BAT1, a putative acyltransferase,modulates brassinosteroid levels in Arabidopsis

Brassinosteroids (BRs) are essential for various aspects of plant development. Cellular BR homeostasis is critical for proper growth and development of plants; however, its regulatory mechanism remains largely unknown. BAT1 (BR‐related acyltransferase 1), a gene encoding a putative acyltransferase, was found to be involved in vascular bundle development in a full‐length cDNA over‐expressor (FOX) screen. Over‐expression of BAT1 resulted in typical BR‐deficient phenotypes, which were rescued by exogenously applied castasterone and brassinolide. Analyses of BR profiles demonstrated that BAT1 alters levels of several brassinolide biosynthetic intermediates, including 6‐deoxotyphasterol, typhasterol and 6‐deoxocastasterone. BAT1 is mainly localized in the endoplasmic reticulum. BAT1 is highly expressed in young tissues and vascular bundles, and its expression is induced by auxin. These data suggest that BAT1 is involved in BR homeostasis, probably by conversion of brassinolide intermediates into acylated BR conjugates.     

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 TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) is a membrane enzyme that drives the final step in the formation of oils using diacylglycerol (DAG) and acyl-CoA to yield triacylglycerol (TAG). We identified a putative plant DGAT gene (TRIACYLGLYCEROL1: TAG1) and demonstrated its function by the cloning of two mutated alleles, designated AS11 (tag1-1) and ABX45 (tag1-2). One allele, AS11, has been previously characterised at the biochemical level. Mutant seeds contained less oil with a modified fatty acid profile and have reduced germination rates compared to wild-type controls. The TAG1 cDNA encodes for a 520-aa protein that possesses multiple putative transmembrane domains and shows 70 % similarity to a human DGAT cDNA.     

The fission yeast ptr1+ gene involved in nuclear mRNA export encodes a putative ubiquitin ligase

Fission yeast ptr1-1 is one of the mRNA transport mutants that accumulate poly(A)+ RNA in the nuclei at the nonpermissive temperature. We found that the ptr1+ gene encodes a homolog of Saccharomyces cerevisiae Tom1p, a hect type ubiquitin ligase. In ptr1-1, a conserved amino acid in the hect domain of Ptr1p is mutated. The ptr1+ gene is essential for growth and its mutation did not affect nuclear protein export. A ptr1-1 rae1-167 double mutant showed a synthetic effect on a growth defect, indicating that Ptr1p functionally interacts with an essential mRNA export factor Rae1p. We also isolated a multi-copy suppressor for ptr1-1 and found that it is the mpd2+ gene isolated as a multi-copy suppressor of cdc7-PD1.     

Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases

We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44°C on ampicillin was isolated. Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells. Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells. Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays. The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42 543. The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids. Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E. coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.     

The accumulation of phenylpropanoid and capsaicinoid compounds in cell cultures and whole fruit of the chilli pepper,Capsicum frutescens Mill

The accumulation of the phenylpropanoid precursors of capsaicin in suspended and immobilised cell cultures of C. frutescens has been studied and compared with accumulation in whole pepper fruit. The use of HPLC techniques has revealed that the phenolic precursors of capsaicin are present in chilli pepper cells at extremely low levels, irrespective of the source of tissue or the developmental state. Radioactive tracer studies have indicated that the majority of the phenolic derivatives of phenylalanine are ultimately bound to the insoluble fraction of the cells. Results from experiments where immobilised cell cultures were grown under conditions which enhance capsaicin yield would suggest that the diversion of compounds into this bound fraction has a considerable influence upon capsaicin biosynthesis in this system.     

Functional characterization of two diacylglycerol acyltransferase 1 genes in Mortierella alpina

Diacylglycerol acyltransferase (DGAT) is a crucial enzyme in the triacylglycerol (TAG) biosynthesis pathway. The oleaginous fungus Mortierella alpina can accumulate large amounts of arachidonic acid (ARA, C20:4) in the form of TAG. Therefore, it is important to study the functional characteristics of its DGAT. Two putative genes MaDGAT1A/1B encoding DGAT1 were identified in M. alpina ATCC 32222 genome by sequence alignment. Sequence alignment with identified DGAT1 homologs showed that MaDGAT1A/1B contain seven conserved motifs that are characteristic of the DGAT1 subfamily. Conserved domain analysis showed that both MaDGAT1A and MaDGAT1B belong to the Membrane-bound O-acyltransferases superfamily. The transforming with MaDGAT1A/1B genes could increase the accumulation of TAG in Saccharomyces cerevisiae to 4·47 and 7·48% of dry cell weight, which was 7·3-fold and 12·3-fold of the control group, respectively, but has no effect on the proportion of fatty acids in TAG. This study showed that MaDGAT1A/1B could effectively promote the accumulation of TAG and therefore may be used in metabolic engineering aimed to increase TAG production of oleaginous fungi.     

The capsanthin-capsorubin synthase gene: a candidate gene for the y locus controlling the red fruit colour in pepper

The red colour of pepper fruits is determined by the y⁺ dominant allele and the yellow colour by the y recessive allele. The capsanthin-capsorubin synthase (CCS) gene is activated specifically during the final stages of pepper fruit ripening. RFLP and specific-PCR polymorphisms derived from the CCS gene were analysed in a F2 progeny of a red by yellow-fruited cross. They cosegregated completely with fruit colour. Our results support the hypothesis that the yellow phenotype might result from a deletion of the CCS gene. These specific markers were integrated into the genetic map and will be useful for marker assisted plant breeding.     

The Drosophila patched gene encodes a putative membrane protein required for segmental patterning

The patched (ptc) gene is one of several segment polarity genes required for correct patterning within every segment of Drosophila. The absence of ptc gene function causes a transformation of the fate of cells in the middle part of each segment so that they form pattern elements characteristic of cells positioned around the segment border. Analysis of the mutant phenotype demonstrates that both segment and parasegment borders are included in the duplicated pattern of ptc mutants. We have cloned the ptc gene and deduced that the product is a 1286 amino acid protein with at least seven putative transmembrane alpha helices. ptc RNA is expressed in embryos in broad stripes of segmental periodicity that later split into two stripes per segment primordium. The pattern of expression does not directly predict the transformation seen in ptc mutant embryos, suggesting that ptc participates in cell interactions that establish pattern within the segment.     

The C1orf9 gene encodes a putative transmembrane member of a novel protein family

Here we report the characterization of a human mRNA encoding a novel protein denoted C1orf9 (chromosome 1 open reading frame 9). The cDNA sequence, derived from a testis cDNA library, contains 5700 bp which encodes an open reading frame of 1254 amino acids. The deduced protein contains a putative N-terminal signal peptide and one putative transmembrane region, indicating membrane localization. No significant homology was found with known characterized proteins. However, a 150 amino acid region has significant homology to deduced protein sequences from other organisms, including Caenorhabditis elegans (43% identity), Saccharomyces cerevisiae (47% identity), Schizosaccharomyces pombe (48% identity), and two proteins from Arabidopsis thaliana (42% and 40% identity), suggesting a novel family of conserved domains. The C1orf9 gene was assigned to chromosome 1q24. The gene spans approximately 78.7 kb and is organized into at least 24 exons. Expression analysis revealed a single C1orf9 mRNA species of approximately 6.0 kb with a predominant expression in pancreas and testis, and only low levels of expression in other tissues examined.     

The GAOLAOZHUANGREN1 gene encodes a putative glycosyltransferase that is critical for normal development and carbohydrate metabolism

Glycosyltransferases are enzymes that catalyze the attachment of a sugar molecule to specific acceptor molecules. These enzymes have been shown to play important roles in a number of biological processes. Whereas a large number of putative glycosyltransferase genes have been identified by genomic sequencing, the functions of most of these genes are unknown. Here we report the characterization of an Arabidopsis mutant, designated gaolaozhuangren1 (glz1), which is allelic to parvus characterized recently. The glz1 mutant exhibited a reduced plant stature, reduced size of organs in the shoot and dark-green leaves, indicating an important role of GLZ1 gene in normal development. The earliest GLZ1 expression appears at the shoot apical region of 4-d-old seedlings, which coincides with the onset of the glz1 morphological phenotypes. GLZ1 is expressed in a tissue-specific and developmentally regulated manner, predominantly in the stem and silique, and moderately in the flower. GLZ1 expression is strong in the midrib of rosette and cauline leaves; however, its expression was not detectable in the midrib of the cotyledon. Further analyses revealed that carbohydrate composition and distribution were aberrant in the glz1 mutant. These, together with the GLZ1 expression pattern, suggest a requirement for the GLZ1 function in normal sink-source transition during plant development.     

Development and application of a suite of non-pungency markers for the Pun1 gene in pepper (Capsicum spp.)

Pungency in peppers is due to the presence of capsaicinoid molecules, which are only produced in Capsicum species. The major gene Pun1 is required for the production of capsaicinoids. Three distinct mutant alleles of Pun1 have been found in three cultivated Capsicum species, one of which has been widely utilized by breeders. Although these mutations have been previously identified, a robust collection of molecular markers for the set of alleles is not available. This has been hindered by the existence of at least one paralogous locus that tends to amplify with Pun1. We present a suite of markers that can differentiate the four Pun1 alleles and test them on a diverse panel of pepper lines and in an F2 population segregating for pungency. These markers will be useful for pepper breeding, germplasm characterization, and seed purity testing.     

Molecular cloning and expression of the hot pepper ERabp1 gene encoding auxin-binding protein

The 22 kDa auxin-binding proteins in higher plants have received considerable attention as candidates for an auxin receptor. A cDNA clone Ca-ERabp1 of hot pepper (Capsicum annum) was isolated using the oligonucleotides as PCR primers. The cDNA codes for a polypeptide related to the major 22 kDa auxin-binding protein from maize and Arabidopsis ERabp1. The deduced amino acid sequence contains an endoplasmic reticulum retention signal, the KDEL sequence located at the C-terminal end, and has two possible auxin-binding sites, HRHSCE and YDDWSVPHTA conserved sequences. Northern hybridization analysis revealed that the Ca-ERabp1 gene is differentially expressed in total RNA isolated from different organs of a pepper plant, showing the highest level of expression in fruits but barely detectable in leaves and roots.     

The C. elegans sex determination gene laf-1 encodes a putative DEAD-box RNA helicase

The Caenorhabditis elegans gene laf-1 is critical for both embryonic development and sex determination. Laf-1 is thought to promote male cell fates by negatively regulating expression of tra-2 in both hermaphrodites and males. We cloned laf-1 and established that it encodes a putative DEAD-box RNA helicase related to Saccharomyces cerevisiae Ded1p and Drosophila Vasa. Three sequenced laf-1 mutations are missense alleles affecting a small region of the protein in or near helicase motif III. We demonstrate that the phenotypes resulting from laf-1 mutations are due to loss or reduction of laf-1 function, and that both laf-1 and a related helicase vbh-1 function in germline sex determination. Laf-1 mRNA is expressed in both males and hermaphrodites and in both the germline and soma of hermaphrodites. It is expressed at all developmental stages and is most abundant in embryos. LAF-1 is predominantly, if not exclusively, cytoplasmic and colocalizes with PGL-1 in P granules of germline precursor cells. Previous results suggest that laf-1 functions to negatively regulate expression of the sex determination protein TRA-2, and we find that the abundance of TRA-2 is modestly elevated in laf-1/+ females. We discuss potential functions of LAF-1 as a helicase and its roles in sex determination.     

The ukb1 gene encodes a putative protein kinase required for bud site selection and pathogenicity in Ustilago maydis

Morphogenesis and pathogenesis are closely associated aspects of the life cycle of the fungal pathogen Ustilago maydis. In this fungus, the dimorphic switch from budding to filamentous growth coincides with the transition from non-pathogenic to pathogenic growth on maize. We have cloned and characterized the ukb1 gene that encodes a putative serine/threonine protein kinase with a role in budding and filamentous growth. Mutants defective in ukb1 were altered in bud site selection and produced lateral buds at a greater frequency than wild-type cells. Dikaryotic cells defective in ukb1 were capable of colonizing host tissue and growing with a filamentous morphology in planta. However, the mutants were incapable of inducing tumor formation and they failed to complete sexual development. In addition, the ukb1 gene influenced the ability of colonies to form aerial mycelia in response to environmental stimuli. Overall, the discovery of ukb1 reinforces the connection between morphogenesis and pathogenesis in U. maydis.     

TRPV1 sustains microglial metabolic reprogramming in Alzheimer's disease

As the brain‐resident innate immune cells, reactive microglia are a major pathological feature of Alzheimer''s disease (AD). However, the exact role of microglia is still unclear in AD pathogenesis. Here, using metabolic profiling, we show that microglia energy metabolism is significantly suppressed during chronic Aβ‐tolerant processes including oxidative phosphorylation and aerobic glycolysis via the mTOR‐AKT‐HIF‐1α pathway. Pharmacological activation of TRPV1 rescues Aβ‐tolerant microglial dysfunction, the AKT/mTOR pathway activity, and metabolic impairments and restores the immune responses including phagocytic activity and autophagy function. Amyloid pathology and memory impairment are accelerated in microglia‐specific TRPV1‐knockout APP/PS1 mice. Finally, we showed that metabolic boosting with TRPV1 agonist decreases amyloid pathology and reverses memory deficits in AD mice model. These results indicate that TRPV1 is an important target regulating metabolic reprogramming for microglial functions in AD treatment.