Stemflow Unveils Trees' Weatherproofing Stratagem During Autumnal Senescence

The cyclic rhythms of nature in forests are partly spawned by abiotic cues of air temperature and photoperiod that trigger the different canopy phenophases – emergence, leafed, senescence, and leafless – over time. Senescence is of particular interest as it marks a time of preparation and involves a host of ecophysiological processes, including nutrient resorption and leaf abscission, as trees ready for lower temperatures and reduced light conditions. A possible key to better understanding the dynamism of senescence is changes in the neutral sugar concentrations of stemflow (the portion of precipitation that reaches the ground by flowing down the stem of a plant) in senescence vis-à-vis other phenophases, as these concentrations may mark particular ecophysiological responses of trees, such as an increase in cold hardiness. This conjecture is consistent with the recognition that the quantification of neutral sugar concentrations in stemflow would be vital to unravelling the ecophysiology of forest environs (Levia and Germer 2015). The problem is that the concentration of neutral sugars in relation to tree species and phenophases is largely unknown, and the connection between stemflow and the temporal variation of woody tissue chemistry is nonexistent.

Neutral sugars are carbohydrates that contain only an alcohol group with their ketone and aldehyde group. These sugars include rhamnose, mannose, glucose, galactose, xylose, arabinose and fucose. Neutral sugars, which are found in appreciable quantities in tree bark (Kofujita et al. 1999), are important to the functional ecology of trees as they provide various functions, including structural support, transport of energy, and storage. Shigihara et al. (2008) recorded concentrations of neutral sugars for Japanese fir (Abies firma Siebold & Zucc.) in rainfall, throughfall, and stemflow while studying the effects of fog deposition on Japanese forests. They found that stemflow had higher concentrations of neutral sugars than throughfall and rainfall. To the authors' knowledge, this effort is the only known study to quantify neutral sugars in stemflow. Herein, we report on the neutral sugar concentration of stemflow with respect to four tree species and canopy phenophase (emergence, leafed, senescence, leafless for deciduous species; and emergence, leafed-spring/summer, senescence, leafed-winter for pine), and we propose a conceptual model to highlight the interplay between higher stemflow neutral sugar concentrations during senescence and ecophysiological changes in bark/wood chemistry that initiate cold hardiness.

Across all tree species – American beech (Fagus grandifolia Ehrh.), sweet birch (Betula lenta L.), yellow poplar (Liriodendron tulipifera L.), and pitch pine (Pinus rigida L.) – and phenophases examined, median stemflow neutral sugar concentrations (µM) were the highest for galactose (15.56 µM) and glucose (13.84 µM) and lowest for rhamnose (4.87 µM) and fucose (1.51 µM) (Supporting Information S1: Table S1, see Supporting Information S1: Table S2 for stemflow dissolved organic carbon [DOC] concentrations). Glucose had the largest range in stemflow concentrations between the first and third quartiles (23.61 µM) and fucose the smallest range (2.41 µM) (Supporting Information S1: Table S1). Likewise, glucose had the largest third quartile concentration (28.87 µM) and fucose the smallest first quartile concentration (0.83 µM) (Supporting Information S1: Table S1). All median stemflow neutral sugar concentrations fell within the ranges of prior literature (Shigihara et al. 2008). In general, median stemflow neutral sugar concentrations tended to be higher for yellow poplar and pitch pine than American beech and sweet birch (Supporting Information S1: Figure S1). Median stemflow concentrations of galactose and glucose were higher than other sugars across tree species (Supporting Information S1: Figure S1).

Median stemflow neutral sugar concentrations by phenophase (all tree species combined) show the clear importance of the senescence phenophase (Figure 1a). In fact, for all sugars except rhamnose, samples from senescence had the highest median stemflow concentrations when compared to the other phenophases (Figure 1a). For rhamnose, samples from the leafed phenophase had the highest median concentrations, followed very closely by those from the senescence phenophase, which had the second highest concentration (Figure 1a). Glucose had the largest interquartile range of the sugars in all phenophases except in the leafless phenophase (Figure 1a). The interquartile range was lowest for fucose across phenophases (Figure 1a). Moving beyond the descriptive statistics of raw concentration data, carbon-normalised (per number of carbon atoms, that is, pentose or hexose sugar) neutral sugar concentrations revealed statistically significant differences (p < 0.05) for all sugars except rhamnose between the senescence phenophase and all other phenophases (Supporting Information S1: Table S4). Figure 1b highlights the clear importance of senescence with regard to the leaching of neutral sugars in stemflow, with senescence having the largest median concentration sums of all phenophases for all four tree species.

Unlike raw stemflow neutral sugar concentrations, larger stemflow sugar yields per unit DOC are found for the thinner-barked species (American beech and sweet birch) than yellow poplar or pitch pine during the senescence and leafless phenophases (Supporting Information S1: Figure S2). The evergreen (pitch pine) shows no difference with phenophase, with yellow poplar showing markedly less variation than American beech or sweet birch during senescence (Supporting Information S1: Figure S2). The stemflow yields as percentage of organic carbon as neutral sugars reinforce these results (Supporting Information S1: Figure S3).

Interestingly, an examination of stemflow neutral sugar yields per unit DOC and yields (as a percentage of organic carbon as sugars, Supporting Information S1: Figure S3), reveals a more nuanced understanding of tree species-phenophase combinations of stemflow that deepens our ecophysiological understanding in light of the role of bark as a thermal insulator (Bär and Mayr 2020). The observation that the largest stemflow sugar leaching yield as a percentage of organic carbon occurred for thinner-barked trees during senescence and the early leafless phenophase but then declined over December, January, and February (Supporting Information S1: Figure S4), when minimum mean air temperatures are lowest (mean minimum air temperature for December 2022, January 2023, and February 2023 was −3.1°C compared to the −0.9°C for March 2023, with fewer days experiencing below freezing air temperatures in March 2023) and the need for optimal cryoprotection is greatest, is consistent with the need for the thinner-barked species to conduct more vigorous cryoprotective measures (depolymerisation to monosaccharides) for improved cold tolerance. Thus, during senescence, dynamic sugar transport toward cold acclimation/weatherproofing occurs, and the sugar composition in the apoplast (especially of the inner bark) is thought to change dramatically. Neutral sugars may then leach into the stemflow accessible to the apoplast.

Soluble oligosaccharides, such as raffinose, are synthesised during senescence and are considered to be important metabolites for cold acclimation (Janská et al. 2010). Biosynthesized soluble oligosaccharides are thought to be stored in intracellular vacuoles (Janská et al. 2010). Soluble oligosaccharides stored in vacuoles are transferred to the apoplast via vesicles during senescence triggered by cold stress (Valluru et al. 2008). The apoplast contains exohydrolase, which degrades some of the transferred soluble oligosaccharides to produce a mixture of various trimeric, dimeric, and monomeric sugars. The arrangement of these different sugar compositions in the apoplast is considered to create an optimal condition for membrane stabilisation (Livingston et al. 2006). Moreover, Davidson et al. (2021) showed that starch and oligosaccharides are among the nonstructural carbohydrates accumulated in the xylem and bark of several tree species, with the decomposition of nonstructural carbohydrates in the bark beginning earlier than in the xylem. The decomposition of these nonstructural carbohydrates and changes in soluble sugar concentrations are attributable to the trees' active acclimation to cold (Davidson et al. 2021).

We propose a conceptual model (Figure 1c), derived from a pairing of our experimental data with mechanisms for sugar transport and translocation from the literature, to explain the interplay between higher stemflow neutral sugar concentrations during senescence and the ecophysiological changes in bark/wood chemistry that initiate cold hardiness. The two mechanisms to account for sugar translocation to the apoplast are via the SWEET (Sugars Will Eventually be Exported Transporter) family of cellular uptake transporters (Selvam et al. 2019) or, in the case of oligosaccharides (e.g., raffinose), by SWEET and/or active transport by vesicles (Valluru et al. 2008). In the present results, the median stemflow galactose concentration was at its maximum during senescence in all tree species, and, similarly, the stemflow glucose concentration was at its maximum in all species, except pitch pine. These results are consistent with the model (Figure 1c) in that soluble sugars such as raffinose are transferred to the apoplast as a cold acclimation response and degraded by exohydrolase, resulting in the release of neutral sugars such as galactose and glucose into the apoplast accessible to the stemflow, which increases the neutral sugar concentration in stemflow during senescence. The leaching of sugars from the bark happens quickly at the intrastorm scale as stemflow is generated because translocation rapidly replenishes sugar stocks in the apoplast via diffusion due to the differential sugar concentrations between the symplast (higher concentration) and apoplast (lower concentration). As long as the concentration gradient is maintained, SWEETs will continue to elute sugars, and those sugars will be leached by stemflow. Thus, stemflow leaching engenders the lower sugar concentrations in the apoplast and triggers SWEET to expel sugars from the symplast.

Our conceptual model links the ecophysiological reasons that trees may begin to accumulate neutral sugars in the apoplast – namely, to ready themselves for the lower air temperatures encountered during winter – to stemflow leaching. The sugar concentrations of bark have been investigated with respect to phenophase (Orihashi et al. 2005). As winter progresses and more sugars are needed to adequately protect against cold, sugar concentrations increase. In larch trees raffinose, glucose, fructose, and sucrose were all reported to increase in concentration in the trunk and twig bark during senescence and winter (Orihashi et al. 2005). The larger stemflow neutral sugar concentrations in senescence could serve as a proxy for the initiation of cold hardiness in plant tissues. We posit that the sugar concentrations in stemflow are larger in senescence than the leafless phenophase because it takes a critical mass of sugars to weatherproof the trees and, as such, those initial translocated sugars are more prone to stemflow leaching than later in winter (Supporting Information S1: Figure S4) once the concentrations of sugars in the apoplast reach a certain threshold and a hydrophobic gating residue from SWEET limits sugar transport (Selvam et al. 2019). Thus, the concentrations of neutral sugars in stemflow may divulge a key link to cold readiness in trees. Tree weatherproofing during senescence and the utilisation of carbohydrates is imprinted in trees' stemflow chemistry. Future iterations of the proposed conceptual model could benefit from including the dynamics of structural carbohydrates such as cellulose, hemicellulose, and pectin, along with other storage sugars. Investigating how these polysaccharides vary in tree bark across different species and phenophases is essential.

The authors declare no conflicts of interest.