Figure 3: TTN5 may be present in punctate structures in seedlings.

Microscopic observations of YFP fluorescence signals were made in a plane underneath the plasma membrane at the cell peripheries. (A), Schematic representation of an Arabidopsis seedling. Images were taken at three different positions of the seedlings and imaged areas are indicated by a red rectangle. (B-J), Analysis of YFP-TTN5, YFP-TTN5^T30N and YFP-TTN5^Q70L Arabidopsis seedlings via fluorescent confocal microscopy. (B-D), Fluorescence signals observed in stomata (indicated by empty white arrowhead) and in the epidermis of cotyledons in punctate structures (indicated by filled white arrowhead). (E-G), Localization in the hypocotyls showed the same pattern of punctate structures. (H-J), Signals were present in punctate structures in the root hair zone and in root hairs (indicated by filled magenta arrowhead). (K), Schematic representation of a N. benthamiana plant, used for leaf infiltration for transient expression. Imaged area is indicated by a red rectangle. (L-N), YFP fluorescence signals in N. benthamiana leaf epidermal cells expressing YFP-TTN5, YFP-TTN5^T30N and YFP-TTN5^Q70L. Signals were present in punctate structures (indicated by white arrowheads) and in the nucleus (indicated by empty magenta arrowheads). Scale bar 50 μm. (O), Root length measurement of HA₃-TTN5, HA₃-TTN5^T30N and HA₃-TTN5^Q70L Arabidopsis lines in comparison with non-transgenic wild type (WT). Seedlings were grown for 10 days on Hoagland plates. Only HA₃-TTN5^T30N showed a slightly reduced root length compared to WT, whereas HA₃-TTN5 and HA₃-TTN5^Q70L did not have a divergent phenotype. Analysis was conducted in replicates (n = 14). One-way ANOVA with Tukey post-hoc test was performed. Different letters indicate statistical significance (p < 0.05). (P), Two representative images of whole-mount immunostaining of HA₃-TTN5 seedling roots in the root differentiation zone (rabbit α-HA primary antibody, Alexa-488-labeled secondary α-rabbit antibody). Alexa-488 signals were present in punctate structures in root cells (indicated by filled white arrowhead) and in root hairs (indicated by filled magenta arrowhead) comparable to YFP signals (Figure 3H–J). Images are presented in a maximum intensity projection of several z-layers for a better visualization. Scale bar 50 μm.