Mutation of the ALBOSTRIANS Ohnologous Gene HvCMF3 Impairs Chloroplast Development and Thylakoid Architecture in Barley

Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained if at least one member of the pair undergoes neo- or sub-functionalization. Phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed them as members of a subfamily of genes coding for CCT motif (CONSTANS, CONSTANS-LIKE and TIMING OF CAB1) proteins characterized by a single CCT domain and a putative N-terminal chloroplast transit peptide. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a yellowish/light green – xantha – phenotype and successively develop pale green leaves. Compared to wild type, plastids of mutant seedlings show a decreased PSII efficiency, impaired processing and reduced amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional domain, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs were localized to plastids and nucleus. Hvcmf3Hvcmf7 double mutants exhibited a xantha-albino or albino phenotype depending on the strength of molecular lesion of the HvCMF7 allele. The chloroplast ribosome deficiency is discussed as the primary observed defect of the Hvcmf3 mutants. Based on our observations, the genes HvCMF3 and HvCMF7 have similar but not identical functions in chloroplast development of barley supporting our hypothesis of neo-/sub-functionalization between both ohnologous genes.

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The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs Involved in Chloroplast Development

Frontiers in Plant Science ◽

10.3389/fpls.2021.681375 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mingjiu Li ◽

Hannes Ruwe ◽

Michael Melzer ◽

Astrid Junker ◽

Goetz Hensel ◽

...

Keyword(s):

Nuclear Localization ◽

Ribosomal Proteins ◽

Protein Import ◽

Chloroplast Development ◽

Transit Peptide ◽

Growth Conditions ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Chloroplast Transit Peptide ◽

Pale Green

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7, or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL, and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.

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The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs involved in Chloroplast Development

10.1101/2021.02.02.429330 ◽

2021 ◽

Author(s):

Mingjiu Li ◽

Hannes Ruwe ◽

Michael Melzer ◽

Astrid Junker ◽

Götz Hensel ◽

...

Keyword(s):

Nuclear Localization ◽

Ribosomal Proteins ◽

Protein Import ◽

Chloroplast Development ◽

Transit Peptide ◽

Growth Conditions ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Chloroplast Transit Peptide ◽

Pale Green

ABSTRACTThe Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7 or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.HIGHLIGHTThe nucleus-localized CCT domain proteins CIA2 and CIL in Arabidopsis and the homologous chloroplast-localized HvCMF3 and HvCMF7 in barley retained partially overlapping functions in chloroplast development.

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Mutation of the ALBOSTRIANS Ohnologous Gene HvCMF3 Impairs Chloroplast Development and Thylakoid Architecture in Barley due to Reduced Plastid Translation

10.1101/756833 ◽

2019 ◽

Author(s):

Mingjiu Li ◽

Goetz Hensel ◽

Michael Melzer ◽

Astrid Junker ◽

Henning Tschiersch ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Chloroplast Development ◽

Higher Plants ◽

Phylogenetic Analyses ◽

Site Directed Mutagenesis ◽

Primary Defect ◽

Functional Region ◽

Transit Peptides ◽

Genes Encoding ◽

Barley Protein

ABSTRACTGene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained only if at least one gene of the pair undergoes neo- or subfunctionalization. Sequence-based phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed that they belong to a newly identified subfamily of genes encoding CCT domain proteins with putative N-terminal chloroplast transit peptides. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a xantha phenotype and successively develop pale green leaves. Compared to the wild type, plastids of the mutant seedlings show decreased PSII efficiency and lower amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional region, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs localized to plastids. Hvcmf3Hvcmf7 double mutants indicated epistatic activity of HvCMF7 over HvCMF3. The chloroplast ribosome deficiency is discussed as the primary defect of the Hvcmf3 mutants. Our data suggests that HvCMF3 and HvCMF7 have similar but not identical functions.One-sentence summaryPhylogenetic and mutant analyses of the barley protein HvCMF3 (ALBOSTRIANS-LIKE) identified, in higher plants, a subfamily of CCT domain proteins with essential function in chloroplast development.

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Chaperone-like protein DAY plays critical roles in photomorphogenesis

Nature Communications ◽

10.1038/s41467-021-24446-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ho-Seok Lee ◽

Ilyeong Choi ◽

Young Jeon ◽

Hee-Kyung Ahn ◽

Huikyong Cho ◽

...

Keyword(s):

Chloroplast Development ◽

Transmembrane Domain ◽

Essential Feature ◽

Chlorophyll Biosynthesis ◽

Transit Peptide ◽

Terrestrial Plants ◽

Chloroplast Transit Peptide ◽

Cell Compartments ◽

Key Enzyme ◽

Chlorophyll Biogenesis

AbstractPhotomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARKANDYELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.

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Identification of Susceptibility Genes in Castanea sativa and Their Transcription Dynamics following Pathogen Infection

Plants ◽

10.3390/plants10050913 ◽

2021 ◽

Vol 10 (5) ◽

pp. 913

Author(s):

Vera Pavese ◽

Andrea Moglia ◽

Paolo Gonthier ◽

Daniela Torello Marinoni ◽

Emile Cavalet-Giorsa ◽

...

Keyword(s):

Phylogenetic Analyses ◽

Castanea Sativa ◽

Transcript Level ◽

Cryphonectria Parasitica ◽

Plant Responses ◽

Functional Domain ◽

Loss Of Function ◽

Pathogen Infection ◽

Domain Identification ◽

S Genes

Castanea sativa is one of the main multipurpose tree species valued for its timber and nuts. This species is susceptible to two major diseases, ink disease and chestnut blight, caused by Phytophthora spp. and Cryphonectria parasitica, respectively. The loss-of-function mutations of genes required for the onset of pathogenesis, referred to as plant susceptibility (S) genes, are one mechanism of plant resistance against pathogens. On the basis of sequence homology, functional domain identification, and phylogenetic analyses, we report for the first time on the identification of S-genes (mlo1, dmr6, dnd1, and pmr4) in the Castanea genus. The expression dynamics of S-genes were assessed in C. sativa and C. crenata plants inoculated with P. cinnamomi and C. parasitica. Our results highlighted the upregulation of pmr4 and dmr6 in response to pathogen infection. Pmr4 was strongly expressed at early infection phases of both pathogens in C. sativa, whereas in C. crenata, no significant upregulation was observed. The infection of P. cinnamomi led to a higher increase in the transcript level of dmr6 in C. sativa compared to C. crenata-infected samples. For a better understanding of plant responses, the transcript levels of defense genes gluB and chi3 were also analyzed.

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Programmed chloroplast destruction during leaf senescence involves 13-lipoxygenase (13-LOX)

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525747113 ◽

2016 ◽

Vol 113 (12) ◽

pp. 3383-3388 ◽

Cited By ~ 15

Author(s):

Armin Springer ◽

ChulHee Kang ◽

Sachin Rustgi ◽

Diter von Wettstein ◽

Christiane Reinbothe ◽

...

Keyword(s):

Leaf Senescence ◽

Critical Role ◽

Transit Peptide ◽

Physiological Changes ◽

Pathogen Defense ◽

Chloroplast Transit Peptide ◽

Selective Destruction ◽

Plastid Envelope ◽

Volatile Aldehydes

Leaf senescence is the terminal stage in the development of perennial plants. Massive physiological changes occur that lead to the shut down of photosynthesis and a cessation of growth. Leaf senescence involves the selective destruction of the chloroplast as the site of photosynthesis. Here, we show that 13-lipoxygenase (13-LOX) accomplishes a key role in the destruction of chloroplasts in senescing plants and propose a critical role of its NH2-terminal chloroplast transit peptide. The 13-LOX enzyme identified here accumulated in the plastid envelope and catalyzed the dioxygenation of unsaturated membrane fatty acids, leading to a selective destruction of the chloroplast and the release of stromal constituents. Because 13-LOX pathway products comprise compounds involved in insect deterrence and pathogen defense (volatile aldehydes and oxylipins), a mechanism of unmolested nitrogen and carbon relocation is suggested that occurs from leaves to seeds and roots during fall.

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Abstract 241: O-GlcNAcylation of Runx2 in Nuclear Matrix Targeting Signal Modulates Vascular Calcification

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.241 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Chang Hyun Byon ◽

Jack Heath ◽

Xia Mao ◽

Yong Sun ◽

Yabing Chen

Keyword(s):

Smooth Muscle ◽

Vascular Calcification ◽

Nuclear Matrix ◽

Kidney Diseases ◽

Site Directed Mutagenesis ◽

Functional Domain ◽

Mortality And Morbidity ◽

Smad Proteins ◽

Targeting Signal

Vascular calcification is prevalent in patients with atherosclerosis, diabetes, and chronic kidney diseases and increases the mortality and morbidity of those patients. Osteogenic differentiation of vascular smooth muscle cells (VSMC) has been recognized as a key feature of the calcification process. Using smooth muscle-specific deletion mouse model, we have demonstrated an essential role of SMC-derived Runx2 in regulating vascular calcification in atherosclerosis in vivo. The present study further defined Runx2 regulation and its functional domains that control osteogenesis of VSMC. Using a serial of Runx2 truncation mutants, we located the domain between amino acids 391 and 432 as responsible region for Runx2 osteogenic function in VSMC. This region contains the nuclear matrix targeting signal, which has been shown to interact with Smad proteins. Accordingly, we determined the contribution of Smad proteins in Runx2-regulated VSMC calcification. By knockdown individual Smad in VSMC, we demonstrated that Smads 1/5/8, but not Smads 2/3, were required for Runx2 osteogenic function and VSMC calcification. In addition, we found the osteogenic function of Runx2 was abolished by inhibition of protein O-GlcNAcylation, a unique posttranslational modification that we have recently reported to be critical in regulating VSMC calcification. Runx2 O-GlcNAc modification was further demonstrated by immunoprecipitation. Based on bioinformatics analysis, we found several putative O-GlcNAcylation sites within Runx2 osteogenic domain. Using site-directed mutagenesis, we demonstrated that mutation on S385, S387, T404, T406, T412, S413, or T427 decreased Runx2 O-GlcNAcylation and Runx2 binding to Smads 1/5/8, and thus decreasing Runx2 activity and VSMC calcification. In summary, we have identified the Runx2 osteogenic functional domain in VSMC; and demonstrated that O-GlcNAc modification of Runx2 in its osteogenic functional domain is critical for Runx2-regualted VSMC calcification. Our studies provide molecular insights into the regulation and function of Runx2 in VSMC calcification, which may shed lights on novel targets that are amenable to drug discovery for vascular calcification.

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Natromarkeyite and pseudomarkeyite, two new calcium uranyl carbonate minerals from the Markey mine, San Juan County, Utah, USA

Mineralogical Magazine ◽

10.1180/mgm.2020.59 ◽

2020 ◽

Vol 84 (5) ◽

pp. 753-765 ◽

Cited By ~ 1

Author(s):

Anthony R. Kampf ◽

Travis A. Olds ◽

Jakub Plášil ◽

Peter C. Burns ◽

Joe Marty

Keyword(s):

Electron Microprobe ◽

San Juan ◽

Secondary Phases ◽

Yellow Colour ◽

Uranyl Carbonate ◽

Mohs Hardness ◽

San Juan County ◽

Pale Green ◽

Light Green ◽

White Streak

AbstractThe new minerals natromarkeyite, Na2Ca8(UO2)4(CO3)13(H2O)24⋅3H2O (IMA2018-152) and pseudomarkeyite, Ca8(UO2)4(CO3)12(H2O)18⋅3H2O (IMA2018-114) were found in the Markey mine, San Juan County, Utah, USA, where they occur as secondary phases on asphaltum. Natromarkeyite properties are: untwinned blades and tablets to 0.2 mm, pale yellow green colour; transparent; white streak; bright bluish white fluorescence (405 nm laser); vitreous to pearly lustre; brittle; Mohs hardness 1½ to 2; irregular fracture; three cleavages ({001} perfect, {100} and {010} good); density = 2.70(2) g cm–3; biaxial (–) with α = 1.528(2), β = 1.532(2) and γ = 1.533(2); and pleochroism is X = pale green yellow, Y ≈ Z = light green yellow. Pseudomarkeyite properties are: twinned tapering blades and tablets to 1 mm; pale green yellow colour; transparent; white streak; bright bluish white fluorescence (405 nm laser); vitreous to pearly lustre; brittle; Mohs hardness ≈ 1; stepped fracture; three cleavages ({10$\bar{1}$} very easy, {010} good, {100} fair); density = 2.88(2) g cm–3; biaxial (–) with α = 1.549(2), β = 1.553(2) and γ = 1.557(2); and it is nonpleochroic. The Raman spectra of markeyite, natromarkeyite and pseudomarkeyite are very similar and exhibit bands consistent with UO22+, CO32– and O–H. Electron microprobe analyses provided the empirical formula Na2.01Ca7.97Mg0.03Cu2+0.05(UO2)4(CO3)13(H2O)24⋅3H2O (–0.11 H) for natromarkeyite and Ca7.95(UO2)4(CO3)12(H2O)18⋅3H2O (+0.10 H) for pseudomarkeyite. Natromarkeyite is orthorhombic, Pmmn, a = 17.8820(13), b = 18.3030(4), c = 10.2249(3) Å, V = 3336.6(3) Å3 and Z = 2. Pseudomarkeyite is monoclinic, P21/m, a = 17.531(3), b = 18.555(3), c = 9.130(3) Å, β = 103.95(3)°, V = 2882.3(13) Å3 and Z = 2. The structures of natromarkeyite (R1 = 0.0202 for 2898 I > 2σI) and pseudomarkeyite (R1 = 0.0787 for 2106 I > 2σI) contain uranyl tricarbonate clusters that are linked by (Ca/Na)–O polyhedra forming thick corrugated heteropolyhedral layers. Natromarkeyite is isostructural with markeyite; pseudomarkeyite has a very similar structure.

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atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins

The Journal of Cell Biology ◽

10.1083/jcb.200311074 ◽

2004 ◽

Vol 165 (3) ◽

pp. 323-334 ◽

Cited By ~ 100

Author(s):

Matthew D. Smith ◽

Caleb M. Rounds ◽

Fei Wang ◽

Kunhua Chen ◽

Meshack Afitlhile ◽

...

Keyword(s):

Plant Cell ◽

Chloroplast Development ◽

Transit Peptide ◽

Chloroplast Biogenesis ◽

Cross Link ◽

Peptide Receptor ◽

Chloroplast Proteins ◽

Transit Peptides ◽

Import Receptor

The members of the Toc159 family of GTPases act as the primary receptors for the import of nucleus-encoded preproteins into plastids. Toc159, the most abundant member of this family in chloroplasts, is required for chloroplast biogenesis (Bauer, J., K. Chen, A. Hiltbunner, E. Wehrli, M. Eugster, D. Schnell, and F. Kessler. 2000. Nature. 403:203–207) and has been shown to covalently cross-link to bound preproteins at the chloroplast surface (Ma, Y., A. Kouranov, S. LaSala, and D.J. Schnell. 1996. J. Cell Biol. 134:1–13; Perry, S.E., and K. Keegstra. 1994. Plant Cell. 6:93–105). These reports led to the hypothesis that Toc159 functions as a selective import receptor for preproteins that are required for chloroplast development. In this report, we provide evidence that Toc159 is required for the import of several highly expressed photosynthetic preproteins in vivo. Furthermore, we demonstrate that the cytoplasmic and recombinant forms of soluble Toc159 bind directly and selectively to the transit peptides of these representative photosynthetic preproteins, but not representative constitutively expressed plastid preproteins. These data support the function of Toc159 as a selective import receptor for the targeting of a set of preproteins required for chloroplast biogenesis.

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Albino mutants of Streptomyces glaucescens tyrosinase

Biochemical Journal ◽

10.1042/bj2740707 ◽

1991 ◽

Vol 274 (3) ◽

pp. 707-713 ◽

Cited By ~ 34

Author(s):

M P Jackman ◽

A Hajnal ◽

K Lerch

Keyword(s):

Active Site ◽

Critical Role ◽

Site Directed Mutagenesis ◽

Mutant Proteins ◽

Carbonyl Derivatives ◽

Hydrogen Bonding Interactions ◽

Albino Phenotype ◽

Derivatives Of ◽

Streptomyces Glaucescens

Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase. Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates. The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site. The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max. displaced more than 25 nm to a longer wavelength compared with native tyrosinase. The remaining histidine mutants display no detectable luminescence. The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme. Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu. The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site. A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.

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