A molecular network of conserved factors keeps ribosomes dormant in the egg.

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1-4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.

eIF4E1b is a non-canonical eIF4E protecting maternal dormant mRNAs.

Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA tails are stable. Here we report that the germline-specific eIF4E paralog, eIF4E1b, is essential for zebrafish oogenesis. eIF4E1b localizes to P-bodies in zebrafish embryos and binds to mRNAs with reported short or no polyA tails, including histone mRNAs. Loss of eIF4E1b results in reduced histone mRNA levels in early gonads, consistent with a role in mRNA storage. Using mouse and human eIF4E1Bs (in vitro) and zebrafish eIF4E1b (in vivo), we show that unlike canonical eIF4Es, eIF4E1b does not interact with eIF4G to initiate translation. Instead, eIF4E1b interacts with the translational repressor eIF4ENIF1, which is required for eIF4E1b localization to P-bodies. Our study is consistent with an important role of eIF4E1b in regulating mRNA dormancy and provides new insights into fundamental post-transcriptional regulatory principles governing early vertebrate development.

The molecular mechanisms underpinning maternal mRNA dormancy.

A large number of mRNAs of maternal origin are produced during oogenesis and deposited in the oocyte. Since transcription stops at the onset of meiosis during oogenesis and does not resume until later in embryogenesis, maternal mRNAs are the only templates for protein synthesis during this period. To ensure that a protein is made in the right place at the right time, the translation of maternal mRNAs must be activated at a specific stage of development. Here we summarize our current understanding of the sophisticated mechanisms that contribute to the temporal repression of maternal mRNAs, termed maternal mRNA dormancy. We discuss mechanisms at the level of the RNA itself, such as the regulation of polyadenine tail length and RNA modifications, as well as at the level of RNA-binding proteins, which often block the assembly of translation initiation complexes at the 5' end of an mRNA or recruit mRNAs to specific subcellular compartments. We also review microRNAs and other mechanisms that contribute to repressing translation, such as ribosome dormancy. Importantly, the mechanisms responsible for mRNA dormancy during the oocyte-to-embryo transition are also relevant to cellular quiescence in other biological contexts.

Constitutive Activation of Leucine-Rich Repeat Receptor Kinase Signaling Pathways by BAK1-INTERACTING RECEPTOR-LIKE KINASE3 Chimera.

Receptor kinases with extracellular leucine-rich repeat domains (LRR-RKs) form the largest group of membrane signaling proteins in plants. LRR-RKs can sense small molecule, peptide, or protein ligands and may be activated by ligand-induced interaction with a shape complementary SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptor kinase. We have previously shown that SERKs can also form constitutive, ligand-independent complexes with the LRR ectodomains of BAK1-INTERACTING RECEPTOR-LIKE KINASE3 (BIR3) receptor pseudokinases, negative regulators of LRR-RK signaling. Here, we report that receptor chimera in which the extracellular LRR domain of BIR3 is fused to the cytoplasmic kinase domains of the SERK-dependent LRR-RKs BRASSINOSTEROID INSENSITIVE1, HAESA and ERECTA form tight complexes with endogenous SERK coreceptors in the absence of ligand stimulus. Expression of these chimeras under the control of the endogenous promoter of the respective LRR-RK leads to strong gain-of-function brassinosteroid, floral abscission, and stomatal patterning phenotypes, respectively. Importantly, a BIR3-GASSHO1 (GSO1)/SCHENGEN3 (SGN3) chimera can partially complement sgn3 Casparian strip formation phenotypes, suggesting that SERK proteins also mediate GSO1/SGN3 receptor activation. Collectively, our protein engineering approach may be used to elucidate the physiological functions of orphan LRR-RKs and to identify their receptor activation mechanism in single transgenic lines.

A genetically validated approach for detecting inorganic polyphosphates in plants.

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. Polyphosphates represent important stores of phosphate and energy, and are abundant in many pro- and eukaryotic organisms. In plants, the existence of polyPs has been established using microscopy and biochemical extraction methods that are now known to produce artifacts. Here we use a polyP-specific dye and a polyP-binding domain to detect polyPs in plant and algal cells. To develop the staining protocol, we induced polyP granules in Nicotiana benthamiana and Arabidopsis cells by heterologous expression of Escherichia coli polyphosphate kinase 1 (PPK1). Over-expression of PPK1 but not of a catalytically impaired version of the enzyme leads to severe growth phenotypes, suggesting that ATP-dependent synthesis and accumulation of polyPs in the plant cytosol is toxic. We next crossed stable PPK1-expressing Arabidopsis lines with plants expressing the polyP-binding domain of E. coli exopolyphosphatase (PPX1c), which co-localized with PPK1-generated polyP granules. These granules were stained by the polyP-specific dye JC-D7 and appeared as electron-dense structures in transmission electron microscopy sections. Using the polyP staining protocol derived from these experiments, we screened for polyP stores in different organs and tissues of both mono- and dicotyledonous plants. While we could not detect polyP granules in higher plants, we could visualize the polyP-rich acidocalcisomes in the green alga Chlamydomonas reinhardtii.

Identity and functions of inorganic and inositol polyphosphates in plants.

Inorganic polyphosphates (polyPs) and inositol pyrophosphates (PP-InsPs) form important stores of inorganic phosphate and can act as energy metabolites and signaling molecules. Here we review our current understanding of polyP and inositol phosphate (InsP) metabolism and physiology in plants. We outline methods for polyP and InsP detection, discuss the known plant enzymes involved in their synthesis and breakdown, and summarize the potential physiological and signaling functions for these enigmatic molecules in plants.

DRACULA2 is a dynamic nucleoporin with a role in regulating the shade avoidance syndrome in Arabidopsis.

When plants grow in close proximity basic resources such as light can become limiting. Under such conditions plants respond to anticipate and/or adapt to the light shortage, a process known as the shade avoidance syndrome (SAS). Following genetic screening using a shade-responsive luciferase reporter line (PHYB:LUC), we identified DRACULA2 (DRA2), which encodes an Arabidopsis homolog of mammalian nucleoporin 98, a component of the nuclear pore complex (NPC). DRA2, together with other nucleoporins, participates positively in the control of the hypocotyl elongation response to plant proximity, a role that can be considered dependent on the nucleocytoplasmic transport of macromolecules (i.e. is transport dependent). In addition, our results reveal a specific role for DRA2 in controlling shade-induced gene expression. We suggest that this novel regulatory role of DRA2 is transport independent and that it might rely on its dynamic localization within and outside of the NPC. These results provide mechanistic insights in to how SAS responses are rapidly established by light conditions. They also indicate that nucleoporins have an active role in plant signaling.

The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling.

Membrane-delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a step that affects receptor function and downstream signaling. In this study we describe a single-subunit RING-type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C-terminal transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent complementation (BiFC) studies showed the RSL1-PYL4 and RSL1-PYR1 interaction is localized to plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma membrane is a process that might affect their function via effect on their half-life, protein interactions or trafficking.

Tomato PYR/PYL/RCAR abscisic acid receptors show high expression in root, differential sensitivity to the abscisic acid agonist quinabactin, and the capability to enhance plant drought resistance.

Abscisic acid (ABA) plays a crucial role in the plant's response to both biotic and abiotic stress. Sustainable production of food faces several key challenges, particularly the generation of new varieties with improved water use efficiency and drought tolerance. Different studies have shown the potential applications of Arabidopsis PYR/PYL/RCAR ABA receptors to enhance plant drought resistance. Consequently the functional characterization of orthologous genes in crops holds promise for agriculture. The full set of tomato (Solanum lycopersicum) PYR/PYL/RCAR ABA receptors have been identified here. From the 15 putative tomato ABA receptors, 14 of them could be grouped in three subfamilies that correlated well with corresponding Arabidopsis subfamilies. High levels of expression of PYR/PYL/RCAR genes was found in tomato root, and some genes showed predominant expression in leaf and fruit tissues. Functional characterization of tomato receptors was performed through interaction assays with Arabidopsis and tomato clade A protein phosphatase type 2Cs (PP2Cs) as well as phosphatase inhibition studies. Tomato receptors were able to inhibit the activity of clade A PP2Cs differentially in an ABA-dependent manner, and at least three receptors were sensitive to the ABA agonist quinabactin, which inhibited tomato seed germination. Indeed, the chemical activation of ABA signalling induced by quinabactin was able to activate stress-responsive genes. Both dimeric and monomeric tomato receptors were functional in Arabidopsis plant cells, but only overexpression of monomeric-type receptors conferred enhanced drought resistance. In summary, gene expression analyses, and chemical and transgenic approaches revealed distinct properties of tomato PYR/PYL/RCAR ABA receptors that might have biotechnological implications.

Molecular characterization of CHAD domains as inorganic polyphosphate-binding modules.

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. Here, we report that bacterial, archaeal, and eukaryotic conserved histidine α-helical (CHAD) domains are specific polyP-binding modules. Crystal structures reveal that CHAD domains are formed by two four-helix bundles, giving rise to a central pore surrounded by conserved basic surface patches. Different CHAD domains bind polyPs with dissociation constants ranging from the nano- to mid-micromolar range, but not nucleic acids. A CHAD-polyP complex structure reveals the phosphate polymer binding across the central pore and along the two basic patches. Mutational analysis of CHAD-polyP interface residues validates the complex structure. The presence of a CHAD domain in the polyPase ygiF enhances its enzymatic activity. The only known CHAD protein from the plant Ricinus communis localizes to the nucleus/nucleolus when expressed in Arabidopsis and tobacco, suggesting that plants may harbor polyPs in these compartments. We propose that CHAD domains may be used to engineer the properties of polyP-metabolizing enzymes and to specifically localize polyP stores in eukaryotic cells and tissues.

Concerted expression of a cell cycle regulator and a metabolic enzyme from a bicistronic transcript in plants.

Eukaryotic mRNAs frequently contain upstream open reading frames (uORFs), encoding small peptides that may control translation of the main ORF (mORF). Here, we report the characterization of a distinct bicistronic transcript in Arabidopsis. We analysed loss-of-function phenotypes of the inorganic polyphosphatase TRIPHOSPHATE TUNNEL METALLOENZYME 3 (AtTTM3), and found that catalytically inactive versions of the enzyme could fully complement embryo and growth-related phenotypes. We could rationalize these puzzling findings by characterizing a uORF in the AtTTM3 locus encoding CELL DIVISION CYCLE PROTEIN 26 (CDC26), an orthologue of the cell cycle regulator. We demonstrate that AtCDC26 is part of the plant anaphase promoting complex/cyclosome (APC/C), regulates accumulation of APC/C target proteins and controls cell division, growth and embryo development. AtCDC26 and AtTTM3 are translated from a single transcript conserved across the plant lineage. While there is no apparent biochemical connection between the two gene products, AtTTM3 coordinates AtCDC26 translation by recruiting the transcript into polysomes. Our work highlights that uORFs may encode functional proteins in plant genomes.