J Plant Res (2003) 116:183–188 © The Botanical Society of Japan and Springer-Verlag Tokyo 2003Digital Object Identifier (DOI) 10.1007/s10265-003-0085-7

Springer-VerlagTokyo10265

0918-9440

1618-0860

30669031Journal of Plant Research

J Plant Res008510.1007/s10265-003-0085-7

Carbon autonomy of reproductive shoots of Siberian alder (

Alnus hirsuta

var.

sibirica

)

ORIGINAL ARTICLE

Received: August 8, 2002 / Accepted: February 3, 2003 / Published online: March 8, 2003

Shigeaki Hasegawa

•

Keisuke Koba

•

Ichiro Tayasu

•

Hiroshi Takeda

•

Hiroki Haga

S. Hasegawa

1

(*

) · I. Tayasu

2

· H. TakedaLaboratory of Forest Ecology, Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

K. KobaDivision of Biosphere Informatics, Graduate School of Informatics, Kyoto University, Japan

H. HagaLake Biwa Museum, Kusatsu, Japan

Present addresses:

1

Laboratory of Regional Ecosystems, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, JapanTel.

+

81-11-7062264; Fax

+

81-11-7064954e-mail: shasegaw@ees.hokudai.ac.jp

2

Research Institute for Humanity and Nature, Kyoto, Japan

Abstract

Carbon autonomy of current-year shoots in flow-ering, and of current-year shoots plus 1-year-old shoots (1-year-old shoot system) in fruiting of Siberian alder (

Alnushirsuta

var.

sibirica

) was investigated using a stable isotopeof carbon,

13

C. The current-year shoot and 1-year-old shootsystems were fed

13

CO

2

and the atom% excess of

13

C inflowers and fruits was determined. The majority of photo-synthate allocated to flower buds was originally assimilatedin the leaves of the flowering current-year shoots. Of all thecurrent-year shoots on fruiting 1-year-old shoots, only thosenearest to the fruits allocated the assimilated photosynthateto fruit maturation. These results indicate that the current-year shoots and 1-year-old shoot systems are carbon-autonomous units for producing flowers and maturingfruits, respectively.

Key words

Alnus hirsuta

var.

sibirica

·

13

C · Current-yearshoot population · Tracer experiment · Translocation ofphotosynthate

Introduction

A current-year shoot of a tree is regarded as a fundamentalunit in reproduction (Lovett Doust and Lovett Doust 1988;Tuomi et al. 1982; Newell 1991). Several studies have beenconducted to investigate the role of current-year shoots inthe reproduction of trees (Tuomi et al. 1982; Hoffmann andAlliende 1984; Cooper and McGraw 1988; Karlsson et al.1996; Obeso 1997; Hasegawa and Takeda 2001; Henriksson2001). These studies are based on the assumption that thecurrent-year shoot is resource-autonomous (Tuomi et al.1988a, 1988b; Sprugel et al. 1991). This assumption is sup-ported by evidence that damage limited to local foliage haspredominantly local effects (Tuomi et al. 1982, 1988a, 1988b,1989; Haukioja and Neuvonen 1985; Haukioja et al. 1990;Ruohomäki et al. 1997).

Many studies have also pointed out that reproductivetissues obtain photosynthates from the nearest vegetativetissues (Kozlowski and Clausen 1966; Hansen 1969;Mooney 1972; Stephenson 1981; Honkanen and Haukioja1994), though they have the ability to derive resources fromvegetative tissues more than 1 m apart (Mooney 1972).Thus, the photosynthates allocated to the reproductive tis-sues are probably assimilated in the leaves on the current-year shoot to which the reproductive tissue is attached.However, there are few studies that show the carbon-autonomy of current-year shoots in reproduction from thedirect evidence of the movement of photosynthate withinand between current-year shoots.

In terms of resource allocation for reproductive activi-ties, two sequential events should be taken into account:flower production and fruit maturation (Stephenson 1981;Willson 1983). Siberian alder (

Alnus hirsuta

Turcz. var.

sibirica

[Fisch.] C. K. Schn.) is a deciduous early-successional tree species, with monoecious sex expression.Male and female flower buds develop on the top part of thecurrent-year shoots and flower in the following year, whichmeans that fertilized female flowers mature to fruit on 1-year-old shoots (Hasegawa and Takeda 2001). These find-ings suggest that flower production and fruit maturation are

184

temporally separated, and the pattern of photosynthateallocation in both flower production and fruit maturationmay require to be checked in Siberian alder.

In this study, we examined the carbon autonomy ofcurrent-year shoots in the reproduction of Siberian alder.Our specific questions are: (1) whether flowers obtain pho-tosynthates assimilated in the leaves on the current-yearshoots to which they are attached, (2) whether the fruitsobtain photosynthates assimilated in the leaves on thecurrent-year shoots developed on the fruiting 1-year-oldshoot, and (3) which current-year shoots on a fruiting 1-year-old shoot are effective for fruit maturation. To answerthese questions, we conducted a tracing experiment usingisotope-labelled CO

2

, a popular technique with which toexamine the movement of photosynthate in a plant body(Rabideau and Burr 1945; Hartt et al. 1963; Geiger andSwanson 1965; Hartt 1965; Hansen 1967, 1969; Davis andSparks 1974; Steer and Pearson 1976; Takeda et al. 1980;Cliquet et al. 1989, 1990; Deléens et al. 1994).

Materials and methods

Species studied

Siberian alder (

Alnus hirsuta

var.

sibirica

) is a deciduousearly-successional tree species. Male and female flowers areproduced from early August. A flowering shoot normallyhas both male and female flower buds at its terminal end.They overwinter and then flower in mid-April of the subse-quent year. Male flowers fall after flowering, whereas fertil-ized female flowers mature to fruit on 1-year-old shoots,and then seeds are dispersed by the wind in November(Hasegawa and Takeda 2001).

Study site and target tree

The study was conducted in a secondary forest (approxi-mately 1,110 m in altitude) at Mt. Norikura in the easternpart of Takayama City, Gifu Prefecture, central Japan. Theonly tree species constituting the canopy layer was Siberianalder. The height of the canopy trees was about 15 m.

Three sample trees were selected from the edge of thestudy site where light conditions appeared to be appropriatefor photosynthetic assimilation. The height of the targettrees was about 11–12 m.

Isotope labeling and carbon stable isotope analysis

In this study,

13

C – a stable isotope of carbon – was used forlabelling carbon dioxide. Using

13

C for tracing experimentsis more advantageous in cost and in handling security prob-lems than

14

C – a radioactive isotope of carbon commonlyused in previous tracing experiments (Deléens et al. 1994).To examine the carbon autonomy of the current-year shootsand 1-year-old shoot systems in reproduction, we traced themovement of photosynthate to the reproductive organs, by

infusing

13

CO

2

onto the leaves and monitoring changes in

13

C content of reproductive organs.We selected flowering current-year shoots and fruiting

1-year-old shoots from the first-order branches (branchesdirectly issuing from the trunk) of target trees. Thesebranches were located about 3 m above the ground, andwere accessible with the help of a ladder.

A nylon bag (40 cm

¥

28 cm) was used as a simple cham-ber. A small hole was opened at the closed side of the nylonbag and a short plastic tube (9 mm in diameter) wasattached, penetrating the hole with adhesives, to allow air-flow. The current-year shoots or 1-year-old shoot systems toreceive the

13

CO

2

were covered with the nylon bags. Theopen side of each nylon bag was sealed with adhesive tape.The air in the nylon bag was roughly purged by hand. Afterpurging the air, the nylon bag was connected to a cylinder(100 ml) via a glass tube (8 mm in diameter, length 27 cm)filled with soda-talc. The soda-talc in the glass tubeabsorbed CO

2

from the air. Thus, pumping the cylinder, wecould feed the air containing a low concentration of CO

2

into the chamber. Then,

13

CO

2

(Syoko-Tsusho, Tokyo,Japan) was injected into the chamber at about 360 ppm.This procedure enabled us to provide a higher proportionof

13

C than is the normal state but with a concentration ofCO

2

equivalent to that of normal air.The current-year shoot or 1-year-old shoot system under

study was exposed to

13

CO

2

during two successive sunnydays. During the daytime period (0800–1800) of

13

CO

2

expo-sure, to maintain the concentration of CO

2

at normal levelswe determined the concentration of CO

2

in the chamberby a one-time CO

2

analyzer, GASTEC No. 2LL (Gastech,Tokyo, Japan) and supplemented any decrease in CO

2

byinjecting

13

CO

2

at 1 h intervals.

13

CO

2

was not supplementedduring the nighttime period of

13

CO

2

exposure.Target flowers and fruits were sampled after exposure

and immediately brought to the laboratory in a cooler boxfilled with ice. At the same time, flowers and fruits that werefar distant from the site of

13

CO

2

addition were sampled ascontrols, to estimate the natural abundance of

13

C in flowersand fruits. Samples were dried at 40°C for 48 h and groundto a fine homogeneous powder using a sample mill TI-100(CMT, Tokyo, Japan). The abundance of

13

C was analyzedusing an automatic nitrogen and carbon analyzer, Integra-CN (European Science, London, UK).

Statistical analysis

The abundance of

13

C was indicated by the index, atom%.Atom% of

13

C is calculated as follows:

(1)

Under natural conditions, the atom% of

13

C is about 1.1%(Deléens et al. 1994). The increment of

13

C in target flowersor fruits from controls was analyzed by the Mann-WhitneyU test, comparing atom% of

13

C of target flowers or fruitswith atom% of

13

C of control intact flowers or fruits. The

atom%amount of C

amount of C amount of C

13

12 13=

+¥ 100

185

Mann-Whitney U test was conducted using SPSS ver. 7.5.1 Jfor Windows (SPSS, Chicago, Ill.).

Experimental design

Each experiment was conducted as follows (Fig. 1). Exper-iment A: (1) leaves of a current-year shoot producing flow-ers were provided with

13

CO

2

, (2) leaves of a non-floweringcurrent-year shoot neighboring a flowering shoot were pro-vided with

13

CO

2

. Experiment B: (1) leaves of a 1-year-oldshoot system with maturing fruits were provided with

13

CO

2

,(2) leaves of a non-fruiting 1-year-old shoot system closestto a fruiting 1-year-old shoot system were provided with

13

CO

2

. Experiment C: (1) leaves of the current-year shootnearest to the fruit in a 1-year-old shoot system were pro-vided with

13

CO

2

, (2) leaves of a current-year shoot furtheraway from fruits were provided with

13

CO

2

.Experiment A was conducted in August 1999. Ten

current-year shoots were selected for each treatment,including the control. Experiments B and C were conductedin August 1998. Ten current-year shoots and ten 1-year-oldshoot systems were selected for each treatment, includingthe control. To check the seasonal changes of translocationof photosynthate to fruits, Experiments B-2 and C were alsoconducted in May, July, August and October 1999. Five 1-

year-old shoot systems were selected for each treatment.We omitted experiment B-1 in 1999 since the transportationof photosynthate from the leaves of 1-year-old shoot systemto fruits could be checked by experiment C-1 and C-2.

Results

Carbon autonomy of current-year shoots

Flowers of current-year shoots whose leaves were providedwith

13

CO

2

showed a significant (

P

<

0.05) rise in atom% of

13

C over the control in August 1998 (Table 1). Flowers onthe current-year shoot adjacent to the current shoot whoseleaves were provided with

13

CO

2

showed no significantincrease in atom% of

13

C in August 1998 (Table 1).

Carbon autonomy of 1-year-old shoot systems

Fruits of 1-year-old shoot systems whose leaves were pro-vided with

13

CO

2

showed a significant rise of atom% of

13

Cover the control. The fruits of the 1-year-old shoots did notshow a significant increase in atom% of

13

C when the leavesof adjacent 1-year-old shoot systems were provided with

13

CO

2

in August 1998 (Table 2). The same pattern wasobserved from May to September 1999 (Table 3).

Effective current shoots within 1-year-old shoot systems

When the leaves of a current-year shoot nearest to thefruits were provided with

13

CO

2

, the fruits of the 1-year-old

Fig. 1.

Schematic diagram of

13

CO

2

addition in Experiments

A

,

B

and

C

.

CS

Current-year shoot,

AS

1-year-old shoot,

FL

flower,

FR

fruit.

Circles

represent the enclosures where

13

CO

2

was provided

Table 1.

Atom% of

13

C in flower buds after exposure to

13

CO

2

inAugust 1999 (mean

±

SD,

n

=

10).

Flowering

Flowering current-yearshoots were provided with

13

CO

2

,

neighboring

current-year shootsneighboring a flowering current-year shoot were provided with

13

CO

2

.Atom% of flowering group was significantly different (

U

=

1.0,

P

<

0.001) from the control according to the Mann-Whitney U test

*

P

<

0.001

Treatment Atom% of

13

C

Flowering 1.182

±

0.059*Neighboring 1.079

±

0.0018Control 1.078

±

0.0010

Table 2. Atom% of 13C in fruits after exposure to 13CO2 in August 1998(mean ± SD, n = 10). Fruiting Fruiting 1-year-old shoot systems wereprovided with 13CO2, neighboring 1-year-old shoot systems closest toa fruiting 1-year-old shoot system were provided with 13CO2. Atom%of the fruiting group was significantly different (U = 0.0, P < 0.001)from the control group according to the Mann-Whitney U test

* P < 0.01

Treatment Atom% of 13C

Fruiting 1.107 ± 0.016*Neighboring 1.076 ± 0.0018Control 1.077 ± 0.0060

186

shoot systems showed a significant increase in atom% of 13Cin August 1998 (Table 4). On the other hand, no significantincrease in atom% of 13C was observed in fruits of 1-year-old shoot systems when the leaves of the current-year shootprovided with 13CO2 were further away from the fruits(Table 4). The same pattern was observed in July and Sep-tember 1999 (Table 5). In August, the increase in atom% of13C in fruits was marginally non-significant when the leavesof the current-year shoot nearest to the fruits were providedwith 13CO2 (U = 5.0, P = 0.058). The pattern was the same asthat in July and September 1999. No significant rise inatom% of 13C occurred either when the leaves of current-year shoots were provided with 13CO2 in May 1999, regard-less of whether the leaves were nearest to the fruits orfurther away (Table 5).

Discussion

Photosynthate allocated to flower production was assimi-lated in the leaves of the current-year shoot bearing theflower (Table 1). Photosynthate allocated to fruit matura-tion was assimilated in the leaves of the 1-year-old shootsystem bearing the fruits throughout the maturation period(Tables 2–5). However, these results are not enough to showthat current-year shoots and 1-year-old shoot systems arecarbon-autonomous units; these results may simply indicatethat reproductive organs obtain photosynthate from thenearest vegetative tissue, as shown by many previousstudies (Kozlowski and Clausen 1966; Hansen 1969;Mooney 1972; Stephenson 1981; Honkanen and Haukioja1994). Thus, long distance translocation of photosynthatemight occur when the photosynthate produced in the leavesof a flowering current-year shoot or a fruiting 1-year-oldshoot is insufficient to supply the flower or fruits, for exam-

ple in a situation where the flowering current-year shoot orfruiting 1-year-old shoot is shaded and photosynthetic activ-ity is limited by light availability.

It has been reported that current-year shoots shorterthan 10 cm do not produce reproductive organs in Siberianalder (Hasegawa and Takeda 2001). The size of a current-year shoot is a good indicator of the amount of photosyn-thate produced in the leaves of the current-year shoot inSiberian alder. Thus, the fact that current-year shootsshorter than 10 cm do not reproduce suggests that current-year shoots that produce insufficient amounts of photosyn-thate to supply the reproductive organs do not reproduce.This explanation is reinforced by the trend that the propor-tion of flowering current-year shoots is higher in the upperpart of the crown, where leaves are able to capture enoughsunlight than in the lower part, where the leaves are shaded(Hasegawa and Takeda 2001). Thus, this minimal thresholdof the size of reproductive current-year shoots indicates thattranslocation of photosynthate over larger distances is notneeded in Siberian alder. Together with these observations,it may be reasonable to consider the results in this studyshow that allocations of photosynthate for reproductiveorgans are very local within current-year shoots and 1-year-old shoots as they demonstrate the carbon autonomy ofcurrent-year shoots and the 1-year-old shoot system inSiberian alder.

It has been reported that the resources available forreproduction vary within a reproductive episode and thatthis affects the pattern of resource allocation (Stephenson1981). Siberian alder is deciduous and the growth patternof shoot elongation and leaf expansion is heterophyllous,i.e., shows continuous expansion for a longer period(Kozlowski 1971; Kikuzawa 1978). Thus, resource availabil-ity for reproduction in Siberian alder is expected to varydrastically. In this experiment, however, allocation of pho-tosynthate from 1-year-old shoot systems other than thefruiting 1-year-old system was not observed throughout thefruit maturation period (Tables 2–5). This may also be oneresults that shows the carbon autonomy of the 1-year-oldshoot system in Siberian alder.

The contribution of carbon stored in the twig, trunk orroots to the maturation of fruits could not be assessed inthis experiment. However, the contribution of stored car-bon to reproduction may be relatively low in Siberian alder.The growth pattern of shoot elongation and leaf expan-sion of Siberian alder is heterophyllous (Kozlowski 1971,Kikuzawa 1978, Hasegawa and Takeda 2001); stock carbonis mainly allocated to expand early leaves, while currentphotosynthate is mainly allocated to expand late leaves in

Table 3. Atom% of 13C in fruits after exposure to 13CO2 in May, July, August and September 1999 (mean ± SD, n = 5). Neighboring Fruiting1-year-old shoot systems were provided with 13CO2. No significant difference (P > 0.05) in atom% was found using the Mann-Whitney U test

Treatment Atom% of 13C

May July August September

Neighboring 1.081 ± 0.0025 1.081 ± 0.0014 1.079 ± 0.00032 1.081 ± 0.0038Control 1.081 ± 0.00066 1.079 ± 0.00091 1.078 ± 0.0010 1.079 ± 0.00036

Table 4. Atom% of 13C in fruits after exposure to 13CO2 in August 1998(mean ± SD, n = 10). Nearest The current-year shoot nearest to fruitswas provided with 13CO2, others current-year shoots further away fromthe fruits were provided with 13CO2. Atom% of the nearest group wassignificantly different (U = 0.0, P < 0.001) from the control groupaccording to the Mann-Whitney U test

*P < 0.001

Treatment Atom% of 13C

Nearest 1.116 ± 0.030*Others 1.080 ± 0.0083Control 1.076 ± 0.0018

187

this type of growth pattern (Kozlowski and Clausen 1966).Thus, the stock carbon may be exhausted earlier in theseason when early leaves expand and current photosynthateis allocated for reproduction. In addition, the maturationpattern of fruits matches well with phenological patterns ofleaf expansion in Siberian alder. The weights of fruits do notincrease from April to June, when the shoots and leaves arevigorously growing, but they increase greatly in July andAugust, when the biomass of leaves reaches a maximum(Hasegawa and Takeda 1998). This coincidence of growthpattern between leaves and fruits does not directly indicatethe relative importance of current photosynthate to storedcarbon; however, the contribution of stored carbon inSiberian alder is suggested to be small.

It has been reported that the reproductive organs ofplants also assimilate photosynthate (Bazzaz et al. 1979;Williams et al. 1985; Reekie and Bazzaz 1987; Ashman1994). In this experiment, not only leaves, but also flowersor fruits were provided with 13CO2 in treatment A-1 andB-1 (Fig. 1). Thus, the contribution of photosynthate assim-ilated in reproductive organs could not be assessed. How-ever, reproductive photosynthesis is counted as one of thefactors promoting carbon autonomy of current-year and1-year old shoot systems (Newell 1991).

Acknowledgments We thank T. Ando, K. Kurumado, N. Miyamotoand all the staff of Takayama Research Station, Institute for BasinEcosystem Studies, Gifu University for their support of field studies.We are grateful to Lake Biwa Museum, Shiga Prefecture for providingfacilities for the stable isotope analysis. We are also grateful to H.Barclay and J. Henriksson for reading through the manuscript andgiving us valuable suggestions, and to H. Nakajima, M. Hirobe, T.Shirota, M. Takagi, H. Kobayashi, Y. Miyazaki and the members ofLaboratory of Forest Ecology, Graduate School of Agriculture, KyotoUniversity for their advice.

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Table 5. Atom% of 13C in fruits after exposure to 13CO2 in May, July, August and September 1999 (mean ± SD, n = 5). Nearest The current-yearshoot nearest to fruits was provided with 13CO2, others current-year shoots away from the fruits were provided with 13CO2. Atom% of the nearestgroup in July and September was significantly different (July U = 0.0, P < 0.05; September U = 0.0, P < 0.05) from the control group accordingto the Mann-Whitney U test

*P < 0.05

Treatment Atom% of 13C

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