The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases

The hy1 mutants of Arabidopsis thaliana fail to make the phytochrome-chromophore phytochromobilin and therefore are deficient in a wide range of phytochrome-mediated responses. Because this defect can be rescued by feeding seedlings biliverdin IXα, it is likely that the mutations affect an enzyme that converts heme to this phytochromobilin intermediate. By a combination of positional cloning and candidate-gene isolation, we have identified the HY1 gene and found it to be related to cyanobacterial, algal, and animal heme oxygenases. Three independent alleles of hy1 contain DNA lesions within the HY1 coding region, and a genomic sequence spanning the HY1 locus complements the hy1–1 mutation. HY1 is a member of a gene family and is expressed in a variety of A. thaliana tissues. Based on its homology, we propose that HY1 encodes a higher-plant heme oxygenase, designated AtHO1, responsible for catalyzing the reaction that opens the tetrapyrrole ring of heme to generate biliverdin IXα.

In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore

Phytochrome B (PhyB), one of the major photosensory chromoproteins in plants, mediates a variety of light-responsive developmental processes in a photoreversible manner. To analyze the structural requirements of the chromophore for the spectral properties of PhyB, we have designed and chemically synthesized 20 analogs of the linear tetrapyrrole (bilin) chromophore and reconstituted them with PhyB apoprotein (PHYB). The A-ring acts mainly as the anchor for ligation to PHYB, because the modification of the side chains at the C2 and C3 positions did not significantly influence the formation or difference spectra of adducts. In contrast, the side chains of the B- and C-rings are crucial to position the chromophore properly in the chromophore pocket of PHYB and for photoreversible spectral changes. The side-chain structure of the D-ring is required for the photoreversible spectral change of the adducts. When methyl and ethyl groups at the C17 and C18 positions are replaced with an n-propyl, n-pentyl, or n-octyl group, respectively, the photoreversible spectral change of the adducts depends on the length of the side chains. From these studies, we conclude that each pyrrole ring of the linear tetrapyrrole chromophore plays a different role in chromophore assembly and the photochromic properties of PhyB.

Feedback Inhibition of Chlorophyll Synthesis in the Phytochrome Chromophore-Deficient aurea and yellow-green-2 Mutants of Tomato

The aurea (au) and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum L.) are unable to synthesize the linear tetrapyrrole chromophore of phytochrome, resulting in plants with a yellow-green phenotype. To understand the basis of this phenotype, we investigated the consequences of the au and yg-2 mutations on tetrapyrrole metabolism. Dark-grown seedlings of both mutants have reduced levels of protochlorophyllide (Pchlide) due to an inhibition of Pchlide synthesis. Feeding experiments with the tetrapyrrole precursor 5-aminolevulinic acid (ALA) demonstrate that the pathway between ALA and Pchlide is intact in au and yg-2 and suggest that the reduction in Pchlide is a result of the inhibition of ALA synthesis. This inhibition was independent of any deficiency in seed phytochrome, and experiments using an iron chelator to block heme synthesis demonstrated that both mutations inhibited the degradation of the physiologically active heme pool, suggesting that the reduction in Pchlide synthesis is a consequence of feedback inhibition by heme. We discuss the significance of these results in understanding the chlorophyll-deficient phenotype of the au and yg-2 mutants.

Convergence of developmental mutants into a single tomato model system: 'Micro-Tom' as an effective toolkit for plant development research

Background: The tomato (Solanum lycopersicum L.) plant is both an economically important food crop and an ideal dicot model to investigate various physiological phenomena not possible in Arabidopsis thaliana. Due to the great diversity of tomato cultivars used by the research community, it is often difficult to reliably compare phenotypes. The lack of tomato developmental mutants in a single genetic background prevents the stacking of mutations to facilitate analysis of double and multiple mutants, often required for elucidating developmental pathways. Results: We took advantage of the small size and rapid life cycle of the tomato cultivar Micro-Tom (MT) to create near-isogenic lines (NILs) by introgressing a suite of hormonal and photomorphogenetic mutations (altered sensitivity or endogenous levels of auxin, ethylene, abscisic acid, gibberellin, brassinosteroid, and light response) into this genetic background. To demonstrate the usefulness of this collection, we compared developmental traits between the produced NILs. All expected mutant phenotypes were expressed in the NILs. We also created NILs harboring the wild type alleles for dwarf, self-pruning and uniform fruit, which are mutations characteristic of MT. This amplified both the applications of the mutant collection presented here and of MT as a genetic model system. Conclusions: The community resource presented here is a useful toolkit for plant research, particularly for future studies in plant development, which will require the simultaneous observation of the effect of various hormones, signaling pathways and crosstalk.

Phytochrome Regulates Gibberellin Biosynthesis during Germination of Photoblastic Lettuce Seeds1

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA1, the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3β-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3β-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3β-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action.

The methylotrophic yeast Pichia pastoris synthesizes a functionally active chromophore precursor of the plant photoreceptor phytochrome.

Induction of the expression of an algal phytochrome cDNA in the methylotrophic yeast Pichia pastoris led to time-dependent formation of photoactive holophytochrome without the addition of exogenous bilins. Both in vivo and in vitro difference spectra of this phytochromic species are very similar to those of higher plant phytochrome A, supporting the conclusion that this species possesses a phytochromobilin prosthetic group. Zinc blot analyses confirm that a bilin chromophore is covalently bound to the algal phytochrome apoprotein. The hypothesis that P. pastoris contains phytochromobilin synthase, the enzyme that converts biliverdin IX alpha to phytochromobilin, was also addressed in this study. Soluble extracts from P. pastoris were able to convert biliverdin to a bilin pigment, which produced a native difference spectrum upon assembly with oat apophytochrome A. HPLC analyses confirm that biliverdin is converted to both 3E- and 3Z-isomers of phytochromobilin. These investigations demonstrate that the ability to synthesize phytochromobilin is not restricted to photosynthetic organisms and support the hypothesis of a more widespread distribution of the phytochrome photoreceptor.

Phytochrome interacting factors 4 and 5 control seedling growth in changing light conditions by directly controlling auxin signaling.

Plant growth is strongly influenced by the presence of neighbors that compete for light resources. In response to vegetational shading shade-intolerant plants such as Arabidopsis display a suite of developmental responses known as the shade-avoidance syndrome (SAS). The phytochrome B (phyB) photoreceptor is the major light sensor to mediate this adaptive response. Control of the SAS occurs in part with phyB, which controls protein abundance of phytochrome-interacting factors 4 and 5 (PIF4 and PIF5) directly. The shade-avoidance response also requires rapid biosynthesis of auxin and its transport to promote elongation growth. The identification of genome-wide PIF5-binding sites during shade avoidance revealed that this bHLH transcription factor regulates the expression of a subset of previously identified SAS genes. Moreover our study suggests that PIF4 and PIF5 regulate elongation growth by controlling directly the expression of genes that code for auxin biosynthesis and auxin signaling components.

Abordagem fotomorfogenética para explorar o estresse abiótico em tomateiro (Solanum lycopersicum L.)

Pós-graduação em Agronomia (Produção Vegetal) - FCAV

Characterization of recombinant phytochrome from the cyanobacterium Synechocystis

The complete sequence of the Synechocystis chromosome has revealed a phytochrome-like sequence that yielded an authentic phytochrome when overexpressed in Escherichia coli. In this paper we describe this recombinant Synechocystis phytochrome in more detail. Islands of strong similarity to plant phytochromes were found throughout the cyanobacterial sequence whereas C-terminal homologies identify it as a likely sensory histidine kinase, a family to which plant phytochromes are related. An ≈300 residue portion that is important for plant phytochrome function is missing from the Synechocystis sequence, immediately in front of the putative kinase region. The recombinant apoprotein is soluble and can easily be purified to homogeneity by affinity chromatography. Phycocyanobilin and similar tetrapyrroles are covalently attached within seconds, an autocatalytic process followed by slow conformational changes culminating in red-absorbing phytochrome formation. Spectral absorbance characteristics are remarkably similar to those of plant phytochromes, although the conformation of the chromophore is likely to be more helical in the Synechocystis phytochrome. According to size-exclusion chromatography the native recombinant apoproteins and holoproteins elute predominantly as 115- and 170-kDa species, respectively. Both tend to form dimers in vitro and aggregate under low salt conditions. Nevertheless, the purity and solubility of the recombinant gene product make it a most attractive model for molecular studies of phytochrome, including x-ray crystallography.

Overexpression of Arabidopsis Phytochrome B Inhibits Phytochrome A Function in the Presence of Sucrose1

Overexpression of phytochrome B (phyB) in Arabidopsis has previously been demonstrated to result in dominant negative interference of phytochrome A (phyA)-mediated hypocotyl growth inhibition in far-red (FR) light. This phenomenon has been examined further in this study and has been found to be dependent on the FR fluence rate and on the availability of metabolizable sugars in the growth medium. Poorly metabolized sugars capable of activating the putative hexokinase sensory function were not effective in eliciting the phytochrome interference response. Overexpressed phyB lacking the chromophore-binding site was also effective at inhibiting the phyA response, especially at higher fluence rates of FR. Overexpressed phyB produces the dominant negative phenotype without any apparent effect on phyA abundance or degradation. It is possible that phyA and phyB interact with a common reaction partner but that either the energy state of the cell or a separate sugar-signaling mechanism modulates the phytochrome-signaling interactions.

Chromophore-bearing NH2-terminal domains of phytochromes A and B determine their photosensory specificity and differential light lability.

In early seedling development, far-red-light-induced deetiolation is mediated primarily by phytochrome A (phyA), whereas red-light-induced deetiolation is mediated primarily by phytochrome B (phyB). To map the molecular determinants responsible for this photosensory specificity, we tested the activities of two reciprocal phyA/phyB chimeras in diagnostic light regimes using overexpression in transgenic Arabidopsis. Although previous data have shown that the NH2-terminal halves of phyA and phyB each separately lack normal activity, fusion of the NH2-terminal half of phyA to the COOH-terminal half of phyB (phyAB) and the reciprocal fusion (phyBA) resulted in biologically active phytochromes. The behavior of these two chimeras in red and far-red light indicates: (i) that the NH2-terminal halves of phyA and phyB determine their respective photosensory specificities; (ii) that the COOH-terminal halves of the two photoreceptors are necessary for regulatory activity but are reciprocally inter-changeable and thus carry functionally equivalent determinants; and (iii) that the NH2-terminal halves of phyA and phyB carry determinants that direct the differential light lability of the two molecules. The present findings suggest that the contrasting photosensory information gathered by phyA and phyB through their NH2-terminal halves may be transduced to downstream signaling components through a common biochemical mechanism involving the regulatory activity of the COOH-terminal domains of the photoreceptors.

Multiscale photoacoustic tomography using reversibly switchable bacterial phytochrome as a near-infrared photochromic probe.

Photoacoustic tomography (PAT) of genetically encoded probes allows for imaging of targeted biological processes deep in tissues with high spatial resolution; however, high background signals from blood can limit the achievable detection sensitivity. Here we describe a reversibly switchable nonfluorescent bacterial phytochrome for use in multiscale photoacoustic imaging, BphP1, with the most red-shifted absorption among genetically encoded probes. BphP1 binds a heme-derived biliverdin chromophore and is reversibly photoconvertible between red and near-infrared light-absorption states. We combined single-wavelength PAT with efficient BphP1 photoswitching, which enabled differential imaging with substantially decreased background signals, enhanced detection sensitivity, increased penetration depth and improved spatial resolution. We monitored tumor growth and metastasis with ∼ 100-μm resolution at depths approaching 10 mm using photoacoustic computed tomography, and we imaged individual cancer cells with a suboptical-diffraction resolution of ∼ 140 nm using photoacoustic microscopy. This technology is promising for biomedical studies at several scales.

De Novo Assembly And Transcriptome Analysis Of The Rubber Tree (hevea Brasiliensis) And Snp Markers Development For Rubber Biosynthesis Pathways.

Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.

Analysis Of The Ergosterol Biosynthesis Pathway Cloning, Molecular Characterization And Phylogeny Of Lanosterol 14 α-demethylase (erg11) Gene Of Moniliophthora Perniciosa.

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.

Isoprenoid biosynthesis in the erythrocytic stages of Plasmodium falciparum

The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic precursors, are excellent targets because they are different or absent in the human host. Isoprenoids are a large and highly diverse group of natural products with many functions and their synthesis is essential for the parasite's survival. During the last few years, the genes, enzymes, intermediates and mechanisms of this biosynthetic route have been elucidated. In this review, we comment on some aspects of the methylerythritol phosphate pathway and discuss the presence of diverse isoprenic products such as dolichol, ubiquinone, carotenoids, menaquinone and isoprenylated proteins, which are biosynthesised during the intraerythrocytic stages of Plasmodium falciparum.