GRAS transcription factors regulate cell division planes in moss overriding the default rule.

Plant cells are surrounded by a cell wall and do not migrate, which makes the regulation of cell division orientation crucial for development. Regulatory mechanisms controlling cell division orientation may have contributed to the evolution of body organization in land plants. The GRAS family of transcription factors was transferred horizontally from soil bacteria to an algal common ancestor of land plants. SHORTROOT (SHR) and SCARECROW (SCR) genes in this family regulate formative periclinal cell divisions in the roots of flowering plants, but their roles in nonflowering plants and their evolution have not been studied in relation to body organization. Here, we show that SHR cell autonomously inhibits formative periclinal cell divisions indispensable for leaf vein formation in the moss Physcomitrium patens, and SHR expression is positively and negatively regulated by SCR and the GRAS member LATERAL SUPPRESSOR, respectively. While precursor cells of a leaf vein lacking SHR usually follow the geometry rule of dividing along the division plane with the minimum surface area, SHR overrides this rule and forces cells to divide nonpericlinally. Together, these results imply that these bacterially derived GRAS transcription factors were involved in the establishment of the genetic regulatory networks modulating cell division orientation in the common ancestor of land plants and were later adapted to function in flowering plant and moss lineages for their specific body organizations.

Quantitative Imaging Reveals Distinct Contributions of SnRK2 and ABI3 in Plasmodesmatal Permeability in Physcomitrella patens.

Cell-to-cell communication is tightly regulated in response to environmental stimuli in plants. We previously used a photoconvertible fluorescent protein Dendra2 as a model reporter to study this process. This experiment revealed that macromolecular trafficking between protonemal cells in Physcomitrella patens is suppressed in response to abscisic acid (ABA). However, it remains unknown which ABA signaling components contribute to this suppression and how. Here, we show that ABA signaling components SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 2 (PpSnRK2) and ABA INSENSITIVE 3 (PpABI3) play roles as an essential and promotive factor, respectively, in regulating ABA-induced suppression of Dendra2 diffusion between cells (ASD). Our quantitative imaging analysis revealed that disruption of PpSnRK2 resulted in defective ASD onset itself, whereas disruption of PpABI3 caused an 81-min delay in the initiation of ASD. Live-cell imaging of callose deposition using aniline blue staining showed that, despite this onset delay, callose deposition on cross walls remained constant in the PpABI3 disruptant, suggesting that PpABI3 facilitates ASD in a callose-independent manner. Given that ABA is an important phytohormone to cope with abiotic stresses, we further explored cellular physiological responses. We found that the acquisition of salt stress tolerance is promoted by PpABI3 in a quantitative manner similar to ASD. Our results suggest that PpABI3-mediated ABA signaling may effectively coordinate cell-to-cell communication during the acquisition of salt stress tolerance. This study will accelerate the quantitative study for ABA signaling mechanism and function in response to various abiotic stresses.

An Aminoacyl tRNA Synthetase, OKI1, Is Required for Proper Shoot Meristem Size in Arabidopsis.

In plants, the stem cells that form the shoot system reside within the shoot apical meristem (SAM), which is regulated by feedback signaling between the WUSCHEL (WUS) homeobox protein and CLAVATA (CLV) peptides and receptors. WUS-CLV feedback signaling can be modulated by various endogenous or exogenous factors, such as chromatin state, hormone signaling, reactive oxygen species (ROS) signaling and nutrition, leading to a dynamic control of SAM size corresponding to meristem activity. Despite these insights, however, the knowledge of genes that control SAM size is still limited, and in particular, the regulation by ROS signaling is only beginning to be comprehended. In this study, we report a new function in maintenance of SAM size, encoded by the OKINA KUKI1 (OKI1) gene. OKI1 is expressed in the SAM and encodes a mitochondrial aspartyl tRNA synthetase (AspRS). oki1 mutants display enlarged SAMs with abnormal expression of WUS and CLV3 and overaccumulation of ROS in the meristem. Our findings support the importance of normal AspRS function in the maintenance of the WUS-CLV3 feedback loop and SAM size.

Abscisic Acid Acts as a Regulator of Molecular Trafficking through Plasmodesmata in the Moss Physcomitrella patens.

In multi-cellular organisms, cell-to-cell communication is crucial for adapting to changes in the surrounding environment. In plants, plasmodesmata (PD) provide a unique pathway for cell-to-cell communication. PD interconnect most cells and generate a cytoplasmic continuum, allowing the trafficking of various micro- and macromolecules between cells. This molecular trafficking through PD is dynamically regulated by altering PD permeability dependent on environmental changes, thereby leading to an appropriate response to various stresses; however, how PD permeability is dynamically regulated is still largely unknown. Moreover, studies on the regulation of PD permeability have been conducted primarily in a limited number of angiosperms. Here, we studied the regulation of PD permeability in the moss Physcomitrella patens and report that molecular trafficking through PD is rapidly and reversibly restricted by abscisic acid (ABA). Since ABA plays a key role in various stress responses in the moss, PD permeability can be controlled by ABA to adapt to surrounding environmental changes. This ABA-dependent restriction of PD trafficking correlates with a reduction in PD pore size. Furthermore, we also found that the rate of macromolecular trafficking is higher in an ABA-synthesis defective mutant, suggesting that the endogenous level of ABA is also important for PD-mediated macromolecular trafficking. Thus, our study provides compelling evidence that P. patens exploits ABA as one of the key regulators of PD function.

SHOOT MERISTEMLESS trafficking controls axillary meristem formation, meristem size and organ boundaries in Arabidopsis.

The shoot stem cell niche, contained within the shoot apical meristem (SAM) is maintained in Arabidopsis by the homeodomain protein SHOOT MERISTEMLESS (STM). STM is a mobile protein that traffics cell-to-cell, presumably through plasmodesmata. In maize, the STM homolog KNOTTED1 shows clear differences between mRNA and protein localization domains in the SAM. However, the STM mRNA and protein localization domains are not obviously different in Arabidopsis, and the functional relevance of STM mobility is unknown. Using a non-mobile version of STM (2xNLS-YFP-STM), we show that STM mobility is required to suppress axillary meristem formation during embryogenesis, to maintain meristem size, and to precisely specify organ boundaries throughout development. STM and organ boundary genes CUP SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3 regulate each other during embryogenesis to establish the embryonic SAM and to specify cotyledon boundaries, and STM controls CUC expression post-embryonically at organ boundary domains. We show that organ boundary specification by correct spatial expression of CUC genes requires STM mobility in the meristem. Our data suggest that STM mobility is critical for its normal function in shoot stem cell control.

Arabidopsis Root-Type Ferredoxin:NADP(H) Oxidoreductase 2 is Involved in Detoxification of Nitrite in Roots.

Ferredoxin:NADP(H) oxidoreductase (FNR) plays a key role in redox metabolism in plastids. Whereas leaf FNR (LFNR) is required for photosynthesis, root FNR (RFNR) is believed to provide electrons to ferredoxin (Fd)-dependent enzymes, including nitrite reductase (NiR) and Fd-glutamine-oxoglutarate aminotransferase (Fd-GOGAT) in non-photosynthetic conditions. In some herbal species, however, most nitrate reductase activity is located in photosynthetic organs, and ammonium in roots is assimilated mainly by Fd-independent NADH-GOGAT. Therefore, RFNR might have a limited impact on N assimilation in roots grown with nitrate or ammonium nitrogen sources. AtRFNR genes are rapidly induced by application of toxic nitrite. Thus, we tested the hypothesis that RFNR could contribute to nitrite reduction in roots by comparing Arabidopsis thaliana seedlings of the wild type with loss-of-function mutants of RFNR2 When these seedlings were grown under nitrate, nitrite or ammonium, only nitrite nutrition caused impaired growth and nitrite accumulation in roots of rfnr2 Supplementation of nitrite with nitrate or ammonium as N sources did not restore the root growth in rfnr2 Also, a scavenger for nitric oxide (NO) could not effectively rescue the growth impairment. Thus, nitrite toxicity, rather than N depletion or nitrite-dependent NO production, probably causes the rfnr2 root growth defect. Our results strongly suggest that RFNR2 has a major role in reduction of toxic nitrite in roots. A specific set of genes related to nitrite reduction and the supply of reducing power responded to nitrite concomitantly, suggesting that the products of these genes act co-operatively with RFNR2 to reduce nitrite in roots.

A model system for analyzing intercellular communication through plasmodesmata using moss protonemata and leaves.

Plant growth, development, and environmental responses require the proper regulation of intercellular movement of signals and nutrients. For this, plants have specialized cytoplasmic channels, the plasmodesmata (PD), which allow the symplasmic movement of micro- and macromolecules between neighboring cells. Internal and external signals spatio-temporally regulate the movement of molecules through the PD to control plant development and environmental responses. Although some aspects of targeted movement of molecules have been revealed, the mechanisms of non-targeted, diffusible flow of molecules through PD, and its regulation and function, remain poorly understood, particularly at the cellular level. Previously, we developed a system to quantitatively analyze non-targeted movement of a photoconvertible fluorescent protein, Dendra2, at the single-cell level in the filamentous protonemata tissue of the moss Physcomitrella patens. In protonemata, one-dimensional intercellular communication can be easily observed and quantitatively analyzed at the cellular level. In this review, we describe how protonemata and leaves of P. patens can be used to study symplasmic movement through PD, and discuss how this system can help improve our understanding of PD regulation and function in development and environmental responses in plants.

Tracking the messengers: Emerging advances in mRNA-based plant communication.

Messenger RNAs (mRNAs) are the templates for protein translation but can also act as non-cell-autonomous signaling molecules. Plants input endogenous and exogenous cues to mobile mRNAs and output them to local or systemic target cells and organs to support specific plant responses. Mobile mRNAs form ribonucleoprotein (RNP) complexes with proteins during transport. Components of these RNP complexes could interact with plasmodesmata (PDs), a major mediator of mRNA transport, to ensure mRNA mobility and transport selectivity. Based on advances in the last two to three years, this review summarizes mRNA transport mechanisms in local and systemic signaling from the perspective of RNP complex formation and PD transport. We also discuss the physiological roles of endogenous mRNA transport and the recently revealed roles of non-cell-autonomous mRNAs in inter-organism communication.

Traveling with purpose: cell-to-cell transport of plant mRNAs.

Messenger RNAs (mRNAs) in multicellular organisms can act as signals transported cell-to-cell and over long distances. In plants, mRNAs traffic cell-to-cell via plasmodesmata (PDs) and over long distances via the phloem vascular system to control diverse biological processes - such as cell fate and tissue patterning - in destination organs. Research on long-distance transport of mRNAs in plants has made remarkable progress, including the cataloguing of many mobile mRNAs, characterization of mRNA features important for transport, identification of mRNA-binding proteins involved in their transport, and understanding of the physiological roles of mRNA transport. However, information on short-range mRNA cell-to-cell transport is still limited. This review discusses the regulatory mechanisms and physiological functions of mRNA transport at the cellular and whole plant levels.

Quantitative imaging of directional transport through plasmodesmata in moss protonemata via single-cell photoconversion of Dendra2.

Cell-to-cell transport of molecules in plants must be properly regulated for plant growth and development. One specialized mechanism that plants have evolved involves transport through plasmodesmata (PD), but when and how transport of molecules via PD is regulated among individual cells remains largely unknown, particularly at the single-cell level. Here, we developed a tool for quantitatively analyzing cell-to-cell transport via PD at a single-cell level using protonemata of Physcomitrella patens and a photoconvertible fluorescent protein, Dendra2. In the filamentous protonemal tissues, one-dimensional intercellular communication can be observed easily. Using this system, we found that Dendra2 was directionally transported toward the apex of the growing protonemata. However, this directional transport could be eliminated by incubation in the dark or treatment with a metabolic inhibitor. Thus, we propose that directional transport of macromolecules can occur via PD in moss protonemata, and may be affected by the photosynthetic and metabolic activity of cells.

A Forward Genetic Approach to Identify Plasmodesmal Trafficking Regulators Based on Trichome Rescue.

Plasmodesmata (PD) are channels in the walls of plant cells which enable cell-to-cell information transfer. This includes the selective transport of specific transcription factors that control cell fate during plant development. KNOTTED1 (KN1) homeobox (KNOX) family transcription factors that are essential for the maintenance and function of stem cells in shoot meristems use this trafficking pathway, but its mechanism is largely unknown. Here we describe a forward genetic approach to the identification of regulators of selective KN1 trafficking through PD, using a trichome rescue system that permits simple visual analysis in Arabidopsis leaves. A KN1 trafficking regulator identified in this approach had the capacity to regulate the transport not only of KN1 but also of another mobile regulatory protein, TRANSPARENT TESTA GLABRA1 (TTG1). Our system could be easily adapted to reveal the mechanism underlying the selective transport of additional mobile signals through PD.

Trafficking and localization of KNOTTED1 related mRNAs in shoot meristems.

Multicellular organisms use transcripts and proteins as signaling molecules for cell-to-cell communication. Maize KNOTTED1 (KN1) was the first homeodomain transcription factor identified in plants, and functions in maintaining shoot stem cells. KN1 acts non-cell autonomously, and both its messenger RNA (mRNA) and protein traffic between cells through intercellular nanochannels called plasmodesmata. KN1 protein and mRNA trafficking are regulated by a chaperonin subunit and a catalytic subunit of the RNA exosome, respectively. These studies suggest that the function of KN1 in stem cell regulation requires the cell-to-cell transport of both its protein and mRNA. However, in situ hybridization experiments published 25 years ago suggested that KN1 mRNA was missing from the epidermal (L1) layer of shoot meristems, suggesting that only the KN1 protein could traffic. Here, we show evidence that KN1 mRNA is present at a low level in L1 cells of maize meristems, supporting an idea that both KN1 protein and mRNA traffic to the L1 layer. We also summarize mRNA expression patterns of KN1 homologs in diverse angiosperm species, and discuss KN1 trafficking mechanisms.

An RNA exosome subunit mediates cell-to-cell trafficking of a homeobox mRNA via plasmodesmata.

Messenger RNAs (mRNAs) function as mobile signals for cell-to-cell communication in multicellular organisms. The KNOTTED1 (KN1) homeodomain family transcription factors act non­cell autonomously to control stem cell maintenance in plants through cell-to-cell movement of their proteins and mRNAs through plasmodesmata; however, the mechanism of mRNA movement is largely unknown. We show that cell-to-cell movement of a KN1 mRNA requires ribosomal RNA­processing protein 44A (AtRRP44A), a subunit of the RNA exosome that processes or degrades diverse RNAs in eukaryotes. AtRRP44A can interact with plasmodesmata and mediates the cell-to-cell trafficking of KN1 mRNA, and genetic analysis indicates that AtRRP44A is required for the developmental functions of SHOOT MERISTEMLESS, an Arabidopsis KN1 homolog. Our findings suggest that AtRRP44A promotes mRNA trafficking through plasmodesmata to control stem cell­dependent processes in plants.

An Optimized Whole-Mount Immunofluorescence Method for Shoot Apices.

The localization of a protein provides important information about its biological functions. The visualization of proteins by immunofluorescence has become an essential approach in cell biology. Here, we describe an easy-to-follow immunofluorescence protocol to localize proteins in whole-mount tissues of maize (Zea mays) and Arabidopsis. We present the whole-mount immunofluorescence procedure using maize ear primordia and Arabidopsis inflorescence apices as examples, followed by tips and suggestions for each step. In addition, we provide a supporting protocol to describe the use of an ImageJ plug-in to analyze colocalization. This protocol has been optimized to observe proteins in 2-5 mm maize ear primordia or in developing Arabidopsis inflorescence apices; however, it can be used as a reference to perform whole-mount immunofluorescence in other plant tissues and species. © 2021 Wiley Periodicals LLC. Basic Protocol: Whole-mount immunofluorescence for maize and Arabidopsis shoot apices Support Protocol: Measure colocalization by JACoP plugin in ImageJ.

Control of Meristem Size.

A fascinating feature of plant growth and development is that plants initiate organs continually throughout their lifespan. The ability to do this relies on specialized groups of pluripotent stem cells termed meristems, which allow for the elaboration of the shoot, root, and vascular systems. We now have a deep understanding of the genetic networks that control meristem initiation and stem cell maintenance, including the roles of receptors and their ligands, transcription factors, and integrated hormonal and chromatin control. This review describes these networks and discusses how this knowledge is being applied to improve crop productivity by increasing fruit size and seed number.

Plasmodesmata-Mediated Cell-to-Cell Communication in the Shoot Apical Meristem: How Stem Cells Talk.

Positional information is crucial for the determination of plant cell fates, and it is established based on coordinated cell-to-cell communication, which in turn is essential for plant growth and development. Plants have evolved a unique communication pathway, with tiny channels called plasmodesmata (PD) spanning the cell wall. PD interconnect most cells in the plant and generate a cytoplasmic continuum, to mediate short- and long-distance trafficking of various molecules. Cell-to-cell communication through PD plays a role in transmitting positional signals, however, the regulatory mechanisms of PD-mediated trafficking are still largely unknown. The induction and maintenance of stem cells in the shoot apical meristem (SAM) depends on PDmediated cell-to-cell communication, hence, it is an optimal model for dissecting the regulatory mechanisms of PD-mediated cell-to-cell communication and its function in specifying cell fates. In this review, we summarize recent knowledge of PD-mediated cell-to-cell communication in the SAM, and discuss mechanisms underlying molecular trafficking through PD and its role in plant development.