Dataset Information

The molecular cloning and clarification of a photorespiratory mutant, oscdm1, using enhancer trapping.

ABSTRACT: Enhancer trap systems have been demonstrated to increase the effectiveness of gene identification in rice. In this study, a chlorophyll-deficient mutant, named oscdm1, was screened and characterized in detail from a T-DNA enhancer-tagged population. The oscdm1 plants were different from other chlorophyll-deficient mutants; they produced chlorotic leaves at the third leaf stage, which gradually died with further growth of the plants. However, the oscdm1 plants were able to survive exposure to elevated CO2 levels, similar to photorespiratory mutants. An analysis of the T-DNA flanking sequence in the oscdm1 plants showed that the T-DNA was inserted into the promoter region of a serine hydroxymethyltransferase (SHMT) gene. OsSHMT1 is a key enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for the interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Full-length OsSHMT1 complemented the oscdm1 phenotype, and the downregulation of OsSHMT1 in wild-type plants by RNA interference (RNAi) produced plants that mimicked the oscdm1 phenotype. GUS assays and quantitative PCR revealed the preferential expression of OsSHMT1 in young leaves. TEM revealed serious damage to the thylakoid membrane in oscdm1 chloroplasts. The oscdm1 plants showed more extensive damage than wild type using an IMAGING-PAM fluorometer, especially under high light intensities. OsSHMT1-GFP localized exclusively to mitochondria. Further analysis revealed that the H2O2 content in the oscdm1 plants was twice that in wild type at the fourth leaf stage. This suggests that the thylakoid membrane damage observed in the oscdm1 plants was caused by excessive H2O2. Interestingly, OsSHMT1-overexpressing plants exhibited increased photosynthetic efficiency and improved plant productivity. These results lay the foundation for further study of the OsSHMT1 gene and will help illuminate the functional role of OsSHMT1 in photorespiration in rice.

SUBMITTER: Wu J

PROVIDER: S-EPMC4490251 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The molecular cloning and clarification of a photorespiratory mutant, oscdm1, using enhancer trapping.

Wu Jinxia J Zhang Zhiguo Z Zhang Qian Q Han Xiao X Gu Xiaofeng X Lu Tiegang T

Frontiers in genetics 20150703

Enhancer trap systems have been demonstrated to increase the effectiveness of gene identification in rice. In this study, a chlorophyll-deficient mutant, named oscdm1, was screened and characterized in detail from a T-DNA enhancer-tagged population. The oscdm1 plants were different from other chlorophyll-deficient mutants; they produced chlorotic leaves at the third leaf stage, which gradually died with further growth of the plants. However, the oscdm1 plants were able to survive exposure to ele ...[more]

PMID: 26191072

Similar Datasets

Project description:Three active-site components in rhodopsin play a key role in the stability and function of the protein: 1) the counter-ion residues which stabilize the protonated Schiff base, 2) water molecules, and 3) the hydrogen-bonding network. The ionizable residue Glu-181, which is involved in an extended hydrogen-bonding network with Ser-186, Tyr-268, Tyr-192, and key water molecules within the active site of rhodopsin, has been shown to be involved in a complex counter-ion switch mechanism with Glu-113 during the photobleaching sequence of the protein. Herein, we examine the photobleaching sequence of the E181Q rhodopsin mutant by using cryogenic UV-visible spectroscopy to further elucidate the role of Glu-181 during photoactivation of the protein. We find that lower temperatures are required to trap the early photostationary states of the E181Q mutant compared to native rhodopsin. Additionally, a Blue Shifted Intermediate (BSI, ?max = 498 nm, 100 K) is observed after the formation of E181Q Bathorhodopsin (Batho, ?max = 556 nm, 10 K) but prior to formation of E181Q Lumirhodopsin (Lumi, ?max = 506 nm, 220 K). A potential energy diagram of the observed photointermediates suggests the E181Q Batho intermediate has an enthalpy value 7.99 KJ/mol higher than E181Q BSI, whereas in rhodopsin, the BSI is 10.02 KJ/mol higher in enthalpy than Batho. Thus, the Batho to BSI transition is enthalpically driven in E181Q and entropically driven in native rhodopsin. We conclude that the substitution of Glu-181 with Gln-181 results in a significant perturbation of the hydrogen-bonding network within the active site of rhodopsin. In addition, the removal of a key electrostatic interaction between the chromophore and the protein destabilizes the protein in both the dark state and Batho intermediate conformations while having a stabilizing effect on the BSI conformation. The observed destabilization upon this substitution further supports that Glu-181 is negatively charged in the early intermediates of the photobleaching sequence of rhodopsin.

Project description:Glucocorticoid-induced gene-1 (Gig1) was identified in a yeast one-hybrid screen for factors that interact with the MyoD core enhancer. The Gig1 gene encodes a novel C2H2 zinc finger protein that shares a high degree of sequence similarity with two known DNA binding proteins in humans, Glut4 enhancer factor and papillomavirus binding factor (PBF). The mouse ortholog of PBF was also isolated in the screen. The DNA binding domain of Gig1, which contains TCF-E-tail CR1 and CR2 motifs shown to mediate promoter specificity of TCF-E-tail isoforms, was mapped to a C-terminal domain that is highly conserved in Glut4 enhancer factor and PBF. In mouse embryos, in situ hybridization revealed a restricted pattern of expression of Gig1 that overlaps with MyoD expression. A nuclear-localized lacZ knockin null allele of Gig1 was produced to study Gig1 expression with greater resolution and to assess Gig1 functions. X-gal staining of Gig1(nlacZ) heterozygous embryos revealed Gig1 expression in myotomal myocytes, skeletal muscle precursors in the limb, and in nascent muscle fibers of the body wall, head and neck, and limbs through E14.5 (latest stage examined). Gig1 was also expressed in a subset of Scleraxis-positive tendon precursors/rudiments of the limbs, but not in the earliest tendon precursors of the somite (syndetome) defined by Scleraxis expression. Additional regions of Gig1 expression included the apical ectodermal ridge, neural tube roof plate and floor plate, apparent motor neurons in the ventral neural tube, otic vesicles, notochord, and several other tissues representing all three germ layers. Gig1 expression was particularly well represented in epithelial tissues and in a number of cells/tissues of neural crest origin. Expression of both the endogenous MyoD gene and a reporter gene driven by MyoD regulatory elements was similar in wild-type and homozygous null Gig1(nlacZ) embryos, and mutant mice were viable and fertile, indicating that the functions of Gig1 are redundant with other factors.

Dataset Information

The molecular cloning and clarification of a photorespiratory mutant, oscdm1, using enhancer trapping.

Publications

The molecular cloning and clarification of a photorespiratory mutant, oscdm1, using enhancer trapping.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets