The Hmx homeobox gene family appears to play a conserved role in CNS development in all animal species examined, and in higher vertebrates has an additional role in sensory organ development. Here, we show that murine Hmx2 and Hmx3 have both overlapping and distinct functions in the development of the inner ear's vestibular system, whereas their functions in the hypothalamic/pituitary axis of the CNS appear to be interchangeable. As in analogous knockin studies of Otx and En function, Drosophila Hmx can rescue conserved functions in the murine CNS. However, in contrast to Otx and En, Drosophila Hmx also rescues significant vertebrate-specific functions outside the CNS. Our work suggests that the evolution of the vertebrate inner ear may have involved (1) the redeployment of ancient Hmx activities to regulate the cell proliferation of structural components and (2) the acquisition of additional, vertebrate-specific Hmx activities to regulate the sensory epithelia.

Calcium acts as a second messenger for signaling to a variety of stimuli including MAMPs (Microbe-Associated Molecular Patterns), such as flg22 and elf18 that are derived from bacterial flagellin and elongation factor Tu, respectively. Here, Arabidopsis thaliana mutants with changed calcium elevation (cce) in response to flg22 treatment were isolated and characterized. Besides novel mutant alleles of the flg22 receptor, FLS2 (Flagellin-Sensitive 2), and the receptor-associated kinase, BAK1 (Brassinosteroid receptor 1-Associated Kinase 1), the new cce mutants can be categorized into two main groups-those with a reduced or an enhanced calcium elevation. Moreover, cce mutants from both groups show differential phenotypes to different sets of MAMPs. Thus, these mutants will facilitate the discovery of novel components in early MAMP signaling and bridge the gaps in current knowledge of calcium signaling during plant-microbe interactions. Last but not least, the screening method is optimized for speed (covering 384 plants in 3 or 10 h) and can be adapted to genetically dissect any other stimuli that induce a change in calcium levels.

The proteasome is an essential protein-degradation machinery in eukaryotic cells that controls protein turnover and thereby the biogenesis and function of cell organelles. Chloroplasts import thousands of nuclear-encoded precursor proteins from the cytosol, suggesting that the bulk of plastid proteins is transiently exposed to the cytosolic proteasome complex. Therefore, there is a cytosolic equilibrium between chloroplast precursor protein import and proteasomal degradation. We show here that a shift in this equilibrium, induced by mild genetic proteasome impairment, results in elevated precursor protein abundance in the cytosol and significantly increased accumulation of functional photosynthetic complexes in protein import-deficient chloroplasts. Importantly, a proteasome lid mutant shows improved photosynthetic performance, even in the absence of an import defect, signifying that functional precursors are continuously degraded. Hence, turnover of plastid precursors in the cytosol represents a mechanism to constrain thylakoid membrane assembly and photosynthetic electron transport.

BackgroundCalcium, as a second messenger, transduces extracellular signals into cellular reactions. A rise in cytosolic calcium concentration is one of the first plant responses after exposure to microbe-associated molecular patterns (MAMPs). We reported previously the isolation of Arabidopsis thaliana mutants with a “changed calcium elevation” (cce) response to flg22, a 22-amino-acid MAMP derived from bacterial flagellin.ResultsHere, we characterized the cce2 mutant and its weaker allelic mutant, cce3. Besides flg22, the mutants respond with a reduced calcium elevation to several other MAMPs and a plant endogenous peptide that is proteolytically processed from pre-pro-proteins during wounding. Downstream defense-related events such flg22-induced mitogen-activated protein kinase activation, accumulation of reactive oxygen species and growth arrest are also attenuated in cce2/cce3. By genetic mapping, next-generation sequencing and allelism assay, CCE2/CCE3 was identified to be ALG3 (Asparagine-linked glycosylation 3). This encodes the α-1,3-mannosyltransferase responsible for the first step of core oligosaccharide Glc3Man9GlcNAc2 glycan assembly on the endoplasmic reticulum (ER) luminal side. Complementation assays and glycan analysis in yeast alg3 mutant confirmed the reduced enzymatic function of the proteins encoded by the cce2/cce3 alleles – leading to accumulation of M5ER, the immature five mannose-containing oligosaccharide structure found in the ER. Proper protein glycosylation is required for ER/Golgi processing and trafficking of membrane proteins to the plasma membrane. Endoglycosidase H-insensitivity of flg22 receptor, FLS2, in the cce2/cce3 mutants suggests altered glycan structures in the receptor.ConclusionProper glycosylation of MAMP receptors (or other exported proteins) is required for optimal responses to MAMPs and is important for immune signaling of host plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0718-3) contains supplementary material, which is available to authorized users.

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The precursors of RNP29 and Ferredoxin (Fd2) were previously identified in the cytosol of ppi2 plant cells with their N-terminal amino acid acetylated. Here, we explore whether precursor accumulation in ppi2 is characteristic for Toc159 client proteins, by characterizing the import properties of the RNP29 precursor in comparison to Fd2 and other Toc159-dependent or independent substrates. We find specific accumulation of the RNP29 precursor in ppi2 but not in wild type or ppi1 protoplasts. With the exception of Lhcb4, precursor accumulation is also detected with all other tested constructs in ppi2. However, RNP29 is clearly different from the other proteins because only precursor but almost no mature protein is detectable in protoplast extracts. Co-transformation of RNP29 with Toc159 complements its plastid import, supporting the hypothesis that RNP29 is a Toc159-dependent substrate. Exchange of the second amino acid in the RNP29 transit peptide to Glu or Asn prevents methionine excision but not N-terminal acetylation, suggesting that different N-acetyltransferases may act on chloroplast precursor proteins in vivo. All different RNP29 constructs are efficiently imported into wild type but not into ppi2 plastids, arguing for a minor impact of the N-terminal amino acid on the import process.

Chloroplast function requires the coordinated action of nuclear- and chloroplast-derived proteins, including several hundred nuclear-encoded pentatricopeptide repeat (PPR) proteins that regulate plastid mRNA metabolism. Despite their large number and importance, regulatory mechanisms controlling PPR expression are poorly understood. Here we show that the Arabidopsis NOT4A ubiquitin-ligase positively regulates the expression of PROTON GRADIENT REGULATION 3 (PGR3), a PPR protein required for translating several thylakoid-localised photosynthetic components and ribosome subunits within chloroplasts. Loss of NOT4A function leads to a strong depletion of cytochrome b6f and NAD(P)H dehydrogenase (NDH) complexes, as well as plastid 30 S ribosomes, which reduces mRNA translation and photosynthetic capacity, causing pale-yellow and slow-growth phenotypes. Quantitative transcriptome and proteome analysis of the not4a mutant reveal it lacks PGR3 expression, and that its molecular defects resemble those of a pgr3 mutant. Furthermore, we show that normal plastid function is restored to not4a through transgenic PGR3 expression. Our work identifies NOT4A as crucial for ensuring robust photosynthetic function during development and stress-response, through promoting PGR3 production and chloroplast translation.

31Chloroplast function requires the coordinated action of nuclear-and chloroplast-derived 32 proteins, including several hundred nuclear-encoded pentatricopeptide repeat (PPR) 33 proteins that regulate plastid mRNA metabolism. Despite their large number and importance, 34 regulatory mechanisms controlling PPR expression are poorly understood. Here we show 35 that the Arabidopsis NOT4A ubiquitin-ligase positively regulates PROTON GRADIENT 3 36 (PGR3), a PPR protein required for translating 30S ribosome subunits and several thylakoid-37 localised photosynthetic components within chloroplasts. Loss of NOT4A function leads to a 38 strong depletion of plastid ribosomes, which reduces mRNA translation and negatively 39 impacts photosynthetic capacity, causing pale-yellow and slow-growth phenotypes. 40Quantitative transcriptome and proteome analyses reveal that these defects are due to a 41 lack of PGR3 expression in not4a, and we show that normal plastid function is restored 42 through transgenic PGR3 expression. Our work identifies NOT4A as crucial for ensuring 43 robust photosynthetic function during development and stress-response, through modulating 44 PGR3 levels to coordinate chloroplast protein synthesis.The synthesis of energy from the sun, photosynthesis, supports organic life on earth. Light 68 harvesting in green plants takes place within the specialized chloroplast organelle, believed 69 to have arisen from engulfment of a photosynthetic prokaryote by an ancestral eukaryotic 70 cell (Archibald, 2015). Coevolution and merging of these organisms has resulted in nuclear 71 and chloroplast genomes separated within cellular compartments. In land plants, the 72 chloroplast genome comprises of ~130 genes, yet chloroplasts contain around 3000 different 73 proteins (Zoschke and Bock, 2018). Consequently, chloroplast function requires expression 74 not only of chloroplast encoded proteins, but a multitude of nuclear encoded genes, which 75 are imported into chloroplasts post-translationally. One such group of nuclear derived factors 76 is the pentatricopeptide repeat domain (PPR) containing proteins. The PPR protein family 77 has significantly expanded in plants (~450 in Arabidopsis, vs <10 in humans and yeast; 78 (Schmitz-Linneweber and Small, 2008)), and members are characterized by a 35-amino acid 79 repeat sequence that facilitates RNA binding and enables them to provide critical gene 80 expression control within chloroplasts and mitochondria (Barkan and Small, 2014). Through 81 binding to organellar RNAs, PPR proteins stabilize gene transcripts, facilitate post-82 transcriptional processing and promote translation of the encoded proteins (Barkan and 83 Small, 2014; Manna, 2015). Whilst their function in the regulation of gene expression control 84 within organelles has been described, including many of the RNA species to which they 85 bind, little is known about how their expression is regulated prior to import. 86Precise, selective removal of proteins is essential to cellular development and response. In 87...