Biology subjects

Bezanilla, M.

Publications and source records attributed to Bezanilla, M..

9 recordsLinked to original sources

bioRxiv2026

The Sulfated PSY Peptide Negatively Regulates Receptor Kinase Activity to Promote Growth

Complex signaling pathways organize cell expansion and proliferation across cells to pattern tissues and organs in plants. The sulfotyrosine peptide hormone family, PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY), contributes to these processes. We identified two plasma membrane-localized receptors, PSYR1 and PSYR2, that are necessary for PSY signaling and regulate growth in Physcomitrium patens. Membrane-associated PSYRs accumulate to high levels in a mutant lacking TYROSYL PROTEIN SULFOTRANSFERASE (TPST). Given that a tpst null mutant ({triangleup}tpst) is impaired in sulfation, this suggests that in the absence of sulfated peptides, PSYRs accumulate on the membrane. A null mutant of the PSY receptors,{triangleup} psyr1/2, showed increased growth and was epistatic to{triangleup} tpst, suppressing defects in gametophore formation and early senescence. The transcriptional profiles comparing wild type to{triangleup} psyr1/2 and{triangleup} psyr1/2/{triangleup}tpst showed 25 to 30 differentially expressed genes between the receptor null mutants and wild type, with a common signature of cell wall remodeling and stress responses. Similarly, a PSYR1 kinase-inactive mutation rescued{triangleup} tpst and relieved the accumulation of membrane-associated PSYRs. In contrast, overexpression of PSYRs inhibited plant growth, with phenotypic severity correlating with the amount of overexpression. These data are consistent with a constitutive activation model in which membrane-associated PSYRs unbound to PSY serve to inhibit growth through an active kinase. In the presence of the PSY peptide, the kinase is inactivated, promoting growth and driving PSY expression. The relationship between growth-repressive PSYR kinase activity and growth-promoting PSYR kinase inactivation in P. patens serves as a model for optimizing plant growth and development.

plant biology↗

bioRxiv2026

Septin-mediated coupling of protein import and division during chloroplast evolution

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 alga Chlamydomonas 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.

cell biology↗

bioRxiv2025

Evidence for Early Evolution of Sulfated Peptide Signaling in Plant Development

In plants, the cell wall fixes the position of each cell; therefore, during development, plants rely on cellular proliferation and expansion for tissue patterning and organ formation. How plant cells communicate with neighboring cells to coordinate expansion for properly patterned tissues and organs is not well understood. In seed plants, organ growth is known to be modulated by sulfotyrosyl peptide signaling. Here, we report that the activity of TYROSYL PROTEIN SULFOTRANSFERASE (TPST), which is responsible for the post-translational modification of sulfotyrosyl peptides, is essential for expansion during development in the non-vascular plant, Physcomitrium patens. Plants that harbor a null mutation in the gene encoding TPST ({Delta}tpst) were smaller, formed fewer caulonemal filaments, and were unable to form expanded gametophores. In {Delta}tpst multiple aspects of gametophore development were affected, including the first division of the gametophore initial, as well as reduced rates of cell division and expansion. Mutational analysis of P. patens TPST identified the residue Histidine 124, a candidate catalytic residue, as essential for TPST function. Notably, addition of the sulfated signaling peptide, PSY1 from either P. patens or Arabidopsis thaliana, rescued all {Delta}tpst developmental deficits. Taken together, these data suggest that TPST functions to sulfate PSY, and this activity is necessary for plant growth and development. Furthermore, since addition of AtPSY fully rescues {Delta}tpst and PpPSY promotes root elongation in Arabidopsis and rice, these findings suggest that PSY signaling is evolutionarily conserved.

developmental biology↗

bioRxiv2022

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. To study the role of this protein in cellular traffic mechanisms in plants, the moss Physcomitrium patens has been selected since it can be studied at the single-cell level. Here, we studied the role of the two moss cornichon homologs in the secretory pathway. Mutant analyzes 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 and trafficking of PINA.

plant biology↗

bioRxiv2021

Cellulose synthase-like D (CSLD) proteins move in the plasma membrane and their targeting to cell tips, but not cell plates, depends on the actin cytoskeleton

Cellulose Synthase-Like D (CSLD) proteins are implicated in cell wall remodeling during tip growth and cell division in plants, and are known to generate {beta}-1,4-glucan. It is unknown whether they form complexes and move in the plasma membrane like members of the Cellulose Synthase (CESA) family. We used the genetically tractable moss Physcomitrium patens, which has a filamentous protonemal stage that undergoes both tip growth and cell division and is amenable to high resolution live cell imaging, to investigate CSLD function and intracellular trafficking. CSLD2 and CSLD6 are highly expressed in gametophores and are redundantly required for gametophore cellular patterning. Live cell imaging revealed that CSLD6 is also expressed in protonemata where it moves in the plasma membrane and localizes to cell plates and cell tips. Notably, delivery to the apical plasma membrane, but not the cell plate, depends on actin. By comparing the behavior of endogenously tagged CSLD6 and CESA10, we discovered that CSLD6 movements in the plasma membrane were significantly faster, shorter in duration and less linear than CESA10 movements and were insensitive to the cellulose synthesis inhibitor isoxaben. These data suggest that CSLD6 and CESA10 function within different structures and may thus produce structurally distinct cellulose microfibrils.

plant biology↗

bioRxiv2020

SABRE populates ER domains essential for cell plate maturation and cell expansion influencing cell and tissue patterning

The SABRE protein, originally identified in plants, is found throughout eukaryotes. In plants, SABRE has been implicated in cell expansion, division plane orientation and planar polarity. However, how SABRE mediates these processes remains an open question. Here, we have taken advantage of the fact that the bryophyte Physcomitrium patens has a single copy of SABRE, is an excellent model for cell biology and is readily amenable to precise genetic alterations to investigate SABREs mechanism of action. We discovered that SABRE null mutants were stunted in both polarized growing and diffusely growing tissues, similar to reported phenotypes in seed plants. However, in polarized growing cells, we observed significant delays in cell plate formation and sometimes catastrophic failures in cell division. We generated a functional SABRE fluorescent fusion protein and determined that it forms dynamic puncta on regions of the endoplasmic reticulum (ER) both in the cytoplasm during interphase and at the new cell plate during division. In the absence of SABRE, ER morphology was severely compromised with large aggregates accumulating in the cytoplasm and abnormal buckling along the developing cell plate late in cytokinesis. In fact, SABRE and the ER maximally accumulated on the developing plate specifically during cell plate maturation, coincident with the timing of the onset of failures in cell plate formation in cells lacking SABRE. Further we discovered that callose deposition is delayed in{Delta} sabre cells, and in cells that failed to divide, abnormal callose accumulations formed at the cell plate. Our findings demonstrated that SABRE functions by influencing the ER and callose deposition, revealing a surprising and essential role for the ER in cell plate maturation. Given that SABRE is conserved, understanding how SABRE influences cell and tissue patterning has profound significance across eukaryotes.

plant biology↗

bioRxiv2020

Regulation of Vacuole Morphology by PIEZO Channels in Spreading Earth Moss

The perception of mechanical force is a fundamental property of most, if not all cells. PIEZO channels are plasma membrane-embedded mechanosensitive calcium channels that play diverse and essential roles in mechanobiological processes in animals1,2. PIEZO channel homologs are found in plants3,4, but their role(s) in the green lineage are almost completely unknown. Plants and animals diverged approximately 1.5 billion years ago, independently evolved multicellularity, and have vastly different cellular mechanics5. Here, we investigate PIEZO channel function in the moss Physcomitrium patens, a representative of one of the first land plant lineages. PpPIEZO1 and PpPIEZO2 were redundantly required for normal growth, size, and shape of tip-growing caulonema cells. Both were localized to vacuolar membranes and facilitated the release of calcium into the cytosol in response to hypoosmotic shock. Loss-of-function ({Delta}Pppiezo1/2) and gain-of-function (PpPIEZO2-R2508K and -R2508H) mutants revealed a role for moss PIEZO homologs in regulating vacuole morphology. Our work here shows that plant and animal PIEZO homologs have diverged in both subcellular localization and in function, likely co-opted to serve different needs in each lineage. The plant homologs of PIEZO channels thus provide a compelling lens through which to study plant mechanobiology and the evolution of mechanoperceptive strategies in multicellular eukaryotes.

plant biology↗

bioRxiv2020

The COPII components Sec23 and Sec24 form isoform specific subcomplexes with Sec23D/E and Sec24C/D essential for tip growth

COPII, a coat of proteins that form vesicles on the ER, mediates vesicle traffic from the ER to the Golgi. In contrast to metazoans that have few genes encoding each COPII component, plants have expanded these gene families leading to the hypothesis that plant COPII has functionally diversified. Here, we analyzed the gene families encoding for the Sec23/24 heterodimer in the moss Physcomitrium (Physcomitrella) patens. In P. patens, Sec23 and Sec24 gene families are each comprised of seven genes. Silencing the Sec23/24 genes revealed isoform specific contributions to polarized growth, with the closely related Sec23D/E and Sec24C/D essential for protonemal development. Focusing on the Sec23 gene family, we discovered that loss of Sec23D alters ER morphology, increases ER stress, inhibits trafficking to the Golgi and to the plasma membrane in tip growing protonemata. In contrast, the remaining five Sec23 genes are dispensable for tip growth. While Sec23A/B/C/F/G do not quantitatively affect ER to Golgi trafficking in protonemata, they do contribute to secretion to the plasma membrane. Of the three highly expressed Sec23 isoforms in protonemata, Sec23G forms ER exit sites that are larger than Sec23B and Sec23D and do not overlap with Sec23D. Furthermore, ER exit sites labeled by Sec23B or Sec23G form in the absence of Sec23D. These data suggest that Sec23D/E form unique ER exit sites contributing to secretion that is essential for tip growing protonemata.

cell biology↗

bioRxiv2019

Efficient and Modular CRISPR-Cas9 Vector System for Physcomitrella patens

CRISPR-Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA-guided nuclease to initiate double-strand breaks. Until recently, classical RAD51-mediated homologous recombination has been a powerful tool for gene targeting in the moss Physcomitrella patens. However, CRISPR-Cas9 mediated genome editing in P. patens was shown to be more efficient than traditional homologous recombination (Collonnier et al. 2017). CRISPR-Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR-Cas9 genome editing in P. patens, here we describe the generation and use of a flexible and modular CRISPR-Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology-directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5 and 3 ends of gene coding regions. With regards to homology-directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a \"stop codon cassette\" with down-stream near-effortless genotyping.

molecular biology↗