The direct estimation of nitrogen (N) in fresh vegetation is challenging due to its weak influence on leaf reflectance and the overlaps with absorption features of other compounds. Different empirical models relate in this work leaf nitrogen concentration ([N]Leaf) on Holm oak to leaf reflectance as well as derived spectral indices such as normalized difference indices (NDIs), the three bands indices (TBIs) and indices previously used to predict leaf N and chlorophyll. The models were calibrated and assessed their accuracy, robustness and the strength of relationship when other biochemicals were considered. Red edge was the spectral region most strongly correlated with [N]Leaf, whereas most of the published spectral indexes did not provide accurate estimations. NDIs and TBIs based models could achieve robust and acceptable accuracies (TBI1310,1720,730: R2 = 0.76, [0.64,0.86]; RMSE (%) = 9.36, [7.04,12.83]). These models sometimes included indices with bands close to absorption features of N bonds or nitrogenous compounds, but also of other biochemicals. Models were independently and inter-annually validated using the bootstrap method, which allowed discarding those models non-robust across different years. Partial correlation analysis revealed that spectral estimators did not strongly respond to [N]Leaf but to other leaf variables such as chlorophyll and water, even if bands close to absorption features of N bonds or compounds were present in the models.This research has been funded by the BIOSPEC project “Linking spectral information at different spatial scales with biophysical parameters of Mediterranean vegetation in the context of Global Change” (http://www.lineas.cchs.csic.es/biospec) (CGL2008-02301/CLI, Ministry of Science and Innovation) and the FLUXPEC project “Monitoring changes in water and carbon fluxes from remote and proximal sensing in a Mediterranean dehesa ecosystem” (http://www.lineas.cchs.csic.es/fluxpec) (CGL2012-34383, Ministry of Economy and Competitiveness). Pacheco-Labrador, J. was sponsored by a JAE-Predoc grant (CSIC)Peer reviewe
