# Recently absorbed nitrogen incorporates into new and old tissues: evidence from a 15 N-labelling experiment in deciduous oaks

Mubarak Mahmud, Tania L Maxwell, Sixtine Cueff, Romain Schroeder, Stéphane Bazot, Nicolas Delpierre, Anne Marmagne, Gaëlle Vincent, Laure Barthes

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

## To cite this version:

Mubarak Mahmud, Tania L Maxwell, Sixtine Cueff, Romain Schroeder, Stéphane Bazot, et al. Recently absorbed nitrogen incorporates into new and old tissues: evidence from a 15 N-labelling experiment in deciduous oaks. Plant and Soil, 2022, 480, pp.407-421. 10.1007/s11104-022-05589-w. hal-04546799

HAL Id: hal-04546799

https://hal.science/hal-04546799

Submitted on 15 Apr 2024

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<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Recently absorbed nitrogen incorporates into new and old tissues: evidence from a 15 N-labelling experiment in deciduous oaks

Mubarak Mahmud, Tania L Maxwell, Sixtine Cueff, Romain Schroeder, Stéphane Bazot, Nicolas Delpierre, Anne Marmagne, Gaëlle Vincent, Laure Barthes

► To cite this version:

Mubarak Mahmud, Tania L Maxwell, Sixtine Cueff, Romain Schroeder, Stéphane Bazot, et al. Recently absorbed nitrogen incorporates into new and old tissues: evidence from a 15 N-labelling experiment in deciduous oaks. Plant and Soil, 2022, 480, pp.407-421. 10.1007/s11104-022-05589-w. hal-04546799

HAL Id: hal-04546799

https://hal.science/hal-04546799v1

Submitted on 15 Apr 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

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

Distributed under a Creative Commons Attribution 4.0 International License
# Recently absorbed nitrogen incorporates into new and old tissues: evidence from a ¹⁵N-labelling experiment in deciduous oaks

Mubarak Mahmud · Tania L. Maxwell · Sixtine Cueff · Romain Schroeder · Stéphane Bazot · Nicolas Delpierre · Anne Marmagne · Gaëlle Vincent · Laure Barthes¹

Received: 10 March 2022 / Accepted: 28 June 2022 / Published online: 12 July 2022

© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022

**Abstract** We quantified the distribution of nitrogen (N), dry-matter (biomass) and of soil-applied ¹⁵N in tree and soil compartments in five naturally-growing 20-year-old oak trees. After applying ¹⁵N solution to soil at the base of the trees in spring, all the trees were felled in the fall, their root system excavated, biomass, nitrogen and ¹⁵N content measured in all compartments. Xylem rings-compartment contains most biomass (47%) while branches and coarse-root contains most nitrogen (29% and 14% respectively). The labelled ¹⁵N absorbed throughout the vegetation season, was found in all compartments except

the heartwood. The majority of recovered ¹⁵N was in the leaves (24%). Some often overlooked compartments (coarse root, stump, xylem and other branches) together recovered 45% of the ¹⁵N. ¹⁵N was found in all the sapwood rings, from the ring formed in the current year up to 10 year-old rings, marking the limit of the heartwood. More ¹⁵N was found in the younger rings compared to older rings. The ¹⁵N allocated to ancient rings can originate from different, non-mutually exclusive, sources: whether directly from the soil via the ¹⁵N uptake throughout the vegetation season and transport in the xylem sap, from the autumnal resorption of ¹⁵N first allocated to the leaves, or from the ¹⁵N mobility once allocated to the forming ring to older rings through ray parenchyma. With about 6% of the initial ¹⁵N retrieved in the microbial biomass at the end of the growing season, we confirmed the role of microbial biomass as forest nitrogen sink.

**Keywords** Nitrogen uptake · Biomass partitioning · Nitrogen allocation · Tree rings · Broadleaf tree · ¹⁵N labeling

**Abbreviations**

DBH Diameter at Breast Height

DOY Day of the Year

Stump The tree's pivot that is inside the soil

AG Aboveground

BG Belowground

EP Experimental plot

Responsible Editor: Xinhua He.

M. Mahmud · T. L. Maxwell · S. Cueff · R. Schroeder · S. Bazot · N. Delpierre · G. Vincent · L. Barthes (✉) Université Paris-Saclay, CNRS, AgroParisTech, Ecologie Systématique et Evolution, 91190 Gif-sur-Yvette, France e-mail: laure.barthes@universite-paris-saclay.fr

M. Mahmud
Kano University of Science and Technology, Wudil,
Nigeria

T. L. Maxwell
INRAE, Bordeaux Sciences Agro, ISPA,
33140 Villenave d'Ornon, France

N. Delpierre
Institut Universitaire de France (IUF), Paris, France

A. Marmagne
Institut Jean-Pierre Bourgin, INRAE-AgroParisTech,
78000 Versailles, France
# Recently absorbed nitrogen incorporates into new and old tissues: evidence from a ¹⁵N-labelling experiment in deciduous oaks

Mubarak Mahmud · Tania L. Maxwell · Sixtine Cueff · Romain Schroeder · Stéphane Bazot · Nicolas Delpierre · Anne Marmagne · Gaëlle Vincent · Laure Barthes¹

Received: 10 March 2022 / Accepted: 28 June 2022 / Published online: 12 July 2022

© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022

**Abstract** We quantified the distribution of nitrogen (N), dry-matter (biomass) and of soil-applied ¹⁵N in tree and soil compartments in five naturally-growing 20-year-old oak trees. After applying ¹⁵N solution to soil at the base of the trees in spring, all the trees were felled in the fall, their root system excavated, biomass, nitrogen and ¹⁵N content measured in all compartments. Xylem rings-compartment contains most biomass (47%) while branches and coarse-root contains most nitrogen (29% and 14% respectively). The labelled ¹⁵N absorbed throughout the vegetation season, was found in all compartments except

the heartwood. The majority of recovered ¹⁵N was in the leaves (24%). Some often overlooked compartments (coarse root, stump, xylem and other branches) together recovered 45% of the ¹⁵N. ¹⁵N was found in all the sapwood rings, from the ring formed in the current year up to 10 year-old rings, marking the limit of the heartwood. More ¹⁵N was found in the younger rings compared to older rings. The ¹⁵N allocated to ancient rings can originate from different, non-mutually exclusive, sources: whether directly from the soil via the ¹⁵N uptake throughout the vegetation season and transport in the xylem sap, from the autumnal resorption of ¹⁵N first allocated to the leaves, or from the ¹⁵N mobility once allocated to the forming ring to older rings through ray parenchyma. With about 6% of the initial ¹⁵N retrieved in the microbial biomass at the end of the growing season, we confirmed the role of microbial biomass as forest nitrogen sink.

**Keywords** Nitrogen uptake · Biomass partitioning · Nitrogen allocation · Tree rings · Broadleaf tree · ¹⁵N labeling

**Abbreviations**

DBH Diameter at Breast Height

DOY Day of the Year

Stump The tree's pivot that is inside the soil

AG Aboveground

BG Belowground

EP Experimental plot

Responsible Editor: Xinhua He.

M. Mahmud · T. L. Maxwell · S. Cueff · R. Schroeder · S. Bazot · N. Delpierre · G. Vincent · L. Barthes (✉)
Université Paris-Saclay, CNRS, AgroParisTech, Ecologie Systématique et Evolution, 91190 Gif-sur-Yvette, France
e-mail: laure.barthes@universite-paris-saclay.fr

M. Mahmud
Kano University of Science and Technology, Wudil,
Nigeria

T. L. Maxwell
INRAE, Bordeaux Sciences Agro, ISPA,
33140 Villenave d'Ornon, France

N. Delpierre
Institut Universitaire de France (IUF), Paris, France

A. Marmagne
Institut Jean-Pierre Bourgin, INRAE-AgroParisTech,
78000 Versailles, France
Introduction

There is an increasing need to improve the accuracy
of forest biomass estimation and its relations with
nitrogen fluxes for several reasons, such as the assess-
ment of carbon pools (Grassi et al. 2017) and also
for studying global biogeochemical cycles, such as
nitrogen, because the amount of nutrients in trees is
also related to the quantity of biomass present (Hen-
drick and Pregitzer 1993). Nutrients are essential for
tree growth and biomass accumulation, and forest
growth can be limited by soil available nutrients, such
as nitrogen (Dickson 1989; LeBauer and Treseder
2008; Rennenberg et al. 2009; Norby et al. 2010;
Vicca et al. 2012; Fernández-Martínez et al. 2014;
Rennenberg and Dannenmann 2015; Eastman et al.
2021). This limitation is further enhanced by CO2
fertilization effect (Goodale et al. 2002) which is the
increased rate of photosynthesis in plant caused by
increased amount of CO2 in the atmosphere (Thara-
mmal et al. 2019; Ueyama et al. 2020). Studies sug-
gest that tree carbon allocation and nitrogen acquisi-
tion are inter-linked processes (Franklin et al. 2012).
For example, Finzi et al. (2015) estimated that up to
one-third of total soil N mineralization may be fuelled
by C allocated to belowground tree compartment and
delivered to soil via rhizodeposition. More under-
standing of the processes of nitrogen nutrition in trees
as it drives their growth, the productivity of forest
ecosystems and their capacity to limit the increase in
atmospheric carbon concentration is crucial.

Oak forests represent an ecologically and eco-
nomically important deciduous tree ecosystem in
France (Timbal and Aussenac 1996). The ability
of deciduous trees to store and internally redistrib-
ute nitrogen resources is a fundamental element
of its N economy, as such Cooke and Weih (2005)
divided the internal redistribution, or cycling, of N
into four phases as follows: (1) primary allocation
of N from N-assimilating source tissues to sink tis-
sues during the growing season; (2) reallocation of
N resources arising from metabolic recycling of N
during the growing season; (3) resorption of N from
senescing tissues and its transport to perennial tis-
sues during autumn senescence; and (4) remobiliza-
tion of N from perennial tissues to actively grow-
ing tissues during spring flush. It is accepted that
the resumption of tree growth in spring is achieved
both from the tree's internal reserves and from the

newly absorbed nitrogen from the soil (Millard and
Grelet 2010). Remobilized stored N and N uptake
from the soil could reach 90% and 27% respectively
in growing leaves and twigs (El Zein et al. 2011a)
with efficiencies that depend on environmental
conditions and competition with the soil microbial
biomass (Maxwell et al. 2020). It has been shown
that absorbed nitrogen is mainly directed to leaves
via xylem vessels (El-Zein et al. 2011a; Bazot et al.
2016). In Oak tree, which is a ring-porous species,
the sap flow coming from the roots is considered
to be restricted to the new, or the most recent few
annual rings, principally in the large vessels of the
early-wood of the current-year ring and in the nar-
row late vessels of the older rings (Čermák et al.
1992; Granier et al. 1994; Tognetti et al. 1996).
The reallocation of nitrogen between the differ-
ent parts of the tree during the growing season or
the transport of nitrogen from senescent leaves into
the bark, ray and axial parenchyma tissues during
the autumn resorption are usually reported to be
ensured through the phloem sap flow (Cooke and
Weih 2005; Millard and Grelet 2010; Plavcová and
Jansen 2015; Sample and Babst 2019).

Some studies of N uptake, remobilization and allo-
cation dynamics during the growing season already
exist in small trees (Millard and Grelet 2010), in
adult trees (El Zein et al. 2011a; Bazot et al. 2016;
Maxwell et al. 2020) and in economically important
fruit trees (Legaz et al. 1995; Quartieri et al. 2002;
Roccuzzo et al. 2017). However, in most of the stud-
ies, few compartments of the trees were investigated.
For example, El Zein et al. 2011a, who highlighted
the importance of N reserves for spring growth in
50-year-old sessile oak trees, only investigated cur-
rent-year shoots (leaves, twigs and buds) and did not
take into account the relative contribution of storage
organs, such as roots, stump, stem and big branches.
In another study, El Zein et al. 2011b compared the
composition of non-structural carbon and nitrogen
compounds of the inner and outer xylem rings where
they highlighted the stability of non-structural nitro-
gen compounds concentration in the stem of sapwood
during the year. Thus, the focus of their study was on
the trunk xylem only.

In this work, we aimed at documenting the alloca-
tion pattern of nitrogen acquired in spring in 20-year
old sessile oak trees using ¹⁵N labelling experiment.
We hypothesized that:

<figure><img src="image_3.png" /></figure>
Introduction

There is an increasing need to improve the accuracy
of forest biomass estimation and its relations with
nitrogen fluxes for several reasons, such as the assess-
ment of carbon pools (Grassi et al. 2017) and also
for studying global biogeochemical cycles, such as
nitrogen, because the amount of nutrients in trees is
also related to the quantity of biomass present (Hen-
drick and Pregitzer 1993). Nutrients are essential for
tree growth and biomass accumulation, and forest
growth can be limited by soil available nutrients, such
as nitrogen (Dickson 1989; LeBauer and Treseder
2008; Rennenberg et al. 2009; Norby et al. 2010;
Vicca et al. 2012; Fernández-Martínez et al. 2014;
Rennenberg and Dannenmann 2015; Eastman et al.
2021). This limitation is further enhanced by CO2
fertilization effect (Goodale et al. 2002) which is the
increased rate of photosynthesis in plant caused by
increased amount of CO2 in the atmosphere (Thara-
mmal et al. 2019; Ueyama et al. 2020). Studies sug-
gest that tree carbon allocation and nitrogen acquisi-
tion are inter-linked processes (Franklin et al. 2012).
For example, Finzi et al. (2015) estimated that up to
one-third of total soil N mineralization may be fuelled
by C allocated to belowground tree compartment and
delivered to soil via rhizodeposition. More under-
standing of the processes of nitrogen nutrition in trees
as it drives their growth, the productivity of forest
ecosystems and their capacity to limit the increase in
atmospheric carbon concentration is crucial.

Oak forests represent an ecologically and eco-
nomically important deciduous tree ecosystem in
France (Timbal and Aussenac 1996). The ability
of deciduous trees to store and internally redistrib-
ute nitrogen resources is a fundamental element
of its N economy, as such Cooke and Weih (2005)
divided the internal redistribution, or cycling, of N
into four phases as follows: (1) primary allocation
of N from N-assimilating source tissues to sink tis-
sues during the growing season; (2) reallocation of
N resources arising from metabolic recycling of N
during the growing season; (3) resorption of N from
senescing tissues and its transport to perennial tis-
sues during autumn senescence; and (4) remobiliza-
tion of N from perennial tissues to actively grow-
ing tissues during spring flush. It is accepted that
the resumption of tree growth in spring is achieved
both from the tree's internal reserves and from the

newly absorbed nitrogen from the soil (Millard and
Grelet 2010). Remobilized stored N and N uptake
from the soil could reach 90% and 27% respectively
in growing leaves and twigs (El Zein et al. 2011a)
with efficiencies that depend on environmental
conditions and competition with the soil microbial
biomass (Maxwell et al. 2020). It has been shown
that absorbed nitrogen is mainly directed to leaves
via xylem vessels (El-Zein et al. 2011a; Bazot et al.
2016). In Oak tree, which is a ring-porous species,
the sap flow coming from the roots is considered
to be restricted to the new, or the most recent few
annual rings, principally in the large vessels of the
early-wood of the current-year ring and in the nar-
row late vessels of the older rings (Čermák et al.
1992; Granier et al. 1994; Tognetti et al. 1996).
The reallocation of nitrogen between the differ-
ent parts of the tree during the growing season or
the transport of nitrogen from senescent leaves into
the bark, ray and axial parenchyma tissues during
the autumn resorption are usually reported to be
ensured through the phloem sap flow (Cooke and
Weih 2005; Millard and Grelet 2010; Plavcová and
Jansen 2015; Sample and Babst 2019).

Some studies of N uptake, remobilization and allo-
cation dynamics during the growing season already
exist in small trees (Millard and Grelet 2010), in
adult trees (El Zein et al. 2011a; Bazot et al. 2016;
Maxwell et al. 2020) and in economically important
fruit trees (Legaz et al. 1995; Quartieri et al. 2002;
Roccuzzo et al. 2017). However, in most of the stud-
ies, few compartments of the trees were investigated.
For example, El Zein et al. 2011a, who highlighted
the importance of N reserves for spring growth in
50-year-old sessile oak trees, only investigated cur-
rent-year shoots (leaves, twigs and buds) and did not
take into account the relative contribution of storage
organs, such as roots, stump, stem and big branches.
In another study, El Zein et al. 2011b compared the
composition of non-structural carbon and nitrogen
compounds of the inner and outer xylem rings where
they highlighted the stability of non-structural nitro-
gen compounds concentration in the stem of sapwood
during the year. Thus, the focus of their study was on
the trunk xylem only.

In this work, we aimed at documenting the alloca-
tion pattern of nitrogen acquired in spring in 20-year
old sessile oak trees using ¹⁵N labelling experiment.
We hypothesized that:
1) Coarse root, stump, xylem rings and other branches which are mostly neglected in the literature would be the most important woody nitrogen sinks.

2) <sup>15</sup>N recovery in xylem rings would depend on the ring age. 3) The soil microbial biomass would contain <sup>15</sup>N at the end of the study period. Therefore, we quantified in a 20-year old oak trees (*Quercus petraea*) the total nitrogen, the distribution pattern of recently incorporated <sup>15</sup>N and the relative stocks of biomass in the different tree compartments: partitioning in leaves, branches of the current and previous years, older branches, fine roots, medium roots, coarse roots, phloem, each ring of the xylem from sapwood to heartwood and stump. Root biomass estimated by direct method (description below) was compared with biomass estimated using allometric equation. In addition to <sup>15</sup>N assessment in the tree, the <sup>15</sup>N in the soil microbial biomass was also quantified and incorporated into the <sup>15</sup>N budget. This exhaustive assessment, taking into account the entirety of the tree compartments, has seldom been done in the literature.

# Material and methods

## Site description

The study was conducted in the Fontainebleau-Barbeau Forest, which is a naturally regenerated forest containing oak trees (*Quercus petraea* (Matt.) Lieb.), located 60 km Southeast of Paris, France (48°29' N, 02°47' E). At the time of the study, the trees were 20-years-old. In 2016, tree heights ranged between 9 and 11 m and the trunk diameter at breast height ranged from 12 to 15.7 cm. A detailed description of the site can be found in (Delpierre et al. 2016; Maxwell et al. 2020). Briefly, the site is at an altitude of 95 m, with an average temperature of 11 °C and annual rainfall of 661 mm (from 1960 to 2016, registered at the station of Meteo France of Melun located 11 km from the Fontainebleau-Barbeau Forest). The soil is a stagnic luvisolisol developed on a clayic bedrock (IUSS Working Group WRB 2015) with a pH ranging between 4 and 6 for the first three horizons. The soil is loamy sand for the first 20 cm and then

loamy clay for the 20–40 cm layer. A dense layer of buhrstone is located at about 60 to 90 cm in depth.

## Field preparation

### Trenching

Five healthy, morphologically similar co-dominant oak trees were selected. On average, the distance between each tree was 20 m. In 2009, a 0.5–0.6 m deep trench was dug around each tree to create the experimental plot (EP) (average area of the EP was 3.4±0.2 m²). Each trench was lined with a polyethylene film and then backfilled with soil. Each EP surface remained bare and soil was undisturbed.

### <sup>15</sup>N labelling

On March 1 2016 (DOY 61), the soil of the EP of each of the five trees was labelled with <sup>15</sup>N by applying a homogenous spray of <sup>15</sup>N labelling solution (prepared with 5 g of <sup>15</sup>NH<sub>4</sub><sup>15</sup>NO<sub>3</sub> containing 98% atoms of <sup>15</sup>N in 20 L of deionised water). Each EP was thus exposed to 1.82 g of the original <sup>15</sup>N, equivalent to 5.35±0.35 kg <sup>15</sup>N /ha. The polyethylene-lining of the EP avoids the uptake of the <sup>15</sup>N applied by the surrounding trees and also allows limits lateral leaching to the surrounding soil.

Sample collection and estimation of the biomass of each compartment

On October 13 2016 (when around 70% of the leaves were yellow) five trees were felled and uprooted, their root system was excavated with a mechanical backhoe. Soil was sampled using a soil stainless steel auger bipartite (Eijkelkamp, SDEC France), with a diameter of 8 cm and a depth of 15 cm, to extract around 1 kg of fresh soil (from which soil microbial biomass was estimated) from one point per EP.

### The leaves, branches and the trunk

The leaves, branches of the year (2016), branches of the previous year (2015) and the remaining branches (categorized as “other branches”) were collected and oven-dried. The trunk of each tree was cut into pieces of around 70 cm. One portion of the trunk, with a thickness of 5 cm taken at the DBH, was
1) Coarse root, stump, xylem rings and other branches which are mostly neglected in the literature would be the most important woody nitrogen sinks.

2) <sup>15</sup>N recovery in xylem rings would depend on the ring age. 3) The soil microbial biomass would contain <sup>15</sup>N at the end of the study period. Therefore, we quantified in a 20-year old oak trees (*Quercus petraea*) the total nitrogen, the distribution pattern of recently incorporated <sup>15</sup>N and the relative stocks of biomass in the different tree compartments: partitioning in leaves, branches of the current and previous years, older branches, fine roots, medium roots, coarse roots, phloem, each ring of the xylem from sapwood to heartwood and stump. Root biomass estimated by direct method (description below) was compared with biomass estimated using allometric equation. In addition to <sup>15</sup>N assessment in the tree, the <sup>15</sup>N in the soil microbial biomass was also quantified and incorporated into the <sup>15</sup>N budget. This exhaustive assessment, taking into account the entirety of the tree compartments, has seldom been done in the literature.

# Material and methods

## Site description

The study was conducted in the Fontainebleau-Barbeau Forest, which is a naturally regenerated forest containing oak trees (*Quercus petraea* (Matt.) Lieb.), located 60 km Southeast of Paris, France (48°29' N, 02°47' E). At the time of the study, the trees were 20-years-old. In 2016, tree heights ranged between 9 and 11 m and the trunk diameter at breast height ranged from 12 to 15.7 cm. A detailed description of the site can be found in (Delpierre et al. 2016; Maxwell et al. 2020). Briefly, the site is at an altitude of 95 m, with an average temperature of 11 °C and annual rainfall of 661 mm (from 1960 to 2016, registered at the station of Meteo France of Melun located 11 km from the Fontainebleau-Barbeau Forest). The soil is a stagnic luvisolisol developed on a clayic bedrock (IUSS Working Group WRB 2015) with a pH ranging between 4 and 6 for the first three horizons. The soil is loamy sand for the first 20 cm and then

loamy clay for the 20–40 cm layer. A dense layer of buhrstone is located at about 60 to 90 cm in depth.

## Field preparation

### Trenching

Five healthy, morphologically similar co-dominant oak trees were selected. On average, the distance between each tree was 20 m. In 2009, a 0.5–0.6 m deep trench was dug around each tree to create the experimental plot (EP) (average area of the EP was 3.4±0.2 m²). Each trench was lined with a polyethylene film and then backfilled with soil. Each EP surface remained bare and soil was undisturbed.

### <sup>15</sup>N labelling

On March 1 2016 (DOY 61), the soil of the EP of each of the five trees was labelled with <sup>15</sup>N by applying a homogenous spray of <sup>15</sup>N labelling solution (prepared with 5 g of <sup>15</sup>NH<sub>4</sub><sup>15</sup>NO<sub>3</sub> containing 98% atoms of <sup>15</sup>N in 20 L of deionised water). Each EP was thus exposed to 1.82 g of the original <sup>15</sup>N, equivalent to 5.35±0.35 kg <sup>15</sup>N/ha. The polyethylene-lining of the EP avoids the uptake of the <sup>15</sup>N applied by the surrounding trees and also allows limits lateral leaching to the surrounding soil.

Sample collection and estimation of the biomass of each compartment

On October 13 2016 (when around 70% of the leaves were yellow) five trees were felled and uprooted, their root system was excavated with a mechanical backhoe. Soil was sampled using a soil stainless steel auger bipartite (Eijkelkamp, SDEC France), with a diameter of 8 cm and a depth of 15 cm, to extract around 1 kg of fresh soil (from which soil microbial biomass was estimated) from one point per EP.

### The leaves, branches and the trunk

The leaves, branches of the year (2016), branches of the previous year (2015) and the remaining branches (categorized as “other branches”) were collected and oven-dried. The trunk of each tree was cut into pieces of around 70 cm. One portion of the trunk, with a thickness of 5 cm taken at the DBH, was
kept in the freezer at -20 °C for subsequent analyses of the phloem and xylem rings. All samples were oven-dried at 60 °C until the dry weight was attained (around 3 months).

## The xylem rings and phloem

For each tree, the size of the xylem rings was measured using a binocular magnifying glass with unit scales. Using the radius from the core to the outer edge and to the inner edge of a ring, the volume of each ring was calculated using the following allometric equation adapted from (Zianis et al. 2005) for *Quercus petraea*:

$$ \text{Ring volume} = \left[ (2 * r_{\text{outer edge}})^{2.0033} - (2 * r_{\text{inner edge}})^{2.0033} \right] * H^{0.85925} * e^{-2.86353} $$

The volume is in dm³, the radius (r) in cm, and the height (H) in m.

The r<sub>outer</sub> edge was measured from the center of the tree to the outer edge of the considered ring. Likewise, the r<sub>inner</sub> edge was measured from the center of the trunk to the inner edge of the considered ring. The height was calculated by measuring the base of the tree to the highest point, when the trees were cut down in autumn.

The volume was then multiplied by the density of the xylem (0.754 g/cm³) in order to have the biomass of each ring (Maxwell et al. 2020). The number of rings in trees 1,2, and 5 were 18 while tree 4 had 22, therefore, for consistency, only 18 rings were considered in each of the trees. The total xylem biomass for each tree was obtained by summing the biomass of the 18 individual rings.

For the biomass of the phloem (which is considered as the inner bark), we used the same approach. The phloem thickness was consistently around 5 mm for each tree and the density was equal to 0.711 g/cm³ (Maxwell et al. 2020). The r<sub>outer</sub> edge was measured from the center of the tree to the outer edge of the phloem and the r<sub>inner</sub> edge was measured from the center of the trunk to the inner edge of the phloem.

## Fine and medium roots

The fine roots (<2 mm in diameter) and medium roots (>2 mm & <1 cm in diameter) as defined by Finzi and Berthrong (2005), were obtained just before

the excavation in the fall. A strip of soil about 20 cm deep, 20 cm wide, and 70 cm long was sampled from a distance of 20 cm away from the trunk of each tree. This strip of soil, which represents approximately 30 kg of fresh soil, was then disaggregated and the fine roots and medium roots were taken out, rinsed with water, separated according to size (Finzi and Berthrong 2005) and placed into an oven at 60 °C to attain dry weight. The quantity of roots per tree was then extrapolated using the area of each EP.

## Coarse roots and stump

The coarse roots (>1 cm in diameter) and the stump were collected after the total excavation of the root system with a mechanical backhoe. The stump and the entire coarse roots were weighed. For each tree, an aliquot of around 600 g (fresh weight) of coarse roots and 300 g of stump was oven dried at 70 °C until the dry weight was attained. The total weight of coarse root and tree stump was calculated using the fresh weight to dried weight ratio of the aliquots.

## The total root system

The total root system biomass was estimated using two methods: (1) by summing the biomass of each root categories (fine roots, medium roots and coarse roots) called "the measured value of total root biomass" (without stump), and (2) by using the regression equation for *Quercus petraea* developed by Drexhage and Colin (2001), and the tree DBH, to calculate the "the estimated value of total root biomass". We then compared the two methods of biomass estimation (Drexhage and Colin 2001).

$$ \text{Log yi} = \beta0 + \beta1 \text{log xi} \quad (1) $$

with,

Log yi logarithmic transformation of root biomass (kg).

log xi log-arithmetic transformation of DBH (cm).

β0 Intercept coefficient (-1.56).

β1 Slope coefficient (2.44).
kept in the freezer at -20 °C for subsequent analyses of the phloem and xylem rings. All samples were oven-dried at 60 °C until the dry weight was attained (around 3 months).

## The xylem rings and phloem

For each tree, the size of the xylem rings was measured using a binocular magnifying glass with unit scales. Using the radius from the core to the outer edge and to the inner edge of a ring, the volume of each ring was calculated using the following allometric equation adapted from (Zianis et al. 2005) for *Quercus petraea*:

$$ \text{Ring volume} = \left[ (2 * r_{\text{outer edge}})^{2.0033} - (2 * r_{\text{inner edge}})^{2.0033} \right] * H^{0.85925} * e^{-2.86353} $$

The volume is in dm³, the radius (r) in cm, and the height (H) in m.

The r<sub>outer</sub> edge was measured from the center of the tree to the outer edge of the considered ring. Likewise, the r<sub>inner</sub> edge was measured from the center of the trunk to the inner edge of the considered ring. The height was calculated by measuring the base of the tree to the highest point, when the trees were cut down in autumn.

The volume was then multiplied by the density of the xylem (0.754 g/cm³) in order to have the biomass of each ring (Maxwell et al. 2020). The number of rings in trees 1,2, and 5 were 18 while tree 4 had 22, therefore, for consistency, only 18 rings were considered in each of the trees. The total xylem biomass for each tree was obtained by summing the biomass of the 18 individual rings.

For the biomass of the phloem (which is considered as the inner bark), we used the same approach. The phloem thickness was consistently around 5 mm for each tree and the density was equal to 0.711 g/cm³ (Maxwell et al. 2020). The r<sub>outer</sub> edge was measured from the center of the tree to the outer edge of the phloem and the r<sub>inner</sub> edge was measured from the center of the trunk to the inner edge of the phloem.

## Fine and medium roots

The fine roots (<2 mm in diameter) and medium roots (>2 mm & <1 cm in diameter) as defined by Finzi and Berthrong (2005), were obtained just before

the excavation in the fall. A strip of soil about 20 cm deep, 20 cm wide, and 70 cm long was sampled from a distance of 20 cm away from the trunk of each tree. This strip of soil, which represents approximately 30 kg of fresh soil, was then disaggregated and the fine roots and medium roots were taken out, rinsed with water, separated according to size (Finzi and Berthrong 2005) and placed into an oven at 60 °C to attain dry weight. The quantity of roots per tree was then extrapolated using the area of each EP.

## Coarse roots and stump

The coarse roots (>1 cm in diameter) and the stump were collected after the total excavation of the root system with a mechanical backhoe. The stump and the entire coarse roots were weighed. For each tree, an aliquot of around 600 g (fresh weight) of coarse roots and 300 g of stump was oven dried at 70 °C until the dry weight was attained. The total weight of coarse root and tree stump was calculated using the fresh weight to dried weight ratio of the aliquots.

## The total root system

The total root system biomass was estimated using two methods: (1) by summing the biomass of each root categories (fine roots, medium roots and coarse roots) called "the measured value of total root biomass" (without stump), and (2) by using the regression equation for *Quercus petraea* developed by Drexhage and Colin (2001), and the tree DBH, to calculate the "the estimated value of total root biomass". We then compared the two methods of biomass estimation (Drexhage and Colin 2001).

$$ \text{Log yi} = \beta0 + \beta1 \log xi \quad (1) $$

with,

Log yi logarithmic transformation of root biomass (kg).

log xi log-arithmetic transformation of DBH (cm).

β0 Intercept coefficient (-1.56).

β1 Slope coefficient (2.44).
## Total aboveground and belowground biomasses

The aboveground biomass was considered as the sum of the biomass of the leaves, the branches of the year, branches of the previous year, the other branches, the sum of the biomass of 18 xylem rings and the phloem. The belowground biomass considered comprises the fine roots, the medium roots, coarse roots (i.e. the total root system) and the stump.

## Analyses

### Tree sample preparation

After tree felling, five samples were taken from each compartment as pseudo-replicates (fine root, medium root, coarse root, stump, branch of the current year, branch of the previous year, other branch, leaf) from each of the five trees. Each compartment was characterized by the mean of five samples for its corresponding tree. The mean of the five trees was subsequently used to represent a given compartment. For phloem and each ring of the xylem, only one sample per tree was taken.

Roots, leaves and branches of the current year (n) and branches of the previous year (n-1) were pre-grinded (chopped into small pieces) using hand-operated secateurs. The stump aliquot and other branches sample where pre-grinded using a mechanical wood shredder (Timberwolf TW PTO/150H). The shredding unit of the mechanical shredder was thoroughly cleaned with air pressure between the shredding operations of each compartment to avoid sample pollution.

To isolate individual xylem rings, a 0.5 cm thick trunk cross section was cut, from which a stick of 2 mm width was removed. The sticks were then freeze-dried (Alpha 1–2 LDplus, Martin Christ). The rings were isolated using a microtome (Sledge microtomes G.S.L.1) with the limit between years being easily visible in this ring-porous species, with a manual magnifier.

Following pre-grinding, all of the samples were grounded to a fine homogenous powder using Mixer Mill MM301, Retsch for 1–6 min in 2 mL microtubes (Sarstedt) with two aluminium balls with pauses to prevent the tube from overheating. The aluminium balls were thoroughly cleaned with ethanol between each sample to avoid contamination.

### Tree sample analysis

The powder was then transferred into tin capsules (D1006, Elemental Microanalysis) using a microgram balance (LE26P, Sartorius) (2 mg for leaves; 3 mg for branches; 4 mg for the stump, other branches and roots: fine, medium and coarse; as well as for xylem and phloem). These samples were then analysed for their total N concentration by dry combustion with an N analyser (TNM-1, Shimadzu, Champs-sur-Marne, France) and for their ¹⁵N concentration by an isotopic mass spectrometer (FLASH 2000 HT/IRMS, Thermo Scientific, INRAE, Versailles).

### Soil and microbial biomass

Soil microbial N was measured using the chloroform fumigation extraction method (Brookes et al. 1985). Two fresh soil subsamples of 5 g sieved at 2 mm were prepared. One subsample was fumigated for 24 h with chloroform vapour, while the other was not fumigated. Nitrogen extraction of both subsamples was performed using 20 ml of 0.5 mol L⁻¹ K₂SO₄ for 30 min under vigorous shaking at 250 rpm for N determination. For ¹⁵N determination, the same procedure was followed, but with an extraction by 20 mL of 0.03 M K₂SO₄ to avoid any interference of the mass spectrometer with the K₂SO₄ salt during analysis. The extracts (fumigated and not fumigated, with 0.5 M or 0.03 M K₂SO₄) were filtered (GFC WHAT-MANN 1.2 µm mesh), then analysed for N content using an N analyser (TNM-1, Shimadzu, Champs-sur-Marne, France) or freeze-dried for subsequent ¹⁵N determination using an isotopic ratio mass spectrometer (FLASH 2000 HT/IRMS, Thermo Scientific).

The microbial biomass N was estimated as [(N in fumigated soil) – (N in non-fumigated soil)]/0.54 (Brookes et al. 1985). The ¹⁵N concentrations were similarly determined from the fumigated and non-fumigated soil extracts. Values from the non-fumigated soils are presented hereafter as the 'soil extractable solution' values. Soil moisture content was determined by drying 3 replicates of around 5 g of fresh soil from each EP, until constant weight at 105 °C. The results expressed per tree (as soil N stock in g N/plot or in mg ¹⁵N/plot) were obtained by multiplying the concentration data (g N or mg ¹⁵N per gram of dry soil) by the soil density (1.1 kg/m³), and then by the volume of
## Total aboveground and belowground biomasses

The aboveground biomass was considered as the sum of the biomass of the leaves, the branches of the year, branches of the previous year, the other branches, the sum of the biomass of 18 xylem rings and the phloem. The belowground biomass considered comprises the fine roots, the medium roots, coarse roots (i.e. the total root system) and the stump.

## Analyses

### Tree sample preparation

After tree felling, five samples were taken from each compartment as pseudo-replicates (fine root, medium root, coarse root, stump, branch of the current year, branch of the previous year, other branch, leaf) from each of the five trees. Each compartment was characterized by the mean of five samples for its corresponding tree. The mean of the five trees was subsequently used to represent a given compartment. For phloem and each ring of the xylem, only one sample per tree was taken.

Roots, leaves and branches of the current year (n) and branches of the previous year (n-1) were pre-grinded (chopped into small pieces) using hand-operated secateurs. The stump aliquot and other branches sample where pre-grinded using a mechanical wood shredder (Timberwolf TW PTO/150H). The shredding unit of the mechanical shredder was thoroughly cleaned with air pressure between the shredding operations of each compartment to avoid sample pollution.

To isolate individual xylem rings, a 0.5 cm thick trunk cross section was cut, from which a stick of 2 mm width was removed. The sticks were then freeze-dried (Alpha 1–2 LDplus, Martin Christ). The rings were isolated using a microtome (Sledge microtomes G.S.L.1) with the limit between years being easily visible in this ring-porous species, with a manual magnifier.

Following pre-grinding, all of the samples were grounded to a fine homogenous powder using Mixer Mill MM301, Retsch for 1–6 min in 2 mL microtubes (Sarstedt) with two aluminium balls with pauses to prevent the tube from overheating. The aluminium balls were thoroughly cleaned with ethanol between each sample to avoid contamination.

### Tree sample analysis

The powder was then transferred into tin capsules (D1006, Elemental Microanalysis) using a microgram balance (LE26P, Sartorius) (2 mg for leaves; 3 mg for branches; 4 mg for the stump, other branches and roots: fine, medium and coarse; as well as for xylem and phloem). These samples were then analysed for their total N concentration by dry combustion with an N analyser (TNM-1, Shimadzu, Champs-sur-Marne, France) and for their ¹⁵N concentration by an isotopic mass spectrometer (FLASH 2000 HT/IRMS, Thermo Scientific, INRAE, Versailles).

### Soil and microbial biomass

Soil microbial N was measured using the chloroform fumigation extraction method (Brookes et al. 1985). Two fresh soil subsamples of 5 g sieved at 2 mm were prepared. One subsample was fumigated for 24 h with chloroform vapour, while the other was not fumigated. Nitrogen extraction of both subsamples was performed using 20 ml of 0.5 mol L⁻¹ K₂SO₄ for 30 min under vigorous shaking at 250 rpm for N determination. For ¹⁵N determination, the same procedure was followed, but with an extraction by 20 mL of 0.03 M K₂SO₄ to avoid any interference of the mass spectrometer with the K₂SO₄ salt during analysis. The extracts (fumigated and not fumigated, with 0.5 M or 0.03 M K₂SO₄) were filtered (GFC WHAT-MANN 1.2 µm mesh), then analysed for N content using an N analyser (TNM-1, Shimadzu, Champs-sur-Marne, France) or freeze-dried for subsequent ¹⁵N determination using an isotopic ratio mass spectrometer (FLASH 2000 HT/IRMS, Thermo Scientific).

The microbial biomass N was estimated as [(N in fumigated soil) – (N in non-fumigated soil)]/0.54 (Brookes et al. 1985). The ¹⁵N concentrations were similarly determined from the fumigated and non-fumigated soil extracts. Values from the non-fumigated soils are presented hereafter as the 'soil extractable solution' values. Soil moisture content was determined by drying 3 replicates of around 5 g of fresh soil from each EP, until constant weight at 105 °C. The results expressed per tree (as soil N stock in g N/plot or in mg ¹⁵N/plot) were obtained by multiplying the concentration data (g N or mg ¹⁵N per gram of dry soil) by the soil density (1.1 kg/m³), and then by the volume of
soil around each tree. This was calculated by multiplying a fixed depth of 15 cm (height of the soil corer) by the specific area around each tree (3.9, 2.8, 3.9, 3.5, and 3.0 m² for trees 1,2,3,4 and 5 respectively).

## Calculations

### Quantity of N and ¹⁵N

The amount of nitrogen present in each compartment expressed per tree, as N stock in g N / tree was found by multiplying the concentration data (g N / gram of compartment) by the estimated or measured compartment’s biomass. That value was then multiplied with the percentage of ¹⁵N to calculate the quantity of ¹⁵N in the compartment.

The same calculation method was used for quantifying N and ¹⁵N in microbial biomass and soil extractable solution.

### Recovered ¹⁵N

The quantity of ¹⁵N from the labelled solution added to the soil March 1 that was recovered at fall in the different compartments of the system in question (tree compartments, microbial biomass and extractable soil solution) was calculated using the following formula:

$$PRN\% = \frac{Recovered^{15}N \text{ in compartment}}{\sum Recovered^{15}N} * 100$$

## Statistical analysis

The data were analysed using the SigmaPlot program. To test for differences between compartments, a one-way Analysis of Variance (ANOVA) (n=5), was done for the biomass, nitrogen quantity and nitrogen concentration, recovered ¹⁵N quantity and as well as its concentration for each compartment. In the case of non-normal residuals, a Kruskal–Wallis One Way ANOVA on ranks was completed as a non-parametric replacement for the ANOVA.

If there was a significant effect of the tree compartment on the variable measured, a Tukey post hoc test was conducted to determine which compartment was significantly different from the other. For the comparison between two means, a t-test or a Welch-test was performed depending on whether the variances were equal or not. The rejection limit was fixed to alpha=5% for all the tests used, and thus any result with a p-value inferior to 0.05 was considered to be significant.

Recovered¹⁵N = Atom%¹⁵N excess compartment * N% compartment * total biomass of compartment

With Atom%¹⁵Nexcess compartment = Atom%¹⁵Ncompartment after labelling – Atom%¹⁵Ncompartment natural abundance

The natural abundance of ¹⁵N in the compartments of each tree and microbial biomass (Atom%¹⁵N natural abundance of the compartment) was measured prior to labelling at DOY 60 except for the leaves, in which the cited natural abundance of 0.36% was used (Deléens et al. 1997). These results were expressed in grams for nitrogen and in milligrams for ¹⁵N.

To quantify the distribution of the applied ¹⁵N between the compartments of the tree, results were expressed in percent (%) of the sum of the total ¹⁵N recovered in those compartments. Percentage of Recovered Nitrogen (PRN) was calculated as follows:

## Results

### Biomass partitioning

Here we present data on biomass partitioning in five 20-year-old oak trees. The average height of the trees was 10.22±0.8 m and the average diameter at breast height was 13.44±1.5 cm (Table 1). The trees had a total biomass of 75 kg±18 kg, ranging from 57.5 kg to 101.6 kg. The distribution of the total biomass was in favor of the aboveground part, which represented 76% of the total biomass (Fig. 1A). The aboveground compartments considered were leaves, branches of the current and previous years, other branches,
soil around each tree. This was calculated by multiplying a fixed depth of 15 cm (height of the soil corer) by the specific area around each tree (3.9, 2.8, 3.9, 3.5, and 3.0 m² for trees 1,2,3,4 and 5 respectively).

## Calculations

### Quantity of N and ¹⁵N

The amount of nitrogen present in each compartment expressed per tree, as N stock in g N / tree was found by multiplying the concentration data (g N / gram of compartment) by the estimated or measured compartment’s biomass. That value was then multiplied with the percentage of ¹⁵N to calculate the quantity of ¹⁵N in the compartment.

The same calculation method was used for quantifying N and ¹⁵N in microbial biomass and soil extractable solution.

### Recovered ¹⁵N

The quantity of ¹⁵N from the labelled solution added to the soil March 1 that was recovered at fall in the different compartments of the system in question (tree compartments, microbial biomass and extractable soil solution) was calculated using the following formula:

$$PRN\% = \frac{Recovered^{15}N \text{ in compartment}}{\sum Recovered^{15}N} * 100$$

## Statistical analysis

The data were analysed using the SigmaPlot program. To test for differences between compartments, a one-way Analysis of Variance (ANOVA) (n=5), was done for the biomass, nitrogen quantity and nitrogen concentration, recovered ¹⁵N quantity and as well as its concentration for each compartment. In the case of non-normal residuals, a Kruskal–Wallis One Way ANOVA on ranks was completed as a non-parametric replacement for the ANOVA.

If there was a significant effect of the tree compartment on the variable measured, a Tukey post hoc test was conducted to determine which compartment was significantly different from the other. For the comparison between two means, a t-test or a Welch-test was performed depending on whether the variances were equal or not. The rejection limit was fixed to alpha=5% for all the tests used, and thus any result with a p-value inferior to 0.05 was considered to be significant.

Recovered¹⁵N = Atom%¹⁵N excess compartment * N% compartment * total biomass of compartment

With Atom%¹⁵Nexcess compartment = Atom%¹⁵Ncompartment after labelling – Atom%¹⁵Ncompartment natural abundance

The natural abundance of ¹⁵N in the compartments of each tree and microbial biomass (Atom%¹⁵N natural abundance of the compartment) was measured prior to labelling at DOY 60 except for the leaves, in which the cited natural abundance of 0.36% was used (Deléens et al. 1997). These results were expressed in grams for nitrogen and in milligrams for ¹⁵N.

To quantify the distribution of the applied ¹⁵N between the compartments of the tree, results were expressed in percent (%) of the sum of the total ¹⁵N recovered in those compartments. Percentage of Recovered Nitrogen (PRN) was calculated as follows:

## Results

### Biomass partitioning

Here we present data on biomass partitioning in five 20-year-old oak trees. The average height of the trees was 10.22±0.8 m and the average diameter at breast height was 13.44±1.5 cm (Table 1). The trees had a total biomass of 75 kg±18 kg, ranging from 57.5 kg to 101.6 kg. The distribution of the total biomass was in favor of the aboveground part, which represented 76% of the total biomass (Fig. 1A). The aboveground compartments considered were leaves, branches of the current and previous years, other branches,
<table><caption>**Table 1** Morphological characteristics of the five trees</caption><thead><tr><th>Tree</th><th>Height of tree (m)</th><th>Diameter at Breast Height (cm)</th></tr></thead><tbody><tr><td>1</td><td>10.6</td><td>15.7</td></tr><tr><td>2</td><td>10.5</td><td>12</td></tr><tr><td>3</td><td>8.96</td><td>13</td></tr><tr><td>4</td><td>11.05</td><td>14</td></tr><tr><td>5</td><td>10</td><td>12.5</td></tr><tr><td>Mean</td><td>10.22±0.8</td><td>13.44±1.5</td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Quantity of dry biomass expressed in kg/tree in above-ground (AB) and belowground (BG) compartments (A), sub-compartments (B) and in the 18 xylem rings (C). Rings 1 and 18 correspond to years 2016 and 1999 respectively. Values are mean and standard deviation of 5 field replicates from five trees. The number above the bars are the percentage of the dry biomass in the considered compartment relative to the whole tree</figcaption></figure>

phloem and xylem, the latter containing nearly 50% of the tree's total biomass and 63% of the above-ground biomass (Fig. 1B). The biomass of the two successive branch compartments (branches of 2015 and branches of 2016) were quite similar representing 0.76±0.3 kg and 0.78±0.2 kg respectively, i.e.

1% of the total biomass of the trees. The trunk biomass associated to individual rings ranged from 0.4±0.6 kg to 3.6±0.9 kg depending on the year. The limits of the sapwood to heartwood varies among the trees (ring 11 for tree 1, ring 11 for tree 2, ring 10 for tree 3, ring 11 for tree 4 and ring 8 for tree 5). The largest rings were those formed in 2016 (ring 1) and 2007 (ring 10) (Fig. 1C). From ring width measurement, we observed a decrease in annual growth from ring 2 to ring 10 (2015 to 2007), a sharp spike in ring 10, and then subsequent decrease from ring 11 to ring 18 (2008 to 2001), with small fluctuations.

The belowground biomass which represents 24% of the total biomass was made of coarse roots and the stump at 90% (Fig. 1B). Fine and medium roots (<1 cm) put together represented 2.6% of the total biomass of the tree, similar to the order of magnitude of the leaves which represented 3.4% of the total biomass of the tree.

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Correlation between the root biomass measured by summing the individual biomass of fine, medium and large roots with the root biomass estimated using DBH and the Eq. 2 from (Drexhage and Colin 2001), with all the trees except the tree 3 (A) or with all the five tree (B). The number under the point refers to the number of the tree (see Table 1)</figcaption></figure>
<table><caption>**Table 1** Morphological characteristics of the five trees</caption><thead><tr><th>Tree</th><th>Height of tree (m)</th><th>Diameter at Breast Height (cm)</th></tr></thead><tbody><tr><td>1</td><td>10.6</td><td>15.7</td></tr><tr><td>2</td><td>10.5</td><td>12</td></tr><tr><td>3</td><td>8.96</td><td>13</td></tr><tr><td>4</td><td>11.05</td><td>14</td></tr><tr><td>5</td><td>10</td><td>12.5</td></tr><tr><td>Mean</td><td>10.22±0.8</td><td>13.44±1.5</td></tr></tbody></table>

<figure><img src="image_2.png" /><figcaption>**Fig. 1** Quantity of dry biomass expressed in kg/tree in above-ground (AB) and belowground (BG) compartments (A), sub-compartments (B) and in the 18 xylem rings (C). Rings 1 and 18 correspond to years 2016 and 1999 respectively. Values are mean and standard deviation of 5 field replicates from five trees. The number above the bars are the percentage of the dry biomass in the considered compartment relative to the whole tree</figcaption></figure>

phloem and xylem, the latter containing nearly 50% of the tree's total biomass and 63% of the above-ground biomass (Fig. 1B). The biomass of the two successive branch compartments (branches of 2015 and branches of 2016) were quite similar representing 0.76±0.3 kg and 0.78±0.2 kg respectively, i.e.

1% of the total biomass of the trees. The trunk biomass associated to individual rings ranged from 0.4±0.6 kg to 3.6±0.9 kg depending on the year. The limits of the sapwood to heartwood varies among the trees (ring 11 for tree 1, ring 11 for tree 2, ring 10 for tree 3, ring 11 for tree 4 and ring 8 for tree 5). The largest rings were those formed in 2016 (ring 1) and 2007 (ring 10) (Fig. 1C). From ring width measurement, we observed a decrease in annual growth from ring 2 to ring 10 (2015 to 2007), a sharp spike in ring 10, and then subsequent decrease from ring 11 to ring 18 (2008 to 2001), with small fluctuations.

The belowground biomass which represents 24% of the total biomass was made of coarse roots and the stump at 90% (Fig. 1B). Fine and medium roots (<1 cm) put together represented 2.6% of the total biomass of the tree, similar to the order of magnitude of the leaves which represented 3.4% of the total biomass of the tree.

<figure><img src="image_3.png" /><figcaption>**Fig. 2** Correlation between the root biomass measured by summing the individual biomass of fine, medium and large roots with the root biomass estimated using DBH and the Eq. 2 from (Drexhage and Colin 2001), with all the trees except the tree 3 (A) or with all the five tree (B). The number under the point refers to the number of the tree (see Table 1)</figcaption></figure>
The result of the root biomass estimated using allometric equation is shown in Fig. 2. The estimated biomass was close to actual (measured) biomass ($R^2=0.7045$) when tree 3 was not included ($r^2=0.3746$, when tree 3 was included).

## Nitrogen distribution

Figure 3 presents the percentage of nitrogen in the tree compartments. Among all of the sub-compartments, the nitrogen percentage was significantly higher in leaves (Fig. 3A), than that in the xylem, phloem and stump. Other than leaves, the compartments with the highest nitrogen were the branches: $0.97 \pm 0.17\%N$ in the current year's branches and $0.72 \pm 0.17\%N$ in the previous year's branches. Fine roots contained $0.63 \pm 0.07\%N$. The percentage of nitrogen ranged from $0.09 \pm 0.01$ to $0.56 \pm 0.01\%N$ for the other compartments (xylem, phloem, stump, coarse root, medium root and other branches). On average, the xylem had the lowest concentration of nitrogen.

<figure><img src="image_6.png" /><figcaption>**Fig. 3** Percentage of nitrogen in sub-compartments (A) and in the 18 xylem rings (B). Rings 1 and 18 correspond to years 2016 and 1999 respectively. Values are mean and standard deviation of 5 field replicates from five trees</figcaption></figure>

In the xylem, the concentration of nitrogen depended on the age of the ring ($p<0.05$): the percentage of nitrogen in the first ring (younger ring) was higher than in the older rings (and significantly higher than in rings 11 to 16) (Fig. 3B). The lowest value of the percentage of nitrogen in the ring was 0.05% (in ring 12). Nitrogen decreases sharply from ring 1 to ring 2 (student T-test $p=0.06$) and then decreased steadily up to ring 12 (which was the maximum inner limit of the sapwood), reaching a minimum after which the N concentration increased up to ring 18 (i.e. throughout the heartwood), reaching 0.75% of the dry mass, i.e. half the average concentration of rings 1–2.

The total quantity of nitrogen in a tree represented $243 \pm 56$ g N / tree, 68% of which was aboveground and 32% belowground (Fig. 4A). The majority of the tree nitrogen was found in the branches and the roots, these two compartments alone accounted for 57.5% of the total tree (Fig. 4B). The trunk (xylem

<figure><img src="image_7.png" /><figcaption>**Fig. 4** Quantity of nitrogen expressed in g N / tree in above and below ground compartments (A), sub-compartments (B) and in the 18 xylem rings (C). Rings 1 and 18 correspond to years 2016 and 1999 respectively. Values are mean and standard deviation of 5 field replicates from five trees. The number above the bars are the percentage of the nitrogen quantity in the considered compartment relative to the whole tree</figcaption></figure>
The result of the root biomass estimated using allometric equation is shown in Fig. 2. The estimated biomass was close to actual (measured) biomass ($R^2=0.7045$) when tree 3 was not included ($r^2=0.3746$, when tree 3 was included).

## Nitrogen distribution

Figure 3 presents the percentage of nitrogen in the tree compartments. Among all of the sub-compartments, the nitrogen percentage was significantly higher in leaves (Fig. 3A), than that in the xylem, phloem and stump. Other than leaves, the compartments with the highest nitrogen were the branches: $0.97 \pm 0.17\%N$ in the current year's branches and $0.72 \pm 0.17\%N$ in the previous year's branches. Fine roots contained $0.63 \pm 0.07\%N$. The percentage of nitrogen ranged from $0.09 \pm 0.01$ to $0.56 \pm 0.01\%N$ for the other compartments (xylem, phloem, stump, coarse root, medium root and other branches). On average, the xylem had the lowest concentration of nitrogen.

<figure><img src="image_4.png" /><figcaption>**Fig. 3** Percentage of nitrogen in sub-compartments (A) and in the 18 xylem rings (B). Rings 1 and 18 correspond to years 2016 and 1999 respectively. Values are mean and standard deviation of 5 field replicates from five trees</figcaption></figure>

In the xylem, the concentration of nitrogen depended on the age of the ring ($p<0.05$): the percentage of nitrogen in the first ring (younger ring) was higher than in the older rings (and significantly higher than in rings 11 to 16) (Fig. 3B). The lowest value of the percentage of nitrogen in the ring was 0.05% (in ring 12). Nitrogen decreases sharply from ring 1 to ring 2 (student T-test $p=0.06$) and then decreased steadily up to ring 12 (which was the maximum inner limit of the sapwood), reaching a minimum after which the N concentration increased up to ring 18 (i.e. throughout the heartwood), reaching 0.75% of the dry mass, i.e. half the average concentration of rings 1–2.

The total quantity of nitrogen in a tree represented $243 \pm 56$ g N / tree, 68% of which was aboveground and 32% belowground (Fig. 4A). The majority of the tree nitrogen was found in the branches and the roots, these two compartments alone accounted for 57.5% of the total tree (Fig. 4B). The trunk (xylem

<figure><img src="image_5.png" /><figcaption>**Fig. 4** Quantity of nitrogen expressed in g N / tree in above and below ground compartments (A), sub-compartments (B) and in the 18 xylem rings (C). Rings 1 and 18 correspond to years 2016 and 1999 respectively. Values are mean and standard deviation of 5 field replicates from five trees. The number above the bars are the percentage of the nitrogen quantity in the considered compartment relative to the whole tree</figcaption></figure>
plus phloem) contained around the same amount of nitrogen as the other branches (23.5% versus 24%). Similar amounts of nitrogen (around 7 g N/tree) were recovered in the 2016 branches and in the latest ring of the xylem (ring 1, also formed in 2016), (Fig. 2B and C). The nitrogen content of this latest ring represents 2.7% of the nitrogen content of the tree and around 20% of the xylem nitrogen. Both the branches of the previous year (2015) and the fine roots contained around 5 g N/tree. Medium roots contained around $6 \pm 4$ g N/tree.

¹⁵ N distribution

Figure 5 presents the percentage of ¹⁵ N in excess in the tree’s compartments. These values indicate the proportion of nitrogen coming from the soil labeling in comparison with the total nitrogen present in the compartment. The percentage of ¹⁵ N in excess was higher in the fine and medium roots (more than 0.4% of ¹⁵ N in excess) (Fig. 5A). The leaves and the newer branches (2016) are the second ¹⁵ N rich

<figure><img src="image_8.png" /><figcaption>**Fig. 5** Percentage of ¹⁵ N in excess in sub-compartments (A) and in the 18 xylem rings (B) in %¹⁵ N in excess. Rings 1 and 18 correspond to years 2016 and 1999 respectively. Values are mean and standard deviation of 5 field replicates from five trees</figcaption></figure>

compartment with values around 0.3±0.16% ¹⁵ N in excess (Fig. 5A). Phloem was also rich in ¹⁵ N with a value of 0.27±0.13%. The other compartments had lower values. In the xylem, ¹⁵ N in excess was found in all the sapwood (from ring 1 to ring 10) and especially in the new ring of 2016, but not in the heartwood (from ring 12 to 18). The ring 11 formed in 2006 was in the sapwood or the heartwood (dependent on the tree and location in the tree) and showed intermediate value (Fig. 5B).

The total tree ¹⁵ N recovered was 475±60 mg ¹⁵ N in excess which represents only 26% of the 1.82 g of ¹⁵ N initially applied to the soil in spring (Fig. 6A). The recovered ¹⁵ N was mainly found in aboveground part (67%) (Fig. 6A) especially in the leaves (24%), then in the stump and coarse root when summed (21%) followed by the trunk (20% when the phloem and xylem were summed) (Fig. 6B). The ¹⁵ N in excess contained in the first

<figure><img src="image_9.png" /><figcaption>**Fig. 6** Quantity of recovered ¹⁵ N expressed in mg ¹⁵ N in excess/tree in aboveground (AG) and belowground (BG) compartments (A), sub-compartments (B) and in the 18 xylem rings (C). Rings 1 and 18 correspond to years 2016 and 1999 respectively. Values are mean and standard deviation of 5 field replicates from five trees. The number above the bars are the percentage of the ¹⁵ N in excess in the considered compartment relative to the whole tree</figcaption></figure>
plus phloem) contained around the same amount of nitrogen as the other branches (23.5% versus 24%). Similar amounts of nitrogen (around 7 g N/tree) were recovered in the 2016 branches and in the latest ring of the xylem (ring 1, also formed in 2016), (Fig. 2B and C). The nitrogen content of this latest ring represents 2.7% of the nitrogen content of the tree and around 20% of the xylem nitrogen. Both the branches of the previous year (2015) and the fine roots contained around 5 g N/tree. Medium roots contained around $6 \pm 4$ g N/tree.

¹⁵ N distribution

Figure 5 presents the percentage of ¹⁵ N in excess in the tree’s compartments. These values indicate the proportion of nitrogen coming from the soil labeling in comparison with the total nitrogen present in the compartment. The percentage of ¹⁵ N in excess was higher in the fine and medium roots (more than 0.4% of ¹⁵ N in excess) (Fig. 5A). The leaves and the newer branches (2016) are the second ¹⁵ N rich

<figure><img src="image_6.png" /><figcaption>**Fig. 5** Percentage of ¹⁵ N in excess in sub-compartments (A) and in the 18 xylem rings (B) in %¹⁵ N in excess. Rings 1 and 18 correspond to years 2016 and 1999 respectively. Values are mean and standard deviation of 5 field replicates from five trees</figcaption></figure>

compartment with values around 0.3±0.16% ¹⁵ N in excess (Fig. 5A). Phloem was also rich in ¹⁵ N with a value of 0.27±0.13%. The other compartments had lower values. In the xylem, ¹⁵ N in excess was found in all the sapwood (from ring 1 to ring 10) and especially in the new ring of 2016, but not in the heartwood (from ring 12 to 18). The ring 11 formed in 2006 was in the sapwood or the heartwood (dependent on the tree and location in the tree) and showed intermediate value (Fig. 5B).

The total tree ¹⁵ N recovered was 475±60 mg ¹⁵ N in excess which represents only 26% of the 1.82 g of ¹⁵ N initially applied to the soil in spring (Fig. 6A). The recovered ¹⁵ N was mainly found in aboveground part (67%) (Fig. 6A) especially in the leaves (24%), then in the stump and coarse root when summed (21%) followed by the trunk (20% when the phloem and xylem were summed) (Fig. 6B). The ¹⁵ N in excess contained in the first

<figure><img src="image_7.png" /><figcaption>**Fig. 6** Quantity of recovered ¹⁵ N expressed in mg ¹⁵ N in excess/tree in aboveground (AG) and belowground (BG) compartments (A), sub-compartments (B) and in the 18 xylem rings (C). Rings 1 and 18 correspond to years 2016 and 1999 respectively. Values are mean and standard deviation of 5 field replicates from five trees. The number above the bars are the percentage of the ¹⁵ N in excess in the considered compartment relative to the whole tree</figcaption></figure>
ring of the xylem (Fig. 6C) (i.e. 14 ± 5 mg ¹⁵ N in excess) is in the same order of magnitude as that in the branches of the current or previous years and represented around 3% of the ¹⁵ N recovered in the tree. As shown in Fig. 6C, the first ring of the xylem accounted for 3% of the ¹⁵ N recovery while the other rings collectively accounted for 4.6%. In relation to the tree trunk (the xylem), the first four rings of the xylem contain 70% of the ¹⁵ N with the first ring taking 38% of it. The other rings of the sapwood (from ring 2 to ring 10) contained 22 ± 4 mg ¹⁵ N in excess that is much lesser than what we found in the stump (48 ± 23 mg ¹⁵ N in excess), fine roots (29 ± 22 mg ¹⁵ N in excess), medium roots (25 ± 19 mg ¹⁵ N in excess) and coarse root (53 ± 13 mg ¹⁵ N in excess). The less studied compartments (coarse root, stump, xylem and other branches) together recovered 45% of the ¹⁵ N.

Out of the 1.82 g of ¹⁵ N that was applied to the trees at the beginning of the experiment, only 0.604 g was recovered in the EP (tree, microbial biomass and the extractable soil solution) in Autumn which represented 33% of the original applied ¹⁵ N (Fig. 7). At this point, most of the recovered ¹⁵ N was found within the tree (79%), while only 17% and 4% were recovered in the microbial biomass and extractable soil solution, respectively.

<figure><img src="image_10.png" /><figcaption>**Fig. 7** Quantity of recovered ¹⁵ N expressed in mg ¹⁵ N in excess/plot in Tree, microbial biomass and soil extractable solution. Values are mean and standard deviation of 5 field replicates. The number above the bars are the percentage of the ¹⁵ N in excess in the considered compartment relative to the ¹⁵ N applied in spring</figcaption></figure>

## Discussion

The objective of the paper was to report an exhaustive assessment of total dry matter (biomass pools), total nitrogen (N), and total ¹⁵ N and distribution in all compartments of the trees as N flux.

### Biomass repartition

The aboveground compartment was found to contain more biomass than the belowground compartment. Similar result have previously been obtained in both deciduous species such as oak (Barbaroux et al. 2003) and evergreen species such as Scots pine (Xiao et al. 2003). While we found that 76% of biomass in the aboveground and 24% belowground, Barbaroux et al. (2003) found these values to be 87% (stem, 51%; branches, 8% and leaves, 2%), and 13% belowground. The proportion of belowground compartment biomass might have been lower in Barbaroux et al. 2003 (13%) compared to our finding (24%), because they did not consider stump (only fine roots, medium roots and coarse roots). Difference in age (20 years in our case and 45 years in Barbaroux et al. 2003) might also explain the difference observed as age-dependent preferential biomass allocation is proven to be higher in the aboveground with increasing age of the tree (Helmisaari et al. 2002; Yang et al. 2019).

The result of more biomass in the aboveground compartment than in the belowground compartment may be due to high NPP allocation to the aboveground compartment (Barbaroux et al. 2003). Biomass variation among rings was likely due to physiological processes and effects of weather and environmental conditions (Verheyden et al. 2004; Delpierre et al. 2016).

Recent advancement in biomass estimation involved the use of allometric equations that employs biomass prediction models to estimate both aboveground (Crow 1978; Cunia 1987; Brown et al. 1989; Houghton et al. 2001; Chave et al. 2001, 2014) and belowground biomass (Zianis et al. 2005) using DBH only (Ter-Mikaelian and Korzukhin 1997; Návar 2009) or DBH-h (DBH and height) equations (Wang 2006). In European species, most of the biomass equations concentrated on aboveground parts, particularly for branches and foliage with very few equations targeting belowground components (Zianis et al. 2005). Currently, for *Quercus petraea*, there is only
ring of the xylem (Fig. 6C) (i.e. 14 ± 5 mg ¹⁵ N in excess) is in the same order of magnitude as that in the branches of the current or previous years and represented around 3% of the ¹⁵ N recovered in the tree. As shown in Fig. 6C, the first ring of the xylem accounted for 3% of the ¹⁵ N recovery while the other rings collectively accounted for 4.6%. In relation to the tree trunk (the xylem), the first four rings of the xylem contain 70% of the ¹⁵ N with the first ring taking 38% of it. The other rings of the sapwood (from ring 2 to ring 10) contained 22 ± 4 mg ¹⁵ N in excess that is much lesser than what we found in the stump (48 ± 23 mg ¹⁵ N in excess), fine roots (29 ± 22 mg ¹⁵ N in excess), medium roots (25 ± 19 mg ¹⁵ N in excess) and coarse root (53 ± 13 mg ¹⁵ N in excess). The less studied compartments (coarse root, stump, xylem and other branches) together recovered 45% of the ¹⁵ N.

Out of the 1.82 g of ¹⁵ N that was applied to the trees at the beginning of the experiment, only 0.604 g was recovered in the EP (tree, microbial biomass and the extractable soil solution) in Autumn which represented 33% of the original applied ¹⁵ N (Fig. 7). At this point, most of the recovered ¹⁵ N was found within the tree (79%), while only 17% and 4% were recovered in the microbial biomass and extractable soil solution, respectively.

<figure><img src="image_8.png" /><figcaption>**Fig. 7** Quantity of recovered ¹⁵ N expressed in mg ¹⁵ N in excess/plot in Tree, microbial biomass and soil extractable solution. Values are mean and standard deviation of 5 field replicates. The number above the bars are the percentage of the ¹⁵ N in excess in the considered compartment relative to the ¹⁵ N applied in spring</figcaption></figure>

## Discussion

The objective of the paper was to report an exhaustive assessment of total dry matter (biomass pools), total nitrogen (N), and total ¹⁵ N and distribution in all compartments of the trees as N flux.

### Biomass repartition

The aboveground compartment was found to contain more biomass than the belowground compartment. Similar result have previously been obtained in both deciduous species such as oak (Barbaroux et al. 2003) and evergreen species such as Scots pine (Xiao et al. 2003). While we found that 76% of biomass in the aboveground and 24% belowground, Barbaroux et al. (2003) found these values to be 87% (stem, 51%; branches, 8% and leaves, 2%), and 13% belowground. The proportion of belowground compartment biomass might have been lower in Barbaroux et al. 2003 (13%) compared to our finding (24%), because they did not consider stump (only fine roots, medium roots and coarse roots). Difference in age (20 years in our case and 45 years in Barbaroux et al. 2003) might also explain the difference observed as age-dependent preferential biomass allocation is proven to be higher in the aboveground with increasing age of the tree (Helmisaari et al. 2002; Yang et al. 2019).

The result of more biomass in the aboveground compartment than in the belowground compartment may be due to high NPP allocation to the aboveground compartment (Barbaroux et al. 2003). Biomass variation among rings was likely due to physiological processes and effects of weather and environmental conditions (Verheyden et al. 2004; Delpierre et al. 2016).

Recent advancement in biomass estimation involved the use of allometric equations that employs biomass prediction models to estimate both aboveground (Crow 1978; Cunia 1987; Brown et al. 1989; Houghton et al. 2001; Chave et al. 2001, 2014) and belowground biomass (Zianis et al. 2005) using DBH only (Ter-Mikaelian and Korzukhin 1997; Návar 2009) or DBH-h (DBH and height) equations (Wang 2006). In European species, most of the biomass equations concentrated on aboveground parts, particularly for branches and foliage with very few equations targeting belowground components (Zianis et al. 2005). Currently, for *Quercus petraea*, there is only
one allometric equation (DBH only) that can be used to estimate root biomass (Drexhage and Colin 2001). The root biomass we estimated using this (Drexhage and Colin 2001) allometric equation was close to actual (measured) biomass (p=0.70) when tree 3 was not included (r²=0.37, when tree 3 was included). The reason why tree 3 reduced the r² was not well understood but we hypothesized that to be due to the lowest height attributed to tree 3 (height=8.96 m) compared to all other trees (mean height=10.22 m). We therefore suggest that for more robust allometric estimation of biomass, DBH-h equations that take into account the height of the tree should be employed.

## Nitrogen distribution

We found 68% of nitrogen in the aboveground compartment and 32% in the belowground compartment. Studies comparing aboveground and belowground nitrogen distribution in deciduous species are insufficient, but studies on evergreen species such as citrus are available and shows similar trend to our deciduous study. Tadashi et al. 1982 found 61% of nitrogen in the aboveground and 39% in the belowground, in citrus trees (Citrus unshiu Marc.) that were 4 and 6 years old. This similarity might be due to the similarities in the diversity of the aboveground compartments measured: Tadashi et al. (1982) studied fruits, current year leaves, current year twigs, 1-year-old leaf, 1-year-old twig, 2-year-old twigs, 3–4-year-old branches, 5–10-year-old branches, while we focused on leaves, branches of the year, branches of the previous year, other branches, phloem and xylem rings in our work. Additionally, similar belowground compartments were studied between both studies, such as the belowground stump, coarse roots, medium roots and fine roots.

Nitrogen quantities and concentration were not stable in the xylem rings but relatively decreased with the aging of the rings. More nitrogen was found in the sapwood (0.107 gN/100gDM) (from ring 1 to ring 10) than in the heartwood (0.064 gN/100gDM) (from ring 12 to 18). This is similar to the finding of Meerts (2002), who found lower nitrogen concentrations in heartwood (0.117 gN/100gDM) than in the sapwood (0.174 gN/100gDM) in 47 angiosperm species. Similarly, Härdtle et al. (2014) and McLauchlan and Craine (2011), respectively found in the heartwood

of *Quercus petraea* and *Quercus alba* around 1 mgN gDM⁻¹ and 1.2 mgN gDM⁻¹ while in the sapwood 1 to 2,2 mgN gDM⁻¹ and 2.22 mgN gDM⁻¹. Fluctuations of nitrogen among rings were observed due to high radial mobility of xylem N within trees (Elhani et al. 2003). There was also a sharp decrease from ring 1 to ring 2 in terms of biomass and N quantity, which might be due to the accumulation of N originating from leaf resorption into ring 1, probably in the form of N reserve (Bazot et al. 2013). Our results show that most of the nitrogen was stored in perennial tissues (trunk, branches, stump and coarse root), which was likely due to the timing of the sampling. In autumn, leaf senescence occurs and nitrogen in the leaves is translocated to perennial storage components (Millard 1995; Aerts 1996; Babst and Coleman 2018). But we still found a large proportion of nitrogen in the leaves (14%) at this date. That could be explained by the delay between the onset of protein degradation and the efficiency of the nitrogen export (Sample and Babst 2019). Nitrogen compounds resulting from the leaf protein degradation could be stored in the vacuole (Sample and Babst 2019) before being withdrawn to vegetative storage components for use in the spring and summer. We estimated that at the sampling date (DOY 287, October 13), the trees were at the mid-period of leaf senescence as the leaf nitrogen percentage was 1.58% at this date, 2.21% at LAI max (data not shown), and the %N in the soil litter around 1% during November (Nicolas Delpierre, personal communication). Following Du et al. (2021) and Vergutz et al. (2012) we calculated the nitrogen resorption efficiency at the sampling date to be 44%. The maximum resorption efficiency was 64% when calculated with the % of N in the soil litter. A similar value was obtained on *Quercus serrata* by Migita et al. (2007).

¹⁵ N distribution

After the soil labelling at budburst, the ¹⁵ N was analyzed in the different tree compartments at the end of the vegetation season in autumn. We found that 67% of the recovered ¹⁵ N within the tree was found in the aboveground compartment, while 33% was found belowground. Zeller et al. (2001) traced a 3 year ¹⁵ N labelled litter decomposition in *Fagus sylvatica* and found 61.1% of recovered ¹⁵ N in the aerial biomass, with 38.8% in the root biomass.
one allometric equation (DBH only) that can be used to estimate root biomass (Drexhage and Colin 2001). The root biomass we estimated using this (Drexhage and Colin 2001) allometric equation was close to actual (measured) biomass (p=0.70) when tree 3 was not included (r²=0.37, when tree 3 was included). The reason why tree 3 reduced the r² was not well understood but we hypothesized that to be due to the lowest height attributed to tree 3 (height=8.96 m) compared to all other trees (mean height=10.22 m). We therefore suggest that for more robust allometric estimation of biomass, DBH-h equations that take into account the height of the tree should be employed.

## Nitrogen distribution

We found 68% of nitrogen in the aboveground compartment and 32% in the belowground compartment. Studies comparing aboveground and belowground nitrogen distribution in deciduous species are insufficient, but studies on evergreen species such as citrus are available and shows similar trend to our deciduous study. Tadashi et al. 1982 found 61% of nitrogen in the aboveground and 39% in the belowground, in citrus trees (Citrus unshiu Marc.) that were 4 and 6 years old. This similarity might be due to the similarities in the diversity of the aboveground compartments measured: Tadashi et al. (1982) studied fruits, current year leaves, current year twigs, 1-year-old leaf, 1-year-old twig, 2-year-old twigs, 3–4-year-old branches, 5–10-year-old branches, while we focused on leaves, branches of the year, branches of the previous year, other branches, phloem and xylem rings in our work. Additionally, similar belowground compartments were studied between both studies, such as the belowground stump, coarse roots, medium roots and fine roots.

Nitrogen quantities and concentration were not stable in the xylem rings but relatively decreased with the aging of the rings. More nitrogen was found in the sapwood (0.107 gN/100gDM) (from ring 1 to ring 10) than in the heartwood (0.064 gN/100gDM) (from ring 12 to 18). This is similar to the finding of Meerts (2002), who found lower nitrogen concentrations in heartwood (0.117 gN/100gDM) than in the sapwood (0.174 gN/100gDM) in 47 angiosperm species. Similarly, Härdtle et al. (2014) and McLauchlan and Craine (2011), respectively found in the heartwood

of *Quercus petraea* and *Quercus alba* around 1 mgN gDM⁻¹ and 1.2 mgN gDM⁻¹ while in the sapwood 1 to 2,2 mgN gDM⁻¹ and 2.22 mgN gDM⁻¹. Fluctuations of nitrogen among rings were observed due to high radial mobility of xylem N within trees (Elhani et al. 2003). There was also a sharp decrease from ring 1 to ring 2 in terms of biomass and N quantity, which might be due to the accumulation of N originating from leaf resorption into ring 1, probably in the form of N reserve (Bazot et al. 2013). Our results show that most of the nitrogen was stored in perennial tissues (trunk, branches, stump and coarse root), which was likely due to the timing of the sampling. In autumn, leaf senescence occurs and nitrogen in the leaves is translocated to perennial storage components (Millard 1995; Aerts 1996; Babst and Coleman 2018). But we still found a large proportion of nitrogen in the leaves (14%) at this date. That could be explained by the delay between the onset of protein degradation and the efficiency of the nitrogen export (Sample and Babst 2019). Nitrogen compounds resulting from the leaf protein degradation could be stored in the vacuole (Sample and Babst 2019) before being withdrawn to vegetative storage components for use in the spring and summer. We estimated that at the sampling date (DOY 287, October 13), the trees were at the mid-period of leaf senescence as the leaf nitrogen percentage was 1.58% at this date, 2.21% at LAI max (data not shown), and the %N in the soil litter around 1% during November (Nicolas Delpierre, personal communication). Following Du et al. (2021) and Vergutz et al. (2012) we calculated the nitrogen resorption efficiency at the sampling date to be 44%. The maximum resorption efficiency was 64% when calculated with the % of N in the soil litter. A similar value was obtained on *Quercus serrata* by Migita et al. (2007).

¹⁵ N distribution

After the soil labelling at budburst, the ¹⁵ N was analyzed in the different tree compartments at the end of the vegetation season in autumn. We found that 67% of the recovered ¹⁵ N within the tree was found in the aboveground compartment, while 33% was found belowground. Zeller et al. (2001) traced a 3 year ¹⁵ N labelled litter decomposition in *Fagus sylvatica* and found 61.1% of recovered ¹⁵ N in the aerial biomass, with 38.8% in the root biomass.
According to El-Zein et al. (2011a) from April 10 (DOY 100), i.e. before bud burst, to May 7 (DOY 166), when bud burst was completed, leaves attracted most of the applied ¹⁵ N (up to 68% in oak), which was lower than the 14% we found the difference might be due to short sampling-labelling time frame such that if sampling would be repeated after relatively longer period, higher values might be recorded. Difference in age of the trees (45 years in El-Zein et al. (2011a, b) and 20 years in our study) might also be a reason for the variation with our finding because translocation of N, P and K were shown to increase with tree age in relation to enlarged stem wood biomass (Colin-Belgrand et al. 1996). Another explanation could be the high turnover of leaf nitrogen over time to maintain the integrity of some enzymes like Rubisco and thus the probable dilution of the ¹⁵ N mobile pool of leaves with unlabeled N. 12% of ¹⁵ N was present in phloem and 8% in the xylem which may be an indication of concomitant resorption (through phloem) and soil nitrogen uptake (through xylem). This is supported by Amponsah et al. (2004); Jordan et al. (2012) and Bazot et al. (2016) which shows the occurrence of autumnal N uptake in Pine, Peach and Oak trees respectively.

We found ¹⁵ N in all the sapwood rings, with increased concentrations in the newest ring, but not in the heartwood ring, probably due to phloem-xylem transfer via the symplast of horizontal ray parenchyma cells (Pfautsch et al. 2015). Bidirectional nitrogen exchange between phloem and xylem has been experimentally proven in Norway spruce trees (Picea abies [L.] Karst; Geßler et al. 2003). That also could be explained by the fact that the latest ring was built during the year 2016, with the ¹⁵ N coming from the labeled soil through sap flow that was particularly high in the ring of the current year (Granier et al. 1994).

The origin of the ¹⁵ N in other rings is undetermined. They may come from the sap flow via the non-embolised tiny vessels of the sapwood (Čermák et al. 1992), through all the most recent and most ancient rings, or from inter-rings transfer during the growing season or during the resorption process. Additionally, it cannot be excluded that the evaporation of the sap-flow during the freeze-drying process has left a residual ¹⁵ N in the sap tissues. Hart and Classen (2003), studying ¹⁵ N trends in rings 14 years after labeling, found that the ¹⁵ N value of rings was a maximum

one year after the labeling and also that the ¹⁵ N value decreases in the most recent rings. While suggesting nitrogen transfer from older to newer rings, their study reported no ¹⁵ N transfer to older rings. Our result also shows that no nitrogen from N absorption has been transported to the heartwood.

The global ¹⁵ N budget repartition between the tree and soil microbial biomass showed that still 18% of the ¹⁵ N retrieved (6% of the initial labeling) was located in the microbial biomass at the end of the growing season, confirming the role of the microbial biomass as nitrogen sink in the forest (Rennenberg and Dannenmann 2015; Bazot et al. 2016; Maxwell et al. 2020). The small percentage of ¹⁵ N retrieved (33%) indicates that other fluxes (such as leaching, denitrification and incorporation into the understorey vegetation) should be measured to obtain a more exhaustive budget. Volatilization was also possible since the soil was not covered after ¹⁵ N labelling as in El-Zein et al. (2011a) for example.

However, in addition to all the physiological and biochemical processes occurring within the tree, possible isotopic exchange between the N pools would be a possible limitation of this study.

## Conclusion

This study has enriched the literature by providing detailed information on the contribution of oak tree compartments to biomass and nitrogen content. The study confirmed the importance of less commonly studied compartments as nitrogen sinks.

While we found ¹⁵ N in all the sapwood and at variable proportions in the xylem rings, the transport mechanism of the ¹⁵ N cannot be fully explained, i.e. to distinguish the source of the ¹⁵ N: whether from the soil via uptake or resorption from leaves to trunk and/or mobility between rings. Further research is needed to better understand this processes especially because N isotopic fractionation associated with uptake and translocation has been shown to affect ¹⁵ N variations in tree rings (Poulson et al. 1995).

Out of the 1.82 g of ¹⁵ N that was applied to the trees at the beginning of the experiment, only 0.604 g (33% of original quantity applied) was recovered in the trees; there is therefore a need for further research
According to El-Zein et al. (2011a) from April 10 (DOY 100), i.e. before bud burst, to May 7 (DOY 166), when bud burst was completed, leaves attracted most of the applied ¹⁵ N (up to 68% in oak), which was lower than the 14% we found the difference might be due to short sampling-labelling time frame such that if sampling would be repeated after relatively longer period, higher values might be recorded. Difference in age of the trees (45 years in El-Zein et al. (2011a, b) and 20 years in our study) might also be a reason for the variation with our finding because translocation of N, P and K were shown to increase with tree age in relation to enlarged stem wood biomass (Colin-Belgrand et al. 1996). Another explanation could be the high turnover of leaf nitrogen over time to maintain the integrity of some enzymes like Rubisco and thus the probable dilution of the ¹⁵ N mobile pool of leaves with unlabeled N. 12% of ¹⁵ N was present in phloem and 8% in the xylem which may be an indication of concomitant resorption (through phloem) and soil nitrogen uptake (through xylem). This is supported by Amponsah et al. (2004); Jordan et al. (2012) and Bazot et al. (2016) which shows the occurrence of autumnal N uptake in Pine, Peach and Oak trees respectively.

We found ¹⁵ N in all the sapwood rings, with increased concentrations in the newest ring, but not in the heartwood ring, probably due to phloem-xylem transfer via the symplast of horizontal ray parenchyma cells (Pfautsch et al. 2015). Bidirectional nitrogen exchange between phloem and xylem has been experimentally proven in Norway spruce trees (Picea abies [L.] Karst; Geßler et al. 2003). That also could be explained by the fact that the latest ring was built during the year 2016, with the ¹⁵ N coming from the labeled soil through sap flow that was particularly high in the ring of the current year (Granier et al. 1994).

The origin of the ¹⁵ N in other rings is undetermined. They may come from the sap flow via the non-embolised tiny vessels of the sapwood (Čermák et al. 1992), through all the most recent and most ancient rings, or from inter-rings transfer during the growing season or during the resorption process. Additionally, it cannot be excluded that the evaporation of the sap-flow during the freeze-drying process has left a residual ¹⁵ N in the sap tissues. Hart and Classen (2003), studying ¹⁵ N trends in rings 14 years after labeling, found that the ¹⁵ N value of rings was a maximum

one year after the labeling and also that the ¹⁵ N value decreases in the most recent rings. While suggesting nitrogen transfer from older to newer rings, their study reported no ¹⁵ N transfer to older rings. Our result also shows that no nitrogen from N absorption has been transported to the heartwood.

The global ¹⁵ N budget repartition between the tree and soil microbial biomass showed that still 18% of the ¹⁵ N retrieved (6% of the initial labeling) was located in the microbial biomass at the end of the growing season, confirming the role of the microbial biomass as nitrogen sink in the forest (Rennenberg and Dannenmann 2015; Bazot et al. 2016; Maxwell et al. 2020). The small percentage of ¹⁵ N retrieved (33%) indicates that other fluxes (such as leaching, denitrification and incorporation into the understorey vegetation) should be measured to obtain a more exhaustive budget. Volatilization was also possible since the soil was not covered after ¹⁵ N labelling as in El-Zein et al. (2011a) for example.

However, in addition to all the physiological and biochemical processes occurring within the tree, possible isotopic exchange between the N pools would be a possible limitation of this study.

## Conclusion

This study has enriched the literature by providing detailed information on the contribution of oak tree compartments to biomass and nitrogen content. The study confirmed the importance of less commonly studied compartments as nitrogen sinks.

While we found ¹⁵ N in all the sapwood and at variable proportions in the xylem rings, the transport mechanism of the ¹⁵ N cannot be fully explained, i.e. to distinguish the source of the ¹⁵ N: whether from the soil via uptake or resorption from leaves to trunk and/or mobility between rings. Further research is needed to better understand this processes especially because N isotopic fractionation associated with uptake and translocation has been shown to affect ¹⁵ N variations in tree rings (Poulson et al. 1995).

Out of the 1.82 g of ¹⁵ N that was applied to the trees at the beginning of the experiment, only 0.604 g (33% of original quantity applied) was recovered in the trees; there is therefore a need for further research
to identify the lost ¹⁵ N through leaching, denitrification and/or volatilization.

**Acknowledgements** Financial support was provided by Laboratoire Ecologie, Systématique et Evolution and the Institut Diversité Ecologie et Evolution du Vivant through the financing of the TIP-TOP project. The authors would like to acknowledge the contributions of Caroline Mauve, Marlène Sibold-Lamothe, Françoise Gilard, Gregory Mouille for their contribution in the TIP-TOP project, as well as Daniel Berveilier, Patricia Le Thuault, Michèle Viel, Lionel Saunois, Sandrine Fontaine, Alain Sévéré, Liliana Pinek and Baptiste Lafont for data collection, field work, and technical assistance. We are grateful to the French National Forest Office (ONF) for allowing us to carry out these experiments at the Fontaineb-leau-Barbeau forest. This work benefited from the French state aid managed by the ANR under the "Investissements d'avenir" programme with the reference ANR-16-CONV-0003 through the financial support provided by CLAND to Mubarak MAHMUD during the period of writing this paper.

**Declarations**

**Conflict of interest** The authors declare that they have no conflict of interest.

**References**

Aerts R (1996) Nutrient resorption from senescing leaves of perennials: are there general patterns? J Ecol 59:608

Amponsah IG, Lieffers VJ, Comeau PG, Landhäusser SM (2004) Nitrogen-15 uptake by *Pinus contorta* seedlings in relation to phenological stage and season. Scand J For Res 19(4):329–338

Babst BA, Coleman GD (2018) Seasonal nitrogen cycling in temperate trees: transport and regulatory mechanisms are key missing links. Plant Sci 270:268–277

Barbaroux C, Bréda N, Dufrêne E (2003) Distribution of above-ground and below-ground carbohydrate reserves in adult trees of two contrasting broad-leaved species (Quercus petraea and Fagus sylvatica). New Phytol 157(3):605–615. https://doi.org/10.1046/j.1469-8137.2003.00681.x

Bazot S, Barthes L, Blanot D, Fresneau C (2013) Distribution of non-structural nitrogen and carbohydrate compounds in mature oak trees in a temperate forest at four key phenological stages. Trees 27(4):1023–1034

Bazot S, Fresneau C, Damesin C, Barthes L (2016) Contribution of previous year's leaf N and soil N uptake to current year's leaf growth in sessile oak. Biogeoosciences 13(11):3475–3484. https://doi.org/10.5194 bg-13-3475-2016

Brookes PC, Landman A, Pruden G, Jenkinson DS (1985) Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol Biochem 17(6):837–842

Brown S, Gillespie A, Lugo AE (1989) Biomass estimation methods for tropical forests with applications to forest inventory data. For Sci 35:881–902

Čermák J, Cienciala E, Kučera J, Hällgren JE (1992) Radial velocity profiles of water flow in trunks of Norway spruce and oak and the response of spruce to severing. Tree Physiol 10(4):367–380

Chave J, Rie'r'a B, Dubois MA (2001) Estimation of biomass in a neotropical forest of French Guiana: spatial and temporal variability. J Trop Ecol 17:79–96

Chave J, Réjou-Méchain M, Búrquez A, Chidumayo E, Colgan MS, Vieilledent DWB, G, et al (2014) Improved allometric models to estimate the aboveground biomass of tropical trees. Glob Change Biol 20(10):3177–3190

Colin-Belgrand M, Ranger J, Bouchon J (1996) Internal nutrient translocation in chestnut tree stemwood: III. Dynamics across an age series of Castanea sativa (Miller). Ann Bot 78(6):729–740

Cooke JEK, Weih M (2005) Nitrogen storage and seasonal nitrogen cycling in *Populus*: Bridging molecular physiology and ecophysiology. New Phytol 167(1):19–30. https://doi.org/10.1111/j.1469-8137.2005.01451.x

Crow TR (1978) Common regressions to estimate tree biomass in tropical stands. For Sci 24:110–114

Cunia T (1987) Error of forest inventory estimates: its main components. In: Wharton EH, Cunia T (eds) Estimating tree biomass regressions and their error. USDA For Serv Gen Tech Rep NE-117, p 303

Deléens E, Morot-Gaudry J, Martin F, Thoreux A, Gojon A (1997) Méthodologie ¹⁵N. In: Morot-Gaudry J (ed) Assimilation de l'azote chez les plantes Aspects physiologique, biochimique et moléculaire. INRA, Paris, pp 265–280

Delpierre N, Berveiller D, Granda E, Dufrêne E (2016) Wood phenology, not carbon input, controls the interannual variability of wood growth in a temperate oak forest. New Phytol 210(2):459–470

Dickson RE (1989) Carbon and nitrogen allocation in trees. In Annales des sciences forestières (Vol. 46, No. Supplement, pp 631s-647s). EDP Sciences

Drexhage M, Colin F (2001) Estimating root system biomass from breast-height diameters. Forestry 74(5):491–497

Du B, Ji H, Liu S, Kang H, Yin S, Liu C (2021) Nutrient resorption strategies of three oak tree species in response to interannual climate variability. Forest Ecosystems 8(1):1–11

Eastman BA, Adams MB, Brzostek ER, Burnham MB, Carrara JE, Kelly C, McNeil BE, Walter CA, Peterjohn WT (2021) Altered plant carbon partitioning enhanced forest ecosystem carbon storage after 25 years of nitrogen additions. New Phytol 230:1435–1448. https://doi.org/10.1111/nph.17256

El Zein R, Bréda N, Gérant D, Zeller B, Maillard P (2011a) Nitrogen sources for current-year shoot growth in 50-year-old sessile oak trees: An in situ ¹⁵N labeling approach. Tree Physiol 31(12):1390–1400. https://doi.org/10.1093/trephys/tpr118

El Zein R, Maillard P, Bréda N, Marchand J, Montpied P, Gérant D (2011b) Seasonal changes of C and N non-struc- tural compounds in the stem sapwood of adult sessile oak and beech trees. Tree Physiol 31(8):843–854
to identify the lost ¹⁵ N through leaching, denitrification and/or volatilization.

**Acknowledgements** Financial support was provided by Laboratoire Ecologie, Systématique et Evolution and the Institut Diversité Ecologie et Evolution du Vivant through the financing of the TIP-TOP project. The authors would like to acknowledge the contributions of Caroline Mauve, Marlène Sibold-Lamothe, Françoise Gilard, Gregory Mouille for their contribution in the TIP-TOP project, as well as Daniel Berveilier, Patricia Le Thuault, Michèle Viel, Lionel Saunois, Sandrine Fontaine, Alain Sévéré, Liliana Pinek and Baptiste Lafont for data collection, field work, and technical assistance. We are grateful to the French National Forest Office (ONF) for allowing us to carry out these experiments at the Fontaineb-leau-Barbeau forest. This work benefited from the French state aid managed by the ANR under the "Investissements d'avenir" programme with the reference ANR-16-CONV-0003 through the financial support provided by CLAND to Mubarak MAHMUD during the period of writing this paper.

**Declarations**

**Conflict of interest** The authors declare that they have no conflict of interest.

**References**

Aerts R (1996) Nutrient resorption from senescing leaves of perennials: are there general patterns? J Ecol 59:608

Amponsah IG, Lieffers VJ, Comeau PG, Landhäusser SM (2004) Nitrogen-15 uptake by *Pinus contorta* seedlings in relation to phenological stage and season. Scand J For Res 19(4):329–338

Babst BA, Coleman GD (2018) Seasonal nitrogen cycling in temperate trees: transport and regulatory mechanisms are key missing links. Plant Sci 270:268–277

Barbaroux C, Bréda N, Dufrêne E (2003) Distribution of above-ground and below-ground carbohydrate reserves in adult trees of two contrasting broad-leaved species (Quercus petraea and Fagus sylvatica). New Phytol 157(3):605–615. https://doi.org/10.1046/j.1469-8137.2003.00681.x

Bazot S, Barthes L, Blanot D, Fresneau C (2013) Distribution of non-structural nitrogen and carbohydrate compounds in mature oak trees in a temperate forest at four key phenological stages. Trees 27(4):1023–1034

Bazot S, Fresneau C, Damesin C, Barthes L (2016) Contribution of previous year's leaf N and soil N uptake to current year's leaf growth in sessile oak. Biogeoosciences 13(11):3475–3484. https://doi.org/10.5194 bg-13-3475-2016

Brookes PC, Landman A, Pruden G, Jenkinson DS (1985) Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol Biochem 17(6):837–842

Brown S, Gillespie A, Lugo AE (1989) Biomass estimation methods for tropical forests with applications to forest inventory data. For Sci 35:881–902

Čermák J, Cienciala E, Kučera J, Hällgren JE (1992) Radial velocity profiles of water flow in trunks of Norway spruce and oak and the response of spruce to severing. Tree Physiol 10(4):367–380

Chave J, Rie'r'a B, Dubois MA (2001) Estimation of biomass in a neotropical forest of French Guiana: spatial and temporal variability. J Trop Ecol 17:79–96

Chave J, Réjou-Méchain M, Búrquez A, Chidumayo E, Colgan MS, Vieilledent DWB, G, et al (2014) Improved allometric models to estimate the aboveground biomass of tropical trees. Glob Change Biol 20(10):3177–3190

Colin-Belgrand M, Ranger J, Bouchon J (1996) Internal nutrient translocation in chestnut tree stemwood: III. Dynamics across an age series of Castanea sativa (Miller). Ann Bot 78(6):729–740

Cooke JEK, Weih M (2005) Nitrogen storage and seasonal nitrogen cycling in *Populus*: Bridging molecular physiology and ecophysiology. New Phytol 167(1):19–30. https://doi.org/10.1111/j.1469-8137.2005.01451.x

Crow TR (1978) Common regressions to estimate tree biomass in tropical stands. For Sci 24:110–114

Cunia T (1987) Error of forest inventory estimates: its main components. In: Wharton EH, Cunia T (eds) Estimating tree biomass regressions and their error. USDA For Serv Gen Tech Rep NE-117, p 303

Deléens E, Morot-Gaudry J, Martin F, Thoreux A, Gojon A (1997) Méthodologie ¹⁵N. In: Morot-Gaudry J (ed) Assimilation de l'azote chez les plantes Aspects physiologique, biochimique et moléculaire. INRA, Paris, pp 265–280

Delpierre N, Berveiller D, Granda E, Dufrêne E (2016) Wood phenology, not carbon input, controls the interannual variability of wood growth in a temperate oak forest. New Phytol 210(2):459–470

Dickson RE (1989) Carbon and nitrogen allocation in trees. In Annales des sciences forestières (Vol. 46, No. Supplement, pp 631s-647s). EDP Sciences

Drexhage M, Colin F (2001) Estimating root system biomass from breast-height diameters. Forestry 74(5):491–497

Du B, Ji H, Liu S, Kang H, Yin S, Liu C (2021) Nutrient resorption strategies of three oak tree species in response to interannual climate variability. Forest Ecosystems 8(1):1–11

Eastman BA, Adams MB, Brzostek ER, Burnham MB, Carrara JE, Kelly C, McNeil BE, Walter CA, Peterjohn WT (2021) Altered plant carbon partitioning enhanced forest ecosystem carbon storage after 25 years of nitrogen additions. New Phytol 230:1435–1448. https://doi.org/10.1111/nph.17256

El Zein R, Bréda N, Gérant D, Zeller B, Maillard P (2011a) Nitrogen sources for current-year shoot growth in 50-year-old sessile oak trees: An in situ ¹⁵N labeling approach. Tree Physiol 31(12):1390–1400. https://doi.org/10.1093/trephys/tpr118

El Zein R, Maillard P, Bréda N, Marchand J, Montpied P, Gérant D (2011b) Seasonal changes of C and N non-struc- tural compounds in the stem sapwood of adult sessile oak and beech trees. Tree Physiol 31(8):843–854
Elhani S, Lema BF, Zeller B, Bréchet C, Guehl JM, Dupouey JL (2003) Inter-annual mobility of nitrogen between beech rings: a labelling experiment. Ann For Sci 60(6):503–508

Fernández-Martínez M, Vicca S, Janssens IA, Sardans J, Luys-saert S, Campioli M, Peñuelas J (2014) Nutrient availability as the key regulator of global forest carbon balance. Nat Clim Chang 4(6):471–476

Finzi AC, Berthrong ST (2005) The uptake of amino acids by microbes and trees in three cold-temperate forests. Ecology 86(12):3345–3353

Finzi AC, Abramoff RZ, Spiller KS, Brzostek ER, Darby BA, Kramer MA, Phillips RP (2015) Rhizosphere processes are quantitatively important components of terrestrial carbon and nutrient cycles. Glob Change Biol 21(5):2082–2094. https://doi.org/10.1111/gcb.12816

Franklin O, Johansson J, Dewar R, Dieckmann U, Mcmurtrie R, Brännström Å, Dybzinski R (2012) Modeling carbon allocation in trees: A search for principles. Tree Physiol 32:648–666. https://doi.org/10.1093/treephys/tpr138

Geßler A, Weber P, Schneider S, Rennenberg H (2003) Bidirectional exchange of amino compounds between phloem and xylem during long-distance transport in Norway spruce trees (*Picea abies* [L] Karst). J Exp Bot 54(386):1389–1397

Goodale CL, Apps MJ, Birdsey RA, Field CB, Heath LS, Houghton RA, Jenkins JC, Kohlmaier GH, Kurz W, Liu S, Nabuurs GJ, Nilsson S, Shvidenko AZ (2002) Forest carbon sinks in the northern hemisphere. Ecol Appl 12(3):891–899. https://doi.org/10.1890/1051-0761(2002)012[0891:FCSTITN]2.0.CO;2

Granier A, Anfodillo T, Sabatti M, Cochard H, Dreyer E, Tomasi M, Bréda N (1994) Axial and radial water flow in the trunks of oak trees: a quantitative and qualitative analysis. Tree Physiol 14(12):1383–1396

Grassi G, House J, Dentener F, Federici S, den Elzen M, Penman J (2017) The key role of forests in meeting climate targets requires science for credible mitigation. Nat Clim Chang 7(3):220–226. https://doi.org/10.1038/nclimate3227

Härdtle W, Niemeyer T, Fichtner A, Li Y, Ries C, Schuldt A, von Oheimb G (2014) Climate imprints on tree-ring $\delta^{15}$N signatures of sessile oak (*Quercus petraea* Liebl.) on soils with contrasting water availability. Ecol Ind 45:45–50

Hart SC, Classen AT (2003) Potential for assessing long-term dynamics in soil nitrogen availability from variations in $\delta^{15}$N of tree rings. Isot Environ Health Stud 39(1):15–28. https://doi.org/10.1080/1025601031000102206

Helmisaari HS, Makkonen K, Kellomäki S, Valtonen E, Mälkönen E (2002) Below-and above-ground biomass, production and nitrogen use in Scots pine stands in eastern Finland. For Ecol Manage 165(1–3):317–326

Hendrick RL, Pregitzer KS (1993) The dynamics of fine root length, biomass, and nitrogen content in two northern hardwood ecosystems. Can J For Res 23(12):2507–2520. https://doi.org/10.1139/x93-312

Houghton RA, Lawrence KL, Hackler JL, Brown S (2001) The spatial distribution of forest biomass in the Brazilian Amazon: a comparison of estimates. Glob Change Biol 7:731–746

IUSS Working Group WRB-FAO (2015) IUSS Working Group WRB. 2015. World Reference Base for Soil Resources 2014, update 2015 International soil classification system for naming soils and creating legends for soil maps. World Soil Resources Reports N°. 106

Jordan MO, Wendler R, Millard P (2012) Autumnal N storage determines the spring growth, N uptake and N internal cycling of young peach trees. Trees 26(2):393–404

LeBauer DS, Treseder KK (2008) Nitrogen limitation of net primary productivity in terrestrial ecosystems is globally distributed. Ecology 89(2):371–379

Legaz F, Serna MD, Primo-Millo E (1995) Mobilization of the reserve N in citrus. Plant Soil 173(2):205–210. https://doi.org/10.1007/BF00011457

Maxwell TL, Bazot S, Marmagne A, Pinek L, Laffont B, Vincent G, Barthes L (2020) In situ fate of mineral N in the tree-soil-microorganism system before and after budburst in 20-year-old *Quercus petraea* (Matt.) Liebl. Plant and Soil 455(1):425–438. https://doi.org/10.1007/s11104-020-04610-4

McLauchlan KK, Craine JM (2011) Species-specific trajectories of nitrogen isotopes in Indiana hardwood forests, USA. Biogeosci Discuss 8(3).

Meerts P (2002) Mineral nutrient concentrations in sap-wood and heartwood: a literature review. Ann For Sci 59(7):713–722

Migita C, Chiba Y, Tange T (2007) Seasonal and spatial variations in leaf nitrogen content and resorption in a *Quercus serrata* canopy. Tree Physiol 27(1):63–70

Millard P (1995) Internal cycling of nitrogen in trees. Acta Horticulturae 383:3–14. https://doi.org/10.17660/ActaHortic.1995.383.1

Millard P, Grelet G (2010) Nitrogen storage and remobilization by trees: Ecophysiological relevance in a changing world. Tree Physiol 30(9):1083–1095. https://doi.org/10.1093/treephys/tpq042

Návar J (2009) Allometric equations for tree species and carbon stocks for forests of northwestern Mexico. For Ecol Manage 257(2):427–434

Norby RJ, Warren JM, Iversen CM, Medlyn BE, Mc Murtrie RE (2010) $CO_2$ enhancement of forest productivity constrained by limited nitrogen availability. Proc Natl Acad Sci 107(45):19368–19373. https://doi.org/10.1073/pnas.1006463107

Pfautsch S, Renard J, Tjoelker MG, Salih A (2015) Phloem as capacitor: radial transfer of water into xylem of tree stems occurs via symplastic transport in ray parenchyma. Plant Physiol 167(3):963–971

Plavcová L, Jansen S (2015) The role of xylem parenchyma in the storage and utilization of nonstructural carbohydrates. In Functional and ecological xylem anatomy (pp. 209–234). Springer, Cham

Poulson SR, Chamberlain CP, Friedland AJ (1995) Nitrogen isotope variation of tree rings as a potential indicator of environmental change. Chem Geol 125(3–4):307–315

Quartieri M, Millard P, Tagliavini M (2002) Storage and remobilisation of nitrogen by pear (*Pyrus communis* L.) trees as affected by timing of N supply. Eur J Agron 17(2):105–110. https://doi.org/10.1016/S1161-0301(01)00141-1

Rennenberg H, Dannenmann M, Gessler A, Kreuzwieser J, Simon J, Papen H (2009) Nitrogen balance in forest soils:

<figure><img src="image_11.png" /></figure>
Elhani S, Lema BF, Zeller B, Bréchet C, Guehl JM, Dupouey JL (2003) Inter-annual mobility of nitrogen between beech rings: a labelling experiment. Ann For Sci 60(6):503–508

Fernández-Martínez M, Vicca S, Janssens IA, Sardans J, Luys-saert S, Campioli M, Peñuelas J (2014) Nutrient availability as the key regulator of global forest carbon balance. Nat Clim Chang 4(6):471–476

Finzi AC, Berthrong ST (2005) The uptake of amino acids by microbes and trees in three cold-temperate forests. Ecology 86(12):3345–3353

Finzi AC, Abramoff RZ, Spiller KS, Brzostek ER, Darby BA, Kramer MA, Phillips RP (2015) Rhizosphere processes are quantitatively important components of terrestrial carbon and nutrient cycles. Glob Change Biol 21(5):2082–2094. https://doi.org/10.1111/gcb.12816

Franklin O, Johansson J, Dewar R, Dieckmann U, Mcmurtrie R, Brännström Å, Dybzinski R (2012) Modeling carbon allocation in trees: A search for principles. Tree Physiol 32:648–666. https://doi.org/10.1093/treephys/tpr138

Geßler A, Weber P, Schneider S, Rennenberg H (2003) Bidirectional exchange of amino compounds between phloem and xylem during long-distance transport in Norway spruce trees (*Picea abies* [L] Karst). J Exp Bot 54(386):1389–1397

Goodale CL, Apps MJ, Birdsey RA, Field CB, Heath LS, Houghton RA, Jenkins JC, Kohlmaier GH, Kurz W, Liu S, Nabuurs GJ, Nilsson S, Shvidenko AZ (2002) Forest carbon sinks in the northern hemisphere. Ecol Appl 12(3):891–899. https://doi.org/10.1890/1051-0761(2002)012[0891:FCSTITN]2.0.CO;2

Granier A, Anfodillo T, Sabatti M, Cochard H, Dreyer E, Tomasi M, Bréda N (1994) Axial and radial water flow in the trunks of oak trees: a quantitative and qualitative analysis. Tree Physiol 14(12):1383–1396

Grassi G, House J, Dentener F, Federici S, den Elzen M, Penman J (2017) The key role of forests in meeting climate targets requires science for credible mitigation. Nat Clim Chang 7(3):220–226. https://doi.org/10.1038/nclimate3227

Härdtle W, Niemeyer T, Fichtner A, Li Y, Ries C, Schuldt A, von Oheimb G (2014) Climate imprints on tree-ring $\delta^{15}$N signatures of sessile oak (*Quercus petraea* Liebl.) on soils with contrasting water availability. Ecol Ind 45:45–50

Hart SC, Classen AT (2003) Potential for assessing long-term dynamics in soil nitrogen availability from variations in $\delta^{15}$N of tree rings. Isot Environ Health Stud 39(1):15–28. https://doi.org/10.1080/1025601031000102206

Helmisaari HS, Makkonen K, Kellomäki S, Valtonen E, Mälkönen E (2002) Below-and above-ground biomass, production and nitrogen use in Scots pine stands in eastern Finland. For Ecol Manage 165(1–3):317–326

Hendrick RL, Pregitzer KS (1993) The dynamics of fine root length, biomass, and nitrogen content in two northern hardwood ecosystems. Can J For Res 23(12):2507–2520. https://doi.org/10.1139/x93-312

Houghton RA, Lawrence KL, Hackler JL, Brown S (2001) The spatial distribution of forest biomass in the Brazilian Amazon: a comparison of estimates. Glob Change Biol 7:731–746

IUSS Working Group WRB-FAO (2015) IUSS Working Group WRB. 2015. World Reference Base for Soil Resources 2014, update 2015 International soil classification system for naming soils and creating legends for soil maps. World Soil Resources Reports N°. 106

Jordan MO, Wendler R, Millard P (2012) Autumnal N storage determines the spring growth, N uptake and N internal cycling of young peach trees. Trees 26(2):393–404

LeBauer DS, Treseder KK (2008) Nitrogen limitation of net primary productivity in terrestrial ecosystems is globally distributed. Ecology 89(2):371–379

Legaz F, Serna MD, Primo-Millo E (1995) Mobilization of the reserve N in citrus. Plant Soil 173(2):205–210. https://doi.org/10.1007/BF00011457

Maxwell TL, Bazot S, Marmagne A, Pinek L, Laffont B, Vincent G, Barthes L (2020) In situ fate of mineral N in the tree-soil-microorganism system before and after budburst in 20-year-old *Quercus petraea* (Matt.) Liebl. Plant and Soil 455(1):425–438. https://doi.org/10.1007/s11104-020-04610-4

McLauchlan KK, Craine JM (2011) Species-specific trajectories of nitrogen isotopes in Indiana hardwood forests, USA. Biogeosci Discuss 8(3).

Meerts P (2002) Mineral nutrient concentrations in sap-wood and heartwood: a literature review. Ann For Sci 59(7):713–722

Migita C, Chiba Y, Tange T (2007) Seasonal and spatial variations in leaf nitrogen content and resorption in a *Quercus serrata* canopy. Tree Physiol 27(1):63–70

Millard P (1995) Internal cycling of nitrogen in trees. Acta Horticulturae 383:3–14. https://doi.org/10.17660/ActaHortic.1995.383.1

Millard P, Grelet G (2010) Nitrogen storage and remobilization by trees: Ecophysiological relevance in a changing world. Tree Physiol 30(9):1083–1095. https://doi.org/10.1093/treephys/tpq042

Návar J (2009) Allometric equations for tree species and carbon stocks for forests of northwestern Mexico. For Ecol Manage 257(2):427–434

Norby RJ, Warren JM, Iversen CM, Medlyn BE, Mc Murtrie RE (2010) $CO_2$ enhancement of forest productivity constrained by limited nitrogen availability. Proc Natl Acad Sci 107(45):19368–19373. https://doi.org/10.1073/pnas.1006463107

Pfautsch S, Renard J, Tjoelker MG, Salih A (2015) Phloem as capacitor: radial transfer of water into xylem of tree stems occurs via symplastic transport in ray parenchyma. Plant Physiol 167(3):963–971

Plavcová L, Jansen S (2015) The role of xylem parenchyma in the storage and utilization of nonstructural carbohydrates. In Functional and ecological xylem anatomy (pp. 209–234). Springer, Cham

Poulson SR, Chamberlain CP, Friedland AJ (1995) Nitrogen isotope variation of tree rings as a potential indicator of environmental change. Chem Geol 125(3–4):307–315

Quartieri M, Millard P, Tagliavini M (2002) Storage and remobilisation of nitrogen by pear (*Pyrus communis* L.) trees as affected by timing of N supply. Eur J Agron 17(2):105–110. https://doi.org/10.1016/S1161-0301(01)00141-1

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**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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