4.2. Investment of C in isoprenoid emissions shifts to heavier
compounds when photosynthesis is reduced in the dry season
From the wet to dry season, we found a general trend of decreasing
photosynthesis rates, coupled with relatively constant total isoprenoid
emission rates (Fig. 4). This combination led to a three-fold average
increase in C allocation to isoprenoid emissions relative to
photosynthesis (though this was variable and non-significant, Fig. 4).
Even so, the C sink to isoprenoid emissions was less than 2% of
photosynthesis in most plants, except in strongly sesquiterpene-emittingP. hebetatum , whose emissions averaged 6.1% of photosynthesis
(occasionally > 10%) in the upland in the dry season.
Associated with this increasing investment in emissions, plants also
allocated more C toward heavier isoprenoid compounds (Fig. 4).
Increasing ‘isoprenoid mass investment’ was a result of reduced isoprene
emissions and increased monoterpene and sesquiterpene emissions in the
dry season (Figs. S2).
Previous studies indicated that seasonality in isoprenoid emissions from
Amazonia is controlled by changes in solar radiation, temperature and
leaf demography and phenology (Kuhn et al. 2004b; Alves et
al. 2016, 2018). It is well known that the central Amazon dry season
reaches maxima in solar radiation, temperature and leaf flushing, and
the latter translates into a higher proportion of canopy young leaves
during this season (Lopes et al. 2016; Wu et al. 2016;
Gonçalves et al. 2020). In terms of changes in chemical
composition of isoprenoid emission capacities, leaf age is an important
factor because the synthesis of these compounds changes with leaf
ontogeny. Isoprene and monoterpenes are produced from dimethylallyl
diphosphate (DMADP), which is catalyzed by isoprene synthase and
monoterpene synthases, in the methylerythritol 4-phosphate (MEP) pathway
in chloroplasts. The DMADP pool size varies with leaf ontogeny, meaning
that in early developmental stages, leaves have low DMADP – due to high
demand for leaf growth and greening, and it increases with leaf
maturation. A low DMADP pool in young leaves may be responsible for a
down-regulation in the isoprene synthesis and up-regulation in the
monoterpene synthesis; this might be in part explained by the fact that
the enzymes involved in the monoterpene synthesis pathway have higher
affinity for DMADP, implying that these enzymes achieve maximum
catalytic efficiency already at low DMADP concentration, which favors
the synthesis of monoterpenes rather than isoprene in young leaves (Kuhnet al. 2004b). As constitutive emission of monoterpenes and
isoprene has been suggested to have similar functional roles to protect
plants against high temperatures and oxidants (Loreto et al.1996), when these stresses - typical of the Amazon dry season - are
imposed to young leaves and isoprene synthesis is constrained by enzyme
deficits or low DMADP concentration (Kuhn et al. 2004b), the
production of monoterpenes might be preferable and then confer the high
demand for leaf protection.
Additionally, the plasticity in emission capacities and chemical
compositions may not exclusively be a response to abiotic stress, but
they may signal up-regulation of defenses against herbivores and
pathogens, to which the plant is more susceptible under abiotic stress
(Monson, Weraduwage, Rosenkranz, Schnitzler & Sharkey 2021).
Monoterpenes and sesquiterpenes have long been recognized as important
compounds for plant defense against herbivores and pathogens (Gershenzon
& Dudareva 2007). Young leaves have more soft tissues and less physical
and structural defenses that make them more palatable and attractive to
predators than mature leaves. In this sense, the increase in emitted
monoterpenes and sesquiterpenes (in P. hebetatum ) during the dry
season might also be interpreted as an increase in the demand for
defense by new leaf tissues of the young leaves.
A higher proportion of young leaves during the dry season is clearly
seen in the study site presented here (Lopes et al. 2016;
Gonçalves et al. 2020). However, it was also observed that
approximately 30% of trees do not have a massive leaf flushing in any
time of the year, meaning that they are possibly flushing leaves
continuously and therefore do not have a distinct seasonal leaf age
distribution (Lopes et al. 2016). As the pattern of leaf
phenology is plant species-specific and we do not have information on
the leaf age for the individuals that we performed our measurements
with, we cannot exclude other reasons for the shift in composition of
isoprenoid emission capacities rather than leaf age. If significant
changes in leaf age is unaccountable (i.e. specifically for these tree
species and in the moments that our measurements were performed), then
what might have been playing a role is the leaf physiological response
to the environment, which is also affected by the environmental
conditions previous to measurements, and not only to the standard
conditions imposed to leaves during measurements.
The dry and wet season environment in central Amazonia is different for
the leaf - the wet season imposes more light-limitation and the dry
season imposes more vapor pressure deficit and higher temperature
limitation to photosynthesis (Wu et al. 2017). The limitations
imposed during the dry season are more stressful for plants, which might
require more or different defense mechanisms. Recent work on the
integration of isoprene, monoterpenes, sesquiterpenes and other
secondary metabolites in signaling networks that mediate growth-defense
tradeoffs (Monson, Weraduwage, Rosenkranz, Schnitzler & Sharkey 2021),
suggests that plasticity and changes in emission composition (as can be
observed seasonally and/or across habitats) could enhance plant chemical
defense and therefore confer to the physiological system more resistance
against different types and levels of stress - e.g., reducing isoprene
and increasing monoterpene and sesquiterpene in the Amazon dry season
might represent a switch in plant gas composition in order to cope with
stresses such as heat and drought, and to prime the plant to associated
biotic stresses.
While the dry-season increase in ‘isoprenoid mass investment’ was most
significant for P. hebetatum , it was mirrored by non-significant
trends within most species and habitats (Fig. 3). We devised the
‘isoprenoid mass investment’ metric to assess whether changing
environmental conditions are associated with shifts in C allocation to
emissions along the spectrum from lighter to heavier isoprenoids. Many
light dependent isoprenoids are evidenced to play related
metabolic-support roles as components of cellular signalling networks
and antioxidant defenses (Vickers, Gershenzon, Lerdau & Loreto 2009;
Riedlmeier et al . 2017; Frank et al. 2021; Zuo et
al. 2019; Monson, Weraduwage, Rosenkranz, Schnitzler & Sharkey 202;
Harrison et al. 2013). While the diversity of specific compounds
is high, there are general trends along the mass gradient from 5C
isoprene through 10C monoterpenes to 15C sesquiterpenes. Notably, along
the low-to-high mass gradient, volatile isoprenoid production has a
decreasing dependency on photosynthesis. Compared to monoterpene
synthase enzymes, isoprene synthase has a low reaction affinity for the
primary isoprenoid substrate DMADP (Harrison et al. 2013; Kuhnet al. 2004b). Isoprene therefore requires a larger substrate
pool in the chloroplast that is derived from photosynthesis (Sharkey &
Monson 2014). This variation in emission capacity with variation in the
supply of photosynthetically derived substrates is likely an explanation
for why species either emit monoterpenes or isoprene at high rates, but
not both (Harrison et al. 2013). Sesquiterpene biosynthesis is at
least partially localized to the cytosol and use Isopentenyl
Pyrophosphate (IPP) and DMADP from the Mevalonate pathway (MVA) (Sallaud
et al. 2009), which further reduces their dependency on photosynthesis
and DMADP pools in the chloroplast, relative to the smaller isoprenoids.
This implies that seasonal changes in photosynthesis may affect isoprene
and higher isoprenoid production, since the amount of DMADP available
for isoprene synthesis is not only dependent on upstream flux through
the MEP pathway, but also its downstream use for the production of
higher compounds, such as monoterpenes and sesquiterpenes (Monson,
Weraduwage, Rosenkranz, Schnitzler & Sharkey 2021), that can be
demanded after distinct biotic and abiotic stresses over seasons
(Harrison et al. 2013). In addition, the heavier compounds tend
to be much more reactive, as evidenced for example by the typical
atmospheric half-lives of isoprene (hours), monoterpenes (minutes), and
sesquiterpenes (minutes) (Atkinson & Arey 2003). Higher reactivity
during the short residence time of volatiles within the cell may allow
for a stronger effect as signaling molecules or even as direct
antioxidants at lower production rates.
We hypothesize that as photosynthesis declines, an investment shift
toward emissions of heavier isoprenoid compounds may allow sustained
metabolic support due to their increasing reactivity and decreasing
dependency on photosynthesis for production. Consistent with this
hypothesis, our results show that when photosynthesis was reduced under
dry season conditions, there was a tendency among all species to shift
emission compositions toward heavier compounds. Interestingly, the only
species to emit detectable sesquiterpenes, P. hebetatum , emitted
them only in the dry season, and showed significantly increasing
emission rates along the soil hydrologic gradient from shallow (wetter)
to deep (drier) water table depths (Fig. 2), suggesting an association
between investment in sesquiterpenes and the seasonal drought conditions
experienced by individual plants. We suggest that our hypothesis is
consistent with our empirical data and general theory, but that further
experimental physiology work is required to demonstrate emission rate
tradeoffs, photosynthetic dependencies, and the relationship between
emission rates and metabolic responses for specific isoprenoid
compounds.
Although there is variability within and among the tree species studied
here and it is not possible to generalize results from three species to
a whole plant community, here we presented results of species that are
widespread in the central Amazon and have meaningful contribution to
plant biomass and productivity (Fauset et al. 2015). Furthermore,
the trend of reduction in isoprene emission with proportional increase
in monoterpene and sesquiterpene emission during the dry season has
implications for atmospheric chemical and physical processes. As for
aerosol formation, laboratory-determined secondary organic aerosol (SOA)
yield from isoprene has been reported as < 6% (Kroll, Ng,
Murphy, Flagan & Seinfeld 2005; Xu, Kollman, Song, Shilling & Ng 2014)
or higher over forested regions when isoprene is emitted in much more
quantities relative to other compounds (Carlton, Wiedinmyer & Kroll
2009). Nonetheless, if other isoprenoids are more emitted, the
contribution to the SOA formation could be also higher, as shown by SOA
yields of ~5-10% for monoterpenes (Griffin, Cocker,
Flagan & Seinfeld 1999a; Griffin, Cocker, Seinfeld & Dabdub 1999b) and
~20-70% for sesquiterpenes (Hoffmann et al.1997; Griffin et al. 1999b; Lee et al. 2006a b; Chen, Li,
McKinney, Kuwata & Martin 2012; Jaoui, Kleindienst, Docherty,
Lewandowski & Offenberg 2013).
In this light, it is important for future studies to consider a wider
range of volatile organic compounds with their synergetic importance in
plant ecophysiological processes and the subsequent impact on the
atmosphere. Our results suggest that emissions of monoterpenes and
sesquiterpenes might be higher than anticipated and indicate a seasonal
change in the composition of the emitted isoprenoids. In fact, seasonal
shifts in monoterpene composition have been already reported in ambient
air (Jardine et al. 2015; Yáñez-Serrano et al. 2018); but
sesquiterpenes might have been underestimated given their high
reactivity with ozone and OH and thereby the difficulty to detect in
ambient air (Yee et al. 2018), indicating that only leaf level
measurements are likely to give us a true measure of forest
sesquiterpene emissions. Even though our observations do not offer
results to make a clear statement of how isoprene, monoterpenes and
sesquiterpenes work together in the face of abiotic stress such as heat
and drought during the dry season, they supports the recent findings
that isoprene does not confer plant tolerance by itself, but rather it
is likely part of an intricate network that involves other plant
metabolic processes and therefore other compounds (Harvey, Li,
Tjellström, Blanchard & Sharkey 2015; Monson, Weraduwage, Rosenkranz,
Schnitzler & Sharkey 2021). This opens more questions in order to
understand what are the processes regulating isoprenoid emission
capacities of Amazonian trees, and the need to consider seasonal shifts
in isoprenoid composition in emission modeling.