Subcellular localization of PpHsp16.4. (A) Confocal microscopy images of: tobacco protoplasts electroporated with 35S:GFP (I) or with 35S:PpHsp16.4-GFP constructs (II); tobacco leaves agroinfiltrated with 35S-PpHsp16.4-GFP construct (III); or Arabidopsis transgenic lines overexpressing PpHsp16.4-GFP fusion proteins (IV and V). (B) Schematic diagram of PpHsp16.4 genomic locus and knock-in construct. Exons (E1 and E2), intron (Int), coding sequence of Citrine (CITR), 5’ and 3’ untranslated regions (UTR), are shown. The position of the primers used for PCR analysis of transgenic lines is indicated by arrows. (C) PCR analysis of transgenic lines (#1, #5 and #7) (D) Immunoblot detection of PpHSP16.4-Citrine fusion proteins in transgenic P. patens. Proteins extracted from lines #1, #5 and #7, treated for 24 hours with 50 μM ABA, were analyzed by Western blot using antibodies α-GFP. As a control, a protein sample from Arabidopsis expressing GFP was included (35S-GFP). Protein sizes in kDa of the molecular marker (M) are shown. Ponceau red (Pc) staining of Rubisco large subunit was used to ensure equal loading of protein samples (E) Immunoblot detection of PpHsp16.4-Citrine P. patens . Samples were prepared from controls (Ctrl) or from plants treated with 50 μM ABA or incubated with 300 mM NaCl (Na) or 500 mM mannitol (Mtl) containing plates, or at 37°C (HS: Heat Shock),. Right panel, plants incubated for 48 hours at 37°C (HS) and allowed to recover for 6 hours (rec). (F) Spatial regulation and subcellular localization of PpHsp16.4-Citrine fusion proteins transgenic line # 5. Confocal microscopy images of untreated protonema (I), leafy gametophyte (II), protonema exposed for 24 hours at 37°C (III), protonema incubated in 500 mM mannitol supplemented plates (IV) and protoplasts (V). White arrows indicate Citrine fluorescence. Green image: GFP or Citrine emission, in red: chloroplast fluorescence, in gray: transmission light microscopy.