Tissue‐Specific Isolation of Tagged Arabidopsis Plastids

Abstract

Plastids are found in all plant cell types. However, most extraction methods to study these organelles are performed at the organ level (e.g., leaf, root, fruit) and do not allow for tissue‐specific resolution, which hinders our understanding of their physiology. Therefore, IPTACT (Isolation of Plastids TAgged in specific Cell Types) was developed to isolate plastids in a tissue‐specific manner in Arabidopsis thaliana (Arabidopsis). Plastids are biotinylated using one‐shot transgenic lines, and tissue specificity is achieved with a suitable promoter as long as such a promoter exists. Cell‐specific biotinylated plastids are then isolated with 2.8‐µm streptavidin beads. Plastids extracted by IPTACT are suitable for RNA or protein isolation and subsequent tissue‐specific OMICs analyses. This method provides the user with a powerful tool to investigate plastidial functions at cell‐type resolution. Furthermore, it can easily be combined with studies using diverse genetic backgrounds and/or different developmental or stress conditions. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Promoter cloning and plant selection Basic Protocol 2: Isolation of biotinylated plastids Basic Protocol 3: Quality control of isolated plastids

Comparison of plastid proteomes points towards a higher plastidial redox turnover in vascular tissues than in mesophyll cells

“…1A ). Note that additional constructs with two other selection markers (hygromycin and kanamycin) have been generated (see ‘Materials and methods’; Boussardon and Keech, 2023 ).…”

“…In contrast to the isolation procedure of plant mitochondria, we used beads with a size of approximately 2.8 µm as indicated by the provider ( Fig. 3A ; Boussardon and Keech, 2023 ). In general, plastids had an ellipsoid shape with a diameter ranging from 3 to 8 µm.…”

“…Images of the full membranes are available in Supplementary Fig. S3 ; the lane for crude extract acting as control was the same as in Boussardon and Keech (2023) .…”

“…Here, we report the application of our IPTACT method ( Boussardon and Keech, 2023 ) to isolate plastids from mesophyll and vascular cells. The method derives from our IMTACT method described earlier ( Boussardon et al , 2020 ; Boussardon and Keech, 2022 ) with the use of a translational fusion of TOC64 containing eGFP and BLRP at the C-terminus, combined with the UBQ10::BirA construct ( Fig.…”

“…A step-by-step protocol can be found in Boussardon and Keech (2023) . Briefly, all steps were performed in a cold room (at 4 °C), on ice, with pre-cooled materials and under green light.…”