Linkage analysis of molecular markers and quantitative trait loci in populations of inbred backcross lines of Brassica napus L.
(25/1068)
Backcross populations are often used to study quantitative trait loci (QTL) after they are initially discovered in balanced populations, such as F(2), BC(1), or recombinant inbreds. While the latter are more powerful for mapping marker loci, the former have the reduced background genetic variation necessary for more precise estimation of QTL effects. Many populations of inbred backcross lines (IBLs) have been developed in plant and animal systems to permit simultaneous study and dissection of quantitative genetic variation introgressed from one source to another. Such populations have a genetic structure that can be used for linkage estimation and discovery of QTL. In this study, four populations of IBLs of oilseed Brassica napus were developed and analyzed to map genomic regions from the donor parent (a winter-type cultivar) that affect agronomic traits in spring-type inbreds and hybrids. Restriction fragment length polymorphisms (RFLPs) identified among the IBLs were used to calculate two-point recombination fractions and LOD scores through grid searches. This information allowed the enrichment of a composite genetic map of B. napus with 72 new RFLP loci. The selfed and hybrid progenies of the IBLs were evaluated during two growing seasons for several agronomic traits. Both pedigree structure and map information were incorporated into the QTL analysis by using a regression approach. The number of QTL detected for each trait and the number of effective factors calculated by using biometrical methods were of similar magnitude. Populations of IBLs were shown to be valuable for both marker mapping and QTL analysis. (+info)
Inhibitor binding studies on enoyl reductase reveal conformational changes related to substrate recognition.
(26/1068)
Enoyl acyl carrier protein reductase (ENR) is involved in fatty acid biosynthesis. In Escherichia coli this enzyme is the target for the experimental family of antibacterial agents, the diazaborines, and for triclosan, a broad spectrum antimicrobial agent. Biochemical studies have suggested that the mechanism of diazaborine inhibition is dependent on NAD(+) and not NADH, and resistance of Brassica napus ENR to diazaborines is thought to be due to the replacement of a glycine in the active site of the E. coli enzyme by an alanine at position 138 in the plant homologue. We present here an x-ray analysis of crystals of B. napus ENR A138G grown in the presence of either NAD(+) or NADH and the structures of the corresponding ternary complexes with thienodiazaborine obtained either by soaking the drug into the crystals or by co-crystallization of the mutant with NAD(+) and diazaborine. Analysis of the ENR A138G complex with diazaborine and NAD(+) shows that the site of diazaborine binding is remarkably close to that reported for E. coli ENR. However, the structure of the ternary ENR A138G-NAD(+)-diazaborine complex obtained using co-crystallization reveals a previously unobserved conformational change affecting 11 residues that flank the active site and move closer to the nicotinamide moiety making extensive van der Waals contacts with diazaborine. Considerations of the mode of substrate binding suggest that this conformational change may reflect a structure of ENR that is important in catalysis. (+info)
Duplication of the Brassica oleracea APETALA1 floral homeotic gene and the evolution of domesticated cauliflower.
(27/1068)
Development of the cauliflower phenotype in Arabidopsis thaliana requires mutations at both the CAULIFLOWER and APETALA1 loci. BoAP1 is the Brassica oleracea orthologue to the Arabidopsis AP1 gene, and is present in two copies in Brassica genomes. The BoAP1-A gene appears to encode a full-length protein, but BoAP1-B alleles in B. oleracea contain insertions that lead to premature translation termination. The BoAP1-B allele found in most B. oleracea subspecies, including B. oleracea ssp. botrytis (domesticated cauliflower) contains a 9 bp insertion in exon 4. This insertion leads to the formation of an in-frame translation termination codon, and these alleles can encode a protein that is truncated at the K domain of this MADS-box transcriptional activator. The allele in B. oleracea ssp. oleracea (wild cabbage) lacks this insertion and instead contains a downstream 4 bp frameshift mutation resulting in the formation of a nonsense mutation. The structure of the BoAP1-B alleles suggests that they are impaired in their ability to perform their floral meristem identity function. These mutations, in conjunction with mutations at the BoCAULIFLOWER (BoCAL) locus, may be associated with the evolution of domesticated cauliflower. (+info)
Structural and transcriptional comparative analysis of the S locus regions in two self-incompatible Brassica napus lines.
(28/1068)
Self-incompatibility (SI) in Brassica is controlled by a single locus, termed the S locus. There is evidence that two of the S locus genes, SLG, which encodes a secreted glycoprotein, and SRK, which encodes a putative receptor kinase, are required for SI on the stigma side. The current model postulates that a pollen ligand recognizing the SLG/SRK receptors is encoded in the genomic region defined by the SLG and SRK genes. A fosmid contig of approximately 65 kb spanning the SLG-910 and SRK-910 genes was isolated from the Brassica napus W1 line. A new gene, SLL3, was identified using a novel approach combining cDNA subtraction and direct selection. This gene encodes a putative secreted small peptide and exists as multiple copies in the Brassica genome. Sequencing analysis of the 65-kb contig revealed seven additional genes and a transposon. None of these seven genes exhibited features expected of S genes on the pollen side. An approximately 88-kb contig of the A14 S region also was isolated from the B. napus T2 line and sequenced. Comparison of the two S regions revealed that (1) the gene organization downstream of SLG in both S haplotypes is highly colinear; (2) the distance between SLG-A14 and SRK-A14 genes is much larger than that between SLG-910 and SRK-910, with the intervening region filled with retroelements and haplotype-specific genes; and (3) the gene organization downstream of SRK in the two haplotypes is divergent. These observations lead us to propose that the SLG downstream region might be one border of the S locus and that the accumulation of heteromorphic sequences, such as retroelements as well as haplotype-unique genes, may act as a mechanism to suppress recombination between SLG and SRK. (+info)
Aluminum fluoride inhibition of cabbage phospholipase D by a phosphate-mimicking mechanism.
(29/1068)
Aluminum fluoride (AlF(4)(-)) inhibited phospholipase D (PLD) purified from cabbage in both PIP(2)-dependent and PIP(2)-independent assays, consistent with its previously observed effect on mammalian PLD. The possibility that AlF(4)(-) may exert this effect through its known phosphate-mimicking property was examined. Inorganic phosphate, as well as two phosphate analogs, beryllium fluoride and orthovanadate, also inhibited cabbage PLD. Enzyme kinetic studies confirmed that PLD followed Hill kinetics, characteristic for allosteric enzymes, with an apparent Hill coefficient (n(app)) of 3.8, indicating positive cooperativity among multiple substrate-binding sites and suggesting possible functional oligomerization of the enzyme. AlF(4)(-) modification of PLD kinetics was consistent with a competitive mode of enzyme inhibition. It is therefore proposed that AlF(4)(-), and other phosphate analogs, inhibits plant PLD by competing with a substrate phosphate group for a substrate-binding site, thereby preventing the formation of an enzyme-phosphatidyl intermediate. This may be a conserved feature of PLD superfamily enzymes. (+info)
A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein.
(30/1068)
Plant virus-encoded movement proteins promote viral spread between plant cells via plasmodesmata. The movement is assumed to require a plasmodesmata targeting signal to interact with still unidentified host factors presumably located on plasmodesmata and cell walls. The present work indicates that a ubiquitous cell wall-associated plant enzyme pectin methylesterase of Nicotiana tabacum L. specifically binds to the movement protein encoded by tobacco mosaic virus. We also show that pectin methylesterase is an RNA binding protein. These data suggest that pectin methylesterase is a host cell receptor involved in cell-to-cell movement of tobacco mosaic virus. (+info)
The male determinant of self-incompatibility in Brassica.
(31/1068)
In the S locus-controlled self-incompatibility system of Brassica, recognition of self-related pollen at the surface of stigma epidermal cells leads to inhibition of pollen tube development. The female (stigmatic) determinant of this recognition reaction is a polymorphic transmembrane receptor protein kinase encoded at the S locus. Another highly polymorphic, anther-expressed gene, SCR, also encoded at the S locus, fulfills the requirements for the hypothesized pollen determinant. Loss-of-function and gain-of-function studies prove that the SCR gene product is necessary and sufficient for determining pollen self-incompatibility specificity, possibly by acting as a ligand for the stigmatic receptor. (+info)
A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants.
(32/1068)
Self-incompatibility, the rejection of self pollen, is the most widespread mechanism by which flowering plants prevent inbreeding. In Brassica, the S receptor kinase (SRK) has been implicated in the self-incompatibility response, but the molecular mechanisms involving SRK are unknown. One putative downstream effector for SRK is ARC1, a protein that binds to the SRK kinase domain. Here it is shown that suppression of ARC1 messenger RNA levels in the self-incompatible Brassica napus W1 line is correlated with a partial breakdown of self-incompatibility, resulting in seed production. This provides strong evidence that ARC1 is a positive effector of the Brassica self-incompatibility response. (+info)