Nucleotide sequence and characterization of a gene encoding the phytochrome polypeptide from Avena.

We have isolated and characterized a gene encoding the phytochrome polypeptide of Avena. Based on nucleotide sequence identity with previously sequenced cDNA clones this gene is designated as type 3 (phy3). The gene is about 5.9 kb long with six exons and five introns, one each of the latter in the 5' and 3'-untranslated regions. The largest exon encodes the entire 74-kDa, chromophore-bearing, N-terminal domain of the photo-receptor postulated to be directly involved in its mechanism of action. The transcription start point, identified by mung-bean nuclease digestion, is located 24 to 35 bp downstream from a tandem TATA box. Sequence elements homologous to a number of motifs implicated as upstream regulatory elements in other genes are present in the 5'-flanking DNA of phy3. Particularly intriguing are three elements at positions -140, -470 and -650. These elements share homology with the 'GT' motif postulated to be a component of the light-regulatory element of genes encoding the small subunit of ribulose bisphosphate carboxylase.

Isolation and characterization of cDNA clones for RNA species induced by substituted benzenesulfonamides in corn.

A search of compounds capable of inducing specific gene expression in plants without affecting growth and development led to the examination of changes in the pattern of gene expression in corn after treatment with substituted benzenesulfonamide herbicide safeners. Following hydroponic treatment of corn with the safener N-(aminocarbonyl)-2-chlorobenzenesulfonamide (2-CBSU), the specific induction of new translatable mRNA species was observed. Replicate copies of a cDNA library made using RNA from 2-CBSU-treated corn roots were differentially screened with cDNA probes made from either the same mRNA fraction used for library construction or mRNA isolated from roots treated with 2-chlorobenzenesulfonamide (2-CBSA), an inactive analog of the safener. Colonies showing hybridization only with the probe made using mRNA from 2-CBSU-treated roots were further characterized to assess the specificity of the induction and decay of the corresponding induced RNA species. RNA blot analyses showed two clones, designated In2-1 and In2-2, contained plasmids that hybridized to RNAs that were induced from an undetectable background in corn roots within 30 minutes after treatment with 2-CBSU. Leaf and meristem tissues showed similar inductions of the In2-1 and In2-2 RNA species after a delay of several hours. In addition, both RNA species were induced in corn by foliar application of 2-CBSU. In contrast, neither RNA species was induced following stress treatments of plants. These results indicate a substituted benzenesulfonamide safener might be used with the promoters from the In2-1 and In2-2 genes to develop a new inducible gene expression system for plants.

Characterization of Tobacco Expressing Functional Oat Phytochrome : Domains Responsible for the Rapid Degradation of Pfr Are Conserved between Monocots and Dicots.

Constitutive expression of a chimeric oat phytochrome gene in tobacco (Nicotiana tabacum) results in the accumulation of a functional 124-kilodalton photoreceptor that markedly alters the phenotype of light-grown tobacco (Keller et al. [1989] EMBO J 8: 1005-1012). Here, we provide a detailed phenotypic and biochemical characterization of homozygous tobacco expressing high levels of oat phytochrome. Phenotypic changes include a substantial inhibition of stem elongation, decreased apical dominance, increased leaf chlorophyll content, and delayed leaf senescence. Oat phytochrome synthesized in tobacco is indistinguishable from that present in etiolated oats, having photoreversible difference spectrum maxima at 665 and 730 nanometers, exhibiting negligible dark reversion of phytochrome-far red-absorbing form (Pfr) to phytochrome-red-absorbing form (Pr), and existing as a dimer with an apparent size of approximately 300 kilodaltons. Heterodimers between the oat and tobacco chromoproteins were detected. Endogenous tobacco phytochrome and transgenically expressed oat phytochrome are rapidly degraded in vivo upon photoconversion of Pr to Pfr. Breakdown of both oat and tobacco Pfr is associated with the accumulation of ubiquitin-phytochrome conjugates, suggesting that degradation occurs via the ubiquitin-dependent proteolytic pathway. This result indicates that the factors responsible for selective recognition of Pfr by the ubiquitin pathway are conserved between monocot and dicot phytochromes. More broadly, it demonstrates that the domain(s) within a plant protein responsible for its selective breakdown can be recognized by the degradation machinery of heterologous species.

Autoregulatory control of translatable phytochrome mRNA levels.

Translatable phytochrome mRNA represents approximately 5 x 10(-3)% of the total poly(A)-RNA present in etiolated Avena seedlings, as determined by incorporation of radioactivity into the immunoprecipitable apoprotein in a cell-free translation system. Irradiation of such seedlings with 5-s red light induces a decline in this mRNA that is detectable within 15-30 min, shows a 50% reduction within 50-60 min, and results in a >95% reduction within 2 hr. The effect of the red light pulse is reversed by an immediately subsequent far-red pulse to the level of the far-red-light control, indicating that phytochrome exerts autoregulatory control over its own translatable mRNA level. This result necessitates revision of existing concepts of how phytochrome concentrations are modulated in vivo. Red-light dose-response curves show that the response is sensitive to very low light levels. Conversion of <1% of the total cellular phytochrome to the biologically active far-red-absorbing form is sufficient to induce approximately 60% of the maximal response, and 20% far-red-absorbing form saturates the response. The observed change in translatable phytochrome mRNA level is one of the most rapid phytochrome-induced alterations in any cellular mRNA yet recorded. Thus, autoregulation of phytochrome mRNA provides an attractive opportunity to examine the early sequence of events in phytochrome control of gene expression.

Phytochrome regulation of phytochrome mRNA abundance.

Pure phytochrome RNA sequence synthesized in an SP6-derived in vitro transcription system has been used as a standard to quantitate phytochrome mRNA abundance in Avena seedlings using a filter hybridization assay. In 4-day-old etiolated Avena seedlings phytochrome mRNA represents ∼0.1% of the total poly(A)(+) RNA. Irradiation of such seedlings with a saturating red-light pulse or continuous white light induces a decline in this mRNA that is detectable within 30 min and results in a 50% reduction by ∼60 min and >90% reduction within 5 h. The effect of the red-light pulse is reversed, approximately to the level of the far-red control, by an immediately subsequent far-red pulse. In seedlings maintained in extended darkness after the red-light pulse, the initial rapid decline in phytochrome mRNA level is followed by a slower reaccumulation such that 50-60% of the initial abundance is reached by 48 h. White-light grown seedlings transferred to darkness exhibit a similar accumulation of phytochrome mRNA that is accelerated by removal of residual Pfr with a far-red light pulse at the start of the dark period. The data establish that previously reported phytochrome-regulated changes in translatable phytochrome mRNA levels result from changes in the physical abundance of this mRNA rather than from altered translatability.

Direct interaction of resistance gene and avirulence gene products confers rice blast resistance.

Rice expressing the Pi-ta gene is resistant to strains of the rice blast fungus, Magnaporthe grisea, expressing AVR-Pita in a gene-for-gene relationship. Pi-ta encodes a putative cytoplasmic receptor with a centrally localized nucleotide-binding site and leucine-rich domain (LRD) at the C-terminus. AVR-Pita is predicted to encode a metalloprotease with an N-terminal secretory signal and pro-protein sequences. AVR-Pita(176) lacks the secretory and pro-protein sequences. We report here that transient expression of AVR-Pita(176) inside plant cells results in a Pi-ta-dependent resistance response. AVR-Pita(176) protein is shown to bind specifically to the LRD of the Pi-ta protein, both in the yeast two-hybrid system and in an in vitro binding assay. Single amino acid substitutions in the Pi-ta LRD or in the AVR-Pita(176) protease motif that result in loss of resistance in the plant also disrupt the physical interaction, both in yeast and in vitro. These data suggest that the AVR-Pita(176) protein binds directly to the Pi-ta LRD region inside the plant cell to initiate a Pi-ta-mediated defense response.

Photoresponses of transgenic tobacco plants expressing an oat phytochrome gene.

The physiological responses of transgenic tobacco (Nicotiana tabacum L.) plants that express high levels of an introduced oat (Avena sativa L.) phytochrome (phyA) gene to various light treatments are compared with those of wild-type (WT) plants. Seeds, etiolated seedlings, and light-grown plants from a homozygous transgenic tobacco line (9A4) constructed by Keller et al. (EMBO J, 8, 1005-1012, 1989) were treated with red (R), far-red (FR), or white light (WL) with or without supplemental FR light, revealing major perturbations of the normal photobiological responses. White light stimulated germination of both WT and transgenic seed, but addition of FR to the WL treatment suppressed germination. In the WT, all fluence rates tested inhibited germination, but in the transgenics, reduction effluence rate partially relieved germination from the FR-mediated inhibition. It is suggested that the higher absolute levels of the FR-absorbing form of phytochrome (Pfr) in the irradiated transgenics, compared to the WT, may be responsible for the reduced FR-mediated inhibition of germination in the former. Hypocotyl extension of dark-grown seedlings of both WT and transgenic lines was inhibited by continuous R or FR irradiation, typical of the high-irradiance response (HIR). After 2 d of de-etiolation in WL, the WT seedlings had lost the FR-mediated inhibition of hypocotyl extension, whereas it was retained in the transgenics. The FR-mediated inhibition of hypocotyl extension in the transgenic seedlings after de-etiolation may reflect the persistence of an, FR-HIR response mediated by the overexpressed oat PhyA phytochrome. Light-grown WT seedlings exhibited typical shade-avoidance responses when treated with WL supplemented with high levels of FR radiation. Internode and petiole extension rates were markedly increased, and the chlorophyll a∶b ratio decreased, in the low-R: FR treatment. The transgenics, however, showed no increases in extension growth under low-R: FR treatments, and at low fluence rates both internode and petiole extension rates were significantly decreased by low R ∶ FR. Interpretation of these data is difficult. The depression of the chlorophyll a∶b ratio by low R ∶ FR was identical in WT and transgenic plants, indicating that not all shade-avoidance responses of light-grown plants were disrupted by the over-expression of the introduced oat phyA gene. The results are discussed in relation to the proposal that different members of the phytochrome family may have different physiological roles.

Cloning and functional expression of the small subunit of acetolactate synthase from Nicotiana plumbaginifolia.

Acetolactate synthase (ALS) is the first committed step of branched-chain amino acid biosynthesis in plants and bacteria. The bacterial holoenzyme has been well characterized and is a tetramer of two identical large subunits (LSUs) of 60 kDa and two identical small subunits (SSUs) ranging in molecular mass from 9 to 17 kDa depending on the isozyme. The enzyme from plants is much less well characterized. Attempts to purify the protein have yielded an enzyme which appears to be an oligomer of LSUs, with the potential existence of a SSU for the plant enzyme remaining a matter of considerable speculation. We report here the discovery of a cDNA clone that encodes a SSU of plant ALS based upon the homology of the encoded peptide with various bacterial ALS SSUs. The plant ALS SSU is more than twice as large as any of its prokaryotic homologues and contains two domains that each encode a full-length copy of the prokaryotic SSU polypeptide. The cDNA clone was used to express Nicotiana plumbaginifolia SSU in Escherichia coli. Mixing a partially purified preparation of this SSU with the LSU of ALS from either N. plumbaginifolia or Arabidopsis thaliana results in both increased specific activity and increased stability of the enzymic activity. These results are consistent with those observed for the bacterial enzyme in similar experiments and represent the first functional demonstration of the existence of a SSU for plant ALS.

Expression of a functional monocotyledonous phytochrome in transgenic tobacco.

A chimeric oat phytochrome structural gene with an uninterrupted coding region was constructed for expression of the monocot protein in transgenic plants. The structural gene was placed under the transcriptional control of either a light-regulated oat phytochrome promoter or the constitutively active cauliflower mosaic virus 35S promoter. These genes were then introduced into Nicotiana tabacum and N.plumbaginifolia. None of the regenerated plants showed expression of oat phytochrome RNA when transcription was controlled by the oat promoter. In contrast, RNA was obtained in plants when the structural gene was functionally linked to the 35S promoter. Transformants expressing oat phytochrome RNA produced a full length 124-kd polypeptide that was recognized by oat-specific anti-phytochrome monoclonal antibodies. The oat protein was a substrate for chromophore addition in tobacco as judged by its red/far-red photoreversible sensitivity to trypsin degradation. Production of oat phytochrome in transgenic plants gave rise to increased phytochrome spectral activity in both light- and dark-grown plants. This increased phytochrome content resulted in phenotypic changes in transformed plants, including semi-dwarfism, darker green leaves, increased tillering and reduced apical dominance. The possible significance of expressing a biologically active phytochrome in transgenic plants is discussed.

Carboxy-terminal deletion analysis of oat phytochrome A reveals the presence of separate domains required for structure and biological activity.

A series of seven carboxy-terminal deletion mutants of oat phytochrome A were stably expressed in transgenic tobacco to localize phytochrome domains involved in chromophore attachment, spectral integrity, photoreversibility between the red light (Pr)- and far-red light (Pfr)-absorbing forms, dimerization, and biological activity. Amino acids necessary for chromophore attachment in vivo were localized to the amino-terminal 398 residues because mutant proteins this small had covalently bound chromophore. Deletion mutants from the carboxy terminus to residue 653 were spectrally indistinguishable from the full-length chromoprotein. In contrast, further truncation to residue 399 resulted in a chromoprotein with a bleached Pfr absorbance spectrum, Pr and Pfr absorbance maxima shifted toward shorter wavelengths, and reduced Pfr to Pr phototransformation efficiency. Thus, residues between 399 ad 652 are required for spectral integrity but are not essential for chromophore attachment. The sequence(s) between residues 919 and 1093 appears to be necessary for dimerization. Carboxy-terminal mutants containing this region behaved as dimers under nondenaturing conditions in vitro, whereas truncations without this region behaved as monomers. None of the plants expressing high levels of deletion mutants lacking the 35 carboxy-terminal amino acids displayed the light-exaggerated phenotype characteristic of plants expressing biologically active phytochrome A, even when the truncated phytochromes were expressed at levels 6- to 15-fold greater than that effective for the full-length chromoprotein. Collectively, these data show that the phytochrome protein contains several separable carboxy-terminal domains required for structure/function and identify a domain within 35 residues of the carboxy terminus that is critical for the biological activity of the photoreceptor in vivo.

Molecular cloning of cDNA for Avena phytochrome.

We have isolated several cDNA clones for phytochrome, a plant regulatory photoreceptor. A cDNA library was constructed by using etiolated Avena poly(A)(+) RNA enriched for phytochrome mRNA by size fractionation. Replicate arrays of colonies were differentially screened with cDNA probes made from poly(A)(+) RNA that had been either enriched in or depleted of phytochrome mRNA. Of the colonies hybridizing preferentially with the enriched probe, several contained plasmids that specifically selected phytochrome mRNA when assayed by hybridization-selection and translation. The largest such plasmid, pAP-2, was used to isolate clones from an Avena genomic library. One of these genomic clones was then used to screen a second cDNA library in an attempt to identify full-length phytochrome clones. The largest of the plasmids thus obtained, pAP-3, contains a 3.4-kilobasepair (kbp) insert, verified to contain phytochrome sequences by hybridization-selection and translation. Sequence analysis of pAP-2 and pAP-3 revealed that the two clones are identical in sequence through a 2.4-kbp region in which they overlap. However, the pAP-2 insert contains, in addition, 1.5 kbp of sequence of unknown origin, the apparent result of a recombination event. Blots of poly(A)(+) RNA hybridized with (32)P-labeled pAP-2 or pAP-3 show a single mRNA band at 4.2 kilobases. Blot analysis of RNA from dark-grown and from red-irradiated tissue demonstrates that a previously reported light-induced decrease in translatable phytochrome mRNA results from a decrease in physical abundance of this mRNA.

Analysis of cloned cDNA and genomic sequences for phytochrome: complete amino acid sequences for two gene products expressed in etiolated Avena.

Cloned cDNA and genomic sequences have been analyzed to deduce the amino acid sequence of phytochrome from etiolated Avena. Restriction endonuclease site polymorphism between clones indicates that at least four phytochrome genes are expressed in this tissue. Sequence analysis of two complete and one partial coding region shows approximately 98% homology at both the nucleotide and amino acid levels, with the majority of amino acid changes being conservative. High sequence homology is also found in the 5'-untranslated region but significant divergence occurs in the 3'-untranslated region. The phytochrome polypeptides are 1128 amino acid residues long corresponding to a molecular mass of 125 kdaltons. The known protein sequence at the chromophore attachment site occurs only once in the polypeptide, establishing that phytochrome has a single chromophore per monomer covalently linked to Cys-321. Computer analyses of the amino acid sequences have provided predictions regarding a number of structural features of the phytochrome molecule.

tA single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.

The rice blast resistance (R) gene Pi-ta mediates gene-for-gene resistance against strains of the fungus Magnaporthe grisea that express avirulent alleles of AVR-Pita. Using a map-based cloning strategy, we cloned Pi-ta, which is linked to the centromere of chromosome 12. Pi-ta encodes a predicted 928-amino acid cytoplasmic receptor with a centrally localized nucleotide binding site. A single-copy gene, Pi-ta shows low constitutive expression in both resistant and susceptible rice. Susceptible rice varieties contain pi-ta(-) alleles encoding predicted proteins that share a single amino acid difference relative to the Pi-ta resistance protein: serine instead of alanine at position 918. Transient expression in rice cells of a Pi-ta(+) R gene together with AVR-Pita(+) induces a resistance response. No resistance response is induced in transient assays that use a naturally occurring pi-ta(-) allele differing only by the serine at position 918. Rice varieties reported to have the linked Pi-ta(2) gene contain Pi-ta plus at least one other R gene, potentially explaining the broadened resistance spectrum of Pi-ta(2) relative to Pi-ta. Molecular cloning of the AVR-Pita and Pi-ta genes will aid in deployment of R genes for effective genetic control of rice blast disease.