SHORT-AND LONG-TERM CARBON ALLOCATION
IN YOUNG WALNUT WITH TWO BRANCHES
GROWN IN DIFFERENT LIGHT ENVIRONMENTS:
A 13 C -14 C DOUBLE TRACING EXPERIMENT

A Lacointe, T Améglio, F A Daudet, J S Frossard, B. Saint-Joanis, B Vandame, C Adam, C Bodet, M Crocombette, S Ploquin, et al.

<figure><img src="image_1.png" /></figure>

To cite this version:

A Lacointe, T Améglio, F A Daudet, J S Frossard, B. Saint-Joanis, et al.. SHORT-AND LONG-TERM CARBON ALLOCATION IN YOUNG WALNUT WITH TWO BRANCHES GROWN IN DIFFERENT LIGHT ENVIRONMENTS: A 13 C -14 C DOUBLE TRACING EXPERIMENT. Acta Horticulturae, 2001, 544, pp.219-226. hal-05368948

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Distributed under a Creative Commons Attribution 4.0 International License
SHORT- AND LONG-TERM CARBON ALLOCATION IN YOUNG WALNUT WITH
TWO BRANCHES GROWN IN DIFFERENT LIGHT ENVIRONMENTS: A ¹³C-¹⁴C
DOUBLE TRACING EXPERIMENT

A. Lacointe, T. Améglio, F.A. Daudet,
J.S. Frossard, B. Saint-Joanis,
M. Vandame, B. Adam, C. Bodet,
M. Crocombette and S. Ploquin
U.A. Bioclimatologie - PIAF,
F-63039 Clermont-Ferrand, France

E. Deléens
and C. Lelarge
I. B. P., bâtiment 630,
Université Paris-sud,
F-91405 Orsay,
France

G. C. Song
Nat. Horticult.
Res. Institute,
Rural Dev. Adm
Suwon 440-310
South Korea

Keywords

assimilate partitioning, heterogeneous environment, labeling, carbohydrates, winter redistribution, branch autonomy, *Juglans regia* L.

Abstract

In order to investigate the effects of an heterogeneous within-crown light environment on carbon allocation, young scions of walnut (*Juglans regia* L.) *cv*. Franquette were trained on two branches, one left in full sunlight and the other shaded resulting in a 67 % reduction in PAR. In September, the two branches were separately labeled with ¹⁴CO₂ and ¹³CO₂ respectively, so that the photosynthates from each branch could be traced independently at the same time.

Although some carbon movements could be detected within 5 days in both directions (including from the shaded branch to the sun branch), between-branch carbon movements were very limited: less than 1% of the diurnal net assimilation of a branch. At this time of the year branch autonomy was nearly total, leading to increased relative respiratory losses and a moderate growth deficit in the shaded branch.

However, more significant carbon import movements into the branches occurred in winter, possibly representing up to 10 % of total branch reserves, which makes branch autonomy more questionable at this time scale. It was even more so regarding reserve mobilization in spring, as new shoots from both branches got carbon mobilized from the whole plant.

From these results, the branch autonomy concept appeared valid for photosynthate allocation in the photosynthetically active season, however no rapid conclusion should be derived regarding the subsequent viability of new shoots the following seasons. New shoots have a new opportunity to develop in spring, even though the bearing shoot was developed under adverse conditions.

As phloem is considered not functional in winter, it is suggested that xylem is involved as the pathway for carbohydrate movements at this time of the year. This is in agreement with other results regarding sugar exchanges between the xylem vessels and the neighboring reserve parenchymas.

Résumé

Afin d'étudier les effets d'un environnement lumineux intra-couronne hétérogène sur la répartition du carbone, de jeunes scions de noyer (*Juglans regia* L.) *cv*. Franquette ont été conduits sur deux branches, l'une maintenue en plein soleil et l'autre sous une ombrière arrêtant 67% du PAR. En septembre, les deux branches ont été marquées séparément, respectivement au ¹⁴CO₂ and ¹³CO₂, afin de pouvoir suivre simultanément et indépendamment le devenir des assimilats issus de chacune d'elles.
5 jours après marquage, on peut détecter des échanges de carbone entre les deux branches et dans les deux sens ; ceux-ci restent cependant très limités, représentant moins de 1% de la photosynthèse nette diurne d'une branche. A cette période, les branches sont donc quasi-autonomes vis-à-vis du carbone, et ceci se traduit, pour la branche ombrée, par une plus grand allocation du carbone assimilé à la respiration, ainsi que par un déficit de croissance modéré.

Cependant, des mouvements de carbone plus importants se produisent en hiver au sein de la plante, une branche pouvant vraisemblablement importer jusqu'à l'équivalent de 10% de ses réserves carbonées. Les branches sont donc moins autonomes à cette échelle de temps. Elles le sont moins encore au printemps suivant, lors de la mobilisation massive des réserves, car les jeunes pousses issues de chaque branche (après suppression de l'ombrière) sont alimentées par du carbone provenant de la mobilisation des réserves de l'ensemble de la plante.

Ainsi, la validité du concept d'autonomie trophique des branches est confirmée pour la phase feuillée (répartition à court terme des photoassimilats), mais elle ne se prolonge pas à plus long terme. Même si la branche-mère s'est développée en conditions défavorables, les nouvelles pousses qu'elle portera l'année suivante auront la possibilité de croître activement si les conditions deviennent plus favorables entre-temps (par ex., si une partie de la couronne a été détruite par une tempête).

Le phloème étant considéré comme non-fonctionnel en hiver, on pense que les mouvements de carbone observés à cette période empruntent la voie xylémienne. Ceci est en accord avec d'autres résultats concernant les échanges de sucres entre les vaisseaux du xylème et les parenchymes de réserves voisins.

# 1. Introduction

Environmental factors affect resource allocation in trees. Such effects are well known at the scale of large tree compartments, e.g. the root:shoot ratio (Lacointe, 1999). However, little is known at lower scales, i.e. the very common situation of within-crown heterogeneity or imbalance in resource acquisition or use, e.g. induced by local fruit load or light environments (Little, 1974; Ryugo et al., 1980). In literature, it is often assumed that individual branches are autonomous regarding carbon balance. This simple “branch autonomy” assumption (Sprugel et al., 1991) has already been included in tree growth models (Ford et al., 1990); however, its validity has still to be tested in different situations.

In this context, the present work was undertaken in young walnut trees of simple architecture, to study the effect of unbalanced carbon supply, as induced by a heterogeneous light environment: full sun vs. shade. The fate of photoassimilates from each branch is investigated in relation to both current shoot growth (year *n*) and carbon availability to new shoots of next generation (year *n+1*).

# 2. Materials and methods

Two-year-old scions of walnut (Juglans regia L.) cv. Franquette were potted in 33-liter containers in late winter 1994-1995. The soil was a 33%:67% (vol:vol) peat:clay mixture complemented with 10 g NH₄NO₃ and continuously drip irrigated to field capacity. At budbreak, the scion was partially debudded so that only two branches were allowed to grow out. In early July, the lower branch of most trees was shaded allowing 33% of the incident PAR, which resulted in a 30% decrease of its diurnal net assimilation (data not shown). The remaining trees were kept in full sunlight as controls.

In mid-September 1995, both branches were separately, quantitatively labeled with *CO₂* in 40-liter chambers according to the procedure reported in Kajji *et al.* (1993). The sun branch (O) was fed with 18 MBq ¹⁴CO₂, the shaded one (●) with 4 mmoles ¹³CO₂ (Fig. 1). The shading system was removed at leaf shedding (November).
<figure><img src="image_3.png" /><figcaption>Fig. 1. Schematic drawing of the 2-branched walnut tree showing the shading system on the lower branch (●), and associated labeling experiments.</figcaption></figure>

Three trees were harvested at each of the 4 following dates:

* 5 days after labeling (end of primary translocation, cf. Lacointe et al., 1995)

* leaf shedding (November 1995)

* early budbreak (April 1996)

* when new shoots had grown to 4 fully displayed leaves (June 1996)

After harvest, the plants were rapidly divided into coarse roots (diameter > 3 mm), fine roots, main stem (including rootstock stem base), branches and new shoots when present. They were immediately frozen in liquid nitrogen and freeze-dried, weighed and ground to pass a 125 µm mesh.

Soluble sugars were extracted in 80% ethanol at 80°C, then purified successively on active charcoal + polyvinylpolypyrrolidone, cation exchanger and anion exchanger resins. Glucose, fructose and sucrose were assayed by the enzymatic method of Boehringer (1988), and the ¹⁴C content of the purified extract was measured by liquid scintillation. After enzymatic extraction from the pellet, starch was quantified as glucose equivalent and its ¹⁴C measured by the same methods.

The ¹⁴C and ¹³C contents of the whole dry matter were measured directly on the same ground powder. ¹⁴C was determined at “infinite thickness” in a low-background argon-methane flow counter (Numelec NU 20). ¹³C was determined in a system composed of an elemental analyser (Carlo Erba NC 1500) connected to an isotope mass spectrometer (Optima). The ¹³C atom % excess for each organ was computed vs. an unlabeled control tree, and the data for both isotopes were expressed as fractions of the total amount of label initially fed to the tree. (1 unit label = 1 ‰ of that amount).

In organs that lost some carbohydrates between 2 sampling dates, the specific label (unit label per mg carbohydrate) of the mobilized carbohydrates was estimated as the ratio of carbohydrate label variation to carbohydrate amount variation.

When appropriate, label values are expressed as scaled for relative organ DMW, which minimizes individual variability:

$$ \text{Scaled label (SL)} = \frac{\text{organ label}(\text{\textperthousand}\text{ total fed to tree at labeling}) \cdot \text{whole tree struct. DMW (g)}}{\text{organ structural DMW (g)}} $$

In the following, results are expressed as average ± standard error values (n = 3 trees per sampling date). Due to the low number of trees per sampling, classical statistical methods can hardly be performed. Therefore, when comparing 2 situations, unless otherwise stated the difference is considered ‘significant’ if all 3 individual values for one situation are either all lower or all higher than all 3 individual values for the situation.
<table><caption>Table 1. Dry matter weight (DMW) and carbohydrate content of the different parts of the trees at leaf shedding, in November. Carbohydrates (soluble sugars + starch) are expressed as mg glucose equivalent per g DMW.</caption><thead><tr><th>Organ</th><th>Total DMW (g)</th><th>Carbohydrates (mg / g DMW)</th></tr></thead><tbody><tr><td>Sun branch (O)</td><td>13 ± 2 (*)</td><td>90 ± 5</td></tr><tr><td>Shaded branch (●)</td><td>9 ± 1 (*)</td><td>88 ± 3</td></tr><tr><td>Main stem</td><td>60 ± 10</td><td>108 ± 1</td></tr><tr><td>Tap root</td><td>200 ± 30</td><td>412 ± 10</td></tr><tr><td>Fine roots</td><td>40 ± 15</td><td>147 ± 7</td></tr></tbody></table>

## 3. Results and discussion

After a description of the global, integrated effect of the differential light environment on growth of each branch, the following sections address more analytical aspects.

### 3.1 Effect of light on branch growth and carbohydrate content

The sun branch exhibited a slightly (but significantly) higher total dry matter weight (DMW) than the shaded one (Table 1). However, as the sun branch was also the upper one whereas the shaded branch was in lower position within the tree architecture, it was not clear which factor this effect was due to. For this reason, control, full sun grown trees were also investigated, and no such difference was found between the 2 branches of these control trees (g DMW: 12 ± 2 for the upper branch vs. 11 ± 2 for the lower one). Therefore, the higher DMW of sun- vs. shaded branches could actually be ascribed to the differential light environment between both branches rather than to their respective position within the tree architecture, showing that more autotrophic C allows more growth.

Regarding carbohydrate concentration, no difference was detected between both branches (Table 1); however, the difference in total DMW resulted in a similar difference in total (absolute) carbohydrate content between branches.

<figure><img src="image_4.png" /><figcaption>Fig. 2. Carbon allocation pattern in September. For each source branch (O: sun-branch; ●: shaded branch), the figure shows the % of total photosynthesates that are recovered either within the source branch (in gray) or in the rest of the tree (export, in black). Assimilates not recovered in either compartment are ascribed to respiratory losses.</figcaption></figure>

<footnote>(*)O:● DMW ratio is greater than 1 (non-param. Wilcoxon significance level: α < 0.05)</footnote>
<table><caption>Table 2. Spatial partitioning of the carbon exported from each source branch, expressed as % of the total exported C that is recovered in each sink part.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Source branch</th></tr><tr><th>Sun (○)</th><th>Shaded (●)</th></tr></thead><tbody><tr><td>Opposite branch</td><td>0.09 ± 0.05*</td><td>0.2 ± 0.1*</td></tr><tr><td>Main stem</td><td>21 ± 2</td><td>22 ± 8</td></tr><tr><td>Roots</td><td>79 ± 2</td><td>78 ± 8</td></tr></tbody></table>

## 3.2 Carbon allocation in September

51 % of the total C assimilated by the shaded branch (i.e. 510 unit label) was lost from the tree within 5 days after labeling, vs. only 34 % for the sun branch (Fig. 2). This difference in relative respiratory losses can be understood in the framework of branch autonomy, as the metabolic requirements of the shaded branch were similar to those of the sun branch whereas the photosynthetic carbon input was lower.

However, both branches exhibited a remarkably similar partitioning pattern: in both cases, *ca.* 60 % of the total C recovered in the whole plant was exported out of the source branch, the other 40 % remaining within it. The patterns of partitioning among sink organs (Table 2) were also identical as in both cases *ca.* 80 % of the exported C was allocated to roots vs. 20 % allocated to the main stem. However, the most interesting result was that from both source branches, regardless of light environment, *ca.* 0.1 % of the exported C was recovered in the opposite branch. It can be calculated that this represented slightly less than 1 % of the branch diurnal net assimilation, showing that the branches were *nearly* but not *strictly* autonomous regarding carbon acquisition.

## 3.3. Autumn and winter dynamics

The sun-branch derived carbon incorporated in the structural fraction of dry matter exhibited no change in the roots or the main stem in fall or winter (Table 3); this was expected as the primary translocation was over. However, there was an increase in both branches, particularly between November and April, which was an indication for some anabolic activity in current-year (but not in older) shoots during the so-called dormant season.

<table><caption>Table 3. Fall and winter variations of sun branch - derived carbon in the extractable carbohydrate (= starch + soluble sugars) and the non-extractable (= 'structural') fractions of each part of the tree. Data expressed as scaled label (SL) values (cf. Material and Methods)</caption><thead><tr><th rowspan="2"></th><th colspan="3">SL in non-extractable structures</th><th colspan="3">SL in mobilizable carbohydrates</th></tr><tr><th>September</th><th>November</th><th>April</th><th>September</th><th>November</th><th>April</th></tr></thead><tbody><tr><td>Source branch</td><td>230 ± 30</td><td>330 ± 40</td><td>* 510 ± 60</td><td>620 ± 30</td><td>* 230 ± 30</td><td>190 ± 40</td></tr><tr><td>Oppos. branch</td><td>6 ± 2</td><td>* 16 ± 3</td><td>* 27 ± 3</td><td>6 ± 0</td><td>* 11 ± 1</td><td>* 21 ± 2</td></tr><tr><td>Main stem</td><td>90 ± 10</td><td>100 ± 10</td><td>100 ± 10</td><td>180 ± 10</td><td>* 74 ± 16</td><td>58 ± 5</td></tr><tr><td>Tap root</td><td>110 ± 30</td><td>110 ± 20</td><td>90 ± 20</td><td>300 ± 30</td><td>170 ± 40</td><td>120 ± 30</td></tr><tr><td>Fine roots</td><td>440 ± 30</td><td>550 ± 120</td><td>480 ± 70</td><td>320 ± 40</td><td>† 190 ± 40</td><td>† 120 ± 70</td></tr></tbody></table>

<footnote>(*): value significantly > 0, *sensu* Material and Methods</footnote>

<footnote>(*): the difference between adjacent columns is significant (*sensu* Material and Methods)</footnote>

<footnote>(†): the difference between September and April is significant (*sensu* Material and Methods), although those between September and November and between November and April are not.</footnote>
<table><caption>Table 4. Estimated specific label of ¹⁴C-carbohydrates mobilized in organs that lost some labeled carbohydrates between November and April.</caption><thead><tr><th>Possible carbon source</th><th>Specific label<br>(unit label / g gluc.equiv.)</th></tr></thead><tbody><tr><td>Main stem soluble sugars</td><td>5.7</td></tr><tr><td>Main stem total carbohydrates</td><td>4.8</td></tr><tr><td>Sun branch soluble sugars</td><td>12.</td></tr><tr><td>Sun branch total carbohydrates</td><td>7.4</td></tr><tr><td>Tap root starch</td><td>1.9</td></tr><tr><td>Tap root total carbohydrates</td><td>4.0</td></tr><tr><td>Fine root starch</td><td>6.5</td></tr><tr><td>Fine root total carbohydrates</td><td>11.</td></tr></tbody></table>

At the same time, the labeled carbohydrates decreased in most organs, particularly between September and November, indicating their possible role as local carbon source, e.g. for above-mentioned increase in source branch structural matter. However, the opposite (= shaded) branch did not fit in this pattern, as this was the one organ where labeled carbohydrates did not decrease, but *increased* significantly, in the same way as labeled structural carbon. When added, both fractions (i.e., the total dry matter) in this branch exhibited a very significant increase of *ca.* 100 % (representing *ca.* 0.5 unit label in absolute terms) between November and April. This shows that some carbon external to the shaded branch was imported into it during winter.

In the different organs that lost some labeled carbohydrates between November and April, and were thus potential carbon sources for the concomitant label increase in the shaded branch, the specific label of mobilized carbohydrates was *ca.* 5 unit label per gram glucose equivalent as an order of magnitude (Table 4). This provides a rough estimate of the (minimum) amount of carbohydrate that has been mobilized and translocated into the shaded branch from other parts of the plant : 0.5 / 5 = 0.1 g carbohydrate as an order of magnitude, which is very low (*ca.* 1 ‰) compared to the total tree reserves, but may be significant (*ca.* 10 ‰) relative to the branch own reserves.

Furthermore, there was an indication that both branches were symmetrical in this respect: in the sun-branch, the total C originating from the opposite (i.e., shaded) branch increased from 6 ± 6 to 18 ± 12 SL units (SL: cf. Material and Methods); however, due to a higher error in ¹³C than ¹⁴C excess evaluation, this increase was not significant.

### 3.4. Spring mobilization

Between April (early budbreak) and June (new shoots became self-sufficient), 67 % of the total tree mobilizable sun-branch - derived carbohydrates were used up (Fig. 3). Of the mobilized

<figure><img src="image_5.png" /><figcaption>Fig. 3: Sun-branch - derived label recovered prior to and after spring mobilization in whole tree carbohydrates and total DM of new shoots, both expressed as % of the total plant carbohydrate label recovered in April.</figcaption></figure>
<table><caption>Table 5. (source branch : opposite branch) ratio of total DM label, in the branches themselves in April (early budbreak), and in the new shoots sprouted in June.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Source branch</th></tr><tr><th>Sun (○)</th><th>Shaded (●)</th></tr></thead><tbody><tr><td>Ratio between bearing branches (April)</td><td>22 ± 6</td><td>40 ± 30</td></tr><tr><td>Ratio between sprouted new shoots (June)</td><td>4.5 ± 1.5</td><td>2.8 ± 0.6</td></tr></tbody></table>

carbon, *ca.* 60 % was recovered in the new shoots as new dry matter, which is a recovery rate similar to that found by Lacointe *et al.* (1993) after labeling whole trees in August. However, the most interesting point is that the new shoots sprouted on the unlabeled branch got a much higher amount of label than expected from the pre-mobilization label content of their mother branch, and the same was true for the shaded branch – derived carbon (Table 5). In both cases, the total label amount recovered in the new shoots sprouted on the source branch, whether that was the sun- or shaded one, was only 3 or 4 times higher than that recovered in the new shoots sprouted on the opposite branch, although the total label in the bearing source branch in April was *ca.* 30 times higher than in the opposite branch.

## 4. Conclusions

In this experiment on young walnut, a heterogeneous within-crown environment yielded contrasting results regarding the branch autonomy principle:

In summer, although some carbon movements could be detected in both directions (including from the shaded branch to the sun branch), between-branch carbon movements were very limited: less than 1% of the diurnal net assimilation of a branch. Thus, at this time of the year branch autonomy was nearly total, leading to increased relative respiratory losses and a moderate growth deficit in the shaded branch.

In winter, however, more significant carbon import movements occurred into the branches, possibly representing up to 10 % of total branch reserves, which makes branch autonomy more questionable at this time scale. It was even more so regarding reserve mobilization in spring, as new shoots from both branches got carbon mobilized from the whole plant.

As a conclusion, branch autonomy appeared a simple and convenient simplifying concept, valid for photosynthate allocation in the photosynthetically active season, at least in the simple 2- branched system that we studied. However no rapid conclusion should be derived regarding the subsequent viability of new shoots the following seasons: new shoots appeared to have a “new chance” in spring, even though the bearing shoot was developed under adverse conditions. This could allow some shaded branches to survive in shaded conditions for several years as weak twigs (which a strict branch autonomy would not allow), and take any opportunity such as a clearing or severe pruning to grow out and become dominant branches in a new crown.

Another interesting question is the pathway for carbohydrate movements in winter. As phloem is considered not functional in winter, this suggests the involvement of the xylem pathway. This is in agreement with other results on walnut regarding sugar exchanges between the xylem vessels and the neighboring reserve parenchymas (Améglio *et al.*, this issue), showing significant sugar influx and efflux movements with major consequences on the water status and likely spring development
Acknowledgements:

This work was supported by the EU Project Fair CT96-1887 W-BRAINS.

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