Electron tomography analysis of the prolamellar body and its transformation into grana thylakoids in the cryofixedArabidopsiscotyledon

The para-crystalline structure of prolamellar bodies (PLBs) and light-induced etioplasts-to-chloroplasts transformation have been investigated with electron microscopy methods. However, these studies suffer from chemical fixation artifacts and limited volumes of three-dimensional reconstruction. We have examined Arabidopsis thaliana cotyledon cells with electron tomography (ET) to visualize etioplasts and their conversion into chloroplasts. We employed the scanning mode of ET for imaging large volumes and high-pressure freezing to improve sample preservation. PLB tubules were arranged in a zinc blende-type lattice like carbon atoms in diamonds. Within 2 hours after illumination, the lattice collapsed from the PLB exterior and the disorganized tubules merged to form thylakoid sheets, a.k.a. pre-granal thylakoids. These pre-granal thylakoids in PLB’s vicinity folded and overlapped with each other to create grana stacks. Since the nascent pre-granal thylakoids had curved membranes in their tips, we examined the expression and localization of CURT1 proteins. CURT1A transcript was most abundant in de-etiolating cotyledon samples, and CURT1A concentrated to the PLB periphery. In curt1a etioplasts, PLB-associated thylakoids were swollen and failed to form grana stacks. By contrast, PLBs had cracks in their lattices in curt1c etioplasts. Our data provide evidence that CURT1A is required for pre-granal thylakoid assembly from PLB tubules during de-etiolation, while CURT1C contributes to the cubic crystal growth under darkness.