OBERON3 and SUPPRESSOR OF MAX2 1-LIKE proteins form a regulatory module driving phloem development.

Spatial specificity of cell fate decisions is central for organismal development. The phloem tissue mediates long-distance transport of energy metabolites along plant bodies and is characterized by an exceptional degree of cellular specialization. How a phloem-specific developmental program is implemented is, however, unknown. Here we reveal that the ubiquitously expressed PHD-finger protein OBE3 forms a central module with the phloem-specific SMXL5 protein for establishing the phloem developmental program in Arabidopsis thaliana. By protein interaction studies and phloem-specific ATAC-seq analyses, we show that OBE3 and SMXL5 proteins form a complex in nuclei of phloem stem cells where they promote a phloem-specific chromatin profile. This profile allows expression of OPS, BRX, BAM3, and CVP2 genes acting as mediators of phloem differentiation. Our findings demonstrate that OBE3/SMXL5 protein complexes establish nuclear features essential for determining phloem cell fate and highlight how a combination of ubiquitous and local regulators generate specificity of developmental decisions in plants.

BD Rhapsody™ Single-Cell Analysis System Workflow: From Sample to Multimodal Single-Cell Sequencing Data.

Advancements in single-cell sequencing have revolutionized our understanding of complex biological systems such as the immune system and allowed us to overcome limitations in various disciplines of life science research such as oncology, developmental biology, or neurobiology (Perkel, Nature 595. https://www.nature.com/articles/d41586-021-01994-w , 2021).The BD Rhapsody™ Single-Cell Analysis System enables us to capture multimodal information from thousands of single cells in parallel ("Multiomics") covering mRNA expression levels, protein expression levels, the immune repertoire for T-cell receptors (TCR) and B-cell receptors (BCR), and the identification of antigen-specific T cells and B cells using dCODE Dextramer® (RiO) from Immudex. The system utilizes microwell-based cartridges that allow to capture a broad range of single cells and an imaging device for sample quality control and workflow quality control (including viability and multiplets). The power of Multiomics relies on simultaneously measuring several aspects of single cells, including gene expression and protein abundance, using next generation sequencing (NGS) as a single readout.Here we describe the complete BD Rhapsody™ Single-Cell Analysis System from the sample preparation including different options for the antibody and/or dCODE Dextramer® staining through to the data analysis.For updated protocols, guides, and technical bulletins, please visit the BD Scomix page: https://scomix.bd.com/hc/en-us or the BDB webpage: https://www.bdbiosciences.com/en-eu .

COP1 dynamics integrate conflicting seasonal light and thermal cues in the control of Arabidopsis elongation.

As the summer approaches, plants experience enhanced light inputs and warm temperatures, two environmental cues with an opposite morphogenic impact. Key components of this response are PHYTOCHROME B (phyB), EARLY FLOWERING 3 (ELF3), and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). Here, we used single and double mutant/overexpression lines to fit a mathematical model incorporating known interactions of these regulators. The fitted model recapitulates thermal growth of all lines used and correctly predicts thermal behavior of others not used in the fit. While thermal COP1 function is accepted to be independent of diurnal timing, our model shows that it acts at temperature signaling only during daytime. Defective response of cop1-4 mutants is epistatic to phyB-9 and elf3-8, indicating that COP1 activity is essential to transduce phyB and ELF3 thermosensory function. Our thermal model provides a unique toolbox to identify best allelic combinations enhancing climate change resilience of crops adapted to different latitudes.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

The Phloem as a Mediator of Plant Growth Plasticity.

Developmental plasticity, defined as the capacity to respond to changing environmental conditions, is an inherent feature of plant growth. Recent studies have brought the phloem tissue, the quintessential conduit for energy metabolites and inter-organ communication, into focus as an instructive developmental system. Those studies have clarified long-standing questions about essential aspects of phloem development and function, such as the pressure flow hypothesis, mechanisms of phloem unloading, and source-sink relationships. Interestingly, plants with impaired phloem development show characteristic changes in body architecture, thereby highlighting the capacity of the phloem to integrate environmental cues and to fine-tune plant development. Therefore, understanding the plasticity of phloem development provides scenarios of how environmental stimuli are translated into differential plant growth. In this Review, we summarize novel insights into how phloem identity is established and how phloem cells fulfil their core function as transport units. Moreover, we discuss possible interfaces between phloem physiology and development as sites for mediating the plastic growth mode of plants.

Bifacial cambium stem cells generate xylem and phloem during radial plant growth.

A reduced rate of stem cell division is considered a widespread feature which ensures the integrity of genetic information during somatic development of plants and animals. Radial growth of plant shoots and roots is a stem cell-driven process that is fundamental for the mechanical and physiological support of enlarging plant bodies. In most dicotyledonous species, the underlying stem cell niche, the cambium, generates xylem inwards and phloem outwards. Despite the importance and intriguing dynamics of the cambium, the functional characterization of its stem cells is hampered by the lack of experimental tools for accessing distinct cambium sub-domains. Here, we use the hypocotyl of Arabidopsis thaliana to map stem cell activity in the proliferating cambium. Through pulse labeling and genetically encoded lineage tracing, we find that a single bifacial stem cell generates both xylem and phloem cell lineages. This cell is characterized by a specific combination of PXY (TDR), SMXL5 and WOX4 gene activity and a high division rate in comparison with tissue-specific progenitors. Our analysis provides a cellular fate map of radial plant growth, and suggests that stem cell quiescence is not a general prerequisite for life-long tissue production.This article has an associated 'The people behind the papers' interview.

A Comprehensive Toolkit for Inducible, Cell Type-Specific Gene Expression in Arabidopsis.

Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell type-specific transactivation in Arabidopsis (Arabidopsis thaliana) based on the well-established combination of the chimeric GR-LhG4 transcription factor and the synthetic pOp promoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of transgenic lines termed GR-LhG4 driver lines targeting most tissues in the Arabidopsis shoot and root with a strong focus on the indeterminate meristems. When we combined these transgenic lines with effectors under the control of the pOp promoter, we observed tight temporal and spatial control of gene expression. In particular, inducible expression in F1 plants obtained from crosses of driver and effector lines allows for rapid assessment of the cell type-specific impact of an effector with high temporal resolution. Thus, our comprehensive and flexible method is suitable for overcoming the limitations of ubiquitous genetic approaches, the outputs of which often are difficult to interpret due to the widespread existence of compensatory mechanisms and the integration of diverging effects in different cell types.

Strigolactone- and Karrikin-Independent SMXL Proteins Are Central Regulators of Phloem Formation.

Plant stem cell niches, the meristems, require long-distance transport of energy metabolites and signaling molecules along the phloem tissue. However, currently it is unclear how specification of phloem cells is controlled. Here we show that the genes SUPPRESSOR OF MAX2 1-LIKE3 (SMXL3), SMXL4, and SMXL5 act as cell-autonomous key regulators of phloem formation in Arabidopsis thaliana. The three genes form an uncharacterized subclade of the SMXL gene family that mediates hormonal strigolactone and karrikin signaling. Strigolactones are endogenous signaling molecules regulating shoot and root branching [1] whereas exogenous karrikin molecules induce germination after wildfires [2]. Both activities depend on the F-box protein and SCF (Skp, Cullin, F-box) complex component MORE AXILLARY GROWTH2 (MAX2) [3-5]. Strigolactone and karrikin perception leads to MAX2-dependent degradation of distinct SMXL protein family members, which is key for mediating hormonal effects [6-12]. However, the nature of events immediately downstream of SMXL protein degradation and whether all SMXL proteins mediate strigolactone or karrikin signaling is unknown. In this study we demonstrate that, within the SMXL gene family, specifically SMXL3/4/5 deficiency results in strong defects in phloem formation, altered sugar accumulation, and seedling lethality. By comparing protein stabilities, we show that SMXL3/4/5 proteins function differently to canonical strigolactone and karrikin signaling mediators, although being functionally interchangeable with those under low strigolactone/karrikin signaling conditions. Our observations reveal a fundamental mechanism of phloem formation and indicate that diversity of SMXL protein functions is essential for a steady fuelling of plant meristems.

Strigolactone versus gibberellin signaling: reemerging concepts?

MAIN CONCLUSION: In this review, we compare knowledge about the recently discovered strigolactone signaling pathway and the well established gibberellin signaling pathway to identify gaps of knowledge and putative research directions in strigolactone biology. Communication between and inside cells is integral for the vitality of living organisms. Hormonal signaling cascades form a large part of this communication and an understanding of both their complexity and interactive nature is only beginning to emerge. In plants, the strigolactone (SL) signaling pathway is the most recent addition to the classically acting group of hormones and, although fundamental insights have been made, knowledge about the nature and impact of SL signaling is still cursory. This narrow understanding is in spite of the fact that SLs influence a specific spectrum of processes, which includes shoot branching and root system architecture in response, partly, to environmental stimuli. This makes these hormones ideal tools for understanding the coordination of plant growth processes, mechanisms of long-distance communication and developmental plasticity. Here, we summarize current knowledge about SL signaling and employ the well-characterized gibberellin (GA) signaling pathway as a scaffold to highlight emerging features as well as gaps in our knowledge in this context. GA signaling is particularly suitable for this comparison because both signaling cascades share key features of hormone perception and of immediate downstream events. Therefore, our comparative view demonstrates the possible level of complexity and regulatory interfaces of SL signaling.

ELF3-PIF4 interaction regulates plant growth independently of the Evening Complex.

The circadian clock plays a pivotal role in the control of Arabidopsis hypocotyl elongation by regulating rhythmic expression of the bHLH factors PHYTOCHROME INTERACTING FACTOR 4 and 5 (PIF4 and 5). Coincidence of increased PIF4/PIF5 transcript levels with the dark period allows nuclear accumulation of these factors, and in short days it phases maximal hypocotyl growth at dawn. During early night, PIF4 and PIF5 transcription is repressed by the Evening Complex (EC) proteins EARLY FLOWERING3 (ELF3), EARLY FLOWERING4 (ELF4), and LUX ARRHYTHMO (LUX). While ELF3 has an essential role in EC complex assembly, several lines of evidence indicate that this protein controls plant growth via other mechanisms that are presently unknown. Here, we show that the ELF3 and PIF4 proteins interact in an EC-independent manner, and that this interaction prevents PIF4 from activating its transcriptional targets. We also show that PIF4 overexpression leads to ELF3 protein destabilization, and that this effect is mediated indirectly by negative feedback regulation of photoactive PHYTOCHROME B (phyB). Physical interaction of the phyB photoreceptor with ELF3 has been reported, but its functional relevance remains poorly understood. Our findings establish that phyB is needed for ELF3 accumulation in the light, most likely by competing for CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-mediated ubiquitination and the proteasomal degradation of ELF3. Our results explain the short hypocotyl phenotype of ELF3 overexpressors, despite their normal clock function, and provide a molecular framework for understanding how warm temperatures promote hypocotyl elongation and affect the endogenous clock.