Mechanistic studies of the phytochromobilin synthase HY2 from Arabidopsis

Phytochromobilin (PPhiB) is an open chain tetrapyrrole molecule that functions as the chromophore of light-sensing phytochromes in plants. Derived from heme, PPhiB is synthesized through an open chain tetrapyrrole intermediate, biliverdin IXalpha (BV), in the biosynthesis pathway. BV is subsequently reduced by the PPhiB synthase HY2 in plants. HY2 is a ferredoxin-dependent bilin reductase that catalyzes the reduction of the A-ring 2,3,3(1),3(2)-diene system to produce an ethylidene group for assembly with apophytochromes. In this study, we sought to determine the catalytic mechanism of HY2. Data from UV-visible and EPR spectroscopy showed that the HY2-catalyzed BV reaction proceeds via a transient radical intermediate. Site-directed mutagenesis showed several ionizable residues that are involved in the catalytic steps. Detailed analysis of these site-directed mutants highlighted a pair of aspartate residues central to proton donation and substrate positioning. A mechanistic prediction for the HY2 reaction is proposed. These results support the hypothesis that ferredoxin-dependent bilin reductases reduce BV through a radical mechanism, but their double bond specificity is decided by strategic placement of different proton-donating residues surrounding the bilin substrate in the active sites.     

A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase

The narB gene from the cyanobacterium Synechococcus sp. PCC 7942 was cloned downstream from the LacI-regulated promoter P_trc in the Escherichia coli vector pTrc99A, rendering plasmid pCSLM1. Addition of isopropyl--D-thiogalactoside to E. coli (pCSLM1) resulted in the parallel expression of a 76 kDa polypeptide and a nitrate reductase activity with properties identical to those known for nitrate reductase isolated from Synechococcus cells. As is the case for nitrate reductase from Synechococcus cells, either reduced methyl viologen or reduced ferredoxin could be used as an electron donor for the reduction of nitrate catalyzed by E. coli (pCSLM1) extracts. This data shows that narB is a cyanobacterial structural gene for nitrate reductase.     

ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase

Polyamines have been implicated in a wide range of biological processes, including growth and development in bacteria and animals, but their function in higher plants is unclear. Here we show that the Arabidopsis: ACAULIS5 (ACL5) gene, whose inactivation causes a defect in the elongation of stem internodes by reducing cell expansion, encodes a protein that shares sequence similarity with the polyamine biosynthetic enzymes spermidine synthase and spermine synthase. Expression of the recombinant ACL5 protein in Escherichia coli showed that ACL5 possesses spermine synthase activity. Restoration of the acl5 mutant phenotype by somatic reversion of a transposon-induced allele suggests a non-cell-autonomous function for the ACL5 gene product. We also found that expression of the ACL5 cDNA under the control of a heat shock gene promoter in acl5 mutant plants restores the phenotype in a heat shock-dependent manner. The results of the experiments showed that polyamines play an essential role in promotion of internode elongation through cell expansion in Arabidopsis: We discuss the relationships to plant growth regulators such as auxin and gibberellins that have related functions.     

Antigenic similarities between ferredoxin-dependent nitrite reductase and glutamate synthase fromChlamydomonas reinhardtii

Polyclonal antisera were prepared against ferredoxin-nitrite reductase (EC 1.7.7.1) and ferredoxin-glutamate synthase (glutamate synthase (ferredoxin); EC 1.4.7.1) from the green algaChlamydomonas reinhardtii. The anti-glutamate synthase antibodies recognized both glutamate synthase and nitrite reductase, but inhibited only the ferredoxin-linked activity of the latter enzyme and not the activity dependent on methyl viologen. Analogously, the anti-nitrite reductase antibodies recognized glutamate synthase and nitrite reductase but the first enzyme was only poorly inhibited. Free ferredoxin protected the nitrite reductase against its inactivation by anti-glutamate synthase antibodies. These results indicate that the ferredoxin-dependent glutamate synthase and nitrite reductase from this alga share common antigenic determinants, and that these are located at the ferrodoxin-binding domains.     

Arabidopsis IRT2 gene encodes a root-periphery iron transporter

Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters. One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast. Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level. IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation. However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast. IRT2 expression is detected only in roots of A. thaliana plants, and is upregulated by iron deficiency. By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter. Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions.     

The Arabidopsis AMP1 gene encodes a putative glutamate carboxypeptidase

Arabidopsis amp1 mutants show pleiotropic phenotypes, including altered shoot apical meristems, increased cell proliferation, polycotyly, constitutive photomorphogenesis, early flowering time, increased levels of endogenous cytokinin, and increased cyclin cycD3 expression. We have isolated the AMP1 gene by map-based cloning. The AMP1 cDNA encodes a 706;-amino acid polypeptide with significant similarity to glutamate carboxypeptidases. The AMP1 mRNA was expressed in all tissues examined, with higher expression in roots, stems, inflorescences, and siliques. Microarray analysis identified four mRNA species with altered expression in two alleles of amp1, including upregulation of CYP78A5, which has been shown to mark the shoot apical meristem boundary. The similarity of the AMP1 protein to glutamate carboxypeptidases, and in particular to N-acetyl alpha-linked acidic dipeptidases, suggests that the AMP1 gene product modulates the level of a small signaling molecule that acts to regulate a number of aspects of plant development, in particular the size of the apical meristem.     

The Arabidopsis AtPNG1 gene encodes a peptide: N-glycanase

Deglycosylation of misfolded proteins by the endoplasmic reticulum-associated degradation (ERAD) pathway is catalyzed by peptide:N-glycanases (PNGases) that are highly conserved among mammals and yeast. The catalytic mechanism of PNGases employs a catalytic triad consisting of Cys, His and Asp residues, which is shared by other enzyme families such as cysteine proteases and protein cross-linking transglutaminases (TGases). In contrast to the yeast and mammalian systems, very little is known about ERAD in plants and the enzymes responsible for proper clearance of misfolded plant proteins. We have used a computer-based modeling approach to identify an Arabidopsis thaliana PNGase (AtPNG1). AtPNG1 is encoded by a single-copy gene and displays high structural homology with known PNGases. Importantly, heterologous expression of AtPNG1 restored N-glycanase activity in a PNGase-deficient Saccharomyces cerevisiae mutant. The AtPNG1 gene is uniformly and constitutively expressed at low levels throughout all developmental stages of the plant, and its expression does not appear to be subject to substantial regulation by external stimuli. Recently, recombinant AtPNG1 produced in Escherichia coli was reported to display TGase activity (Della Mea et al., Plant Physiol. 135, 2046-54, 2004). However, inactivation of the AtPNG1 gene did not result in decreased TGase activity in the mutant plant, and recombinant AtPNG1 produced in S. cerevisiae exhibited only residual TGase activity. We propose that the AtPNG1 gene encodes a bona fide peptide:N-glycanase that contributes to ERAD-related protein quality control in plants.     

Saccharomyces cerevisiae ACR2 gene encodes an arsenate reductase

The ACR2 gene of Saccharomyces cerevisiae was disrupted by insertion of a HIS3 gene. Cells with the disruption were sensitive to arsenate. This phenotype could be complemented by ACR2 on a plasmid. The ACR2 gene was cloned and expressed in Escherichia coli as a malE gene fusion with a C-terminal histidine tag. The combination of chimeric MBP-Acr2-6H protein and yeast cytosol from an ACR2-disrupted strain exhibited arsenate reductase activity.     

The specificity of biliverdin reductase. The reduction of biliverdin XIII isomers

The substrate specificity of the different molecular forms of biliverdin reductase (bilirubin:NAD(P)+ oxidoreductase, EC 1.3.1.24) using biliverdin XIII alpha, XIII beta and XIII gamma was examined. It was found that molecular form 1 (the major form in normal rat liver) reduced biliverdin XIII alpha at a much higher rate than the other two isomers. Molecular form 2 (the minor form) reduced isomers XIII alpha and XIII beta at similar rates, while molecular form 3 (the major form induced by CoCl2 treatment) reduced the XIII beta isomer at a slightly higher rate than the XIII alpha isomer. Molecular forms 2 and 3, both reduced isomer XIII gamma more slowly than they reduced the XIII alpha and XIII beta isomers. These results are similar to those obtained previously using biliverdins IX alpha, IX beta and IX gamma, suggesting that biliverdin reductase specificity is related to the type of the isomer rather than to the series (IX or XIII) of the isomer.     

Localization of Blvr, biliverdin reductase, on mouse chromosome 2

Biliverdin reductase, Blvr, has been mapped on mouse chromosome 2, using an electrophoretic variant. The gene order obtained from a five-point cross and calculating genetic distance as percentage recombination +/- SE was Blvr-3.7 +/- 1.8-pa-0.9 +/- 0.9-we-5.6 +/- 2.2-un-2.8 +/- 1.6-a. Thus, Blvr must be closely linked to several genes, limb deformity (ld), Strong's luxoid, (1st), and small eye (Sey), involved in limb and/or craniofacial development. In man, GCPS, Greig cephalopolysyndactyly syndrome, associated with limb anomalies and craniofacial dysmorphism has been assigned to 7p13. Thus GCPS probably maps near to BLVR, the human homolog of Blvr, and also to TCRG, T-cell receptor gamma. However, in mouse, Blvr and Tcrg are asyntenic, and the proposed murine homolog of GCPS is extra toes (Xt), closely linked to Tcrg on chromosome 13. Possibly in the common ancestor of man and mouse there was a cluster of genes for craniofacial and limb development, which remains linked to BLVR and TCRG in man, but has become broken up in the mouse with the loss of synteny of Blvr and Tcrg, although linkage of some genes in the cluster to Blvr and Tcrg has been retained.     

The pgp1 mutant locus of Arabidopsis encodes a phosphatidylglycerolphosphate synthase with impaired activity

Phosphatidylglycerol is a ubiquitous phospholipid that is also present in the photosynthetic membranes of plants. Multiple independent lines of evidence suggest that this lipid plays a critical role for the proper function of photosynthetic membranes and cold acclimation. In eukaryotes, different subcellular compartments are competent for the biosynthesis of phosphatidylglycerol. Details on the plant-specific pathways in different organelles are scarce. Here, we describe a phosphatidylglycerol biosynthesis-deficient mutant of Arabidopsis, pgp1. The overall content of phosphatidylglycerol is reduced by 30%. This mutant carries a point mutation in the CDP-alcohol phosphotransferase motif of the phosphatidylglycerolphosphate synthase (EC 2.7.8.5) isoform encoded by a gene on chromosome 2. The mutant shows an 80% reduction in plastidic phosphatidylglycerolphosphate synthase activity consistent with the plastidic location of this particular isoform. Mutant plants are pale green, and their photosynthesis is impaired. This mutant provides a promising new tool to elucidate the biosynthesis and function of plastidic phosphatidylglycerol in seed plants.     

Human herpesvirus 8 gene encodes a functional thymidylate synthase

We demonstrate that human herpesvirus 8, obtained from the lymphoma cell line BC-3 as well as from Kaposi's sarcoma lesions, carries a gene that encodes a functional thymidylate synthase (TS). The particular characteristics of this enzyme are studied and compared to the characteristics of TSs encoded by other organisms.     

Detection of spatial-specific phytochrome responses using targeted expression of biliverdin reductase in Arabidopsis

To regulate levels of holophytochrome in a spatial-specific manner and investigate the major sites of action of phytochromes during seedling development, we constructed transgenic Arabidopsis (Arabidopsis thaliana) plant lines expressing plastid-targeted mammalian biliverdin IXα reductase (pBVR) under regulatory control of CAB3 and MERI5 promoters. Comparative photobiological and phenotypic analyses indicated that spatial-specific expression of pBVR led to the disruption of distinct subsets of phytochrome-regulated responses for different promoters. pBVR expression in photosynthetic tissues (CAB3pBVR lines) had intermediate effects on chlorophyll accumulation, carotenoid production, anthocyanin synthesis, and leaf development responses in white-light conditions. CAB3pBVR expression, however, resulted in distinctive phenotypes in far-red (FR) conditions. A number of FR high irradiance responses were disrupted in CABpBVR lines, including FR-dependent inhibition of hypocotyl elongation and stimulation of anthocyanin accumulation. By contrast, preferential expression of pBVR in the shoot apical meristem in MERI5pBVR lines resulted in a phytochrome-deficient, leaf development phenotype under short-day growth conditions. These results implicate leaf-localized phytochrome A as having a unique role in regulating FR-mediated hypocotyl elongation and meristem- and/or leaf primordia-localized phytochromes as having a novel role in phytochrome-dependent responses. Taken together, these studies demonstrate the efficacy of selectively inactivating distinct phytochrome-mediated responses by regulated expression of BVR in transgenic plants, a novel means to investigate the sites of phytochrome photoperception and to regulate specifically light-mediated plant growth and development.     

A chlorate-resistant mutant defective in the regulation of nitrate reductase gene expression in Arabidopsis defines a new HY locus.

Light acts both directly as a signal and indirectly through photosynthesis to regulate the expression of genes encoding nitrate reductase (NR). Here, we report the isolation and characterization of a novel chlorate-resistant mutant that is defective in the regulation of NR gene expression. The response of NR2, but not NR1 or the gene encoding nitrate reductase (NiR), to light signals was impaired in this Arabidopsis mutant, designated cr88. In addition to NR2, the light regulation of the genes encoding the chlorophyll a/b binding protein (CAB) and the small subunit of ribulose bisphosphate carboxylase (RBCS) was also impaired in this mutant. These results suggest that the pathway through which light regulates the expression of NR2, CAB, and RBCS genes is different from those that regulate the expression of NR1 and NiR. An examination of the deetiolation process under different light spectrum showed that cr88 is defective in red light-mediated deetiolation. Complementation tests with various long hypocotyl (hy) mutants indicated that CR88 identifies a new HY locus. The possible functions of CR88 are discussed.