Plant Physiology Preview. Published on August 19, 2014, as DOI:10.1104/pp.114.246207
Dani et al (2014)
Revised Research Article for Plant Physiology_Aug14-2014
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Running head (<50 characters):
Drought, photosynthesis and isoprenoid emission
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Corresponding authors:
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Kaidala Ganesha Srikanta Dani,
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Brian James Atwell,
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Department of Biological Sciences,
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Macquarie University,
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North Ryde,
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Sydney, NSW 2109
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Australia
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Primary research area:
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Ecophysiology and Sustainability
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Secondary research area:
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Photosynthesis and biogenic volatile isoprenoids
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Dani et al (2014)
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Footnotes:
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Financial source:
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Macquarie University Higher Degrees Research grant (HDR 40310819); Postgraduate Research
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Fund (PGRF Round 2- 2012); and International Postgraduate Research Scholarship from The
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Department of Industry, Innovation, Science, Research and Tertiary Education (DIISRTE),
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Commonwealth of Australia
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Experiment station:
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Macquarie University plant growth facility and glass house complex, Building F5A;
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The Light House Lab, Department of Biological Sciences (E7B and E8A);
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Department of Chemistry and Biomolecular Sciences (F7B);
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Macquarie University, North Ryde, Sydney, NSW 2109, Australia
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Corresponding authors; email [email protected] ; [email protected]
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Title of article (<150 characters)
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Increased ratio of electron transport to net assimilation rate supports
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elevated isoprenoid emission rate in eucalypts under drought
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Kaidala Ganesha Srikanta Dani*, Ian McLeod Jamie, Iain Colin Prentice, Brian James
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Atwell*
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Department of Biological Sciences, Macquarie University, North Ryde, Sydney, NSW 2109,
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Australia (K.G.S.D., I.C.P., B.J.A.);
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Macquarie University, North Ryde, Sydney, NSW 2109, Australia (K.G.S.D., I.M.J.); and AXA
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Chair of Biosphere and Climate Impacts, Grantham Institute for Climate Change and Grand
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Challenges in Ecosystems and Environment, Department of Life Sciences, Imperial College London,
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Silwood Park Campus, Buckhurst Road, Ascot SL5 7PY, United Kingdom (I.C.P.)
Department of Chemistry and Biomolecular Sciences,
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*Corresponding authors; email [email protected] ; [email protected]
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Summary (<200 characters):
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Volatile isoprenoids emitted by plants have a significant influence on ozone pollution and global
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climate. We show how changes in photosynthesis cause increased isoprenoid emissions in plants
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under drought.
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Abstract (<250 words):
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Plants undergoing heat and low CO2 stresses emit large amounts of volatile isoprenoids compared
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with those in stress-free conditions. One hypothesis posits that the balance between reducing power
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availability and its use in carbon assimilation determines constitutive isoprenoid emission rates in
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plants and potentially even their maximum emission capacity under brief periods of stress. To test
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this, we used abiotic stresses to manipulate the availability of reducing power. Specifically, we
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examined the effects of mild to severe drought on photosynthetic electron transport rate (ETR) and
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net carbon assimilation rates (NAR) and the relationship between estimated energy pools and
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constitutive volatile isoprenoid emission rates in two species of eucalypts: Eucalyptus occidentalis
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(drought-tolerant) and Eucalyptus camaldulensis (drought-sensitive). Isoprenoid emission rates were
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insensitive to mild drought and the rates increased when decline in NAR reached a certain species-
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specific threshold. ETR was sustained under drought and the ETR/NAR ratio increased, driving
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constitutive isoprenoid emission until severe drought caused carbon limitation of the MEP pathway.
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The estimated residual reducing power unused for carbon assimilation, based on the energetic status
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model, significantly correlated with constitutive isoprenoid emission rates across gradients of
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drought (r2 >0.8) and photorespiratory stress (r2 >0.9). Carbon availability could critically limit
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emission rates under severe drought and photorespiratory stresses.
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moderate abiotic stress-levels, increased isoprenoid emission rates compete with photorespiration for
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the residual reducing power not invested in carbon assimilation. A similar mechanism also explains
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the individual positive effects of low CO2, heat and drought stress on isoprenoid emission.
Under most instances of
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Introduction (<1000 words):
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The emission of volatile isoprenoids by plants (globally amounting to ~1000 TgC yr-1, of which
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isoprene constitutes ~500 TgC yr-1) plays a significant role in tropospheric oxidation chemistry (The
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Royal Society Report- Fowler et al., 2008). Plant isoprenoid emission at the ecosystem scale is
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determined not only by intrinsic biochemical and physiological controls but also by the relative
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abundances of species, each with characteristic baseline emission capacities and each subject to
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modification by environmental conditions (e.g. Harrison et al., 2013). While the effects of most
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environmental factors on isoprenoid emission have been documented (Loreto and Schnitzler, 2010),
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their interactions are likely to be complex and hold the key to accurate projections of global
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emissions (Arneth et al., 2007; Squire et al., 2014). The effect of soil water availability on plant
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volatile isoprenoid emission is crucial to the projections, especially as rainfall patterns are
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themselves subject to the impacts of climate change.
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Volatile isoprenoid emission is notably insensitive to moderate drought (when fraction of available
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soil water ranges from 40 to 70%; Fortunati et al., 2008; Centritto et al., 2011). Given the various
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other consequences of drought for plant function viz., stomatal closure leading to reduced
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photosynthesis (Lawlor & Cornic, 2002); increased leaf temperature (Jones 2004); leaf shedding
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(Tyree et al., 1993); reduced growth and potential hydraulic failure (Maherali et al., 2004); reduced
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shoot-to-root ratio (Poorter et al., 2012); increased oxidative stress due to the activation of reactive
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oxygen species (Mittler & Zilinskas, 1994); and an increased sucrose-to-starch ratio, affecting
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osmotic adjustment (Chaves, 1991), it is not surprising that there are large variations in experimental
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and field measurements of isoprenoid emission in response to drought (reviewed in
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Laothawornkitkul et al., 2009; Niinemets, 2010).
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Isoprene emission involves an energy-intensive biosynthesis through the methylerythritol phosphate
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(MEP) pathway in chloroplasts (reviewed in Sharkey & Yeh, 2001). Photosynthesis contributes the
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required carbon skeletons and reducing power for isoprenoid biosynthesis, at least under stress-free
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conditions (reviewed in Loreto & Schnitzler, 2010). The photosynthetic energy and reducing power
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output of a chloroplast is shared in unequal proportions among co-localized pathways (Table 1).
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Under favourable conditions photosynthetic carbon reduction (PCR) is the largest energy sink
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(~50%). Abiotic stresses enhance the supply of energy and reducing power to non-PCR sinks in the
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chloroplast (Haupt-Herting & Fock, 2002). Examples include (a) increased photorespiration under
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drought, at least until Rubisco is directly affected by stress (e.g. Lawlor, 1976; Noctor et al., 2002);
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(b) increased photorespiration under low-CO2, low-light or high-light stress (Kozaki & Takeba 1996)
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and (c) increased photo-reduction of oxygen under photo-oxidative stress (Makino et al., 2002). It
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has been posited that isoprenoid emission is a mechanism to consume surplus reducing power in
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stressful high-light and/or low-CO2 environments (Niinemets et al., 1999; Way et al., 2011).
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Increased accumulation of secondary metabolites such as phenols, alkaloids and isoprenoids have
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been documented in plants under abiotic stresses (reviewed in Wilhelm and Selmar, 2011).
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Decreased carboxylation and increased oxidative stress due to oversupply of reducing equivalents is
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seen as the main driver of increased secondary metabolism under drought (Selmar and Kleinwächter,
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2013). Post illumination behaviour of isoprene emission (primary and secondary bursts) under O2-
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free, pure N2 atmospheres have been attributed to availability of reducing equivalents (Rasulov et al.,
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2011; Li & Sharkey, 2013). It has further been proposed that the MEP pathway competes with other
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sinks for reducing power, so that the flow of reducing power to isoprenoid biosynthesis is
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proportional to the energy unused for primary metabolism (Morfopoulos et al., 2013, 2014; Dani et
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al., 2014). However, the MEP pathway’s requirement for reducing power is very small relative to
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that of photorespiration (Sharkey et al., 2008) and given the diversity in the relative sink strengths of
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intra-plastidic processes (Table 1), it is still unclear how the demands of these different processes
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influence one another.
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Eucalypts have been used as model systems to study plant isoprenoid emission since the early years
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of isoprenoid research (e.g. Guenther et al., 1991; Brilli et al., 2013).
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monoterpenes as well as constitutively emit isoprene and some monoterpenes (e.g. He et al., 2000).
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Eucalyptus camaldulensis subsp. camaldulensis (River Red Gum) is a drought-avoiding mesic
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species that is tolerant to waterlogging and distributed in riparian, temperate south-eastern Australia
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(Farrell et al., 1996). E. occidentalis (Swamp Yate) is a drought-tolerant species found in saline
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environments in mediterranean south-western Australia (Benyon et al., 1999; Searson et al., 2004).
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E. camaldulensis subsp. obtusa is the most widespread eucalyptus in subtropical Australia (Butcher
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et al., 2009). Exploiting this ecological contrast, we empirically tested the hypothesis that the
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isoprene emission is driven by ATP and NADPH availability (Loreto and Sharkey, 1993; Niinemets,
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1999) and could potentially compete for the same with carbon assimilation (Harrison et al., 2013;
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Morfopoulos et al., 2014). We manipulated the energy source-sink dynamics by imposing various
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abiotic stresses including drought, heat, low CO2, and high O2. We investigated the relationship
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between three plastidic biochemical processes (carbon assimilation, photorespiration and volatile
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isoprenoid emission) in eucalypts acclimated to drought for four to six months. Interactive effects of
All eucalypts store
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short-term exposure to five CO2 concentrations, three O2 levels and heat stress on isoprenoid
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emission rates were also analysed. It was hypothesised that the relative sink strength of various
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processes requiring reducing power in the chloroplasts could determine the variations of isoprenoid
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emission in plants experiencing abiotic stress. We started with a premise that the light-dependent and
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light-independent reaction components of photosynthesis have different susceptibilities to abiotic
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stress (particularly to drought) and that these susceptibilities vary across species.
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Results:
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The results are from three independent experiments (see methods, Table 2).
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Photosynthesis
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(a) Acclimation to drought in paired species (at 20% O2): E. occidentalis had a significantly
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higher stomatal conductance (gs, P = 0.043) when watered to field capacity (FC) than E.
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camaldulensis subsp. camaldulensis but the difference in transpiration rates was not
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significant (Tr, P = 0.193). Net assimilation rate (NAR) of both E. occidentalis (16.8 ± 2.28
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µmol m-2 s-1) and E. camaldulensis subsp. camaldulensis (18.1 ± 1.86 µmol m-2 s-1) were
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comparable (test of equal means, P = 0.001). During acclimation to severe drought stress (FC
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≤ 50%), E. camaldulensis subsp. camaldulensis showed a significant decline in all
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photosynthetic parameters (P < 0.001) whereas net assimilation in E. occidentalis (15.3 ±
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1.81 µmol m-2 s-1) although decreased, remained comparable to control values despite a
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significant decrease in stomatal conductance (Fig. 1A, 1D). Under well-watered conditions,
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estimates of photosynthetic linear electron transport rate (ETR) based on chlorophyll
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fluorescence for E. occidentalis and E. camaldulensis subsp. camaldulensis showed a
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consistent proportionality with their respective NARs. For E. occidentalis the ETR/NAR
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ratio remained unchanged even at 50% FC while the ratio significantly increased (doubled)
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for E. camaldulensis (Fig. 2A). The carbon cost of isoprenoid emission as a proportion of net
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assimilation increased >10 fold as drought intensified and was highest in both species at 25%
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FC (Fig. 2B). ETR was measured independently using fluorescence and was not coupled with
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LiCor measurements (Experiment 1, Table 2). Hence, the observations should be treated
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only as indicative and not as absolute. ETR/NAR ratios (~7:1 under 20% O2) were more
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realistic when obtained after fitting A-Ci curves (Experiment 3; Fig. S3B). ETR/NAR ratio
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across drought treatments follow a near-significant quadratic regression with total emission
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rates (r2 = 0.75; P = 0.12; Fig. 2C).
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(b) Response to heat and CO2 in E. camaldulensis subsp. obtusa: Heat caused a significant
decline in photosynthesis under elevated CO2 (≥1000 µmol mol-1, P < 0.001). Leaves at 38ºC
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transpired at a significantly higher rate than those at 28 ºC (P < 0.0001). Heat did not cause a
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significant change in either gs or Tr under normal O2 in plants under drought (50% FC). This
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was true across most of the CO2 range (400 to 1800 µmol mol-1), except at 180 µmol mol-1
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CO2 and at the photorespiratory compensation point (60 µmol mol-1, normal O2). Leaves of
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well-watered plants did not show a decline in NAR when subjected to 38 ºC under present-
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day ambient CO2 (400 µmol mol-1) and normal O2 (Fig. 3A). Heat had a generally positive
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effect on Tr, under normal O2 at 100% FC (but not under drought) and it was pronounced at
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CO2 ≤ 400 µmol mol-1 (Fig. S1).
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(c) Response to varying O2 concentration: Net assimilation rate in both species significantly
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increased (>30%) during low O2. The gain was proportional to their respective basal rates
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under normal O2 except for E. camaldulensis subsp. camaldulensis at FC ≤ 50% (Fig. 1B,
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S3). During low O2, Tr did not change significantly at 100% FC (equal means, P = 0.002)
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despite decreases in gs and (as a result) a decrease in leaf-internal CO2 (Ci). Tr was insensitive
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to CO2 concentrations under low O2 in E. camaldulensis subsp. obtusa (Fig. S1, P > 0.1).
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Low O2 had a significant negative effect on transpiration and net assimilation in E.
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camaldulensis subsp. camaldulensis only under acute water deficit (Fig. 1C, FC ≤ 50%, P <
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0.0001, also see Fig. S3A). High O2 (50% O2) caused significant increase in Ci, severe
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decline in net assimilation rate in E. camaldulensis subsp. camaldulensis and the effect was
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persistent and amplified under drought (Table S1).
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Volatile isoprenoid emission:
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(a) Response to drought: Branch-level basal isoprene emission rate (Ie) at 25 ºC, 1200 µmol m-2
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s-1 PAR, and present day CO2 and normal O2 was 40% higher (P = 0.004) in E. camaldulensis
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subsp. camaldulensis (5.9 ± 1.48 nmol m-2 s-1) than in E. occidentalis (3.4 ± 1.99 nmol m-2 s-
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) and the rates remained unchanged in both species despite acclimation to moderate drought
(70% FC). The trends were conserved in leaf level measurements. There was a tiny decrease
(relative to control) in net assimilation rate in E. occidentalis at 50% FC (∆NAR= −1.5 µmol
m-2 s-1) and it was accompanied by a marginally significant increase in isoprene emission rate
(∆Ie= +1.4 nmol m-2 s-1; Fig. 1E; P=0.05; Note: There is three orders of magnitude difference
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between amounts of carbon fixed via photosynthesis and carbon lost via emission). The
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biggest increase in Ie for these two species occurred at two different drought intensities. Ie
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peaked significantly at 50% FC for E. camaldulensis (P < 0.005, Fig. 1E), and at 25% FC for
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E. occidentalis (P < 0.001).
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increase in their respective ETR/NAR ratios (Fig. 2A, 2C) but the emission declined for E.
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camaldulensis subsp. camaldulensis although ETR/NAR increased further at 25% FC.
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Constitutive monoterpene emission rate, Me (pinenes and d-limonene) in E. camaldulensis
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subsp. camaldulensis behaved in a manner similar to isoprene while Me in E. occidentalis did
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not respond to drought even at 25% FC.
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camaldulensis subsp. camaldulensis were comparable in magnitude across the drought
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gradient (Fig. 1E and 1F). Ie to Me molar ratio was roughly 10:1 in both species. The
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subspecies obtusa showed a basal Ie/Me molar ratio of ~2:1.
These emission peaks coincided with the first noticeable
Ie and Me in both E. occidentalis and E.
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(b) Response to heat and CO2 in E. camaldulensis subsp. obtusa: Heat was the most significant
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factor causing an increase in Ie (P < 0.0001) and Me (P < 0.001) across all treatments. At 28
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ºC, 100% FC (N = 6) and 20% O2, Ie showed a significant peak at 180 µmol mol-1 CO2 (P <
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0.001) and almost full inhibition at the saturating CO2 level of 1800 µmol mol-1 (Fig 3B and
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4). This response completely disappeared at 38 ºC (Fig. 3B). Under normal O2, the Ie
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response at 50% FC without heat stress (28 ºC) was equivalent in magnitude to Ie observed in
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the well-watered plants subjected to heat stress (38 ºC; P < 0.001) irrespective of CO2
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acclimation (Fig. S5). Plants acclimated to drought (50% FC) and exposed to heat stress (38
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ºC) showed the highest isoprenoid emission. High-CO2 induced inhibition of isoprene
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emission at 28 ºC disappeared at 38 ºC and was accompanied by a significantly low stomatal
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conductance and low leaf internal CO2 (Fig. S1).
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(c) Response to varying O2 concentration: Exposure to 2% O2 (10 min) resulted in a marginal
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increase in Ie and Me across the drought gradient (Fig. 1E and 1F) and these trends were
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conserved in both E. occidentalis and E. camaldulensis subsp. camaldulensis, except that the
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latter showed no significant change in Ie at 100% and 70% FC. Low O2 on its own did not
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significantly affect Me in E. occidentalis (Fig. 1f) or in E. camaldulensis subsp. obtusa (P >
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0.6, Fig. 3C). Low O2 significantly increased Ie at CO2 = 1800 µmol mol-1 (no heat stress)
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compared to lower CO2 levels, which was opposite to the effect under normal O2 (P <
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0.0001). Low O2 also had a positive effect on the Ie of well-watered plants at 38 ºC. In E.
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camaldulensis subsp. camaldulensis Ie increased when well-watered plants were exposed to
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50% O2 (relative to plants at 20% O2). Emission rate decreased significantly when the plants
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simultaneously experienced drought (50% FC) and high oxygen (50% O2). The effect of low
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O2 on Ie was more pronounced than on Me in all three taxa (P < 0.001) and increased Ie under
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low O2 resulted in a decreased Me in well-watered plants (Fig. 5A, 5C).
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The relationship between leaf energetic status and isoprenoid emission rate: The estimated electron
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transport rate not used for light independent reactions (Jr) correlated significantly and positively with
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isoprenoid emission rate in both species (r2 = 0.81; P = 0.014) and it was consistent across drought
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gradient (Fig. 4A).
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isoprenoid emission rate (Fig. 4B) was consistent with the ETR/NAR ratio peaking with increased
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emission in both species. The relationship between Jr and Ie estimated at three different oxygen
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concentrations was significantly positive (r2 > 0.91; P = 0.02) for E. camaldulensis subsp.
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camaldulensis (Fig. 5A). Plants experiencing drought (50% FC) exposed to 50% O2 were the only
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exception to the trend and showed a significant decline in isoprenoid emission despite large Jr (Fig.
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5A). E. camaldulensis subsp. camaldulensis exposed to extreme photorespiratory stress without
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drought (100% FC, 50% O2) and drought without extreme photorespiration (50% FC, 20% O2)
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resulted in large increases in J/Vcmax (Fig. 5B).
The positive relationship between J/Vcmax (derived from A-Ci curves) and
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Discussion:
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Drought, ETR/NAR ratio and constitutive isoprenoid emission
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The insensitivity of ETR to drought in both species in this study confirmed that the photosystems and
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the electron transport chain are not susceptible to moderate drought stress (Ben et al., 1987) and the
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relative decrease in ETR under drought is proportionately less when compared to decrease in CO2
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assimilation (Cornic and Briantais, 1991, Bota et al., 2004). The absolute rates of isoprene and
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constitutive monoterpene emission did not change provided that the simultaneous assimilation rate of
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CO2 remained unchanged.
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The mediterranean species Eucalyptus occidentalis maintained almost an unchanged NAR and
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isoprene emission rate even at 50% FC (Fig. 1D and 1E). E. camaldulensis subsp. camaldulensis
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showed a gradual and more marked decline in NAR with increasing water deficit. ETR/NAR ratio
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significantly increased at 25% FC for the former and at 50% FC for the latter, the point where the
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species showed its highest isoprene and monoterpene emission rates (Fig. 1, 2A, 2C). We attribute
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the increased isoprene emission rates under drought to increased ETR/NAR ratio and increased
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availability of reducing power to the MEP pathway among other non-PCR sinks (Fig. 4). Under
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severe drought (25% FC) despite a favourable ETR status of its leaves, isoprenoid emissions of E.
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camaldulensis subsp. camaldulensis declined suggesting carbon limitation despite 2% oxygen (see
decline in NAR at %FC ≤50; Fig. S3A). It was later confirmed that low O2 exposure did not
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significantly affect ETR/NAR ratios both under well-watered and droughted conditions (Fig. S3B).
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Similarly, imposing severe photorespiratory stress (50% O2) on well-watered plants resulted in
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increased isoprene emission rate and the rates plummeted under drought despite a large pool of
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residual reducing power (Fig. 5).
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photorespiration and the MEP pathway) compete for reducing power not allocated to carbon
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assimilation reactions under situations of sub-optimal carbon assimilation due to abiotic stress.
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Although eucalypts may only have a modest capacity for non-photochemical quenching (NPQ) due
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to their typically high photosynthetic capacities and acclimation to high-light habitats, the proportion
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of energy dissipated through NPQ, which is one of the primary mechanisms to mitigate oxidative
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stress, increased in E. camaldulensis subsp. camaldulensis as drought intensified (Fig. S2). NPQ is
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likely to be a significant sink for ETR and may also account for reduced emissions under severe
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stress (Fig. 5A).
The results suggested that non-PCR reactions (especially
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Drought, photorespiration and constitutive isoprenoid emission
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The direction of change in the rates of isoprene emission and photorespiration in response to many
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environmental factors are same, despite absence of a biochemical (carbon based) link between the
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two pathways (e.g. Monson and Fall, 1989; Hewitt et al., 1990; Loreto and Sharkey, 1990). In this
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study, net assimilation and isoprene emission rates increased by a small yet significant extent in well-
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watered E. occidentalis exposed to short-term low O2 (Fig. 1D, 1E; in agreement with Hewitt et al.
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1990) and such an increase persisted under drought. Increased de novo carbon pool and decreased
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competition for ATP and NADPH may explain the small increase in isoprene emission under low O2
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in well-watered plants, given that ATP could be limiting emissions when carbon is plentiful (Loreto
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and Sharkey, 1993). Although increased isoprene emission in low O2 under most conditions may be
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physiologically important, it is not comparable to the large difference in emission between well-
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watered plants (low emission) and plants experiencing drought (high emission) under 20% O2 (Fig.
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1E). Increased emission under drought is sustained so long as the intensity of drought is within a
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species-specific tolerance threshold. When such a threshold is exceeded, isoprene emission rate
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significantly decreased (at 25% FC for E. camaldulensis subsp. camaldulensis; Fig. 1E). Since we
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did not directly estimate photorespiration rates under drought (which is known to significantly
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increase under abiotic stress), we increased photorespiratory stress in well-watered plants to mimic
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alternative scenarios. When photorespiratory stress is extreme (50% O2) and it is coupled with
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368
reduced carbon assimilation capacity due to drought (50% FC), the MEP pathway suffers a ‘double
369
jeopardy’ and is likely deprived of both carbon and reducing power (Fig. 5A).
370
371
The cost of carbon due to isoprenoid emission increased nearly 15 fold from 0.46% of freshly fixed
372
carbon in well-watered plants to 7.2% in severely stressed E. camaldulensis subsp. camaldulensis
373
(Fig. 2B). Although alternative carbon imported from the cytosol avoids carbon-limitation of the
374
MEP pathway under moderate abiotic stress (Brilli et al., 2007; Funk et al. 2004; Trowbridge et al.,
375
2012), under extreme stress carbon could be limiting due to irreparable biochemical impairment of
376
both the PCR cycle and rates of carbon import into plastids. Whether drought just inhibits carbon
377
assimilation rate through stomatal diffusional limitation or there is clear biochemical down-
378
regulation is highly debatable (e.g. Flexas et al., 2004). Under severe drought stress (especially for E.
379
camaldulensis subsp. camaldulensis) even those limited number of leaves that were retained by the
380
plants could not have recovered fully if the plants were re-watered. In such cases, diffusional
381
limitation was likely compounded by impairment of photosynthetic biochemical machinery. Limited
382
catalytic activity of the MEP pathway, particularly isoprene synthase (Brilli et al., 2007), could have
383
contributed to decreased emissions under extreme stress.
384
385
Drought, low CO2, heat and constitutive isoprenoid emission
386
In E. camaldulensis subsp. obtusa, short-term acclimation to heat stress (38 ºC) caused significantly
387
higher emissions with no significant change in net assimilation despite drought (50% FC, Fig. 3).
388
CO2 inhibition of isoprene emission also disappeared at high temperatures (as reviewed in Sharkey
389
and Monson, 2014). It is known that moderate heat stress can suppress ETR yet increase both Vcmax
390
and isoprene emission rate (e.g. Dreyer et al., 2001; Darbah et al., 2008). Cold and heat treatments
391
are shown to selectively suppress PS II (linear electron transport) and thus reduce NADPH
392
availability and up-regulate PS I (cyclic electron transport) to increase ATP production (e.g. Huner et
393
al., 1993; Zhang and Sharkey 2009). All of these observations appear to contradict the view that
394
reducing power availability (however small may the requirement be) influences variation in volatile
395
isoprenoid emission. For the moment if we ignore cold stress, which is not relevant to isoprene
396
emission, at least to the extent we understand the phenomenon today, the energetic status model may
397
be inadequate to explain emission behaviour at high temperatures for the following reasons (a)
398
prolonged heat stress reduces net assimilation rate (despite an increase in Vcmax), primarily due to
399
decreased CO2 solubility and decreased Rubisco–CO2 affinity (Sage and Kubien, 2007); (b)
400
prolonged heat and drought stress (when imposed together) reduce emission, possibly due to heat
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401
sensitivity of the cytosolic carbon pool (Fortunati et al., 2008; Centritto et al., 2011); and (c) extreme
402
stress not only reduces net assimilation rates but also increases photorespiratory drain on carbon and
403
reducing power (Fig. 5).
404
405
Unlike isoprene emission, the response of constitutive monoterpene emission did not follow a
406
consistent pattern across any of the treatments (Fig. 1F and 3C).
407
camaldulensis, constitutively emitted monoterpenes behaved like isoprene while in E. occidentalis,
408
monoterpene emission was not sensitive even to severe drought. This could be partly due to
409
sustained monoterpene synthase activity during drought, as reported in evergreen oaks (Lavoir et al.,
410
2009). Oddly, isoprene emission increased in leaves simultaneously exposed to 28 ºC, 1800 µmol
411
mol-1 CO2 (not saturating for eucalypts) and 2% O2 (Fig. 3B). For reasons unknown, the same leaves
412
also showed a significant (16%) decrease in net assimilation rate (Fig. 3A). We speculate that the
413
possible (temporary) inhibition/down-regulation of other non-PCR sinks of reducing power such as
414
photoassimilation of nitrogen under very high CO2 (Bloom et al., 2002) could have also contributed
415
to increased isoprene emission. However, it is acknowledged that the PCR cycle and nitrate
416
assimilation, both do not directly compete for reducing power (Robinson, 1988).
417
between isoprene and monoterpene emissions in response to CO2 (28 ºC; Fig. S4) and low O2 (Fig.
418
5C) indicates that emission of isoprene and monoterpenes is inversely related (also see Harrison et
419
al., 2013).
In E. camaldulensis subsp.
A trade-off
420
421
Increased secondary metabolism and specifically increased isoprene emission under drought could
422
be involved in protecting photosystems against transient periods of oxidative and heat stress, as seen
423
in some transgenic studies, although the mechanisms are unclear (Behnke et al., 2007; Velikova et
424
al., 2011; Selmar and Kleinwächter, 2013; Ryan et al., 2014). However, the tiny increase in isoprene
425
emission when photorespiration (the largest photoprotective sink) is suppressed, despite down-
426
regulation of PCR under drought, suggests that the MEP pathway has limited capacity to oxidize the
427
pool of excess reductants available under abiotic stress. The increased isoprenoid emission rates
428
among plants experiencing drought (without heat stress), heat (without drought stress; Fig. S5) and
429
artificially increased photorespiratory stress (without drought and without heat at 50% O2) were
430
quantitatively equivalent and more experiments are needed to differentiate the underlying
431
mechanisms between these responses.
432
433
Conclusions:
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434
The energy (ATP) and reducing power (NADPH) budget of the chloroplast are used in a hierarchical
435
fashion. The PCR cycle dominates while, possibly, all other reducing sequences co-localized in the
436
chloroplasts must compete for the remaining pool (Table 1). The equilibrium between the source
437
(light reactions) and major sinks (carbon reduction and photorespiration) of energy, as well as
438
sources (de novo and stored) and sinks (all anabolic processes) of carbon, becomes distorted under
439
drought stress. Drought-induced reduction in the PCR cycle is accompanied by an increase in
440
ETR/NAR ratio and a significant increase in volatile isoprenoid emission. The qualitative response
441
of isoprenoid emission under drought may be similar among species, but the degree of drought-
442
induced shift in the J/Vcmax ratio is species-specific. While energy-availability is clearly the common
443
factor that underpins the individual effects of low CO2 (Morfopoulos et al., 2014), heat, drought and
444
photorespiratory stress (this study) on isoprenoid emission, the complex interactive effects of heat,
445
CO2 and drought seem to defy simple assumptions and remain largely uncertain (Fig. 3). Variation
446
in atmospheric O2 concentration between 10 to 35% during the last 200 million years (Falkowski et
447
al., 2005) and its influence on carboxylation efficiency could have also played an important role in
448
regulating global isoprene emissions on a macroevolutionary time scale. All of these indicate a need
449
for a wider experimental analysis across different functional plant types and ecosystems if we are to
450
reliably ‘scale up’ volatile isoprenoid emissions from plants to large regions.
451
452
Materials and methods:
453
The study included three independent and yet mutually supporting experiments (Table 2).
454
Rationale for selecting species: A group of 15 species of eucalypts belonging to distinct biomes
455
within Australia were screened for photosynthetic performance and isoprenoid emission potential in
456
March 2012. The first experiment involved a paired comparison of isoprenoid emission rates and
457
photosynthesis in Eucalyptus occidentalis and Eucalyptus camaldulensis subsp. camaldulensis in
458
response to drought acclimation. E. camaldulensis subsp. camaldulensis and E. occidentalis were
459
studied as a pair in Experiment 1, as both had comparable photosynthetic capacities and both
460
predominantly emitted isoprene and some monoterpenes at significant levels. A desirable contrast in
461
the physiology of their water-relations is already highlighted (see introduction).
462
The second experiment tested whether known CO2 and heat responses of isoprenoid emission are
463
consistent under drought acclimation. The idea was to test any potential pathway discrimination
464
towards either isoprene or monoterpene emission under abiotic stresses. E. camaldulensis subsp.
465
obtusa was selected for Experiment 2, because it emitted comparable quantities of isoprene and
466
constitutive monoterpenes.
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467
The third experiment was a supplementary exercise that was inspired by the results of the first
468
experiment. The third experiment explicitly tested the relationship between photorespiration and
469
isoprenoid emission under drought in E. camaldulensis subsp. camaldulensis.
470
Eucalypts store monoterpenes and it is difficult to estimate instantaneous carbon and energy invested
471
in monoterpenoid biosynthesis. However, we took necessary precautions to rule-out monoterpene
472
emissions from stored pools (from leaf glands). Even if one considers both constitutive and stored
473
monoterpenes together, the quantities are smaller (in the paired species of experiment 1) than that of
474
isoprene emission by an order of magnitude. Besides, it is recently shown that stored monoterpenes
475
are quantitatively insensitive to drought stress in eucalypts although there are clear qualitative
476
variations and inconsistent trends in secondary metabolite accumulation (phenolics and terpenoids)
477
in various plants under drought (Brilli et al., 2013; Selmar and Kleinwächter, 2013).
478
479
Plant Material: Seeds of Eucalyptus camaldulensis subsp. camaldulensis (Dehnh), E. occidentalis
480
(Endl) were obtained from the Australian Tree Seed Centre at CSIRO (Canberra) and germinated in
481
May 2012. Two-to three-month-old seedlings (N = 8 per species) were transplanted to large pots
482
comprising ~80 kg of red soil (from the Robertson area in NSW) and the required quantities of
483
Osmocote® slow release fertilizer.
484
(Blakely) (N = 6) and was established in similar large pots. The plants were grown under the open
485
sun with regular watering. Six-month-old saplings (Dec 2012) were transferred to and kept until the
486
end of the experiment in a glasshouse maintained at a 25 °C/18 °C diurnal temperature cycle and
487
natural photoperiodic regime.
488
camaldulensis (N = 5) were germinated in March 2013 and grown in a similar manner for one year
489
(used for experiment 3). These plants were maintained at 100% FC and isoprenoid emission rates
490
were determined at three different oxygen levels (2, 20 and 50%) in April-May 2014. After the
491
measurements were complete, the plants were droughted to achieve 50% FC and maintained (over 10
492
days). Gas exchange measurements and volatile sampling were repeated.
An independent group of E. camaldulensis subsp. obtusa
A third independent group of Eucalyptus camaldulensis subsp.
493
494
Water relations: The soil water holding capacity was determined by water saturation and weighing.
495
Five-month old saplings were watered and weighed (pot + plant) to obtain the 100% field capacity
496
(FC) reference point. Plants were grouped into two sets of four biological replicates per species.
497
One set was maintained at 100% FC throughout the experiment while the other received reduced
498
water to achieve 70% FC (within two weeks) and maintained thereafter for three months. After
499
volatiles were sampled from plants acclimated to 70% FC, watering was further reduced to achieve
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500
50% FC and acclimated for two weeks followed by volatile sampling (repeated for 25% FC). One
501
batch of E. camaldulensis subsp. obtusa was acclimated to 50% FC for one month before volatile
502
sampling. The difference in acclimation period between experiment 2 and 3 is due to the species
503
involved. In experiment 2 we had E. camaldulensis subsp. obtusa, which comes from lower latitudes
504
of Australia and it grows in some of the driest places on the continent. It could endure longer periods
505
of drought and also took longer to acclimate (checked by measuring stomatal conductance on
506
randomly selected leaves) while E. camaldulensis subsp. camaldulensis stabilised more quickly as
507
well as reflecting the effects of drought sooner. In experiment 3 we had only E. camaldulensis and
508
we could shorten the acclimation period. The duration of severe stress was shorter than the duration
509
at 70% FC to avoid severe defoliation (especially in E. camaldulensis subsp. camaldulensis) that
510
would have otherwise hampered emission measurements. Leaf water potential was determined
511
(experiment 1) using a 12-channel thermocouple psychrometer (JRD Merril Specialty Equipment,
512
Logan, Utah, USA) calibrated at 25 °C using standard sodium chloride solutions (Lang 1967). Leaf
513
discs (diameter = 0.5 cm) were bored out at predawn from one half of a fully expanded leaf and
514
transferred and sealed in the psychrometer (10 leaves per treatment group). The chambers were
515
equilibrated in a water bath at 25 °C for 3 hr before signal acquisition. The procedure was repeated
516
on the following midday using a leaf disc punched out from the other half of the same leaf.
517
Measurements were repeated at three time points during drought treatment (see Fig. 6 for the
518
relationship between leaf water potential and % field capacity for a physiological assessment of
519
drought intensity).
520
521
Monitoring photosynthesis under drought:
522
(a) Photosynthetic gas-exchange measurements were made using a LI-6400XT (Li-Cor
523
Biosciences, Lincoln, Nebraska, USA) infrared gas analyser. During branch-level sampling,
524
photosynthesis was measured between 12 noon and 3 pm in parallel to isoprenoid sampling
525
on independent branches of the same plant. Leaf temperature was 25 °C, light intensity was
526
1200 µmol m-2 s-1, and relative humidity ranged from 39 to 47%.
527
A-Ci curves were obtained from one or two of the best performing healthiest leaves from each
528
biological replicate (N=4) per treatment group using a Li-Cor6400 at 25 °C and normal O2
529
(also at 2% and 50% O2 for experiment 3). Vcmax and J were estimated using the curve-fitting
530
tool described in Sharkey et al., 2007 (minimised errors) and the mean values were used as
531
model input parameters. Jr (the portion of the electron transport rate not used for dark
16
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Revised Research Article for Plant Physiology_Aug14-2014
532
reactions of photosynthesis, given Ci and Vcmax) was calculated following Harrison et al.,
533
(2013) and Morfopoulos et al., (2014).
୴ 4ୡ୫ୟ୶ ୧ 2Γ
‫כ‬
୧ ୫
୰ ୴
1 2
534
Given Vcmax, we calculated Jv and then substituted (1) in (2) where, Jv = proportion of
535
electron transport used for dark reactions; Vcmax = maximum carboxylation rate by Rubisco;
536
Km= effective Michaelis-Menten coefficient for carboxylation by Rubisco (700 µmol mol-1 at
537
538
25 ºC); Γ*=photorespiratory compensation point (60 µmol mol-1); Ci = species-specific leaf
internal CO2 concentration (µmol mol-1) at ambient CO2 = 400 µmol mol-1.
539
540
(b) Chlorophyll fluorescence was monitored using a Pocket PAM chlorophyll fluorescence meter
541
(Gademann Instruments GmbH, Wurzburg, Germany).
542
induction curves were obtained from dark adapted leaves (pre-dawn) between 3 and 5 am and
543
replicated on 15 fully expanded leaves per treatment group (N=4, biological replicates). The
544
measurements were made in a dark room (PPFD < 10 µmol m-2 s-1). Pre-dawn leaf
545
temperature was 15.5 ± 0.3 °C. During the day (between 2 and 4 pm), leaves experiencing
546
moderate light levels (350 < PPFD < 450 µmol m-2 s-1) were used to estimate linear electron
547
transport rates using steady-state light induction curves by gradually increasing the pulse
548
intensity from 0 to 1500 µmol m-2 s-1. Pre-dusk leaf temperature was 24.5 ± 0.8 °C. The data
549
for ETR/NAR was measured only after the dry-group had been acclimated to 50% FC and the
550
control group (100% FC plants) was retained through-out the experiment.
551
Kautsky dark-light fluorescence
Volatile isoprenoid sampling
552
(a) Branch enclosure method: 25 L capacity Tedlar® bags (Sigma) were modified to make a
553
volatile collection chamber with a PTFE (polytetrafluoroethylene) base for air tight sealing.
554
The gas exchange line was plumbed with Teflon tubing and stainless steel air tight connectors
555
(Swagelok®). High-purity instrument-grade air (BOC; 78% N2, 21% O2, 1% Ar) was mixed
556
with CO2 (beta mix 5% ± 0.1% in N2) to achieve ambient CO2 (400 ± 10 µmol mol-1)
557
concentration in the head space containing the branch. The unit was flushed at 20 L min-1 for
558
10 min before each sampling to remove memory effects (after Niinemets et al., 2011). A
559
branch was inserted into the chamber and sealed around at the base. Plants were provided
560
with natural PAR at 800 to 1200 µmol m-2 s-1 during sampling. The chamber temperature
17
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Revised Research Article for Plant Physiology_Aug14-2014
561
was maintained at 30 ± 2 °C and leaf temperature was 26 ± 2 °C measured using an infra-red
562
thermometer (Agri-Therm IIITM, Everest Interscience Inc, USA). Relative humidity varied
563
from 29 to 52%. Sterile fritted glass thermal desorption (TD) tubes comprising Carboxen
564
1016 and Carbopack X adsorbents (Sigma Supelco®) were conditioned at 250 °C through
565
helium purging (100 mL min-1; 60 min). Volatiles were collected on to TD tubes in April and
566
May 2013 using an oil-free pump connected to a mass flow controller (Brooks® 5850E).
567
Chamber blank control, TD tube secondary desorption control, branch memory effect (pre-
568
flush blank) screenings were also performed.
569
(b) Cuvette based leaf level sampling: A LI6400 XT portable gas exchange system was suitably
570
modified to sample volatiles directly from the leaf cuvette onto the thermal adsorbent bed
571
described above. The LiCor was supplied with volatile free humidified air mixed with CO2.
572
Within-cuvette ambient CO2 was 400 µmol mol-1; leaf temperature was 25 °C; humidity
573
ranged from 40 to 62% and the light intensity was 1200 µmol m-2 s-1. Isoprenoids were also
574
sampled in a low oxygen (2%), ambient CO2 (400 µmol mol-1) atmosphere generated by
575
mixing nitrogen, high-purity O2 and CO2 at the required ratio and humidified to achieve 40 to
576
50% RH in the cuvette. Leaves were exposed to low O2 or high O2 for 5 to 10 min (stable
577
readings) prior to sampling. The effect of varying oxygen exposure on photosynthesis was
578
also studied independently (although simultaneously) on many leaves.
579
(c) Sampling from E. camaldulensis subsp. obtusa under controlled CO2, O2, temperature, and
580
water availability: One-year-old saplings (N = 6) were maintained at 100% FC (volatiles
581
were sampled) and then gradually dried down to 50% FC, again acclimated for 15 days
582
(volatiles were sampled again). Before sampling, individual leaves were exposed for 10 min
583
to five possible atmospheric CO2 concentrations (60, 180, 400, 1000 and 1800 µmol mol-1),
584
two temperatures (28 °C and 38 °C), two O2 concentrations (20% and 2%) and saturating light
585
intensity (1500 µmol m-2 s-1). CO2 compensation point remained close to 60 µmol mol-1 in all
586
treatments except in well-watered plants at 28 °C (low oxygen) where it was 10 µmol mol-1.
587
CO2 treatment was randomised so that on a given day some leaves from different biological
588
replicates received low CO2 treatment while others received high CO2 treatment to avoid
589
either stimulation or limitation of photosynthesis.
590
591
Thermal Desorption GC-MS analysis: A Shimadzu GCMS-QP 2010 fitted with an auto thermal
592
desorption system (TD-20) was used for off-line volatile analysis. Ultrapure helium (BOC) was used
593
as the carrier gas. Isoprene and d-limonene (analytical grade, Sigma) were injected into sterile 5 L
18
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Revised Research Article for Plant Physiology_Aug14-2014
594
Tedlar® bags comprising nitrogen to generate standard mixing ratios. Then, the standard mixture was
595
adsorbed onto TD tubes (described earlier), which were used to calibrate the instrument at regular
596
intervals. Isoprene sampled from the plant chamber was desorbed from the TD tube at 220 °C (60
597
mL min-1 for 5 min). A 30 m, 25mm ID, 25 µm RTX-5 Sil MS (Restek) capillary column was used
598
for GC. The temperature regime for GC run was 28 °C (3min) to 110 °C (3 min) at 5 °C min-1 and
599
finally to 180 °C at 5 °C min-1. The chromatographic peaks were identified by comparing them to
600
isoprene and monoterpene standards (α-pinene and d-limonene) and reference mass spectrographs in
601
the NIST Standard Reference Database 1A (NIST 2008). Isoprene emission rates were calculated by
602
utilizing sampling flow rate, total leaf area in the sampling chamber (for branches) and quantified
603
isoprene standards.
604
605
Statistical Analysis: The statistical tests were performed using Minitab (v16 statistical package,
606
Minitab Inc; PA, USA). Equality of means in responses within species between two treatments was
607
analysed using paired t-tests. Differences in mean responses between two species to the same
608
treatment were subjected to two-sample t-tests. The CO2, O2, temperature and drought intensity
609
interactions were analysed using a multilevel general full factorial model with ANOVA
610
(Montgomery, 2004). Experiments had 4 (between species) to 6 (sequential) biological replicates, 8
611
to 15 independent leaf level measurements (technical replicates) per treatment group.
612
613
Acknowledgements:
614
We thank Mr Shuangxi Zhou, Dr Christopher McRae (CBMS), Dr Ante Jerkovic (ASAM), Mr
615
Walther Adendorff (METS), Mr Muhammad Masood (PGF) and Dr Ian Wright’s lab group. We are
616
indebted to Dr Craig Barton and Dr Julia Cooke (University of Western Sydney) for lending us
617
additional infra-red gas analysers and to Mr Marco Michelozzi (CNR, Italy) for preliminary
618
screening of stored and constitutive monoterpenes. We thank Dr Roger Hiller (MQ), Dr Francesco
619
Loreto, Dr Mauro Centritto (CNR, Italy) for critically reading the manuscript. We also thank Dr
620
Marianne Peso, Dr Simon Griffith (MQ) for discussions and Dr Thomas Sharkey (MSU, US) for
621
suggestions on the modelled relationship between reducing power and isoprenoid emission rates.
622
623
Figure captions:
624
Fig. 1: Photosynthesis and isoprenoid emission rates over a drought gradient in two eucalypts,
625
Eucalyptus occidentalis (solid line, diamonds) and Eucalyptus camaldulensis subsp. camaldulensis
626
(dotted line, triangles) subjected to 20% O2 (left panel) and 2% O2 (right panel): (A) stomatal
19
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Dani et al (2014)
Revised Research Article for Plant Physiology_Aug14-2014
627
conductance (B) leaf internal CO2 concentration (C) transpiration rate (D) net assimilation rate (E)
628
isoprene emission rate (F) constitutive monoterpene emission rate (each point represents N=4 plants;
629
mean ± 1 SE; *P ≤ 0.05; ** P < 0.01; ***P< 0.001, comparison within species relative to 100% FC
630
control).
Note: The pronounced decline in NAR with drought in E. camaldulensis subsp.
631
camaldulensis reflected the fact that its stomata were sensitive to soil water status, a mechanism that
632
presumably achieves a minimum necessary transpiration rate per unit leaf area during drought stress
633
(White et al., 2000).
634
635
Fig. 2: (A) ETR/NAR ratio and (B) Carbon cost (C) ETR/NAR ratio vs. total isoprenoid
636
emission rates at 20% O2.
637
assimilation rate (NAR) in two eucalypts over a drought gradient. Note the greater decline in NAR
638
in E. camaldulensis subsp. camaldulensis at 50% FC, which results in a large ETR/NAR ratio.
639
Emission is almost twice more expensive under all conditions on carbon basis in E. camaldulensis
640
(the drought sensitive species). The regression fits in Figure 2C encompasses data points from both
641
species (N=4; mean ± 1SE for emission rates, Quadratic: P=0.12; Linear: P=0.18). If E. occidentalis
642
Relative response of linear electron transport rate (ETR) and net
were subjected to harsher droughts (% FC ≤10%), it is also likely to have followed a quadratic
643
regression with peak emission at 25% FC and declining emission thereafter due to carbon limitation
644
despite favourable ETR/NAR ratio.
645
646
Fig. 3: (A) Photosynthesis, (B) isoprene and (C) constitutive monoterpene emission rates
647
response to short-term heat stress under 20% O2 (left panel) and 2% O2 (right panel) over a CO2
648
concentration span (60 µmol mol-1 to 1800 µmol mol-1) in Eucalyptus camaldulensis subsp. obtusa
649
(experiment 2) acclimated to well-watered condition (100% FC) and drought (50% FC) at two
650
independent temperature treatments (28 ºC and 38 ºC) (N=6, means ± 1 SE).
651
Fig. 4: The energetic status model: The relationship between residual electron transport rate Jr
652
(not used for light-independent reactions of photosynthesis) and isoprenoid emission rate at different
653
drought stress levels (A) E. occidentalis (solid rhomboids) and E. camaldulensis subsp.
654
camaldulensis (solid triangles); (B) J/Vcmax vs. isoprenoid emission rate in both species. (N=3 for Jr
655
and J/Vcmax ; means ± SD, N=4 for isoprenoid emission rate; means ± 1 SE, P<0.05)
656
Fig. 5: Photorespiration, drought and isoprenoid emission: (A) Jr vs. Ie (r2=0.91; P=0.02) (B)
657
J/Vcmax vs. Ie (r2=0.85; P=0.06) in E. camaldulensis subsp. camaldulensis (experiment 3) acclimated
658
to well-watered (100% FC) and droughted (50% FC) conditions and exposed to three different levels
20
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Revised Research Article for Plant Physiology_Aug14-2014
659
of oxygenated atmospheres. 2% and 50% O2 exposure were maintained for 10 to 15 minutes before
660
volatile sampling (N=5; means ± 1 SE). The correlation remains significant (at P<0.05) even when
661
both isoprene and monoterpenes are considered together. (C) Response of constitutive monoterpene
662
emission rate (Me) in response to drought and varying oxygen levels. Drought (50% FC) had a
663
significant positive impact on Me at both 2% and 20% O2, which was consistent with isoprene like
664
response of monoterpenes in E. camaldulensis subsp. camaldulensis (Fig. 1f) (N=5; means ± 1 SE;
665
**P<0.05).
666
Fig. 6: Relationship between % Field Capacity and leaf water potential in E. occidentalis and E.
667
camaldulensis subsp. camaldulensis (experiment 1)
668
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Revised Research Article for Plant Physiology_Aug14-2014
854
Table 1: Major sinks for energy and reducing power generated by the light reactions of photosynthesis
Biochemical pathway
Key steps
ATPs
NADPHs
Reference
% Quantum
Share *
(or equivalents)
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Calvin cycle or
Photosynthetic Carbon Reduction cycle
(PCR cycle without photorespiration)
10 RuBP + 10 CO2
10 reduced C + 10 RuBP
30
20
Ogren, 1984
49 to 53
Photorespiration
(PCO cycle, including RuBP recovery)
10 RuBP + 10 O2 10 PGA … 20 Gly 10 CO2 + 10 NH3
10 NH3 + 10 Glmt 10 Glu
… … 10 PGly 5 Glycerate
10 PGA (from oxygenase activity) + 5 Glycerate 10 RuBP
47.5
(17.5)
30
(10)
Peterhansel et al. 2010
23 to 29 #
Nitrate reduction (photo-assimilation)
NO3- NO2- NH4+
NH4+ + Glu Gln
Gln + 2 oxyGlutarate
1
10
Noctor and Foyer, 1998
2 to 5
Carbohydrate biosynthesis
6CO2
19
12
Skillman, 2008
6 to 10
Mehler reaction ‡ (water-water cycle)
10 H2O + 5 O2 10 H2O2
10 H2O2 20 Asc 20 MDA+ 20 H2O
20 MDA+ 10 H2O 5 O2
0‡
10
Heber, 2002
3 to 13
Other reducing sequences (include lipid biosynthesis, sulphate reduction, the MEP pathway)
20
10
2 to 6
TOTAL
~90
72
~100
†
C6H12O6 …
2 Glu
C12H24O12
* From Haupt-Herting and Fock (2002) and Skillman (2008); the share is determined by not only the absolute demand per pathway but also the actual instantaneous rates of each
process. E.g. Nitrate reduction needs a lot of reducing power but its actual processing rate is very slow compared to core reactions of photosynthesis
If one assumes a linear electron transport (ETR) rate of 100 µmol m-2 s-1 then approximately 50 µmol m-2 s-1 would be spent on reducing carbon (net assimilation rate ≈12 µmol m-2 s-1,
given 4 electrons are needed per mol of CO2 fixed). Similarly, photorespiration accounts for ~25 µmol m-2 s-1 and the remaining processes utilise 25 µmol m-2 s-1
†
Both photorespiration and nitrate reduction have access to reducing power from extra-plastid sources
#
PCO cycle (per se) requires 17.5 ATPS and 10 NADPH equivalents when we discount the energy consumed by PCR cycle during RuBP recovery via photorespiratory route.
Therefore, the % quantum share of PCO cycle is roughly half of that of Calvin cycle
‡
The Mehler reaction utilises NADH instead of NADPH. It does not consume ATPs rather adds to the pool of ATPs (Heber, 2002)
27
Dani et al (2014)
Revised Research Article for Plant Physiology_Aug14-2014
855
856
Table 2: Experimental lay out
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
Experiment
1
2
3.
Focus
Paired set
(Fig. 1)
(Fig. 2)
(Fig. 4)
Effect of drought
tolerance of
photosynthesis on
isoprenoid emission
Individual
species
(Fig. 3)
Interactive effects of
heat, CO2 and drought
on isoprenoid emission
Individual
species
Relationship between
photorespiration, net
assimilation and
isoprenoid emission
(Fig. 5)
Name of the species
Eucalyptus occidentalis
(Drought tolerant)
Eucalyptus camaldulensis subsp.
camaldulensis (Drought sensitive)
Ie to Me
molar ratio
10:1
10:1
Drought
acclimation
100% FC
70% FC (3 months)
50% FC (15 days)
25% FC (15 days)
CO2 concentration
(µmol mol-1)
Growing
condition
Exposure before
and during
sampling
400
400
Temperature
(ºC)
25
Oxygen (%)
Growing
condition
Exposure
before and
during
sampling
20
2, 20
Eucalyptus camaldulensis subsp.
obtusa
2:1
100% FC
50% FC (1 month)
400
60, 180, 400,
1000, 1800
28,
38 (only exposure)
20
2, 20
Eucalyptus camaldulensis subsp.
camaldulensis
10:1
100% FC
50% FC (10 days)
400
400
25
20
2, 20 and
50
857
858
859
860
861
28
Dani et al (2014)
Revised Research Article for Plant Physiology_Aug14-2014
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
29
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