Source–sink manipulations did not greatly regulate leaf photosynthesis and morphology
As expected, with the highest values in the middle-wet season, the seasonal patterns of Pn and gs in P. volubilis plants are consistent with our previous results [27, 28]. At the leaf level, we did not find evidence that photosynthetic regulation compensated for leaf or fruit loss in the defoliated or deflorated plants, respectively (Fig. 1A). A lack of photosynthetic upregulation and reduced leaf area in the defoliated plants were also reported in black oak trees (Quercus velutina Lam.) . The physiological mechanism that drives the change in Pn following defoliation appears to vary widely between species [14, 30, 31]. Although leaf N content increased by defoliation, the stable Pn was probably due to the changes in within-cell partitioning of N within the leaf to decrease Rubisco levels (thus lower PNUE; Fig. 1E) [28, 32]. At the whole-plant level, defoliation can reduce the source:sink ratio for photosynthethate and increase the inhibition on Pn through carbohydrate feedback. Moreover, low soil nutrient availability may restrict the potential for compensatory photosynthesis and growth after defoliation . But this does not seem to be the case in our study, as all plants were sufficiently supplied with fertilizer to avoid growth limitation by soil nutrients. Alternatively, the defoliated plants were less water-stress than those of control plants, which is indicated by higher leaf WUEi values (Fig. 1D, but ), largely because of a reduced transition surface. In a young Eucalyptus nitens plantation, the aboveground biomass production per unit transpiration (i.e., stand water-use efficiency) was also increased by shoot pruning . No significant variations in photosynthetic rate in the deflorated plants were found compared with control, suggesting that the sink effect was moderate in 2.5-year-old P. volubilis plants bearing approximately hundred fruits.
Across all seasons, the lowest SLA found in the dry season supported the consumption that SLA in leaves that develop under water-deficit conditions was generally lower than those develop under wet conditions . Whereas SLA did not change greatly between different source or sink manipulations (Fig. 1F). It was found that severe fruit pruning did not change leaf morphology (leaf size and area) in oil palm (Elaeis guineensis) . Therefore, a relatively stable SLA of P. volubilis plant in response to disturbance is important to maintain leaf structural function and adapt to drought [25, 27].
C And N Differently Responded To Source–sink Manipulations
Carbohydrates stored in woody tissues of perennial plants are essential for a sustainable crop production because of their role in nutrient (mainly N) uptake and assimilation, leaf area formation, and inflorescence induction, particularly under stress situations . Since P. volubilis plants have extremely small root biomass fraction (ca. 6%) and store only very little NSC in roots [27, 34], vegetative aboveground tissues have to assume these functions. NSC contents across plant tissues on diurnal and seasonal scales are generally assumed to be indicative of the C balance, i.e., the difference between C supply (photosynthesis) and demand (export and growth) [5, 8, 31]. The highest and lowest NSC contents occurred in the middle-wet season and in the dry season, respectively (Fig. 2D), reflecting the phenomenon of ‘refill’ and ‘carbon depletion’ during the wet and dry season, respectively. Water stress in the dry season can cause substantial inhibition of photosynthesis and may have necessitated a decrease in carbohydrate mobilization from storage tissues [27, 35]. Whereas in a pot experiment with four grapevine varieties, it was found that an increase rather than a decrease in carbohydrate levels in wood tissues under prolonged drought because of the decreased demand of aboveground growth .
NSC contents (especially sugar) were sharply reduced within a short period (three weeks) after defoliation applied in the early-wet season, but were completely recovered in the late-wet season and were maintained in the dry season (Fig. 2B, D). Our findings are consistent with the consumption that fast-growing species (e.g., P. volubilis), typically having low leaf construction costs and more flexible growth strategies, respond rapidly to defoliation, through changes in carbohydrate contents . Defoliated plants reduced NSC contents, although temporally, either by reducing the photosynthetic apparatus and/or by triggering new foliage growth, but not by the compensatory photosynthesis observed (Fig. 1A). On the other hand, defloration increased available resources through sink limitation, as indicated by the continuous increase in NSC reserves (Fig. 2D). This was probably the cause of the less resource competitions between the remaining fruits and also between vegetative and reproductive tissues. Thus, carbohydrate reserves in vegetative tissues of P. volubilis plants served to buffer sink–source imbalances that may result from temporary reductions in demand for assimilates (e.g., defloration) or shortfalls in carbon assimilation (e.g., defoliation) [9, 10, 38].
After defoliation, C and N limitations are assumed to result in the decreased plant growth and even plant mortality, especially in evergreen species that they have abundant nutrition reserves (e.g., N and P) in leaves year-round [1, 31]. Compared with control, the defoliated P. volubilis plants had a temporally reduced NSC contents and lower long-term growth (Fig. 5C). This was consistent with the result of a large-scale manipulative field experiment in a hybrid poplar plantation, where repeated defoliation lead to long-term growth decline but only transient C storage reduction occurred . On the other hand, defoliation lead to the increased N contents in both remaining leaves (Fig. 1C) and branches (Fig. 3A). This was probably resulted from the facts that the incensement of the internal N availability contributed to the initiation of new axes and the functional biomass partition between woody tissues and/or the enhanced N uptake from soils increased N contents in plant tissues [2, 7, 38, 39]. Thus, C limitation, but not N remobilization, is a source-driven growth process in P. volubilis plants [cf. 1]. But for a winter-deciduous temperate adult tree (Nothofagus pumilio), the greatly reduced plant growth induced by extreme defoliation was due to growth limitations (i.e., preventative C allocation to storage), not to insufficient C or N availability . A more integrated understanding of the possible shifts in C and/or N limitation on growth and the yield that occur during the lifetime of P. volubilis plants, is required.
Source–sink Manipulations Differently Regulated Reproductive Traits
After defoliation, either the advanced or delayed budburst was found in some temperate deciduous fruit trees (e.g., peach, ; walnut, ), because of the dose-dependent response of the supply of sugars (especially sucrose) involved in budburst processes. For the recurrent woody plants that bloom and fruit continuously in tropical areas, flowering and fruit maturation date show a marked yearly rhythm but the control of their periodicity is not well understood. Largely determined by temperature and photoperiod in plants without a vernalization requirement within a growing season , the phonological development of P. volubilis plants, i.e., initial peak mature time, was not affected by source–sink regulations (Fig. 4A), which was contrary to its response to long-term exposure to shade . But the dynamic pattern of the yield in the most peak mature dates differed between different treatments. The highest mature fruits were harvested earlier in the deflorated plants than the control; the reverse is true in the defoliated plants (Fig. 4A). A decrease in assimilate supply, due to leaf removal, might increase early fruit abortion thus delayed fruit maturation in evergreen trees .
Combined with a small fraction of fruit sets (< 40%, Fig. 3; ), the well-developed reproductive tissues (i.e., relatively large flower numbers per plant; ) indicated that flower initiation did not limit the number of fruits produced. But the fruit retention and development are the limiting factors for the yield of the wind-pollinated P. volubilis plants. Fruit (seed) number is a dominant factor for seed yield in P. volubilis plants because seed size in each harvested date and throughout the growing season did not differ significantly between three treatments (Fig. 4B; Fig. 5B). Along with our previously studies that fruit (seed) number, rather than seed size, responded greatly to agricultural management practices (e.g., fertilizers, irrigation and plant density; [25, 26, 27]), our results supported that, from an evolutionary perspective, plants would have much less plasticity in seed weight than in seed number . Increased fruit (seed) production following artificial defoliation or herbivory (i.e., overcompensation) has been found in some wild plant species and herb crops (e.g., potatoes), but the potential negative or zero effect of source-reducing on crop yield depends on the studied crop species and plant size, environmental conditions, and the frequency and intensity of defoliation . Fruit development can be supplied with C imported either from current photoassimilates of neighboring branches or from C reserves stored in the woody tissues. Defloration slightly increased, whereas defoliation greatly decreased the total seed yield (Fig. 5B). This implied that source–sink regulations affected fruit (seed) yield mainly by decreasing flower bud number and/or inducing fruit abortion, rather than individual fruit growth (size).
The alteration of the whole-plant carbon balance induced by source–sink regulations affect nutrition and carbon reserve in leaves and the reproductive tissues (flowers and fruitlets), leading to abscission [10, 43]. In addition, fruit abortion not only depends on the source strength but also on the sink strength of competing tissues [9, 11, 46]. Within a plant system, the smallest and less developed fruitlets generally undergo abortion, as the strongest fruitlets are positively selected against the weakest ones. In our study, defoliation reduced, but defloration increased the percentage of fruit abortion, especially in the earlier reproductive stage (Fig. 3). Thus, high fruit set of P. volubilis plants was involved an increased photosynthetic input and thus increased resource limitation (no defoliation), and less sink strength of competing reproductive tissues (deflorated plants). It was also reported that carbohydrate shortage, especially sugar, leads to dramatically accelerated fruitlet abortion in lychee, a recurrent tropical fruit tree . Generally, plants with a larger plant size (stem diameter) had the higher total seed yield across all treatments (Fig. 5C), indicating that the amount of stored resources (i.e., carbohydrate and N) from maternal plants mainly determine the seeds (fruits) numbers during the reproductive stage [27, 42]. Compared with control, the reduced amount of current and stored carbohydrates by defoliation during the reproductive stage may restrict the numbers of fruits (fruit load; but see olive tree ), especially in the dry season when water was limited . Carbohydrates (especially sugar) play a key role in flower bud formation and their levels can be directly correlated with floral induction in fruit trees . Our results may indicate that fruit retention and thereafter mature fruit number, rather than individual seed development (size), is strongly source limited in P. volubilis plants [c.f. 27]. However, in response to source–sink manipulations applied in the early-wet season, we still cannot know whether the varied mature fruit number (load) is due to a direct effect of fruit set, or an indirect effect caused by changes on the total fruit number per plant, or a complex carbohydrate and hormone signalling crosstalk controlling flower/fruitlet abscission .
On the other hand, to promote the understanding of fruit (seed) development and their metabolic regulations in P. volubilis plants, we characterized the primary and secondary metabolome in fruitlets after 24 days of source/sink regulation applied. The source or sink regulation had a substantial effect on the metabolites in fruitlets, as a significant differentiation occurred in the samples between defoliated and deflorated groups (Fig. S2). Compared with control, fewer metabolites in fruits showed relatively lower fluctuations in the defoliated plants than in the deflorated plants, especially for the secondary metabolites (Table 1, 2). Secondary metabolites are essential in the interactions with the environmental stressors, rather than playing a key role in plant growth . Given that defloration is known to reduce the competition for carbohydrate source and thus promote fruit size and carbohydrate content in several fruit trees including peach  and apple , it can be expected that the assimilated photosynthetic carbohydrate in the deflorated P. volubilis plants might be diverged to growth rather than to secondary metabolite synthesis in fruitlets. Defoliation downregulated the TCA cycle and carbohydrate metabolism in fruitlets (Fig. 8), resulting in carbon starvation and insufficient energy metabolism, thus inhibiting the growth and enhancing fruit abortion [11, 47]. Moreover, at the whole-plant level, regulation of primary metabolism determines the C:N balance and also affects sink strength [3, 21]. The decreased content of malic acid by defoliation may limit the energetic cost for fruit development, as it serves as a crucial intermediate involved in several metabolic pathways, such as TCA cycle, amino acid metabolism and biosynthesis of plant secondary metabolites. Interestingly, compared with control, both defoliation and defloration decreased lyxose content (Table 1, 2). Direct regulatory role of this compound is unclear. It was found that xylose is a major component of the pericarp cell walls of tomato fruits ; the metabolism of this hemi-cellulose is key for wall loosening linked with cell expansion . Moreover, fruit xylose was connected to leaf trehalose, possibly linking leaf sugar sensing to fruit expansion . On the other hand, the lipid production stage of oilseed development in requires significant demand on carbon sources, such as sugars produced by photosynthesis in the leaves . Defoliation, but not defloration, reduced the content of glycerol (Table 2), a product from lipid metabolism, implying that the reduced lipid accumulation occurred in seeds. Overall, this study indicated potentially important metabolites that are correlated to the potential fruit abortion and fruit development would provide a basis for further study on the process of fruit maturation during various developmental stages.