More than 95% of fertilized Drosophila oocytes from outbred stocks develop fully regardless of maternal age, in contrast to human oocytes, which frequently generate non-viable aneuploid embryos. Since Drosophila oocytes are normally stored only briefly prior to ovulation, unlike their human counterparts, we investigated the effects of storage on oocyte quality. Using a novel system to acquire oocytes held for known periods, we analyzed by ribosome profiling how translation and cellular function change over time. Oocyte developmental capacity decays in a precise temperature-dependent manner over 1-4 weeks, due to a progressive inability to complete meiosis. Meiotic metaphase genes, the Fmr1 translational regulator, and the small heat shock protein chaperones Hsp26 and Hsp27 are preferentially translated during storage, and oocytes lacking Hsp26 and Hsp27 age prematurely. However translation falls generally 2.3-fold with age despite constant mRNA levels, and this inability to maintain translational equilibrium correlates with oocyte functional decline. These findings show that meiotic chromosome segregation in Drosophila oocytes is uniquely sensitive to prolonged quiescence, and suggest that the extended storage of mature human oocytes contributes to their chromosome instability. If so, then these problems may be more amenable to intervention than previously supposed.

Ovarian murine somatic cells are essential to form first wave medullar follicles and second wave primordial follicles. Using single cell RNA sequencing we characterized the transcriptomes of both somatic and germline ovarian cells during fetal and early neonatal development. Wnt4-expressing somatic cells we term "escort-like cells (ELCs)" interact with incoming germ cells and early developing cysts of both sexes. In the medullar region, ELCs differentiate into the granulosa cells of fast-growing first wave follicles. In contrast, after E12.5, Lgr5+ pre-granulosa cells ingress from the ovarian surface epithelium and replace cortical escort-like cells. These surface-derived cells become the main population of granulosa cells supporting primordial follicles, and differ in transcription from ELC derivatives. Reflecting their different cellular origins, ablation of Lgr5+ cells at E16.5 using Lgr5-DTR-EGFP eliminates second wave follicles, but first wave follicles continue to develop normally and support fertility. Our findings provide striking evidence that somatic cell behavior supporting germline cyst development in mice and Drosophila has been evolutionarily conserved.

Mutations in Fmr1, encoding the RNA binding protein FMRP, are leading causes of intellectual disability, autism, and female infertility, but FMRP’s mechanism of action is controversial. In contrast to its previously postulated function as a translation repressor acting by stalling elongation, we recently found that FMRP activates translation initiation of large proteins in Drosophila oocytes up to ~2-fold. We report here that FMRP’s function as a translational activator is conserved in the mammalian brain. Reanalysis of mouse cortex ribosome profiling data shows that translation of large proteins in Fmr1 mutants is down-regulated 2.0-1.2-fold; ribosome stalling appears not to influence FMRP target protein translation in either cortex or oocyte tissue. Consistent with an activator function, most FMRP targets are associated with clinical syndromes when reduced, but not when over-expressed. Fmr1-dependent translation of one target, the N-end rule E3 ligase Poe/UBR4, occurs in microscopically visible ribonucleoprotein particles. These "Poe particles" require FMRP for their formation, are distinct from P bodies, and depend on actively elongating ribosomes, as indicated by their dissolution following a brief puromycin treatment. N-end rule-mediated proteolysis via Poe/UBR4 restrains cell growth and limits MAPK signaling in nervous tissue. Thus, loss of FMRP reduces production of an important growth repressor.

FMR1 enhances translation of large neural/oocyte proteinsMutations in the highly conserved Fragile X mental retardation gene (Fmr1) cause the most common inherited human intellectual disability/autism spectrum disorder. Fmr1 is also needed for ovarian follicle development, and lesions are the largest genetic cause of premature ovarian failure (POF). FMR1 associates with ribosomes and is thought to repress translation, but identifying functional targets has been difficult. We analyzed FMR1s role in quiescent Drosophila oocytes stored prior to ovulation, cells that depend entirely on translation of stored mRNA. Ribosome profiling revealed that in quiescent oocytes FMR1 stimulates the translation of large proteins, including at least twelve proteins whose human homologs are associated with dominant intellectual disability disorders, and 25 others associated with neural dysfunction. Knockdown of Fmr1 in unstored oocytes did not affect embryo development, but more than 50% of embryos derived from stored oocytes lacking FMR1 developed severe neural defects. Fmr1s previously unappreciated role promoting the translation of large proteins from stored mRNAs in oocytes and neurons may underlie POF as well as multiple aspects of neural dysfunction.

Accurate relative quantification is critical in proteomic studies. The incorporation of stable isotope 15N to plant-expressed proteins in vivo is a powerful tool for accurate quantification with a major advantage of reducing preparative and analytical variabilities. However, 15N labeling quantification has several challenges. Less identifications are often observed in the heavy labeled samples because of incomplete labeling, resulting in missing values in reciprocal labeling experiments. Inaccurate quantification can happen when there is contamination from co-eluting peptides or chemical noise in the MS1 survey scan. These drawbacks in quantification can be more pronounced in less abundant but biologically interesting proteins, which often have very few identified peptides. Here we demonstrate the application of parallel reaction monitoring (PRM) to 15N labeled samples on a high resolution, high mass accuracy Orbitrap mass spectrometer to achieve reliable quantification even of low abundance proteins in samples.

Stomata, cellular valves found on the surface of aerial plant tissues, present a paradigm for studying cell fate and patterning in plants. A highly conserved core set of related basic helix-loop-helix (bHLH) transcription factors (TFs) regulate aspects of stomatal cell identity in diverse species. We characterized BdFAMA in the temperate grass, Brachypodium distachyon, and found this late-acting TF was necessary and sufficient for specifying stomatal GC fate, and unexpectedly could also induce the recruitment of subsidiary cells in the absence of its paralogue, BdMUTE. The overlap in function is paralleled by an overlap in expression pattern and by unique regulatory relationships between BdMUTE and BdFAMA. To better appreciate the relationships among the Brachypodium stomatal bHLHs, we characterized the diversity of bHLH complexes through in vivo proteomics in developing leaves and found evidence for multiple shared interaction partners. The ability of BdFAMA to compensate for BdMUTE then prompted us to reexamine the roles of these paralogues in Arabidopsis. By testing genetic sufficiency within and across species, we found that, while BdFAMA and AtFAMA can rescue stomatal production in Arabidopsis fama and mute mutants, only AtFAMA can participate in the specification of Brassica-family-specific myrosin idioblasts. Taken together, our findings further our understanding of how the molecular framework of stomatal development has been reprogrammed across the monocot/dicot divide, as well as within the grasses, to produce distinct stomatal forms and patterns, and compel us to refine the current models of stomatal bHLH function and regulatory feedbacks amongst paralogues.

Metabolic labeling using stable isotopes is widely used for the relative quantification of proteins in proteomic studies. In plants, metabolic labeling using 15N has great potential, but the associated complexity of data analysis has limited its usage. Here, we present the 15N stable-isotope labeled protein quantification workflow utilizing open-access web-based software Protein Prospector. Further, we discuss several important features of 15N labeling required to make reliable and precise protein quantification. These features include ratio adjustment based on labeling efficiency, median and interquartile range for protein ratios, isotope cluster pattern matching to flag incorrect monoisotopic peak assignment, and caching of quantification results for fast retrieval.

How the membrane trafficking system spatially organizes intracellular activities and intercellular signaling networks is not well understood in plants. The Transport Protein Particle (TRAPP) complexes are known to play key roles in selective delivery of membrane vesicles to various subcellular compartments in yeast and animals, but remain to be fully characterized in plants. Here we interrogate the TRAPP complexes in Arabidopsis using quantitative proteomic approaches. TRS33 is a component shared by all TRAPP complexes in yeast and animals, and the Arabidopsis AtTRS33 is essential for the subcellular dynamics of other TRAPP components. Affinity purification of AtTRS33 followed by quantitative mass spectrometry identified fourteen interacting proteins; these include not only thirteen homologs of all known TRAPP components in yeast and mammals but also a novel protein we named TRAPP-interacting plant protein (TRIPP), which is conserved in multi-cellular photosynthetic organisms. Proteomic and molecular analyses showed that TRIPP specifically associates with the TRAPPII complex in vivo and directly interacts with the TRAPPII-specific subunits but not the subunits shared with TRAPPIII. TRIPP co-localizes with a subset of TRS33 compartments, and its localization is disrupted in the trs33 mutant. Loss-of-function tripp mutation caused growth and reproductive development defects, including partial photomorphogenesis in the dark. Our study demonstrates that plants possess at least two distinct TRAPP complexes similar to metazoan, and identifies TRIPP as a novel plant-specific component of the TRAPPII complex with important functions in plant growth and development.

Hundreds of leucine-rich repeat receptor kinases (LRR-RKs) have evolved to control diverse processes of growth, development, and immunity in plants; the mechanisms that link LRRRKs to distinct cellular responses are not understood. Here we show that two LRR-RKs, the brassinosteroid hormone receptor BRI1 (BRASSINOSTEROID INSENSITIVE 1) and the flagellin receptor FLS2 (FLAGELLIN SENSING 2), regulate downstream glycogen synthase kinase 3 (GSK3) and mitogen-activated protein (MAP) kinases, respectively, through phosphocoding of the BRI1-SUPPRESSOR1 (BSU1) phosphatase. BSU1 was previously identified as a component that inactivates GSK3s in the BRI1 pathway. We found surprisingly that loss of the BSU1 family phosphatases activates effector-triggered immunity (ETI) and impairs flagellin-triggered MAP kinase activation and immunity. The flagellinactivated BOTRYTIS-INDUCED KINASE 1 (BIK1) phosphorylates BSU1 at serine-251. Mutation of serine-251 reduces the ability of BSU1 to mediate flagellin-induced MAP kinase activation and immunity, but not its abilities to suppress ETI and interact with GSK3, which is enhanced through the phosphorylation of BSU1 at serine-764 upon brassinosteroid signaling. These results demonstrate that BSU1 plays an essential role in immunity and transduces brassinosteroid-BRI1 and flagellin-FLS2 signals using different phosphorylation sites. Our study illustrates that phosphocoding in shared downstream components provides signaling specificities for diverse plant receptor kinases.

Transient protein-protein interactions (PPIs), such as those between posttranslational modifying enzymes and their substrates, play key roles in cellular regulation, but are difficult to identify. Here we demonstrate the application of enzyme-catalyzed proximity labeling (PL), using the engineered promiscuous biotin ligase TurboID, as a sensitive method for characterizing PPIs in signaling networks. We show that TurboID fused with the GSK3-like kinase BIN2 or a PP2A phosphatase biotinylates their known substrate, the BZR1 transcription factor, with high specificity and efficiency. We optimized the protocol of biotin labeling and affinity purification in transgenic Arabidopsis expressing a BIN2-TurboID fusion protein. Subsequent quantitative mass spectrometry (MS) analysis identified about three hundred proteins biotinylated by BIN2-TurboID more efficiently than the YFP-TurboID control. These include a significant subset of previously proven BIN2 interactors and a large number of new BIN2-proximal proteins that uncover a broad BIN2 signaling network. Our study illustrates that PL-MS using TurboID is a powerful tool for mapping signaling networks, and reveals broad roles of BIN2 kinase in cellular signaling and regulation in plants.Impact StatementTurboID-mediated proximity labeling is a powerful tool for protein interactomics in plants.