The tomato photomorphogenetic mutant, aurea, is deficient in phytochromobilin synthase for phytochrome chromophore biosynthesis

The aurea mutants of tomato have been widely used as phytochrome-deficient mutants for photomorphogenetic and photobiological studies. By expressed sequence tag (EST)-based screening of sequence databases, we found a tomato gene that encodes a protein homologous to Arabidopsis HY2 for phytochromobilin synthase catalyzing the last step of phytochrome chromophore biosynthesis. The tomato protein expressed in Escherichia coli showed phytochromobilin synthase activity. The corresponding loci in all aurea mutants tested have nucleotide substitutions, deletions or DNA rearrangements. These results indicate that aurea is a mutant of phytochromobilin synthase in tomato. We also discuss a phylogenetic analysis of phytochromobilin synthases in the bilin reductase family.     

Multiple heme oxygenase family members contribute to the biosynthesis of the phytochrome chromophore in Arabidopsis

The oxidative cleavage of heme by heme oxygenases (HOs) to form biliverdin IXalpha (BV) is the committed step in the biosynthesis of the phytochrome (phy) chromophore and thus essential for proper photomorphogenesis in plants. Arabidopsis (Arabidopsis thaliana) contains four possible HO genes (HY1, HO2-4). Genetic analysis of the HY1 locus showed previously that it is the major source of BV with hy1 mutant plants displaying long hypocotyls and decreased chlorophyll accumulation consistent with a substantial deficiency in photochemically active phys. More recent analysis of HO2 suggested that it also plays a role in phy assembly and photomorphogenesis but the ho2 mutant phenotype is more subtle than that of hy1 mutants. Here, we define the functions of HO3 and HO4 in Arabidopsis. Like HY1, the HO3 and HO4 proteins have the capacity to synthesize BV from heme. Through a phenotypic analysis of T-DNA insertion mutants affecting HO3 and HO4 in combination with mutants affecting HY1 or HO2, we demonstrate that both of the encoded proteins also have roles in photomorphogenesis, especially in the absence of HY1. Disruption of HO3 and HO4 in the hy1 background further desensitizes seedlings to red and far-red light and accelerates flowering time, with the triple mutant strongly resembling seedlings deficient in the synthesis of multiple phy apoproteins. The hy1/ho3/ho4 mutant can be rescued phenotypically and for the accumulation of holo-phy by feeding seedlings BV. Taken together, we conclude that multiple members of the Arabidopsis HO family are important for synthesizing the bilin chromophore used to assemble photochemically active phys.     

Light-induced global structural changes in phytochrome A regulating photomorphogenesis in plants

Phytochromes are photoreceptor proteins that monitor the light environment and regulate a variety of photomorphogenic responses to optimize the growth and development of plants. Phytochromes comprise N-terminal photosensory and C-terminal regulatory domains. They are mutually photoconvertible between a red-light-absorbing (Pr) and a far-red-light-absorbing (Pfr) form. Their interconversion by light stimuli initiates downstream signaling cascades. Here we report the molecular structures of pea phytochrome A lacking the N-terminal 52 amino-acid residues in the Pr and Pfr forms studied by small-angle X-ray scattering. A new purification protocol yielded monodispersive sample solutions. The molecular mass and the maximum dimension of Pr determined from scattering data indicated its dimeric association. The molecular structure of Pr predicted by applying the ab initio simulation method to the scattering profile was approximated as a stack of two flat bodies, comprising two lobes assignable to the functional regions. Scattering profiles recorded under red-light irradiation showed small but definite changes from those of Pr. The molecular dimensions and predicted molecular structure of Pfr suggest global structural changes such as movement of the C-terminal domains in the Pr-to-Pfr phototransformation. Red-light-induced structural changes in Pfr were reversible, mostly due to thermal relaxation processes.     

Root-localized phytochrome chromophore synthesis is required for photoregulation of root elongation and impacts root sensitivity to jasmonic acid in Arabidopsis

Plants exhibit organ- and tissue-specific light responses. To explore the molecular basis of spatial-specific phytochrome-regulated responses, a transgenic approach for regulating the synthesis and accumulation of the phytochrome chromophore phytochromobilin (PΦB) was employed. In prior experiments, transgenic expression of the BILIVERDIN REDUCTASE (BVR) gene was used to metabolically inactivate biliverdin IXα, a key precursor in the biosynthesis of PΦB, and thereby render cells accumulating BVR phytochrome deficient. Here, we report analyses of transgenic Arabidopsis (Arabidopsis thaliana) lines with distinct patterns of BVR accumulation dependent upon constitutive or tissue-specific, promoter-driven BVR expression that have resulted in insights on a correlation between root-localized BVR accumulation and photoregulation of root elongation. Plants with BVR accumulation in roots and a PΦB-deficient elongated hypocotyl2 (hy2-1) mutant exhibit roots that are longer than those of wild-type plants under white illumination. Additional analyses of a line with root-specific BVR accumulation generated using a GAL4-dependent bipartite enhancer-trap system confirmed that PΦB or phytochromes localized in roots directly impact light-dependent root elongation under white, blue, and red illumination. Additionally, roots of plants with constitutive plastid-localized or root-specific cytosolic BVR accumulation, as well as phytochrome chromophore-deficient hy1-1 and hy2-1 mutants, exhibit reduced sensitivity to the plant hormone jasmonic acid (JA) in JA-dependent root inhibition assays, similar to the response observed for the JA-insensitive mutants jar1 and myc2. Our analyses of lines with root-localized phytochrome deficiency or root-specific phytochrome depletion have provided novel insights into the roles of root-specific PΦB, or phytochromes themselves, in the photoregulation of root development and root sensitivity to JA.     

Expression and biochemical properties of a ferredoxin-dependent heme oxygenase required for phytochrome chromophore synthesis

The HY1 gene of Arabidopsis encodes a plastid heme oxygenase (AtHO1) required for the synthesis of the chromophore of the phytochrome family of plant photoreceptors. To determine the enzymatic properties of plant heme oxygenases, we have expressed the HY1 gene (without the plastid transit peptide) in Escherichia coli to produce an amino terminal fusion protein between AtHO1 and glutathione S-transferase. The fusion protein was soluble and expressed at high levels. Purified recombinant AtHO1, after glutathione S-transferase cleavage, is a hemoprotein that forms a 1:1 complex with heme. In the presence of reduced ferredoxin, AtHO1 catalyzed the formation of biliverdin IXalpha from heme with the concomitant production of carbon monoxide. Heme oxygenase activity could also be reconstituted using photoreduced ferredoxin generated through light irradiation of isolated thylakoid membranes, suggesting that ferredoxin may be the electron donor in vivo. In addition, AtHO1 required an iron chelator and second reductant, such as ascorbate, for full activity. These results show that the basic mechanism of heme cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of heme oxygenases differ substantially.     

Dictyostelium PAKc is required for proper chemotaxis

We have identified a new Dictyostelium p21-activated protein kinase, PAKc, that we demonstrate to be required for proper chemotaxis. PAKc contains a Rac-GTPase binding (CRIB) and autoinhibitory domain, a PAK-related kinase domain, an N-terminal phosphatidylinositol binding domain, and a C-terminal extension related to the Gbetagamma binding domain of Saccharomyces cerevisiae Ste20, the latter two domains being required for PAKc transient localization to the plasma membrane. In response to chemoattractant stimulation, PAKc kinase activity is rapidly and transiently activated, with activity levels peaking at approximately 10 s. pakc null cells exhibit a loss of polarity and produce multiple lateral pseudopodia when placed in a chemoattractant gradient. PAKc preferentially binds the Dictyostelium Rac protein RacB, and point mutations in the conserved CRIB that abrogate this binding result in misregulated kinase activation and chemotaxis defects. We also demonstrate that a null mutation lacking the PAK family member myosin I heavy chain kinase (MIHCK) shows mild chemotaxis defects, including the formation of lateral pseudopodia. A null strain lacking both PAKc and the PAK family member MIHCK exhibits severe loss of cell movement, suggesting that PAKc and MIHCK may cooperate to regulate a common chemotaxis pathway.     

Dystroglycan is required for proper retinal layering

Dystroglycan (DG) is a transmembrane receptor linking the extracellular matrix to the internal cytoskeleton. Its structural function has been mainly characterized in muscle fibers, but DG plays signaling and developmental roles also in different tissues and cell types. We have investigated the effects of dystroglycan depletion during eye development of Xenopus laevis. We have injected a specific morpholino (Mo) antisense oligonucleotide in the animal pole of one dorsal blastomere of embryos at four cells stage. Mo-mediated loss of DG function caused disruption of the basal lamina layers, increased apoptosis and reduction of the expression domains of specific retinal markers, at early stages. Later in development, morphants displayed unilateral ocular malformations, such as microphtalmia and retinal delayering with photoreceptors and ganglion cells scattered throughout the retina or aggregated in rosette-like structures. These results recall the phenotypes observed in specific human diseases and suggest that DG presence is crucial at early stages for the organization of retinal architecture.     

The manganese-stabilizing protein is required for photosystem II assembly/stability and photoautotrophy in higher plants

Interfering RNA was used to suppress the expression of two genes that encode the manganese-stabilizing protein of photosystem II in Arabidopsis thaliana, MSP-1 (encoded by psbO-1, At5g66570), and MSP-2 (encoded by psbO-2, At3g50820). A phenotypic series of transgenic plants was recovered that expressed high, intermediate, and low amounts of these two manganese-stabilizing proteins. Chlorophyll fluorescence induction and decay analyses were performed. Decreasing amounts of expressed protein led to the progressive loss of variable fluorescence and a marked decrease in the fluorescence quantum yield (F(v)/F(m)) in both the absence and the presence of dichloromethylurea. This result indicated that the amount of functional photosystem II reaction centers was compromised in the plants that exhibited intermediate and low amounts of the manganese-stabilizing proteins. An analysis of the decay of the variable fluorescence in the presence of dichlorophenyldimethylurea indicated that charge recombination between Q ((A-)) and the S(2) state of the oxygen-evolving complex was seriously retarded in the plants that expressed low amounts of the manganese stabilizing proteins. This may have indicated a stabilization of the S(2) state in the absence of the extrinsic component. Immunological analysis of the photosystem II protein complement indicated that significant losses of the CP47, CP43, and D1 proteins occurred upon the loss of the manganese-stabilizing proteins. This indicated that these extrinsic proteins were required for photosystem II core assembly/stability. Additionally, although the quantity of the 24-kDa extrinsic protein was only modestly affected by the loss of the manganese-stabilizing proteins, the 17-kDa extrinsic protein dramatically decreased. The control proteins ribulose bisphosphate carboxylase and cytochrome f were not affected by the loss of the manganese-stabilizing proteins; the photosystem I PsaB protein, however, was significantly reduced in the low expressing transgenic plants. Finally, it was determined that the transgenic plants that expressed low amounts of the manganese-stabilizing proteins could not grow photoautotrophically.     

The Arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme oxygenase

The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids. Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis.     

Mutant analyses define multiple roles for phytochrome C in Arabidopsis photomorphogenesis

The analysis of Arabidopsis mutants deficient in the A, B, D, and E phytochromes has revealed that each of these phytochrome isoforms has both distinct and overlapping roles throughout plant photomorphogenesis. Although overexpression studies of phytochrome C (phyC) have suggested photomorphogenic roles for this receptor, conclusive evidence of function has been lacking as a result of the absence of mutants in the PHYC locus. Here, we describe the isolation of a T-DNA insertion mutant of phyC (phyC-1), the subsequent creation of mutant lines deficient in multiple phytochrome combinations, and the physiological characterization of these lines. In addition to operating as a weak red light sensor, phyC may perform a significant role in the modulation of other photoreceptors. phyA and phyC appear to act redundantly to modulate the phyB-mediated inhibition of hypocotyl elongation in red light and to function together to regulate rosette leaf morphology. In addition, phyC performs a significant role in the modulation of blue light sensing. Several of these phenotypes are supported by the parallel analysis of a quadruple mutant deficient in phytochromes A, B, D, and E, which thus contains only active phyC. Together, these data suggest that phyC has multiple functions throughout plant development that may include working as a coactivator with other phytochromes and the cryptochrome blue light receptors.     

Cellulose biosynthesis and deposition in higher plants

The plant cell wall is central to plant development. Cellulose is a major component of plant cell walls, and is the world's most abundant biopolymer. Cellulose contains apparently simple linear chains of glucose residues, but these chains aggregate to form immensely strong microfibrils. It is the physical properties of these microfibrils that, when laid down in an organized manner, are responsible for both oriented cell elongation during plant growth and the strength required to maintain an upright growth habit. Despite the importance of cellulose, only recently have we started to unravel details of its synthesis. Mutational analysis has allowed us to identify some of the proteins involved in its synthesis at the plasma membrane, and to define a set of cellulose synthase enzymes essential for cellulose synthesis. These proteins are organized into a very large plasma membrane-localized protein complex. The way in which this protein complex is regulated and directed is central in depositing cellulose microfibrils in the wall in the correct orientation, which is essential for directional cell growth. Recent developments have given us clues as to how cellulose synthesis and deposition is regulated, an understanding of which is essential if we are to manipulate cell wall composition.     

Purine and pyrimidine biosynthesis in higher plants

Purine and pyrimidine nucleotides have important functions in a multitude of biochemical and developmental processes during the life cycle of a plant. In higher plants the processes of nucleotide metabolism are poorly understood, but it is in principle accepted that nucleotides are essential constituents of fundamental biological functions. Despite of its significance, higher plant nucleotide metabolism has been poorly explored during the last 10–20 years (Suzuki and Takahashi 1977, Schubert 1986, Wagner and Backer 1992). But considerable progress was made on purine biosynthesis in nodules of ureide producing tropical legumes, where IMP-synthesis plays a dominant role in primary nitrogen metabolism (Atkins and Smith 2000, Smith and Atkins 2002). Besides these studies on tropical legumes, this review emphasises on progress made in analysing the function in planta of genes involved in purine and pyrimidine biosynthesis and their impact on metabolism and development.     

The ArcB leucine zipper domain is required for proper ArcB signaling

The Arc two-component system modulates the expression of numerous genes in response to respiratory growth conditions. This system comprises ArcA as the response regulator and ArcB as the sensor kinase. ArcB is a tripartite histidine kinase whose activity is regulated by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Here, we report that the ArcB protein segment covering residues 70-121, fulfills the molecular characteristics of a leucine zipper containing coiled coil structure. Also, mutational analyses of this segment reveal three different phenotypical effects to be distributed along the coiled coil structure of ArcB, demonstrating that this motif is essential for proper ArcB signaling.     

Complete loss of photoperiodic response in the rice mutant line X61 is caused by deficiency of phytochrome chromophore biosynthesis gene

In rice (Oryza sativa), a short-day plant, photoperiod is the most favorable external signal for floral induction because of the constant seasonal change throughout the years. Compared with Arabidopsis, however, a large part of the regulation mechanism of the photoperiodic response in rice still remains unclear due mainly to the lack of induced mutant genes. An induced mutant line X61 flowers 35 days earlier than its original variety Gimbozu under a natural photoperiod in Kyoto (35°01′N). We attempted to identify the mutant gene conferring early heading to X61. Experimental results showed that the early heading of X61 was conferred by a complete loss of photoperiodic response due to a novel single recessive mutant gene se13. This locus interacts with two crucial photoperiod sensitivity loci, Se1 and E1. Wild type alleles at these two loci do not function in coexistence with se13 in a homozygous state, suggesting that Se13 is an upstream locus of the Se1 and E1 loci. Linkage analysis showed that Se13 is located in a 110 kb region between the two markers, INDEL3735_1 and INDEL3735_3 on chromosome 1. A database search suggested that the Se13 gene is identical to AK101395 (=OsHY2), which encodes phytochromobilin synthase, a key enzyme in phytochrome chromophore biosynthesis. Subsequent sequence analysis revealed that X61 harbors a 1 bp insertion in exon 1 of OsHY2, which induces a frame-shift mutation producing a premature stop codon. It is therefore considered that the complete loss of photoperiodic response of X61 is caused by a loss of function of the Se13 (OsHY2) gene involved in phytochrome chromophore biosynthesis.     

Adequate connexin-mediated coupling is required for proper insulin production

To assess whether connexin (Cx) expression contributes to insulin secretion, we have investigated normal and tumoral insulin-producing cells for connexins, gap junctions, and coupling. We have found that the glucose-sensitive cells of pancreatic islets and of a rat insulinoma are functionally coupled by gap junctions made of Cx43. In contrast, cells of several lines secreting insulin abnormally do not express Cx43, gap junctions, and coupling. After correction of these defects by stable transfection of Cx43 cDNA, cells expressing modest levels of Cx43 and coupling, as observed in native beta-cells, showed an expression of the insulin gene and an insulin content that were markedly elevated, compared with those observed in both wild-type (uncoupled) cells and in transfected cells overexpressing Cx43. These findings indicate that adequate levels of Cx-mediated coupling are required for proper insulin production and storage.     

NuMA is required for the proper completion of mitosis

NuMA is a 236-kD intranuclear protein that during mitosis is distributed into each daughter cell by association with the pericentrosomal domain of the spindle apparatus. The NuMA polypeptide consists of globular head and tail domains separated by a discontinuous 1500 amino acid coiled-coil spacer. Expression of human NuMA lacking its globular head domain results in cells that fail to undergo cytokinesis and assemble multiple small nuclei (micronuclei) in the subsequent interphase despite the appropriate localization of the truncated NuMA to both the nucleus and spindle poles. This dominant phenotype is morphologically identical to that of the tsBN2 cell line that carries a temperature-sensitive mutation in the chromatin-binding protein RCC1. At the restrictive temperature, these cells end mitosis without completing cytokinesis followed by micronucleation in the subsequent interphase. We demonstrate that the wild-type NuMA is degraded in the latest mitotic stages in these mutant cells and that NuMA is excluded from the micronuclei that assemble post-mitotically. Elevation of NuMA levels in these mutant cells by forcing the expression of wild-type NuMA is sufficient to restore post-mitotic assembly of a single normal-sized nucleus. Expression of human NuMA lacking its globular tail domain results in NuMA that fails both to target to interphase nuclei and to bind to the mitotic spindle. In the presence of this mutant, cells transit through mitosis normally, but assemble micronuclei in each daughter cell. The sum of these findings demonstrate that NuMA function is required during mitosis for the terminal phases of chromosome separation and/or nuclear reassembly.     

The essential GTPase YqeH is required for proper ribosome assembly in Bacillus subtilis

Recent work with bacteria and eukaryotes has shown that GTPases play important roles in ribosome assembly. Here we show that the essential GTPase YqeH is required for proper 70S ribosome formation and 30S subunit assembly/stability in Bacillus subtilis.