Stemi 2000

Images of mature plants were taken with a Cannon Stylus 1010 digital camera set on a tripod for stability. Close-up leaf, inflorescence, and silique images were collected on a Zeiss Stemi-2000 stereo microscope (Carl Zeiss, Germany) using Lumenera Infinity2 software (Lumenera Corp.). Seedling and root cell file images were collected on a Leica DMI6000B using LAS imaging software (Leica Co., Germany) with the exception of amiRNA1334 and abcb6-1 abcb20-1 which were collected on a Zeiss LSM 780 using a 10× objective. Root length measurements were made from high-resolution scanned images. All seedling measurements were made using ImageJ (Schneider et al., 2012 (link)). Measurements for mature aerial tissues were done manually. Brightness and contrast were adjusted using Adobe Photoshop to enhance visualization of the cell profiles equally within each image set as noted in the respective figure legends. All assays were conducted at least three times with similar results.

To sample the leaf mesofauna, 25 leaves per month and plot were sampled in 2014 (June–October) randomly in the middle of the canopy. The samples (n = 4 per treatment) were taken in the morning from the middle of an inner row (e.g., row 4 of 7) of each plot by taking the leaves from 12 to 15 different plants (see Figure 2B). Leaves were transferred into water with detergent and washed according to the method of [30 ]. All arthropods were then identified at the family level and Eriophyid mites to the species level with a stereomicroscope (Zeiss Stemi 2000, Carl-Zeiss GmbH, Oberkochen, Germany). For species determination of Phytoseiidae and Tydeidae, random samples of adult predatory mites were separated and stored in 70% ethanol. These adult mites were then cleared with lactic acid and mounted on slides using Hoyers Fixative for embedding [31 ]. The mites were identified using a fluorescent light microscope (Leica DM 4000 B, Leica Microsystems, Wetzlar, Germany) following the key provided by [32 ]. The total number of mite individuals determined to the family level was 26,968. The number of Eriophyid mites determined to species level was 796, of Tydeidae 162 and of Typhlodromidae 762, meaning that 6.4% of the total mites were determined to the species level. As the species composition did not differ between the variants, we chose not to display these results in detail.

To isolate mouse or mouse embryonic tissues, the mouse was first sacrificed by cervical dislocation. After cleaning with 70% ethanol, the abdominal wall and peritoneum were opened to expose the organs or embryos, which were removed with forceps and sharp scissors, washed in PBS and fixed ON in 4% PFA at 4°C or in Roti-Histofix (4% formaldehyde, Roth) at RT. To isolate the brains from E18.5 embryos, the heads were removed and immersed in PBS in a Petri dish. The skulls were carefully removed with fine forceps. This procedure was performed under a stereomicroscope (Zeiss Stemi 2000; Zeiss, Oberkochen, Germany). After fixation, the tissues were washed in PBS and incubated for 30 minutes at 4°C in 30% ethanol, followed by a transfer to 50% ethanol, processing and paraffin embedding. The paraffin-embedded tissues were then cut into 5-µm-thick slices with a microtome (Microm, Cavriago, Italy). The slices were mounted on glass slides and dried overnight at 55°C. Hematoxylin and eosin (H&E) staining or immunohistochemistry (IHC) were then performed. All of the procedures performed in this study were approved by the Ethical Committee of Animal Research at the Leibniz Institute for Age Research.