery of ~4C from the apical half of the lamina was relatively greater in young importing leaves than in mature ones. I n the basal half of the lamina, 14C recovery was related to the age of the source leaf, and the recovery curves changed in shape from L P I 3.0 to 8.0. ~4C recoveries from the petiole and midvein of importing leaves were fairly

106

P. 1~. Larson and R. E. Dickson:

Table 3. Ratio of 14C distribution in the right and left halves of assayed leaves a L P I importing leaf

L P I source leaf 3.0

4.0

5.0

6.0

Leaf No. b

7.0

8.0

1.95 0.11 5.33 1.05 0.59 1.83 0.92 1.51 *

1.00 1.76 0.20 2.56 1.18 0.89 1.54 1.13 1.84 *

10 9 8 7 6 5 4 3 2 1

1.38 0.12 10.90 0.80 0.83 1.88 0.43 3.93 *

1.03 2.70 0.13 4.35 0.40 1.28 2.09 0.72 1.59 *

10 9 8 7 6 5 4 3 2 1

1.38 0.25 7.70 0.78 0.09 1.67 0.46 100.00 *

0.53 0.42 0.11 11.82 0.58 0.42 1.46 1.65 0.66 *

10 9 8 7 6 5 4 3 2 1

24-h series

--1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

0.52 1.24 1.52 1.46 *

2.06 0.96 1.27 0.23 6.81 *

4.26 1.10 0.62 3.83 0.22 1.49 *

--1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

1.25 0.74 0.69 1.59 *

1.40 1.76 1.78 0.60 0.68 *

3.17 0.63 0.83 1.30 0.29 10.24 *

- - 1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

--1.36 1.12 *

-100.00 0.35 0.36 1.97 *

2.18 1.30 2.01 2.97 0.38 1.02 *

0.16 7.26 0.95 0.77 4.68 0.18 1.46 *

12-h series

1.74 4.74 1.40 0.34 2.23 0.21 3.18 *

2-h series

0.76 6.35 1.04 0.27 1.97 1.21 0.76 *

a The data are the ratio R/L. All trees have been arithmetically transposed to left phyllotaxy for comparison. Data for petiole and midvein were deleted in computing lamina ratios. b Leaf numbers above the source leaf conform to Fig. 6. The position of each source leaf (No. 1) is marked by an asterisk (*), and the number of the leaf above the source leaf is found by reading diagonally upward from right to left.

c o n s i s t e n t w h e n t h e s o u r c e leaf w a s L P [ 3.0. H o w e v e r , w h e n t h e s o u r c e leaf w a s L P I 5.0 o r 8.0, t h e p e r c e n t a g e of laC r e c o v e r e d f r o m t h e p e t i o l e a n d m i d v e i n i n c r e a s e d s t e a d i l y w i t h a g e of t h e i m p o r t i n g leaf.

Distribution of Imported ~O in Leaves SOURCE

LEAF

107

LPI 3.0

80-

60-

40~ x T+UM 20-

x/x

E "x. 80 -

SOURCE L E A F

LPI 5.0

I- 60> l40(J, IZ

20-

I.g L)

~ - x ~ x ~

n,.. LU 0,.

. . . .

"''-'-~x

SOURCE L E A F

SO"

'

T*UM

LPI 8.0 / P+MV

6040LM+B 20-

T+UM

LPI

OF I M P O R T I N G

LEAF

Fig. 7. Longitudinal distribution of 140 within importing leaves from source leaves of LPI 3.0, 5.0, and 8.0. T d-UM represents the apical half of the lamina (see Fig. 1). L~[d-B represents the basal half of the lamina. Pd-MW represents the petiole and midvein combined. Percentage data were computed on a dpm/mg basis to adjust for differences in size and weight of the lamina sectors

] n general, t h e r e l a t i v e recoveries of 14C from different sectors of i m p o r t i n g leaves older t h a n L P I 1.0 changed a p p r e c i a b l y as t h e age of t h e source leaf increased from L P I 3.0 to 8.0. This change was m o s t p r o n o u n c e d when t h e age of t h e i m p o r t i n g leaf r e a c h e d L P I 3.0, a n d t h e r e c o v e r y of 14C from t h e petiole a n d m i d v e i n exceeded t h a t of t h e o t h e r l a m i n a sectors (compare source leaves L P I 3.0 a n d 8.0).

108

P.R. Larson and R. E. Dickson:

Discussion The amount of 14C02 presented and the presentation time were deliberately kept low in this investigation to clarify the distribution patterns of 14C. High levels of 14C tend to mask these patterns. Gross recovery data were included primarily to place all other data in proper perspective and to aid interpretations of the 1~C import patterns in the assayed leaves. The data showed t h a t a leaf about one-third expanded at L P I 3.0 fixed a small fraction of the ~4CO~presented. With increasing leaf age up to maturity at L P I 6.0 and beyond, both 14C fixation and export increased (Fig. 3). These patterns are typical of most dicotyledonous species and they are well-documented in the literature (Kricdemann, 1968; Thrower, 1962; Hansen, 1967). x4C import by a developing leaf varied according to the position of the importing leaf in the phy]lotactic sequence and the stage of development of both the source leaf and the importing leaf. Withinan 8-1eafgrowth unit, import was highest by those leaves having the central and one lateral vascular bundle in contact with one lateral and the central bundle, respectively, of the source leaf. A moderately high level of 14C import was attained by leaves with one lateral bundle in contact with one lateral of the source leaf. A moderately low level of import was achieved by leaves with one lateral lying between, but not in direct contact with, the central and one lateral bundle of the source ]earl Leaves inserted on the opposite side of the stem and having no direct contact with the source leaf imported very low levels of ~4C. The foregoing ~40 distribution patterns were modified by the developmental stage of both the source and the importing leaves. A fully mature source leaf, such as L P I 8.0, exported aeropeta]ly to a complete 8-leaf growth unit. When two leaves within the growth unit had similar bundle contacts with the source leaf, the younger leaf in the most rapid phase of expansion invariably imported the most 140. Irrespective of source leaf age, the highest 14C import was b y leaves from L P I 1.0 to 3.0. During this phase of development, intercellular spaces and maturation of the photosynthetic system in a cottonwood leaf are developing basipetally into the expanding lamina base, while maturation of the major vein system is proceeding acropetally (Isebrands and Larson, 1972). laC was also translocated preferentially to either the right or left half of the lamina depending on the position of the importing leaf in the phyllotactic sequence and its stage of development(Figs. 8, 9). A similar vascular organization m a y explain why those leaves not directly above the source leaf were found to be more radioactive in the half nearest the source leaf of tobacco (Jones et al., 1959) and sugar beet (Joy, 1964). Downward export did not necessarily conform to the phy]lotactic sequence, regardless of source leaf age. Although downward export m a y enter mature leaves, it is more common for a mature leaf to receive 14C

Distribution of Imported 14C in Leaves

109

from a source leaf situated below it. Previous reports on import by mature leaves are contradictory. Some investigators found that mature leaves do not import ~4C (Jones et al., 1959; Joy, 1964)whereas others found that they may (Thrower, 1962; Quinlan, 1965; Khan and Sagar, 1969). Most evidence suggests that 14C enters mature leaves via the xylem (Biddulph and Cory, 1965), and that considerable cross-transfer from phloem to xylem can occur (Webb and Gorham, 1965; Peel, 1967). Autoradiography (Fig. t0), together with the high level of 14C activity incorporated into the midvein and petiole of mature leaves found in the present study (Fig. 7), strongly support xylem transport. The data also support the finding of K6cher and Leonard (1971) that the petiole and veins act as sinks for photosynthates, and the contention of Carr and Pate (1967) that imported photosynthate is utilized in the synthesis of structural tissues, since development of the petiole and major veins, particularly the xylem, continues long after mesophyll maturation. The lamina tip of cottonwood obviously matures precociously both in a structural and in a functional sense. It was previously concluded that 14CO2 fixed by the precociously mature lamina tip was utilized either in 8itu or by the immediately adjacent tissues. The small quantity exported moved directly out of the leaf and none was redistributed to other parts of the lamina (Larson et al., 1972). In a similar way, imported ~4C is distributed primarily to differentiating tissues and little or none to the mature tip (Fig. 11). Hence, the mature tip is physiologically analogous to a mature leaf; most of the import that does occur is incorporated into the vein system (Fig. 11). These data further confirm the suggestion of Carr and Pate (1967) that imported photosynthate is utilized principally in synthesizing structural tissue. The foregoing discussion describes a static situation based on data derived from individual leaves on many different plants. However, a cottonwood plant represents a dynamic system and every leaf progresses through each of the importing and exporting stages as its lamina expands. Consequently, a leaf develops from a heterotrophic organ when it first emerges from the apex to an essentially autotrophic one when it attains full expansion and maturity. Initial import of photosynthates is to the precociously maturing lamina tip, then basipetally and laterally as leaf development progresses. Investigation of this continuum at any one point in time shows that the photosynthate imported by a given leaf is compartmentalized, with different exporting leaves supplying photosynthate to rather restricted regions of the lamina. Such localization within the importing leaf depends on its vascular connections with each of the exporting leaves, and these are predictable from a knowledge of vascular phyllotaxy. As the leaf in question develops through the continuum and is replaced by younger leaves appearing above, its relative role as an importer and an exporter continuously changes. I t is

1 i0

P.R. Larson and 1%. E. Dickson:

Figs. 8--11

Distribution of Imported 14C in Leaves

111

Figs. 8--11. Autoradiographs of developing leaves above a leaf fed 14002. - Fig. 8. Right bundle (2R) of importing leaf (LPI 4.0) in contact with left bundle (1 L) of source leaf (LPI 5.0). - - Fig. 9. Left bundle (3 L) of importing leaf (LPI 4.0) lying between the central (1C) and one lateral (11~) of the source leaf (LPI 6.0). - - Fig. 10. A recently matured leaf (LPI 6.0) above the source leaf (LPI 8.0). Heavy labeling of the main veins and lack of movement into the mesophyll suggest xylem transport. - - Fig. 11. 14C imported by the precociously matured lamina tip is generally confined to the veins suggesting xylem transport and/or incorporation into the vein system. The remainder of the leaf is heavily labeled because one lateral (4R) and the central bundle (4C) of the importing leaf (LPI 3.0) are in contact with the central bundle (1C) and one lateral (1L), respectively, of the source leaf (LPI 6.0). In the autoradiograph, the heavy label contributed by the central bundle masks the preponderance of 14C present in the right lamina half

impossible to investigate the c o n t i n u u m of i m p o r t - e x p o r t functions of a n i n d i v i d u a l leaf in situ. However, to gain a perspective of this process, t h e p a t t e r n s shown b y the d a t a of T a b l e 2 a n d 3 m a y be visualized as a single leaf developing t h r o u g h the c o n t i n u u m of a n ontogenetic series d o w n the stem. The authors wish to acknowledge Mr. Gary Garton who assisted with the 14C analyses.

References Aronoff, S.: Transloeation from soybean leaves. Plant Physiol. 30, 184-t85 (1955). Baldy, C. IV[.,LeBuhan, J. P. : R6partition de la photesynth~se nette dans les feuilles de tabac. Phot~)synthetica 5, 421-423 (1971). Biddulph, 0., Cory, R. : Translocation of C14 metabolites in the phloem of the bean plant. Plant Physiol. 40, 119-129 (1965). Brown, K. J. : Translocation of carbohydrate in cotton: Movement to the fruiting bodies. Ann. Bot. 82, 703-713 (1968). Carr, D. J., Pate, J. S. : Ageing in the whole plant. Symp. Soc. exp. Bot. 21,559-599 (1967). Crafts, A. S., Yamaguchi, S. : The autoradiography of plant materials. Calif. Agr. Expt. Sta. Extens. Serv. Manual No. 35 (1964). Eriekson, R. O., Michelini, F. J.- The plastochron index. Amer. J. Bot. 44, 297-305 (1957). Hale, C. R., Weaver, R. J. 9The effect of developmental stage on direction of translocation of photosynthate in Vitis vini]era. Hilgardia 33, 89-131 (1962). Hansen, P.: 14C-studies on apple trees. II. Distribution of photosynthates from top and base leaves from extension shoots. Physiol. Plantarum 20, 720-725 (1967). Hardwiek, K., Wood, M., Woolhouse, H. W.: Photosynthesis and respiration in relation to leaf age in Perilla [ructescens (L.) Britt. New Phytologist 67, 79-86 (1968). Ho, L. C., Peel, A. J. : Transport of ~C-labelled assimilates and 32P-labelled phosphate in Saliz viminalis in relation to phyllotaxis and leaf age. Ann. Bot. 83, 743-751 (1969). Isebrands, J. G., Larson, P. R. : Anatomical changes during leaf ontogeny in Populus deltoides Bartr. Amer. J. Bot., in press (1972). 8 Planta (Berl,), ~3d.11i

112 P . R . Larson and 1~. E. Diekson: Distribution of Imported 14C in Leaves Jones, H., Eagles, J . E . : Transloeation of l~Carbon within and between leaves. Ann. Bet. 26, 505-510 (1962). Jones, H., Martin, I~. V., Porter, H . K . : Translocation of laCarbon in tobacco following assimilation of l~Carbon dioxide by a single leaf. Ann. Bet. 23, 493-508 (1959). Joy, K. W.: Transloeation in sugar beet. I. Assimilation of 14C0~ and distribution of materials from leaves. J. expt. Bet. 15, 485-494 (1964). Khan, A.A., Sagar, G. R.:Changingpatterns of distribution of the products of photosynthesis in the tomato plant with respect to time and to the age of a leaf. Ann. Bet. 83, 763-779 (1969). K6eher, H., Leonard, O. A. : Transloeation and metabolic conversion of 14C-labeled assimilates in detached and attached leaves of Phaesotus vulgaris L. in different phases of leaf expansion. Plant Physiol. 47, 212-216 (1971). Kriedemann, P. E. : 14C transloeation patterns in peach and apricot shoots. Aust. J. Agr. Res. 19, 775-780 (1968). Larson, P. R., Gordon, J. C. : Leaf development, photosynthesis, and C1~ distribution in Populus deltoldes seedlings. Amer. J. Bet. 56, 1058-1066 (1969). Larson, P. R., Isebrands, J. G.: The plastochron index as applied to developmental studies of cottonwood. Canad. J. For. Res. 1, 1-11 (1971). Larson, P. R., Isebrands, J. G., Dickson, R. E.: Fixation patterns of 14C within developing leaves of eastern cottonwood. Planta (Berl.) 107, 307-314 (1972). Peel, A. J. : Demonstration of solute movement from the extraeambial tissues into the xylem stream in willow. J. exp. Bet. 18, 600-606 (1967). Penny, P., Nelson, C. D. : Movement within leaves and plants of 1~C applied as laCO~. Canad. J. Bet. 48, 1033-1037 (1970). Quinlan, J. D. : The pattern of distribution of 14Carbon dioxide by a single leaf. East Malling Res. Sta. Rept. 1964, p. 117-118 (1965). Rinne, R. W., Langston, R. G.: Studies on lateral movement of phosphorus-32 in peppermint. Plant Physiol. 35, 216-219 (1960). Shiroya, M., Lister, G. R., Nelson, C.D., Krotkov, G.: Translocation of ~4C in tobacco at different stages of development following assimilation of 14CO2 by a single leaf. Canad. J. Bet. 39, 855-864 (1961). Thaine, R., Ovenden, S. L., Turner, J. S.: Translocation of labelled assimilates in the soybean. Aust. J. biol. Sci. 12, 349-372 (1959). Thrower, S. A. : Translocation of labelled assimilates in the soybean. II. The pattern of transloeation in intact and defoliated plants. Aust. J. biol. Sci. 1~, 629-649 (1962). Vasilevskaya, V. K., Ermolaeva, E.Y.: Interrelation between the movement of assimilates from leaves of different stories and the structure of the vascular system of the sunflower. Vestn. Leningrad Univ., Ser. biol. 25, 33-42 (1970). Wada, Y., Kuroda, S.: Changes in the photosynthetic activity during aging in different parts of intact tobacco plants [Jap. with Engl. summ.]. Bet. Mag. 81, 226-331 (1968). Wardlaw, I. F. : The control and pattern of movement of carbohydrates in plants. Bet. Rev. 84, 79-105 (1968). Webb, J.A., Gorham, P. R.: Radial movement of Cl*-translocates from squash phloem. Canad. J. Bet. 43, 97-103 (1965). Yamamoto, T.: The distribution of carbon-14 assimilated by a single leaf in tobaeeo plant. Plant Cell Physiol. 8, 353-362 (1967). u T., Sekiguchi, S., Ozeki, K. : The transloeation of photosynthetic products from mesophyll into midrib in flue-cured tobacco plants. I. The effect of sinks on the translocation of total laC. [Jap. with Engl. suture.]. Prec. Crop Sol. See. Jap. 88, 489-494 (1969).