Expression of an anther-specific chalcone synthase-like gene is correlated with uninucleate microspore development in Nicotiana sylvestris

Two cDNA clones, specifically expressed in Nicotiana sylvestris anthers during uninucleate microspore development, were isolated using a subtractive hybridization approach. Sequence analysis showed that one of them, NSCHSLK, displayed a high level of similarity to several anther-specific chalcone synthase-like (CHSLK) proteins and an ORF from chromosome 1 of Arabidopsis thaliana. A lower, but significant, similarity to chalcone synthases and closely related enzymes (CHSRE) was also detected. The structure of the nschslk gene was found to be typical of the chalcone (chs) / stilbene (sts) synthase family. Expression of NSCHSLK mRNA was confined to microspores and tapetal cells. UV-irradiation or infection with Phytophthora parasitica var. nicotianae of transgenic Nicotiana benthamiana plants carrying a chimeric nschslk/GUS gene indicated that the nschslk promoter exhibits the same anther-specific, developmentally regulated expression pattern. Comparison of CHSRE and CHSLK polypeptide sequences revealed some important similarities and differences between the two groups. The data presented in this study, suggest that the anther-specific chslk genes represent a separate sub-family of plant polyketide synthases related to chs/sts in terms of gene structure, polypeptide sequence and the possible catalytic mechanism, but differing in substrate/product specificity. The putative role of CHSLK enzymes in anther development and particularly in exine synthesis is discussed.     

Arabidopsis indole synthase, a homolog of tryptophan synthase alpha, is an enzyme involved in the Trp-independent indole-containing metabolite biosynthesis

The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.     

Dark is a Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway

Here we identify a new gene, dark, which encodes a Drosophila homologue of mammalian Apaf-1 and Caenorhabditis elegans CED-4, cell-death proteins. Like Apaf-1, but in contrast to CED-4, Dark contains a carboxy-terminal WD-repeat domain necessary for interactions with the mitochondrial protein cytochrome c. Dark selectively associates with another protein involved in apoptosis, the fly apical caspase, Dredd. Dark-induced cell killing is suppressed by caspase-inhibitory peptides and by a dominant-negative mutant Dredd protein, and enhanced by removal of the WD domain. Loss-of-function mutations in dark attenuate programmed cell deaths during development, causing hyperplasia of the central nervous system, and other abnormalities including ectopic melanotic tumours and defective wings. Moreover, ectopic cell killing by the Drosophila cell-death activators, Reaper, Grim and Hid, is substantially suppressed in dark mutants. These findings establish dark as an important apoptosis effector in Drosophila and raise profound evolutionary considerations concerning the relationship between mitochondrial components and the apoptosis-promoting machinery.     

Crystal structure of chalcone synthase,a key enzyme for isoflavonoid biosynthesis in soybean

Isoflavonoid is one of the groups of flavonoids that play pivotal roles in the survival of land plants. Chalcone synthase (CHS), the first enzyme of the isoflavonoid biosynthetic pathway, catalyzes the formation of a common isoflavonoid precursor. We have previously reported that an isozyme of soybean CHS (termed GmCHS1) is a key component of the isoflavonoid metabolon, a protein complex to enhance efficiency of isoflavonoid production. Here, we determined the crystal structure of GmCHS1 as a first step of understanding the metabolon structure, as well as to better understand the catalytic mechanism of GmCHS1.     

DnaJA4 is a SREBP-regulated chaperone involved in the cholesterol biosynthesis pathway

Using subtractive hybridization technique in 3T3-L1 adipocytes overexpressing constitutively active SREBP2, we have identified a DnaJ/Hsp40 chaperone, DnaJA4, as a new SREBP-responsive gene. SREBP2 regulation was demonstrated by changes in DnaJA4 mRNA under conditions of altered sterol status that were strictly parallel to that of well-characterized SREBP targets (LDL receptor and HMG-CoA reductase). The role of SREBP2 was further established using adenoviral overexpression of a dominant negative SREBP2, which abolished cholesterol-regulated changes in DnaJA4 expression. To determine the functional significance of this regulation, DnaJA4 was overexpressed in COS cells, which induced a specific increase in the synthesis of cholesterol from acetate. We also observed that DnaJA4 overexpression increased the activity and the protein content of HMG-CoA reductase, the rate limiting enzyme in this pathway. At the molecular level, DnaJA4 overexpression did not alter HMG-CoA reductase stability or mRNA levels, suggesting a co-translational effect of the chaperone. In the DnaJ/Hsp40 family, DnaJA4 uniquely exhibited SREBP-regulated expression, and also responded to heat shock. Through its responsiveness to SREBP, and its stimulatory effect on cholesterol synthesis, the DnaJA4 chaperone can be viewed as a new player in cholesterol synthesis. These data suggest a link between molecular chaperones, heat stress and cholesterol synthesis.     

beta-Cyanoalanine synthase is a mitochondrial cysteine synthase-like protein in spinach and Arabidopsis

beta-Cyano-alanine synthase (CAS; EC 4.4.1.9) plays an important role in cyanide metabolism in plants. Although the enzymatic activity of beta-cyano-Ala synthase has been detected in a variety of plants, no cDNA or gene has been identified so far. We hypothesized that the mitochondrial cysteine synthase (CS; EC 4.2.99. 8) isoform, Bsas3, could actually be identical to CAS in spinach (Spinacia oleracea) and Arabidopsis. An Arabidopsis expressed sequence tag database was searched for putative Bsas3 homologs and four new CS-like isoforms, ARAth;Bsas1;1, ARAth;Bsas3;1, ARAth;Bsas4;1, and ARAth;Bsas4;2, were identified in the process. ARAth;Bsas3;1 protein was homologous to the mitochondrial SPIol;Bsas3;1 isoform from spinach, whereas ARAth;Bsas4;1 and ARAth;Bsas4;2 proteins defined a new class within the CS-like proteins family. In contrast to spinach SPIol;Bsas1;1 and SPIol;Bsas2;1 recombinant proteins, spinach SPIol;Bsas3;1 and Arabidopsis ARAth;Bsas3;1 recombinant proteins exhibited preferred substrate specificities for the CAS reaction rather than for the CS reaction, which identified these Bsas3 isoforms as CAS. Immunoblot studies supported this conclusion. This is the first report of the identification of CAS synthase-encoding cDNAs in a living organism. A new nomenclature for CS-like proteins in plants is also proposed.     

The evolutionarily conserved porcupine gene family is involved in the processing of the Wnt family.

The Drosophila segment polarity gene product Porcupine (Porc) was first identified as being necessary for processing Wingless (Wg), a Drosophila Wnt (Wnt) family member. Mouse and Xenopus homologs of porc (Mporc and Xporc) were identified and found to encode endoplasmic reticulum (ER) proteins with multiple transmembrane domains. In contrast with porc, four different types of Mporc and Xporc mRNA (A-D) are generated from a single gene by alternative splicing. Mporc mRNA is differentially expressed during embryogenesis and in various adult tissues, demonstrating that the alternative splicing is regulated to synthesize the specific types of Mporc. In transfected mammalian cells, all Mporc types affect the processing of mouse Wnt 1, 3A, 4, 6, and 7B but not 5A. Furthermore, all Mporc types are co-immunoprecipitated with various Wnt proteins. These results suggest that Mporc may function as a chaperone-like molecule for Wnt. Interestingly, all Mporc types can substitute for Porc, as they are able to rescue the phenotypes of Drosophila porc embryos. Consistent with this observation, Mporc, like Porc, modifies the processing of Wg expressed in mammalian cells. These results demonstrate that the porc gene family encodes the multitransmembrane ER proteins, which are evolutionarily well conserved and involved in processing the Wnt family.     

Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway

Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile.     

Regulation of RNA interference by Hsp90 is an evolutionarily conserved process

RNAi is a highly conserved mechanism in almost every eukaryote with a few exceptions including the model organism Saccharomyces cerevisiae. A recent study showed that the introduction of the two core components of canonical RNAi systems, Argonaute and Dicer, from another budding yeast, Saccharomyces castellii, restores RNAi in S. cerevisiae. We report here that a functional RNAi system can be reconstituted in yeast with the introduction of only S. castellii Dicer and human Argonaute2. Interestingly, whether or not TRBP2 was present, human Dicer was unable to restore RNAi with either S. castellii or human Argonaute. Contrary to previous reports, we find that human Dicer, TRBP2 and Argonaute2 are not sufficient to reconstitute RNAi in yeast when bona fide RNAi precursors are co-expressed. We and others have previously reported that Hsp90 regulates conformational changes in human and Drosophila Argonautes required to accommodate the loading of dsRNA duplexes. Here we show that the activities of both human and S. castellii Argonaute are subject to Hsp90 regulation in S. cerevisiae. In summary, our results suggest that regulation of the RNAi machinery by Hsp90 may have evolved at the same time as ancestral RNAi.     

Evidence for an evolutionarily conserved interaction between cell wall biosynthesis and flowering in maize and sorghum

Background  

Factors that affect flowering vary among different plant species, and in the grasses in particular the exact mechanism behind this transition is not fully understood. The brown midrib (bm) mutants of maize (Zea mays L.), which have altered cell wall composition, have different flowering dynamics compared to their wild-type counterparts. This is indicative of a link between cell wall biogenesis and flowering. In order to test whether this relationship also exists in other grasses, the flowering dynamics in sorghum (Sorghum bicolor (L.) Moench) were investigated. Sorghum is evolutionarily closely related to maize, and a set of brown midrib (bmr) mutants similar to the maize bm mutants is available, making sorghum a suitable choice for study in this context.     

Plumbing the depths of PUFA biosynthesis: a novel polyketide synthase-like pathway from marine organisms

Polyunsaturated fatty acids (PUFAs) are involved in determining the biophysical properties of membranes as well as being precursors for signalling molecules. C(20+) PUFA biosynthesis is catalysed by sequential desaturation and fatty acyl elongation reactions. This aerobic biosynthetic pathway was thought to be taxonomically conserved, but an alternative anaerobic pathway for the biosynthesis of polyunsaturated fatty acids is now known to exist that is analogous to polyketide synthases (PKS). These novel PKS genes could be used to direct the synthesis of PUFAs in heterologous hosts, as well as exploiting the combinatorial chemistry of PKSs to make unusual fatty acids.     

Three-dimensional structure of 6-pyruvoyl tetrahydropterin synthase, an enzyme involved in tetrahydrobiopterin biosynthesis.

The crystal structure of rat liver 6-pyruvoyl tetrahydropterin synthase has been solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.4% at 2.3 A resolution. 6-Pyruvoyl tetrahydrobiopterin synthase catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. The functional enzyme is a hexamer of identical subunits. The 6-pyruvoyl tetrahydropterin synthase monomer folds into a sequential, four-stranded, antiparallel beta-sheet with a 25 residue, helix-containing insertion between strands 1 and 2 at the bottom of the molecule, and a segment between strands 2 and 3 forming a pair of antiparallel helices, layered on one side of the beta-sheet. Three 6-pyruvoyl tetrahydropterin synthase monomers form an unusual 12-stranded antiparallel beta-barrel by tight association between the N- and C-terminal beta-strands of two adjacent subunits. The barrel encloses a highly basic pore of 6-12 A diameter. Two trimers associate in a head-to-head fashion to form the active enzyme complex. The substrate-binding site is located close to the trimer-trimer interface and comprises residues from three monomers: A, A' and B. A metal-binding site in the substrate-binding pocket is formed by the three histidine residues 23, 48 and 50 from one 6-pyruvoyl tetrahydropterin synthase subunit. Close to the metal, but apparently not liganding it, are residues Cys42, Glu133 (both from A) and His89 (from B), which might serve as proton donors and acceptors during catalysis.     

Insulin/IGF-I-signaling pathway: an evolutionarily conserved mechanism of longevity from yeast to humans

Although the underlying mechanisms of longevity are not fully understood, it is known that mutation in genes that share similarities with those in humans involved in the insulin/insulin-like growth factor I (IGF-I) signal response pathway can significantly extend life span in diverse species, including yeast, worms, fruit flies, and rodents. Intriguingly, the long-lived mutants, ranging from yeast to mice, share some important phenotypic characteristics, including reduced insulin signaling, enhanced sensitivity to insulin, and reduced IGF-I plasma levels. Such genetic homologies and phenotypic similarities between insulin/IGF-I pathway mutants raise the possibility that the fundamental mechanism of aging may be evolutionarily conserved from yeast to mammals. Very recent findings also provide novel and intriguing evidence for the involvement of insulin and IGF-I in the control of aging and longevity in humans. In this study, we focus on how the insulin/IGF-I pathway controls yeast, nematode, fruit fly, and rodent life spans and how it is related to the aging process in humans to outline the prospect of a unifying mechanism in the genetics of longevity.