Dataset Information

Arabidopsis RIBA proteins: two out of three isoforms have lost their bifunctional activity in riboflavin biosynthesis.

ABSTRACT: Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprised of GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). Angiosperms share a small RIBA gene family consisting of three members. A reduction of AtRIBA1 expression in the Arabidopsis rfd1mutant and in RIBA1 antisense lines is not complemented by the simultaneously expressed isoforms AtRIBA2 and AtRIBA3. The intensity of the bleaching leaf phenotype of RIBA1 deficient plants correlates with the inactivation of AtRIBA1 expression, while no significant effects on the mRNA abundance of AtRIBA2 and AtRIBA3 were observed. We examined reasons why both isoforms fail to sufficiently compensate for a lack of RIBA1 expression. All three RIBA isoforms are shown to be translocated into chloroplasts as GFP fusion proteins. Interestingly, both AtRIBA2 and AtRIBA3 have amino acid exchanges in conserved peptides domains that have been found to be essential for the two enzymatic functions. In vitro activity assays of GCHII and DHBPS with all of the three purified recombinant AtRIBA proteins and complementation of E. coli ribA and ribB mutants lacking DHBPS and GCHII expression, respectively, confirmed the loss of bifunctionality for AtRIBA2 and AtRIBA3. Phylogenetic analyses imply that the monofunctional, bipartite RIBA3 proteins, which have lost DHBPS activity, evolved early in tracheophyte evolution.

SUBMITTER: Hiltunen HM

PROVIDER: S-EPMC3509567 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Arabidopsis RIBA proteins: two out of three isoforms have lost their bifunctional activity in riboflavin biosynthesis.

Hiltunen Hanna-Maija HM Illarionov Boris B Hedtke Boris B Fischer Markus M Grimm Bernhard B

International journal of molecular sciences 20121031 11

Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprised of GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). Angiosperms share a small RIBA gene family consisting of three members. A reduction of AtRIBA1 expression in the Arabidopsis rfd1mutant and in RIBA1 antisense lines is not com ...[more]

PMID: 23203051

Similar Datasets

Project description:BackgroundThe CDC20 and Cdh1/CCS52 proteins are substrate determinants and activators of the Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligase and as such they control the mitotic cell cycle by targeting the degradation of various cell cycle regulators. In yeasts and animals the main CDC20 function is the destruction of securin and mitotic cyclins. Plants have multiple CDC20 gene copies whose functions have not been explored yet. In Arabidopsis thaliana there are five CDC20 isoforms and here we aimed at defining their contribution to cell cycle regulation, substrate selectivity and plant development.Methodology/principal findingsStudying the gene structure and phylogeny of plant CDC20s, the expression of the five AtCDC20 gene copies and their interactions with the APC/C subunit APC10, the CCS52 proteins, components of the mitotic checkpoint complex (MCC) and mitotic cyclin substrates, conserved CDC20 functions could be assigned for AtCDC20.1 and AtCDC20.2. The other three intron-less genes were silent and specific for Arabidopsis. We show that AtCDC20.1 and AtCDC20.2 are components of the MCC and interact with mitotic cyclins with unexpected specificity. AtCDC20.1 and AtCDC20.2 are expressed in meristems, organ primordia and AtCDC20.1 also in pollen grains and developing seeds. Knocking down both genes simultaneously by RNAi resulted in severe delay in plant development and male sterility. In these lines, the meristem size was reduced while the cell size and ploidy levels were unaffected indicating that the lower cell number and likely slowdown of the cell cycle are the cause of reduced plant growth.Conclusions/significanceThe intron-containing CDC20 gene copies provide conserved and redundant functions for cell cycle progression in plants and are required for meristem maintenance, plant growth and male gametophyte formation. The Arabidopsis-specific intron-less genes are possibly "retrogenes" and have hitherto undefined functions or are pseudogenes.

Project description:BackgroundProline (Pro) accumulation is a widespread response of prokaryotic and eukaryotic cells subjected to osmotic stress or dehydration. When the cells are released from stress, Pro is degraded to glutamate by Pro-dehydrogenase (ProDH) and Pyrroline-5-carboxylate dehydrogenase (P5CDH), which are both mitochondrial enzymes in eukaryotes. While P5CDH is a single copy gene in Arabidopsis, two ProDH genes have been identified in the genome. Until now, only ProDH1 (At3g30775) had been functionally characterised.ResultsWe demonstrate vasculature specific expression of the Arabidopsis ProDH2 gene (At5g38710) as well as enzymatic activity and mitochondrial localisation of the encoded protein. Expression levels of ProDH2 are generally low, but increased in senescent leaves and in the abscission zone of floral organs. While sucrose represses ProDH2 expression, Pro and NaCl were identified as inducers. Endogenous ProDH2 expression was not able to overcome Pro sensitivity of ProDH1 mutants, but overexpression of a GFP-tagged form of ProDH2 enabled the utilisation of Pro as single nitrogen source for growth. Amongst two intronic insertion mutants, one was identified as a null allele, whereas the other still produced native ProDH2 transcripts.ConclusionsArabidopsis possesses two functional ProDHs, which have non-redundant, although partially overlapping physiological functions. The two ProDH isoforms differ with respect to spatial, developmental and environmental regulation of expression. While ProDH1 appears to be the dominant isoform under most conditions and in most tissues, ProDH2 was specifically upregulated during salt stress, when ProDH1 was repressed. The characterisation of ProDH2 as a functional gene requires a careful re-analysis of mutants with a deletion of ProDH1, which were so far considered to be devoid of ProDH activity. We hypothesise that ProDH2 plays an important role in Pro homeostasis in the vasculature, especially under stress conditions that promote Pro accumulation.

Project description:The actinobacterium Microbacterium maritypicum splits riboflavin (vitamin B2) into lumichrome and d-ribose. However, such degradation by other bacteria and the involvement of a two-component flavin-dependent monooxygenase (FMO) in the reaction remain unknown. Here we investigated the mechanism of riboflavin degradation by the riboflavin-assimilating alphaproteobacterium Devosia riboflavina (formerly Pseudomonas riboflavina). We found that adding riboflavin to bacterial cultures induced riboflavin-degrading activity and a protein of the FMO family that had 67% amino acid identity with the predicted riboflavin hydrolase (RcaE) of M. maritypicum MF109. The D. riboflavina genome clustered genes encoding the predicted FMO, flavin reductase (FR), ribokinase, and flavokinase, and riboflavin induced their expression. This finding suggests that these genes constitute a mechanism for utilizing riboflavin as a carbon source. Recombinant FMO (rFMO) protein of D. riboflavina oxidized riboflavin in the presence of reduced flavin mononucleotide (FMN) provided by recombinant FR (rFR), oxidized FMN and NADH, and produced stoichiometric amounts of lumichrome and d-ribose. Further investigation of the enzymatic properties of D. riboflavina rFMO indicated that rFMO-rFR coupling accompanied O2 consumption and the generation of enzyme-bound hydroperoxy-FMN, which are characteristic of two-component FMOs. These results suggest that D. riboflavina FMO is involved in hydroperoxy-FMN-dependent mechanisms to oxygenize riboflavin and a riboflavin monooxygenase is necessary for the initial step of riboflavin degradation.IMPORTANCE Whether bacteria utilize either a monooxygenase or a hydrolase for riboflavin degradation has remained obscure. The present study found that a novel riboflavin monooxygenase, not riboflavin hydrolase, facilitated this process in D. riboflavina The riboflavin monooxygenase gene was clustered with flavin reductase, flavokinase, and ribokinase genes, and riboflavin induced their expression and riboflavin-degrading activity. The gene cluster is uniquely distributed in Devosia species and actinobacteria, which have exploited an environmental niche by developing adaptive mechanisms for riboflavin utilization.

Project description:Ethylene (C(2)H(4)) is a unique plant-signaling molecule that regulates numerous developmental processes. The key enzyme in the two-step biosynthetic pathway of ethylene is 1-aminocyclopropane-1-carboxylate synthase (ACS), which catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC, the precursor of ethylene. To understand the function of this important enzyme, we analyzed the entire family of nine ACS isoforms (ACS1, ACS2, ACS4-9, and ACS11) encoded in the Arabidopsis genome. Our analysis reveals that members of this protein family share an essential function, because individual ACS genes are not essential for Arabidopsis viability, whereas elimination of the entire gene family results in embryonic lethality. Phenotypic characterization of single and multiple mutants unmasks unique but overlapping functions of the various ACS members in plant developmental events, including multiple growth characteristics, flowering time, response to gravity, disease resistance, and ethylene production. Ethylene acts as a repressor of flowering by regulating the transcription of the FLOWERING LOCUS C. Each single and high order mutant has a characteristic molecular phenotype with unique and overlapping gene expression patterns. The expression of several genes involved in light perception and signaling is altered in the high order mutants. These results, together with the in planta ACS interaction map, suggest that ethylene-mediated processes are orchestrated by a combinatorial interplay among ACS isoforms that determines the relative ratio of homo- and heterodimers (active or inactive) in a spatial and temporal manner. These subunit isoforms comprise a combinatorial code that is a central regulator of ethylene production during plant development. The lethality of the null ACS mutant contrasts with the viability of null mutations in key components of the ethylene signaling apparatus, strongly supporting the view that ACC, the precursor of ethylene, is a primary regulator of plant growth and development.

Dataset Information

Arabidopsis RIBA proteins: two out of three isoforms have lost their bifunctional activity in riboflavin biosynthesis.

Publications

Arabidopsis RIBA proteins: two out of three isoforms have lost their bifunctional activity in riboflavin biosynthesis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure