Faculteit Bio-ingenieurswetenschappen
Academiejaar 2013 – 2014
Internal CO2 gradients and transport in tree roots of
American beech and yellow poplar
Bryan De Bel
Promotor: Prof. dr. ir. Kathy Steppe
Tutor: dr. ir. Jasper Bloemen
Masterproef voorgedragen tot het behalen van de graad van
Master in de bio-ingenieurswetenschappen: Milieutechnologie
Copyright
De auteur en de promotor geven toelating om deze masterproef voor consultatie beschikbaar te
stellen en delen van de masterproef te kopiëren voor persoonlijk gebruik. Voor elk ander gebruik
gelden de beperkingen van het auteursrecht en dient bijgevolg toelating te worden verkregen van de
auteur. Bij het aanhalen van resultaten uit deze masterproef moet de bron verplicht uitdrukkelijk
vermeld worden.
The author and the promoter give permission to use this thesis for consultation and to copy parts of
the work for personal use. Every other use is subject to copyright laws and therefore needs explicit
permission of the author. When using results of this thesis the source must be extensively specified.
Gent, June 2014
The promoter
Prof. dr. ir. Kathy Steppe
The tutor
Dr. ir. Jasper Bloemen
The author
Bryan De Bel
Acknowledgements
As I look back at this eventful year, I realize that I have learned a lot on both a scientific and a
personal level. It is already almost a year ago that I started my first real journey in the scientific
world. The fact that those first steps were in the States, made it even more exciting. Although there
was a lot of work to do and time was limited, I was surrounded by professionals who were always
ready to help. I therefore want to take the time to thank these people and everyone else that helped
me make this thesis to what it is today.
Let’s start at the very beginning. I cannot thank my promoter, Kathy Steppe, enough for giving me
the opportunity to go to the States and be a part of this fundamental scientific research. Kathy, you
are the most enthusiastic person I have ever met. You also have no problem transferring these
positive vibes to other people and making them feel passionate about research. I remember the
meeting we had when we were back from the States. Jonas, Thomas and I were a bit disappointed
with our data but you rallied our spirits with your passion and enthusiasm and made us realize that
our data were actually very good. But let’s not forget my tutor Jasper Bloemen! I am very grateful for
the many questions you answered, the texts you corrected and the tips you gave me. It is
unbelievable that you did this for the three of us while finishing your Ph.D. and working at another
university. It would have been a hard time without your guidance! I also want to thank Roberto for
his invaluable tips on statistical analysis.
Now let’s travel to all the great people in the States! Firstly, I want to express my gratitude to Bob
Teskey and Mary Anne McGuire. You both are very kind, experienced and helpful people. Bob, you
were always calm and positive. Your knowledge and experience were one of the key ingredients for
the success of my -and probably a lot of other- experiments. MAM, what would we have done
without you? I think your technical expertise and handiness are the main reasons why we were able
to get these good data on such a short time. Next, I want to say thanks to Ridwan Bhuiyan and Miles
Ingwers. Ridwan, you were such an awesome personal taxi! Just kidding, it felt like we were friends
from the moment I met you. It was fun driving around Whitehall Forest with you! Also thanks for the
help with my experiments and the nice food! Miles, you helped us out more than a few times and let
us taste a real American BBQ! Lastly, special thanks to Ed, Dominique, Isabel, Alex and Cody as they
helped us get supplies and really made us enjoy our stay in the States.
Finally, I want to thank the people closer to home. To start with, I want to thank all of my friends for
the unforgettable times in Ghent and all the crazy stories we will be able to tell about our student
i
years. These will undoubtedly stay with me forever! Furthermore, I would like to thank my entire
family for their support. In particular my parents, as I could not have asked for more understanding
and supportive mothers and fathers. Thank you for your unconditional support and love and the
opportunities you have given me. I hope I made you proud. I also want to thank my little brother
Killian for his support. I know I haven’t always had a lot of time for you with my busy ‘kot’ life but I
always enjoyed your company! Next, I would like to thank my amazing girlfriend Magali for her
support and cheerfulness. I will never forget the good times we had when road tripping through the
Eastern US. I am very excited to see what the future will bring us! To end with, I want to thank Jonas
and Thomas. It would not have been the same without you guys! Thanks for helping to dig up my
roots and for all the fun times!
Gent, June 2014
Bryan
ii
Samenvatting
Bosecosystemen hebben een belangrijke invloed op de globale koolstofcyclus door fotosynthese en
respiratie. Bijgevolg is het doorgronden van de processen gerelateerd aan ecosysteemrespiratie van
primair belang. Boomwortels spelen hierbij een belangrijke rol. Recent onderzoek suggereert immers
dat een deel van de lokaal gerespireerde CO2 intern getransporteerd kan worden naar andere delen
van de plant via de transpiratiestroom. CO2-effluxwaarden houden echter geen rekening met dit
intern transport maar worden doorgaans toch gebruikt als maat voor lokale respiratie van wortels,
stammen en takken bij bomen. Dit zou betekenen dat bodem efflux metingen wortelrespiratie
onderschatten en bovengrondse efflux metingen lokale respiratie overschatten. Er is tot op heden
echter zeer weinig onderzoek verricht naar de interne CO2 concentratie ([CO2]) in volwassen wortels
en de relatie met de bodem.
Het onderzoek werd uitgevoerd in Whitehall Forest (het experimentele proefbos van de University of
Georgia, Athens, USA) op vier volwassen Amerikaanse beuken (Fagus grandifolia) en vier volwassen
Amerikaanse tulpenbomen (Liriodendron tulipifera). Twee wortels per boom werden uitgerust met
drie soorten sensoren op een afstand van nul, een halve en één meter van stam basis. Er werden
non-dispersive infrared (NDIR) CO2-sensoren in de wortels geïnstalleerd om interne [CO2] te meten.
Daarnaast werden thermal dissipation probes (TDP) geïnstalleerd om de sapstroom (FS) te meten.
Ten slotte werden eveneens thermokoppels geplaatst om de worteltemperatuur na te gaan. Naast
de sensoren in de wortels werd de bodem telkens voorzien van een NDIR CO2-sensor en een
thermokoppel om respectievelijk bodem [CO2] en bodemtemperatuur te bepalen.
De resultaten gaven aan dat de wortels van beide soorten veel hogere [CO2] bevatten dan de bodem.
Bij de beuk werd een gemiddelde interne wortel [CO2] van 8.5% ± 2.0% gemeten terwijl in de bodem
veel lagere waarden geobserveerd werden (gemiddeld 1.0% ± 0.4%). Analoog waren wortel [CO2] van
de tulpenboom (gemiddeld 5.2% ± 1.9%) substantieel hoger dan in de bodem (1.3% ± 1.3%). Dit
toont aan dat de hoge interne [CO2] in bomen afkomstig is van wortelrespiratie en niet van opname
uit de bodem. Daarnaast werd een sterke CO2-gradiënt waargenomen, waarbij de [CO2] omgekeerd
evenredig was tot de afstand van de stam. Dit wijst erop dat gerespireerd CO2 getransporteerd kan
worden vanuit de wortels naar de stam. Er kan besloten worden dat een belangrijk deel van wortelgerespireerd CO2 intern blijft en wordt getransporteerd doorheen de boom met de sapstroom.
Daarenboven heeft de bodem weinig effect op de interne wortel [CO2]. Wortelrespiratie zal dus een
belangrijke rol spelen bij de koolstofhuishouding van bomen.
iii
Summary
Forest ecosystems have an important influence on the global carbon cycle through photosynthesis
and respiration. Understanding the processes of ecosystem respiration (Reco) is therefore of great
importance. Tree roots are expected to play a vital role in these processes. Yet, presently there are
few studies that measure internal CO2 concentration ([CO2]) in mature roots and investigate its
relation with soil [CO2]. Soil and aboveground CO2 efflux measurements are generally being used to
estimate local respiration of roots, stem and branches. Recent studies suggest however that part of
locally respired CO2 can be internally transported with the transpiration stream to other plant parts.
This would mean that soil CO2 efflux (Esoil) measurements underestimate root respiration and
aboveground efflux measurements overestimate local respiration.
This study was conducted in Whitehall Forest (i.e. an experimental forest of the University of
Georgia, Athens, USA) on four mature American beeches (Fagus grandifolia) and four mature
American yellow poplars (Liriodendron tulipifera). Two roots of every tree were equipped with three
sensor types at a distance of zero, a half and one meter to the stem base. The first sensors installed
in the roots were non-dispersive infrared (NDIR) CO2 sensors to measure internal [CO2]. Thermal
dissipation probes (TDP) were then installed to measure sap flow rate (FS). Thermocouples were
subsequently installed to monitor root temperature. Besides the sensors installed in the root, the soil
was also provided with NDIR CO2 sensors and thermocouples to measure [CO2] and soil temperature
respectively.
The results of the internal CO2 measurements indicated that roots of both species have a
substantially higher [CO2] than the soil. For beech the mean root [CO2] was 8.5% ± 2.0%, while much
lower values were observed in the surrounding soil (mean 1.0% ± 0.4%). Similarly, [CO2] were much
higher in the roots of yellow poplar (mean 5.2% ± 1.9%) than in the surrounding soil (1.3% ± 1.3%).
This indicates that high internal [CO2] in trees are derived from root respiration and not from uptake
from the soil. Furthermore, a strong CO2 gradient was observed for which the concentration was
inversely proportional to the distance to the stem. These results suggest that respired CO 2 is being
transported from the roots to the stem.
It can be concluded that a substantial part of the root-respired CO2 stays in the root and is
transported through the tree with the xylem sap. In addition, the surrounding soil will most likely
have little effect on the internal root [CO2]. Root respiration will consequently play an important role
in the carbon cycle of trees.
iv
Contents
Acknowledgements ________________________________________________________________ i
Samenvatting _____________________________________________________________________iii
Summary ________________________________________________________________________iv
List of abbreviations and symbols ___________________________________________________ vii
Introduction and outline of the thesis _________________________________________________ ix
I.
Revealing the significance of roots for tree physiology ________________________________ 1
1.
The importance of forest ecosystem respiration for global carbon budgets ______________ 1
2.
Root anatomy _______________________________________________________________ 3
3.
Sources of internal CO2 in roots _________________________________________________ 5
3.1.
Root respiration _________________________________________________________ 5
3.2.
Factors that affect root respiration __________________________________________ 6
3.2.1.
Biotic factors________________________________________________________ 6
3.2.2.
Abiotic factors ______________________________________________________ 7
3.3.
4.
5.
II.
Dissolved soil derived CO2 _________________________________________________ 9
Fate of internal CO2 in roots ___________________________________________________ 11
4.1.
Transport of internal respired CO2 with the transpiration stream _________________ 11
4.2.
Diffusion of root-respired CO2 into the soil environment ________________________ 13
4.3.
Diffusion of internally transported CO2 out of the stem _________________________ 15
4.4.
Refixation by corticular and woody tissue photosynthesis _______________________ 15
Research objectives _________________________________________________________ 16
Materials and methods ________________________________________________________ 19
1.
Study site _________________________________________________________________ 19
2.
Experimental setup _________________________________________________________ 21
v
3.
III.
Internal root CO2 concentration ____________________________________________ 21
2.2.
Root sap flow __________________________________________________________ 24
2.3.
Soil CO2 concentration ___________________________________________________ 26
Data, statistical and graphical analysis ___________________________________________ 28
Results ___________________________________________________________________ 29
1.
Internal root CO2 concentration ________________________________________________ 29
2.
Root CO2 gradients __________________________________________________________ 30
3.
Root sap flux density and sap flow rate __________________________________________ 33
4.
Diurnal variation in root CO2 concentration and sap flow ____________________________ 35
5.
Influence of vapor pressure deficit on root sap flow rate ____________________________ 38
6.
Influence of soil CO2 concentration on root CO2 concentration _______________________ 39
7.
Influence of PAR and relationship with the stem ___________________________________ 40
IV.
V.
2.1.
Discussion _________________________________________________________________ 43
1.
Internal root CO2 concentration ________________________________________________ 43
2.
Root sap flow rate __________________________________________________________ 45
3.
Transport of root-respired CO2_________________________________________________ 46
4.
Relationship between root CO2 concentration and the soil environment ________________ 48
5.
Directions for future research _________________________________________________ 50
Conclusion ________________________________________________________________ 51
References ______________________________________________________________________ 53
vi
List of abbreviations and symbols
Abbreviation
Meaning
Unit
ANOVA
Analysis of variance
-
CO2
Carbon dioxide
-
DIC
Dissolved inorganic carbon
-
DOY
Day of year
-
Esoil
Soil CO2 efflux
mg C m-2 h-1
Estem
Stem CO2 efflux
µmol CO2 m-2 s-1
FS
Sap flow rate
L h-1
NDIR
Non-dispersive infrared
-
PAR
Photosynthetic active radiation
W m-2
PTFE
Polytetrafluoroethene
-
Ra
Autotrophic component of respiration
mg C m-2 h-1
Reco
Ecosystem respiration
mg C m-2 h-1
Rh
Heterotrophic component of respiration
mg C m-2 h-1
RH
Relative humidity
%
SFD
Sap flux density
cm3 cm-2 h-1
STD
Standard deviation
-
TA
Air temperature
°C
TDP
Thermal dissipation probe
-
VPD
Vapor pressure deficit
kPa
[CO2]
Gaseous CO2 concentration
%
[CO2*]
Dissolved CO2 concentration
mmol L-1
[O2]
Gaseous O2 concentration
%
vii
Introduction and outline of the thesis
The global carbon cycle undoubtedly has an important influence on climate change. As forest
ecosystems play a vital role in this cycle, it is imperative to understand all processes related to these
ecosystems. An important and insufficiently studied part of these ecosystems are mature roots.
Recent studies suggest that CO2 produced by root respiration can be dissolved in the transpiration
stream and internally transported to other plant organs. This could have serious implications for the
use of efflux-based respiration measurements as they rely on the paradigm that all root-respired CO2
immediately diffuses to the surrounding soil. This study will focus on these mature roots by
measuring internal root CO2 concentration ([CO2]) and root sap flow (FS). It will try to confirm if rootrespired CO2 is indeed internally transported and consequently soil CO2 efflux (Esoil) measurements
are underestimating belowground respiration. Lastly, the relationship with the surrounding soil will
be studied to verify if internal root [CO2] are derived from root respiration and/or uptake of the soil.
Chapter I contains the literature study. Section 1 describes the importance of forest ecosystems and
gives an overview of current developments in the research around internal [CO2] and transport in
trees. Section 2 describes the general root anatomy of trees. While looking at tree root anatomy, it is
indicated why roots could contain high internal [CO2] and transport CO2 internally. Section 3 gives the
different sources of internal CO2 in roots and describes the influence of some biotic and abiotic
factors that have an effect on these sources. Previous research is consulted to see if internal CO2
originates in the root itself or is taken up from the surrounding soil. In Section 4, the sinks of internal
root CO2 are identified and the relative importance of internal transport is considered.
In Chapter II, the materials and methods that were used in the experiments, conducted in Whitehall
Forest (Athens, GA, USA), are discussed. Chapter III contains the results of this study. Firstly, the
results of the internal [CO2] and gradients are presented. Thereafter, the sap flux density (SFD) and FS
results are depicted. Lastly, the influence of some weather conditions is presented. The discussion of
these results can be found in Chapter IV. Previous research is used to understand and critically
discuss the obtained results. Some future research and methods will also be suggested. Finally,
Chapter V will concisely summarize the most important findings of this study.
ix
I. Revealing the significance of roots
for tree physiology
1. The importance of forest ecosystem respiration for global
carbon budgets
Forest ecosystems are responsible for the largest part of the primary terrestrial productivity and
consequently have a very important impact on the global carbon budget (Jobbágy & Jackson, 2000;
Geider et al., 2001; Aubrey & Teskey, 2009). The earth’s climate is probably directly impacted by this
global carbon budget. Understanding the baseline processes of these forest ecosystems is therefore
vital to the expansion of our knowledge on this fundamental worldwide problem. Net ecosystem
production is the result of two large ecosystem fluxes: photosynthesis and respiration. Carbon mainly
enters the ecosystem in the form of carbon dioxide (CO2) through photosynthesis while it exits as CO2
through respiration. Respiration is a collective term for a variety of processes by which CO2 is
produced and released to the environment (Trumbore, 2006). There are other processes for carbon
to be released into the atmosphere (e.g. fire, leaching and erosion) but they are out of the scope of
this thesis (Fig. I.1).
Photosynthesis and respiration represent vast carbon fluxes that globally are roughly in balance
(Schimel, 1995; Trumbore, 2006). The balance between these two fluxes is referred to as the net
ecosystem exchange (Schuur & Trumbore, 2006). Ecosystem respiration (Reco) is responsible for
releasing approximately 16-18 times the CO2 annually emitted by use of fossil fuels in the 1990s
(Prentice et al., 2001). It is comprised of an autotrophic (leaves, stems and roots) and heterotrophic
(fungi, bacteria and animals) component. About 77-85% of the annual global gross primary
productivity in forest ecosystems is consumed by Reco (Law et al., 2002; Luyssaert et al., 2007;
Baldocchi, 2008; Aubrey & Teskey, 2009). Variations in Reco are responsible for the regional patterns
in net ecosystem exchange (Valentini et al., 2000). Fig. I.1 illustrates the carbon cycle of a terrestrial
forest ecosystem. After photosynthesis, soil CO2 efflux (Esoil) is the second largest carbon flux in most
ecosystems, accounting for 60-90% of total Reco (Goulden et al., 1996; Longdoz et al., 2000; Kuzyakov,
2006).
1
Fig. I.1. Different respiration pathways after uptake of carbon through photosynthesis (after Trumbore, 2006).
Plant physiologists generally assume that root-respired CO2 immediately diffuses to the outside of
the root and is released into the soil atmosphere (Trumbore, 2006; Aubrey & Teskey, 2009).
However, CO2 formed by respiration does not only contribute to soil Esoil but also to an internal flux.
Locally respired CO2 can dissolve and be internally transported via the transpiration stream to other
parts of the plant. Through the transpiration stream, it is internally relocated and can efflux out of
another plant part or be used for refixation (Stringer & Kimmerer, 1993; Aubrey & Teskey, 2009;
McGuire et al., 2009; Bloemen et al., 2013). This refixation compensates for some of the respiratory
carbon losses (Teskey et al., 2008). Measuring Esoil does not account for internal fluxes and hence it is
most likely that conventional methods for measuring belowground respiration underestimate the
actual rates. It has been estimated that twice the amount of respired CO2 enters the xylem stream as
is diffused into the soil environment (Teskey et al., 2008; Aubrey & Teskey, 2009).
The xylem in trees often contains CO2 concentrations ([CO2]) of 3-10% (up to 20%), while the
atmosphere only holds about 0.04% (Teskey et al., 2008 and references therein). Furthermore, high
[CO2] are found at the base of tree stems, indicating that root respiration contributes to the [CO2] in
the xylem downstream (Teskey & McGuire, 2007; Aubrey & Teskey, 2009). The relative importance
and fate of this internally transported CO2 is still not entirely certain (Aubrey & Teskey, 2009;
Bloemen et al., 2013) and there is still very little information on root [CO2] (Greenway et al., 2006;
Teskey et al., 2008). Identifying the importance of internal CO2 transport, root and stem [CO2] is
therefore crucial to expand our comprehension of the carbon balance of trees and to make global
climate models more accurate (Teskey et al., 2008). In the following sections the contribution of root
respiration to internal [CO2] in trees will be highlighted.
2
2. Root anatomy
Root anatomy, compared with stem anatomy, is typically rather simple because there are no nodes
and internodes present. Young roots are usually composed of an epidermis, cortex and vascular
tissues which results from primary growth. The epidermis absorbs water and minerals from the
surrounding soil. Fine root hairs on the epidermis contribute to the large root surface area and
increase the surface root uptake. These root hairs are found in the maturation zone of a growing root
and are formed in the elongation zone. Mycorrhizae are often utilized by vascular plants to extend
their network and acquire larger volumes of water and nutrients from the soil. Mycorrhizae are a
mutually beneficial symbiosis between the roots of vascular plants and fungi (Raven et al., 2013). The
cortex usually takes up a big part of the root and mostly has a storage function. Besides storing
starch, it allows symplastic and apoplastic transport of substances (Fig. I.2). The cortex is composed
of living parenchyma cells that contribute to the internal root [CO2] by their respiration. At the center
of the root, the cortex forms a densely arranged layer referred to as endodermis. This endodermis
holds Casparian strips that contain suberin and occasionally lignin. Because of this suberin, water and
ions have to utilize the symplastic plasmodesmata or cross the plasma membrane to get across the
endodermis (Raven et al., 2013). The vascular tissues and the non-vascular tissue layers surrounding
it, called the pericycle, make up the vascular cylinder. Both tissues originate from the procambium.
The protoxylem poles and protophloem poles are enclosed by the pericycle (Raven et al., 2013). Fig.
I.3 provides an overview of the general anatomy of a root tip.
Fig. I.2. Schematic view of symplastic and apoplastic transport pathways through the epidermis, cortex and
endodermis (after Raven et al., 2013).
3
As the young root ages, it will usually experience secondary growth that leads to the creation of
secondary vascular tissues from the vascular cambium and a periderm from the cork cambium. In
woody roots, the periderm is formed and replaces the epidermis as a protective layer. The periderm
is made out of three tissue types: cork, cork cambium and phelloderm. Parts of the periderm are
often suberized and could be responsible for the prevention of efflux of internal CO2 to the
surrounding soil environment and therefore contribute to a higher internal root [CO2]. Some regions
of the periderm do allow gas exchange between root and surrounding soil environment through
lenticels. Lenticels are spongy areas with numerous intercellular spaces that permit passage of air
(Raven et al., 2013). Fig. I.3 depicts the different tissues of a root with secondary growth. In the next
section, the sources of internal root CO2 will be discussed.
A
B
Fig. I.3. (A) Anatomical structure of the different root tip zones (adapted from Raven et al., 2013) (B) Cross
section of a root after its first year of growth (after Raven et al., 2013).
4
3. Sources of internal CO2 in roots
3.1.
Root respiration
Internal CO2 in roots mostly originates from the respiration of living cells inside the root. These live
cells are found in the vascular cambium, the parenchyma cells of phloem and xylem rays (Raven et
al., 2013). Respiration in living plant cells contributes to the autotrophic component of Reco (Ra).
Besides Ra, there is also the heterotrophic component of Reco (Rh) which refers to respiration of
heterotrophic organisms. The process of respiration, which uses sugars to provide metabolic cell
energy, is used for different metabolic processes like growth and maintenance and hence, processes
are generally categorized in either growth or maintenance respiration. The simplified equation for
respiration is (Eq. I.1; Stiles & Leach, 1936):
(Eq. I.1.)
Maintenance respiration includes protein turnover, maintenance of ion and metabolite gradients,
and physiological adaption to a changing environment (Penning de Vries, 1975). Growth respiration,
on the other hand, refers to processes generating energy for the synthesis of plant matter (McCree,
1970). Ryan (1990) reported that environmental differences have a much larger effect on
maintenance respiration than on growth respiration.
The living cells, where respiration occurs, are found in the vascular cambium, the parenchyma cells of
phloem and in xylem rays. Xylem is, however, for a significant part comprised of non-living cells. The
xylem and phloem originate in the roots and are extended throughout the plant. The cork cambium,
cork and phelloderm which compose the periderm, form a protective exodermis in tree roots of
woody plants. This exodermis is composed of suberin (Taiz & Zeiger, 2002). Suberin in mature root
regions makes it impermeable for water. Hence, water uptake takes place in the root tips. Although
water uptake is prevented in mature root regions, gas exchange (e.g. O2 and CO2) between the root
and soil is possible through lenticels in the periderm (Kramer & Boyer, 1995; Zwieniecki et al., 2002;
Bloemen, 2013).
Belowground respiration can be separated into Ra and Rh. Without this separation between both
component fluxes, it is difficult to understand and model carbon fluxes in ecosystems (Trumbore,
2006). While Rh is linked to underground carbon accumulation and nutrient dynamics (Ryan & Law,
2005; Kuzyakov, 2006; Wang & Yang, 2007), Ra is related to plant activity (Sulzman et al., 2005;
5
Binkley et al., 2006; Kuzyakov & Gavrichkova, 2010; Agneessens, 2012) and contributes to the
internal [CO2] in roots.
The high [CO2] probably present in tree roots may be due to an oxygen concentrating trait in roots of
wetland species (Aubrey & Teskey, 2009). Physical barriers in roots hinder radial CO2 diffusion. This
might cause a buildup of respired CO2 in the root system, especially in older parts of the roots that
are more suberized (Kramer, 1979). Yet, there are no previous studies that have measured root [CO2]
to confirm this (Greenway et al., 2006). This accumulation could interfere with efflux-based
estimations of root and belowground respiration. Studies often underestimate root respiration due
to this buildup and internal transport of root-respired CO2 as it might be partly released through
efflux out of the stem (Aubrey & Teskey, 2009; Hanson & Gunderson, 2009). Levy et al. (1999)
observed that for Betula pendula and some tropical species there was a positive correlation between
sap flow rate (FS) and stem efflux (Estem). They hypothesized that dissolved internal CO2 was being
imported from the roots via the xylem sap. If root-respired CO2 is indeed being transported to the
stem, it most likely does not only efflux to the atmosphere but is also used in woody tissue
photosynthesis. McGuire et al. (2009) reported that xylem-transported CO2 can indeed be
assimilated in photosynthetic cells in woody tissue. Root respiration could therefore not only
contribute to Esoil but also to other plant processes.
3.2.
Factors that affect root respiration
There are a multitude of ecophysiological/biotic and abiotic factors that alter the root respiration
rate. Next section will discuss the most important factors shortly.
3.2.1.
Biotic factors
Carbohydrate supply
Carbohydrate supply is a key factor. Root respiration is dependent on the supply of carbohydrates to
roots (Millenaar et al., 2000; Poorter et al., 1991). From 8 to 52% of carbohydrates produced by
herbaceous plants may be transported and used by the root system (van der Werf et al., 1988;
Poorter et al., 1991; Lambers et al., 1996; Lipp & Andersen, 2003). The average root system utilizes
about one-quarter to two-thirds of this carbon, that is allocated from the leaves to the roots, for
respiration (Lambers, 1987; Williams & Farrar, 1990; Moyano et al., 2009). The remainder of the
allocated carbon is used for root growth, is stored or is released as root exudates into the
rhizosphere. This means that root respiration is closely dependent on the supply of carbohydrates
originating from carbon assimilation at leaf level (Hanson et al., 2000).
6
Due to the dependence of carbohydrate supply on solar radiation, especially deciduous tree will be
influenced by seasonal variability. When growing, roots will use carbohydrates allocated from the
leaves for respiration and growth. When dormant, root reserves are used for maintenance
respiration (Moyano et al., 2008). However, regardless whether the belowground respired CO2
originated from root reserves or relocated carbohydrates, a portion of this CO2 will be transported
internally with the transpiration stream (Aubrey & Teskey, 2009).
Morphology and age
Roots that grow and age will not always retain the same physiological functions and morphological
traits (Eissenstat et al., 2000; Chen et al., 2010). This results in a large variety of respiration rates
throughout the different roots of the system. Because of physiological functions like water uptake,
fine roots are assumed to have higher respiration rates. This is caused by a more active metabolism
of fine roots (George et al., 2003; Gill & Jackson, 2000). In contrast, larger roots will have a less active
metabolism due to the structural function and transport of water and nutrients. Thanks to this
slower metabolism, they will have a lower respiration rate than the fine roots (Fig. I.4; Pregitzer et
al., 1998).
B
A
Fig. I.4. (A) Root respiration (at 24 °C, quantified as O2 uptake) by diameter and soil depth for Acer sacharum
for two Michigan northern hardwood forests (after Pregitzer et al., 1998). (B) Sugar maple roots from the upper
10 cm of soil. Roots are drawn to scale whereas diameters are proximate (after Pregitzer et al., 1998).
3.2.2.
Abiotic factors
Temperature
Temperature is an important environmental factor that is commonly associated with respiration. As
mentioned before, total respiration can be subdivided into growth and maintenance respiration.
7
Changes in temperature may greatly affect maintenance respiration (Amthor, 1989; Atkin et al.,
2000). However, soil temperature will vary less than air temperature because of the slower warming
up and cooling down of the soil. This reduces the effect on the respiration of roots. Still, soil
temperature variations occur on short- and long-term and induce varying root respiration rates at
daily to seasonal scale. It should also be noted that global warming could have an effect on (root)
respiration in the future as it might change some of these seasonal and diurnal variations (Atkin et
al., 2000).
Soil moisture content
Another important abiotic factor influencing the root respiration rate is soil moisture. Elevated soil
moisture levels will lower oxygen availability which will lead to slower root metabolism and less
respiration (Moyano et al., 2009). Droughts, on the other hand, will reduce root cell turgor. This
slows cell growth and reduces growth respiration rates (Bryla et al., 1997; Burton et al., 1998;
Moyano et al., 2007). Further studies also reported negative effects of drought on root respiration
(Palta & Nobel, 1989; Rochette et al., 1991; Kosola & Eissenstat, 1994; Bouma et al., 1997).
Soil nitrogen content
Tree roots supply the tree with essential nutrients from the soil (Jungk et al., 2002). One of the most
important of these nutrients is nitrogen. Consequently, a change in soil nitrogen concentration will
influence root metabolism and therefore also root respiration. Especially root maintenance
respiration is assumed to be affected by internal nitrogen concentration (Comas & Eissenstat, 2004;
Atkinson et al., 2007; Wang et al., 2010). This is caused by the fact that nitrogen is an essential
element in many enzymes or co-factors (Reich et al., 2008). These enzymes or co-factors are closely
linked with cellular activity (Ryan et al., 1996). When soil nitrogen is more readily available, root
respiration increases (Wang et al., 2010).
Soil CO2 concentration
As described in section 1, internal [CO2] in the roots and stem is considerably higher than the
atmospheric concentration. Soil [CO2] are however also substantially higher than atmospheric
conditions. The soil concentration varies by growth season and even by diurnal variation (Bouma &
Bryla, 2000; Sands et al., 2000). In literature, there are conflicting opinions concerning the sensitivity
of root CO2 efflux and root respiration to varying soil [CO2] (Lipp & Andersen, 2003; Subke et al.,
2006). Some studies report an inhibition or alteration of the total root respiration rate (Burton et al.,
1997; Clinton & Vose, 1999) and the maintenance respiration with elevated soil [CO2] (Qi et al., 1994;
8
McDowell et al., 1999). Similarly, there was an increase in root CO2 efflux with decrease in soil [CO2]
(Ryan et al., 1996). However, other authors observed very little to no sensitivity with citrus and bean
plants (Bouma et al., 1997). In nine different tree species, Burton & Pregitzer (2002) found no
influence of soil CO2 on root respiration. Although the exact mechanisms remain unclear, they seem
to be dependent on the taxon (Subke et al., 2006). When using efflux measurement for the
determination of root respiration, they should be performed at concentrations approximately equal
to the original soil (Hanson et al., 2000; Subke et al., 2006).
Soil O2 concentration
On the other hand, there is more consensus surrounding the effect of soil O2 concentrations ([O2]) on
root respiration rate. When soil O2 is limited (for example for wetlands plants in flooded soils), this
has a negative effect on the plant’s metabolism. This is caused by the slower gas diffusivity in water
than in air (Nobel, 1999; Abiko et al., 2012). Hence, O2 needs to be internally transported from
aboveground tissues to the roots via high porosity tissues and aerenchyma in the corticular root and
stem tissues (Armstrong, 1979; Colmer, 2003). This transportation route may also be used for
transport of internally root-respired CO2 to aboveground tissues for use in photosynthesis (Wetzel &
Grace, 1983). Often, wetland species have strongly suberized walls and/or a thickened exodermis (De
Simone et al., 2003) preventing O2 molecules of diffusing to the soil (Colmer, 2003). In addition to
preventing O2 diffusion, these barriers will also limit the efflux of root-respired CO2 and sediment
derived CO2. This will result in an accumulation of CO2 in the roots (Brix, 1990; Li & Jones, 1995;
Colmer, 2003; Aubrey & Teskey, 2009). At the tip zone, loss of O2 to the soil occurs, due to lower
barrier effects, but this efflux is useful to detoxify reduced components and maintain an oxidized
rhizosphere (Drew, 1997).
3.3.
Dissolved soil derived CO2
As mentioned, one of the main functions of tree roots is to take up soil water. As gaseous CO2 in the
soil is in equilibrium with dissolved CO2, according to the law of Henry, soil water will contain
dissolved inorganic carbon (DIC). Some studies hypothesize that DIC could influence plant growth by
altering the [CO2] gradient from the roots to the soil or by supplying substrate for aboveground plant
fixation after uptake by roots (Enoch & Olesen, 1993; Ford et al., 2007; Bloemen, 2013). [CO2] in the
soil environment in forest ecosystems are typically higher than atmosphere concentrations and vary
between <0.1% and 2% (Amundson & Davidson, 1990; Yavitt et al., 1995; Pumpanen et al., 2003;
Jassal et al., 2005). Several studies suggest that roots are able to take up CO2 from the soil through
soil water (Arteca & Poovaiah, 1982; Amiro & Ewing, 1992; Enoch & Olesen, 1993). Ford et al. (2007)
9
confirmed this with an experiment with 13C labeled DIC but found out that the uptake of DIC only
contributed less than 1% to the total net carbon assimilation of Pinus taeda seedlings. Another study
by Aubrey & Teskey (2009) on Populus deltoides reported that about 8% of the internal root [CO2]
was due to uptake from the surrounding soil. The remaining 92% originated from root respiration.
These findings are in line with another study, undertaken by Teskey & McGuire (2007), that
measured the [CO2] at the base of a Platanus occidentalis tree and of the surrounding soil. They
reported concentrations of 7.6% at the base of the tree and 1.2% in the soil. As concentrations at
stem base were substantially higher than in the surrounding soil, they concluded that only a small
part of the root [CO2] could have hailed from the CO2 dissolved in the soil water.
A more recent study performed by Anné (2014) showed that that dissolved carbon in soil water has
little effect on the internal [CO2] of Pinus taeda and Quercus rubra seedlings at natural occurring soil
concentrations. Yet, when soil concentrations were very high (> 5%), there appeared to be an
influence of the soil on the internal concentration. However, soil concentrations rarely exceed 2%
and almost never reach 4-5% (Amundson & Davidson, 1990). Fig. I.5 depicts the relationship between
soil [CO2] and Estem from the study of Anné (2014). To confirm this relationship for mature tree roots
more research is needed surrounding internal root [CO2] and the relationship with the soil. In the
following section, the fate of root-respired internal CO2 will be discussed.
Fig. I.5. Relationship between soil CO2 concentration ([CO2]) and stem efflux (Estem) for Pinus taeda and Quercus
rubra seedlings (after Anné, 2014).
10
4. Fate of internal CO2 in roots
Once CO2 is respired at root level, there are multiple pathways it can follow. There are at least 3
pathways that have been described in literature. Firstly, internal CO2 can diffuse out of the root and
enter the soil environment and thereby contribute to the CO2 efflux from soils. Secondly, CO2 can
also be transported internally with the transpiration stream to other parts of the plant. It is then
assimilated in tissues containing chlorophyll. Finally, it can diffuse to the atmosphere through stem
or branches after internal transport. Fig. I.6 illustrates the different pathways and sinks for rootrespired CO2.
B
A
Fig. I.6. (A) Different fates of root-respired CO2. Path 1 shows the transport of CO2 with the transpiration
stream. Path 2 indicates the diffusion of root-respired CO2 out of the root into the soil matrix. Soil CO 2 can
efflux to the atmosphere (Esoil) (adapted from Cruiziat & Tyree, 1990). (B) Schematic overview of the different
sinks for root-respired CO2. CO2 can efflux out of the roots into the soil environment, can be transported and
efflux out of the stem into the atmosphere or can be refixed in chlorophyll containing tissues (adapted from
Cruiziat & Tyree, 1990).
4.1.
Transport of internal respired CO2 with the transpiration stream
Boysen-Jensen (1933) & Johansson (1933) already speculated about the relationship between
internal [CO2] and transpiration early 20th century as the greater part of respiring cells and tissues are
either perfused with xylem parenchyma or immediately adjacent to the vascular cambium (Saveyn,
2007). Because of respiration in root cells and a high radial CO2 diffusion resistance of the
surrounding tissues, root [CO2] could potentially be very high, in particular in older root sections
where root tissue often is more suberized (Aubrey & Teskey, 2009). The high internal [CO2] could
11
increase the fraction of respired CO2 that dissolved in the xylem sap. The dissolved CO2 concentration
[CO2*] can be determined using Henry’s law by converting gaseous [CO2] measurements.
A study by Teskey & McGuire (2007) reported that over a 24h period, on average 34% of the CO2
released by respiring cells in the stem remained within the tree and was transported in the
transpiration stream. They also found that stem efflux consisted of 55% CO2 derived from local cells
and 45% CO2 transported internally in the xylem. They determined that a portion of this transported
CO2 must have originated in the root system because of the fact that the [CO2] at the stem base was
high. However they lacked measurements of internal CO2 at root level to confirm the contribution of
root respiration to internal CO2 transport. Still, this indicates that root-respired CO2 can dissolve into
xylem sap and can be transported downstream to stem and branches. While measuring Fs, stem
[CO2], and soil [CO2] simultaneously, Aubrey & Teskey (2009) observed the contributions of root
respiration to soil Esoil. They estimated that about twice the amount of root-respired CO2 was
transported with the transpiration stream than diffused into the soil and contributed to Esoil.
Grossiord et al. (2012) found for Eucalyptus that internally transported root-respired CO2 accounted
for 24% of total root-respired CO2. Yet, it is still generally accepted to use Esoil measurements to
estimate for soil respiration, based on the paradigm that all root-respired CO2 will diffuse through the
soil and efflux to the atmosphere (Aubrey & Teskey, 2009). Recently, Aubrey & Teskey (2009) and
Grossiord et al. (2012) suggested that using Esoil measurements underestimates soil respiration. Both
studies reported that part of the root-respired CO2 does not efflux out of the root but is internally
transported instead. A study by Bloemen et al. (2014) confirmed this by comparing the Esoil and flux
of root-respired CO2 transported in the transpiration stream in girdled and non-girdled oak trees.
Fs influences xylem sap [CO2*]. McGuire & Teskey (2002) observed a negative relationship between Fs
and sap [CO2*] in stems of Liriodendron tulipifera trees. At night, when FS was zero, xylem [CO2*] was
elevated. During morning, xylem [CO2*] decreased as Fs increased. In the afternoon, Fs decreased and
xylem [CO2*] increased again. Even during brief periods of cloudiness, the effect was visible in Fs and
sap [CO2*]. Another study by McGuire et al. (2007) reported, under controlled temperature
conditions, that sap [CO2*] in branches of Platanus occidentalis decreased with increasing FS. This
indicates that high FS can dilute xylem sap [CO2*] which leads to an inverse correlation between Fs
(i.e. transpiration) and diurnal variation in [CO2*] (Bloemen, 2013). However, the largest amount of
xylem-dissolved CO2 will be transported at high FS when expressed in total quantity because higher Fs
will compensate for decrease in xylem sap [CO2*] due to dilution (McGuire et al., 2007; Bloemen,
2013). A similar relationship was found between sap flux (SFD) and xylem sap [CO2*] in Pinus taeda
(Maier & Clinton, 2006). They discovered that removing most of the foliage, and thereby reducing
SFD to near zero, caused the xylem [CO2*] to increase significantly. Saveyn et al. (2008) observed
12
analogous results when xylem [CO2*] was compared between sunny and rainy days for Populus
deltoides. On rainy days, sap flux was lower and xylem [CO2*] higher than on sunny days.
This demonstrates that factors like Fs influence how much CO2 can be transported from roots to stem
and branches (McGuire & Teskey, 2002; Maier & Clinton, 2006). The continuous use of efflux based
measurements and underestimation of carbon needed to sustain belowground tissues in literature
shows that our understanding of belowground respiration and root system processes is not yet
developed enough and needs further research (Aubrey & Teskey, 2009; Hanson & Gunderson, 2009;
Bloemen, 2013). McGuire & Teskey (2004) used a mass balance approach for estimating local stem
respiration in a stem section that takes into account stem efflux, internal CO2 transport and storage
in the stem section. The proposed method takes internal fluxes into account, unlike efflux
measurements. Perhaps such a method could be developed and utilized for belowground respiration.
We also currently lack detailed measurement of internal [CO2] at root level. Studying internal root
[CO2] could potentially shed light on different hypotheses (i.e. Esoil measurements underestimate
belowground respiration, root-respired CO2 contributes to Estem, surrounding soil has little effect on
internal root [CO2], etc.).
4.2.
Diffusion of root-respired CO2 into the soil environment
Plants are the most important autotrophic organisms contributing to Esoil by root respiration. Other
organisms like algae and chemolithotrophs are of minor importance (Kuzyakov, 2006). Yet,
belowground autotrophic and heterotrophic contributions to Esoil are difficult to separate (Hanson et
al., 2000; Subke et al., 2006). Hanson et al. (2000) reported a range of 10-90% for the contribution of
Ra to Esoil. Recent studies have estimated that about twice the amount of CO2 is dissolved and
transported with the xylem sap than is diffused to the soil environment (Aubrey & Teskey, 2009).
This could be caused by the high [CO2] environment, which hinders radial CO2 diffusion from the
roots to the soil environment (Qi et al., 1994; Saveyn, 2007). Another reason could be the physical
barriers that limit CO2 diffusion, as described in section 4.1. They induce a CO2 buildup in the roots
that facilitates the dissolving in xylem sap (Saveyn, 2007). This high internal [CO2] has been observed
in earlier studies (Clements, 1921; Rakonczay et al., 1997).
13
Fig. I.7. Histogram of the percent root contribution to total soil CO2 efflux (Esoil) for forests (adapted from
Hanson et al., 2000).
Disentangling the autotrophic and heterotrophic components of belowground respiration is
challenging as mentioned before. Despite this fact, it is essential for the comprehension and
modeling of carbon fluxes in ecosystems (Trumbore, 2006). In literature, there are a variety of
techniques used for the partitioning of Esoil. Each with their own advantages and disadvantages
(Hanson et al., 2000; Högberg et al., 2001; Lee et al., 2003; Ryan & Law, 2005; Subke et al., 2006;
Kuzyakov, 2006; Saveyn, 2007). These techniques can be subdivided into 4 types: (1) root exclusion,
(2) physical separation of soil respiration components, (3) isotope techniques and (4) indirect
methods (Subke et al., 2006).
Firstly, there is root exclusion, which can be achieved through trenching, girdling, clipping and gap
analysis. Root exclusion methods are techniques that measure soil respiration both with and without
the presence of roots. Besides root exclusion, physical separation of soil respiration components can
be utilized to partition Esoil into Ra and Rh. Physical separation can be obtained by component
analysis, root excising and live root respiration measurements. Isotope techniques can be subdivided
into isotopic labelling and radiocarbon analysis. Indirect techniques are the fourth kind. They include
modelling, mass-balance, subtraction and root mass regression (Subke et al., 2006). These
techniques will not be discussed in this thesis but it becomes clear, there are many techniques
available to separate Ra from total soil respiration but most of them do not account for internal
transport of root-respired CO2 to the stem. Almost all of these methods rely on the paradigm that all
root-respired CO2 immediately diffuses to the soil (Aubrey & Teskey, 2009). As this has not been
verified and recent study has indicated that root-respired CO2 can be transported to the stem, these
methods could be underestimating belowground respiration.
14
4.3.
Diffusion of internally transported CO2 out of the stem
Levy et al. (1999) hypothesized that part of Estem originated from belowground respiration. As
mentioned before, Aubrey & Teskey (2009) estimated that twice the amount of root-respired CO2
enters the xylem, and is transported up to stem and branches, than diffuses into the soil
environment. Moreover, Teskey & McGuire (2007) reported that about 45% of the Estem had been
transported through the xylem stream. This means that a considerable part of root-respired CO2
might be released by stems (Aubrey & Teskey, 2009; Hanson & Gunderson, 2009). More research is
needed to confirm this as little is known about internal root [CO2] (Greenway et al., 2006) and
internal transport of root-respired CO2 to the stem. If indeed part of Estem is composed of rootrespired CO2, then this would verify that Estem does not accurately estimate local stem respiration. It
would imply that belowground respiration has continuously been underestimated by Esoil
measurements and aboveground respiration has been continuously overestimated by Estem.
4.4.
Refixation by corticular and woody tissue photosynthesis
Photosynthesis has been measured in petioles, green fruits, stem tissues and even roots. Several
studies pose that there is no doubt that photosynthesis of green tissues other than leaf mesophyll
will positively contribute to the overall carbon budget of plants (Weiss et al., 1988; Blanke & Lenz,
1989; Hetherington, 1997; Pfanz et al., 2002). Most woody species’ stems, branches and twigs still
have green-ish bark sub-layers where woody-tissue photosynthesis can occur (Fig. I.8; Pfanz et al.,
2002). There is also chlorophyll present in ray cells of the wood (Rentzou & Psaras, 2008) and in the
pith (van Cleve et al., 1993).
Fig. I.8. Peeled peridermal layer of a Sambucus nigra tree to show bark chlorenchyma (after Pfanz et al., 2002).
15
Several studies found that artificially or naturally illuminating the stem caused a drop in E stem,
indicating that internal CO2 was being used for photosynthetic assimilation by chlorophyll containing
tissues in the stem (Foote & Schaedle, 1976a, 1976b; Kharouk et al., 1995; Pfanz, 1999; Pfanz et al.,
2002). This could be attributed to the fact that refixating respired CO2 recovers energy that otherwise
would have been lost to the atmosphere (Cernusak & Hutley, 2011). Another cause for internal
recycling of carbon might be to reduce carbon loss during drought or winter periods (Pfanz & Aschan,
2001). Cernusak & Marshall (2000) reported two major advantages of refixation over leaf
photosynthesis. Firstly, there is little to no water loss as no stomata are involved, which greatly
increases the plant’s resilience to drought. Secondly, as a consequence of high internal [CO2], there
will be less photorespiration. An additional advantage of corticular and woody tissue photosynthesis
could be the production of O2 that avoids the development of anoxic conditions in these organs
(Pfanz et al., 2002).
According to some studies, a large part of internal CO2 is refixed. Aschan et al. (2001) reported that
the chlorophyll in Populus tremula twigs refixed about 80% of its internal CO2. Owing to the fact that
refixation uses internal CO2, it should be noted that it is very likely that the process also utilizes
internally transported root-respired CO2. This is supported by a recent study of McGuire et al. (2009),
who reported that CO2 transported by the xylem stream can be assimilated by chlorophyll containing
cells in both woody tissues and leaves. They used 13C-enriched water that was taken up by branches
of Platanus occidentalis and observed an average assimilation of 35% of the label. This indicates that
root-respired CO2 could easily be transported to woody tissues in the stem or even to the leaves for
refixation. Yet, no studies on the internal root [CO2] and internal transport of root-respired CO2 have
been conducted to confirm this.
5. Research objectives
In this thesis, we hypothesized that root-respired CO2 does not only efflux to the surrounding soil
environment but is also internally transported to the stem. One of the main objectives of this study
was therefore to verify if root-respired CO2 contributed to a high internal [CO2] and did not only
efflux to the soil environment. This would prove that efflux-based measurements are not an accurate
estimation of belowground respiration. To achieve this, internal [CO2] in mature tree roots of
American beech (Fagus grandifolia) and yellow poplar (Liriodendron tulipifera) were measured.
Another objective was to investigate if this root-respired CO2 was being transported internally to the
stem. By installing three CO2 sensors along the root, gradients could be measured. These measured
gradients would then be an indication of the size and direction of the transport.
16
An additional hypothesis of this thesis was that these high internal root [CO2] are almost entirely
derived from root respiration and the contribution soil DIC uptake is very small. To confirm this, soil
[CO2] was measured simultaneously with internal root [CO2]. This way, we could see if there was a
significant difference between the root and the soil [CO2] and if changes in soil [CO2] had an effect on
the root [CO2].
17
II. Materials and methods
1. Study site
Field experiments were conducted at Whitehall Forest from July 15th until September 15th 2013 (day
of year (DOY) 196-259). This experimental forest is property of the University of Georgia (Athens, GA,
USA). Whitehall forest covers approximately 340 ha and consists of natural, planted, and hardwood
pine, upland hardwood and bottomland hardwood species (Warnell School of Forestry and Natural
Resources, 2013). The forest is located at N 33° 53' 5.4234" W 83° 21' 27.5754". Athens lies in a
humid sub-tropical zone and has a temperate four-season climate with mild winters and hot, humid
summers. It has a relatively high average annual rainfall of about 1,220 mm (48.0 inches; compared
to global land average of about 715 mm) and high average temperature of 16.6 °C (61.9 °F; global
14.7 °C) (University of Georgia, 2012).
Micro-climatological measurements were collected by an on-site weather station. Data of the air
temperature (TA, °C), relative humidity (RH, %), photosynthetic active radiation (PAR, W m-2) and
precipitation (mm) were taken every 15 minutes. The vapor pressure deficit (VPD, kPa) was then
calculated using TA and RH collected from the on-site weather station with following equation (Eq.
II.1):
(
)(
)
(Eq. II.1)
with a, b and c empirical derived constants being 613.75, 17.50 and 240.97 respectively (Jones,
1992), TA the air temperature (°C) and RH the relative humidity (%).
American beech (Fagus grandifolia) and yellow poplar (Liriodendron tulipifera) were used for this
thesis. American beech is the sole species of its genus in North America. Although the species was
widespread throughout the United States before the glacial period, it is now only found in the east of
the country. It is a slow growing, deciduous tree that attains ages of 300 to 400 years old. Beech is
primarily found in regions with annual precipitation from 760 mm to 1270 mm and mean annual
temperatures from 4° to 21° Celsius. They are a mesophytic species. This means beeches are plants
which are adapted to neither predominantly warm nor predominantly wet environments. They still
require a lot more water for transpiration and growth processes than some drought resistant oaks
and pines. Fagus grandifolia has a thin, smooth bark that probably has a lower resistance to CO2
19
efflux than some other species with ticker bark tissues. Beech is a shade tolerant species. It has a
very low transpiration rate and a quick stomatal reaction to light. The stomata open very quickly
when light intensity increases and close very rapidly when light intensity decreases. Beeches are
more tolerant of shade than yellow poplars (Loach, 1967; Woods & Turner, 1971; Walters & Yawney,
2004).
Yellow poplar is a fast growing, tall hardwood tree with a high commercial value. It is widely found in
the Eastern United States and its range stretches from southern New England to Louisiana (Walters &
Yawney, 2004). Yellow poplar flourishes under a variety of climatic conditions. It can grow in regions
with 760 mm to 2030 mm rainfall and with a very wide mean temperature range. A moderately
moist, well drained and loose textured soil is the spot where yellow poplar grows best (Walters &
Yawney, 2004). When aging, stems of yellow poplars become deeply furrowed with thick cracked
bark (Gilman & Watson, 1993). These cracks most likely decrease their resistance to CO2 efflux. It is
probable that the bark of yellow poplar has a lower CO2 resistance than the bark of American beech
because of its cracks. A recent study confirmed this by reporting that CO2 diffusion resistance was 9%
higher for Fagus grandifolia than for Liriodendron tulipifera (Wittocx, 2014).
Of each species, 4 adult trees were used. Of which two roots were selected which were relatively
shallow, to limit the impact of root excavation on tree physiology. Due to equipmental limitations,
one tree at a time was measured. It should be noted that a second experiment was simultaneously
performed on the stem of the trees at a height of 1.25 meter. However, the measurements of this
experiment were expected to have no impact on the measurements performed during this
experiment.
For the measurements on the different trees, the following procedure was used: the first day, roots
were uncovered, sensor holes were drilled, and sensors were installed, after which measurements
started around 18:00 on the first day. The second day was entirely reserved for measurements.
Usually, during the second day increment cores were taken of the previous tree and xylem sap pH
was measured. The last day at around 18:00 the installed sensors were retrieved, holes were closed
with corks, and the next tree was equipped with sensors.
20
2. Experimental setup
2.1.
Internal root CO2 concentration
When a tree was selected, two of its roots were uncovered carefully by shovel, by hand shovel and
by cleaning brush. This was done cautiously as damaging the roots could have had an influence on its
internal CO2 concentration ([CO2]). Larger roots were selected, to minimize the impact of drilling
holes on the internal water transport and CO2 build-up. Roots were subdivided into 3 zones: zone 1
at stem base, zone 2 at 0.5 m from stem base and zone 3 at 1 m from stem base. Due to these three
zones, gradients could be potentially measured. Fig. II.1 is an example of a root used for the
experiment. A hole of 20 mm wide and 50 mm deep was drilled in each zone to install a nondispersive infrared (NDIR) CO2 sensor (Fig. II.2; model GMM220; Vaisala Inc., Woburn, MA, USA).
Fig. II.1. Schematic overview of the sensors used in the root and the surrounding soil. Non-dispersive infrared
(NDIR) CO2 sensors, thermal dissipation probes (TDP) and thermocouples were used.
Solid state NDIR CO2 sensors were used to measure the internal [CO2] in roots. The sensors were
equipped with polytetrafluoroethene (PTFE) membranes (Model 200-07-S-4; International Polymer
Engineering, Tempe, AZ, USA) to repel water (or tree sap), as it would have influenced the results.
CO2 and other gases were able to diffuse through this membrane. It was attached to the sensor with
Plasti-Dip (Plasti-Dip International; Blaine, MN, USA). PVC tubing was also used to protect the sensor
21
from dirt and to make it possible to air tighten it when placed in the tree. It was attached to the
sensor using glue (Model Surebonder 725R510; FPC Corporation, Wauconda, IL, USA) with a glue gun
(Model Surebonder PR02-100; FPC Corporation, Wauconda, IL, USA). Once the sensor was inserted in
a hole, it was air-tightened with silicon sealant (Model GE5000; GE, Huntersville, MN, USA). This
prevented CO2 from leaking to the atmosphere and thereby biasing the measured internal [CO2]. The
NDIR CO2 sensors measured the [CO2] of the gas in the headspace of the holes. This gas is in
equilibrium with the xylem sap (Levy et al., 1999). NDIR sensors are known for their long-term
stability and accuracy but are sensitive to moisture. They are widely used for real-time
measurements of CO2. As they use a physical sensing principle such as gas absorption at a particular
wavelength (426 nm), the sensors have a high selectivity and sensitivity. CO2 has a strong absorbance
at that wavelength with negligible interference.
Fig. II.2. Picture of the non-dispersive infrared (NDIR) CO2 sensors used in the experiments. The
polytetrafluoroethene (PTFE) membrane and Plasti-Dip used are also visible. The plastic tubing is not depicted.
At about 10 cm of the NDIR CO2 sensor, a copper-constantan thermocouple (Type T, Model PP-T-24SLE, Omega Engineering, Stamford, CT, USA) was also installed in each zone of the root. Tissue
temperature measurements were needed to be able to calculate the dissolved CO2 concentration
([CO2*]). Thermocouples were also sealed and held into place with silicon sealing.
After installation, sensors were covered with double reflective insulation (Reflectix; Reflectix,
Markleville, IN, USA) that reflected solar irradiance. This was applied to avoid influence of
temperature changes by diurnal variations in irradiance. A tarp was also used to protect the
experimental setup from rainfall. Measurements were taken every 5 minutes for a 24 hour period
22
and stored on a datalogger (model CR23X; Campbell Scientific, Logan, UT, USA). The data were then
transferred to a PC later. After the measuring period, all sensors were carefully removed from the
tree. The holes were then closed with corks. An increment core (Haglöf Sweden AB, Langsele,
Sweden) was also taken from every zone of each root for xylem sap pH measurements and sapwood
area estimations. The sap was collected from the cores using a vise and pipette. The pH of this sap
was then measured using a solid state pH microsensor connected to a pH-meter (Fig. II.3; Model RedLine standard sensor and Argus meter; Sentron Europe BV, Roden, The Netherlands).
Fig. II.3. Image of the core sap extraction through vise and pipette. After the extraction, the sap pH was
measured using a solid state pH microsensor connected to a pH-meter.
Calculations
Xylem sap and soil [CO2*] can be calculated from the gaseous [CO2] with Henry’s law when
temperature, pH and the sap or soil [CO2] in the gaseous phase are known. Using the equation:
[
]
[
]
[
]
[
]
(Eq. II.2)
where [CO2*] is total dissolved carbon (mmol l-1) and [CO2]aq, [HCO3-] and [CO32-] are dissolved forms
of CO2 and:
[
]
(Eq. II.3)
23
[
[
]
(Eq. II.4)
]
(
)
(Eq. II.5)
where pCO2 is the gaseous [CO2] and KH, K1 and K2 are solubility constants. The constants are
temperature dependent and can be calculates for temperature between 0 and 50 °C with following
equations:
(Eq. II.6)
(
)
(
(
)(
)
(
)
)
(Eq. II.7)
(Eq. II.8)
where T is temperature in °C (McGuire & Teskey, 2002).
Xylem sap [CO2*] will be affected by pH as becomes clear from above equations (Eq. II.3; Eq. II.4; Eq.
II.5). Below pH 8, the contribution of [CO32-] is negligible and can be left out of the calculation. Below
pH 5, [HCO3-] can also be left out (McGuire & Teskey, 2002). Temperature also has an effect on xylem
[CO2*] (Eq. II.6; Eq. II.7; Eq. II.8).
2.2.
Root sap flow
Additional holes were drilled in each zone for the installation of thermal dissipation probes (TDP)
(model TDP-50; DynaMax Inc., Houston, TX, USA). These probes measured sap flux density (SFD) of
the sap in the xylem at a depth of 25 mm. The TDP was installed 10 cm down the root of the other
two sensors. TDP sensors were insulated with rubber self-seal pipe wrap insulation (Model Armaflex
HST05812; Armacell LLC, Mebane, NC, USA). Data from the TDP were also sent to the datalogger. A
picture of the experimental set-up (without soil sensors, tarp and reflective foil) is given by Fig. II.4.
Fig. II.1 gives a schematic overview of the installation of the different sensors.
24
B
C
A
Fig. II.4. A picture of a yellow poplar root fitted with (A) thermal dissipation probes (TDPs), (B) thermocouples
and (C) non-dispersive infrared (NDIR) CO2 sensors. All data is sent to and logged by the datalogger, visible in
the upper left of the photo.
TDP sensors are frequently used because of their simplicity, high degree of accuracy, reliability and
relatively low cost. Fig. II.5 gives a schematic view of a TDP set-up. The temperature difference
between the heated probe and reference probe (i.e. wood and sap) is related to SFD.
Fig. II.5. Schematic overview of the thermal dissipation probe (TDP) (or Granier) method (after Lu et al., 2004).
25
The diameter of every zone of each root was measured or estimated. This information was combined
with estimates of sapwood diameter from the samples taken with the increment core to calculate
total sapwood area for each of the zones. Sapwood area was then used for the calculation of FS from
SFD. Bark thickness was also measured from the increment core samples.
Calculations
Granier (1985, 1987) found a relation between SFD and the ΔT measured with TDP sensors, given by:
(Eq. II.9)
with 0.0119 and 1.231 empirically determined coefficients and the dimensionless flow index K given
by:
(Eq. II.10)
where ΔT is the temperature difference between the heated needle and the reference needle and
ΔTm is the maximum temperature difference between the two needles (i.e. ΔT at zero flow
conditions) (Steppe et al., 2010).
FS (L h-1) can then be calculated using the estimated sapwood areas (obtained the through diameter
measurements and increment cores) with following equation:
(Eq. II.11)
with As the sapwood area (cm2) of the root, 10-3 a conversion factor (cm3 to L).
2.3.
Soil CO2 concentration
To be able to compare internal [CO2] with surrounding soil [CO2], NDIR CO2 sensors were installed in
a hole in the top soil adjacent to the root. One soil NDIR CO2 sensor was installed per root (i.e. per
three zones). The sensors were placed at a depth of 30 cm at an approximate distance of 20 cm of
root zone 3 (i.e. 1 meter from stem base). The NDIR sensor was installed in plastic tubing with holes
drilled at the bottom to protect the sensor without limiting the diffusion of CO 2 to the sensor head
(Fig. II.6). Thermocouples were also installed in the soil to monitor temperature of the surrounding
soil and to be able to calculate soil [CO2*]. Soil samples were taken, using a soil sampler (Model G;
Oakfield Apparatus, Fond du Lac, WI, USA), to allow for soil pH measurements. Soil samples were
kept in paper bags and were dried for several days in a gravity flow convection oven (Model Isotemp
26
637G; Fisher Scientific, Waltham, MA, USA). Each sample was then homogenized and 10 g of soil was
weighed three times with an analytical scale (Model A200S; Sartorius GmbH, Göttingen, Germany).
These three subsamples were each dissolved in 20 mL of deionized water. After 30 minutes, the pH
of the samples was measured using a solid state pH microsensor connected to a pH-meter (Red-Line
standard sensor, Argus meter; Sentron, Roden, The Netherlands). A schematic overview of the whole
set-up is given by Fig. II.7.
A
B
Fig. II.6. (A) Picture of the thermal dissipation probe (TDP), the non-dispersive infrared (NDIR) CO2 sensors and
the soil NDIR CO2 sensors. (B) Picture of a measurement with a soil NDIR CO2 sensor.
Fig. II.7. Schematic overview of the experimental set-up. As shown, there are 2 roots per tree and three
measuring zones per root. The first zone is located at the stem base. The second zone is 0.5 meter downroot
from the first zone and the third zone is 1 meter from the first. The soil temperature and CO 2 concentration
([CO2]) were also measured.
27
3. Data, statistical and graphical analysis
Data were processed using Excel 2010 (Microsoft Inc., Redmond, WA, USA). Measurement data were
confined to second day data collected for 24 hour. Graphical analysis was performed using Sigmaplot
11.0 (Systat Software Inc., San Jose, CA, USA). RStudio Version 0.98.507 (RStudio Inc., Boston, MA,
USA) was utilized for the statistical data analysis. RStudio used R version 3.1.0 (R foundation) with α =
0.05. For the statistical analysis, days were split in 6 periods of 4 hours. The mean values of those 4
hour period measurements were used.
A one-way analysis of variance (ANOVA) was used to test the significance of the difference between
the two species. This was achieved using the linear model function (lm) of R. The significance in
difference of FS between both species was tested with a similar model and ANOVA. A linear mixed
effect model for a one-way ANOVA was used to test the significance of the influence of the distance
(i.e. different root zones) to the stem base on root [CO2]. Distance was taken as the treatment on
[CO2]. ‘Tree’ was taken as a random subject factor and ‘root’ was nested in ‘tree’. By doing this, the
influence of the different roots (n = 2) of each tree and the different trees (n = 4) of each species (n =
2) was taken into account. To test this for each species, subsets were used. A similar ANOVA was
used to test the influence of FS on the root [CO2]. For this test, ‘distance’ to the stem was nested in
‘root’ as distance to the stem had a significant effect on root [CO2]. The significance of the influence
of soil [CO2] on root [CO2] was also tested by a similar linear mixed effect model and a one-way
ANOVA. Lastly, to analyze the difference between soil [CO2] and root [CO2], another linear mixed
effect model was used with a one-way ANOVA. ‘Tree’ was also taken as a random subject factor with
‘root’ nested in ‘tree’ and ‘distance’ nested in ‘root’.
28
III. Results
1. Internal root CO2 concentration
Both species had a significantly higher internal root CO2 concentration ([CO2]) relative to the soil
[CO2] (Table III.1; [CO2]~Soil; beech: p < 0.0001; yellow poplar: p <0.0001). The mean internal root
[CO2] for beech was 8.5% ± 2.0%. Yellow poplar had a mean internal root [CO2] of 5.2% ± 1.9% (Table
III.1). The difference in mean internal root [CO2] between American beech and yellow poplar was also
significant (Fig. III.1; [CO2]~Species; p < 0.0001). Mean internal root [CO2] was higher in beech than in
yellow poplar. The highest concentrations measured in American beech and yellow poplar roots were
13.4% and 8.8%, respectively. American beech had a more profound diurnal variation in internal
[CO2] than yellow poplar with a decrease in internal [CO2] observed during daytime, when high sap
flux density (SFD) occurred (Fig. III.2; Fig. III.3).
Fig. III.1. Comparison of the internal root CO2 concentration ([CO2]) of American beech and yellow poplar
during a 24 hour measuring period averaged for 4 trees per species and 2 roots per tree. Standard deviations
(STDs) are given by the dashed lines.
29
2. Root CO2 gradients
For both American beech and yellow poplar there was a clear [CO2] gradient visible along the root
(Fig. III.2; Fig. III.3). The internal root [CO2] was highest at the base of the stem and decreased the
further from the stem base (Fig. III.4). For beech the mean concentrations at zone 1, zone 2 and zone
3 were 9.6% ± 1.5%, 8.9% ± 1.6% and 7.2% ± 2.0% respectively. For yellow poplar these internal root
[CO2] were 5.6% ± 1.7%, 5.1% ± 1.8% and 4.7% ± 2.0%. American beech showed a more typical
diurnal pattern in internal root [CO2] than yellow poplar, with increase of internal CO2 during daytime
and decrease during nighttime. The mean soil [CO2] concentrations were 1.0% ± 0.4% for beech and
1.3% ± 1.3% for yellow poplar (Table III.1). There was a significant difference between soil and root
[CO2] both for beech (p < 0.0001) and yellow poplar (p < 0.0001). The large standard deviation (STD)
for the soil mean was due to a very high mean soil [CO2] of 4.6% ± 0.1% measured near one of the
two roots of yellow poplar 4.
As visible in Fig. III.2 and Fig. III.3, each of the root zones followed a similar pattern. Yet, the soil did
not follow this pattern as it remained relatively stable. For beech there was an inversion of zone 1
and zone 2 at 18:00. This was caused by a sudden increase in [CO2] of zone 2 for beech 2.
Table III.1. Mean internal root CO2 concentration ([CO2]), mean root zone [CO2] and soil [CO2] of each species
with standard deviation (STD).
Species
American beech
Yellow poplar
Internal root
[CO2] ± STD (%)
8.5 ± 2.0
5.2 ± 1.9
Root zone
Root [CO2] ± STD
Soil [CO2] ± STD
(%)
(%)
1
9.6 ± 1.5
2
8.9 ± 1.6
3
7.2 ± 2.0
1
5.6 ± 1.7
2
5.1 ± 1.8
3
4.7 ± 2.0
30
1.0 ± 0.4
1.3 ± 1.3
Fig. III.2. Mean root CO2 concentration ([CO2]) per zone and soil [CO2] of American beech. Soil [CO2] was
measured in the soil near root zone 3 during the 24 hour measuring period. Root zone 1 is at stem base, zone 2
is at 0.5 m from stem base and zone 3 is at 1 m from stem base.
Fig. III.3. Mean root CO2 concentration ([CO2]) per zone and soil [CO2] of yellow poplar. Soil [CO2] was
measured in the soil near root zone 3 during the 24 hour measuring period. Root zone 1 is at stem base, zone 2
is at 0.5 m from stem base and zone 3 is at 1 m from stem base.
31
Fig. III.4 shows that there is an internal gradient in roots for both species. Closer to the stem base,
there was a significantly higher internal [CO2] than further down the root ([CO2]~Distance; beech: p <
0.0001; yellow poplar: p < 0.0001). This indicates that respired CO2 was internally transported to the
stem. The R2 (0.947 for American beech; 0.998 for yellow poplar) are very high which indicates that
the relationship between internal root [CO2] and distance to the stem can be accurately
approximated using linear regression. The large STD can be explained by the diurnal variation and the
[CO2] between different trees of the same species. Fig. III.5 confirms this as averaging for one tree of
each species results in much smaller STDs.
Fig. III.4. Mean internal root CO2 concentration ([CO2]) of each species at different distances to the stem. Linear
regression was applied to indicate that there is a linear relationship between internal root [CO 2] and distance to
the stem.
32
Fig. III.5. Mean internal root CO2 concentration ([CO2]) at different distances to the stem for American beech 3
and yellow poplar 2. Linear regression was applied to indicate that there is a linear relationship between
internal root [CO2] and distance to the stem.
3. Root sap flux density and sap flow rate
The SFDs of both species were similar in magnitude and followed a similar pattern but peaked at a
different time of the day (Fig. III.6). American beech peaked between 16:00 and 18:00 at around 14.4
cm3 cm-2 h-1, while yellow poplar seemed to maximize SFD between 12:00 and 14:00 at around at
around 13.6 cm3 cm-2 h-1. As there was a substantial variation in SFD between different roots of the
same tree and different trees of the same species, STDs were not included in Fig. III.6. The large
variation between different trees was most likely caused by different weather conditions (i.e. VPD
and PAR) as each tree was measured on another day.
33
Fig. III.6. Mean sap flux density (SFD) of American beech and yellow poplar for the 24 hour measurement. Both
species follow similar patterns but their peak in SFD occurs at another time of the day.
American beeches had a significantly higher sap flow rate (Fs) than yellow poplars (Fig. III.7;
Sap~Species; p < 0.0001). Because of the higher FS, more water was transported daily by American
beech than yellow poplar (Table III.2), which accounts for the larger daytime dynamics in internal
root [CO2] (Fig. III.1). Additionally, variation in Fs was observed among individuals within a single
species. This was expected as not all trees were of the same size and weather conditions during
measurements differed among trees. Note that, although the SFDs of both species were similar in
magnitude, the FS were not. This is due to large differences in sapwood area between the species
(Table III.2), which was much larger for American beech than for yellow poplar. Mean estimated
sapwood area was larger for beech than for yellow poplar (Table III.2). This is most likely a
consequence of the bigger roots of American beech. The larger sapwood areas for beech lead to the
higher internal water transport than yellow poplar. STDs of internal water transport were not
included as they were not relevant due to the differences in sapwood area between trees of the
same species and due to the different weather conditions as each tree was measured on another day
(Table III.2).
34
Table III.2. Mean sapwood area, bark thickness of the roots of each species with standard deviation (STD). And
mean daily internal water transport per root for each species.
Species
Mean sapwood area ±
STD (cm2)
Mean bark thickness ±
STD (cm)
Mean daily water
transport (L d-1)
American beech
941.4 ± 587.5
0.6 ± 0.3
114.5
Yellow poplar
226.7 ± 150.9
1.2 ± 0.4
24.1
Fig. III.7. Mean sap flow rate (FS) of American beech and yellow poplar for the 24 hour measuring period.
4. Diurnal variation in root CO2 concentration and sap flow
Fig. III.8 shows that the internal root dissolved CO2 concentration ([CO2*]) followed the same pattern
as internal root [CO2]. All previous graphs with internal root [CO2] would thus follow the same diurnal
pattern when plotted for internal root [CO2*]. This is due to very stable xylem pH and temperature.
35
Fig. III.8. Comparison of the pattern for internal root CO2 concentration ([CO2]) and the xylem dissolved CO2
*
concentration ([CO2 ]) for beech 3.
As mentioned previously, beech had a more profound diurnal variation for internal root [CO2] than
yellow poplar (Fig. III.1). Fig. III.9 and Fig. III.10 show the relationship between Fs and internal root
[CO2]. For beech, there was a clear but unsignificant relationship (beech; [CO2]~FS; p = 0.11). When FS
increased, internal [CO2] decreased as internal CO2 was transported within the root. Although it was
(almost but still) not significant, a trend can be reported. For yellow poplar, there was a similar
relationship that was significant (yellow poplar; [CO2]~FS; p < 0.01). When FS was high, root [CO2]
stagnated while it increased when FS was low.
FS started to increase at around 08:00 to 09:00 for both species. For beech, it peaked between 16:00
and 18:00, while for yellow poplar it peaked between 12:00 and 14:00. Internal root [CO2] increased
between 18:00 and 9:00 for beech and between 20:00 and 12:00 for yellow poplar. It decreased from
09:00 to 18:00 in beech, when FS was high. In yellow poplar, it stagnated between 12:00 and 20:00.
36
Fig. III.9. Relationship between mean internal root CO 2 concentration ([CO2]) and mean sap flow rate (FS) of
American beech during the 24 hour measurement.
Fig. III.10. Relationship between mean internal root CO2 concentration ([CO2]) and mean sap flow rate (FS) of
yellow poplar during the 24 hour measurement.
37
5. Influence of vapor pressure deficit on root sap flow rate
A linear relationship was observed between vapor pressure deficit (VPD) and FS (Fig. III.11). The R2
found were very good (0.899 for American beech; 0.877 for yellow poplar). This means that there is a
very close linear relationship between atmospheric VPD and root FS. Yet, the increase in FS should
stagnate at high VPDs. A hysteresis curve was clearly observed for both species and was very
pronounced for beech. The two slopes of the hysteresis are the delayed increase before noon and
the delayed decrease in the afternoon. When looking at only one of the two slopes, the stagnation at
high VPDs is more pronounced. The relationship can then be approximated by an exponential curve
rising to a maximum. Although the relation found in this experiment is to be expected, there are no
reports of this for roots and it demonstrates that VPD has an effect on root water uptake.
Fig. III.11. Relationship between mean vapor pressure deficit (VPD) and mean sap flow rate (FS) for every tree
for the 24 hour measurements. Although a linear relationship was found, FS should stagnate at higher VPD.
When looking at only one slope of the observed hysteresis, it becomes clear that F S does stagnate and can be
approximated by an exponential function rising to a maximum.
38
6. Influence of soil CO2 concentration on root CO2
concentration
Fig. III.12 depicts the relationship between soil and internal root [CO2]. For American beech there
seemed to be no clear pattern. This could be due to the little variation in soil [CO 2]. Although there
was no visible relationship, there was a slightly significant effect of the soil on the root (beech:
[CO2]~Soil; p = 0.036). For yellow poplar there appeared to be a non-linear relationship between soil
and internal root [CO2]. Yet, this relationship was not significant (yellow poplar [CO2]~Soil; p = 0.54).
The relationship could therefore be coincidence as there were only 2 data points with relatively high
soil [CO2] (≥ 2%) for yellow poplar and almost all data points of beech were concentrated in one spot.
Fig. III.12. Relationship between the internal root CO2 concentration ([CO2]) and the surrounding soil [CO2] for
both species. Each data point is the mean daily [CO2] of one root of a tree.
39
7. Influence of PAR and relationship with the stem
Fig. III.13 shows the relationship between photosynthetic active radiation (PAR), root and stem FS,
and internal root [CO2] for a selected American beech root. This day was selected because PAR
showed strong dynamics. It becomes clear that PAR exerted a similar influence on the root FS as on
the stem FS. When PAR increased, during the morning, FS also increased but with a slight delay. FS
increased even faster than PAR after the initial delay. This could indicate that stomata were
anticipating the future increase of PAR. When there was a period with lower PAR (because of
cloudiness; after the vertical line in Fig. III.13), FS lowered accordingly with only a very small delay.
The effect of the variation in PAR on root and stem FS was almost exactly the same. When the clouds
were gone and PAR increased again, there was a longer delay before FS increased.
Another interesting relationship in Fig. III.13 is internal root [CO2] versus root FS. As mentioned
before, [CO2] increased when Fs was low and decreased when FS was high. From this figure it
becomes clear that this is a very close relationship. Firstly, [CO2] increased when there was no FS (i.e.
at night) and decreased when FS increased (i.e. in the morning). But when FS suddenly dropped at
around 14:00, due to low PAR, [CO2] started to increase again. When later FS returned to earlier
levels, [CO2] stopped increasing until nightfall (i.e. when FS stops).
40
Fig. III.13. Relationship between photosynthetic active radiation (PAR), root and stem sap flow rate (FS), and
internal root CO2 concentration ([CO2]) for American beech 2. Stem FS was acquired from a study conducted by
Wittocx (2014) on the same tree at the same time. PAR was measured using the on-site weather station.
41
IV. Discussion
1. Internal root CO2 concentration
Previous research on internal CO2 in trees has primarily focused on the internal CO2 concentration
([CO2]) in stems. Concentrations of these measurements ranged from < 1% to over 26% indicating
that there are barriers preventing diffusion from the stem to the surrounding atmosphere (Teskey et
al., 2008). Although it has been hypothesized that roots have an influence on the internal [CO2] inside
the stem (Teskey & McGuire, 2007), no studies have yet been conducted measuring the internal
[CO2] in roots (Greenway et al., 2006). Results of this thesis indicated that the internal root [CO2]
were also within the range of < 1 - 26%. For American beech and yellow poplar, a mean of 8.5% ±
2.0% and 5.2% ± 1.9% was measured, respectively. The high internal root [CO2] reported in this thesis
indicate that barriers were present in the root similar to the ones observed at stem level (Aubrey &
Teskey, 2009). These barriers allow CO2 to accumulate inside the root resulting in [CO2] substantial
higher than the atmospheric one. This high internal root [CO2] concentration was likely almost
entirely derived from root respiration. As concentrations in the surrounding soil were much lower, it
is indeed doubtful that a substantial fraction of the root internal CO2 was derived from uptake of CO2
from the surrounding soil (Fig. III.2; Fig. III.3). Ford et al. (2007) reported that there was a 0.8%
contribution of soil dissolved inorganic carbon (DIC) to the net carbon (C) gain of Pinus taeda
seedlings, implying low uptake of soil CO2. For the belowground C gain this was about 1.6%. Other
researchers found values in the range of 1-3% (Schäfer, 1988; Stringer & Kimmerer, 1993). Ford et al.
(2007) concluded that soil DIC only has a very minor contribution to the total seedling and
belowground carbon gain. They also hypothesized that this would be similar for trees in natural
systems. The results reported in this thesis confirm that the influence of soil carbon on root internal
[CO2] in natural systems is limited and therefore root respiration will be the main source of high
internal root [CO2].
The difference in internal root [CO2] between the two species was probably caused by physiological
differences. The beech trees used were larger with larger roots than those of the yellow poplar. Yet,
the mean root bark thickness was higher for yellow poplar (1.2 cm ± 0.4 cm) than for American beech
(0.6 cm ± 0.3 cm). When plotting the relationship between root bark thickness and internal root
[CO2] for both species, there was no clear pattern visible. Although American beech’s root bark was
thinner than yellow poplar’s, it was much smoother and had almost no cracks. Previous studies
43
reported that CO2 diffusion is facilitated by cracks (Grosse, 1997; Langenfeld-Heyser, 1997). This
could be one of the reasons why the internal root [CO2] was higher in beech as some of the CO2
might be able to efflux out of the cracks of yellow poplar bark. This hypothesis was confirmed by
Wittocx (2014) who found that stems of American beech had a 9% higher CO2 diffusion resistance
than yellow poplar. Wittocx (2014) also found that the internal stem [CO2] was higher for American
beech (9.9% ± 2.0%) than for yellow poplar (5.0% ± 1.6%). These results are similar to the ones
measured in this thesis for roots. In a potential follow up experiment, measuring CO2 efflux out of the
roots of different species could be interesting as it would shed light on the question whether CO 2
does efflux out of the root into the soil environment and also what effect the different root bark
types have on this efflux is. In the study by Wittocx (2014), efflux was measured using cuvettes. This
technique could also be used to measure root efflux by installing cuvettes on the root surface.
There was also a noticeable difference in internal root [CO2] between trees of the same species and
even between two roots of a single tree. The mean values of each American beech root were
between 7.8% and 11.1%. For yellow poplar these ranged from 3.0% to 7.2%. The cause of these
interroot and intraspecies differences in internal [CO2] could be the different levels of respiration and
different weather conditions (i.e. vapor pressure deficit (VPD) and photosynthetic active radiation
(PAR)) that influenced sap flow rate (FS) and thus also internal root [CO2]. Mean daytime (i.e. 8:00 to
20:00) VPD values for each tree ranged from 0.7 kPa to 1.6 kPa for beech and from 0.8 kPa to 1.5 kPa
for yellow poplar. PAR measurements ranged from 15.4 kW m-2 day-1 to 38.8 kW m-2 day-1 for beech
and from 17.8 kW m-2 day-1 to 34.9 kW m-2 day-1 for yellow poplar. As these are very large
differences, they will have a large impact on FS and therefore also on internal root [CO2] (Fig. III.13).
The variations in [CO2] observed between roots of the same tree are likely to be derived from
different respiration rates and/or diffusion resistances. The differences in respiration rates could be
caused by temperature differences due to the presence of more shade on one of the two roots and
therefore less respiration (Amthor, 1989). Another reason for a lower root respiration rate is a lower
moisture content (Moyano et al., 2009) and/or lower nitrogen content (Wang et al., 2010) of the
surrounding soil. The diffusion resistance of roots is likely to be dependent on root age. As older
roots are typically more suberized (Raven et al., 2013), this could increase their CO2 diffusion
resistance and therefore lead to a higher internal [CO2].
Measuring the internal [CO2] seemed to affect the concentration itself. This effect was particularly
pronounced for yellow poplar. Although about 6 hours of adaption time was foreseen, the internal
root [CO2] concentration was always higher after the 24 hour measuring period than at the start of
the period. The mean internal root [CO2] for yellow poplar at the begin of the 24 hour measurements
was 3.8% ± 1.7%, while it was 6.0% ± 1.4% at the end of the 24 hour measurements. The same
44
pattern can be reported for American beech. The mean first measurement for beech was 6.9% ±
1.8% and the mean last measurement was 9.3% ± 1.8% (Fig. III.1). This indicates that the installation
of non-dispersive infrared (NDIR) CO2 sensors in roots, and possibly in other plant parts, induced
processes that alter the normal internal [CO2]. Most likely these processes are related to wound
reaction as the holes that were needed for the installation of the sensors were quite large (20 mm
diameter; 50 mm deep). Teskey & McGuire (2005) hypothesized that increase in CO2 efflux from
injured trees could partly be derived from increase in respiration of the injured cells (i.e. wound
respiration) and partly from the removal of barriers to diffusion. Wound respiration could thus have
influenced the measurements taken in this thesis. More research is needed to confirm this however.
2. Root sap flow rate
As was to be expected but -until now- unverified for roots, sap flux density (SFD) increased during the
morning, peaked throughout the day and decreased to zero at night. SFD of both species peaked at
around 14 cm3 cm-2 h -1, but there was a time shift when maximal SFD was observed. Yellow poplar
peaked at around noon, while American beech peaked later. As each tree was measured on another
day, this could be caused by the fact that on average PAR peaked at around 13:00 for yellow poplar
and around 16:00 for American beech. The late PAR peak for American beech can be contributed to
the weather conditions that were cloudier when the main part of the beech trees was measured.
While SFD was similar in magnitude for both species, FS was not (Fig. III.6; Fig. III.7). This is due to the
fact that American beech had larger roots with a larger sapwood area. Their average sapwood area
was multiple times larger as the one measured for yellow poplar (Table III.2), allowing them to
transport more water in the transpiration stream from the roots to the stem. This can also explain
why the internal [CO2] concentration was more dynamic when looking at the results of beech (Fig.
III.9; Fig. III.10). More water transport also means that more CO2 can be dissolved and transported
via the transpiration stream. Yet, a larger root could also imply higher respiration rates and
consequently a cancelling out of the sapwood effect. When looking at the results in this thesis it
seems that the larger sapwood area had a greater effect than extra respiration due to the larger
roots.
As FS has never before been measured in roots, it was interesting to see that VPD had a direct impact
on root FS (Fig. III.11). Although the relation found in this experiment was to be expected, there were
no previous reports of this for roots and it showed that certain parameters have an influence on the
45
whole tree. This was confirmed by the close relationship between stem and root FS (Fig. III.13). Both
curves followed almost exactly the same pattern.
It should be noted that radial variation in SFD was not accounted for in this thesis. This gives an
overestimation of the total water transport. Wullschleger & King (2000) found that sap velocity was
dependent on sapwood depth in yellow poplar stems. They reported that sap velocity gradually
lowered with sapwood depth because of lower water content of the inner sapwood. At the deepest
measurement (i.e. inner sapwood area) the sap velocity was only 0.35 times as high as at the outer
sapwood area. Other studies reported the same relationship (Cohen et al., 1981; Ford et al., 2004).
Yet, this overestimation might be (partly) nullified by the fact that thermal dissipation probes (TDP)
tend to underestimate the SFD. For example, a correction factor of 2.5 is to be used to acquire the
correct SFD of American beech trees when utilizing TDP sensors based on a gravimetric sap flow
analysis (Steppe et al., 2010).
3. Transport of root-respired CO2
Studies generally rely on the paradigm that all root-respired CO2 immediately diffuses out of the root
into the soil environment when measuring total belowground respiration (Teskey et al., 2008). This
way, they can use soil CO2 efflux (Esoil) measurements to estimate total belowground respiration.
However, a study by Teskey & McGuire (2007) hypothesized that part of the root-respired CO2 is
transported up to the stem and thus is unaccounted for by Esoil measurements. The results of this
study strongly indicated that this is indeed the case which means that belowground and more
precisely root respiration have been underestimated in many previous studies.
Results in this thesis showed that there was a clear gradient along the root (Fig. III.2; Fig. III.3; Fig.
III.4). The large standard deviations (STD) were caused by the diurnal and intraspecies variation in
internal CO2 (Fig. III.4). When the mean concentrations of only one tree (Fig. III.5) were plotted, the
STDs were much smaller and only depended on diurnal variation and differences between both
roots. For American beech, the mean xylem [CO2] of 0, 0.5 and 1 meter of the stem were 9.6% ±
1.5%, 8.9% ± 1.6% and 7.2% ± 2.0% respectively. For yellow poplar, they were 5.6% ± 1.7%, 5.1% ±
1.8% and 4.7% ± 2.0%. These gradients support the hypothesis that root-respired CO2 is dissolved
into the xylem sap and transported from the root tip to the stem base. The concentration increases
as CO2 dissolves into the xylem sap along the root. The average increase in internal [CO2] for
American beech from root zone 3 to zone 2 was about 24% and from root zone 2 to zone 1 was
about 8%. For yellow poplar, these were about 9% and 9% respectively. The root-respired CO2 is then
46
transported further with the transpiration stream to the stem, branches and leaves. There, the
internally transported CO2 can then efflux or be refixed.
Another clear indication of the internal transportation of root-respired CO2 was the diurnal variation
in internal root [CO2]. When FS increased in the morning, internal root [CO2] started to decrease. This
relationship shows that root-respired CO2 was dissolved into the transpiration stream and was
internally transported away from site of respiration. This could be explained by a sort of dilution
effect of the higher FS. When FS increases more xylem sap, containing low levels of dissolved CO2,
flows through the root and consequently more CO2 can be dissolved. This leads to a decrease in
internal root [CO2] as it is transported away from the root. The results of this thesis indicated that FS
and internal root [CO2] were strongly coupled (Fig. III.13). As FS decreased to near zero due to a
passing cloud at around 14:00, internal root [CO2] increased practically without any delay. When later
the transpiration picked up again (at somewhat lower levels), the internal root [CO2] was being
transported away again and therefore stopped increasing. When there was no FS, there seemed to be
no noteworthy sink for the root-respired CO2. Although such a strong coupling between FS and
internal [CO2] has never been reported before for roots, it has been previously observed at stem
level (Teskey & McGuire, 2002). The close relationship between FS and internal root [CO2] strongly
indicated that the greater part of the root-respired CO2 was transported with the transpiration
stream to the stem and that there were few other significant sinks. In a girdling study, Bloemen et al.
(2014) observed that stem girdling decreased stem xylem [CO2] what confirms that a considerable
fraction of stem internal CO2 is derived from internally transported root-respired CO2. Hence,
estimating total belowground and root respiration using efflux-based techniques could consequently
underestimate actual respiration rates substantially.
When looking at the results of this thesis, it is very likely that part of the root-respired CO2 was
internally transported to stem, branches, leaves, and other aboveground plant organs for refixation.
This recycling mechanism is assumed to positively contribute to the overall carbon gain of trees
(Aschan & Pfanz, 2003; Teskey et al., 2008). By recycling large fractions of root-respired CO2, trees
avoid water loss due to transpiration because of open stomata. The high internal [CO2] also leads to
less photorespiration (Cernusak & Marshall, 2000). Aubrey & Teskey (2009) estimated that up to 50%
of the root-respired CO2 is transported via the transpiration stream to other plant parts. The results
in this study suggest that possibly even more than 50% is transported as they indicated that sap flow
is a major sink for root-respired CO2 (Fig. III.9, Fig. III.10 and Fig. III.13). Hanson & Gunderson (2009)
concluded that if a large fraction of the root-respired CO2 indeed reaches the leaves through
transport with the transpiration stream, it could be impacting the availability of CO2 for leaf
photosynthesis. As a study by Bloemen et al. (2013) confirmed that, using isotopic labeling, CO2 can
47
be transported all the way up to the leaves, it becomes clear that the impact of root respiration on
the plant carbon gain has been continuously underestimated. Bloemen et al. (2013) also estimated
that up to 47% of root-respired CO2, that was internally transported, diffused to the atmosphere
from stem and branches. Teskey & McGuire (2007) estimated that 45% of stem efflux (Estem) is
composed of xylem-transported CO2. This means that Estem measurements are for a substantial part
composed of internally transported root-respired CO2 and therefore do not accurately estimate local
stem respiration.
It should be mentioned that there were a few roots where inverse gradients (i.e. highest [CO2]
farthest from the stem) were measured. This was not visible when averaging for all the roots of a
species but was nonetheless observed in individual roots of a few trees. A possible explanation for
this phenomenon could be hydraulic redistribution. For example, the FS of yellow poplar 3 (i.e. a tree
where a reversed gradient was measured in one root) was not zero at night but around 0.8 L h -1. This
could mean that the xylem sap was flowing in opposite direction to the root tip. Although this is
possible, there was no way to verify this as TDPs can only measure FS and cannot distinguish between
a normal and an inverse FS (Burgess et al., 2000). Burgess et al. (2000) therefore suggested using
methods that can measure slow and reversed FS for roots of woody plants. Another reason why
hydraulic redistribution is a possibility was the dry period of seven days before the measurement on
this tree. In this period only 15 mm of rain fell what could have resulted in a very dry soil. Trees are
known to be able to redistribute water from one root to another at night in dry periods (Nadezhdina
et al., 2006; Bauerle et al., 2008; Domec et al., 2010). Although this is a viable theory, more research
is needed to confirm it.
4. Relationship between root CO2 concentration and the soil
environment
For American beech, no clear relationship between soil [CO2] and root [CO2] was observed. This was
due to the narrow range of soil [CO2] measured in the experiments. Future experiments could try to
artificially increase soil [CO2] near the roots of adult trees while measuring internal root [CO2]. This
could increase our knowledge of the processes and barriers involved in the root uptake of carbon
from the soil. An experiment by Anné (2014) added different [CO2] solutions to the soils of seedlings
to monitor the uptake and transport of this soil CO2 molecules through efflux from the stems. While
they reported that adding [CO2] solutions increased the stem efflux, they hypothesized that for low
concentrations this was due to changing gradients and that only with high [CO2] solutions (> 5%)
there was uptake from the soil by the roots. Soil [CO2] is usually < 2% in field conditions. In the
48
experiments conducted in this thesis, soil [CO2] was also in that range. Yet, there were two soil
measurements that exceeded 2%. Both those roots were of yellow poplar trees. For yellow poplar,
there seemed to be a relationship between soil [CO2] and root [CO2]. The non-linear regression can
be described by an exponential function rising to a maximum. This indicated that there might be a
limited effect of soil [CO2] on internal root [CO2]. However, this effect stopped at higher
concentrations which is not in line with the research conducted by Anné (2014). An explanation for
the divergent results could be the age and maturation of the trees used in the experiments. As roots
often undergo suberizing and lignification of tissues when aging and maturing (Raven et al., 2013),
there will be a lower uptake from the soil and less diffusion to the soil. The relation found could
however be coincidence as there were only two data points with > 2% soil [CO2]. More research on
this topic is needed to understand these relationships. Especially for adult trees as they are very
different from seedlings. A problem with the soil measurements used in this thesis was that the
uncovering of the roots meant that they were not in full contact with the soil what could have led to
an efflux to the atmosphere instead of to the soil and a limited uptake of soil [CO 2] to the root. An
experiment where the roots would not be uncovered, except for where the root NDIR CO2 sensor is
installed, could be more accurate in predicting the influence of the soil on the root and vice versa.
Ford et al. (2007) and Jones et al. (2009) reported that DIC from soil water had very little impact on
the overall carbon budgets of plants. The results of this thesis support this as internal root [CO2] was
multiple times as high as soil [CO2] (Fig. III.2; Fig. III.3; Table III.1), indicating that only a small quantity
of CO2 could have originated from the surrounding soil. Ford et al. (2007) and Ubierna et al. (2009)
tested if soil CO2 was taken up by roots of loblolly pine seedlings and mature conifers, respectively,
by adding enriched CO2 solutions to the soil and measuring isotopic composition of stem CO2 efflux
(Estem). They did not observe any influence of this enrichment what, in combination with our reported
results, confirms one of the main hypotheses of this thesis: that internal root [CO2] is almost entirely
derived from root respiration. As internal root [CO2] is substantially higher than surrounding soil
[CO2], it can also be concluded that root respiration is the main source of internal CO2 measured at
stem base level.
Soil [CO2], measured in this thesis, had no diurnal variation (Fig. III.2; Fig. III.3). This could be
indicating that the impact of root respiration on surrounding soil [CO2] is very limited. As higher
internal root [CO2] at night would mean more efflux and thus more CO2 in the soil environment, soil
[CO2] should increase when internal root [CO2] increases. As mean internal root [CO2] was
substantially higher at the end of the 24 hour measurement period than at the beginning, it would be
expected that mean soil [CO2] would also be higher if efflux occurred. Yet, mean soil [CO2] at the end
of the measuring period (1.2%) was lower than at the beginning (1.3%). It can therefore be concluded
49
that root-respired CO2 almost does not efflux to the soil environment and thus root respiration has
limited impact on soil [CO2].
An increasing number of studies (Aubrey & Teskey, 2009; Grossiord et al., 2012; Bloemen et al.,
2013) are suggesting that transportation of root-respired CO2 via the transpiration stream and efflux
to the atmosphere may have important implications for the assessment of above- and belowground
metabolism. Together with refixation in aboveground plant organs, this could account for a
substantial part of the total root-respired CO2. Esoil measurements for estimation of belowground
respiration depend on the assumption that all root-respired CO2 immediately diffuses to the
surrounding soil environment. As this assumption is likely to be incorrect when looking at recent
studies (Hanson et al., 2000; Kuzyakov, 2006; Trumbore, 2006; Aubrey & Teskey, 2009) and the
results of this thesis, the current methods will have to be adjusted to include internal transport of
root-respired CO2 or another method will have to be developed. Perhaps the mass balance method
developed by McGuire & Teskey (2004) can be used to measure root respiration and then be
combined with Esoil measurements for an accurate estimation of the contribution of root respiration
to the total belowground respiration.
5. Directions for future research
Measuring soil [CO2] and root [CO2] simultaneously in this research made clear that there is very little
influence of the root on the soil and vice versa. Future research could use cuvettes to measure root
[CO2] efflux and further quantify the CO2 contribution of the root to the soil. Together with these
root efflux measurements, NDIR CO2 sensors and TDPs could be placed along the root, stem base and
stem to increase our knowledge of internal transport of root-respired CO2 throughout the whole
tree. Additionally, Estem could also be measured. An accurate estimation of the fraction of rootrespired CO2 that contributes to root efflux and to internal stem [CO2] could then be made.
Another future experiment could be the artificial increase of soil [CO2] close to mature roots in a
natural system while simultaneously measuring internal root [CO2]. The influence of soil carbon on
mature suberized roots could then be studied.
By using TDPs on roots, there was no possibility of measuring reverse flows. As reverse flows
probably occur, it should be studied if other methods are to be used for roots. Burgess et al. (2000)
already suggested using methods capable of measuring reverse and slow FS rates in roots.
50
V. Conclusion
The results of this study indicate that CO2 concentrations ([CO2]) in roots of mature trees are
substantially higher than [CO2] in the atmosphere and the soil. The buildup of these high [CO2] shows
that there are barriers present in roots that prevent CO2 diffusion out of the root. The differences in
internal [CO2] between species are therefore most likely caused by dissimilarities in these barriers
and consequently different CO2 diffusion resistances. Furthermore, this study proves that a
substantial fraction of the root-respired CO2 is dissolved in the transpiration stream and transported
to the stem and other plant parts. The amount of CO2 transported internally is highly dependent on
FS. High FS at midday can transport a substantial amount of root-respired CO2 and therefore cause
midday depressions in internal root [CO2]. The transported CO2 can then be refixed in leaves and
woody tissue or efflux out of the stem and branches.
In this study there seemed to be little influence from mature roots of adult trees on the surrounding
soil or vice versa. The high internal root [CO2] as compared to surrounding soil [CO2] implies that
root-respiration will be the main source of internal CO2 measured at stem base level.
This study also provides evidence that internal transport of root-respired CO2 has to be taken in
account when estimating above- and belowground respiration. Therefore, the assumption that CO2
efflux equals respiration has to be reconsidered. Firstly, as a large part of the root-respired CO2 is
internally transported and diffuses out of the stem, stem CO2 efflux (Estem) measurements do not
accurately reflect local stem respiration. This means that current efflux methods should be replaced
by more accurate methods, like the mass balance approach of McGuire & Teskey (2004), that include
internal transport. Secondly, this thesis verifies that soil CO2 efflux (Esoil) measurements cannot be
used to accurately estimate total belowground respiration as a large fraction of the root-respired CO2
does not immediately diffuse to the soil environment but is internally transported to the stem
instead. Consequently, by not including internal transport of root-respired CO2 in efflux-based
measurements, there will be an underestimation of the root respiration and an overestimation of the
aboveground respiration.
It becomes clear that root respiration has an important impact on tree physiology. It is therefore
essential that we gain more insight in root physiology as it will allow us to estimate and model the
total ecosystem carbon cycles more accurately.
51
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