The hy3 Long Hypocotyl Mutant of Arabidopsis Is Deficient in Phytochrome B.

The six long hypocotyl (hy) complementation groups of Arabidopsis (hy1, hy2, hy3, hy4, hy5, and hy6) share the common feature of an elongated hypocotyl when grown in white light. The varied responses of these mutants to irradiations of differing wavelengths have suggested that some of the lines may lack elements of the phytochrome signal transduction pathway. We have performed immunoblot and RNA gel blot analyses of the multiple types of phytochrome present in wild-type and mutant Arabidopsis and provide evidence that mutations at the HY3 locus cause a specific deficiency in phytochrome B. Using an Escherichia coli overexpression system, we have developed and identified monoclonal antibodies that selectively recognize phytochromes A, B, and C from Arabidopsis. In wild-type plants, phytochrome A is highly abundant in etiolated tissue, but rapidly decreases about 200-fold upon illumination. Phytochromes B and C are present at much lower levels in etiolated tissue but are unaffected by up to 24 hr of red light illumination, and together predominate in green seedlings. These data establish that phytochromes B and C are "type 2" or photostable phytochromes. Levels of phytochromes A, B, and C similar to those of the wild type are observed in strains containing mutations at the HY4 and HY5 loci. In contrast, all four hy3 mutant alleles tested here exhibit a modest (twofold to threefold) reduction in phyB transcript and a severe (20- to 50-fold) deficiency in phyB-encoded protein, relative to levels in wild-type plants. The levels of phyA- and phyC-encoded mRNA and protein, however, are indistinguishable from the wild type in these mutants. We conclude that the phenotype conferred by hy3 is due to the reduced levels of the light-stable phytochrome B.

Coordination of phytochrome levels in phyB mutants of Arabidopsis as revealed by apoprotein-specific monoclonal antibodies.

Accumulating evidence indicates that individual members of the phytochrome family of photoreceptors have differential but interactive roles in controlling plant responses to light. To investigate possible cross-regulation of these receptors, we have identified monoclonal antibodies that specifically detect each of the five Arabidopsis phytochromes, phyA to phyE (phytochrome A holoprotein; PHYA, phytochrome A apoprotein; PHYA, phytochrome A gene; phyA, mutant allele of phytochrome A gene), on immunoblots and have used them to analyze the effects of phyA and phyB null mutations on the levels of all five family members. In phyB mutants, but not in phyA mutants, a four- to six-fold reduction in the level of phyC is observed in tissues grown either in the dark or in the light. Coordinate expression of phyB and phyC is induced in the phyB mutant background by the presence of a complementing PHYB transgene. However, in transgenic lines that overexpress phyB 15- to 20-fold, phyC is not similarly overexpressed. In these overexpressor lines, the levels of phyA, phyC, and phyD are increased two- to four-fold over normal in light-grown but not dark-grown seedlings. These observations indicate that molecular mechanisms for coordination or cross-regulation of phytochrome levels are active in Arabidopsis and have implications for the interpretation of phytochrome mutants and overexpressor lines.

Nucleotide and amino acid sequence of a Cucurbita phytochrome cDNA clone: identification of conserved features by comparison with Avena phytochrome.

The amino acid (aa) sequence of Cucurbita phytochrome has been deduced from the nucleotide (nt) sequence of a cDNA clone which was initially identified by hybridization to an Avena phytochrome cDNA clone. Cucurbita, a dicot, and Avena, a monocot, represent evolutionarily divergent groups of plants. The Cucurbita phytochrome polypeptide is 1123 aa in length, corresponding to 125 kDa. Overall, the Cucurbita and Avena phytochrome sequences are 65% homologous at both the nt and aa levels but this sequence conservation is not evenly distributed. Most of the N-terminal two-thirds of the aligned polypeptide chains exhibits localized regions of high conservation, while the extreme N terminus and the C-terminal one-third are less homologous. Comparison of the predicted hydropathic properties of these polypeptides also indicates conservation of domains of phytochrome structure. The possible correlation of these conserved structural features with previously identified functional domains of phytochrome is discussed.

Arabidopsis phytochromes C and E have different spectral characteristics from those of phytochromes A and B.

The red/far-red light absorbing phytochromes play a major role as sensor proteins in photomorphogenesis of plants. In Arabidopsis the phytochromes belong to a small gene family of five members, phytochrome A (phyA) to E (phyE). Knowledge of the dynamic properties of the phytochrome molecules is the basis of phytochrome signal transduction research. Beside photoconversion and destruction, dark reversion is a molecular property of some phytochromes. A possible role of dark reversion is the termination of signal transduction. Since Arabidopsis is a model plant for biological and genetic research, we focussed on spectroscopic characterization of Arabidopsis phytochromes, expressed in yeast. For the first time, we were able to determine the relative absorption maxima and minima for a phytochrome C (phyC) as 661/725 nm and for a phyE as 670/724 nm. The spectral characteristics of phyC and E are strictly different from those of phyA and B. Furthermore, we show that both phyC and phyE apoprotein chromophore adducts undergo a strong dark reversion. Difference spectra, monitored with phycocyanobilin and phytochromobilin as the apoprotein's chromophore, and in vivo dark reversion of the Arabidopsis phytochrome apoprotein phycocyanobilin adducts are discussed with respect to their physiological function.

The Arabidopsis phytochrome A gene has multiple transcription start sites and a promoter sequence motif homologous to the repressor element of monocot phytochrome A genes.

We have determined the sequence of the phytochrome A gene (PHYA) and its flanking DNA from Arabidopsis thaliana and have identified transcription start sites for three nested transcripts of increasing length. The overall structure of the gene is similar as regards exon/intron organization to other angiosperm PHY genes characterized. The triple transcription start site arrangement is similar to that of pea PHYA but different from the single start site of oat, rice and maize PHYA genes, indicating a possible monocot-dicot difference. Comparison of the Arabidopsis PHYA promoter sequence with others available indicates that both pea and Arabidopsis promoters contain a DNA element with a core sequence motif identical to one conserved in all existing monocot PHYA sequences and defined by functional assay in the oat PHYA gene as repressor element, RE1, responsible for negative light regulation.

Novel phytochrome sequences in Arabidopsis thaliana: structure, evolution, and differential expression of a plant regulatory photoreceptor family.

Phytochrome is a plant regulatory photoreceptor that mediates red light effects on a wide variety of physiological and molecular responses. DNA blot analysis indicates that the Arabidopsis thaliana genome contains four to five phytochrome-related gene sequences. We have isolated and sequenced cDNA clones corresponding to three of these genes and have deduced the amino acid sequence of the full-length polypeptide encoded in each case. One of these proteins (phyA) shows 65-80% amino acid sequence identity with the major, etiolated-tissue phytochrome apoproteins described previously in other plant species. The other two polypeptides (phyB and phyC) are unique in that they have low sequence identity (approximately 50%) with each other, with phyA, and with all previously described phytochromes. The phyA, phyB, and phyC proteins are of similar molecular mass, have related hydropathic profiles, and contain a conserved chromophore attachment region. However, the sequence comparison data indicate that the three phy genes diverged early in plant evolution, well before the divergence of the two major groups of angiosperms, the monocots and dicots. The steady-state level of the phyA transcript is high in dark-grown A. thaliana seedlings and is down-regulated by light. In contrast, the phyB and phyC transcripts are present at lower levels and are not strongly light-regulated. These findings indicate that the red/far light-responsive phytochrome photoreceptor system in A. thaliana, and perhaps in all higher plants, consists of a family of chromoproteins that are heterogeneous in structure and regulation.

Intergenic suppressors of temperature-sensitive sporulation in Bacillus subtilis are allele non-specific.

The Bacillus subtilis mutant cal1 carries a non-reverting mutation in ribosomal protein L17 (r-protein L17) that causes both resistance to the antibiotic chalcomycin (Calr) and temperature-sensitive sporulation (Spots). Second-site suppressor (rev) mutations that relieve the Spots phenotype have been isolated from cal1. Three suppressor mutations - rev4, rev10, rev11 - each increase the sporulation frequency of cal1 at the non-permissive temperature from 3% to 95% of the wild-type level. The cal1 rev strains remain resistant to chalcomycin and two-dimensional gel electrophoresis analysis indicates that they contain the same altered r-protein L17 as the original cal1 strain and no additional altered r-proteins. The three rev mutations have been mapped at a single locus between narA and sacA on the B. subtilis chromosome and recombination indexes for the rev mutations indicate that they are tightly linked to one another. Antibiotic resistance Spots mutations that cause temperature-sensitive sporulation have previously been isolated in RNA polymerase, in the 30S and 50S subunits of the ribosome, and in elongation factor G. The rev4, 10, and 11 suppressor mutations are non-specific in their action in that they restore significant levels of sporulation at the non-permissive temperature in all of the Spots strains that we have tested. This result suggests that Spots mutations in components of the B. subtilis transcription and translation systems share a common molecular basis for their sporulation-defective phenotypes.

Molecular and phenotypic specificity of an antisense PHYB gene in Arabidopsis.

The family of phytochrome photoreceptors plays an essential role in regulating plant growth and development in response to the light environment. An antisense PHYB transgene has been introduced into wild-type Arabidopsis and shown to inhibit expression of the PHYB sense mRNA and the phyB phytochrome protein 4- to 5-fold. This inhibition is specific to phyB in that the levels of the four other phytochromes, notably the closely related phyD and phyE phytochromes, are unaffected in the antisense lines. Antisense-induced reduction in phyB causes alterations of red light effects on seedling hypocotyl elongation, rosette leaf morphology, and chlorophyll content, similar to the phenotypic changes caused by phyB null mutations. However, unlike the phyB mutants, the antisense lines do not flower early compared to the wild type. Furthermore, unlike the phyB mutants, the antisense lines do not show a reduction in phyC level compared to the wild type, making it possible to unequivocally associate several of the photomorphogenic effects seen in phyB mutants with phytochrome B alone. These results indicate that an antisense transgene approach can be used to specifically inhibit the expression and activity of a single member of the phytochrome family and to alter aspects of shade avoidance responses in a targeted manner.

Cell sampling and analysis (SiCSA): metabolites measured at single cell resolution.

By using a fine oil-filled glass microcapillary mounted on a micromanipulator, the solutes of individual plant cells can be sampled. These samples can then be analysed using a range of physical and chemical methods. Hydrostatic pressure (cell pressure probe), osmotic pressure (picolitre osmometer), organic solutes (enzyme-linked fluorescence microscope spectrometry or capillary electrophoresis), inorganic solutes (X-ray microdroplet analysis or capillary electrophoresis), (14)C (mass spectrometry), proteins (microdroplet immunoblotting), and mRNA (rt PCR) have been measured. Collectively, the battery of techniques is called single cell sampling and analysis (SiCSA) and all of the techniques have relevance to the study of plant metabolism at the resolution of the individual cell. This review summarizes the techniques for SiCSA and presents examples of applications used in this laboratory, in particular those relating to cell metabolism.

The phytochrome gene family in grasses (Poaceae): a phylogeny and evidence that grasses have a subset of the loci found in dicot angiosperms.

The phytochrome nuclear gene family encodes photoreceptor proteins that mediate developmental responses to red and far red light throughout the life of the plant. From studies of the dicot flowering plant Arabidopsis, the family has been modeled as comprising five loci, PHYA-PHYE. However, it has been shown recently that the Arabidopsis model may not completely represent some flowering plant groups because additional PHY loci related to PHYA and PHYB of Arabidopsis apparently have evolved independently several times in dicots, and monocot flowering plants may lack orthologs of PHYD and PHYE of Arabidopsis. Nonetheless, the phytochrome nucleotide data were informative in a study of organismal evolution because the loci occur as single copy sequences and appear to be evolving independently. We have continued our investigation of the phytochrome gene family in flowering plants by sampling extensively in the grass family. The phytochrome nuclear DNA data were cladistically analyzed to address the following questions: (1) Are the data consistent with a pattern of differential distribution of phytochrome genes among monocots and higher dicots, with homologs of PHYA, B, C, D, and E present in higher dicots, but of just PHYA, B, and C in monocots, and (2) what phylogenetic pattern within Poaceae do they reveal? Results of these analyses, and of Southern blot experiments, are consistent with the observation that the phytochrome gene family in grasses comprises the same subset of loci detected in other monocots. Furthermore, for studies of organismal phylogeny in the grass family, the data are shown to provide significant support for relationships that are just weakly resolved by other data sets.

Macrolide and aminoglycoside antibiotic resistance mutations in the bacillus subtilis ribosome resulting in temperature-sensitive sporulation.

Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spots). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes. Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spots. Representative mutations, neo162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map a single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.

Intergenic suppression of spoO phenotypes by the Bacillus subtilis mutation rvtA.

A collection of intergenic suppressors of the Bacillus subtilis spoOF221 mutation has been isolated. One of these suppressors, rvtA, has been mapped between lys-1and aroD. The rvtA suppressor restores spoOF sporulation to wild type levels and substantially improves the sporulation efficiencies of spoOB and spoOE strains. The rvtA gene does not affect the Spo phenotype of spoOH, spoOJ or spoOK mutants. The rvtA gene also prevents the induction by aliphatic alcohols of SpoO phenocopies in wild type B. subtilis cells.

The phytochrome apoprotein family in Arabidopsis is encoded by five genes: the sequences and expression of PHYD and PHYE.

Two novel Arabidopsis phytochrome genes, PHYD and PHYE, are described and evidence is presented that, together with the previously described PHYA, PHYB and PHYC genes, the primary structures of the complete phytochrome family of this plant are now known. The PHYD- and PHYE-encoded proteins are of similar size to the other phytochrome apoproteins and show sequence similarity along their entire lengths. Hence, red/far-red light sensing in higher plants is mediated by a diverse but structurally conserved group of soluble photoreceptors. The proteins encoded by the PHYD and PHYE genes are more closely related to phytochrome B than to phytochromes A or C, indicating that the evolution of the PHY gene family in Arabidopsis includes an expansion of a PHYB-related subgroup. The PHYB and PHYD phytochromes show greater than 80% amino acid sequence identity but the phenotypes of phyB null mutants demonstrate that these receptor forms are not functionally redundant. The five PHY mRNAs are, in general, expressed constitutively under varying light conditions, in different plant organs, and over the life cycle of the plant. These observations provide the first description of the structure and expression of a complete phytochrome family in a higher plant.

Inhibitory effect of high-level transcription of the bacteriophage lambda nutL region on transcription of rRNA in Escherichia coli.

Transcription of the bacteriophage lambda nutL region from the PL promoter on a multicopy plasmid in Escherichia coli causes a reduction in growth rate and in transcription of rRNA relative both to total transcription and to transcription of tRNAs that are not encoded in rRNA operons. These observations support the hypothesis, previously based on nut site DNA sequence homology, that the phage lambda and rRNA antitermination systems are related.

Defective antitermination of rRNA transcription and derepression of rRNA and tRNA synthesis in the nusB5 mutant of Escherichia coli.

The nusB5 mutant of Escherichia coli was originally selected for reduced ability to support the antitermination of transcription that is mediated by the gene N product of bacteriophage lambda. By analyzing pulse-labeled RNA with an RNA.DNA filter hybridization technique, we have shown that, in the nusB5 mutant, the ratio of promoter-proximal rRNA transcripts to promoter-distal transcripts is increased at least by a factor of 1.6; that is, in the absence of the functional nusB gene product, premature transcription termination takes place within rRNA operons. These results demonstrate that rRNA transcription in E. coli utilizes an antitermination mechanism that has at least one factor in common with the phage lambda system, the nusB gene product. We have also observed that the transcription initiation frequency at rRNA promoters is increased in the nusB5 strain and that this strain accumulates 30S and 50S ribosomal subunits at approximately the same rate as the parent. Thus, it appears that E. coli compensates for premature termination of rRNA transcription by derepressing rRNA operon expression. The increase in rRNA promoter activity in the nusB5 mutant is accompanied by a parallel derepression of synthesis of tRNAs that are not encoded by rRNA operons. These results are consistent with a model for negative feedback regulation of rRNA and tRNA synthesis by products of rRNA operons.

Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation.

Two genomic clones (lambda Ubi-1 and lambda Ubi-2) encoding the highly conserved 76 amino acid protein ubiquitin have been isolated from maize. Sequence analysis shows that both genes contain seven contiguous direct repeats of the protein coding region in a polyprotein conformation. The deduced amino acid sequence of all 14 repeats is identical and is the same as for other plant ubiquitins. The use of transcript-specific oligonucleotide probes shows that Ubi-1 and Ubi-2 are expressed constitutively at 25 degrees C but are inducible to higher levels at elevated temperatures in maize seedlings. Both genes contain an intron in the 5' untranslated region which is inefficiently processed following a brief, severe heat shock. The transcription start site of Ubi-1 has been determined and a transcriptional fusion of 0.9 kb of the 5' flanking region and the entire 5' untranslated sequence of Ubi-1 with the coding sequence of the gene encoding the reporter molecule chloramphenicol acetyl transferase (CAT) has been constructed (pUBI-CAT). CAT assays of extracts of protoplasts electroporated with this construct show that the ubiquitin gene fragment confers a high level of CAT expression in maize and other monocot protoplasts but not in protoplasts of the dicot tobacco. Expression from the Ubi-1 promoter of pUBI-CAT yields more than a 10-fold higher level of CAT activity in maize protoplasts than expression from the widely used cauliflower mosaic virus 35S promoter of a 35S-CAT construct. Conversely, in tobacco protoplasts CAT activity from transcription of pUBI-CAT is less than one tenth of the level from p35S-CAT.

Differential patterns of expression of the Arabidopsis PHYB, PHYD, and PHYE phytochrome genes.

The Arabidopsis thaliana phyB, phyD, and phyE phytochrome apoproteins show higher amino acid sequence similarity to each other than to phyA or phyC, they are the most recently evolved members of this photoreceptor family, and they may interact in regulating photomorphogenesis. The expression patterns of translational fusions of the 5' upstream regions of the PHYB, PHYD, and PHYE genes to the beta-glucuronidase (GUS) coding sequence were compared. PD-GUS and PE-GUS fusions were 5- to 10-fold less active than a PB-GUS fusion, but all three promoter regions drove expression of the reporter gene in all stages of the plant's life cycle. Over the first 10 d of seedling growth, the PHYB and PHYD promoters were more active in the dark than in the light, whereas the opposite was true of the PHYE promoter. Unlike the PB-GUS construct, which was expressed in most parts of seedlings and mature plants, the PD-GUS and PE-GUS transgenes showed differential expression, notably in leaves, flower organs, and root tips. Tissue sections showed that the three promoters are coexpressed in at least some leaf cells. Hence, the PHYB, PHYD, and PHYE genes differ in expression pattern but these patterns overlap and interaction of these receptor forms within individual cells is possible.

Transgenic complementation of the hy3 phytochrome B mutation and response to PHYB gene copy number in Arabidopsis.

A recombinant PHYB minigene (mPHYB) consisting of the complete Arabidopsis PHYB cDNA sequence fused to 2.3 kb of upstream PHYB promoter sequence has been introduced into wild-type Arabidopsis and into a strain containing the Bo64 allele of the hy3 mutation. The Bo64 mutant has previously been shown to contain a nonsense mutation in the PHYB coding sequence. Transformation of this strain with the mPHYB gene results in complementation of all of the mutant phenotypic characteristics tested including hypocotyl length and hypocotyl cell size, response to end-of-day far-red light, leaf morphology, chlorophyll level, and flowering time. Presence of the mPHYB transgene in a wild-type genetic background causes exaggeration of this same set of phenotypic characteristics, indicating that these diverse photo-morphogenic responses are sensitive to the copy number of the PHYB gene. The transgene inserts in the Bo64(mPHYB) and WT(mPHYB) lines are shown to be single locus and single copy and the immunologically detectable level of phytochrome B is shown to vary linearly with PHYB gene copy number. These results demonstrate a complex role for phytochrome B in Arabidopsis photo-morphogenesis and suggest that the expression level of this phytochrome gene is an important determinant of the intensity of light-induced plant responses.

Feedback regulation of rRNA and tRNA synthesis and accumulation of free ribosomes after conditional expression of rRNA genes.

We have constructed a conditional rRNA gene expression system by fusing a plasmid-encoded rrnB operon to the lambda PL promoter/operator. It was thereby possible to study the events that lead to the regulation of chromosomal rRNA and tRNA synthesis after overproduction of rRNA. rRNA induction resulted in a 2-fold increase in 30S and 50S free ribosomal subunits, whereas the polysome fraction was unaffected. Overproduction of rRNA and "free" ribosomes produced a large repression of rRNA and tRNA synthesis from chromosomal genes and a smaller increase in the concentration of guanosine tetraphosphate. These results lend support to the ribosome feedback regulation model: rRNA and tRNA operons are negatively regulated, either directly or through some intermediate, by free, nontranslating ribosomes.