Constitutive overexpression of <i>NtCP14</i> leads to cell death at the two-celled proembryo stage.

<p>(A) Enhanced expression of <i>NtCP14</i> under the control of <i>proZC1</i> promoter in transgenic lines, as measured by RT-qPCR. The expression level of <i>NtCP14</i> in the WT was set to l. (B) Cathepsin H-like proteolytic activity towards substrate Bz-FVR-AMC in the two-celled proembryos from WT and <i>NtCP14</i>-overexpressing line L14-4. Data represent the mean ± SE, with 45 to 65 two-celled proembryos in WT and line L14-4, respectively. (C) The frequency of aborted seeds in WT and <i>NtCP14</i>-overexpressing lines. Data represent the mean ± SE from three independent experiments, with 300 to 400 seeds per line analysed in each experiment. (D) Cell viability in the two-celled proembryos from WT and <i>NtCP14</i>-overexpressing line analysed by staining with FDA and PI. Scale bars, 10 µm. (E) The frequency of two-celled proembryos with PI-positive apical and basal cells in WT and <i>NtCP14</i>-overexpressing lines. Data represent the mean ± SE from four independent experiments, with 50 proembryos per line analysed in each experiment (<i>n</i> = 200). (F) Nuclear DNA fragmentation in the two-celled proembryos from WT and <i>NtCP14</i>-overexpressing line L14-4 stained with TUNEL. Scale bars, 10 µm. (G) The frequency of two-celled proembryos with TUNEL-positive apical and basal cells in WT and <i>NtCP14</i>-overexpressing line L14-4. Data represent the mean ± SE from three independent experiments, with 30 proembryos per line analysed in each experiment (<i>n</i> = 90). (H) Caspase-like activities in the two-celled proembryos from WT and <i>NtCP14</i>-overexpressing line L14-4. Data represent the mean ± SE, with 40 to 65 two-celled proembryos in WT and L14-4, respectively. (I) Cathepsin H-like activity of NtCP14 in the WT embryo proper versus suspensor at developmental stages 3 and 4. Data represent the mean ± SE, with 85 to 100 cells of embryo proper or suspensor analysed at stages 3 and 4. (J) Cathepsin H-like activity of NtCP14 in the basal cell lineage at successive stages of embryogenesis in WT plants. Data represent the mean ± SE, with 85 to 100 suspensor cells. * and ** indicate statistical difference compared to WT or as shown otherwise (<i>t</i>-test, <i>p</i><0.05 or 0.01, respectively).