Balancing the nutrient needs: Optimizing growth in Malus sieversii seedlings through tailored nitrogen and phosphorus effects

The impact of nitrogen (N) and phosphorus (P) on the physiological and biochemical processes crucial for tree seedling growth is substantial. Although the study of plant hydraulic traits in response to N and P is growing, comprehensive research on their combined effects remains limited. Malus sieversii, a key ancestral species of modern apples and a dominant species in Xinjiang's Tianshan wild fruit forest, is witnessing a decline due to climate change, pests and diseases, compounded by challenges in seedling regeneration. Addressing this, a four-year study was conducted to determine the optimal fertilization method for it. The experiment explored varying levels of N (N10, N20, N40) and P (P2, P4, P8), and their combined effects (N20Px: N20P2, N20P4, N20P8; NxP4: N10P4, N20P4, N40P4), assessing their impact on gas exchange, hydraulic traits, and the interplay among functional traits in Tianshan Mountains' M. sieversii seedlings. Our study revealed that all nitrogen treatments enhanced gas exchange, while phosphorus addition negatively impacted it. N10 significantly increasing leaf hydraulic conductivity. All phosphorus-inclusive fertilizers adversely affected hydraulic conductivity. P8, N20P4 and N20P8 notably increased seedlings' vulnerability to embolism. Seedlings can adaptively adjust multiple functional traits in response to nutrient changes. The research suggests N10 and N20 as the most effective fertilization treatments for M. sieversii seedlings in this region, while fertilization involving phosphorus is less suitable. This study contributes valuable insights into the specific nutrient needs of it, vital for conservation and cultivation efforts in the Tianshan region.

Methods
Select sun-oriented, fully expanded leaves. Gas exchange parameters, including net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci), were measured using a Li-Cor 6400 portable photosynthesis system under a photosynthetic photon flux density of 1500 μmol m-2 s-1 between 9:00-13:00 BST. The leaf chamber used was a 2×3 cm unit with a red and blue light source. CO2 concentration was set to 400 ppm with a high flow rate of 500 μmol s-1 to minimize stabilization time for Pn and Gs measurements. 

Predawn water potential (ΨPd) and the midday water potential (ΨM) were measured at predawn (5:00–7:00) and midday (13:00–14:00) using a pressure chamber (Model 3500, PMS Instrument Company, Albany, USA). The evening before measurements, select and mark healthy, north-south oriented branches with colored tape. The next day, measure the water potential of the marked branches. Place the cut branches in the pressure chamber, open the control valve, gradually increase pressure until liquid appears at the cut surface, then close the valve and record the water potential.

In August 2020, we selected one healthy sample plant from each plot and marked them. In the evening, when transpiration tension is lowest, cut sun-facing branches about 50 cm underwater. Transport the branches back to the laboratory and store them at low temperatures, keeping them immersed in water. Cut the branches into 25 cm stems, removing 2-3 cm from both ends. Use a xylem hydraulic conductivity and embolism measurement system (Xylem embolism meter, Bronkhorst, Montigny-les-cormeilles, France) to measure initial hydraulic conductivity (K0) and maximum hydraulic conductivity (Kmax). K0 represents the xylem's hydraulic conductivity (Kh). Measure Kh using degassed pure water at low pressure (0.5-1.0 kPa) through a 0.45 μm filter until a stable flow rate is achieved. Calculate specific hydraulic conductivity (Kw) by normalizing Kh to the branch cross-sectional area (SA) and leaf-specific hydraulic conductivity (Kl) by normalizing Kmax to the total leaf area (LA).

Flush the catheter with the same solution three times at high pressure (0.2 MPa) until hydraulic conductivity stabilizes. Then, measure the xylem's hydraulic conductivity again at low pressure (0.5-1.0 kPa) to determine maximum hydraulic conductivity (Kmax). Gradually increase the branch xylem tension using an external cavitation chamber (PMS Model 3500, PMS Instrument Company, Albany, USA), maintaining each pressure level for 5 minutes. Immerse the branches in water and measure hydraulic conductivity (Kh). Continue adjusting the pressure gradient and measuring until hydraulic conductivity reaches 0.Create a xylem embolism vulnerability curve for plants based on varying pressures and the corresponding loss of hydraulic conductivity at these different pressure levels.