Discussion

Effects of N and P addition on leaf NSC

Compared with starch, soluble sugars are relatively more mobilizable, and participate in many physiological and metabolic activities, including the regulation of cell osmotic pressure and transport systems involved in plant growth (Hoch et al. 2003, Millard and Grelet 2010). However, starch is supposed to be the storage compound, and its pool could be depleted and changeable under the adverse environmental conditions (Martínez-Vilalta et al. 2016, Ivanov et al. 2019). Exogenous N fertilization has been proven to increase the plant growth rates and productivity in global tropical forests (Pasquini and Santiago 2012, Selene and Jürgen 2018), and also alters the NSC allocation pattern in tropical trees (Burslem et al. 1996). Surprisingly, we found that continuous N fertilization did not change the leaf NSC concentrations (Table 2). This may be due to the relatively higher leaf N contents and ‘N-saturation’ in the studied forest (Mo et al. 2015, Lu et al. 2018). The slightly positive or negative response in leaf N concentrations to N fertilization across 2012, 2015, and 2017 also revealed that the tropical plants were exposed to a sufficient N supply, and that the leaves were failed to absorb excess N (Fig. S2 and S3).
In contrast, P addition significantly altered leaf NSC concentrations (both soluble sugars and starch) in the three sampled years. Overall, +P reduced the soluble sugar and starch concentrations by 12.78% and 38.10%, respectively. These results indicate that the NSC dynamics are more sensitive to exogenous P addition rather than to N addition in this forest, which is in agreement with our hypothesis one. Our findings also demonstrate that the tropical trees prefer to lower their starch pools for allocating relatively larger mobile C to enhance plant growth under sufficient P availability (Li et al. 2016b). Additionally, our results reveal that continuous P addition down-regulated both leaf soluble sugars and starch concentrations of the majority of the species evaluated in this P-limiting tropical forest (Fig. S1). This further supports our hypothesis that the leaf NSCs would be primarily regulated by P availability rather than N availability in this low-P availability forest. A previous study reported that plant NSC storage was mediated by nutrient availability, with a larger allocation to storage when growth was limited by nutrients (Knox and Clarke 2005). In this P-deficient tropical forest, the higher leaf NSC storage was observed in CK plots, and exogenous P addition reduced the allocation of leaf soluble sugars and starch in most of the studied species in the tropical forest, which is consistent with a previous study (Knox and Clarke 2005). The reduced NSC concentrations may be converted into structural C to form the tissue biomass, which would further enhance plant productivity (Li et al. 2016b).

Species-specific effects on leaf NSCs

Leaf soluble sugars, starch, and total NSC (soluble sugars and starch) concentrations generally vary greatly among different plant species (Hoch et al. 2003, Almeida et al. 2015). The soluble sugar/starch ratios can reflect the NSC allocation pattern in leaves, which is essential for understanding the plant nutrient utilization strategies (Martínez-Vilalta et al. 2016, Xie et al. 2018). However, the leaf soluble sugars, starch, and NSC concentrations varied greatly across different regions (Druege et al. 2004, Guo et al. 2016, Li et al. 2016a, Martínez-Vilalta et al. 2016, Liu et al. 2018). In our study, the concentrations of leaf soluble sugar, starch, and NSCs in three of four species were similar to the results from a neighboring tropical forest on Hainan Island (Li et al. 2016a),S. octophylla was the exception to this pattern, as in this species leaf soluble sugar and starch concentrations were two to four times higher than those of the other three species evaluated in this tropical forest. Plant life history and ecological strategies primarily determine the quantity and allocation of carbohydrates in plants exposed to similar environmental conditions (Newell et al. 2002, Palacio et al. 2007, Hartmann et al. 2018). In this tropical forest, all studied species were understory shade-tolerant species that well adapted to the shade environment. The C assimilation of these tree species was lower due to their understory shade conditions, while their photosynthetic C assimilation would be primarily limited by continuous nutrient supply status, but not by light availability (Liu et al. 2018, Xie et al. 2018). These findings may provide profound insights into seedling regeneration and ecosystem functions in tropical forests (Santiago et al. 2012).
Leaf expansion may also be closely linked with the storage of NSCs because the long-distance transport of nutrients could consume a larger amount of energy derived from NSCs (Zhao and Oosterhuis 2000). Previous studies suggested that a higher concentration of NSCs may be closely related to a larger leaf area (Zhao and Oosterhuis 2000, Almeida et al. 2015). In 2015, we measured the LMA of these four species included in this study, and found thatS. octophylla had significantly lower LMA than those of S. bullockii and P. rubra (Mo et al. 2019). Hence, the lower LMA of S. octophylla may be attributed to its higher soluble sugars and starch concentrations. In addition, previous studies have suggested that plants with higher initial NSC reserves are more likely to survive when exposed to the adverse environmental conditions (Canham et al. 1999, Imaji and Seiwa 2010). The higher amount of seedling individuals of S. octophylla could be explained by the relatively higher leaf NSC storage compared to that of the other species studied in this tropical forest (Table 1).

Leaf NSC and plant adaptation to lower P availability

Previous studies have shown that both N and P addition reduced the leaf NSC (soluble sugar and starch) concentrations in some herbaceous plants, due to the carbohydrate consumption associated with relatively higher plant growth rates under N and P fertilization (Ai et al. 2017, Wang et al. 2017b). At this study site, we did not observe a significant change in the rate of photosynthesis under N or P addition in these plants (Mo et al. 2019). Nonetheless, we found that P addition significantly decreased the leaf soluble sugar concentrations in our study species. The reduced soluble sugar concentrations may be converted into the leaf tissue, which was explained by the negative correlation between the leaf soluble sugar concentrations and LMA (Table 3). Moreover, the increased structural P fraction further confirmed that P addition enhanced leaf expansion (Mo et al. 2019). Therefore, under P-insufficient conditions, tropical trees tend to maintain lower P concentrations and higher NSC contents, which may be one potential evolutionary mechanism by which tropical plants have adapted to P-deficient soil.
Although leaf NSCs are mainly regulated by P availability rather than N availability in this tropical forest, plants may also tend to transform and reallocate NSCs among leaves, branches, stems and roots to adapt to the adverse environmental conditions. Given that only leaf NSCs were evaluated in this study, the NSC responses to continuous N and P addition and changes in NSC concentrations among different plant organs could be far more complex than the results on leaves reported in this study, more detailed studies are still needed to investigate the leaf NSC responses to N and P availability in a wider range of plant species in tropical forests.