Cytokinin and ALOG proteins regulate pluripotent stem cell identity in the moss Physcomitrium patens.

The shoot apical meristem (SAM) contains pluripotent stem cells that produce all the aerial parts of the plant. Stem cells undergo asymmetric cell divisions to self-renew and to produce differentiating cells. Our research focused on unraveling the mechanisms governing the specification of these two distinct cell fates following the stem cell division. For this purpose, we used the model organism Physcomitrium patens, which features a singular pluripotent stem cell known as the gametophore apical cell. We show that the activity of cytokinins, critical stem cell regulators, is restricted to the gametophore apical cell due to the specific localization of PpLOG, the enzyme responsible for cytokinin activation. In turn, PpTAW, which promotes differentiating cell identity of the merophyte, is excluded from the gametophore apical cell by the action of cytokinins. We propose a cytokinin-based model for the establishment of asymmetry in the pluripotent stem cell division.

Contrasting and conserved roles of NPR pathways in diverged land plant lineages.

The NPR proteins function as salicylic acid (SA) receptors in Arabidopsis thaliana. AtNPR1 plays a central role in SA-induced transcriptional reprogramming whereby positively regulates SA-mediated defense. NPRs are found in the genomes of nearly all land plants. However, we know little about the molecular functions and physiological roles of NPRs in most plant species. We conducted phylogenetic and alignment analyses of NPRs from 68 species covering the significant lineages of land plants. To investigate NPR functions in bryophyte lineages, we generated and characterized NPR loss-of-function mutants in the liverwort Marchantia polymorpha. Brassicaceae NPR1-like proteins have characteristically gained or lost functional residues identified in AtNPRs, pointing to the possibility of a unique evolutionary trajectory for the Brassicaceae NPR1-like proteins. We find that the only NPR in M. polymorpha, MpNPR, is not the master regulator of SA-induced transcriptional reprogramming and negatively regulates bacterial resistance in this species. The Mpnpr transcriptome suggested roles of MpNPR in heat and far-red light responses. We identify both Mpnpr and Atnpr1-1 display enhanced thermomorphogenesis. Interspecies complementation analysis indicated that the molecular properties of AtNPR1 and MpNPR are partially conserved. We further show that MpNPR has SA-binding activity. NPRs and NPR-associated pathways have evolved distinctively in diverged land plant lineages to cope with different terrestrial environments.

Identification of a novel viral factor inducing tumorous symptoms by disturbing vascular development in planta.

Plant viruses induce various disease symptoms that substantially impact agriculture, but the underlying mechanisms of viral disease in plants are poorly understood. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Here, we show that a gene fragment of gentian Kobu-sho-associated virus, which is designated as Kobu-sho-inducing factor (KOBU), induces gall formation accompanied by ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. Transgenic gentian expressing KOBU exhibited tumorous symptoms, confirming the gall-forming activity of KOBU. Surprisingly, KOBU expression can also induce differentiation of an additional leaf-like tissue on the abaxial side of veins in normal N. benthamiana and gentian leaves. Transcriptome analysis with Arabidopsis thaliana expressing KOBU revealed that KOBU activates signaling pathways that regulate xylem development. KOBU protein forms granules and plate-like structures and co-localizes with mRNA splicing factors within the nucleus. Our findings suggest that KOBU is a novel pleiotropic virulence factor that stimulates vascular and leaf development. IMPORTANCE While various mechanisms determine disease symptoms in plants depending on virus-host combinations, the details of how plant viruses induce symptoms remain largely unknown in most plant species. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Our findings demonstrate that a gene fragment of gentian Kobu-sho-associated virus (GKaV), which is designated as Kobu-sho-inducing factor, induces the gall formation accompanied by the ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. The molecular mechanism by which gentian Kobu-sho-associated virus induces the Kobu-sho symptoms will provide new insight into not only plant-virus interactions but also the regulatory mechanisms underlying vascular and leaf development.

Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface.

Many secretory enzymes acquire essential zinc ions (Zn2+) in the Golgi complex. ERp44, a chaperone operating in the early secretory pathway, also binds Zn2+ to regulate its client binding and release for the control of protein traffic and homeostasis. Notably, three membrane transporter complexes, ZnT4, ZnT5/ZnT6 and ZnT7, import Zn2+ into the Golgi lumen in exchange with protons. To identify their specific roles, we here perform quantitative Zn2+ imaging using super-resolution microscopy and Zn2+-probes targeted in specific Golgi subregions. Systematic ZnT-knockdowns reveal that ZnT4, ZnT5/ZnT6 and ZnT7 regulate labile Zn2+ concentration at the distal, medial, and proximal Golgi, respectively, consistent with their localization. Time-course imaging of cells undergoing synchronized secretory protein traffic and functional assays demonstrates that ZnT-mediated Zn2+ fluxes tune the localization, trafficking, and client-retrieval activity of ERp44. Altogether, this study provides deep mechanistic insights into how ZnTs control Zn2+ homeostasis and ERp44-mediated proteostasis along the early secretory pathway.

Control of vegetative reproduction in Marchantia polymorpha by the KAI2-ligand signaling pathway.

In vegetative reproduction of Marchantia polymorpha (M. polymorpha), propagules, called gemmae, are formed in gemma cups. Despite its significance for survival, control of gemma and gemma cup formation by environmental cues is not well understood. We show here that the number of gemmae formed in a gemma cup is a genetic trait. Gemma formation starts from the central region of the floor of the gemma cup, proceeds to the periphery, and terminates when the appropriate number of gemmae is initiated. The MpKARRIKIN INSENSITIVE2 (MpKAI2)-dependent signaling pathway promotes gemma cup formation and gemma initiation. The number of gemmae in a cup is controlled by modulating the ON/OFF switch of the KAI2-dependent signaling. Termination of the signaling results in the accumulation of MpSMXL, a suppressor protein. In the Mpsmxl mutants, gemma initiation continues, leading to the formation of a highly increased number of gemmae in a cup. Consistent with its function, the MpKAI2-dependent signaling pathway is active in gemma cups where gemmae initiate, as well as in the notch region of the mature gemma and midrib of the ventral side of the thallus. In this work, we also show that GEMMA CUP-ASSOCIATED MYB1 works downstream of this signaling pathway to promote gemma cup formation and gemma initiation. We also found that the availability of potassium affects gemma cup formation independently from the KAI2-dependent signaling pathway in M. polymorpha. We propose that the KAI2-dependent signaling pathway functions to optimize vegetative reproduction by adapting to the environment in M. polymorpha.

NARROW AND DWARF LEAF 1, the Ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN, Mediates Leaf Development and Maintenance of the Shoot Apical Meristem in Oryza sativa L.

The molecular basis for leaf development, a major focus in developmental biology, remains unclear in the monocotyledonous grass, rice (Oryza sativa). Here, we performed a mutant screen in rice and identified an AP2-type transcription factor family protein, NARROW AND DWARF LEAF1 (NDL1). NDL1 is the ortholog of Arabidopsis thaliana (subsequently called Arabidopsis) ENHANCER OF SHOOT REGENERATION1 (ESR1)/DORNRÖSCHEN (DRN) and mediates leaf development and maintenance of the shoot apical meristem (SAM). Loss of function of NDL1 results in bladeless leaves and SAMs that are flat, rather than dome-shaped, and lack cell proliferation activity. This loss of function also causes reduced auxin signaling. Moreover, as is the case with Arabidopsis ESR1/DRN, NDL1 plays crucial roles in shoot regeneration. Importantly, we found that NDL1 is not expressed in the SAM but is expressed in leaf primordia. We propose that NDL1 cell autonomously regulates leaf development, but non-cell autonomously regulates SAM maintenance in rice.

The bryophytes Physcomitrium patens and Marchantia polymorpha as model systems for studying evolutionary cell and developmental biology in plants.

Bryophytes are nonvascular spore-forming plants. Unlike in flowering plants, the gametophyte (haploid) generation of bryophytes dominates the sporophyte (diploid) generation. A comparison of bryophytes with flowering plants allows us to answer some fundamental questions raised in evolutionary cell and developmental biology. The moss Physcomitrium patens was the first bryophyte with a sequenced genome. Many cell and developmental studies have been conducted in this species using gene targeting by homologous recombination. The liverwort Marchantia polymorpha has recently emerged as an excellent model system with low genomic redundancy in most of its regulatory pathways. With the development of molecular genetic tools such as efficient genome editing, both P. patens and M. polymorpha have provided many valuable insights. Here, we review these advances with a special focus on polarity formation at the cell and tissue levels. We examine current knowledge regarding the cellular mechanisms of polarized cell elongation and cell division, including symmetric and asymmetric cell division. We also examine the role of polar auxin transport in mosses and liverworts. Finally, we discuss the future of evolutionary cell and developmental biological studies in plants.

NARROW AND DWARF LEAF 1, the Ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN, Mediates Leaf Development and Maintenance of the Shoot Apical Meristem in Oryza sativa L.

The molecular basis for leaf development, a major focus in developmental biology, remains unclear in the monocotyledonous grass, rice (Oryza sativa). Here, we performed a mutant screen in rice and identified an AP2-type transcription factor family protein, NARROW AND DWARF LEAF1 (NDL1). NDL1 is the ortholog of Arabidopsis thaliana (subsequently called Arabidopsis) ENHANCER OF SHOOT REGENERATION1 (ESR1)/DORNRÖSCHEN (DRN) and mediates leaf development and maintenance of the shoot apical meristem (SAM). Loss of function of NDL1 results in bladeless leaves and SAMs that are flat, rather than dome-shaped, and lack cell proliferation activity. This loss of function also causes reduced auxin signaling. Moreover, as is the case with Arabidopsis ESR1/DRN, NDL1 plays crucial roles in shoot regeneration. Importantly, we found that NDL1 is not expressed in the SAM but is expressed in leaf primordia. We propose that NDL1 cell autonomously regulates leaf development, but non-cell autonomously regulates SAM maintenance in rice.

Major components of the KARRIKIN INSENSITIVE2-dependent signaling pathway are conserved in the liverwort Marchantia polymorpha.

KARRIKIN INSENSITIVE2 (KAI2) was first identified as a receptor of karrikins, smoke-derived germination stimulants. KAI2 is also considered a receptor of an unidentified endogenous molecule called the KAI2 ligand. Upon KAI2 activation, signals are transmitted through the degradation of D53/SMXL proteins via MAX2-dependent ubiquitination. Although components in the KAI2-dependent signaling pathway, namely MpKAI2A and MpKAI2B, MpMAX2, and MpSMXL, exist in the genome of the liverwort Marchantia polymorpha, their functions remain unknown. Here, we show that early thallus growth is retarded and gemma dormancy in the dark is suppressed in Mpkai2a and Mpmax2 loss-of-function mutants. These defects are counteracted in Mpkai2a Mpsmxl and Mpmax2 Mpsmxl double mutants indicating that MpKAI2A, MpMAX2, and MpSMXL act in the same genetic pathway. Introduction of MpSMXLd53, in which a domain required for degradation is mutated, into wild-type plants mimicks Mpkai2a and Mpmax2 plants. In addition, the detection of citrine fluorescence in Nicotiana benthamiana cells transiently expressing a SMXL-Citrine fusion protein requires treatment with MG132, a proteasome inhibitor. These findings imply that MpSMXL is subjected to degradation, and that the degradation of MpSMXL is crucial for MpKAI2A-dependent signaling in M. polymorpha. Therefore, we claim that the basic mechanisms in the KAI2-dependent signaling pathway are conserved in M. polymorpha.

AGC kinases and MAB4/MEL proteins maintain PIN polarity by limiting lateral diffusion in plant cells.

Polar subcellular localization of the PIN exporters of the phytohormone auxin is a key determinant of directional, intercellular auxin transport and thus a central topic of both plant cell and developmental biology. Arabidopsis mutants lacking PID, a kinase that phosphorylates PINs, or the MAB4/MEL proteins of unknown molecular function display PIN polarity defects and phenocopy pin mutants, but mechanistic insights into how these factors convey PIN polarity are missing. Here, by combining protein biochemistry with quantitative live-cell imaging, we demonstrate that PINs, MAB4/MELs, and AGC kinases interact in the same complex at the plasma membrane. MAB4/MELs are recruited to the plasma membrane by the PINs and in concert with the AGC kinases maintain PIN polarity through limiting lateral diffusion-based escape of PINs from the polar domain. The PIN-MAB4/MEL-PID protein complex has self-reinforcing properties thanks to positive feedback between AGC kinase-mediated PIN phosphorylation and MAB4/MEL recruitment. We thus uncover the molecular mechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development.

Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana.

Cell and tissue polarization is fundamental for plant growth and morphogenesis. The polar, cellular localization of Arabidopsis PIN-FORMED (PIN) proteins is crucial for their function in directional auxin transport. The clustering of PIN polar cargoes within the plasma membrane has been proposed to be important for the maintenance of their polar distribution. However, the more detailed features of PIN clusters and the cellular requirements of cargo clustering remain unclear. Here, we characterized PIN clusters in detail by means of multiple advanced microscopy and quantification methods, such as 3D quantitative imaging or freeze-fracture replica labeling. The size and aggregation types of PIN clusters were determined by electron microscopy at the nanometer level at different polar domains and at different developmental stages, revealing a strong preference for clustering at the polar domains. Pharmacological and genetic studies revealed that PIN clusters depend on phosphoinositol pathways, cytoskeletal structures and specific cell-wall components as well as connections between the cell wall and the plasma membrane. This study identifies the role of different cellular processes and structures in polar cargo clustering and provides initial mechanistic insight into the maintenance of polarity in plants and other systems.

Diversity of Pectin Rhamnogalacturonan I Rhamnosyltransferases in Glycosyltransferase Family 106.

Rhamnogalacturonan I (RG-I) comprises approximately one quarter of the pectin molecules in land plants, and the backbone of RG-I consists of a repeating sequence of [2)-α-L-Rha(1-4)-α-D-GalUA(1-] disaccharide. Four Arabidopsis thaliana genes encoding RG-I rhamnosyltransferases (AtRRT1 to AtRRT4), which synthesize the disaccharide repeats, have been identified in the glycosyltransferase family (GT106). However, the functional role of RG-I in plant cell walls and the evolutional history of RRTs remains to be clarified. Here, we characterized the sole ortholog of AtRRT1-AtRRT4 in liverwort, Marchantia polymorpha, namely, MpRRT1. MpRRT1 had RRT activity and genetically complemented the AtRRT1-deficient mutant phenotype in A. thaliana. However, the MpRRT1-deficient M. polymorpha mutants showed no prominent morphological changes and only an approximate 20% reduction in rhamnose content in the cell wall fraction compared to that in wild-type plants, suggesting the existence of other RRT gene(s) in the M. polymorpha genome. As expected, we detected RRT activities in other GT106 family proteins such as those encoded by MpRRT3 in M. polymorpha and FRB1/AtRRT8 in A. thaliana, the deficient mutant of which affects cell adhesion. Our results show that RRT genes are more redundant and diverse in GT106 than previously thought.

The origin and evolution of the ALOG proteins, members of a plant-specific transcription factor family, in land plants.

The Arabidopsis LSH1 and Oryza G1 (ALOG) protein is a family of plant-specific transcription factors that regulate reproductive growth in angiosperms. Despite their importance in plant development, little research has been conducted on ALOG proteins in basal land plants and the processes involved in their evolution remain largely unknown. Here, we studied the molecular evolution of ALOG family proteins. We found that ALOG proteins are absent in green algae but exist in all land plants analyzed as well as in some Charophycean algae, closest relatives of land plants. Multiple sequence alignments identified the high sequence conservation of ALOG domains in divergent plant lineages. Phylogenetic analyses also identified a distinct clade of ALOG protein member of lycophytes and bryophytes, including two of Marchantia polymorpha LATERAL ORGAN SUPPRESOR (MpLOS1 and MpLOS2) with a long branch length in MpLOS2. Consistent with this, the function of MpLOS1 was replaceable by Phycomitrella patens ALOG proteins, whereas MpLOS2 failed to replace the molecular function of MpLOS1. Moreover, the rice ALOG proteins, OsTAW1 and OsG1, were not able to replace the molecular function of MpLOS1 although we previously found that the function of OsG1 was replaceable by MpLOS1. Altogether, these findings suggest that ALOG proteins emerged before the evolution of land plants and that they exhibit functional conservation and diversification during the evolution of land plants. The finding that MpLOS1 is able to complement rice ALOG mutants but not vice versa also suggest the existence of conserved and the partly divergent functions of ALOG proteins in bryophytes and angiosperms.

A conserved regulatory mechanism mediates the convergent evolution of plant shoot lateral organs.

Land plant shoot structures evolved a diversity of lateral organs as morphological adaptations to the terrestrial environment, with lateral organs arising independently in different lineages. Vascular plants and bryophytes (basally diverging land plants) develop lateral organs from meristems of sporophytes and gametophytes, respectively. Understanding the mechanisms of lateral organ development among divergent plant lineages is crucial for understanding the evolutionary process of morphological diversification of land plants. However, our current knowledge of lateral organ differentiation mechanisms comes almost entirely from studies of seed plants, and thus, it remains unclear how these lateral structures evolved and whether common regulatory mechanisms control the development of analogous lateral organs. Here, we performed a mutant screen in the liverwort Marchantia polymorpha, a bryophyte, which produces gametophyte axes with nonphotosynthetic scalelike lateral organs. We found that an Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and Oryza G1 (ALOG) family protein, named M. polymorpha LATERAL ORGAN SUPRESSOR 1 (MpLOS1), regulates meristem maintenance and lateral organ development in Marchantia. A mutation in MpLOS1, preferentially expressed in lateral organs, induces lateral organs with misspecified identity and increased cell number and, furthermore, causes defects in apical meristem maintenance. Remarkably, MpLOS1 expression rescued the elongated spikelet phenotype of a MpLOS1 homolog in rice. This suggests that ALOG genes regulate the development of lateral organs in both gametophyte and sporophyte shoots by repressing cell divisions. We propose that the recruitment of ALOG-mediated growth repression was in part responsible for the convergent evolution of independently evolved lateral organs among highly divergent plant lineages, contributing to the morphological diversification of land plants.

BLADE-ON-PETIOLE genes are not involved in the transition from protonema to gametophore in the moss Physcomitrella patens.

The timing of the transition between developmental phases is a critical determinant of plant form. In the moss Physcomitrella patens, the transition from protonema to gametophore is a particularly important step as it results in a change from two-dimensional to three-dimensional growth of the plant body. It is well known that this transition is promoted by cytokinin (CK), however, the underlying mechanisms are poorly understood. Previously, it was reported that P. patens orthologs of BLADE-ON-PETIOLE (BOP) genes (PpBOPs) work downstream of CK to promote the transition to gametophore. To further understand the role of PpBOPs in the control of this transition, we performed functional analyses of PpBOP genes. We simultaneously disrupted the function of all three PpBOP genes in P. patens using CRISPR technology, however, no abnormal phenotypes were observed in the triple mutant during either the gametophytic or the sporophytic growth stages. CK treatment did not alter the phase change in the triple mutant. We conclude that PpBOP genes are unnecessary in the control of P. patens development under normal conditions. We propose that BOP genes are not involved in the control of developmental processes in bryophytes and other basal land plants, but may function in physiological processes such as in the defense response.

Cytokinin Signaling Is Essential for Organ Formation in Marchantia polymorpha.

Cytokinins are known to regulate various physiological events in plants. Cytokinin signaling is mediated by the phosphorelay system, one of the most ancient mechanisms controlling hormonal pathways in plants. The liverwort Marchantia polymorpha possesses all components necessary for cytokinin signaling; however, whether they respond to cytokinins and how the signaling is fine-tuned remain largely unknown. Here, we report cytokinin function in Marchantia development and organ formation. Our measurement of cytokinin species revealed that cis-zeatin is the most abundant cytokinin in Marchantia. We reduced the endogenous cytokinin level by overexpressing the gene for cytokinin oxidase, MpCKX, which inactivates cytokinins, and generated overexpression and knockout lines for type-A (MpRRA) and type-B (MpRRB) response regulators to manipulate the signaling. The overexpression lines of MpCKX and MpRRA, and the knockout lines of MpRRB, shared phenotypes such as inhibition of gemma cup formation, enhanced rhizoid formation and hyponastic thallus growth. Conversely, the knockout lines of MpRRA produced more gemma cups and exhibited epinastic thallus growth. MpRRA expression was elevated by cytokinin treatment and reduced by knocking out MpRRB, suggesting that MpRRA is upregulated by the MpRRB-mediated cytokinin signaling, which is antagonized by MpRRA. Our findings indicate that when plants moved onto land they already deployed the negative feedback loop of cytokinin signaling, which has an indispensable role in organogenesis.

BLADE-ON-PETIOLE genes temporally and developmentally regulate the sheath to blade ratio of rice leaves.

Axis formation is a fundamental issue in developmental biology. Axis formation and patterning in plant leaves is crucial for morphology and crop productivity. Here, we reveal the basis of proximal-distal patterning in rice leaves, which consist of a proximal sheath, a distal blade, and boundary organs formed between these two regions. Analysis of the three rice homologs of the Arabidopsis BLADE-ON-PETIOLE1 (BOP1) gene indicates that OsBOPs activate proximal sheath differentiation and suppress distal blade differentiation. Temporal expression changes of OsBOPs are responsible for the developmental changes in the sheath:blade ratio. We further identify that the change in the sheath:blade ratio during the juvenile phase is controlled by the miR156/SPL pathway, which modifies the level and pattern of expression of OsBOPs. OsBOPs are also essential for differentiation of the boundary organs. We propose that OsBOPs, the main regulators of proximal-distal patterning, control temporal changes in the sheath:blade ratio of rice leaves.

Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane.

The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses.