4 Discussion
With increasing drought duration, tree
mortality increased (Fig. 1), leaf
water potential and photosynthesis decreased for the two species (Fig.
2), which is similar to those results found in drought intensity
experiments with trees (Schönbeck et al. , 2018; Lauder et
al. , 2019; Archambeau et al. , 2020; Schönbeck et al. ,
2020a; Ouyang et al. , 2021). For instance, extreme drought was
found to significantly decrease predawn water potential and
net-photosynthetic rates and to increase the mortality for bothPinus sylvestris (Schönbeck et al. , 2020a) andQuercus pubescens saplings (Ouyang et al. , 2021). Drought,
both severe drought intensity and long drought duration, decreases soil
water availability and plant leaf water potential, and thus results in
stomatal closure to prevent transpiration exceeding root water uptake
capacity, which caused declined
photosynthesis
and CO2 uptake (Li et al. , 2020). Duan et
al. (2019) found that severe drought intensity with short duration led
to a stronger decrease in leaf water potential and photosynthesis of
three tree species (Syzygium rehderianum, Castanopsis chinensis
and Schima superba ) than moderate drought with longer duration. The
water potential of Robinia pseudoacacia exhibited a linear
decline with increasing drought duration, while Quercus
acutissima’s water potential remained relatively stable during the
first month of mild drought (Li et al. , 2020), and thus Liet al. (2020) concluded that the two tree species differ in their
sensitivity to drought (Bhusal et al. , 2021), which confirmed
that Quercus species are anisohydric plants (Sade et al. ,
2012).
However, in spite of decreased photosynthesis (Fig. 2c-f) and NSC levels
(Fig. 3b, g, c, h) with increasing drought duration, the relative tree
height increment of the two species did not differ among the drought
treatments in our study (Fig. 2g, h). Li et al. (2013), Schönbecket al. (2018, 2020a), and Ouyang et al. (2021) found that
drought-stressed trees maintained relatively stable NSC levels at the
expense of growth, implying an active process of NSC storage (Li, M-Het al. , 2018). For example, drought declined the growth but did
not decrease tissue NSC level in Quercus faginea and Pinus
halepensis (Sanz-Perez et al. , 2009). The present study,
however, seemed to support the view of Martínez‐Vilalta (2016) that NSC
storage is mainly a passive process following the growth
priority, because the growth did not
vary with drought duration (Fig. 2g,h) while the NSC levels in storage
tissues, especially in shoots, decreased with increasing drought
duration (Fig. 3b,c,d,e,g,h,I,j). In this case, for example, the
mortality (Fig. 1b) of fertilized beech under D2 and D3 is thus mainly a
result of carbon limitation that was confirmed by very low leaf
photosynthetic rate (Fig. 2d, f) and near-zero NSC level at the
end-season (Fig. 3g, h). McDowell et
al. (2011) proposed that NSC concentrations can increase initially
under drought due to the faster decline of growth than photosynthesis,
but NSC concentrations may decline later on due to the prolonged
suppression of photosynthesis and the utilization of stored C for
meeting C demands especially under extreme drought.
Similar to results gained from most drought intensity experiments with
trees (Li et al. , 2013; Schönbeck et al. , 2018; Schönbecket al. , 2020a; Zhang et al. , 2020; Ouyang et al. ,
2021), the present study found that the longer drought duration
treatments (D2, D3) did not decrease the end-season leaf NSC
(pre-winter) levels (Fig. 3a,f), although the D2 and D3 treatment
significantly decreased leaf photosynthesis of the two species (Fig.
2c-f). This might be explained by the osmoregulation strategy of plants
suffering from drought stress on the one hand (O’Brien et al. ,
2014; Dickman et al. , 2015), and on the other hand, it may be a
result of basipetal carbon translocation failure (Rowland et al. ,
2015), if the phloem function becomes impaired and carbon translocation
gets limited or stopped by hydraulic
failure caused by severe or long drought stress (Griffin-Nolan et
al. , 2021). In this case, lower NSC levels in the sink tissues of
shoots, and especially roots, and thus carbon limitation may be
expected. Recently, this expectation has been repeatedly confirmed in
severe drought-stressed trees in controlled drought intensity experiment
(e.g. Schönbeck et al. , 2020a; Ouyang et al. , 2021), and
also in trees under longer drought duration (D2, D3) treatment in the
present study (Fig. 3b,g,c,h). Therefore, it may be speculated that a
hydraulic failure induced carbon limitation seems to be the
physiological mechanism underlying the high mortality of beech saplings,
particularly the N-fertilized D2 and D3 beech (Fig. 1b) which had very
low end-season shoot and root NSC levels close to zero (Fig. 3g,h).
These results also seem to exclude that limited sink activity, e.g. in
root tissues as a result of drought is responsible for reduced sugar
transport from the leaves to the sink tissues as in that case increased
NSC concentrations are to be expected in both, roots and shoots
(Hagedorn, F. et al. , 2016; Gessler & Grossiord, 2019).
We found that the responses of end-season NSC level to drought duration
seemed to be both species- and tissue type-dependent (Fig. 3a-b, Fig.
3f-h). For instance, leaf NSC increased (Fig. 3a) but shoot NSC
decreased (Fig. 3b) in oak with increased drought duration, while they
did not change in beech (Fig. 3f,g). Similar to beech, root NSC reserve
of aspen (Populus tremuloides ) seedlings didn’t change over a
3-month period of severe drought (Galvez et al. , 2011). However,
moderate drought was found to increase NSC in stems and roots ofQuercus pubescens saplings (Ouyang et al. 2021). Experiments with
more vs. less precipitation found that extreme drought (no irrigation
for two consecutive years) reduced shoot and root NSC, whereas
intermediate drought levels did not affect shoot and root NSC forPinus sylvestris saplings (Schönbeck et al. 2020a).
Less is known about winter NSC consumption of trees previously exposed
to drought of various intensities or duration. Trees, as exemplified by
the deciduous species in the present study, consume NSC storage for
maintenance respiration over winter (Sperling et al. , 2015).
Therefore, we found that the post-winter NSC levels were lower that the
pre-winter level in each tissue for both species (Fig. 3b vs. 3d; Fig.
3c vs. 3e; Fig. 3g. vs. 3i; Fig. 3h vs. 3j). The winter temperature was
beyond 5°C in the greenhouse of the present study (Fig. S1), but even
near freezing winter temperatures were found to significantly increase
stem respiration by 10% to 170% in 13 out of 15 species studied in the
western US, according to Sperling et al. (2015). Sperlinget al. (2015) further calculated that “frost-induced respiration
accelerated stem NSC consumption by 8.4 mg (glucose eq.)
cm-3 yr-1 on average
(cm-3 stem wood basis) in the western US, a level of
depletion that may continue to significantly affect spring NSC
availability”. This is agreement with findings that in temperate
deciduous trees, tissue NSC concentrations decline during winter
dormancy. This decrease is more pronounced in stem than in roots as
observed for aspen (Populus grandidentata ) and oak (Quercus
rubra ) (Gough et al. , 2010). The present study, for the first
time, indicated that the over-winter NSC consumption was not affected by
drought duration for the two species but it was significantly decreased
by N-fertilization for beech across the four drought treatments (Table
S1; Fig. 4a – d). This result, may indicate on the one hand a common
response of winter NSC consumption of tree species that is independent
on the previously imposed drought duration. On the other hand, our
results suggest a species-specific sensitivity of winter NSC consumption
to other environmental change such as nutrient availability. We can only
speculate why the NSC consumption was lower in fertilized beech but it
is known that free amino acids and soluble proteins can increase stress
resistance of beech (Stajner et al. , 2013). Thus, an increased N
availability might reduce stress-induced respiration in this species
under winter temperature conditions (Fig. S1).
The over-winter changes (post-winter vs. pre-winter) in the sugar/starch
ratio (Fig. 4e – h) indicated that starch to sugar conversion occurred
in oak saplings (Fig. 4e, g; Figs. S4 and S6), whereas a strong sugar
consumption and depletion were the main reasons for decreased tissue
sugar/starch ratio in beech saplings (Fig. 4f – h; Figs. S5 and S7).
Similarly,
starch
concentrations were reduced and soluble sugars increased in Prunus
dulcis during winter, and the NSC concentration (starch + sugars) were
only slight reduced (Sperling et al. , 2019). In winter, increased
sugar concentrations in the xylem are important to avoid or reduce the
number of freeze-thaw embolization cycles, because sugars increase the
osmotic potential of xylem and thus lowering its freezing point (Sauteret al. , 1973; Thierry et al. , 2004; Li et al. ,
2018).
Previous season drought duration treatment did not affect photosynthesis
of the two species after rewetting in the next year (Table 1). The
decreased photosynthesis determined in the longer duration drought
treatments in 2018 (Fig. 2c – f) recovered and all treatments showed
the same level of photosynthesis in June 2019 (Fig. 5a, b). This
recovery indicates that there is no legacy of previous drought duration
on photosynthetic carbon assimilation. Previous studies found that
drought stress can result in incomplete and lagged growth recovery
(Anderegg et al. , 2013; Pederson et al. , 2014; Huanget al. , 2018). Extreme drought caused drought legacy response
with reduced growth for deep−rooted forests for up to 4 years (Wuet al. , 2018), and negative drought legacy was found to last
about one year for different plant functional types in Tibetan Plateau
(Li et al. , 2020). However, fast recovery of carbon acquisition
and allocation to different plant organs after drought release was also
observed in different tree species (Hagedorn, Frank et al. , 2016;
Joseph et al. , 2020). In the present study, small saplings with
large plasticity may be one reason for the quick recovery after
rewetting leading to a lack of legacy of past drought. In addition, the
longest drought duration (2 months) applied here may be still not long
(or severe) enough to impair the physiological processes on the longer
term. For example, an open top chamber experiment with 40 cm soil depth
but without any watering for 2 years resulted only in a mortality rate
of 60% for Pinus sylvestris (Schönbeck et al. 2020a) and 50%
for Quercus pubescens saplings (Ouyang et al. 2021). In line with
the findings of Schönbeck et al. (2020a) for pine, we found a
fertilization-induced higher mortality rate for beech but not for oak
saplings (Fig. 1). Ouyang et al. (2021) showed a lower mortality
rate (32%) in fertilized compared to non-fertilized (50%) Q.
pubescens saplings under extreme drought. In oak species fertilization
might thus not enhance drought effects but genus and species-specific
mechanism still need to be elucidated.
The height growth of the two species was not correlated with shoot NSC
storage but significantly positively correlated with both pre-winter (P
= 0.07) and post-winter (P = 0.01) root storage (Fig. 6). The small
values of R2 (Fig. 6) suggest that root NSC is not the
only or the most important factor determining the recovery growth.
However, this result (Fig. 6) in conjunction with low photosynthetic
rate (Fig. 2c-f), low pre- and post-winter NSC levels in both shoots and
roots of D2 and D3 saplings suggests a root carbon limitation that
determines the high mortality rate of D2 and D3 saplings for the two
species, particularly for beech (Fig. 1). This result, i.e. root carbon
limitation, supports a recent hypothesis that the alpine climatic
treeline is determined by a winter root carbon limitation as proposed by
Li et al. (2018) recently. Our result is also supported by data
for P. sylvestris (Schönbeck et al. , 2020a) and Q.
pubescens saplings (Ouyang et al. , 2021) under extreme drought
that also showed low root NSC. Indeed, root carbon shortage has been
widely found in various tree and shrub species in stressed conditions
(Shi et al. , 2006; Li et al. , 2008a; Li et al. ,
2008b; Genet et al. , 2011; Zhu et al. , 2012a; Zhu et
al. , 2012b; Ouyang et al. , 2021; Wang et al. , 2021).
Interactions between drought duration and N-fertilization were found
only for gas exchange rate during the treatment period (Table 1),
indicating that the effects of N-fertilization vary in direction (or
magnitude) with drought duration only for photosynthesis but not on
other parameters studied (Tables 2 and S1). Schönbeck et al.(2020a) found that a mitigating effect of N-fertilization on the
negative drought effects on P. sylvestris saplings occurred only
when the drought stress was relatively mild. In a summer drought
experiment (no rainfall for 2 summer months during two consecutive
years) it was found that the negative effects of drought on beech growth
were amplified by N fertilization (Dziedek et al. , 2016), which
is similar to our results that drought-induced mortality of beech was
amplified by N-fertilization (Fig. 1b). Theoretically, increases in N
availability may promote the formation of xylem structures that
transport water more efficiently in humid conditions (Borghetti et
al. , 2017) but may also easily lead to xylem embolism – due to larger
cross section and bigger tracheids or vessels – in dry conditions, and
therefore, further studies are needed to clarify the N-fertilization
effects (e.g. addition rate, amount and frequency) in relation to
drought intensity or duration not only on seedlings and saplings but
also adult trees.
Now we can go back to our research questions to see whether they have
been answered. We found that longer drought duration decreased the
physiological performance (e.g. leaf water potential, photosynthetic
capacity, NSC levels) but not the growth rate (question 1). We,
therefore, speculate that growth is a higher priority than resource
storage for the saplings of the two species stressed by long-lasting
drought below a certain threshold, as the longest drought duration in
the present study was 2 months only. Previous growing season drought
seems to not affect the tissue NSC over winter, but the over-winter NSC
is considerable in saplings for both species (question 2). The
post-winter root NSC level (Fig. 3i, j) plays a more important role in
determining the growth (Fig. 6) and survival (Fig. 1) for both species
(question 3), suggesting a root carbon limitation in severe
drought-stressed saplings, particularly for beech. In line with recent
findings (Schönbeck et al. , 2018;
Schönbeck et al. , 2020a;
Ouyang et al. , 2021),
N-fertilization did not play a role to mitigating the negative drought
effects on saplings of the two species (question 4). Compared to oak,
beech had lower levels of physiological parameters and growth (Figs. 2,
3, 5) but showed higher winter NSC consumption (Fig. 4) and especially
higher mortality rate with increasing drought duration in combination
with fertilization (Fig. 1), indicating that beech is more sensitive to
drought and N-deposition. The present study, in a physiological
perspective, experimentally confirmed the view of Ellenberg (2009) that
the European beech, compared to oak, may be more strongly affected by
future environmental changes.