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.