A cornichon protein controls polar localization of the PINA auxin transporter in Physcomitrium patens.

Newly synthesized membrane proteins pass through the secretory pathway, starting at the endoplasmic reticulum and packaged into COPII vesicles, to continue to the Golgi apparatus before reaching their membrane of residence. It is known that cargo receptor proteins form part of the COPII complex and play a role in the recruitment of cargo proteins for their subsequent transport through the secretory pathway. The role of cornichon proteins is conserved from yeast to vertebrates, but it is poorly characterized in plants. Here, we studied the role of the two cornichon homologs in the secretory pathway of the moss Physcomitrium patens. Mutant analyses revealed that cornichon genes regulate different growth processes during the moss life cycle by controlling auxin transport, with CNIH2 functioning as a specific cargo receptor for the auxin efflux carrier PINA, with the C terminus of the receptor regulating the interaction, trafficking and membrane localization of PINA.

COPII Sec23 proteins form isoform-specific endoplasmic reticulum exit sites with differential effects on polarized growth.

Coat Protein complex II (COPII), a coat protein complex that forms vesicles on the endoplasmic reticulum (ER), mediates trafficking to the Golgi. While metazoans have few genes encoding each COPII component, plants have expanded these gene families, leading to the hypothesis that plant COPII has functionally diversified. In the moss Physcomitrium (Physcomitrella) patens, the Sec23/24 gene families are each composed of seven genes. Silencing Sec23/24 revealed isoform-specific contributions to polarized growth, with the closely related Sec23D/E and Sec24C/D essential for protonemal development. Focusing on Sec23, we discovered that Sec23D/E mediate ER-to Golgi transport and are essential for tip growth, with Sec23D localizing to presumptive ER exit sites. In contrast, Sec23A, B, C, F, and G are dispensable and do not quantitatively affect ER-to-Golgi trafficking. However, Δsec23abcfg plants exhibited reduced secretion of plasma membrane cargo. Of the four highly expressed protonemal Sec23 genes, Sec23F/G are members of a divergent Sec23 clade specifically retained in land plants. Notably, Sec23G accumulates on ER-associated foci that are significantly larger, do not overlap with, and are independent of Sec23D. While Sec23D/E form ER exit sites and function as bona fide COPII components essential for tip-growing protonemata, Sec23G and the closely related Sec23F have likely functionally diversified, forming separate and independent ER exit sites and participating in Golgi-independent trafficking pathways.

A glossary of plant cell structures: Current insights and future questions.

In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.

ATAD3 Proteins: Unique Mitochondrial Proteins Essential for Life in Diverse Eukaryotic Lineages.

ATAD3 proteins (ATPase family AAA domain-containing protein 3) are unique mitochondrial proteins that arose deep in the eukaryotic lineage but that are surprisingly absent from the Fungi and Amoebozoa. These ~600 amino acid proteins are anchored in the inner mitochondrial membrane and are essential in metazoans and Arabidopsis thaliana. ATAD3s comprise a C-terminal AAA+ matrix domain and an ATAD3_N domain that is located primarily in the inner membrane space but potentially extends into cytosol to interact with the ER. Sequence and structural alignments indicate ATAD3 proteins are most similar to classic chaperone unfoldases in AAA+ family, suggesting that they operate in mitochondrial protein quality control. A. thaliana has four ATAD3 genes in two distinct clades that appear first in the seed plants, and both clades are essential for viability. The four genes are generally coordinately expressed, and transcripts are highest in growing apices and imbibed seeds. Plants with disrupted ATAD3 have reduced growth, aberrant mitochondrial morphology, diffuse nucleoids and reduced oxidative phosphorylation complex I. These and other pleiotropic phenotypes are also observed in ATAD3 mutants in metazoans. Here we discuss the distribution of ATAD3 proteins as they have evolved in the plant kingdom, their unique structure, what we know about their function in plants, and the challenges in determining their essential roles in mitochondria.

A Fully Functional ROP Fluorescent Fusion Protein Reveals Roles for This GTPase in Subcellular and Tissue-Level Patterning.

Rho of Plants (ROPs) are GTPases that regulate polarity and patterned wall deposition in plants. As these small, globular proteins have many interactors, it has been difficult to ensure that methods to visualize ROP in live cells do not affect ROP function. Here, motivated by work in fission yeast (Schizosaccharomyces pombe), we generated a fluorescent moss (Physcomitrium [Physcomitrella] patens) ROP4 fusion protein by inserting mNeonGreen after Gly-134. Plants harboring tagged ROP4 and no other ROP genes were phenotypically normal. Plants lacking all four ROP genes comprised an unpatterned clump of spherical cells that were unable to form gametophores, demonstrating that ROP is essentially for spatial patterning at the cellular and tissue levels. The functional ROP fusion protein formed a steep gradient at the apical plasma membranes of growing tip cells. ROP also predicted the site of branch formation in the apical cell at the onset of mitosis, which occurs one to two cell cycles before a branch cell emerges. While fluorescence recovery after photobleaching studies demonstrated that ROP dynamics do not depend on the cytoskeleton, acute depolymerization of the cytoskeleton removed ROP from the membrane only in recently divided cells, pointing to a feedback mechanism between the cell cycle, cytoskeleton, and ROP.

The Moss Physcomitrium (Physcomitrella) patens: A Model Organism for Non-Seed Plants.

Since the discovery two decades ago that transgenes are efficiently integrated into the genome of Physcomitrella patens by homologous recombination, this moss has been a premier model system to study evolutionary developmental biology questions, stem cell reprogramming, and the biology of nonvascular plants. P patens was the first non-seed plant to have its genome sequenced. With this level of genomic information, together with increasing molecular genetic tools, a large number of reverse genetic studies have propelled the use of this model system. A number of technological advances have recently opened the door to forward genetics as well as extremely efficient and precise genome editing in P patens Additionally, careful phylogenetic studies with increased resolution have suggested that P patens emerged from within Physcomitrium Thus, rather than Physcomitrella patens, the species should be named Physcomitrium patens Here we review these advances and describe the areas where P patens has had the most impact on plant biology.

In vivo analysis of formin dynamics in the moss P. patens reveals functional class diversification.

Formins are actin regulators critical for diverse processes across eukaryotes. With many formins in plants and animals, it has been challenging to determine formin function in vivo We found that the phylogenetically distinct class I integral membrane formins (denoted For1) from the moss P.patens enrich at sites of membrane turnover, with For1D more tightly associated with the plasma membrane than For1A. To probe formin function, we generated formin-null lines with greatly reduced formin complexity. We found that For1A and For1D help to anchor actin near the cell apex, with For1A contributing to formation of cytosolic actin, while For1D contributes to plasma membrane-associated actin. At the cortex, For1A and For1D localized to motile puncta and differentially impacted actin dynamics. We found that class I cortical formin mobility depended on microtubules and only moderately on actin, whereas class II formin (denoted For2) mobility solely depended on actin. Moreover, cortical For2A tightly correlated with the puncta labeled by the endocytic membrane dye FM4-64, and null mutants in class I formins did not affect uptake of a similar dye, FM1-43, suggesting that class I and II formins are involved in distinct membrane trafficking pathways.

Slip slidin' away: Bristle-driven gliding by Tetradesmus deserticola (Chlorophyta) in microfluidic chambers.

Microalgae within the Scenedesmaceae are often distinguished by spines, bristles, and other wall characteristics. We examined the dynamic production and chemical nature of bristles extruded from the poles of Tetradesmus deserticola previously isolated from microbiotic crust. Rapidly growing cells in a liquid growth medium were established in polydimethylsiloxane microfluidic chambers specially designed to maintain aerobic conditions over time within a chamber 6-12 µm deep. This geometry enabled in-focus imaging of single cells over long periods. Differential interference contrast (DIC) imaging revealed that after multiple fission of mother cells, the newly released, lemon-shaped daughter cells began extruding bristles from each pole. In some instances, the bristles became stuck to either the glass floor or polydimethylsiloxane (PDMS) walls of the chamber, and the force by which the new bristle was extruded was sufficient to propel the cells across the field of view at ~1.2 µm · h-1 . Confocal fluorescence and DIC imaging of cells stained with pontamine fast scarlet and calcofluor, and treated with proteinase K, suggested that bristles are proteinaceous and may also host carbohydrate modifications. The polar bristles extruded by this desert-derived T. deserticola may simply be relics of bristles produced by an aquatic ancestor for flotation or predator deterrence. But, their tendency to attach to glass (silicate) and/or PDMS surfaces suggests a potential role in tethering cells in place or binding soil particles. T. deserticola is closely related to T. obliquus, which is of interest for biofuels development; extruded bristles in T. deserticola may offer tethers for industrial use of these stress-tolerant algae.

Simultaneous imaging and functional studies reveal a tight correlation between calcium and actin networks.

Tip-growing cells elongate in a highly polarized manner via focused secretion of flexible cell-wall material. Calcium has been implicated as a vital factor in regulating the deposition of cell-wall material. However, deciphering the molecular and mechanistic calcium targets in vivo has remained challenging. Here, we investigated intracellular calcium dynamics in the moss Physcomitrella patens, which provides a system with an abundant source of genetically identical tip-growing cells, excellent cytology, and a large molecular genetic tool kit. To visualize calcium we used a genetically encoded cytosolic FRET probe, revealing a fluctuating tipward gradient with a complex oscillatory profile. Wavelet analysis coupled with a signal-sifting algorithm enabled the quantitative comparison of the calcium behavior in cells where growth was inhibited mechanically, pharmacologically, or genetically. We found that cells with suppressed growth have calcium oscillatory profiles with longer frequencies, suggesting that there is a feedback between the calcium gradient and growth. To investigate the mechanistic basis for this feedback we simultaneously imaged cytosolic calcium and actin, which has been shown to be essential for tip growth. We found that high cytosolic calcium promotes disassembly of a tip-focused actin spot, while low calcium promotes assembly. In support of this, abolishing the calcium gradient resulted in dramatic actin accumulation at the tip. Together these data demonstrate that tipward calcium is quantitatively linked to actin accumulation in vivo and that the moss P. patens provides a powerful system to uncover mechanistic links between calcium, actin, and growth.

Conditional genetic screen in Physcomitrella patens reveals a novel microtubule depolymerizing-end-tracking protein.

Our ability to identify genes that participate in cell growth and division is limited because their loss often leads to lethality. A solution to this is to isolate conditional mutants where the phenotype is visible under restrictive conditions. Here, we capitalize on the haploid growth-phase of the moss Physcomitrella patens to identify conditional loss-of-growth (CLoG) mutants with impaired growth at high temperature. We used whole-genome sequencing of pooled segregants to pinpoint the lesion of one of these mutants (clog1) and validated the identified mutation by rescuing the conditional phenotype by homologous recombination. We found that CLoG1 is a novel and ancient gene conserved in plants. At the restrictive temperature, clog1 plants have smaller cells but can complete cell division, indicating an important role of CLoG1 in cell growth, but not an essential role in cell division. Fluorescent protein fusions of CLoG1 indicate it is localized to microtubules with a bias towards depolymerizing microtubule ends. Silencing CLoG1 decreases microtubule dynamics, suggesting that CLoG1 plays a critical role in regulating microtubule dynamics. By discovering a novel gene critical for plant growth, our work demonstrates that P. patens is an excellent genetic system to study genes with a fundamental role in plant cell growth.

A model suite of green algae within the Scenedesmaceae for investigating contrasting desiccation tolerance and morphology.

Microscopic green algae inhabiting desert microbiotic crusts are remarkably diverse phylogenetically, and many desert lineages have independently evolved from aquatic ancestors. Here we worked with five desert and aquatic species within the family Scenedesmaceae to examine mechanisms that underlie desiccation tolerance and release of unicellular versus multicellular progeny. Live cell staining and time-lapse confocal imaging coupled with transmission electron microscopy established that the desert and aquatic species all divide by multiple (rather than binary) fission, although progeny were unicellular in three species and multicellular (joined in a sheet-like coenobium) in two. During division, Golgi complexes were localized near nuclei, and all species exhibited dynamic rotation of the daughter cell mass within the mother cell wall at cytokinesis. Differential desiccation tolerance across the five species, assessed from photosynthetic efficiency during desiccation/rehydration cycles, was accompanied by differential accumulation of intracellular reactive oxygen species (ROS) detected using a dye sensitive to intracellular ROS. Further comparative investigation will aim to understand the genetic, ultrastructural and physiological characteristics supporting unicellular versus multicellular coenobial morphology, and the ability of representatives in the Scenedesmaceae to colonize ecologically diverse, even extreme, habitats.

Geometric cues forecast the switch from two- to three-dimensional growth in Physcomitrella patens.

During land colonization, plants acquired a range of body plan adaptations, of which the innovation of three-dimensional (3D) tissues increased organismal complexity and reproductivity. In the moss, Physcomitrella patens, a 3D leafy gametophore originates from filamentous cells that grow in a two-dimensional (2D) plane through a series of asymmetric cell divisions. Asymmetric cell divisions that coincide with different cell division planes and growth directions enable the developmental switch from 2D to 3D, but insights into the underlying mechanisms coordinating this switch are still incomplete. Using 2D and 3D imaging and image segmentation, we characterized two geometric cues, the width of the initial cell and the angle of the transition division plane, which sufficiently distinguished a gametophore initial cell from a branch initial cell. These identified cues were further confirmed in gametophore formation mutants. The identification of a fluorescent marker allowed us to successfully predict the gametophore initial cell with > 90% accuracy before morphological changes, supporting our hypothesis that, before the transition division, parental cells of the gametophore initials possess different properties from those of the branch initials. Our results suggest that the cell fate decision of the initial cell is determined in the parental cell, before the transition division.

SECONDARY WALL ASSOCIATED MYB1 is a positive regulator of secondary cell wall thickening in Brachypodium distachyon and is not found in the Brassicaceae.

Grass biomass is comprised chiefly of secondary walls that surround fiber and xylem cells. A regulatory network of interacting transcription factors in part regulates cell wall thickening. We identified Brachypodium distachyon SECONDARY WALL ASSOCIATED MYB1 (SWAM1) as a potential regulator of secondary cell wall biosynthesis based on gene expression, phylogeny, and transgenic plant phenotypes. SWAM1 interacts with cellulose and lignin gene promoters with preferential binding to AC-rich sequence motifs commonly found in the promoters of cell wall-related genes. SWAM1 overexpression (SWAM-OE) lines had greater above-ground biomass with only a slight change in flowering time while SWAM1 dominant repressor (SWAM1-DR) plants were severely dwarfed with a striking reduction in lignin of sclerenchyma fibers and stem epidermal cell length. Cellulose, hemicellulose, and lignin genes were significantly down-regulated in SWAM1-DR plants and up-regulated in SWAM1-OE plants. There was no reduction in bioconversion yield in SWAM1-OE lines; however, it was significantly increased for SWAM1-DR samples. Phylogenetic and syntenic analyses strongly suggest that the SWAM1 clade was present in the last common ancestor between eudicots and grasses, but is not in the Brassicaceae. Collectively, these data suggest that SWAM1 is a transcriptional activator of secondary cell wall thickening and biomass accumulation in B. distachyon.

Systematic survey of the function of ROP regulators and effectors during tip growth in the moss Physcomitrella patens.

Rho/Rac of plants (ROP) GTPases are plant-specific small GTPases that regulate cell morphology. ROP activity is controlled by several families of regulatory proteins. However, how these diverse regulators contribute to polarized growth remains understudied. In a system-wide approach, we used RNAi to silence each gene family of known ROP regulators in the juvenile tissues of the moss Physcomitrella patens. We found that the GTPase activating proteins, but not the ROP enhancers, are essential for tip growth. The guanine exchange factors (GEFs), which are comprised of ROPGEFs and Spikes, both contribute to growth. However, silencing Spikes results in less-polarized plants as compared to silencing ROPGEFs, suggesting that Spikes contribute more to establishing cell polarity. Silencing the single-gene family of guanine dissociation inhibitors also inhibits growth, resulting in small, unpolarized plants. In contrast, silencing the ROP effector ROP-interactive CRIB-containing (RIC) protein, which is encoded by a single gene, results in plants larger than the controls, suggesting that RIC functions to inhibit tip growth in moss. Taken together, this systematic loss-of-function survey provides insights into the function of ROP regulators during polarized growth.

Non-model model organisms.

Model organisms are widely used in research as accessible and convenient systems to study a particular area or question in biology. Traditionally only a handful of organisms have been widely studied, but modern research tools are enabling researchers to extend the set of model organisms to include less-studied and more unusual systems. This Forum highlights a range of 'non-model model organisms' as emerging systems for tackling questions across the whole spectrum of biology (and beyond), the opportunities and challenges, and the outlook for the future.

A family of ROP proteins that suppresses actin dynamics, and is essential for polarized growth and cell adhesion.

In plants, the ROP family of small GTPases has been implicated in the polarized growth of tip-growing cells, such as root hairs and pollen tubes; however, most of the data derive from overexpressing ROP genes or constitutively active and dominant-negative isoforms, whereas confirmation by using loss-of-function studies has generally been lacking. Here, in the model moss Physcomitrella patens, we study ROP signaling during tip growth by using a loss-of-function approach based on RNA interference (RNAi) to silence the entire moss ROP family. We find that plants with reduced expression of ROP genes, in addition to failing to initiate tip growth, have perturbed cell wall staining, reduced cell adhesion and have increased actin-filament dynamics. Although plants subjected to RNAi against the ROP family also have reduced microtubule dynamics, this reduction is not specific to loss of ROP genes, as it occurs when actin function is compromised chemically or genetically. Our data suggest that ROP proteins polarize the actin cytoskeleton by suppressing actin-filament dynamics, leading to an increase in actin filaments at the site of polarized secretion.

Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development.

Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers. Because the PDMS chambers are bonded to a coverslip, it is possible to image P. patens development at high resolution over long time periods. Using PDMS chambers, we report that wild-type protonemal tissue grows at the same rate as previously reported for growth on solid medium. Using long-term imaging, we highlight key developmental events, demonstrate compatibility with high-resolution confocal microscopy, and obtain growth rates for a slow-growing mutant. By coupling the powerful genetic tools available to P. patens with long-term growth and imaging provided by PDMS microfluidic chambers, we demonstrate the capability to study cellular and subcellular developmental events in plants directly and in real time.

Phosphatase and Tensin Homolog Is a Growth Repressor of Both Rhizoid and Gametophore Development in the Moss Physcomitrella patens.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase implicated in cellular proliferation and survival. In animal cells, loss of PTEN leads to increased levels of phosphatidylinositol (3,4,5)-trisphosphate, stimulation of glucose and lipid metabolism, cellular growth, and morphological changes (related to adaptation and survival). Intriguingly, in plants, phosphatidylinositol (3,4,5)-trisphosphate has not been detected, and the enzymes that synthesize it were never reported. In this study we performed a genetic, biochemical, and functional characterization of the moss Physcomitrella patens PTEN gene family. P. patens has four PTENs, which are ubiquitously expressed during the entire moss life cycle. Using a knock-in approach, we show that all four genes are expressed in growing tissues, namely caulonemal and rhizoid cells. At the subcellular level, PpPTEN-green fluorescent protein fusions localized to the cytosol and the nucleus. Analysis of single and double knockouts revealed no significant phenotypes at different developmental stages, indicative of functional redundancy. However, compared with wild-type triple and quadruple pten knockouts, caulonemal cells grew faster, switched from the juvenile protonemal stage to adult gametophores earlier, and produced more rhizoids. Furthermore, analysis of lipid content and quantitative real-time polymerase chain reaction data performed in quadruple mutants revealed altered phosphoinositide levels [increase in phosphatidylinositol (3,5)-bisphosphate and decrease in phosphatidylinositol 3-phosphate] and up-regulation of marker genes from the synthesis phase of the cell cycle (e.g. P. patens proliferating cell nuclear antigen, ribonucleotide reductase, and minichromosome maintenance) and of the retinoblastoma-related protein gene P. patens retinoblastoma-related protein1. Together, these results suggest that PpPTEN is a suppressor of cell growth and morphogenic development in plants.