Advances in retrieval of solar-induced chlorophyll fluorescence (SIF) provide a promising and independent approach for quantifying gross primary production (GPP) across spatial scales. Recent studies have highlighted the prominent role of qL, the fraction of open Photosystem II (PSII) reaction centers, in mechanistically modeling GPP from remote sensing SIF. However, due to the limited availability of simulated and experimental data, a comprehensive understanding of qL responses to environmental and physiological variations has yet to emerge, and as a consequence, prediction of qL across leaf and canopy scales is still in an early stage. Based on a global sensitivity analysis of a recently developed mechanical model of photosynthesis, we find that the broadband total SIF emitted from PSII (SIFTOT_FULL_PSII) and leaf temperature (TLeaf) are the two major predictors of qL. A leaf-level instrument is designed to obtain concurrent measurements of qL, SIFTOT_FULL_PSII, and TLeaf over a wide range of environmental conditions. From these measurements, we show that qL can be modelled as a hyperbolic function of SIFTOT_FULL_PSII with only one temperature-related parameter m which increases with temperature, but decreases rapidly as temperatures exceed the optimum temperature. It is suggested that m can be mathematically modelled by a peaked function. The results of the leaf-level experiments on winter wheat demonstrate that the proposed model predicts qL with high accuracy (R2 ≥ 0.91, rRMSE ≤ 8.46%) under diverse light and temperature conditions. The essential steps necessary to apply it at canopy scale, including estimating the escape fraction, removing fluorescence emitted from Photosystem I, and reconstructing SIFTOT_FULL_PSII from top-of-canopy (TOC) narrowband SIF, are also presented. Our results confirm that estimated GPP using SIF-informed qL agrees well with measured GPP at a winter wheat site (R2 = 0.81, rRMSE = 12.03%). The key benefit of SIF-informed approach is that SIFTOT_FULL_PSII provides critical information on the collective influence of the sub-canopy light environment on qL, avoiding the requirement to explicitly estimate qL at different canopy depths, potentially promoting the ability of SIF to mechanistically quantify photosynthetic CO2 assimilation at large scales.