ABSTRACT
Root elongation depends on the action of the gibberellin (GA) growth hormones, which promote cell production in the root meristem and cell expansion in the elongation zone. Sites of GA biosynthesis in the roots of 7-d-old Arabidopsis thaliana seedlings were investigated using tissue-specific GA inactivation in wild-type (Col-0) or rescue of GA-deficient dwarf mutants. Tissue-specific GA depletion was achieved by ectopic expression of the GA-inactivating enzyme AtGA2ox2, which is specific for C19 -GAs, and AtGA2ox7, which acts on C20 -GA precursors. In addition, tissue-specific rescue of ga20ox triple and ga3ox double mutants was shown. Furthermore, GUS reporter lines for major GA20ox, GA3ox and GA2ox genes were used to observe their expression domains in the root. The effects of expressing these constructs on the lengths of the root apical meristem and cortical cells in the elongation zone confirmed that roots are autonomous for GA biosynthesis, which occurs in multiple tissues, with the endodermis a major site of synthesis. The results are consistent with the early stages of GA biosynthesis within the root occurring in the meristematic region and indicate that the penultimate step of GA biosynthesis, GA 20-oxidation, is required in both the meristem and elongation zone.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant , Gibberellins , Meristem/metabolismABSTRACT
EGF receptor (EGFR) endocytosis is induced by stress in a manner dependent on the p38 MAPK family. Ligand and stresses such as X-rays, reportedly promote nuclear trafficking of endocytosed EGFR for regulation of gene transcription and DNA repair. We fail to detect EGFR endocytosis or nuclear transport following X-ray treatment of HeLa or head and neck cancer cells, despite extensive DNA damage induction. Apparent nuclear staining with EGFR extracellular domain antibody remained present despite reduced/absent EGFR expression, and so did not represent nuclear EGFR. UVB and UVC, but not X-ray or UVA, treatment induced p38 activation and EGFR endocytosis, although all of these stresses induced DNA damage, indicating that DNA damage alone is not sufficient to induce EGFR endocytosis. Increased reactive oxygen species (ROS) levels following UVB treatment, compared to that seen with X-rays, do not alone explain differences in p38 activation. UVB, like UVC, induced EGFR accumulation predominantly in perinuclear endosomes, rather than in the nucleus. Our morphological techniques identifying major changes in receptor distribution do not exclude the possibility that small but biologically relevant amounts of EGFR enter the nucleus. This study highlights the importance and limitations of morphological analyses of receptor distribution in understanding signaling outcome.
Subject(s)
Cell Nucleus/metabolism , Endocytosis/radiation effects , ErbB Receptors/metabolism , Ultraviolet Rays , X-Rays , p38 Mitogen-Activated Protein Kinases/metabolism , Active Transport, Cell Nucleus/radiation effects , Animals , Enzyme Activation/radiation effects , HeLa Cells , Humans , Mice , NIH 3T3 CellsABSTRACT
BACKGROUND: The gibberellin (GA) pathway plays a central role in the regulation of plant development, with the 2-oxoglutarate-dependent dioxygenases (2-ODDs: GA20ox, GA3ox, GA2ox) that catalyse the later steps in the biosynthetic pathway of particularly importance in regulating bioactive GA levels. Although GA has important impacts on crop yield and quality, our understanding of the regulation of GA biosynthesis during wheat and barley development remains limited. In this study we identified or assembled genes encoding the GA 2-ODDs of wheat, barley and Brachypodium distachyon and characterised the wheat genes by heterologous expression and transcript analysis. RESULTS: The wheat, barley and Brachypodium genomes each contain orthologous copies of the GA20ox, GA3ox and GA2ox genes identified in rice, with the exception of OsGA3ox1 and OsGA2ox5 which are absent in these species. Some additional paralogs of 2-ODD genes were identified: notably, a novel gene in the wheat B genome related to GA3ox2 was shown to encode a GA 1-oxidase, named as TaGA1ox-B1. This enzyme is likely to be responsible for the abundant 1ß-hydroxylated GAs present in developing wheat grains. We also identified a related gene in barley, located in a syntenic position to TaGA1ox-B1, that encodes a GA 3,18-dihydroxylase which similarly accounts for the accumulation of unusual GAs in barley grains. Transcript analysis showed that some paralogs of the different classes of 2-ODD were expressed mainly in a single tissue or at specific developmental stages. In particular, TaGA20ox3, TaGA1ox1, TaGA3ox3 and TaGA2ox7 were predominantly expressed in developing grain. More detailed analysis of grain-specific gene expression showed that while the transcripts of biosynthetic genes were most abundant in the endosperm, genes encoding inactivation and signalling components were more highly expressed in the seed coat and pericarp. CONCLUSIONS: The comprehensive expression and functional characterisation of the multigene families encoding the 2-ODD enzymes of the GA pathway in wheat and barley will provide the basis for a better understanding of GA-regulated development in these species. This analysis revealed the existence of a novel, endosperm-specific GA 1-oxidase in wheat and a related GA 3,18-dihydroxylase enzyme in barley that may play important roles during grain expansion and development.
Subject(s)
Biosynthetic Pathways/genetics , Genes, Plant , Gibberellins/biosynthesis , Mixed Function Oxygenases/genetics , Multigene Family , Poaceae/enzymology , Poaceae/genetics , Biocatalysis , Brachypodium/enzymology , Brachypodium/genetics , Gene Expression Regulation, Plant , Hordeum/enzymology , Hordeum/genetics , Oryza/enzymology , Oryza/genetics , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, RNA , Signal Transduction/genetics , Triticum/enzymology , Triticum/geneticsABSTRACT
The introduction of the Reduced height (Rht)-B1b and Rht-D1b semidwarfing genes led to impressive increases in wheat (Triticum aestivum) yields during the Green Revolution. The reduction in stem elongation in varieties containing these alleles is caused by a limited response to the phytohormone gibberellin (GA), resulting in improved resistance to stem lodging and yield benefits through an increase in grain number. Rht-B1 and Rht-D1 encode DELLA proteins, which act to repress GA-responsive growth, and their mutant alleles Rht-B1b and Rht-D1b are thought to confer dwarfism by producing more active forms of these growth repressors. While no semidwarfing alleles of Rht-A1 have been identified, we show that this gene is expressed at comparable levels to the other homeologs and represents a potential target for producing novel dwarfing alleles. In this study, we have characterized additional dwarfing mutations in Rht-B1 and Rht-D1. We show that the severe dwarfism conferred by Rht-B1c is caused by an intragenic insertion, which results in an in-frame 90-bp insertion in the transcript and a predicted 30-amino acid insertion within the highly conserved amino-terminal DELLA domain. In contrast, the extreme dwarfism of Rht-D1c is due to overexpression of the semidwarfing Rht-D1b allele, caused by an increase in gene copy number. We show also that the semidwarfing alleles Rht-B1d and Rht-B1e introduce premature stop codons within the amino-terminal coding region. Yeast two-hybrid assays indicate that these newly characterized mutations in Rht-B1 and Rht-D1 confer "GA-insensitive" dwarfism by producing DELLA proteins that do not bind the GA receptor GA INSENSITIVE DWARF1, potentially compromising their targeted degradation.
Subject(s)
Gibberellins/pharmacology , Plant Growth Regulators/pharmacology , Plant Proteins/genetics , Triticum/growth & development , Triticum/genetics , Alleles , Amino Acid Sequence , Base Sequence , DNA, Plant/chemistry , DNA, Plant/genetics , Molecular Sequence Data , Mutation , Phenotype , Plant Proteins/metabolism , Plant Stems/genetics , Plant Stems/growth & development , Polyploidy , RNA, Messenger/genetics , RNA, Plant/genetics , Sequence Alignment , Sequence Analysis, DNA , Triticum/metabolism , Two-Hybrid System TechniquesABSTRACT
The composition of proanthocyanidins in the testa (seed coat) of bread wheat was analyzed by thiolysis of PA oligomers from developing grain and found to consist of (+)-catechin monomers, with a small amount of (+)-gallocatechin. The average chain length of soluble PA stayed relatively constant between 10 and 20 days post-anthesis, whereas that of unextractable PA increased over the same period, suggesting that increases in chain length might account for the insolubility of PAs from mature wheat grain. We carried out RNA-Seq followed by differential expression analysis from dissected tissues of developing grain from red- and white-grained near-isogenic lines differing in the presence of an active R gene that encodes a MYB transcription factor involved in control of PA biosynthesis. In addition to genes already identified encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, and dihydroxyflavonoid 4-reductase, we showed that wheat genes encoding phenylalanine ammonia lyase, flavonoid 3',5'-hydroxylase, leucoanthocyanidin reductase, and a glutathione S-transferase (the orthologue of maize Bronze-2) were more highly expressed in the red NIL. We also identified candidate orthologues of other catalytic and regulatory components of flavonoid biosynthesis in wheat.
ABSTRACT
Stress exposure triggers ligand-independent EGF receptor (EGFR) endocytosis, but its post-endocytic fate and role in regulating signalling are unclear. We show that the p38 MAP kinase-dependent, EGFR tyrosine kinase (TK)-independent EGFR internalization induced by ultraviolet light C (UVC) or the cancer therapeutic cisplatin, is followed by diversion from the canonical endocytic pathway. Instead of lysosomal degradation or plasma membrane recycling, EGFR accumulates in a subset of LBPA-rich perinuclear multivesicular bodies (MVBs) distinct from those carrying EGF-stimulated EGFR. Stress-internalized EGFR co-segregates with exogenously expressed pre-melanosomal markers OA1 and fibrillar PMEL, following early endosomal sorting by the actin polymerization-promoting WASH complex. Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs. In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention. EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance.
Subject(s)
DNA-Binding Proteins/metabolism , Endocytosis , Endosomal Sorting Complexes Required for Transport/metabolism , ErbB Receptors/metabolism , Microfilament Proteins/metabolism , Transcription Factors/metabolism , HeLa Cells , Humans , Oxidative Stress , Protein Transport , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The central engines of disc-accreting stellar-mass black holes appear to be scaled down versions of the supermassive black holes that power active galactic nuclei. However, if the physics of accretion is universal, it should also be possible to extend this scaling to other types of accreting systems, irrespective of accretor mass, size, or type. We examine new observations, obtained with Kepler/K2 and ULTRACAM, regarding accreting white dwarfs and young stellar objects. Every object in the sample displays the same linear correlation between the brightness of the source and its amplitude of variability (rms-flux relation) and obeys the same quantitative scaling relation as stellar-mass black holes and active galactic nuclei. We also show that the most important parameter in this scaling relation is the physical size of the accreting object. This establishes the universality of accretion physics from proto-stars still in the star-forming process to the supermassive black holes at the centers of galaxies.
ABSTRACT
Progress in astronomy comes from interpreting the signals encoded in the light received from distant objects: the distribution of light over the sky (images), over photon wavelength (spectrum), over polarization angle and over time (usually called light curves by astronomers). In the time domain, we see transient events such as supernovae, gamma-ray bursts and other powerful explosions; we see periodic phenomena such as the orbits of planets around nearby stars, radio pulsars and pulsations of stars in nearby galaxies; and we see persistent aperiodic variations ('noise') from powerful systems such as accreting black holes. I review just a few of the recent and future challenges in the burgeoning area of time domain astrophysics, with particular attention to persistently variable sources, the recovery of reliable noise power spectra from sparsely sampled time series, higher order properties of accreting black holes, and time delays and correlations in multi-variate time series.
ABSTRACT
Cellular senescence is a program of irreversible cell cycle arrest that cells undergo in response to a variety of intrinsic and extrinsic stimuli including progressive shortening of telomeres, changes in telomeric structure or other forms of genotoxic and non-genotoxic stress. The role of nuclear factor-κB in cellular senescence is controversial, as it has been associated with both proliferation and tumour progression, and also with growth arrest and ageing. This research perspective focuses on the evidence for a functional relationship between NF-κB and senescence, and how disruption of the NF-κB pathway can lead to its bypass.
Subject(s)
Cellular Senescence/physiology , NF-kappa B/metabolism , Cell Cycle/physiology , Humans , Poly(ADP-ribose) Polymerases/metabolism , Signal Transduction/physiology , Telomere/metabolismABSTRACT
Many insect and fungal pathogens posing agronomically important threats specifically target the roots in strawberry. The use of a root-specific promoter to confer expression of resistance genes in a targeted manner has the potential appreciably to benefit the genetic improvement of commercial strawberry varieties. A novel gene, FaRB7, was isolated from strawberry (Fragariaxananassa Duch.) and found to contain motifs characteristic of tonoplast intrinsic proteins (TIPs). Phylogenetic analysis revealed that FaRB7 represents an RB7-type TIP. In strawberry, this gene is expressed predominantly in roots, with very low expression in petioles. A 2.843 kb region representing the FaRB7 gene upstream regulatory sequence was isolated and found to share a number of sequence motifs with the promoter of the Nicotiana tabacum TobRB7 root-specific RB7-type TIP. When cloned upstream of the gusA reporter gene and introduced into strawberry plants, the FaRB7 promoter was shown to direct strong, near root-specific expression with expression patterns very similar to that of the endogenous gene. Furthermore, the FaRB7 promoter was found to confer constitutive expression, comparable to that produced by the cauliflower mosaic virus (CaMV) 35S RNA promoter, in tobacco. Thus, the FaRB7 promoter may be used to achieve near-root-specific transgene expression in strawberry and also represents an alternative to the CaMV 35S promoter for producing constitutive foreign gene expression in heterologous hosts. The FaRB7 full-length genomic sequence and 5' upstream regulatory region have been submitted to the EMBL/GenBank database under accession number DQ178022.
Subject(s)
Fragaria/genetics , Plant Proteins/genetics , Promoter Regions, Genetic , Amino Acid Sequence , Fragaria/anatomy & histology , Fragaria/metabolism , Genes, Reporter , Glucuronidase/analysis , Molecular Sequence Data , Phylogeny , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Plants, Genetically Modified/anatomy & histology , Plants, Genetically Modified/metabolism , Recombinant Fusion Proteins/analysis , Nicotiana/geneticsABSTRACT
Recently, an S haplotype-specific F-box (SFB) gene has been proposed as a candidate for the pollen-S specificity gene of RNase-mediated gametophytic self-incompatibility in Prunus (Rosaceae). We have examined two pollen-part mutant haplotypes of sweet cherry (Prunus avium). Both were found to retain the S-RNase, which determines stylar specificity, but one (S3' in JI 2434) has a deletion including the haplotype-specific SFB gene, and the other (S4' in JI 2420) has a frame-shift mutation of the haplotype-specific SFB gene, causing amino acid substitutions and premature termination of the protein. The loss or significant alteration of this highly polymorphic gene and the concomitant loss of pollen self-incompatibility function provides compelling evidence that the SFB gene encodes the pollen specificity component of self-incompatibility in Prunus. These loss-of-function mutations are inconsistent with SFB being the inactivator of non-self S-RNases and indicate the presence of a general inactivation mechanism, with SFB conferring specificity by protecting self S-RNases from inactivation.