The mobilization and transport of newly fixed carbon are driven by plant water use in an experimental rainforest under drought

Abstract Non-structural carbohydrates (NSCs) are building blocks for biomass and fuel metabolic processes. However, it remains unclear how tropical forests mobilize, export, and transport NSCs to cope with extreme droughts. We combined drought manipulation and ecosystem 13CO2 pulse-labeling in an enclosed rainforest at Biosphere 2, assessed changes in NSCs, and traced newly assimilated carbohydrates in plant species with diverse hydraulic traits and canopy positions. We show that drought caused a depletion of leaf starch reserves and slowed export and transport of newly assimilated carbohydrates below ground. Drought effects were more pronounced in conservative canopy trees with limited supply of new photosynthates and relatively constant water status than in those with continual photosynthetic supply and deteriorated water status. We provide experimental evidence that local utilization, export, and transport of newly assimilated carbon are closely coupled with plant water use in canopy trees. We highlight that these processes are critical for understanding and predicting tree resistance and ecosystem fluxes in tropical forest under drought.

Table S3.Two-way ANOVA testing effects of drought and species and their interactions on concentrations of soluble sugars, starch and total NSCs in leaves.
Numbers represent p-values.ANOVA was conducted with Type I sums of squares.NSCs, nonstructural carbohydrates.
Table S4.Two-way ANOVA testing effects of drought and species and their interactions on the amount of the 13 C label.
Numbers represent p-values.ANOVA was conducted with Type I sums of squares.The 13 C label represents the excess 13 C relative to the pre-labeling 13 C values in atom % in leaves (at day 0), stem phloem (at day 4) and roots (at day 4).The amount of excess 13 C (atom %) in leaves after labeling under drought was divided by 2.1 prior to test to account for the differences in excess 13 C in the atmosphere before (1.30 atom %) and during drought (2.73 atom %) (Werner et al., 2021).
Table S5.Correlation coefficient (R) and significance (P) of the linear regressions (see Fig. S2) of leaf sugars, starch, total NSCs versus time for the three canopy species.
Table S6.Correlation coefficient (R) and significance (P) of the linear regressions (see Fig. S3) of leaf sugars, starch, total NSCs versus time for the five understory species.
Fig. S1.The drought treatment, labeling and sampling timeline.To impose drought treatment, rainfall and irrigation were withheld from 8 Oct, 2019 to 2 December 2019 (c. 8 weeks).We conduced 13 CO2-pulse labeling under pre-drought conditions (5 Oct, 2019; left table) and under drought conditions (23 Nov, 2019; right table).Samples were collected for NSC analysis and isotopic tracing during each labeling campaign: 1) leaves were sampled before labeling and 0, 1, 3 and 5 days after labeling; 2) stem phloem samples from the canopy trees were collected before labeling, and 3 to 9 days after labeling; 3) roots were collected before labeling, and 3 to 4 days after the labeling.Samples were immediately frozen in liquid nitrogen or dry ice to stop metabolic activity, and stored at -20 °C.Note that post-labeling root samples collected from the canopy trees under drought were lost during shipping process and not available for analyses.All samples were transported on dry ice by car, freeze-dried, and ground to fine powder before metabolite and isotope analysis.S5 for correlation coefficient (R) and significance (P).Note that we added twice as much 13 CO2 label to the atmosphere to compensate for the reduction in photosynthesis under drought.

Fig. S2 .
Fig. S2.Changes in concentrations of soluble sugars, starch, and total nonstructural carbohydrates (NSCs; soluble sugars + starch) in the leaves and stem phloem over the course of the experiment in the three canopy tree species: Clitoria fairchildiana (CF), Phytolacca dioica (PD), Pachira aquatica (PA).Values are the means of 3 or 4 plants per species, and error bars represent standard errors.Background shadings indicate the drought days.See TableS5for correlation coefficient (R) and significance (P).

Fig. S3 .
Fig. S3.Changes in concentrations of soluble sugars, starch, and total nonstructural carbohydrates (NSCs; soluble sugars + starch) in the leaves and roots over the course of the experiment in the five understory tree species: Piper auritum (PI), Hibiscus rosa sinensis (HR), Calathea sp.(CA), Syngonium sp.(SY), Dieffenbachia sp.(DI).Values are the means of 3 or 4 plants per species, and error bars represent standard errors.Background shadings indicate the drought days.See TableS6for correlation coefficient (R) and significance (P).

Fig. S4 .
Fig. S4.Relative changes in the ratio of soluble sugars to nonstructural carbohydrates (sugars/NSCs) in the leaves (a) and stem phloem (b) under drought, expressed as percent deviations from pre-drought values (n = 3 or 4 plants per species).Data are shown for the three canopy species including Clitoria fairchildiana (CF), Phytolacca dioica (PD), Pachira aquatica (PA), and five understory species including Piper auritum (PI), Hibiscus rosa sinensis (HR), Calathea sp.(CA), Syngonium sp.(SY), Diefenbachia sp.(DI), as well as averaged (AVG) across the species means (grey).Percent deviations are not computed (NS) for PD phloem due to low starch concentrations (<0.5%;Fig. S2).Positive values represent increase under drought and negative values represent decrease.Error bars represent standard errors.Significant within-species (Student's t-test) and cross-species (two-way ANOVA) differences between pre-drought and drought were calculated based on the raw concentrations and indicated by an asterisk (P < 0.05).

Fig. S5 .
Fig. S5.Changes in δ 13 C of leaf soluble carbon for the three canopy species (Clitoria fairchildiana, CF; Phytolacca dioica, PD; Pachira aquatica, PA) under pre-drought and drought conditions (n = 3 or 4 plants per species).Values are the means of 3 or 4 plants per species, and error bars represent standard errors.Note that we added twice as much 13 CO2 label to the atmosphere to compensate for the reduction in photosynthesis under drought.

Fig. S6 .
Fig. S6.Changes in δ 13 C of leaf soluble carbon for the five understory species (Piper auritum, PI; Hibiscus rosa sinensis, HR; Calathea sp., CA; Syngonium sp., SY; Dieffenbachia sp., DI) under pre-drought and drought conditions (n = 3 or 4 plants per species).Values are the means of 3 or 4 plants per species, and error bars represent standard errors.Note that we added twice as much 13 CO2 label to the atmosphere to compensate for the reduction in photosynthesis under drought.

Fig. S7 .
Fig. S7.Changes in δ 13 C of phloem soluble carbon for the three canopy species (Clitoria fairchildiana, CF; Phytolacca dioica, PD; Pachira aquatica, PA) under pre-drought and drought conditions (n = 3 or 4 plants per species).Values are the means of 3 or 4 plants per species, and error bars represent standard errors.

Fig. S8 .
Fig. S8.Changes in δ 13 C of root soluble carbon for the three canopy species (Clitoria fairchildiana, CF; Phytolacca dioica, PD; Pachira aquatica, PA) under pre-drought and drought conditions (n = 3 or 4 plants per species).Values are the means of 3 or 4 plants per species, and error bars represent standard errors.

Fig. S9 .
Fig. S9.Changes in δ 13 C of root soluble carbon for the five understory species (Piper auritum, PI; Hibiscus rosa sinensis, HR; Calathea sp., CA; Syngonium sp., SY; Dieffenbachia sp., DI) under pre-drought and drought conditions (n = 3 or 4 plants per species).Values are the means of 3 or 4 plants per species, and error bars represent standard errors.

Fig. S10 .
Fig. S10.Changes in stomatal conductance (Gs) over the course of the experiment in five species: Clitoria fairchildiana (CF), Phytolacca dioica (PD), Pachira aquatica (PA), Piper auritum (PI), Hibiscus rosa sinensis (HR).Values are the means of 3 or 4 plants per species, expressed as a percentage of pre-drought.Error bars represent standard errors.Background shadings indicate the drought days.Grey lines indicate the pulse-labeling events.

Fig. S11 .
Fig. S11.Changes in δ 13 C of leaf soluble carbon versus non-soluble carbon for the three canopy species (Clitoria fairchildiana, CF; Phytolacca dioica, PD; Pachira aquatica, PA) and five understory species (Piper auritum, PI; Hibiscus rosa sinensis, HR; Calathea sp., CA; Syngonium sp., SY; Dieffenbachia sp., DI) under pre-drought and drought conditions (n = 3 or 4 plants per species).See Method S3 for details.Values are the means of 3 or 4 plants per species, and error bars represent standard errors.Across canopy and understory species, there were significant correlations and these correlations were not affected by drought, indicating that drought did not affect partitioning of recent photosynthates into soluble vs. non-soluble carbon pools.

Fig. S12 .
Fig. S12.The relationships between midday leaf water potential and the absolute MRT (hours) of the 13 C label in leaf soluble carbon for the three canopy species (Clitoria fairchildiana, CF; Phytolacca dioica, PD; Pachira aquatica, PA) and two understory species (Piper auritum, PI; Hibiscus rosa sinensis, HR) under pre-drought (blue) and drought (yellow) conditions (n = 3 or 4 plants per species).

Fig. S13 .
Fig. S13.Changes in gross primary productivity (GPP) during the imposed drought (yellow background shading) and recovery (blue background shading) in 2002 (left panel) and 2019 (right panel).Grey lines indicate the pulse-labeling period in the 2019 experiment.In both experiments, GPP decreased during drought and then increased after re-watering.

Table S2 .
Absolute values of plant hydraulics under pre-drought and under drought.