Substrate-specificity studies on protochlorophyllide reductase in barley (Hordeum vulgare) etioplast membranes.
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ABSTRACT:
SUBMITTER: Griffiths WT
PROVIDER: S-EPMC1161527 | biostudies-other | 1980 Jan
REPOSITORIES: biostudies-other
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Project description:The assimilatory nitrite reductase (ferredoxin: nitrite oxidoreductase, EC 1.7.7.1) from barley (Hordeum vulgare L.) leaves has been purified over 1500-fold with a recovery of 30% and a specific activity of 84 mumol of nitrite reduced/min per mg of protein. The purification procedure includes (NH4)2SO4 fractionation, ion-exchange and molecular-sieve chromatographies and, finally, ferredoxin-Sepharose-4B affinity chromatography. The enzyme appears homogeneous by polyacrylamide gel electrophoresis and consists of a single polypeptide chain with an Mr of 61 000. The absorption spectrum of the pure enzyme was typical of a haem-containing protein. The enzyme showed low thermostability and was specific for ferredoxin (Km 0.4 microM), although reduced Methyl Viologen (Km 120 microM) was also effective. The same Km value for nitrite (250 microM) was obtained with both electron carriers. Cyanide acted as a powerful pure competitive inhibitor of enzyme with respect to nitrite (Ki 40 microM). Thiol-blocking agents also caused considerable inhibition, but only the ferredoxin-driven activity was significantly inhibited by sulphite and hydroxylamine.
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Project description:Laevulinic acid (Lev) was used to control the rate of protocholorophyllide (PChl) regeneration in the leaves of dark-grown seedlings of barley (Hordeum vulgare) after a brief light treatment. In the leaves given Lev, at concentrations that severely block the resynthesis of protochlorophyllide, there was a massive overproduction of delta-aminolaevulinic acid (AmLev) that was well in excess of that required for the regeneration of PChl observed in the control leaves. Lev, at low concentrations, slightly delayed regeneration and held up, rather than inhibited, the utilization of the AmLev, which accumulated in the tissues. The overproduction and uncontrolled formation of AmLev also occurred in dark-grown leaves treated with a high concentration of Lev and given a light treatment of just sufficient energy to photoreduce only small quantities of the endogenous PChl. Experiments in which a high level of free PChl was induced by incubating the leaves in AmLev indicated that the active species of PChl was that associated with, and bound to, the PChl reductase protein. The results strongly demonstrate a close relationship between the PChl-protein complex and the ability of the leaves to synthesize AmLev.
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Project description:Gene expression in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. The division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear due to a lack of comprehensive information on promoter usage. Here we present a thorough investigation into the distribution of PEP and NEP promoters within the plastid genome of barley (Hordeum vulgare L). Using a novel differential RNA sequencing approach, which discriminates between primary and processed transcripts, we obtained a genome-wide map of transcription start sites in plastids of mature first leaves. PEP-lacking plastids of the albostrians mutant allowed for the unambiguous identifications of NEP promoters. We observed that the chloroplast genome contains many more promoters than genes. According to our data, most genes (including genes coding for photosynthesis proteins) have both PEP and NEP promoters. We also detected numerous transcription start sites within operons indicating transcriptional uncoupling of genes in polycistronic gene clusters. Moreover, we mapped many transcription start sites in intergenic regions, as well as opposite to annotated genes demonstrating the existence of numerous non-coding RNA candidates.
2011-11-23 | GSE33773 | GEO
Project description:Small angle X-ray scattering was used to study the solution conformation of the C hordeins of barley (Hordeum vulgare), a group of proteins whose primary structure consists predominantly of an octapeptide repeat motif. Measurements on the protein in 0.1 M-acetic acid at 25 degrees C are consistent with a model for the protein conformation of a stiff coil, the so-called 'worm-like' chain. The characteristic parameters (the Kuhn statistical segment length and the contour length) of the protein were calculated as 5.11 and 71.5 nm respectively.
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Project description:BACKGROUND: Endogenous phytase plays a crucial role in phytate degradation and is thus closely related to nutrient efficiency in barley products. The understanding of genetic information of phytase in barley can provide a useful tool for breeding new barley varieties with high phytase activity. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative trait loci (QTL) analysis for phytase activity was conducted using a doubled haploid population. Phytase protein was purified and identified by the LC-ESI MS/MS Shotgun method. Purple acid phosphatase (PAP) gene was sequenced and the position was compared with the QTL controlling phytase activity. A major QTL for phytase activity was mapped to chromosome 5 H in barley. The gene controlling phytase activity in the region was named as mqPhy. The gene HvPAP a was mapped to the same position as mqPhy, supporting the colinearity between HvPAP a and mqPhy. CONCLUSIONS/SIGNIFICANCE: It is the first report on QTLs for phytase activity and the results showed that HvPAP a, which shares a same position with the QTL, is a major phytase gene in barley grains.
| S-EPMC3080886 | biostudies-literature
Project description:BackgroundSequencing analysis of mitochondrial genomes is important for understanding the evolution and genome structures of various plant species. Barley is a self-pollinated diploid plant with seven chromosomes comprising a large haploid genome of 5.1 Gbp. Wild barley (Hordeum vulgare ssp. spontaneum) and cultivated barley (H. vulgare ssp. vulgare) have cross compatibility and closely related genomes, although a significant number of nucleotide polymorphisms have been reported between their genomes.ResultsWe determined the complete nucleotide sequences of the mitochondrial genomes of wild and cultivated barley. Two independent circular maps of the 525,599 bp barley mitochondrial genome were constructed by de novo assembly of high-throughput sequencing reads of barley lines H602 and Haruna Nijo, with only three SNPs detected between haplotypes. These mitochondrial genomes contained 33 protein-coding genes, three ribosomal RNAs, 16 transfer RNAs, 188 new ORFs, six major repeat sequences and several types of transposable elements. Of the barley mitochondrial genome-encoded proteins, NAD6, NAD9 and RPS4 had unique structures among grass species.ConclusionsThe mitochondrial genome of barley was similar to those of other grass species in terms of gene content, but the configuration of the genes was highly differentiated from that of other grass species. Mitochondrial genome sequencing is essential for annotating the barley nuclear genome; our mitochondrial sequencing identified a significant number of fragmented mitochondrial sequences in the reported nuclear genome sequences. Little polymorphism was detected in the barley mitochondrial genome sequences, which should be explored further to elucidate the evolution of barley.
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Project description:It is not known to what degree aquaporin-facilitated water uptake differs between root developmental regions and types of root. The aim of this study was to measure aquaporin-dependent water flow in the main types of root and root developmental regions of 14- to 17-d-old barley plants and to identify candidate aquaporins which mediate this flow. Water flow at root level was related to flow at cell and plant level. Plants were grown hydroponically. Hydraulic conductivity of cells and roots was determined with a pressure probe and through exudation, respectively, and whole-plant water flow (transpiration) determined gravimetrically in response to the commonly used aquaporin inhibitor HgCl(2). Expression of aquaporins was analysed by real-time PCR and in situ hybridization. Hydraulic conductivity of cortical cells in seminal roots was largest in lateral roots; it was smallest in the fully mature zone and intermediate in the not fully mature 'transition' zone along the main root axis. Adventitious roots displayed an even higher (3- to 4-fold) cortical cell hydraulic conductivity in the transition zone. This coincided with 3- to 4-fold higher expression of three aquaporins (HvPIP2;2, HvPIP2;5, HvTIP1:1). These were expressed (also) in cortical tissue. The largest inhibition of water flow (83-95%) in response to HgCl(2) was observed in cortical cells. Water flow through roots and plants was reduced less (40-74%). It is concluded that aquaporins contribute substantially to root water uptake in 14- to 17-d-old barley plants. Most water uptake occurs through lateral roots. HvPIP2;5, HvPIP2;2, and HvTIP1;1 are prime candidates to mediate water flow in cortical tissue.
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Project description:Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.
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Project description:Heading date (HD) of cereals is an important trait for adaptation to diverse environments and is critical for determining yield and quality and the number of genes and gene combinations that confer earliness in barley under short days is limited. In our study, a QTL for early flowering was identified from the cross between an Australian malting barley cultivar and a Chinese landrace. Four sets of near isogenic lines (NILs) were developed with a QTL located on chromosome 5H at the interval of 122.0-129.0 cM. Further experiments were conducted to investigate how this gene was regulated by photoperiod using the NILs with three sowing dates from autumn to summer. The NILs carrying the earliness allele were significantly earlier than the late genotype at all sowing dates. This gene was different from previously reported vernalisation genes that are located at a similar position as no vernalisation was required for all the NILs. The difference between this gene and Eam5 (HvPHYC) locus which also located between two co-segregated markers (3398516S5, 122.5 cM, and 4014046D5, 126.1 cM), is that with the existence of Ppd-H1 (Eam1), Eam5 has no effect on ear emergence under long days while the gene from TX9425 still reduced the time to ear emergency. The locus showed no pleiotropic effects on grain pasting properties and agronomic traits except for spike length and number of spikelets per spike, and thus can be effectively used in breeding programs. The array of early heading dates caused by interactions of Eam5 gene with other maturity genes provides an opportunity to better fine tune heading dates with production environments, which can be critical factor in barley breeding.
| S-EPMC6049932 | biostudies-literature
Project description:The photoreduction of protochlorophyllide (Pchl) in dark-grown leaves of barley (Hordeum vulgare) brings about the synthesis of delta-aminolaevulinic acid (AmLev). Manipulation of the Pchl level in the leaves by incubation in AmLev indicated that the production of AmLev was intimately related to the state of the Pchl reductase ternary complex. Free Pchl reductase that is unassociated with substrate/product appeared at first to be essential for the photoinduction of AmLev synthesis. Experiments on the photoreduction of Pchl in dark-grown leaves exposed to low-energy red-light, however, showed that photoreduction and AmLev synthesis would occur when the Pchl reductase, together with substrate, was maintained at relatively high endogenous concentration. Under such conditions the availability of free reductase protein would be negligible. An alternative scheme is presented, therefore, that can explain many, if not all, of the observations on AMLev synthesis and its close relationship to Pchl reduction, and which is based on a common supply of NADPH for the reduction of glutamate to AmLev and the synthesis of chorophyll(-ide).
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