Abstract
Vacuum infiltration-centrifugation (VIC) is the most reproducible technique for the isolation of apoplast washing fluid (AWF) from leaves, but its effectiveness depends on the infiltration-centrifugation conditions and the anatomical and physiological peculiarities of leaves. This study aimed to elaborate an optimal procedure for AWF isolation from the leaves of Tartary buckwheat grown in in vivo and in vitro conditions and reveal the leaf anatomical and physiological traits that could contribute to the effectiveness of AWF isolation. Here, it was demonstrated that leaves of buckwheat plants grown in vitro could be easier infiltrated, were less sensitive to higher forces of centrifugation (900× g and 1500× g), and produced more AWF yield and apoplastic protein content than in vivo leaves at the same forces of centrifugation (600× g and 900× g). The extensive study of the morphological, anatomical, and ultrastructural characteristics of buckwheat leaves grown in different conditions revealed that in vitro leaves exhibited significant plasticity in a number of interconnected morphological, anatomical, and physiological features, generally driven by high RH and low lighting; some of them, such as the reduced thickness and increased permeability of the cuticle of the epidermal cells, large intercellular spaces, increase in the size of stomata and in the area of stomatal pores, higher stomata index, drop in density, and area of calcium oxalate druses, are beneficial to the effectiveness of VIC. The size of stomata pores, which were almost twice as large in in vitro leaves as those in in vivo ones, was the main factor contributing to the isolation of AWF free of chlorophyll contamination. The opening of stomata pores by artificially created humid conditions reduced damage to the in vivo leaves and improved the VIC of them. For Fagopyrum species, this is the first study to develop a VIC technique for AWF isolation from leaves.