*4.1. Leaf* δ*13C Changed with Leaf Age and Tree Age*

*P. crassifolia* is widely distributed throughout the arid zone of northwest China [22]. In our work, leaf <sup>δ</sup>13C varied from <sup>−</sup>28.22‰ to <sup>−</sup>24.09‰, with a mean of <sup>−</sup>26.76‰, nearly identical to that reported by Pei et al. [27] (−28.58‰ to −25.02‰) and Yun et al. [28] (−28.9‰ to −25.4‰) for *P. crassifolia* in the Qilian Mountains. Moreover, the findings were almost the same as average values reported for *Pinus tabulaeformis* in northwest China with values of −26.82‰, which fell within the range of −28.6‰ to −25.02‰ [21].

Previous studies have confirmed that the rate of photosynthesis and respiration declines with leaf aging [13]. In this study, a significant difference in δ13C between current-year leaves and other old leaves was observed. The δ13C values achieved a relative maximum at the current-year leaves (−25.54‰) and lower values occurred in other old leaves. Since carbon was directly assimilated from the air or remobilized from reserve carbohydrates [13,29], we assumed that the isotopic patterns reported in our study might be caused by several effects. First of all, variations in leaf δ13C values were related with *P. crassifolia* interior biochemical processes. In the initial developmental stages, a 'hungry' state of intercellular CO2 concentrations exists because plants grow relatively rapidly and need to synthesize large amounts of organic matter to meet the demands of development and construction, leading to reduced distinguishing and exclusion of 13CO2 [18]. Thus, the values become enriched. When the exterior morphology, interior structure and physiological metabolism functions are mature, plants have the ability to adjust physiological and biochemical reactions. Therefore, they can efficiently distinguish and exclude 13CO2, and the δ13C values are expected to be depleted [30]. More importantly, photosynthetic capacity is the central process that coordinates carbon isotope discrimination, with more photosynthetically active leaves being relatively 13C-enriched [16].

Second, it was associated with leaf development stage [13]. Due to the changing growth rate between different aged leaves, the allocated proportions of the structural, functional and storage components within plants varied significantly to meet the leaves' nutrient needs. Previous 13CO2 tracer studies have reported that there were two leaf developmental stages including heterotrophic and autotrophic stages [14]. During the heterotrophic growth stage, where organic carbon was imported from elsewhere in the plant, enrichment in 13C was most evident in this stage and supposedly a result of the heterotrophic carbon source for growth [13,31]. This stage was more obvious for current-year-old leaves. Furthermore, Cernusak et al. [32] discussed six hypotheses regarding the explanation for the 13C enrichment of heterotrophic versus autotrophic plant organs. Based on these hypotheses, newly expanded leaves might need to synthesize large amounts of organic matter to meet the demands of development and construction, which leads to a reduction in distinguishing among sources of C and allows the leaves to obtain C from 13CO2. Moreover, young new leaves contained more 13C-enriched cellulose and import carbon from older leaves, while the old leaves had more 13C-depleted lipids and lignin and export carbon to the younger leaves [33]. As a consequence of these effects, an enrichment of 13C in current-year leaves was observed. During the autotrophic growth stage, carbon was assimilated and exported to other plant organs [17], which was most evident for older leaves [14]. The lighter carbon isotope was preferentially assimilated and used to produce the lipids and lignin, while the heavier carbon isotope was transported as 13C-enriched sucrose to the young new leaves [13]. As a result, the old leaves were expected to be 13C-depleted.
