Fig 3. Characterization of zfp2, bhlh99, pap2 and at1g66810 mutants.

(A) Oregon Green 488 dextran-derived fluorescence upon infiltration of the full expanded leaves with the apoplastic pH indicator. In comparison to Col-0, more intense fluorescence emission was observed in the four mutants, and was particularly intense in pap2 plants, indicative of enhanced alkalinization of the apoplast in mutants compared to Col-0 plants. (B) Estimation of plasma membrane-anchored (GPI-AGP) and free AGP content in Col-0 and zfp2, bhlh99, pap2, and at1g66810 insertion mutant plants using β-d-Glucosyl Yariv reagent. AGP content was calculated with respect to a regression curve obtained by a radial diffusion assay in agarose plates containing Yariv regent and increasing amounts of gum Arabic (calibrating curve on the left). (C) Scanning electron microscopy (SEM) of leaf epidermis in Col-0 and zfp2, bhlh99, pap2, and at1g66810 mutants. Only pap2 exhibited a slight increase in epidermal cell size which was variable among different leaves of different plants. (D) Histochemical detection of lignin in proximal stem sections of Col-0 and zfp2, bhlh99, pap2 and at1g66810 mutant stems. Stem sections were stained with phloroglucinol-HCl (red color) for lignin detection in the interfascicular fiber walls and xylem cells, as observed with light microscopy. if, Interfascicular fibers; pi, Pith parenchyma; x, Xylem. (E-F) Cotyledon vein patterns were altered in zfp2, bhlh99, pap2, and at1g66810 insertion mutants. E, magnified pictures showing pattern defects exhibited by some mutants (e.g., 2 loops; right picture) compared to the most common 4 loop pattern observed in Col-0 (left picture). F, columns 2–7: number of cotyledons displaying the venation pattern depicted at the top, which ranged from the most common 4 loops observed in Col-0 to the less common phenotype of 2+1 or 2 loops, but prevalent in the pap2, bhlh99, or zfp2 mutants. “n” = total number of scored cotyledons.