1. Nitrogen (N) resorption is a strategy for plant N conservation through which plants withdraw N from senescing leaves prior to litterfall and its underlying mechanisms are important for better understanding of N cycling. However, most current studies focused on the impacts of soil and leaf nutrients on leaf N resorption efficiency (NRE), and plant physiological regulation that is species-dependent is still unclear.

2. Here, we conducted a field experiment to investigate the variations of leaf NRE along an altitudinal gradient in a temperate forest of Northeastern China.

3. Results showed that leaf NRE of Q. mongolica and F. mandshurica increased with altitude, while leaf NRE of T. amurensis, A. mono and A. pseudosieboldianum exhibited an opposite trend, although the relationships were not significant for F. mandshurica and A. mono. The inconsistent responses of leaf NRE of different species to increasing altitude were primarily due to the effect of leaf Glutamate dehydrogenase (GDH), an enzyme responsible for N translocation. Leaf GDH activity in senescing leaves explained the variation of NRE more than soil and climate factors did, suggesting that different plant species had different physiological regulation strategies for their N conservation under similar environment.

4. Synthesis. Our study highlights the role of leaf enzyme as a pivotal regulator of leaf NRE and helps us better understand and predict N cycling under climate change in forest ecosystems.

One tree per species per plot was selected, and the selected trees of the same species should have similar heights and diameter at breast height (DBH). In order to calculate leaf NRE, green mature leaves were sampled from the selected trees by climbing the trees during the peak growing season from July 20 to 23, 2019, while senesced leaves were collected from October 18 to 21, 2019. Healthy green mature leaves were collected from lower and middle outer canopy. To minimize sample contamination, we collected green mature and senesced leaves with nitrile gloves. Each sampled tree was marked to facilitate collection of senesced leaves at the end of growing season. Senesced leaves were collected either directly from recently fallen leaves on the ground, or by gently shaking the branches and collecting fallen leaves. In all cases, non-degraded senesced leaves were acquired from the marked trees. The DBH of each sampled tree was measured to estimate leaf biomass.

Another set of green mature and senescing leaves were collected separately for GDH measurement. Leaves were recognized as senescing leaves when they just started to turn yellow. Both green mature and senescing leaves were collected on the trees by climbing the trees. Green mature leaves were all sampled in July. Due to the different phenology of different species, it took two months from September to October to collect senescing leaves. All senescing leaves were collected at the same canopy position as that of green mature leaves from high to low altitude. Senescing leaves of Q. mongolica, F. mandshurica, T. amurensis, A. mono and A. pseudosieboldianum were obtained on October 21, September 27, October 5, and October 10 to 11, respectively, in 2019. The harvested green mature leaves were divided into two parts: one was used to measure leaf area immediately and the other was frozen in liquid nitrogen immediately and stored at -80 ℃ for GDH analysis.

Because different soil horizons in temperate forests generally have distinct soil properties including soil nutrient, microbial community and soil texture, it is necessary to divide forest soil into organic (O) and mineral (A) layers. In each plot, after removing litter, five soil cores were acquired randomly using a soil auger (20 cm in depth). The O and A horizons were separated by visual observation of soil color. Generally, the depth of O horizon was approximately 5 cm, while that of A horizon was approximately 15 cm. The five soil cores were combined into one composite sample for each soil horizon. Additionally, two paired soil cores were obtained using stainless steel cores of 5 cm height and 100 cm3 volume in each plot from the O horizon and the A horizon, respectively. These soil cores were used for gross N ammonification (GA) and nitrification (GN) measurement using the 15N pool dilution mesocosm incubation method.