After the onset of drought stress in Pt and Pb seedlings, our results suggest that a cascade of responses occurred in the drought-exposed seedlings: (1) soil desiccation limited A and gs, which (2) limited stem growth and, with that, the production of new leaves and possibly fine roots; (3) as seedlings stopped growing, photoassimilates were initially re-directed to accumulate in stem and root tissues; and (4) although hydraulic conductivity continued to be compromised and the season progressed towards dormancy, reserves did not continue to accumulate substantially and were only half that of the non-drought-exposed CON seedlings during the dormant season. However, a decrease in photosynthesis and an increase in reserves in woody tissues also appear to be natural seasonal processes in seedlings growing under outside conditions. Under well-watered conditions, assimilation also declined in these seedlings throughout the growing season, whereas NSC in stem and root tissues accumulated once height growth had terminated (Fig. 5).
After shoot growth had ceased, root growth increased in the CON seedlings. The cues for these seasonal changes in Populus are probably the shortened day length and cooler night temperatures, which are known to induce tissue hardening and dormancy (Ibáñez et al., 2010). Similar late-seasonal root growth has been observed in mature boreal aspen stands (Landhäusser & Lieffers, 2003). At the end of the growing season and before the dormant season, NSC reserves in the drought-exposed seedlings did not reach or surpass the NSC levels of the CON seedlings. This pattern was in contrast with an earlier study that described a substantial increase in NSC reserves in seedlings growing under glasshouse conditions (Galvez et al., 2011). In that study, the well-watered Pt seedlings did not have environmental cues to terminate height growth, and therefore grew continuously, and the newly acquired carbon was probably used to maintain height growth, whereas the drought-exposed seedlings stopped growing as a result of the drought stress and started to accumulate NSC reserves in their tissues. This substantial difference between these two studies clearly highlights the importance of phenological stage as a controlling factor for seasonal variation in NSC acquisition, accumulation and allocation. In ecophysiological field and glasshouse studies, this impact is commonly overlooked, in particular when investigating complex perennial plants such as trees (Landhäusser & Lieffers, 2012).
Drought-exposed seedlings in the current study showed a clear decline in starch concentration of the root system; however, its potential impacts were only revealed after the dormant period. After re-watering following the dormant period, it became clear that the roots in the drought-exposed seedlings were dead. This could not have been detected prior to or during the dormant season measurement, as roots did not show any visible signs of necrosis of their tissues, even after they had thawed out after the dormant season. However, shortly thereafter, the root systems turned black and no live root segments, new root tips or root suckers were detected on these root systems. Interestingly, even at this stage, root NSC reserves were not depleted completely, supporting the hypothesis that minimum reserve requirements exist in plant tissues (McDowell, 2011; Sala et al., 2012).
The low concentration of NSC and the heavily embolized stems and roots probably compromised the re-initiation of leaves on the shoot and/or the development of root suckers, and new root growth after the dormant period. Although no PLC measurements were performed on roots, it is possible that roots suffered severe embolization prior to or during the dormant period (Pittermann & Sperry, 2006), compromising root function. However, the low NSC reserves in the roots could also have resulted in poor frost protection of the root tissues. No profound conversion of starch reserves to soluble sugars during the dormant period was detected in the DRY seedlings, whereas CON seedlings demonstrated this conversion. This conversion has been described for many different perennial plant species, and is considered to be a mechanism for the frost protection of tissues (Levitt, 1980).
Combining the effect of hydraulic failure and disruption of carbon reserve accumulation
By the end of the first growing season, the drought-exposed seedlings had suffered catastrophic hydraulic failure; PLC was above 90% in Pt and above 80% in Pb (Fig. 3c). At that time, we observed substantial stem necrosis in both species, which affected a large portion of the stem, but remained in all seedlings well above the root collar. Stem necrosis and phloem tissue damage are well-known responses of woody stems to severe drought stress, but these alone were not sufficient to kill seedlings in the short term (Lu et al., 2010). In the light of the heavily embolized stems, the likelihood of flushing from these necrotic shoots in both species was very small; however, as the non-necrotic portions of the lower stem had lateral buds and the coarse root system did not show signs of necrosis (e.g. maintained the potential to the root sucker), seedlings should have been able to re-sprout from these living tissues (Lu et al., 2010). Lu et al. (2010), however, only desiccated 1-yr-old Pt seedlings in a short-term drought until all leaves had been shed and the stems were necrotic (PLC averaged 90%); when re-watered, these plants were able to re-sprout from axial buds or from their roots. The shoot symptoms matched the conditions of our drought-exposed seedlings in September 2011. However, in their study, seedling carbon reserves and the performance after a dormant season were not measured. Both studied Populus species have the ability to regenerate from adventitious root sprouts; therefore, a still functioning root system with sufficient NSC reserves should have been able to produce new shoots (Landhäusser et al., 2006; Snedden et al., 2010). Clearly, this was not the case. To our knowledge, this is the first study to present experimental data detailing the complex set of physiological processes interacting with seasonal abiotic signals. Further, our results indicate that hydraulic damage during drought impairs carbon accumulation in the roots, which might, in turn, have hampered root survival of winter conditions if the stress survived that long. Nevertheless, uncertainty remains with regard to the exact timing of seedling death, which may have occurred before the dormant period. Under this scenario, changes in NSC could be interpreted as a consequence of death, rather than a mechanism leading to mortality. Interestingly, the change in total NSC (i.e. soluble sugars + starch) concentration at the whole-plant scale (TNSCplant) as a result of drought was predominantly driven by the NSC concentration in the root tissues (Figs 4, 5); hence, any stress affecting NSC accumulation in the roots of the seedlings probably has a direct effect on the whole-plant performance (Landhäusser & Lieffers, 2012).
The average leaf soluble sugar concentration (SugConcleaf) in DRY seedlings increased during the first 4 wk of the experiment, even though A in DRY seedlings remained at least 50% lower than that in CON seedlings. This initial increase in SugConcleaf may suggest the onset of osmotic adjustment, a well-documented response to drought in Populus species (Gebre et al., 1994, 1998) and other tree species (Tschaplinski et al., 1998). We hypothesized that the increase in SugConcstem of DRY seedlings, measured during the growth period, could also have been an osmotic response that could have been used to up-regulate the xylem pressure potential, which has been suggested as a potential mechanism for the repair of xylem embolism (Secchi et al., 2011). The maintenance of NSC in the stems is also important because shoots need access to reserves for the new leaf flush after the dormant season (Landhäusser, 2011). The fact that, by the end of the experiment, only CON seedlings had produced new leaves and increased their root volume highlights the relevance of starch accumulation during the growing season and starch-to-sugar conversion during the cold hardening period (Levitt, 1980; Sauter, 1988; Fig. 4). Starch is a compound with no other known biological function in plants apart from storage, and it is needed to buffer periods of stress (Kozlowski & Pallardy, 2002). Starch-to-sugar conversion is a temperature-dependent adaptive mechanism well studied in Populus (Sauter, 1988; Sauter & van Cleve, 1991). This conversion plays an important role in the maintenance of cell membranes at low temperatures and increases freezing tolerance (Levitt, 1980). Recently, Hennon et al. (2012) have suggested that frost damage to fine roots as a result of reduced snow depth and soil drainage could be a main driver of the extensive yellow cedar die-off events in Alaska. Snow cover is probably an important factor protecting root systems from freezing, even in boreal forests (Hennon et al., 2012). In our study, after CON seedlings had thawed, SugConcroot in CON seedlings declined as apical and lateral meristems became active and new leaves expanded, a process previously observed in other Populus and Salix species (Sauter, 1988; Von Fircks & Sennerby-Forsse, 1998).
Although Pt and Pb can overlap on sites with similar mesic, edaphic and climatic conditions (Peterson & Peterson, 1992; Landhäusser et al., 2002; Landhäusser & Lieffers, 2003), they thrive in distinctive habitats (Burns & Honkala, 1990). Pt forms extensive stands in mesic to dry mesic upland sites, whereas Pb occupies the moister (flood plains or seepage areas) and cooler extremes (Rood et al., 2003, 2007). This difference in habitat is also reflected by the different hydraulic (Tyree et al., 1994) and stomatal adaptations to drought. Stomatal behavior in response to drought was distinctively different between Pt and Pb seedlings during the desiccation process (Fig. 1). During the drying period, gs in DRY Pt seedlings declined earlier and more rapidly than in DRY Pb seedlings. The decline in gs in DRY Pt seedlings suggests a more isohydric behavior (i.e. leaf stomatal conductance decreased as soil desiccation progressed; Tardieu & Simonneau, 1998), in comparison with a much slower response in DRY Pb seedlings, which maintained similar gs to well-irrigated CON seedlings for a period of 8 d before gs started to decrease (Larchevêque et al., 2011).
To our knowledge, our work is the first study to present detailed experimental data illustrating a complex feedback between stomatal behavior, gas exchange, water relations and carbon reserve accumulation dynamics. These responses and feedbacks are clearly influenced by seasonality, making the role of drought stress as a driver of plant mortality dependent on the interaction between phenology and physiology, and very likely the ontogeny of plants.