Non-destructive X-ray analysis of Arabidopsis embryo mutants

1993 ◽  
Vol 3 (3) ◽  
pp. 167-170 ◽  
Author(s):  
R. J. Bino ◽  
J. W. Aartse ◽  
W. J. van der Burg
Keyword(s):  
Arabidopsis Thaliana ◽  
Double Mutant ◽  
Embryo Morphology ◽  
Wild Type ◽  
X Ray ◽  
Non Destructive ◽  
Mature Seeds

AbstractX-radiography is a simple, rapid and non-destructive method for analysing the morphology of embryos in dry, mature seeds of Arabidopsis thaliana. In wild type seeds, the cotyledons, hypocotyl and radicle tip can be readily distinguished. In seeds of the mutant types knolle, keule, and the double mutant keulelgnom, aberrations in embryo morphology can be visualized. X-radiography may therefore be useful in the isolation of embryo mutants from Arabidopsis seed samples.

scholarly journals Overexpression of Avocado (Persea americana Mill.) PaRAP2.1 Promotes Fatty Acid Accumulation in Arabidopsis thaliana

2021 ◽  
Vol 13 (6) ◽  
pp. 1
Author(s):  
Weihong Ma ◽  
Xiaoping Zang ◽  
Yuanzheng Liu ◽  
Lixia Wang ◽  
Jiashui Wang ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Persea Americana ◽  
Wild Type ◽  
Acid Biosynthesis ◽  
Fatty Acid Accumulation ◽  
Avocado Fruit ◽  
Acid Accumulation ◽  
Mature Seeds

Fatty acids in avocado fruit (Persea americana Mill.) are vital composition affecting flavour and nutritive value. Hence, horticulturalists are interested in illustrating the functions of transcription factors on fatty acid accumulation in avocado fruit. In the present study, the APETALA2/ethylene-responsive transcription factor gene, PaRAP2.1, was cloned from avocado mesocarp, and the subcellular localization demonstrated that PaRAP2.1 was located in the cytoplasm and nucleus. The PaRAP2.1 was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation. Furthermore, PaRAP2.1 were functionally verified its effect on fatty acid biosynthesis. Histological analyses of lipid droplets displayed that the striking difference in the lipid droplets in the mature seeds between PaRAP2.1-overexpressing transgenic and wild-type Arabidopsis thaliana lines were revealed based on confocal microscopy images. Subsequently, fatty acid analyses of PaRAP2.1-overexpressing Arabidopsis thaliana lines displayed the significantly higher contents of fatty acids than those in the wild-type plants. Meanwhile, expression amount of ten genes involving in fatty acid biosynthesis dramatically up-regulated in the mature seeds of PaRAP2.1-overexpressing lines than those of wild-type plants. These results provide a theoretical basis for future research in regard to the function of PaRAP2.1 on fatty acid biosynthesis.

The early-flowering mutant efs is involved in the autonomous promotion pathway of Arabidopsis thaliana

Development ◽  
1999 ◽  
Vol 126 (21) ◽  
pp. 4763-4770 ◽  
Author(s):  
W.J. Soppe ◽  
L. Bentsink ◽  
M. Koornneef
Keyword(s):  
Arabidopsis Thaliana ◽  
Flowering Time ◽  
Double Mutant ◽  
Plant Size ◽  
Early Flowering ◽  
Wild Type ◽  
Late Flowering ◽  
Long Day ◽  
Short Days ◽  
Transition To Flowering

The transition to flowering is a crucial moment in a plant's life cycle of which the mechanism has only been partly revealed. In a screen for early flowering, after mutagenesis of the late-flowering fwa mutant of Arabidopsis thaliana, the early flowering in short days (efs) mutant was identified. Under long-day light conditions, the recessive monogenic efs mutant flowers at the same time as wild type but, under short-day conditions, the mutant flowers much earlier. In addition to its early-flowering phenotype, efs has several pleiotropic effects such as a reduction in plant size, fertility and apical dominance. Double mutant analysis with several late-flowering mutants from the autonomous promotion (fca and fve) and the photoperiod promotion (co, fwa and gi) pathways of flowering showed that efs reduces the flowering time of all these mutants. However, efs is completely epistatic to fca and fve but additive to co, fwa and gi, indicating that EFS is an inhibitor of flowering specifically involved in the autonomous promotion pathway. A vernalisation treatment does not further reduce the flowering time of the efs mutant, suggesting that vernalisation promotes flowering through EFS. By comparing the length of the juvenile and adult phases of vegetative growth for wild-type, efs and the double mutant plants, it is apparent that efs mainly reduces the length of the adult phase.

Elements present in mineral nutrient reserves in dry Arabidopsis thaliana seeds of wild type and pho1, pho2, and man1 mutants

2001 ◽  
Vol 79 (11) ◽  
pp. 1292-1296 ◽  
Author(s):  
John NA Lott ◽  
M Marcia West
Keyword(s):  
Arabidopsis Thaliana ◽  
P Uptake ◽  
Mineral Nutrient ◽  
Wild Type ◽  
Nutrient Storage ◽  
X Ray ◽  
Spherical Inclusions ◽  
Nutrient Reserves ◽  
Total P ◽  
Different Tissues

Comparison of wild type and mutants of Arabidopsis thaliana offers an opportunity to study the genetic control of nutrient storage in seeds. We used energy dispersive X-ray analysis to determine the elements present and their relative amounts in globoids of dry wild-type seeds, as well as seeds of a reduced total P uptake mutant (pho1), a phosphate accumulator (pho2), and a metal accumulator (man1). Globoids are spherical inclusions, rich in phytate that function as a store for inositol, P, K, Mg, Ca, Fe, and Zn. Key findings of this study were the following: (i) globoids in protein bodies from nine different tissues and (or) organs in dry Arabidopsis thaliana seeds contained P, K, Mg, and Ca, and sometimes traces of Fe and Zn; (ii) globoids contained higher Ca and lower Mg amounts than occur in globoids in seeds of most other plant species; (iii) globoids in comparable tissue and (or) organ regions of seeds were very similar in elemental composition for wild type and all mutant plants.Key words: Arabidopsis, dry seeds, phytate, mineral nutrient mutants, phosphorus, globoids.

scholarly journals Enhancing ubiquitin crystallization through surface-entropy reduction

2014 ◽  
Vol 70 (10) ◽  
pp. 1434-1442 ◽  
Author(s):  
Patrick J. Loll ◽  
Peining Xu ◽  
John T. Schmidt ◽  
Scott L. Melideo
Keyword(s):  
Surface Density ◽  
Double Mutant ◽  
High Surface ◽  
Wild Type ◽  
Mutant Proteins ◽  
X Ray ◽  
Surface Entropy ◽  
Lysine Residues ◽  
Entropy Reduction ◽  
Packing Interactions

Ubiquitin has many attributes suitable for a crystallization chaperone, including high stability and ease of expression. However, ubiquitin contains a high surface density of lysine residues and the doctrine of surface-entropy reduction suggests that these lysines will resist participating in packing interactions and thereby impede crystallization. To assess the contributions of these residues to crystallization behavior, each of the seven lysines of ubiquitin was mutated to serine and the corresponding single-site mutant proteins were expressed and purified. The behavior of these seven mutants was then compared with that of the wild-type protein in a 384-condition crystallization screen. The likelihood of obtaining crystals varied by two orders of magnitude within this set of eight proteins. Some mutants crystallized much more readily than the wild type, while others crystallized less readily. X-ray crystal structures were determined for three readily crystallized variants: K11S, K33S and the K11S/K63S double mutant. These structures revealed that the mutant serine residues can directly promote crystallization by participating in favorable packing interactions; the mutations can also exert permissive effects, wherein crystallization appears to be driven by removal of the lysine rather than by addition of a serine. Presumably, such permissive effects reflect the elimination of steric and electrostatic barriers to crystallization.

scholarly journals The Drosophila melanogaster DmRAD54 Gene Plays a Crucial Role in Double-Strand Break Repair after P-Element Excision and Acts Synergistically with Ku70 in the Repair of X-Ray Damage

1999 ◽  
Vol 19 (9) ◽  
pp. 6269-6275 ◽  
Author(s):  
Rolf Kooistra ◽  
Albert Pastink ◽  
José B. M. Zonneveld ◽  
Paul H. M. Lohman ◽  
Jan C. J. Eeken
Keyword(s):  
Drosophila Melanogaster ◽  
Homologous Recombination ◽  
Crucial Role ◽  
Double Mutant ◽  
Strand Break ◽  
P Element ◽  
Slight Reduction ◽  
Wild Type ◽  
Dsb Repair ◽  
X Ray

ABSTRACT The RAD54 gene has an essential role in the repair of double-strand breaks (DSBs) via homologous recombination in yeast as well as in higher eukaryotes. A Drosophila melanogasterstrain deficient in the RAD54 homolog DmRAD54is characterized by increased X-ray and methyl methanesulfonate (MMS) sensitivity. In addition, DmRAD54 is involved in the repair of DNA interstrand cross-links, as is shown here. However, whereas X-ray-induced loss-of-heterozygosity (LOH) events were completely absent in DmRAD54 −/− flies, treatment with cross-linking agents or MMS resulted in only a slight reduction in LOH events in comparison with those in wild-type flies. To investigate the relative contributions of recombinational repair and nonhomologous end joining in DSB repair, aDmRad54 −/−/DmKu70 −/−double mutant was generated. Compared with both single mutants, a strong synergistic increase in X-ray sensitivity was observed in the double mutant. No similar increase in sensitivity was seen after treatment with MMS. Apparently, the two DSB repair pathways overlap much less in the repair of MMS-induced lesions than in that of X-ray-induced lesions. Excision of P transposable elements inDrosophila involves the formation of site-specific DSBs. In the absence of the DmRAD54 gene product, no male flies could be recovered after the excision of a single P element and the survival of females was reduced to 10% compared to that of wild-type flies. P-element excision involves the formation of two DSBs which have identical 3′ overhangs of 17 nucleotides. The crucial role of homologous recombination in the repair of these DSBs may be related to the very specific nature of the breaks.

scholarly journals The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography

2004 ◽  
Vol 385 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Barbara PETSCHACHER ◽  
Stefan LEITGEB ◽  
Kathryn L. KAVANAGH ◽  
David K. WILSON ◽  
Bernd NIDETZKY
Keyword(s):  
Double Mutant ◽  
Profile Analysis ◽  
Xylose Reductase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binary Complex ◽  
Wild Type ◽  
Coenzyme Specificity ◽  
X Ray ◽  
X Ray Crystallography

CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2′-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme–NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274→Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R–N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 °C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 Å resolution.

scholarly journals Identification of an arabinopyranosyltransferase fromPhyscomitrella patensinvolved in the synthesis of the hemicellulose xyloglucan

2017 ◽  
Author(s):  
Lei Zhu ◽  
Murali Dama ◽  
Markus Pauly
Keyword(s):  
Arabidopsis Thaliana ◽  
Transgenic Plants ◽  
Linkage Analysis ◽  
Double Mutant ◽  
Physcomitrella Patens ◽  
Proper Function ◽  
Side Chain ◽  
Glycosidic Linkage ◽  
Nmr Analysis ◽  
Wild Type

AbstractThe hemicellulose xyloglucan consists of a backbone of a β-1,4 glucan substituted with xylosyl moieties and many other, diverse sidechains that are important for its proper function. Many, but not all glycosyltransferases involved in the biosynthesis of xyloglucan have been identified. Here, we report the identification of an hitherto elusive xyloglucan:arabinopyranosyltransferase. This glycosyltransferase was isolated from the mossPhyscomitrella patens, where it acts as aXyloglucan “D”-side-chainTransferase (XDT). Heterologous expression ofXDTin theArabidopsis thalianadouble mutantmur3.1 xlt2, where xyloglucan consists of a xylosylated glucan without further glycosyl substituents, results in the production of the arabinopyranose-containing “D” side chain as characterized by oligosaccharide mass profiling, glycosidic linkage analysis, and NMR analysis. In addition, expression of a relatedPhyscomitrellaglycosyltransferase hortholog ofXLT2leads to the production of the galactose-containing “L” side chain. The presence of the “D” and “L” xyloglucan side chains inPpXDT mur3.1 xlt2andPpXLT2 mur3.1 xlt2transgenic plants, respectively, rescue the dwarfed phenotype of untransformedmur3.1 xlt2mutants to nearly wild-type height. Expression ofPpXDTandPpXLT2in the Arabidopsismur3.1 xlt2mutant also enhanced root growth.

scholarly journals Separate Ion Pathways in a Cl−/H+ Exchanger

2005 ◽  
Vol 126 (6) ◽  
pp. 563-570 ◽  
Author(s):  
Alessio Accardi ◽  
Michael Walden ◽  
Wang Nguitragool ◽  
Hariharan Jayaram ◽  
Carole Williams ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Transport Mechanism ◽  
Double Mutant ◽  
Wild Type ◽  
Extracellular Solution ◽  
X Ray ◽  
Single Mutant ◽  
Cl Transport ◽  
Alternating Access

CLC-ec1 is a prokaryotic CLC-type Cl−/H+ exchange transporter. Little is known about the mechanism of H+ coupling to Cl−. A critical glutamate residue, E148, was previously shown to be required for Cl−/H+ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Cl− transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Cl− transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl− transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl− and one for H+. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes.

scholarly journals Involvement of THH1, an Arabidopsis thaliana homologue of the TOM1 gene, in tobamovirus multiplication

2006 ◽  
Vol 87 (8) ◽  
pp. 2397-2401 ◽  
Author(s):  
Koki Fujisaki ◽  
Gerald B. Ravelo ◽  
Satoshi Naito ◽  
Masayuki Ishikawa
Keyword(s):  
Arabidopsis Thaliana ◽  
Coat Protein ◽  
Double Mutant ◽  
Detectable Level ◽  
Expression Level ◽  
Wild Type ◽  
Homologous Proteins ◽  
Single Mutant ◽  
Mutant Lines

The TOM1 and TOM3 genes of Arabidopsis thaliana encode homologous proteins that are required for tobamovirus multiplication. Although the A. thaliana genome encodes another TOM1-like gene, THH1, the tobamovirus coat protein (CP) does not accumulate to a detectable level in the tom1 tom3 double mutant. Here, double and triple mutants of tom1, tom3 and thh1 were generated to investigate whether THH1 functions to support tobamovirus multiplication. In the tom1 thh1 double mutant, the tobamovirus CP accumulated to a level that was detectable, but lower than that in the tom1 single mutant. In tom1 tom3 double-mutant lines overexpressing THH1, the tobamovirus CP accumulated to a level similar to that observed in wild-type plants. These results suggest that THH1 supports tobamovirus multiplication, but to a lesser extent than TOM1 and TOM3. The expression level of THH1 is lower than that of TOM1 and TOM3, which might explain the smaller contribution of THH1 to tobamovirus multiplication.

scholarly journals Temperature-Sensitive, Vitamin-Requiring Mutants of Arabidopsis Thaliana

1965 ◽  
Vol 18 (2) ◽  
pp. 311 ◽  
Author(s):  
J Langridge
Keyword(s):  
Arabidopsis Thaliana ◽  
High Temperatures ◽  
Temperature Sensitive ◽  
Wild Type ◽  
X Ray ◽  
Recessive Mutations ◽  
Naturally Occurring ◽  
Induced Mutant

Three mutants of Arabidopsi8 requiring specific vitamins for growth at certain temperatures are described. All are due to recessive mutations of single wild-type genes. One X-ray-induced mutant is unable to synthesize thiamine at low tempera-tures because of a block in the phosphorylation or coupling of the immediate precursors. Two mutants which require biotin for growth at high temperatures are determined by recessive alleles of the same gene. These mutants comprise naturally occurring ecotypes in Spain and Austria. Experiments indicate that an inability to make biotin at high temperatures may be adaptively advantageous. The consequent cessation in growth is a balanced one, readily reversible by a lowering of temperature, which allows the plant to escape the irreversible sterilizing effect of heat.

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