Recent overview of the Mg branch of the tetrapyrrole biosynthesis leading to chlorophylls

In plants, chlorophylls (chlorophyll a and chlorophyll b) are the most abundant tetrapyrrole molecules and are essential for photosynthesis. The first committed step of chlorophyll biosynthesis is the insertion of Mg²⁺ into protoporphyrin IX, and thus subsequent steps of the biosynthesis are called the Mg branch. As the Mg branch in higher plants is complex, it was not until the last decade—after many years of intensive research—that most of the genes encoding the enzymes for the pathway were identified. Biochemical and molecular genetic analyses have certainly modified the classic metabolic map of tetrapyrrole biosynthesis, and only recently have the molecular mechanisms of regulatory pathways governing chlorophyll metabolism been elucidated. As a result, novel functions of tetrapyrroles and biosynthetic enzymes have been proposed. In this review, I summarize the recent findings on enzymes involved in the Mg branch, mainly in higher plants.     

Phospholipase D is a negative regulator of proline biosynthesis in Arabidopsis thaliana

Accumulation of proline has been observed in a large number of plant species in response to drought and salt stresses, suggesting a key role of this amino acid in plant stress adaptation. Upstream components of the proline biosynthesis signal transduction pathways are still poorly defined. We provide experimental evidence that phospholipase D (PLD) is involved in the regulation of proline metabolism in Arabidopsis thaliana. The application of primary butyl alcohols, which divert part of PLD-derived phosphatidic acid by transphosphatidylation, stimulated proline biosynthesis even without hyperosmotic constraints. Moreover, application of primary butyl alcohols enhanced the proline responsiveness of seedlings to mild hyperosmotic stress. These data indicate that some PLDs are negative regulators of proline biosynthesis and that plants present a higher proline responsiveness to hyperosmotic stress when this regulator is abolished. We clearly demonstrate that PLD signaling for proline biosynthesis is similar to RD29A gene expression and different from the abscisic acid-dependent RAB18 gene expression. Our data reveal that PLDs play positive and negative roles in hyperosmotic stress signal transduction in plants, contributing to a precise regulation of ion homeostasis and plant salt tolerance.     

D-cbl, a negative regulator of the Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis

During Drosophila oogenesis, asymmetrically localized Gurken activates the EGF receptor (Egfr) and determines dorsal follicle cell fates. Using a mosaic follicle cell system we have identified a mutation in the D-cbl gene which causes hyperactivation of the Egfr pathway. Cbl proteins are known to downregulate activated receptors. We find that the abnormal Egfr activation is ligand dependent. Our results show that the precise regulation of Egfr activity necessary to establish different follicle cell fates requires two levels of control. The localized ligand Gurken activates Egfr to different levels in different follicle cells. In addition, Egfr activity has to be repressed through the activity of D-cbl to ensure the absence of signaling in the ventral most follicle cells.     

Arabidopsis ICX1 is a negative regulator of several pathways regulating flavonoid biosynthesis genes

Flavonoid biosynthesis gene expression is controlled by a range of endogenous and environmental signals. The Arabidopsis icx1 (increased chalcone synthase expression 1) mutant has elevated induction of CHS (CHALCONE SYNTHASE) and other flavonoid biosynthesis genes in response to several stimuli. We show that ICX1 is a negative regulator of the cryptochrome 1, phytochrome A, ultraviolet (UV)-B, low temperature, sucrose, and cytokinin induction of CHS expression and/or anthocyanin accumulation, demonstrating that these pathways are regulated either directly or indirectly by at least one common component. Expression analysis of CHS and other genes (LTP, CAB, and rbcS) indicates that ICX1 functions in both seedlings and mature leaf tissue and acts principally in the epidermis, consistent with the alterations in epidermal development seen in icx1. The mutant was unaltered in the synergistic interactions between UV-B, blue, and UV-A light that regulate CHS and we propose a model of action of ICX1 in these responses.     

Concurrent interactions of heme and FLU with Glu tRNA reductase (HEMA1), the target of metabolic feedback inhibition of tetrapyrrole biosynthesis, in dark- and light-grown Arabidopsis plants

The regulation of tetrapyrrole biosynthesis in higher plants has been attributed to metabolic feedback inhibition of Glu tRNA reductase by heme. Recently, another negative regulator of tetrapyrrole biosynthesis has been discovered, the FLU protein. During an extensive second site screen of mutagenized flu seedlings a suppressor of flu, ulf3, was identified that is allelic to hy1 and encodes a heme oxygenase. Increased levels of heme in the hy1 mutant have been implicated with inhibiting Glu tRNA reductase and suppressing the synthesis of delta-aminolevulinic acid (ALA) and Pchlide accumulation. When combined with hy1 or ulf3 upregulation of ALA synthesis and overaccumulation of protochlorophyllide in the flu mutants were severely suppressed supporting the notion that heme antagonizes the effect of the flu mutation by inhibiting Glu tRNA reductase independently of FLU. The coiled-coil domain at the C-terminal end of Glu tRNA reductase interacts with FLU, whereas the N-terminal site of Glu tRNA reductase that is necessary for the inhibition of the enzyme by heme is not required for this interaction. The interaction with FLU is specific for the Glu tRNA reductase encoded by HEMA1 that is expressed in photosynthetically active tissues. FLU seems to be part of a second regulatory circuit that controls chlorophyll biosynthesis by interacting directly with Glu tRNA reductase not only in etiolated seedlings but also in light-adapted green plants.     

BANYULS, a novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat

A mutant of Arabidopsis that accumulates a high level of red pigments within the seed coat has been isolated from a population of T-DNA-transformed plants. Genetic analysis revealed that the mutation is recessive and affects maternal seed tissues only. Due to the color of the immature seeds, this mutation was named banyuls ( ban ). Pigments accumulated continuously from early seed development to the desiccation stage in the seed coat of the mutant. The phenotype of the double mutant banyuls/transparent testa confirmed the flavonoid nature of the pigments and enabled assignment of the regulatory TT ( Transparent Testa ) genes to two groups according to their epistatic relationship to ban . The flavonoid content of germinated ban and wild-type seedlings was similar. Plants harbouring the ban mutation had a normal formation of trichomes and root hairs and were not affected in their responses to light. The seeds of ban plants exhibited reduced germination compared to wild-type which may be a direct consequence of the high level of pigments. These results suggest that BANYULS functions as a negative regulator of flavonoid biosynthesis that prevents accumulation of pigments in the seed coat during early embryogenesis in Arabidopsis .     

Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop

We have previously observed that metabolic oxidative stress-induced death domain-associated protein (Daxx) trafficking is mediated by the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. The relocalized Daxx from the nucleus to the cytoplasm during glucose deprivation participates in a positive regulatory feedback loop by binding to apoptosis signal-regulating kinase (ASK) 1. In this study, we report that Akt1 is involved in a negative regulatory feedback loop during glucose deprivation. Akt1 interacts with c-Jun NH(2)-terminal kinase (JNK)-interacting protein (JIP) 1, and Akt1 catalytic activity is inhibited. The JNK2-mediated phosphorylation of JIP1 results in the dissociation of Akt1 from JIP1 and subsequently restores Akt1 enzyme activity. Concomitantly, Akt1 interacts with stress-activated protein kinase/extracellular signal-regulated kinase (SEK) 1 (also known as MKK4) and inhibits SEK1 activity. Knockdown of SEK1 leads to the inhibition of JNK activation, JIP1-JNK2 binding, and the dissociation of Akt1 from JIP1 during glucose deprivation. Knockdown of JIP1 also leads to the inhibition of JNK activation, whereas the knockdown of Akt1 promotes JNK activation during glucose deprivation. Altogether, our data demonstrate that Akt1 participates in a negative regulatory feedback loop by interacting with the JIP1 scaffold protein.     

Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes--VI. De novo purine nucleotide biosynthesis is limited to the final steps of the pathway in vitro

1. High pressure liquid radiochromatography was used to show the incorporation of [14C] formate with Z-compounds into ATP and GTP in opossum erythrocytes. 2. The use of Z-riboside with [14C] formate resulted in more extensive labeling of ATP than the Z-base/[14C] formate combination as substrates for nucleotide biosynthesis. 3. Substantial accumulation of ZMP and ZTP, but no ZDP was detected in the chromatograms. 4. ATP was unstable in red cells metabolizing in the presence of Z-compounds under an atmosphere of air as gas phase in these experiments.     

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab-GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild-type (Rab23-GFP), constitutively active Rab23 (Rab23Q68L-GFP), and inactive Rab23 (Rab23S23N-GFP) in a range of mammalian cell types. Rab23-GFP and Rab23Q68L-GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N-GFP was predominantly cytosolic. Vesicular Rab23-GFP colocalized with Rab5Q79L and internalized transferrin-biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23-GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23-GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor-positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA-positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild-type nor mutant Rab23-GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.     

PpsR, a regulator of heme and bacteriochlorophyll biosynthesis, is a heme-sensing protein

Heme-mediated regulation, presented in many biological processes, is achieved in part with proteins containing heme regulatory motif. In this study, we demonstrate that FLAG-tagged PpsR isolated from Rhodobacter sphaeroides cells contains bound heme. In vitro heme binding studies with tagless apo-PpsR show that PpsR binds heme at a near one-to-one ratio with a micromolar binding constant. Mutational and spectral assays suggest that both the second Per-Arnt-Sim (PAS) and DNA binding domains of PpsR are involved in the heme binding. Furthermore, we show that heme changes the DNA binding patterns of PpsR and induces different responses of photosystem genes expression. Thus, PpsR functions as both a redox and heme sensor to coordinate the amount of heme, bacteriochlorophyll, and photosystem apoprotein synthesis thereby providing fine tune control to avoid excess free tetrapyrrole accumulation.     

Sodium is a negative allosteric regulator of the ghrelin receptor

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