Satoshi Fujita

SUMMARY: Bioactive molecules such as hormones play crucial roles throughout the life cycle of organisms. Due to their profound effects, these molecules are tightly regulated at multiple levels, including biosynthesis, storage, transport, modification, and signaling. Recent technical advances in plant sciences have added dynamics of hormones and receptors as an emerging tier in such regulation, and we will use brassinosteroid (BR) phytohormones to showcase these latest developments. In this review, we first focus on the intercellular dynamics of BRs and their intermediates through plasmodesmata. Upon the completion of biosynthesis, ABCB1/19 transporters export bioactive BRs toward the extracellular space where BRs are perceived by the BRASSINOSTEROID‐INSENSITIVE 1 (BRI1) receptor. BRI1 activity is tightly regulated by its redistribution within cells through membrane trafficking and the formation of nanodomains at the plasma membrane, mechanisms also influenced by environmental cues. Additionally, the relocalization of downstream signaling components enables the spatiotemporal control of the BR signaling pathway, which helps guide optimal plant development. Finally, we discuss the possible evolutionary dynamics underlying the establishment of BR signaling and perception, which may have contributed to the diversification and expansion of signaling networks in plants. Taken together, the dynamics of molecules at multiple layers shape their physiological outputs, thus impacting plant growth and development.

Cells harbour numerous receptor pathways to respond to diverse stimuli, yet often share common downstream signalling components. Mitogen-activated protein kinase (MPK) cascades are an example of such common hubs in eukaryotes. How such common hubs faithfully transduce distinct signals within the same cell-type is insufficiently understood, yet of fundamental importance for signal integration and processing in plants. We engineered a unique genetic background allowing direct comparisons of a developmental and an immunity pathway in one cell-type, the Arabidopsis root endodermis. We demonstrate that the two pathways maintain distinct functional and transcriptional outputs despite common MPK activity patterns. Nevertheless, activation of different MPK kinases and MPK classes led to distinct functional readouts, matching observed pathway-specific readouts. On the basis of our comprehensive analysis of core MPK signalling elements, we propose that combinatorial activation within the MPK cascade determines the differential regulation of an endodermal master transcription factor, MYB36, that drives pathway-specific gene activation.

Adjusting the microenvironment around the cell surface is critical to responding to external cues or endogenous signals and to maintaining cell activities. In plant cells, the plasma membrane is covered by the cell wall and scaffolded with cytoskeletal networks, which altogether compose the cell surface. It has long been known that these structures mutually interact, but the mechanisms that integrate the whole system are still obscure. Here we spotlight the brassinosteroid (BR) plant hormone receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) since it represents an outstanding model for understanding cell surface signalling and regulation. We summarize how BRI1 activity and dynamics are controlled by plasma membrane components and their associated factors to fine-tune signalling. The downstream signals, in turn, manipulate cell surface structures by transcriptional and post-translational mechanisms. Moreover, the changes in these architectures impact BR signalling, resulting in a feedback loop formation. This Review discusses how BRI1 and BR signalling function as central hubs to integrate cell surface regulation.

Significance: Pollen viability depends on a tough external barrier called the pollen wall. Pollen wall components are produced by tapetum cells, which surround developing pollen grains within the anther. Precise coordination of tapetum activity with pollen grain development is required to ensure effective pollen wall formation. Here, we reveal that this is achieved through a multidirectional dialogue involving three distinct cell types. We show that peptide precursors from the tapetum are activated by proteases produced stage specifically in developing pollen grains. Unexpectedly, we found that activated peptides are perceived not in the tapetum, but in the middle layer, which encloses the developing tapetum and pollen grains, revealing an unsuspected role for this enigmatic cell layer in the control of tapetum development.

Precise coordination between cells and tissues is essential for differential growth in plants. During lateral root formation in Arabidopsis thaliana , the endodermis is actively remodeled to allow outgrowth of the new organ. Here, we show that microtubule arrays facing lateral root founder cells display a higher order compared to arrays on the opposite side of the same cell, and this asymmetry is required for endodermal remodeling and lateral root initiation. We identify that MICROTUBULE ASSOCIATED PROTEIN 70-5 (MAP70-5) is necessary for the establishment of this spatially defined microtubule organization and endodermis remodeling and thus contributes to lateral root morphogenesis. We propose that MAP70-5 and cortical microtubule arrays in the endodermis integrate the mechanical signals generated by lateral root outgrowth, facilitating the channeling of organogenesis.

Multiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Use of CRISPR-Cas9 has increased rapidly in recent years and is becoming a routine method for generating single and higher order Arabidopsis mutants. To facilitate rapid and efficient use of CRISPR/Cas9 for Arabidopsis research, we developed a CRISPR/Cas9-based toolbox for generating large deletions at multiple genomic loci, using two-color fluorescent seed selection. In our system, up-to eight gRNAs can be routinely introduced into a binary vector carrying either FastRed, FastGreen or FastCyan fluorescent seed selection cassette. Both, FastRed and FastGreen binary vectors, can be co-transformed as a cocktail via floral dip to introduce sixteen gRNAs at the same time. The seeds can be screened either for red or green fluorescence, or for the presence of both colors at the same time. Our approach provides fast and flexible cloning, avoids very big constructs and enables screening different order mutants in the same generation. Importantly, in the second generation after transformation, Cas9 free plants are identified simply by screening the dark, non-fluorescent seeds. Our collection of binary vectors allows to choose between two widely-used promoters to drive Cas enzymes, either the egg cell-specific (pEC1.2) or ubiquitous promoter (PcUBi4-2). Available enzymes are “classical” Cas9, a recently reported, intron-optimized version or Cpf1 (Cas12a). Finally, we have taken care to introduce convenient restriction sites flanking promoter, Cas9 and fluorescent selection cassette in the final T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system.

Lignin is a complex polymer deposited in the cell wall of specialised plant cells, where it provides essential cellular functions. Plants coordinate timing, location, abundance and composition of lignin deposition in response to endogenous and exogenous cues. In roots, a fine band of lignin, the Casparian strip encircles endodermal cells. This forms an extracellular barrier to solutes and water and plays a critical role in maintaining nutrient homeostasis. A signalling pathway senses the integrity of this diffusion barrier and can induce over-lignification to compensate for barrier defects. Here, we report that activation of this endodermal sensing mechanism triggers a transcriptional reprogramming strongly inducing the phenylpropanoid pathway and immune signaling. This leads to deposition of compensatory lignin that is chemically distinct from Casparian strip lignin. We also report that a complete loss of endodermal lignification drastically impacts mineral nutrients homeostasis and plant growth.

Lignin has enabled plants to colonize land, grow tall, transport water within their bodies, and protect themselves against various stresses. Consequently, this polyphenolic polymer, impregnating cellulosic plant cell walls, is the second most abundant polymer on Earth. Yet, despite its great physiological, ecological, and economical importance, our knowledge of lignin biosynthesis in vivo, especially the polymerization steps within the cell wall, remains vague—specifically, the respective roles of the two polymerizing enzymes classes, laccases and peroxidases. One reason for this lies in the very high numbers of laccases and peroxidases encoded by 17 and 73 homologous genes, respectively, in Arabidopsis. Here, we have focused on a specific lignin structure, the ring-like Casparian strips (CSs) within the root endodermis. By reducing candidate numbers using cellular resolution expression and localization data and by boosting stacking of mutants using CRISPR-Cas9, we mutated the majority of laccases in Arabidopsis in a nonuple mutant—essentially abolishing laccases with detectable endodermal expression. Yet, we were unable to detect even slight defects in CS formation. By contrast, we were able to induce a complete absence of CS formation in a quintuple peroxidase mutant. Our findings are in stark contrast to the strong requirement of xylem vessels for laccase action and indicate that lignin in different cell types can be polymerized in very distinct ways. We speculate that cells lignify differently depending on whether lignin is localized or ubiquitous and whether cells stay alive during and after lignification, as well as the composition of the cell wall. Significance Lignin is a defining polymer of vascular plants and of great physiological, ecological, and economical importance. Yet, its polymerization in the cell wall is still not understood. Lignin polymerizing enzymes, laccases and peroxidases, exist in vast numbers in plant genomes. By focusing on a specific lignin structure, the ring-like Casparian strips (CSs), we reduced candidate numbers and abolished essentially all laccases with detectable endodermal expression. Yet, not even slight defects in CS formation were detected. By contrast, a quintuple peroxidase mutant displayed a complete absence of CS. Our findings suggest that cells lignify differently depending on whether lignin is localized or ubiquitous and whether cells stay alive during and after lignification, as well as the composition of the cell wall.

Lignin is a complex polymer precisely deposited in the cell wall of specialised plant cells, where it provides essential cellular functions. Plants coordinate timing, location, abundance and composition of lignin deposition in response to endogenous and exogenous cues. In roots, a fine band of lignin, the Casparian strip encircles endodermal cells. This forms an extracellular barrier to solutes and water and plays a critical role in maintaining nutrient homeostasis. A signalling pathway senses the integrity of this diffusion barrier and can induce over-lignification to compensate for barrier defects. Here, we report that activation of this endodermal sensing mechanism triggers a transcriptional reprogramming strongly inducing the phenylpropanoid pathway and immune signaling. This leads to deposition of compensatory lignin that is chemically distinct from Casparian strip lignin. We also report that a complete loss of endodermal lignification drastically impacts mineral nutrients homeostasis and plant growth.