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G C Whitelam

Publications and source records attributed to G C Whitelam.

At least 37 records · Page 2Linked to original sources
PubMed1998

Phytochrome E influences internode elongation and flowering time in Arabidopsis.

From a screen of M2 seedlings derived from gamma-mutagenesis of seeds doubly null for phytochromes phyA and phyB, we isolated a mutant lacking phyE. The PHYE gene of the selected mutant, phyE-1, was found to contain a 1-bp deletion at a position equivalent to codon 726, which is predicted to result in a premature stop at codon 739. Immunoblot analysis showed that the phyE protein was undetectable in the phyE-1 mutant. In the phyA- and phyB-deficient background, phyE deficiency led to early flowering, elongation of internodes between adjacent rosette leaves, and reduced petiole elongation. This is a phenocopy of the response of phyA phyB seedlings to end-of-day far-red light treatments. Furthermore, a phyE deficiency attenuated the responses of phyA phyB seedlings to end-of-day far-red light treatments. Monogenic phyE mutants were indistinguishable from wild-type seedlings. However, phyB phyE double mutants flowered earlier and had longer petioles than did phyB mutants. The elongation and flowering responses conferred by phyE deficiency are typical of shade avoidance responses to the low red/far-red ratio. We conclude that in conjunction with phyB and to a lesser extent with phyD, phyE functions in the regulation of the shade avoidance syndrome.

Amino Acid Sequence↗
PubMed1997

The Brassica rapa elongated internode (EIN) gene encodes phytochrome B.

The elongated internode (ein) mutation of Brassica rapa leads to a deficiency in immunochemically detectable phytochrome B. Molecular analysis of the PHYB gene from ein indicates a deletion in the flanking DNA 5' of the ATG start codon, which could interfere either with PHYB transcription or processing of the PHYB transcript. Restriction fragment length polymorphisms and inverse PCR fragments generated from the PHYB gene of wild-type and ein seedlings demonstrate the deletion to be 500 bp in length. Seedlings of heterozygote, EIN/ein, contain about 50% of the level of immunochemically detectable phytochrome B of equivalent wild-type EIN/EIN seedlings. Etiolated seedlings of EIN/ein show a responsiveness to red light almost intermediate between that of ein/ein and EIN/EIN homozygotes. Furthermore, whereas the ein/ein homozygote is poorly responsive to low red/far-red ratio light, the presence of one functional allele of EIN in the heterozygote confers an elongation response intermediate between that of the homozygotes EIN/EIN and ein/ein in these light conditions. The partial dominance of ein indicates a close relationship between phytochrome B level and phenotype.

Brassica↗
PubMed1997

Expression of heterologous phytochromes A, B or C in transgenic tobacco plants alters vegetative development and flowering time.

In this study, oat phytochrome A (phyA), Arabidopsis phytochrome B (phyB) or Arabidopsis phytochrome C (phyC) were expressed in both day-neutral and photo-period-sensitive (short-day) tobacco (Nicotiana tabacum cv. Hicks). Introgression of the Maryland Mammoth (MM) gene into cv Hicks was used to confer short-day photo-periodic sensitivity. Expression of oat phyA led to characteristic hypersensitivity of hypocotyls to red light (R) and far-red light (FR) and an overall dwarfing of the mature plant. Expression of Arabidopsis phyB enhanced the sensitivity of hypocotyls to R and caused even more marked dwarfing of the mature plant. In contrast, the expression of Arabidopsis phyC had no detectable consequences for the photocontrol of hypocotyl elongation. However, phyC expression did lead to a R-dependent increase in cotyledon expansion in de-etiolating seedlings and to a significant increase in leaf area in mature plants. This provides the first experimental evidence that phyC is biologically active. The flowering time of cv Hicks plants grown under 8 h photoperiods was virtually unaffected by a 30 min white light (W) night break given 8 h into the dark period. In contrast, cv Hicks MM plants responded to a night break with a delay in flowering. Expression of phyA or phyB led to a night break-dependent delay in flowering in cv Hicks plants. For cv Hicks MM plants, the expression of any of phyA, phyB or phyC caused a marked enhancement of the flower-delaying effect of a night break. These observations indicate that transgenic phyA, phyB or phyC can interact with the endogenous mechanisms controlling flowering time in tobacco.

Apoproteins↗
PubMed1996

The ELONGATED gene of Arabidopsis acts independently of light and gibberellins in the control of elongation growth.

A novel elongated mutant has been isolated from EMS-mutagenized populations of the Arabidopsis thaliana ga4 mutant. After backcrossing with the Landsberg erecta (Ler) wild-type (WT) followed by selfing, the mutant phenotype was identified in the GA4 background. Seedlings of the mutant, which has been named elg (elongated), are characterized by elongated hypocotyls and petioles, leaves that are narrow and somewhat epinastic and early flowering. Allelism tests with the hy1-hy5 mutants indicate that elg is not allelic with any of these long-hypocotyl mutants. From linkage analyses, the location of elg on chromosome 4, between cer2 and ap2 has been established. The pleiotropic phenotype of elg seedlings is suggestive of a disruption of phytochrome and/or gibberellin (GA) function. Although the elg mutant displays a light-dependent long-hypocotyl phenotype, elg seedlings retain a full range of photomorphogenic responses and the elg mutation acts additively with the photomorphogenic mutants phyB, hy1 and hy2. This suggests that ELG acts independently of phytochrome action. The elg mutation partially suppresses the effect of GA-deficiency on elongation growth, and, although elg ga1 seedlings are more elongated than ga1 seedlings, both genotypes respond in the same way to applied GA. That applied GA and the elg mutation interact additively suggests that ELG acts independently of GA action.

Arabidopsis↗
PubMed1996

The rosette habit of Arabidopsis thaliana is dependent upon phytochrome action: novel phytochromes control internode elongation and flowering time.

A major function of phytochromes in light-grown plants involves the perception of changes in the relative amounts of red and far-red light (R:FR ratio) and the initiation of the shade-avoidance response. In Arabidopsis thaliana, this response is typified by increased elongation growth of petioles and accelerated flowering and can be fully induced by end-of-day far-red light (EOD FR) treatments. Phytochrome B-deficient (phyB) mutants, which have a constitutive elongated-petiole and early-flowering pheno-type, do not display a petiole elongation growth response to EOD FR, but they do respond to EOD FR by earlier flowering. Seedlings deficient in both phytochrome A and phytochrome B (phyA phyB), have a greatly reduced stature compared with wild-type or either monogenic mutant. The phyA phyB double null mutants also respond to EOD FR treatments by flowering early, suggesting the operation of novel phytochromes. Contrary to the behaviour of wild-type or monogenic phyA or phyB seedlings, petiole elongation in phyA phyB seedlings is reduced in response to EOD FR treatments. This reduction in petiole elongation is accompanied by the appearance of elongated internodes such that under these conditions the plants no longer display a rosette habit.

Arabidopsis↗
PubMed1996

fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression.

Physiological analysis of the fhy1 mutant of Arabidopsis has led to the proposal that the mutant is deficient in a downstream component of the phytochrome A signal transduction pathway. To define this lesion at the molecular level, we have examined the expression of a range of phytochrome-regulated genes in fhy1. In far-red light, the regulation of genes such as CHS and CHI is blocked in fhy1, whereas the induction of CAB and NR genes is affected minimally. In contrast, the induction of all genes tested is blocked in a phytochrome A-deficient mutant, confirming that gene expression in far-red light is regulated solely by phytochrome A. Thus, fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression. Contrary to the general opinion that responses to continuous red light are mediated by phytochrome B and other photostable phytochromes, we have shown also that red light-induction of CHS is mediated almost entirely by phytochrome A. Furthermore, phytochrome A-mediated induction of CHS by red light is blocked in fhy1. The induction of CHS by blue light, however, is normal in fhy1, suggesting that although FHY1 is a component of the phytochrome A signaling pathway, it is not a component of the blue-light signaling pathway for CHS expression.

Acyltransferases↗
PubMed1996

Study of antibody-antigen interaction through site-directed mutagenesis of the VH region of a hybrid phage-antibody fragment.

An important aspect of the study of antibody structure-function relationships involves analysis of natural or synthetic mutations of antigen-combining sites. The anti-hen egg lysozyme monoclonal antibody HyHEL-10 has been a focus for antibody structure-function studies. We have displayed on bacteriophage of a hybrid single chain Fv, containing the light chain variable region of HyHEL-10 and the heavy chain variable region of a structurally related but functionally distinct antibody, AS32. By using a combination of site-directed mutagenesis, complementary determining region grafting and molecular modeling, we have identified a number of contact and non-contact residues that are important in the affinity of HyHEL-10 for lysozyme. In particular, the heavy chain variable region framework residue at position 94 was shown to be an important determinant of high-affinity binding. The phage display approach eliminates the need for purification of antibodies and, when used in combination with polymerase chain reaction for variable region sequence mutagenesis, facilitates the rapid generation and characterization of mutant antibodies.

Animals↗
PubMed1996

Far-red light blocks greening of Arabidopsis seedlings via a phytochrome A-mediated change in plastid development.

We have characterized a far-red-light response that induces a novel pathway for plastid development in Arabidopsis seedlings. This response results in the inability of cotyledons to green upon subsequent white light illumination, and the response is suppressed by exogenous sucrose. Studies with mutants showed that this far-red block of greening is phytochrome A dependent and requires an intact downstream signaling pathway in which FHY1 and FHY3 may be components but in which HY5 is not. This highlights a previously undefined branchpoint in the phytochrome signaling pathway. Ultrastructural analysis showed that the far-red block correlates with both the failure of plastids to accumulate prolamellar bodies and the formation of vesicles in the stroma. We present evidence that the far-red block of greening is the result of severe repression of protochlorophyllide reductase (POR) genes by far-red light coupled with irreversible plastid damage. This results in the temporal separation of phytochrome-mediated POR; repression from light-dependent protochlorophyllide reduction, two processes that normally occur in coordination in white light.

Arabidopsis↗
PubMed1995

Impaired splicing of phytochrome B pre-mRNA in a novel phyB mutant of Arabidopsis.

Phytochrome is the red/far-red absorbing photoreceptor active in photomorphogenesis, the apoprotein of which is encoded by a small gene family (PHYA, PHYB, PHYC, PHYD and PHYE). A novel phytochrome B-deficient mutant, phyB-103, was isolated from a screen of EMS-mutagenised Arabidopsis M2 seed. phyB-103 carries a G-to-A base substitution at the 5' splice site +1 G nucleotide of intron 1 of PHYB. The phyB-103 PHYB transcript is larger than the wild-type PHYB transcript and DNA sequence analysis showed that the entire intron is retained in the mature PHYB transcript of phyB-103. Thus the phyB-103 G-to-A substitution prevents intron splicing. The retained intron contains within it an in-frame stop codon, and the predicted PHYB-003 apoprotein thus terminates prematurely. phyB-103 is therefore likely to be a null allele of PHYB, consistent with the observation that the phenotype conferred by phyB-103 is as severe as that conferred by previously described phyB null alleles.

Arabidopsis↗
PubMed1995

Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh.

The long-day plant Arabidopsis thaliana (L.) Heynh. flowers early in response to brief end-of-day (EOD) exposures to far-red light (FR) following a fluorescent short day of 8 h. FR promotion of flowering was nullified by subsequent brief red light (R) EOD exposure, indicating phytochrome involvement. The EOD response to R or FR is a robust measure of phytochrome action. Along with their wild-type (WT) parents, mutants deficient in either phytochrome A or B responded similarly to the EOD treatments. Thus, neither phytochrome A nor B exclusively regulated flowering, although phytochrome B controlled hypocotyl elongation. Perhaps a third phytochrome species is important for the EOD responses of the mutants and/or their flowering is regulated by the amount of the FR-absorbing form of phytochrome, irrespective of the phytochrome species. Overexpression of phytochrome A or phytochrome B resulted in differing photoperiod and EOD responses among the genotypes. The day-neutral overexpressor of phytochrome A had an EOD response similar to all of the mutants and WTs, whereas R EOD exposure promoted flowering in the overexpressor of phytochrome B and FR EOD exposure inhibited this promotion. The comparisons between relative flowering times and leaf numbers at flowering of the over-expressors and their WTs were not consistent across photoperiods and light treatments, although both phytochromes A and B contributed to regulating flowering of the transgenic plants.

Arabidopsis↗
PubMed1994

Antigen detection using recombinant, bifunctional single-chain Fv fusion proteins synthesised in Escherichia coli.

A gene fusion approach has been used to produce antibody conjugates for use in immunoassays. Escherichia coli expression vectors encoding fusions between the outer membrane protein A signal peptide, an anti-phytochrome single-chain Fv protein, and either Escherichia coli alkaline phosphatase or Staphylococcal protein A downstream of the T7 O10 promoter were constructed. A crude lysate from cells expressing the single-chain Fv-alkaline phosphatase fusion protein could be used directly for the sensitive and specific staining of phytochrome on protein blots by a single-step immunoassay procedure. Following purification by immunoglobulin G affinity chromatography, the Staphylococcal protein A-single-chain Fv fusion protein was also used for selective immunostaining of phytochrome on protein blots by a two-step procedure in which a rabbit immunoglobulin-alkaline phosphatase conjugate was used to detect antigen-bound Staphylococcal protein A. Recombinant antibody conjugates of the types described here are simple and inexpensive to produce and are a realistic alternative to conventional antibody conjugates.

Alkaline Phosphatase↗
PubMed1994

Endoplasmic reticulum targeting of active modified beta-glucuronidase (GUS) in transgenic tobacco plants.

We have investigated targeting to the endoplasmic reticulum (ER) of wild-type GUS and a modified form (GUS S358) by making an N-terminal fusion of the beta-glucuronidase (GUS) enzyme with the wheat alpha-amylase signal peptide. In vitro studies demonstrated that the modified GUS (S358) lacked the glycosylation site present within the wild-type enzyme. Analysis of transgenic tobacco plants revealed that the modified GUS enzyme retained activity upon passage to the ER. When further experiments were carried out to determine the cellular location of the modified GUS enzyme, it was found that (contrary to expectation) the majority of GUS activity was retained within the cell and was not secreted to the cell surface via the default pathway. The data indicated that the modified GUS enzyme is an unsuitable reporter enzyme for studying protein secretion.

Amino Acid Sequence↗
PubMed1993

Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures.

A synthetic gene encoding an anti-phytochrome single-chain Fv (scFv) antibody bearing an N-terminal signal peptide has been used to transform tobacco plants. Immunoblot analysis showed that transformed plants accumulate high levels of scFv protein, accounting for up to 0.5% of the total soluble protein fraction, which could be extracted by simple infiltration and centrifugation of leaf tissue. A substantial proportion of the scFv protein extracted in this way was found to possess antigen-binding activity. Callus cell suspension cultures derived from transformed plants secrete functional scFv protein into the surrounding medium. Compared with the levels of scFv protein observed in plants expressing the native scFv gene, the incorporation of an N-terminal signal peptide, to target the scFv to the apoplast, results in elevated accumulation of the protein.

Amino Acid Sequence↗
PubMed1993

Phytochrome A null mutants of Arabidopsis display a wild-type phenotype in white light.

Phytochrome is a family of photoreceptors that regulates plant photomorphogenesis; the best-characterized member of this family is phytochrome A. Here, we report the identification of novel mutations at three Arabidopsis loci (fhy1, fhy2, and fhy3) that confer an elongated hypocotyl in far-red but not in white light. fhy2 mutants are phytochrome A deficient, have reduced or undetectable levels of PHYA transcripts, and contain structural alterations within the PHYA gene. When grown in white light, fhy2 mutants are morphologically indistinguishable from wild-type plants. Thus, phytochrome A appears to be dispensable in white light-grown Arabidopsis plants. fhy2 alleles confer partially dominant phenotypes in far-red light, suggesting that the relative abundance of phytochrome A can affect the extent of the far-red-mediated hypocotyl growth inhibition response. Plants homozygous for the recessive fhy1 and fhy3 mutations have normal levels of functional phytochrome A. The FHY1 and FHY3 gene products may be responsible for the transduction of the far-red light signal from phytochrome A to downstream processes involved in hypocotyl growth regulation.

Arabidopsis↗
PubMed1992

Production and secretion of a bifunctional staphylococcal protein A::antiphytochrome single-chain Fv fusion protein in Escherichia coli.

A bifunctional molecule was genetically engineered which contained the secretory signal and four Fc-binding domains of Staphylococcus aureus protein A (FcA), fused to a single-chain Fv (scFv) derived from an immunoglobulin (Ig) G1 mouse monoclonal antibody (AS32) directed against the plant regulatory photoreceptor protein, phytochrome. The FcA::AS32scFv sequence was encoded in a single synthetic gene and expressed as a 60-kDa periplasmic protein in Escherichia coli. The bifunctionality of the fusion protein was established by its ability to bind to both IgG-agarose and phytochrome-sepharose. Growth of cultures, producing the FcA::AS32scFv, at 37 degrees C, resulted in a decrease in the periplasmic accumulation of the fusion protein, and an increased accumulation of an assumed degradation product which retained Fc-binding activity. Growth of cultures at lower temperatures favoured the accumulation of undegraded fusion protein. The recombinant fusion protein could be purified to homogeneity by a simple, rapid chromatography procedure.

Animals↗