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Polarized growth drives the morphogenesis of elongated cellular structures. In plants, polarized growth depends on actin and a tip focused ionic calcium gradient. How the calcium gradient is maintained remains unclear. We discovered that autoinhibitory calcium ATPases (ACAs) redundantly contribute to the steepness of the calcium gradient. ACA1 and ACA2 localize to the subapical plasma membrane and ACA5 to the vacuole membrane, providing spatial regulation of calcium efflux. Tip-growing plant cells also exhibit apical calcium fluctuations. Even though Δaca1/2/5 cells have a diminished calcium gradient, they exhibit normal fluctuations and actin but have significantly reduced apical secretion. Furthermore, cells lacking apical actin retain a strong calcium gradient but have reduced apical secretion. Suppression of both the calcium gradient and apical actin dramatically impairs growth, supporting a model where two independent and parallel processes, the calcium gradient and apical actin, promote rapid polarized growth. 

Similar to cellulose synthases (CESAs), cellulose synthase–like D (CSLD) proteins synthesize β-1,4-glucan in plants. CSLDs are important for tip growth and cytokinesis, but it was unknown whether they form membrane complexes in vivo or produce microfibrillar cellulose. We produced viable CESA-deficient mutants of the mossPhyscomitrium patensto investigate CSLD function without interfering CESA activity. Microscopy and spectroscopy showed that CESA-deficient mutants synthesize cellulose microfibrils that are indistinguishable from those in vascular plants. Correspondingly, freeze-fracture electron microscopy revealed rosette-shaped particle assemblies in the plasma membrane that are indistinguishable from CESA-containing rosette cellulose synthesis complexes (CSCs). Our data show that proteins other than CESAs, most likely CSLDs, produce cellulose microfibrils inP. patensprotonemal filaments. The data suggest that the specialized roles of CSLDs in cytokinesis and tip growth are based on differential expression and different interactions with microtubules and possibly Ca²⁺, rather than structural differences in the microfibrils they produce. 

Abstract ATPase family AAA domain–containing 3 (ATAD3) proteins are unique mitochondrial proteins that arose deep in the eukaryotic lineage but that are surprisingly absent in 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 ATPases Associated with a variety of cellular Activities (AAA+) matrix domain and an ATAD3_N domain, which is located primarily in the inner membrane space but potentially extends to the cytosol to interact with the ER. Sequence and structural alignments indicate that ATAD3 proteins are most similar to classic chaperone unfoldases in the 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. 

Cellulose Synthase-Like D (CSLD) proteins, important for tip growth and cell division, are known to generate β-1,4-glucan. However, whether they are propelled in the membrane as the glucan chains they produce assemble into microfibrils is unknown. To address this, we endogenously tagged all eight CSLDs in Physcomitrium patens and discovered that they all localize to the apex of tip-growing cells and to the cell plate during cytokinesis. Actin is required to target CSLD to cell tips concomitant with cell expansion, but not to cell plates, which depend on actin and CSLD for structural support. Like Cellulose Synthase (CESA), CSLD requires catalytic activity to move in the plasma membrane. We discovered that CSLD moves significantly faster, with shorter duration and less linear trajectories than CESA. In contrast to CESA, CSLD movement was insensitive to the cellulose synthesis inhibitor isoxaben, suggesting that CSLD and CESA function within different complexes possibly producing structurally distinct cellulose microfibrils. 

ABSTRACT Chloroplast biogenesis depends on both protein import and organelle division, yet how their coordination emerged during evolution remains unclear. Here, we show that the single septin SEP1 links these pathways in the green algaChlamydomonas reinhardtii. SEP1 forms a filamentous network on the chloroplast envelope during interphase and reorganizes into a ring at the chloroplast division site during cytokinesis. Loss of SEP1 selectively impairs import of chloroplast-division proteins and causes mispositioning of the division ring, without impairing bulk chloroplast protein import. SEP1 physically associates with outer-envelope TOC GTPases through evolutionarily related GTPase domains. Phylogenetic analysis places TOC GTPases within an algal septin-derived clade, and heterologous expression of SEP1 in land plants, in which septins are absent, shows conservation of its chloroplast targeting and TOC binding. Together, these findings identify septins as coordinators of plastid protein import and division and suggest that this coupling emerged early in chloroplast evolution. 

Abstract 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. 

Abstract 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. 

In animals, PIEZOs are plasma membrane–localized cation channels involved in diverse mechanosensory processes. We investigated PIEZO function in tip-growing cells in the moss Physcomitrium patens and the flowering plant Arabidopsis thaliana . Pp PIEZO1 and Pp PIEZO2 redundantly contribute to the normal growth, size, and cytoplasmic calcium oscillations of caulonemal cells. Both Pp PIEZO1 and Pp PIEZO2 localized to vacuolar membranes. Loss-of-function, gain-of-function, and overexpression mutants revealed that moss PIEZO homologs promote increased complexity of vacuolar membranes through tubulation, internalization, and/or fission. Arabidopsis PIEZO1 also localized to the tonoplast and is required for vacuole tubulation in the tips of pollen tubes. We propose that in plant cells the tonoplast has more freedom of movement than the plasma membrane, making it a more effective location for mechanosensory proteins.