Determining photosynthetic responses of forest species to elevated
[CO2]: alternatives to FACE
E. A Pinkard1,2*, C. L. Beadle1,2, D. S. Mendham1, J. Carter1 and M Glen1,2
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CSIRO Sustainable Ecosystems and CSIRO Climate Adaptation Flagship
Private Bag 12
Hobart 7000
Australia
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2
Tasmanian Institute of Agricultural Research
Private Bag 54
Hobart 7000
Australia
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*corresponding author:
Ph 61 3 62375656
Email [email protected]
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Abstract
Free air CO2 enrichment (FACE) experiments are considered the most
reliable approach for quantifying our expectations of forest ecosystem
responses to changing atmospheric CO2 concentrations [CO2]. Because very
few Australian tree species have been studied in this way, or are likely to be
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studied in the near future because of the high installation and maintenance
costs of FACE, there are no clear answers to questions such as: (1) which
species will be the winners in Australia’s natural forests and what are the
implications for biodiversity and carbon (C) sequestration; and (2) which will
be the most appropriate species or genotypes to ensure the sustainability of
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Australia’s plantation forests.
We examined possible experimental approaches that may provide insights
into, and more rapid assessment of, responses to elevated [CO2]. Our main
conclusions were: (1) better understanding the extent to which species are Climited could indicate when elevated [CO2] might be expected to increase
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photosynthesis and biomass production. Plant tissue carbohydrate
concentrations can be used to assess any C limitation. Consistently high
levels of carbohydrates indicate that plants are not C limited, but rather that
growth is determined by other limiting resources or by rates of cell
development and expansion; (2) historical examination of forest responses to
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increasing atmospheric [CO2] using stable isotopes in wood cores can provide
clues as to which species may respond favourably to increasing [CO2],
although it may remain difficult to distinguish between the environmental
conditions under which favourable responses occurred. Undertaking stable
isotope studies close to anthropogenic CO2 sources has the potential to
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provide insights into how species may respond to the higher [CO2] that is
predicted during this century; (3) by focusing on genetic and metabolomic
regulation of source and sink activity, selection for greater biomass production
under elevated [CO2] is possible.
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Key words: sink limitation; stable isotopes; metabolomics; genetics;
photosynthetic upregulation, elevated [CO2]
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1. Introduction
Forests are a major reservoir of terrestrial carbon (C) (Körner, 2003).
Importantly, forests can provide long-term capacity for C sequestration and
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storage, and this has raised their profile as a potential means of mitigating
rising atmospheric CO2 concentrations ([CO2]). Does elevated [CO2] affect
the capacity of existing and newly-planted forests to store C? This question is
becoming pertinent to forest managers who in making the right choices for
sustaining timber yields, must at the same time consider increasingly stringent
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requirements for conserving C, water yields and biodiversity in their forest
estates.
The amount of C stored in a forest is a function of rates of C assimilation,
losses associated with respiration and leaching (Long et al., 2004b; Ainsworth
and Long, 2005; Hyvonen et al., 2008), and allocation of C between mobile
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pools and biomass (Körner, 2006; Millard et al., 2007). While increases in
rates of C assimilation are frequently reported in response to elevated [CO2],
they may be short-lived, particularly if resources other than CO2 are limiting
growth (Ainsworth and Long, 2005). Modelling studies have suggested that
elevated [CO2] may have little effect on forest growth rates and total biomass
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where water or nutrient supplies are low (Oren et al., 2001; Nowak et al.,
2004), and may influence the distribution of species in such a way that total
biomass, or rates of biomass accumulation, are reduced (Li et al., 2003;
Chiang et al., 2008). For example, Chiang et al. (2008) predicted that a
transition from spruce-fir to deciduous forest in western Maine, USA,
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associated with changing climate, would reduce total biomass by up to 12%.
Australia has approximately 150M ha of natural forests and woodlands;
80% of the forested area is dominated by eucalypt species (Montreal Process
Implementation Group for Australia, 2008). Australian eucalypts also dominate
planted forests outside Australia, particularly in the tropics and subtropics; the
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global area of plantation eucalypts is currently 19.5M ha (Iglesias &
Wistermann, 2008). Australia has 2.0M ha of planted forest (Gavran and
Parsons, 2009). A number of studies have examined the inter- and intraspecific rainfall and temperature requirements of Australian tree species
(Booth and Pryor, 1991; Hughes et al., 1996; Booth and Jovanovic, 2005;
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Costa e Silva et al., 2006) to predict where these species may grow as
exotics. In contrast, there is very little understanding of Australian tree
species responses to elevated [CO2], even though a lack of understanding of
inter- and intra-specific variation in CO2 responses, and the influence of
environment on these responses, have been identified as major impediments
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to adaptive management in the plantation industry (Battaglia et al., 2009).
Hence there is currently little information to assist forest managers in making
decisions about:
(1) Which species are best adapted to changing climates in natural forests
and what the longer-term implications of species shifts are for forest
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biomass and wood production;
(2) Which species or genotypes would be most appropriate to ensure the
sustainability of plantation forests under changing environments.
A variety of experimental techniques are used to study the responses of
trees to elevated [CO2]. The most common are open- and closed-top
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chambers, branch bags in which only a part of the tree is exposed to elevated
[CO2], free air CO2 enrichment (FACE), and natural CO2 vents (Saxe et al.,
1998a). Elevated [CO2] experiments are high cost (Saxe et al., 1998), and
have a number of technological issues that may limit their relevance to trees
growing in situ (Ainsworth and Long, 2005). They are also generally restricted
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to individual tree species rather than ecosystems, and to relatively short
timeframes.
In this review, we examine whether there might be alternative approaches
to determine tree responses to elevated [CO2]. If there are, can they be used
in conjunction with the techniques described above to improve our
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understanding of species and ecosystem responses to rising [CO2], and to
assist in making management decisions today that protect forest biodiversity
and biomass production and the viability of the commercial forest industry into
the future. First, we examine the approaches currently used to investigate
responses of trees to elevated [CO2], and their relevance to forest
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management. Second, we explore factors that influence a tree’s capacity for
photosynthetic upregulation and increased growth and biomass under
elevated [CO2]. We then discuss possible experimental approaches that may
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provide insights into, and more rapid assessment of, responses to elevated
[CO2]. While our focus is on Australian forests, the approaches described
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have relevance more generally to forest ecosystem management.
2. Current experimental methodology for assessing tree responses to
elevated [CO2]
Current techniques for assessing responses to elevated [CO2] can be
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broadly divided into two categories, increasing the [CO2] (1) around a leaf,
leaves, branches, whole plant(s) or trees in a sealed or semi-sealed chamber;
and (2) around numerous individuals over a large area in a FACE
environment or near natural CO2 vents. Table 1 summarises the advantages
and disadvantages of each. In the first category, glasshouses and growth
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chambers are useful for minimising the effects of all variables except the one
in question, but they are otherwise totally artificial environments and not suited
to the study of the effects of elevated [CO2] on forests in situ (Figure 1, Table
1). The approach is suited to examining mechanisms associated with
physiological responses to elevated [CO2], and provides a valuable adjunct to
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FACE experiments. It will generally not be suited to broad screening of
species and ecosystem responses to elevated [CO2].
Experiments with open-top chambers have been used extensively in the
field, especially in agricultural crops, but these are also subject to
shortcomings. The microenvironment within chambers is often markedly
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different to the surrounding environment, bringing into question the relevance
of results to the real world. For example, Whitehead et al. (1995) found large
open-topped chambers had air temperatures up to 4.3°C and air saturation
deficit up to 0.8 kPa greater than ambient at peak irradiance; mean light
transmittance was 74-81% of ambient and there was a 13-21% higher
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proportion of diffuse irradiation in the chambers than outside. Open-top
chambers also effectively remove any wind effect, which can reduce rainfall
interception and change plant-atmosphere coupling. Collectively, these
shortcomings may reduce the value of any measured responses to elevated
[CO2] as they can potentially have a physiological effect equivalent to doubling
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of ambient [CO2] (Long et al. 2004). In addition, it is recognised that results
from chambers cannot adequately represent the behaviour of mature and
highly diverse forest ecosystems, although they have been shown to
adequately represent the climatic conditions of understorey species in humid
tropical forests (Würth et al., 1998). This is because chambers can only
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physically enclose a limited number of small trees, and thus prevent
interactions between trees, including the tight coupling with the atmosphere
referred to above. Whilst chambers have been particularly suited for studying
younger trees, the responses of small plants to elevated [CO2] are likely to be
quite different to those of older trees for a range of reasons: younger trees
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often have different photosynthetic and allocation responses to environment
(James and Bell, 2000), and there may be less competition for water,
nutrients, and light (Drake et al. 1997). Older trees also have slower rates of
growth and their physiological responses to elevated [CO2] may be less
plastic than those of faster-growing juvenile forms.
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FACE overcomes many of the disadvantages associated with chamber
and glasshouse experiments, enabling long-term in situ study at the tree and
ecosystem scale (Table 1). FACE provides a detailed understanding of
physiological processes at tree and ecosystem scales, and accounts for
interactions between soil, atmosphere and ecosystem. It remains the method
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of choice for elevated [CO2] studies. Early FACE systems started operation in
1989 (Lewin et al., 1994), and there are now over 30 installations listed on the
Oak Ridge National Laboratory website global list (http://public.ornl.gov/face/
global_face.shtml). FACE systems in forests typically surround a circular area
of 25-30 m diameter with vertically elevated pipes which emit CO2 at a
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controlled rate to keep the area within the circle at a specified atmospheric
[CO2]. Delivery systems for CO2 need to be very sophisticated and
instantaneously account for variable wind speed across the experiment and
also vertical air movements within the forest canopy.
FACE systems continue to service invaluable experiments for
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understanding plant and ecosystem responses to elevated [CO2]. However
FACE brings its own set of challenges for understanding effects of elevated
[CO2] on ecosystem function. The first is that of cost. A fully functioning FACE
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system represents a very significant investment in infrastructure. Raison et al.
(2007) estimated that a single installation would have an establishment cost of
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up to AU$0.5M, and ongoing annual CO2 and maintenance costs of AU$1.8$4M. Location of FACE systems also needs to take into account accessibility
for installation, maintenance, and CO2 delivery, and availability of services
such as electricity and accommodation for permanent staffing. A FACE
installation probably needs to run for at least 10 years (Raison et al. 2007) to
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gain an adequate understanding of changes to carbon, water and nutrient
cycling in forest systems. These logistical and time constraints prohibit large
numbers of sites and replication within sites, so any installation needs to be
highly targeted and can necessarily only represent a very small proportion of
the world’s forest ecosystems. Problems with lack of replication can present
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an issue with detecting small but important changes in physiological function
(Rogers et al. 2006).
A range of scientific issues associated with FACE systems is also
recognised. For example, the relatively abrupt increase in [CO2] imposed by
FACE experiments may result in a different plant response to that following a
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more gradual change in atmospheric [CO2]. In a modelling analysis, Luo and
Reynolds (1999) showed that a step change in [CO2] increased N demand by
4.1 g N m-2 y-1, and this contrasted with 0.6-1.7 g N m-2 y-1 increase in N
demand under a slowly increasing [CO2] scenario. The difference was due to
the increased system cycling of N in the latter case.
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Short term variation in [CO2] may also present problems in interpreting
outcomes. McLeod and Long (1999) showed that one-minute averaged [CO2]
typically varied by ±10% for 90% of the time in arable-cropping and ±20% for
90% of the time in forest FACE systems . These measurements are usually
taken from the centre of the ring, and there is likely to be much greater
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variation closer to the edge, where CO2 may be less well mixed with
surrounding air. The effect of short-scale temporal variation in [CO2] is
uncertain but potentially of concern because of the non-linear response of
stomata to atmospheric [CO2].
A third issue that has become apparent with FACE systems is that influx of
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CO2 can lead to a break-up of an inversion layer under still conditions, which
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maintains a warmer temperature compared to outside the FACE ring structure
(McLeod and Long, 1999). This effect can be reduced by turning off the CO2
injection at night when an inversion layer is likely to form, but the decrease in
[CO2] may then affect rates of dark respiration (Drake et al. 1997). McLeod
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and Long (1999) also identified a “FACE Island” effect; two phenomena are
described. First, within the island or high [CO2] ring, the difference in the
surrounding humidity leads to a gradient across the ring in the use of site
resources. Second, the island may differentially attract or repel insects and
fungi.
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So while chamber and FACE experiments have shown us that trees and
forest ecosystems can be highly responsive to changes in [CO2], it is also
clear that there is a need for alternative experimental techniques to assess the
effects of elevated [CO2]. This is particularly because these current systems
lack capacity to cover a large number of ecosystems and replication within
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ecosystems.
3. Photosynthetic up-regulation and acclimation under elevated CO2
3.1. Fundamentals
Carbon dioxide and water are the substrates that, through the light and
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dark reactions of photosynthesis, are combined into dry mass. Thus [CO2]
can be a major factor limiting photosynthesis (Hall and Rao, 1992). Stomata
regulate the diffusion of CO2 into leaves; stomata can respond sensitively to
[CO2] as part of a proportionate response to the CO2 requirement for
photosynthesis; increasing concentrations are therefore associated with a
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closing response and vice-versa. Thus elevated [CO2] is anticipated to
increase or up-regulate photosynthesis, decrease stomatal conductance and
increase intrinsic water-use efficiency i.e. the ratio of leaf photosynthesis to
stomatal conductance (Long et al., 2004). Many factors other than [CO2]
determine photosynthetic rate, and the law of limiting factors (von Liebig,
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1840) will ultimately determine photosynthetic responses to [CO2]; i.e. more
than one limiting factor may be involved (Bloom et al., 1985; Chapin and
Shaver, 1985). In Australian environments, marked dry seasons, extended
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periods of drought and poor soils can dominate patterns and rates of growth
of most vegetation types, at least during part of their life cycle.
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3.2. What is photosynthetic up-regulation?
Up-regulation of photosynthesis refers to a significant increase in the lightsaturated rate of photosynthesis (Amax), the rate of photosynthesis under
ambient light (A), and/or diurnal photosynthesis (A’) (Ainsworth and Rogers,
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2007). Elevated [CO2] up-regulates photosynthesis by increasing the
carboxylation rate, Vc, of ribulose bisphosphate carboxylase (Rubisco) and
competitively inhibiting the oxygenation of ribulose bisphosphate (RuBP),
thereby reducing photorespiration (Drake et al., 1997; Long et al., 2004).
Elevated [CO2] is also associated with the expression of several other
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changes that affect photosynthesis. The common observation of reduced
stomatal conductance, gs, will tend to dampen the extent to which any upregulation is expressed at a leaf-scale, but may conserve water such that
stand-scale responses are positive (Ainsworth and Rogers, 2007).
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3.3. What is photosynthetic acclimation or down-regulation to elevated [CO2]?
Photosynthetic acclimation refers to longer-term adaptive changes in the
photosynthetic responses to external stimuli that reduce the net level of the
initial response; acclimation is also referred to as down-regulation.
Acclimation is commonly observed, and arises from the plant’s need to
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balance all resources that are allocated to photosynthetic processes, including
the external [CO2] (Sage, 1990; Gunderson and Wullschleger, 1994). For
elevated [CO2], acclimation is mechanistically linked to decreased maximum
apparent carboxylation velocity (Vc,max) and reduced investment in Rubisco
(Rogers and Humphries, 2000), and an associated reduction in N content;
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these changes are linked to a decrease in control of Amax by Vc,max but an
increase by Jmax, that is by the rate of regeneration of RuBP (Long and Drake,
1992). There is also an increase in starch and sugar content.
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3.4. Photosynthetic response of forests to elevated [CO2]: Evidence from
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FACE
Ainsworth and Long (2005) conducted a meta-analysis of published data
from 12 FACE experiments, five experiments of which had one or more tree
species (Table 1). This demonstrated increased rates of photosynthesis
across a range of species growing at elevated [CO2]. For the 12 tree species
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across these five sites, there was a 47% increase in Amax with growth under
elevated [CO2], which was a larger increase than for other functional types;
however diurnal carbon assimilation (A’) increased less than for some other
functional types (29%; Table 2). That Amax of tree species can be strongly
enhanced by elevated [CO2] has been noted in studies of trees in enclosures
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(Medlyn et al. 1999). However, maximum carboxylation rate (Vc,max) was
decreased by 6% only and there was no change in Jmax, and as a result only a
small decrease (3%) in the ratio of Vc,max/Jmax. There was a 10% reduction in
foliar N on a mass basis but none on an area basis. Elevated [CO2] was
associated with a 16% reduction in stomatal conductance and as a result
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there was a very large increase in intrinsic water-use efficiency (74%).
In properly interpreting the functional responses summarised in Table 2, it
must be noted that these are averages and that there were large differences
between FACE sites and between species examined. At two sites examining
Pinus taeda and understorey hardwood species in North Carolina, and
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Populus alba in Italy, there was no significant change in Vc,max. There was
some evidence of significant increases in Jmax and Vc,max/Jmax but the
response was variable. Down-regulation of Vc,max where it did occur, was
associated with small sink capacity (Hovenden, 2003) or distance from active
sinks (Takeuchi et al., 2001). Such acclimation may be related to low N
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supply that restricts sink development (Stitt and Krapp, 1999) as well as the
specific decrease in Rubisco that appears to occur at elevated [CO2]
(Ainsworth and Long, 2005). Thus it is likely that marked down-regulation of
photosynthesis in response to elevated [CO2] will feature prominently in many
Australian environments.
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3.5. The mechanics of the response
All forest tree species are C3 and their schematic response to increasing
[CO2] has two phases (Long and Hallgren, 1985). In the first phase there is a
marked and linear increase in Amax with increasing [CO2] that occurs between
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0 and around 300-to-350 µmol mol-1 i.e. somewhat below current ambient
levels of [CO2] (380 µmol mol-1). The relationship then becomes curvi-linear
to a greater or lesser extent, with saturating [CO2] for Amax occurring around
1000 µmol mol-1. The slope of the linear phase is associated with the
efficiency of carboxylation or activity of Rubisco measured as Vc or Vc,max;
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beyond the point of inflection, the flat part of the curve represents limitations
by the supply of RuBP measured as the maximum rate of RuBP regeneration
(Jmax). Thus increases in [CO2] that have been occurring for around the last
50 years and that will occur into the future have less relative influence per unit
increase in [CO2]. At very low light flux density, elevated [CO2] has the
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potential to reduce the light compensation point and permit plants to grow in
greater shade (Körner, 2006).
4. Does photosynthetic up-regulation translate into increased tree
growth?
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In the short term, rising [CO2] increases photosynthesis in many of the
woody species that have been studied (Ainsworth and Long, 2005; Körner,
2006), which has the potential to yield significant increases in rates of
biomass accumulation. Ainsworth and Long (2005) found that allocation of dry
mass to above-ground parts in forest FACE experiments increased 28% on
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average; this includes a greater allocation to woody components (Table 2). In
general, larger responses in growth, biomass and leaf area index to elevated
[CO2] have been observed in trees than other functional types (Curtis and
Wang, 1998; Saxe et al., 1998; Ainsworth and Long, 2005)
However there is often a poor correlation between photosynthetic capacity
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measured as Amax and total biomass production (Gifford and Evans, 1981;
Wardlaw, 1990; Oren et al., 2001). For example, Schimel (2006) found in a
meta analysis of agricultural crops that high-yielding cultivars often have lower
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Amax than the lower-yielding parent material. While this does not exclude the
possibility that Amax of individual species may respond positively to elevated
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[CO2] and produce more total biomass (Table 2), it suggests that allocation of
biomass after the C has been fixed, as well as its turnover, will ultimately
determine how any benefit is delivered. Hence, while increases in both net
primary productivity (NPP, biomass accumulation per unit time) and total
biomass have been reported in elevated [CO2] studies (Saxe et al., 1998;
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Nowak et al., 2004; Ainsworth and Long, 2005; Hyvonen et al., 2008), a 1:1
translation of photosynthetic responses to growth responses cannot be
assumed, and responses can vary from no change to large increases in
growth (Körner, 2006) (Table 2). Some of the reasons why photosynthetic upregulation may not translate into increased growth include:
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1.
Increased exudates of non structural carbohydrates (NSCs).
Emissions of NSC from leaves, root exudates, and transfer to
symbionts can account for significant losses of C (Millard et al.,
2007a). For example, studies of ectomycorrhizal associations with
conifers have demonstrated that up to 30% of total C assimilated
can be transferred to symbiotic fungi (Sodenstrom, 2002). These
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sorts of emissions have been found to increase under elevated
[CO2] in some species (Millard et al. 2007).
2.
Increased allocation of NSCs to organs with high rates of turnover,
such as fine roots and leaves. Under elevated [CO2], patterns of
biomass allocation may change to promote leaf and fine root
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development (Hyvonen et al., 2008), both of which constitute
temporary stores of biomass.
3.
Age-related decline in responses to elevated [CO2]. Trees can
capitalise most rapidly on elevated [CO2] when they are in the
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exponential growth phase prior to canopy closure (Idso, 1999;
Körner, 2006), and hence an initial large increase in biomass is
commonly reported, followed by a rapid decline once canopy
closure has occurred (Figure 2, from Idso, 1999). This response
pattern is supported by long-term dendrochronological studies of
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trees growing near natural CO2 vents compared to those growing
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under ambient [CO2], where increased stem diameter growth
associated with elevated [CO2] was largest when trees were young
(Hattenschwiler et al., 1997). When atmospheric [CO2] was
experimentally increased in mature forest stands (~100 years old) in
Switzerland, no effect was measured on stem basal area increment
380
after four years of increased CO2 exposure (Asshoff et al., 2006).
4.
Competition for resources. Whether post-canopy closure forests
exhibit an increase in biomass at elevated [CO2] depends at least in
part on whether resources other than C are limiting growth.
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Experiments with maturing pines found that increases in biomass
associated with elevated [CO2] were related to nitrogen availability,
with little or no increase in biomass when N was limiting but large
increases when nutrients were added (Oren et al., 2001). Elevated
[CO2] may accelerate the development of nutrient limitations
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because of more rapid initial growth rates (Saxe et al., 1998). A
faster depletion of available water also can reduce C assimilation
and offset the benefits of elevated [CO2], despite improvements in
leaf-level water-use efficiency (Messinger et al., 2006; Hyvonen et
al., 2008) and whole-tree water use (Cech et al., 2003) that have
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been observed.
Tree species differ in their inherent rates of growth and the times during
their life cycle when their highest growth rates occur. Because of this,
elevated [CO2] is likely to favour more responsive taxa in mixed forest
systems. However, other factors, like soil type, may ultimately determine
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which species become dominant (Körner, 2006).
In an analysis of carbon limitation in trees at several different scales,
Millard et al. (2007) came to a similar conclusion to Körner (2006) and stated
“that the growth of trees is not carbon-limited, with the key to understanding
future responses to climate change being turnover of soil organic matter and
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nutrient cycling”.
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5. How can we determine tree responses to elevated [CO2]: alternatives
to FACE
5.1. Dendrochronology and stable isotopes
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Dendrochronology: Dendrochronological studies assess longer-term
responses of trees to historical increases in ambient [CO2] (recently reviewed
by Huang et al., 2007). Growth rates recorded in the annual rings are
functionally ascribed to tree age- or size-related growth trends, climate
records, and disturbance factors, including changes in [CO2]. Advantages of
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this approach include that the effects of increased [CO2] on growth of older
trees may be examined (Hattenschwiler et al., 1997a; Voelker et al., 2006), in
situ measurements are possible, and there is potential for high levels of
replication (Table 1, Figure 1). The method is well-developed, although
further development is required in order to use it to investigate responses to
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elevated [CO2]. An important disadvantage of the method is that historical
changes in [CO2] may not reflect future changes, meaning that interpolative
power may be limited.
While enhanced growth rates from tree-ring analysis during the latter half
of the 20th century are correlated with the rise in atmospheric [CO2] (e.g.
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(LaMarche et al., 1984; Voelker et al., 2006; Knapp and Soule, 2008; Leal et
al., 2008), there is the inevitable difficulty of distinguishing responses to
elevated [CO2] from those to other environmental variables that increase
growth. To provide a clearer separation, dendrochronological studies have
been made on trees growing adjacent to natural CO2 springs, mostly in the
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form of CO2-emitting vents at former volcanic sites (Paoletti et al., 2004).
These sites, in principle, allow spatial study of the long-term impacts of [CO2]
gradients, although the vents may alter the soil and air temperatures and pH
which also affect tree growth (Paoletti et al., 2004). In addition, these springs
are often associated with saline groundwater and H2S emissions which are
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both toxic to plants (Van Gardingen et al., 1997). These effects and fluctuating
[CO2] (Van Gardingen et al., 1997) may explain why some papers report
increased growth (Miglietta et al., 1993; Hattenschwiler et al., 1997b) and
others no differences in growth rates of trees adjacent to springs compared to
trees at control sites (Körner and Miglietta, 1994; Tognetti et al., 2000).
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Carbon- (C-) isotope chronologies: Several studies link tree ring width
sequences with measurements of C-isotope ratios to assess how rates of
photosynthesis (A) and stomatal conductance (gs) have responded to
historical increases in atmospheric [CO2], and in association with CO2 springs
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or industrial CO2 sources ( Miglietta et al., 1996;Dusquesnay et al., 1998;
Feng, 1998, 1999; Tognetti and Penuelas, 2003; Saurer et al., 2004; Betson
et al., 2007; Liu et al., 2007). Changes in A and/or gs can lead to changes in
the ratio of intercellular (Ci) to ambient (Ca) [CO2]; Ci:Ca is linearly related to
the C-isotope ratio in leaves (Farquhar et al., 1982). Therefore,
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measurements of the C-isotope ratio in plant tissue can inform photosynthetic
and stomatal functioning over various time scales, which can then be related
to changes in atmospheric [CO2].
Carbon in CO2 has two possible stable isotopes, 12C and 13C. 12C is by far
the most prevalent, with a natural abundance of 98.89% compared with 1.11%
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for 13C. Discrimination occurs against the heavier 13CO2 during diffusion into
stomata and during fixation of CO2 by the enzyme Rubisco, so that plant
tissue is depleted in 13C relative to the atmosphere (Ehleringer and Rundel,
1989). The ratio of stable isotopes is expressed in delta notation, δ, measured
in parts per thousand (‰), and relative to an internationally accepted standard
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sample (PeeDee belemnite for 13C/12C). A more negative δ13C value indicates
greater discrimination against CO2 with the 13C isotope; the values for C3
plants range from -20 to -35‰ (Ehleringer, 1989). The ratio Ci:Ca, and hence
δ13C, is determined by the balance between CO2 supply, measured through
gs, and CO2 assimilation measured as A. In general terms, high gs relative to
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Amax results in high Ci:Ca and more negative δ13C (lower instantaneous wateruse efficiency), while low gs relative to Amax results in low Ci:Ca and less
negative δ13C (higher instantaneous water-use efficiency).
Combining C- and oxygen(O-) isotope ratios to separate stomatal and direct
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photosynthetic responses to environment: Although changes in C- isotope
ratios can reflect changes in Ci:Ca, this provides limited information on the
15
extent to which Ci is dependent on stomatal control (supply of C), or changes
in photosynthetic capacity (demand for C). Concurrent measurements of Cisotope ratio and the ratio of 16O (the more abundant oxygen isotope) to 18O
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(the less abundant isotope) can help to separate these processes, as δ18O in
plant material is influenced by gs, but not by Amax (Farquhar and Lloyd, 1993).
The negative relationship between gs and δ18O can provide insight into the
relative contributions that stomatal and non-stomatal control make to
variations in Ci:Ca as evidenced by δ13C (Farquhar and Lloyd, 1993). A
480
positive relationship between δ18O and δ13C indicates that gs is driving both
the variation in ambient:intercellular vapour pressure (Ea:Ei) and Ci:Ca (Saurer
et al., 1997; Scheidegger et al., 2000; Barbour et al., 2002); a negative or no
correlation between δ18O and δ13C, indicates that changes in Ci:Ca are driven
more by biochemical effects (Yakir and Israeli, 1995; Scheidegger et al.,
485
2000).
The relationship between δ13C and δ18O in tree rings has been used to
assess tree responses to elevated [CO2] and their relative impact on stomatal
conductance and photosynthesis (Saurer et al., 2003). Measurements of 14C
in wood of Quercus ilex trees were used to assess the relative uptake of C
490
from a CO2 spring in Italy and atmospheric CO2. The responses to elevated
[CO2] experienced by trees close to the spring were compared with control
trees that experienced ambient [CO2]. Trees near the spring had lower δ13C
and water-use efficiency than controls, but there were no differences in δ18O;
Saurer et al. (2003) concluded that gs was unaffected by elevated [CO2] and
495
that increased Ci:Ca was caused by reduced photosynthetic capacity. The
authors suggested this down-regulation of photosynthesis might have been
associated with a poor sink strength and low soil nitrogen availability at the
CO2 spring site.
Can a similar approach be used to investigate physiological responses to
500
historical changes in atmospheric [CO2]? Saurer et al. (2008) noted that
increases in atmospheric [CO2] in the last 30 years of the 20th century were
associated with divergence in relationships between tree ring δ18O and δ13C
and other climatic variables, causing problems for climate reconstruction.
They noted that corrections had been proposed for the CO2 fertilization effect,
16
505
but that these would be species-specific. Trends in tree ring δ18O and δ13C
could be used to infer how particular species have responded to historical
increases in [CO2].
While there are a number of methodological disadvantages associated
with interpretation of results from dendrochronological and stable isotope
510
studies, including appropriate timescales (Leavitt and Long, 1982, 1986;
Gessler et al., 2009), changing isotopic signals of the source (Thorburn and
Walker, 1993; McCarroll and Loader, 2004) and phenological effects on
isotopic composition (Bert et al., 1997; Schafer et al., 2000; McCarroll and
Loader, 2004), these techniques have the advantage of enabling in situ
515
assessment of large numbers of samples, and are applicable to mature trees
and ecosystems. The methods are well understood and developed, but
further development will be required if they were to be applied routinely in the
context of elevated [CO2]. There is scope to use the methods in association
with anthropogenic CO2 sources to explore likely response of species to [CO2]
520
higher than ambient.
5.2 Screening for indicator genes/metabolites
Recent advances in genomic sequencing (e.g. full genome sequence for
Populus trichocarpa, Tuskan et al. 2006; draft genome sequence for
525
Eucalyptus grandis, http://eucalyptusdb.bi.up.ac.za/) have prompted
discussion about the potential to breed or engineer trees to improve forest
productivity under elevated [CO2]. Gene expression influences C uptake and
utilization, biomass allocation, plant defence strategies and stress responses
(Cseke et al. 2009), which may influence how a species responds to elevated
530
[CO2] and determine whether photosynthetic upregulation translates into
increased growth. Micro-array studies have revealed altered levels of
expression in large numbers of genes in response to an increase of
atmospheric [CO2] in Arabidopsis thaliana (Li et al., 2006), soybean
(Ainsworth et al., 2006), sugarcane (De Souza et al., 2008), rice (Fukayama et
535
al., 2009) and poplar (Taylor et al., 2005). Genetic and phenotypic responses
17
vary both within a species (Li et al., 2006; Watkinson et al., 2008; Cseke et al.,
2009) and among species (Li et al., 1999).
Transcription patterns in leaves of two Populus tremuloides clones known
to have contrasting growth responses to elevated [CO2] were examined by
540
Cseke et al.(Cseke et al., 2009). While both clones had similar physiological
response to elevated [CO2] in terms of photosynthesis, stomatal conductance
and leaf area index, one grew much faster than the other. Total Rubisco was
significantly reduced in the non-responsive clone, but was not significantly
different in the responsive clone. The responsive clone had delayed leaf
545
senescence, resulting in a growing period 2 weeks longer than the
unresponsive clone, and accumulated ~50% more stem biomass than the
unresponsive clone. Micro-array and real-time RT-PCR analysis of transcript
abundance indicated that the responsive clone appeared to partition C into
active stress responses associated with carbohydrate and starch
550
biosynthesis, with subsequent enhanced growth; the unresponsive clone
increased passive defences such as lignin, phenylpropanoid and thickened
cell walls. Of the 183 genes that were differentially expressed, there was little
overlap between the two clones. The high level of intraspecific variation in
response to elevated [CO2] indicates that there is potential to select genetic
555
material with greater biomass yields under elevated [CO2], at least for shortrotation tree crops.
Downregulation of photosynthesis associated with acclimation to elevated
[CO2] has been linked to a reduced sink demand for assimilate (Ainsworth et
al. 2004). Genetic control of sink regulation of photosynthesis has not been
560
studied extensively in trees, but studies with other functional plant types may
offer some insights. For spinach, wheat and Beta vulgaris (sugarbeet), sink
regulation has been studied by feeding detached leaves (Krapp et al., 1991;
Kilb et al., 1996; Lee and Daie, 1997) or intact plants (Jones et al., 1996) with
sugars. These experiments demonstrated reduced transcription of nuclear
565
and chlorophyll genes, reductions in Rubisco protein, chlorophyll and the D1
protein of PSII, and increased leaf levels of sugars and starches. There is also
some evidence to indicate that hexose sugars initiate the signalling pathway
that leads to reduced expression of photosynthetic genes (Smith and Stitt,
18
2007). The key message is that similar responses have been observed in
570
wheat (Zhang et al., 2008), rice (Gesch et al., 1998) and several other plant
species, including trees (Van Oosten and Besford, 1996), in response to
elevated [CO2]. Direct comparisons of the effects of sugar feeding and
elevated atmospheric [CO2] on transcription of several nuclear and chloroplast
genes involved in photosynthesis have been undertaken in tomato (Van
575
Oosten and Besford, 1994). This study demonstrated parallel responses to
sugar feeding and elevated [CO2] in levels of transcripts for the Rubisco small
subunit, Rubisco activase, two proteins associated respectively with PSI and
PSII, and ADP glucose pyrophosphorylase. Parallel micro-array and
metabolomic studies directly comparing the effects of sugar feeding with those
580
of elevated atmospheric [CO2] would allow a more robust examination of the
potential to use sugar feeding as a surrogate for elevated [CO2] studies.
Taking into account intraspecific variation in [CO2] response, the use of clonal
plants would be advisable for this type of study. Screening for indicator
genes/metabolites has the potential to provide insights into the control of sink
585
regulation in trees. The approach has application for in situ assessment of
both small and large trees, and there is scope for high levels of replication and
rapid assessment (Table 1). A disadvantage is that processes are poorly
understood for trees, requiring methodological development before the
approach can be routinely applied.
590
5.3. Carbon limitation
Körner (2003) postulated that tree growth is not limited by C supply but
rather by intrinsic developmental rates and supply of water, nutrients and light.
In support of this there is considerable evidence that trees store large
595
quantities of C as mobile non-soluble carbohydrates (NSC’s), and that even in
times of peak sink activity, such as bud burst in deciduous species, these
stores are rarely depleted (Körner, 2006; Millard et al., 2007a).
Concentrations of NSC’s in plant tissues can be considered a measure of
C storage or supply for growth, that is, sink demand for C (Handa et al., 2005;
600
Sala and Hoch, 2009). A reduction in NSC concentrations indicates that
either C demand exceeds supply or that both source and sink activity are low
19
(Körner, 2003). A steady, high NSC concentration suggests that
photosynthesis fully meets, or exceeds, the C requirements for growth (Li et
al., 2002; Handa et al., 2005).
605
Sink limitation can be expected to become more important as source
strength increases under elevated [CO2] (Uddling et al., 2008). A large sink
capacity has been identified as critical for maximising plant production in
elevated [CO2] environments (Drake et al., 1997b; McCormick et al., 2008),
and significant feedbacks to source activity due to sink limitation can be
610
expected for plants with low sink strength. Ainsworth et al. (2004)
demonstrated acclimation of photosynthesis at elevated [CO2] in Glycine max
genetically modified to have low sink strength.
Better understanding sink limitations in tree species may assist in
identifying which species are likely to increase rates of biomass accumulation
615
in response to increasing atmospheric [CO2] and how this is modified by
growing conditions (Körner, 2006). Concentration of NSC as a measure of
the degree to which growth is C-limited (Körner, 2003) has been used to
examine growth limitations at tree-lines (Li et al., 2002; Handa et al., 2005),
and age-related growth decline in Pinus ponderosa (Sala and Hoch, 2009).
620
Understanding the temporal dynamics of NSC’s, particularly in roots and
stems, provides an approach for examining sink limitation on a whole tree
basis that can be applied in situ to trees at varying stages of development and
in varying growing conditions (Körner, 2003). If a plant is sink-limited under
current atmospheric [CO2], as defined by high carbohydrate concentrations in
625
stems and roots, then it is unlikely to respond positively to elevated [CO2]
unless other resources currently limiting growth become more freely available,
for example through improved water-use efficiency. Thus a measure of sink
limitation now offers an approach for screening the potential for growth
responses to increasing [CO2]. While this does not necessarily provide direct
630
evidence of actual growth responses to increased availability of C assimilates
(Körner, 2003), it provides a method for rapid screening of plants for potential
responses to elevated [CO2] (Table 1). The methods are well-developed
(Table 1, Figure 1), but further development will be required in order to
interpret spatial and temporal variation in carbohydrate concentrations in the
20
635
context of likely tree responses to elevated [CO2] and to determine inter- and
intra-specific variation in carbohydrate concentrations indicative of C
limitation.
6. Conclusions
640
While literature suggests that many species may benefit from elevated
[CO2] in terms of increased photosynthetic rates and water-use efficiency
(Long et al., 2004; Hyvonen et al., 2007), large increases in rates of biomass
accumulation and total biomass may not occur in mature forests in Australia
where growth is primarily limited by water and nutrient supply. Tree species
645
are likely to benefit most from elevated [CO2] during early growth and prior to
canopy closure when water and nutrient supplies may be less limiting to
growth. Commercial plantations may also benefit most from elevated [CO2] in
locations where other resources are not limiting growth.
It is likely that experiments examining responses to elevated [CO2] will
650
continue to focus on FACE and similar methods, although in many places
such as Australia costs have largely ruled out the use of FACE for forest
ecosystem studies. Our study has highlighted a number of experimental
approaches that can be used in conjunction with elevated [CO2] studies; these
may provide more rapid indications of likely effects of increasing [CO2] on
655
forest growth and biomass, thereby assisting forest managers in making
decisions today that bring about desirable outcomes in the future:
(1) Identify inter-and intra-specific variation in carbohydrate concentrations
in the context of where and when C, as opposed to other resources such as
water and nutrients, are likely to limit growth. Trees with high carbohydrate
660
concentrations are unlikely to be limited by C supply and hence are unlikely to
respond favourably to increasing CO2 availability. This will help to separate
the winners from the losers under future climates from a biodiversity
perspective in native forests, and to provide indications of appropriate
genotype x environment matching to maximise productivity in commercial
665
plantations. Körner (2003) suggested that examining carbohydrate dynamics
of wood and roots will help to provide insights into how to make correct
21
choices. The same approach has been used previously to examine limitations
to growth in extreme environments (Li et al., 2002; Handa et al., 2005).
(2) Use stable isotopes in wood cores to examine the history of forest
670
responses to increasing atmospheric [CO2] to provide clues as to which
species may respond favourably to increasing [CO2]. If they have exhibited
more rapid growth with increasing [CO2] in the past, then this suggests that
they may respond favourably as [CO2] continues to rise. However, teasing
apart the environmental conditions under which favourable responses occur
675
remains a challenge. Undertaking stable isotope studies close to
anthropogenic CO2 sources has the potential to provide insights into how
species may respond to the higher [CO2] that are predicted over the next
century. The methodologies are well-established.
(3) Select for greater biomass production under elevated [CO2] by focusing
680
on genetic and metabolomic regulation of source and sink activity. The
method offers potential for rapid screening of genotypes for improved
production under elevated [CO2]. While this approach has been used to some
extent in crop species, the methods are poorly developed for trees. Recent
advances in mapping tree genomes and in technology and capacity for rapid
685
screening of physiological traits and metabolites in young (seedling) trees
(http://www.plantphenomics.org.au) both improve our capacity to undertake
these kinds of studies. Feeding of sugars to intact plants may be a useful
surrogate for elevated [CO2] that would allow selection of genotypes most
appropriate for future [CO2] environments, and warrants further attention.
690
We argue that expenditure of a fraction of the money invested in FACE
and elevated [CO2] experiments could make a large contribution to the further
development of the above methods and provide useful information on
environmental constraints to growth and production of forest ecosystems
relevant to understanding responses to elevated [CO2] and forest responses
695
to environment more generally. The key advantages of the methods are that
they allow for high replication, can be performed in situ, and can provide
information pertinent to large as well as small trees. They all offer scope as
possible screening tools to help address management questions around
species choice to promote the sustainability of commercial forests under
22
700
changing environments, and biodiversity implications of changing climate. In
the absence of FACE, these approaches offer a way of understanding
potential forest ecosystem responses to elevated [CO2].
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30
List of tables and figures
1030
Table 1. Summary of the advantages and disadvantages of a range of
methods for assessing tree responses to elevated [CO2]
Table 2. A meta-analysis of tree-FACE effects on light-saturated CO2 uptake
(Amax), diurnal carbon assimilation (A’), stomatal conductance (gs),
instantaneous water-use efficiency (Amax/gs), maximum carboxylation rate
1035
(Vc,max), maximum rate of electron transport (Jmax), ratio of Vc,max/Jmax, N
content per area or mass, sugar content, starch content, plant height, leafarea index, specific leaf area, total yield and above-ground dry matter
production. The effect size is the ratio of performance in elevated to
atmospheric [CO2] (from Appendix 2, (Ainsworth and Long, 2005b)
1040
Figure 1. Comparison of existing and potential methods for examining tree
and ecosystem responses to elevated [CO2], in terms of ecosystem
coverage and the degree of realism provided by the system (e.g. in terms of
whether in situ measurements are possible; whether the method can be
used on small and large trees; whether the method allows for coupling
1045
between environment and ecosystem). The current state of methodological
development of each method is also presented.
Figure 2. Per cent enhancement of standing tree biomass over time
produced by an approximate 300 ppm increase in atmospheric CO2
concentration from ambient (from (Idso, 1999)). Data for loblolly pine (Pinus
1050
taeda), evergreen oak (Quercus ilex), and sour orange (Citrus aurantium)
trees growing out-of-doors and rooted in the ground were included in the
original study
31
Table 1. Summary of the advantages and disadvantages of a range of methods for assessing tree responses to elevated [CO2]
1055
Method
Questions that can be
addressed
Advantages
Disadvantages
Method development
required?
FACE
Detailed understanding of
physiological processes at tree
and ecosystem scales
Can include ecosystems
Expensive to establish and maintain
Well-established methods
In situ measurements are
possible
Generally low replication
Interactions between soil,
atmosphere and ecosystem
Good coupling to environment
Most studies focused on young and
not mature forests
Long timeframe for understanding
Growth responses
Some methodological issues eg
effects of step versus gradual
change in [CO2]
Natural CO2 vents
Detailed understanding of
physiological processes at tree
and ecosystem scales
Interactions between soil,
atmosphere and ecosystem
In situ measurements are
possible
Issues with effects of non-CO2
emissions on ecosystem processes
Can include mature forest
systems
Limitations in selecting controls
Good coupling to environment
Growth responses to elevated
[CO2]
Whole tree
chambers/glassho
use studies
Detailed understanding of
physiological processes at tree
scale
Dendrochronology
and stable isotope
Historical growth responses to
increasing atmospheric [CO2]
Long timeframe for understanding
Low replication
Good control of all variables
Artificial environment
Rapid results
Small plants only: will results be
relevant to mature forests and the
surrounding ecosystem?
Effects on older trees can be
examined
Difficulty in interpreting results due
to high levels of variability
Growth responses to elevated
[CO2]
32
Well established methods
Well established methods
Data collection and
interpretation requires
Method
studies
Questions that can be
addressed
Advantages
Disadvantages
Interaction of growth and
environment
Historical changes can be
examined to assess possible
future responses
Isotopic composition influenced by
factors other than CO2 responses
(eg phenology, changing isotopic
signal of source, other
environmental factors influencing
the tree response)
Photosynthetic drivers of growth
responses to elevated [CO2]
In situ measurements are
possible
Rapid results
Large numbers of
sites/replications possible
Indicator genes
and metabolites
Historical studies may not reflect
future changes in [CO2]
Studies near CO2 vents or
anthropogenic CO2 sources may be
influenced by factors other than
CO2 such as other gases and
salinity
Genetic control of sink regulation
under elevated [CO2]
In situ measurements are
possible
Processes poorly understood for
trees
Potential to breed or engineer
trees more productive under
elevated [CO2]
Rapid results
Requires broad screening within
ecosystems
Potential to use sugar feeding as
a surrogate for elevated
[CO2]studies
Provides fundamental
understanding of controllers of
photosynthetic responses to
environment, relevant more
widely than to eCO2
Method development
required?
further development to deal
with factors such as within
and between-ring
variability, effects of
changing isotopic signals
from source, phenological
effects on isotopic
composition
Requires development for
trees
Effects on older trees can be
examined
Large numbers of
sites/replications possible
Carbon limitation
Identification of species likely to
increase rates of biomass
In situ measurements are
possible
33
Does not provide direct evidence of
eCO2 responses
Methods are welldeveloped; interpretation of
Method
Questions that can be
addressed
accumulation in response to
elevated [CO2] through better
understanding of sink limitation
Role of other environmental
constraints on growth responses
to elevated [CO2]
Advantages
Disadvantages
Rapid results
Requires broad screening within
ecosystems
Effects on older trees can be
examined
Provides fundamental
understanding of limitations to
productivity relevant more widely
than to eCO2
Large numbers of
sites/replications possible
34
Method development
required?
results requires further
development.
1060
Table 2. A meta-analysis of tree-FACE effects on light-saturated CO2 uptake (Amax), diurnal
carbon assimilation (A’), stomatal conductance (gs), instantaneous water-use efficiency
(Amax/gs), maximum carboxylation rate (Vc,max), maximum rate of electron transport (Jmax), ratio
of Vc,max/Jmax, N content per area or mass, sugar content, starch content, plant height, leafarea index, specific leaf area, total yield and above-ground dry matter production. The effect
is the ratio of performance in elevated to atmospheric [CO2] (from Appendix 2, Ainsworth and
Long, 2005b)
1065
Variable
df
No of
No of Sites
Effect size (95% CI)
species
Amax
126
12
5
1.47 (1.43-1.52)
A’
19
5
2
1.29 (1.18-1.41)
gs
78
6
3
0.84 (0.79-0.89)
Amax/gs
26
4
3
1.74 (1.60-1.89)
Vc,max
71
11
4
0.94 (0.89-0.99)
Jmax
57
9
4
1.00 (0.96-1.04)
Vc,max/Jmax
61
11
4
0.97 (0.95-0.99)
N (mass/area)
36
3
3
1.02 (0.98-1.07)
N (mass/mass)
56
6
3
0.90 (0.87-0.93)
Sugar (mass/area)
10
2
2
1.11 (0.90-1.34)
Starch (mass/area)
10
2
2
1.37 (1.1-1.72)
Height
44
4
2
1.06 (1.04-1.09)
Leaf-area index
15
6
3
1.21 (1.04-1.40)
Specific leaf area
56
5
4
0.92 (0.89-0.95)
DM production
9
7
2
1.28 (1.06-1.54)
above-ground
35
Higher
C-limitation
Dendrochronology +
stable isotopes
Genomics and
metabolomics
Lower
Relative numbers of systems that can be explored
Ecosystem coverage
1070
e[CO2]
using
chamber or
glasshouse
FACE
Realism
(e.g. in situ measurements possible; appropriate for small and large trees; coupling between
environment and ecosystem)
Extent of methodological development
Methodology is well developed
Methodology is well developed but requires
further development to understand climate
change effects
Methodology at early stage of development
1075
Figure 1. Comparison of existing and potential methods for examining tree and ecosystem
responses to elevated [CO2], in terms of ecosystem coverage and the degree of realism
provided by the system (e.g. in terms of whether in situ measurements are possible; whether
the method can be used on small and large trees; whether the method allows for coupling
between environment and ecosystem). The current state of methodological development of
each method is also presented.
1080
36
Enhancement of standing
biomass (% difference over
control)
300
250
200
150
100
50
0
0
10
20
30
40
Duration of CO2 enrichment (years)
1085
Figure 2. Per cent enhancement of standing tree biomass over time produced by an
approximate 300 ppm increase in atmospheric CO2 concentration from ambient (from Idso,
1999). Data for loblolly pine (Pinus taeda), evergreen oak (Quercus
ilex), and sour orange (Citrus aurantium) trees growing out-of-doors
and rooted in the ground were included in the original study.
1090
37
38