Branch MeOH and AA emission responses to experimental
drought
During the drought experiment, a subset of drought (N = 6) and control
(N = 6) plants were transported to the analytical laboratory in the
morning and analyzed for ‘snap-shot’ branch MeOH and AA emissions for 1
hour in a constant light and temperature environment (Fig.
2a-c ). Control plants had high average rates of MeOH emissions (2.3-4.4
nmol m-2 s-1) and low, but
detectable levels of AA emissions (0.1 nmol m-2s-1). In contrast, drought stressed trees showed low
MeOH emissions (0.3 nmol m-2 s-1)
while also showing higher average AA emissions (0.2 nmol
m-2 s-1). This pattern resulted in
the branch ‘snap-shot’ AA/MeOH emission ratio for the control (10 +/-
10%) being lower than the drought stressed plants (84 +/- 57%).
In contrast to the greenhouse drought experiments which showed rapid
negative leaf physiological effects. A second set of drought experiments
occurred in a cooler lab, where limited lighting was provided
artificially via a grow-light. To confirm the pattern of decreased MeOH
emissions and increased AA emissions, and consequently high AA/MeOH
emission ratios during the greenhouse drought, real-time MeOH and AA
emissions were characterized before, during and after the onset of
drought impacts on leaf gas exchange. Five well-watered control
individuals were sequentially transported to the laboratory and studied
for real-time diurnal branch VOC emissions in the absence of additional
soil moisture additions under a daytime (6:00-22:00) light pattern that
mimicked the greenhouse lighting conditions. While variability in the
timing and magnitudes of the MeOH and AA emissions was observed between
the five individuals, the same general emission pattern was observed
during the real-time emission studies as those from the ‘snap-shot’
studies with drought inducing a pattern of decreasing branch MeOH
emissions and increasing AA emissions together with high AA/MeOH
emissions ratios (Figure 2d-f, Figure 3 and supplementaryFigures S2-5 ).
When the temporal patterns of branch gas exchange during drought was
analyzed in more detail, four distinct phases could be described. The
first ‘growth phase’ with physiologically active foliage is
characterized by high rates of transpiration, net photosynthesis, and
MeOH emissions, with low AA emissions and values of AA/MeOH emission
ratios < 30% (e.g. the first three days in Figure
3 ). During this ‘growth phase’, high light-dependent emissions of
isoprene linked with photosynthetic CO2 assimilation
occur during the day (6:00-22:00). Despite the constant daytime light
environment and relatively stable laboratory temperature, a strong
circadian gas exchange pattern was observed with maximum gas exchange
fluxes near mid-day of transpiration (enhancing H2O
concentrations in the branch chamber), net photosynthesis (drawing down
the CO2 concentrations in the branch chamber), and MeOH
and AA emissions. However, the high MeOH emissions relative to AA
emissions from physiologically active branches in the ‘growth phase’
constrain daytime AA/MeOH emission ratios to low values, reaching
maximum mid-day values of 6% (e.g. day 3 in Figure 4 ). The
second phase of drought response consists of a strong suppression in
MeOH emissions, apparently occurring prior to any reductions in stomatal
conductance and CO2 and H2O gas exchange
(e.g. day 4 in Figure 3 ). Although AA emissions remained low,
similar to the well-watered active growth conditions, branch AA/MeOH
emission ratios during this ‘MeOH suppression’ phase increased slightly
from 18% on day 4 to 24% on day 5. The third phase of plant response
to drought stress is characterized by a reduction in stomatal
conductance with a suppression of transpiration and net photosynthesis
rates, a continued strong suppression of MeOH emissions, together with
the activation of aerobic fermentation including high branch emissions
of the fermentation volatiles acetaldehyde, ethanol, acetic acid (AA),
and acetone (e.g. initiated on day 5 in Figure 3 ). High rates
of fermentation VOC emissions were found to be initiated both during the
day and the night, depending on the individual, and so is not directly
considered a light-dependent process. Emissions of acetaldehyde during
this phase was far higher than those of the other fermentation products,
whose emissions in general tracked the rise and fall of acetaldehyde
emissions in this fermentation phase. Elevated branch fermentation VOC
emissions from the individual shown in Figure 3 continued for
three days, with the peak in AA/MeOH emission ratio (444%) occurring on
beginning of day 6. Throughout this ‘aerobic fermentation’ phase,
daytime transpiration and net photosynthesis continued to decline,
likely as a consequence of decreasing stomatal conductance, with a loss
of positive net carbon assimilation evident by day 7 and strongly
reduced daytime isoprene emissions. Following this ‘fermentation phase’,
a final ‘senescence phase’ was observed (day 7-10), likely associated
with irreversible damage to cellular components including photosynthetic
membranes and greatly compromised cellular structure and function. In
this senescence phase which inevitably resulted in the leaves dying and
falling off the tree, isoprene emissions were essentially eliminated,
while MeOH emissions increased again to a high level with AA emissions
also continuing at an elevated rate resulting in AA/MeOH emission ratios
declining but remaining elevated reaching a low value of 50% by day 10.
To test for the potential reversibility of the branch MeOH emission
suppression during drought, when another drought-stressed potted tree
showed strong suppression of MeOH emissions in the laboratory,
re-watering of the soil with 100 ml additions on day 4 (red arrows in
supplementary Figure S6 ), resulted in a rapid
(~15 min) return of high branch MeOH emissions and a
dramatic reduction of the AA/MeOH emission ratios to around 1%. As the
soil continued to dry through the experiment, the suppression of MeOH
emissions was again rapidly relieved by a 100 ml soil moisture addition,
regardless if it was added during the day or night. This watering effect
of the drought stressed plant, completely altered the normal diurnal
cycle in MeOH emissions which normally peak around mid-day in
well-watered individuals. While the drought-stimulated aerobic
fermentation emissions were observed on day 5, they were greatly reduced
with maximum AA/MeOH emission ratios of 12%. This is in contrast to the
five trees for which water was completely withheld (Figure 3and S2-5 ) which showed strong drought-stimulated aerobic
fermentation emissions and high maximum AA/MeOH emission ratios ranging
from 400-3500%.