scholarly journals Heterologous Expression of the Barley (Hordeum vulgare L.) Xantha-f, -g and -h Genes that Encode Magnesium Chelatase Subunits

2020 ◽  
Vol 39 (5) ◽  
pp. 554-562
Author(s):  
Rabab Mahdi ◽  
David Stuart ◽  
Mats Hansson ◽  
Helmy M. Youssef
Keyword(s):  
Hordeum Vulgare ◽  
Recombinant Proteins ◽  
Structural Information ◽  
Recombinant Expression ◽  
Expression System ◽  
Protoporphyrin Ix ◽  
Buffer System ◽  
Enzymatic Reactions ◽  
Hordeum Vulgare L ◽  
Magnesium Chelatase

AbstractBiosynthesis of chlorophyll involves several enzymatic reactions of which many are shared with the heme biosynthesis pathway. Magnesium chelatase is the first specific enzyme in the chlorophyll pathway. It catalyzes the formation of Mg-protoporphyrin IX from the insertion of Mg2+ into protoporphyrin IX. The enzyme consists of three subunits encoded by three genes. The three genes are named Xantha-h, Xantha-g and Xantha-f in barley (Hordeum vulgare L.). The products of the genes have a molecular weight of 38, 78 and 148 kDa, respectively, as mature proteins in the chloroplast. Most studies on magnesium chelatase enzymes have been performed using recombinant proteins of Rhodobacter capsulatus, Synechocystis sp. PCC6803 and Thermosynechococcus elongatus, which are photosynthetic bacteria. In the present study we established a recombinant expression system for barley magnesium chelatase with the long-term goal to obtain structural information of this enigmatic enzyme complex from a higher plant. The genes Xantha-h, -g and -f were cloned in plasmid pET15b, which allowed the production of the three subunits as His-tagged proteins in Escherichia coli BL21(DE3)pLysS. The purified subunits stimulated magnesium chelatase activity of barley plastid extracts and produced activity in assays with only recombinant proteins. In preparation for future structural analyses of the barley magnesium chelatase, stability tests were performed on the subunits and activity assays were screened to find an optimal buffer system and pH.

Construction of a genetic map of barley (Hordeum vulgare L.) cross 'Azumamugi' × 'Kanto Nakate Gold' using a simple and efficient amplified fragment-length polymorphism system

Genome ◽  
2001 ◽  
Vol 44 (2) ◽  
pp. 284-292 ◽  
Author(s):  
Y Mano ◽  
S Kawasaki ◽  
F Takaiwa ◽  
T Komatsuda
Keyword(s):  
Hordeum Vulgare ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Recombinant Inbred ◽  
Fragment Length Polymorphism ◽  
Recombinant Inbred Lines ◽  
Amplified Fragment Length Polymorphism ◽  
Buffer System ◽  
Hordeum Vulgare L ◽  
Sequence Tagged Sites

We have devised a simple and efficient amplified fragment-length polymorphism (AFLP) system consisting of small slab gels, a discontinuous buffer system, and silver staining. Using this system, a single worker developed a barley map with 227 polymorphic fragments in 2 months. As a mapping population, 99 recombinant inbred lines of barley cultivars 'Azumamugi' × 'Kanto Nakate Gold' were used. Most of the 227 AFLP fragments showed a Mendelian segregation ratio of 1:1, and all were assigned to the seven barley chromosomes. Thus, these fragments are useful as molecular markers. They were integrated with 40 previously characterized sequence-tagged sites, 3 isozymes, and 2 morphological markers to construct an integrated map. The resulting map covered 925.6 cM with 272 markers (detecting 150 loci) at an average interval of 6.5 cM/locus. This system greatly simplifies map construction.Key words: amplified fragment length polymorphism (AFLP), Hordeum vulgare, linkage map, recombinant inbred line (RIL), sequence-tagged site (STS).

scholarly journals Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare L.)

1997 ◽  
Vol 322 (3) ◽  
pp. 815-822 ◽  
Author(s):  
Mette D. ANDERSEN ◽  
Arne JENSEN ◽  
Jon D. ROBERTUS ◽  
Robert LEAH ◽  
Karen SKRIVER
Keyword(s):  
Hordeum Vulgare ◽  
Recombinant Protein ◽  
Heterologous Expression ◽  
Specific Activity ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Hordeum Vulgare L ◽  
Sequence Alignments ◽  
Natural Form

To investigate structure–function relationships in plant chitinases, we have developed a heterologous expression system for the 26 kDa endochitinase from Hordeum vulgare L. (barley). Escherichia coli cells harbouring the gene in a T7 RNA polymerase-based expression vector synthesized completely insoluble recombinant protein under standard induction conditions at 37 °C. However, a concentration of soluble recombinant protein of approx. 15 mg/l was achieved by inducing bacteria at low temperature (15 °C). Recombinant endochitinase was purified to homogeneity and shown to be structurally and functionally identical to the seed protein. An average of three disulphide bonds are present in the recombinant enzyme, consistent with the number found in the natural form. The seed and recombinant proteins showed the same specific activity towards a high-molecular-mass substrate and exhibited similar anti-fungal activity towards Tricoderma reesei. Site-directed mutagenesis was used to replace residues that are likely to be involved in the catalytic event, based on structural similarities with lysozyme and on sequence alignments with related chitinases. The Glu67 → Gln mutation resulted in a protein with undetectable activity, while the Glu89 → Gln mutation yielded an enzyme with 0.25% of wild-type specific activity. This suggests that two acidic residues are essential for catalytic activity, similar to the situation with many other glycosyl hydrolases. Examination of conserved residues stretching into the proposed substrate binding cleft suggests that Asn124 also plays an important functional role.

Magnesium Chelatase of Hordeum vulgare L. Is Not Activated by Light but Inhibited by Pheophorbide

1997 ◽  
Vol 52 (3-4) ◽  
pp. 144-152 ◽  
Author(s):  
Gerhard Pöpperl ◽  
Ulrike Oster ◽  
Inge Bios ◽  
Wolfhart Rüdiger
Keyword(s):  
Enzyme Activity ◽  
Hordeum Vulgare ◽  
White Light ◽  
Envelope Membrane ◽  
Strong Inhibition ◽  
Hordeum Vulgare L ◽  
Magnesium Chelatase ◽  
Metal Free ◽  
Plastid Envelope

AbstractThe enzyme activity of magnesium chelatase was determined in intact etioplasts of barley (Hordeum vulgare L.) seedlings. Irradiation of isolated plastids with white light for 15 min does not lead to any activation of the enzyme but to a decrease in activity, especially in etioplasts. The enzyme was inhibited by chlorophyllide and zinc pheophorbide only to a certain extent. Strong inhibition was observed with the metal-free pheophorbide (Ki = 0.92 μM) but not with pheophytin or chlorophyll. Penetration of chlorophyllide through the envelope membrane was confirmed by the chlorophyll synthase reaction that occurs in the inner membranes of etioplasts and chloroplasts. The possible role of inhibition of magnesium chelatase by pheophorbide in senescent leaves and tetrapyrrole transport across the plastid envelope are discussed.

Ultrastructure and Senescence in an Achloroplastic Mutant of Hordeum vulgare L. cv. Dyan

1992 ◽  
Vol 50 (1) ◽  
pp. 844-845
Author(s):  
R.H.M. Cross ◽  
C.E.J. Botha ◽  
A.K. Cowan ◽  
B.J. Hartley
Keyword(s):  
Cell Wall ◽  
Hordeum Vulgare ◽  
Leaf Tissue ◽  
Ultrastructural Changes ◽  
Hordeum Vulgare L ◽  
Mesophyll Cells ◽  
Lethal Mutant ◽  
Cytoplasmic Matrix ◽  
Close Proximity ◽  
Pinocytotic Vesicles

Senescence is an ordered degenerative process leading to death of individual cells, organs and organisms. The detection of a conditional lethal mutant (achloroplastic) of Hordeum vulgare has enabled us to investigate ultrastructural changes occurring in leaf tissue during foliar senescence.Examination of the tonoplast structure in six and 14 day-old mutant tissue revealed a progressive degeneration and disappearance of the membrane, apparently starting by day six in the vicinity of the mitochondria associated with the degenerating proplastid (Fig. 1.) where neither of the plastid membrane leaflets is evident (arrows, Fig. 1.). At this stage there was evidence that the mitochondrial membranes were undergoing retrogressive changes, coupled with disorganization of cristae (Fig. 2.). Proplastids (P) lack definitive prolamellar bodies. The cytoplasmic matrix is largely agranular, with few endoplasmic reticulum (ER) cisternae or polyribosomal aggregates. Interestingly, large numbers of actively-budding dictysomes, associated with pinocytotic vesicles, were observed in close proximity to the plasmalemma of mesophyll cells (Fig. 3.). By day 14 however, mesophyll cells showed almost complete breakdown of subcellular organelle structure (Fig. 4.), and further evidence for the breakdown of the tonoplast. The final stage of senescence is characterized by the solubilization of the cell wall due to expression and activity of polygalacturonase and/or cellulose. The presence of dictyosomes with associated pinocytotic vesicles formed from the mature face, in close proximity to both the plasmalemma and the cell wall, would appear to support the model proposed by Christopherson for the secretion of cellulase. This pathway of synthesis is typical for secretory glycoproteins.

ИЗМЕНЧИВОСТЬ ЯЧМЕНЯ (Hordeum vulgare L.) РАЗНОГО ГЕОГРАФИЧЕСКОГО ПРОИСХОЖДЕНИЯ ПО ЭЛЕМЕНТАМ СТРУКТУРЫ УРОЖАЯ

2012 ◽  
pp. 33-40
Author(s):  
А.В. ЖЕЛЕЗНОВ ◽  
◽  
Н.Б. ЖЕЛЕЗНОВА ◽  
Т.В. КУКОЕВА ◽  
Н.В. БУРМАКИНА ◽  
...  
Keyword(s):  
Hordeum Vulgare ◽  
Hordeum Vulgare L

ВНУТРИВИДОВОЙ ПОЛИМОРФИЗМ ЯРОВОГО ЯЧМЕНЯ (Hordeum vulgare L.) ПО УСТОЙЧИВОСТИ К ДЕЙСТВИЮ СВИНЦА

2014 ◽  
pp. 78-87 ◽  
Author(s):  
А.В. ДИКАРЕВ ◽  
◽  
В.Г. ДИКАРЕВ ◽  
Н.С. ДИКАРЕВА ◽  
С.А. ГЕРАСЬКИН ◽  
...  
Keyword(s):  
Hordeum Vulgare ◽  
Hordeum Vulgare L

Using AMMI approach to delineate genotype by environment interaction and stability of barley (Hordeum vulgare L.) genotypes under northern Indian shivalik hill conditions

2020 ◽  
Vol 80 (03) ◽  
Author(s):  
Om Prakash Yadav ◽  
A. K. Razdan ◽  
Bupesh Kumar ◽  
Praveen Singh ◽  
Anjani K. Singh
Keyword(s):  
Hordeum Vulgare ◽  
Grain Yield ◽  
Principal Component ◽  
Genotype By Environment Interaction ◽  
Environment Interaction ◽  
Hordeum Vulgare L ◽  
Genotype By Environment ◽  
Biplot Analysis ◽  
Principal Component Axis ◽  
Ammi Analysis

Genotype by environment interaction (GEI) of 18 barley varieties was assessed during two successive rabi crop seasons so as to identify high yielding and stable barley varieties. AMMI analysis showed that genotypes (G), environment (E) and GEI accounted for 1672.35, 78.25 and 20.51 of total variance, respectively. Partitioning of sum of squares due to GEI revealed significance of interaction principal component axis IPCA1 only On the basis of AMMI biplot analysis DWRB 137 (41.03qha–1), RD 2715 (32.54qha–1), BH 902 (37.53qha–1) and RD 2907 (33.29qha–1) exhibited grain yield superiority of 64.45, 30.42, 50.42 and 33.42 per cent, respectively over farmers’ recycled variety (24.43qha–1).

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