Plant morphogenesis relies on the accurate positioning of the partition (cell plate) between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which consists of microtubules, actin filaments, membrane compartments and associated proteins. The phragmoplast forms between daughter nuclei during the transition from anaphase to telophase. As cells are commonly larger than the originally formed phragmoplast, the construction of the cell plate requires phragmoplast expansion. This expansion depends on microtubule polymerization at the phragmoplast forefront (leading zone) and loss at the back (lagging zone). Leading and lagging zones sandwich the ‘transition’ zone. A population of stable microtubules in the transition zone facilitates transport of building materials to the midzone where the cell plate assembly takes place. Whereas microtubules undergo dynamic instability in all zones, the overall balance appears to be shifted towards depolymerization in the lagging zone. Polymerization of microtubules behind the lagging zone has not been reported to date, suggesting that microtubule loss there is irreversible. In this Review, we discuss: (1) the regulation of microtubule dynamics in the phragmoplast zones during expansion; (2) mechanisms of the midzone establishment and initiation of cell plate biogenesis; and (3) signaling in the phragmoplast.

The performance of the photosynthesis machinery in plants, including light harvesting, electron transport, and protein repair, is controlled by structural changes in the thylakoid membrane system inside the chloroplasts. In particular, the structure of the stacked grana area of thylakoid membranes is highly dynamic, changing in response to different environmental cues such as light intensity. For example, the aqueous thylakoid lumen enclosed by thylakoid membranes in grana has been documented to swell in the presence of light. However, light‐induced alteration of the stromal gap in the stacked grana (partition gap) and of the unstacked stroma lamellae has not been well characterized. Light‐induced changes in the entire thylakoid membrane system, including the lumen in both stacked and unstacked domains as well as the partition gap, are presented here, and the functional implications are discussed. This structural analysis was made possible by development of a robust semi‐automated image analysis method combined with optimized plant tissue fixation techniques for transmission electron microscopy generating quantitative structural results for the analysis of thylakoid ultrastructure.Significance Statement: A methodical pipeline ranging from optimized leaf tissue preparation for electron microscopy to quantitative image analysis was established. This methodical development was employed to study details of light‐induced changes in the plant thylakoid ultrastructure. It was found that the lumen of the entire thylakoid system (stacked and unstacked domains) undergoes light‐induced swelling, whereas adjacent membranes on the stroma side in stacked grana thylakoid approach each other.

Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H₂O₂ to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H₂O₂ content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H₂O₂ and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H₂O₂ by 3,3′-diaminobenzidine (DAB) staining suggested that H₂O₂ could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H₂O₂-based method. Moreover, HyB induced overproduction of H₂O₂ in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.