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Plants produce new organs post-embryonically throughout their entire life cycle. This is due to stem cells present in the shoot and root apical meristems, the SAM and RAM, respectively. In the SAM, stem cells are located in the central zone where they divide slowly. Stem cell daughters are displaced laterally and enter the peripheral zone, where their mitotic activity increases and lateral organ primordia are formed. How the spatial arrangement of these different domains is initiated and controlled during SAM growth and development, and how sites of lateral organ primordia are determined in the peripheral zone is not yet completely understood. We found that the SHORT-ROOT (SHR) transcription factor together with its target transcription factors SCARECROW (SCR), SCARECROW-LIKE23 (SCL23) and JACKDAW (JKD), promotes formation of lateral organs and controls shoot meristem size. SHR, SCR, SCL23, and JKD are expressed in distinct, but partially overlapping patterns in the SAM. They can physically interact and activate expression of key cell cycle regulators such as CYCLIND6;1 (CYCD6;1) to promote the formation of new cell layers. In the peripheral zone, auxin accumulates at sites of lateral organ primordia initiation and activates SHR expression via the auxin response factor MONOPTEROS (MP) and auxin response elements in the SHR promoter. In the central zone, the SHR-target SCL23 physically interacts with the key stem cell regulator WUSCHEL (WUS) to promote stem cell fate. Both SCL23 and WUS expression are subject to negative feedback regulation from stem cells through the CLAVATA signaling pathway. Together, our findings illustrate how SHR-dependent transcription factor complexes act in different domains of the shoot meristem to mediate cell division and auxin dependent organ initiation in the peripheral zone, and coordinate this activity with stem cell maintenance in the central zone of the SAM.

Understanding stem cell regulatory circuits is the next challenge in plant biology, as these cells are essential for tissue growth and organ regeneration in response to stress. In the Arabidopsis primary root apex, stem cell-specific transcription factors BRAVO and WOX5 co-localize in the quiescent centre (QC) cells, where they commonly repress cell division so that these cells can act as a reservoir to replenish surrounding stem cells, yet their molecular connection remains unknown. Genetic and biochemical analysis indicates that BRAVO and WOX5 form a transcription factor complex that modulates gene expression in the QC cells to preserve overall root growth and architecture. Furthermore, by using mathematical modelling we establish that BRAVO uses the WOX5/BRAVO complex to promote WOX5 activity in the stem cells. Our results unveil the importance of transcriptional regulatory circuits in plant stem cell development.

Spatial organization of chromatin contributes to gene regulation of many cellular processes and includes a connection of chromatin with the nuclear lamina (NL). The NL is a protein mesh that resides underneath the inner nuclear membrane and consists of lamins and lamina-associated proteins. Chromatin regions associated with lamins in animals are characterized mostly by constitutive heterochromatin, but association with facultative heterochromatin mediated by Polycomb-group (PcG) proteins has been reported as well. In contrast with animals, plant NL components are largely not conserved and NL association with chromatin is poorly explored. Here, we present the connection between the lamin-like protein, CROWDED NUCLEI1 (CRWN1), and the chromatin- and PcG-associated component, PROLINE-TRYPTOPHANE-TRYPTOPHANE-PROLINE INTERACTOR OF POLYCOMBS1, in Arabidopsis (Arabidopsis thaliana). We show that PWO1 and CRWN1 proteins associate physically with each other, act in the same pathway to maintain nuclear morphology, and control expression of a similar set of target genes. Moreover, we demonstrate that transiently expressed PWO1 proteins form foci located partially at the subnuclear periphery. Ultimately, as CRWN1 and PWO1 are plant-specific, our results argue that plants might have developed an equivalent, rather than homologous, mechanism of linking chromatin repression and NL.

Plants exhibit an intriguing morphological and physiological plasticity that enables them to thrive in a wide range of environments. To understand the cell biological basis of this unparalleled competence, a number ofmethodologies have been adapted or developed over the last decades that allow minimal or non-invasive live-cell imaging in the context of tissues. Combined with the ease to generate transgenic reporter lines in specific genetic backgrounds or accessions, we are witnessing a blooming in plant cell biology. However, the imaging of plant cells entails a number of specific challenges, such as high levels of autofluorescence, light scattering that is caused by cell walls and their sensitivity to environmental conditions. Quantitative live-cell imaging in plants therefore requires adapting or developing imaging techniques, as well as mounting and incubation systems, such as micro-fluidics. Here, we discuss some of these obstacles, and review a number of selected state-of-the-art techniques, such as two-photon imaging, light sheet microscopy and variable angle epifluorescence microscopy that allow high performance and minimal invasive live-cell imaging in plants.

Protein complex formation has been extensively studied using Förster resonance energy transfer (FRET) measured by Fluorescence Lifetime Imaging Microscopy (FLIM). However, implementing this technology to detect protein interactions in living multicellular organism at single-cell resolution and under native condition is still difficult to achieve. Here we describe the optimization of the labeling conditions to detect FRET-FLIMin living plants. This study exemplifies optimization procedure involving the identification of the optimal position for the labels either at the N or C terminal region and the selection of the bright and suitable, fluorescent proteins as donor and acceptor labels for the FRET study. With an effective optimization strategy, we were able to detect the interaction between the stem cell regulators SHORT-ROOT and SCARECROW at endogenous expression levels in the root pole of living Arabidopsis embryos and developing lateral roots by FRET-FLIM. Using this approach we show that the spatial profile of interaction between two transcription factors can be highly modulated in reoccurring and structurally resembling organs, thus providing new information on the dynamic redistribution of nuclear protein complex configurations in different developmental stages. In principle, our optimization procedure for transcription factor complexes is applicable to any biological system.