Chloroplast biogenesis. XXIX. The occurrence of several novel chlorophyll a and b chromophores in higher plants.

With the use of low temperature spectrofluorometry and matrix calculations it was demonstrated that the chlorophyll a pool of higher plants is made up of four different chlorophyll a chromophores. The latter were segregated by high pressure liquid chromatography on a silica column. They were designated Chl a (E432 F664), Chl a (E436 F670), Chl a (E443 F672) and Chl a (E446 F674), where E refers to the Soret excitation maximum and F to the fluorescence emission maximum at 77 K in ether. Likewise the Chl b pool was shown to consist of at least four different Chl b chromophores which were designated: Chl b (E465), Chl b (E470), Chl b (E475) and Chl b (E485). It was proposed that the various chlorophyll chromophores differed by the degree of oxidation of their side chains at the 2 and 4 positions of the macrocycle. It was also suggested that the chemical modifications at the 2 and 4 positions of the macrocycle may play an important role in positioning the different chlorophyll chromophores in the thylakoid membranes.

Molecular basis for allelic polymorphism of the maize Globulin-1 gene.

An abundant protein in maize (Zea mays L.) embryos is a storage globulin encoded by the polymorphic Glb1 gene. Several Glb1 protein size alleles and a null allele have been described. Here we report the isolation and nucleotide sequence analysis of genomic clones corresponding to two Glb1 size alleles (Glb1-L and Glb1-S) and to the Glb1-0 null allele. The Glb1-L and Glb1-0 alleles differ from Glb1-S by the presence of small nucleotide insertions which are imperfect or perfect duplications, respectively, of adjacent sequences. In the case of Glb1-L, the insertion is in-frame and results in a protein larger than that encoded by Glb1-S, whereas in Glb1-0 the insertion causes a translational frameshift which introduces a premature termination codon. Although steady-state levels of Glb1-0 transcripts are extremely low in Glb1-0/0 embryos, nuclear transcription assays indicate that the Glb1-0 gene is transcribed at a level comparable to that of Glb1-L. This suggests that the low amounts of Glb1-0 transcripts in the cytoplasm may be due to mRNA instability.

Purification and Characterization of an Endophytic Fungal Proteinase That Is Abundantly Expressed in the Infected Host Grass.

A novel Acremonium typhinum proteinase that is expressed during endophytic infection of the grass Poa ampla Merr. was purified from endophyte-infected leaf sheath tissue. It is a thiol-containing serine alkaline endoproteinase with bound carbohydrate. In the infected host tissue, this proteinase is an abundant protein localized within fungal membrane vesicles and in the plant and/or fungal cell walls. This proteinase was not expressed constitutively during fungus culture. Rather, its expression appeared to be induced by nutrient depletion. Expression of an antigenically similar proteinase was detected in five other endophyte-infected Poa species. The regulated expression of the proteinase in culture and its abundance in infected plant tissue suggest that its expression may be involved in the symbiotic interaction of the plant and the fungus.

A Novel Fungal Protease Expressed in Endophytic Infection of Poa Species.

The fungus Acremonium typhinum produces a novel endoprotease during symbiotic endophytic infection of the grass, Poa ampla. This protease is unusual because it is highly active in the presence of sodium dodecyl sulfate. The enzyme is a thiol-containing serine protease and is localized to a crude membrane fraction. Similar protease activity has been detected in endophyte-infected Poa autumnalis and Poa sylvestris plants. Expression of this protease may be important in endophytic infection of Poa spp., because similar activity has not been detected in endophyte-infected Festuca arundinacea or Lolium perenne.

Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host.

Infection by pathogenic fungi involves breaching the outer layer of the host by either mechanical or enzymatic means. Subtilisin-like proteinases are considered to be important in the infection process of entomopathogenic, nematophagous, and mycoparasitic fungi. Little is known regarding the expression of such proteinases by plant pathogenic fungi. Magnaporthe poae, a fungal pathogen of Kentucky bluegrass, expressed a subtilisin-like proteinase, proteinase Mp1, in the infected roots. Antibody was produced against the purified enzyme. From immunoblot analysis, expression of the proteinase in infected roots correlated with increasing severity of disease symptoms. Sequence analysis of a genomic clone indicated proteinase Mp1 was homologous to other fungal subtilisin-like proteinases. DNA gel blot analysis indicated proteinase Mp1 was encoded by a small gene family.

Phosphoglycolate phosphatase: purification and preparation of antibodies.

Phosphoglycolate phosphatase was partially purified from leaves of Nicotiana rustica using ion exchange and chromatofocusing columns. The native molecular weight of the enzyme was determined to be about 58 kD from Ferguson plots, with a subunit size of about 32 kD. The native enzyme is thus likely to be a dimer. A polyclonal antibody prepared against the LDS denatured enzyme cross reacted with proteins from Nicotiana tabacum, Glycine max, Spinacea oleracea and Arabidopsis thaniana. There was little or no reaction with an Arabidopsis mutant lacking phosphoglycolate phosphatase activity, indicating a much reduced level of phosphoglycolate phosphatase protein in the mutant.

Molecular characterization of the major maize embryo globulin encoded by the glb1 gene.

One of the most abundant proteins in maize (Zea mays L.) embryos is the molecular weight 63,000 globulin encoded by the Glb1 gene. To obtain DNA clones corresponding to Glb1, a cDNA library corresponding to RNA from developing maize embryos was constructed in a lambda expression vector and screened with antibodies specific for Glb1-encoded proteins. Here we report the complete nucleotide sequence, as determined from two overlapping clones, of pcGlb 1S, a 2009 base pair clone containing the entire translated region of Glb1. The deduced amino acid sequence of pcGlb 1S shows similarities to 7S-type seed storage proteins of wheat and legumes. Southern blot analysis of maize DNA confirms previous genetic studies which had indicated the presence of a single copy of Glb1 per haploid genome. Northern blot analysis indicates that Glb1 transcripts are present throughout most of embryo development and that expression of this gene is limited to seed tissues. Embryos homozygous for a Glb1 null allele, in which Glb1-encoded proteins are not detectable, contain low levels of Glb1 transcripts which are a different size from those encoded by functional alleles. This suggests that the defect in the null allele is at the level of gene transcription or RNA processing.

Chloroplast biogenesis. Detection of divinyl protochlorophyllide in higher plants.

It is shown that the protochlorophyllide pool of etiolated higher plants is made up of both monovinyl and divinyl protochlorophyllide. Although the two pigments exhibited similar emission maxima, they were distinguishable by their Soret excitation maxima, which were found at 436 to 437 and 443 to 444 nm, respectively, in ether at 77 K. The two pigments were partially separated on thin layers of polyethylene. They were shown to be accompanied by two unknown fluorescent compounds. The latter were designated compound (E451 F626) and compound (E453 F640) where E refers to the Soret excitation maxima and F to the fluorescence emission maxima of the two unknown compounds. Furthermore, it was shown under several different growth conditions that divinyl protochlorophyllide constituted the major component of the protochlorophyllide pool.

Chloroplast biogenesis. Detection of monovinyl magnesium-protoporphyrin monoester and other monovinyl magnesium-porphyrins in higher plants.

It is shown that the magnesium-protoporphyrin-6-ester pool of plants is made up of monovinyl and divinyl components which were separated by chromatography on thin layers of polyethylene. At 77K, in ether, monovinyl-Mg-protoporphyrin-6-ester exhibited fluorescence emission and excitation maxima at 589 and 417 nm, respectively. The divinyl-Mg-protoporphyrin-6-ester component exhibited red-shifted emission and excitation maxima at 591 and 424 nm respectively. Demetallation of the monovinyl and divinyl Mg-protoporphyrin ester components converted them into monovinyl and divinyl-protoporphyrin IX free bases. Catalytic Mg-mesoporphyrin ester and the demetallated free bases into mesoporphyrin ester, Monovinyl-Mg-porphyrin chemical species were also detected in the Mg-protoporphyrin IX and the Mg-protoporphyrin IX diester pools of plants. It is proposed that the Mg-protoporphyrin IX, Mg-protoporphyrin IX-6-ester, and Mg-protoporphyrin IX-diester pools of plants are heterogenous and are made up of monovinyl and divinyl chemical species.

Chloroplasts biogenesis: detection of divinylprotochlorophyllide ester in higher plants.

It is shown that the protochlorophyllide ester pool of etiolated higher plants is a faithful copy of the protochlorophyllide pool. It is made up of both monovinyl- and divinylprotochlorophyllide esters. Although the two tetrapyrroles exhibited similar emission maxima, they were distinguishable by their Soret excitation maxima, which were found at 436-437 and 443-444 nm, respectively, in ether at 77K. The two pigments were partially separated on thin layers of polyethylene. They were accompanied by two unknown fluorescent compounds. It was also shown that during greening, the protochlorophyllide ester pool maintained a constant qualitative composition. This was in sharp contrast with the drastic qualitative changes undergone by the protochlorophyllide pool of etiolated tissues grown under identical conditions.

Heat shock inhibits release of the signal recognition particle from the endoplasmic reticulum in barley aleurone layers.

When barley (Hordeum vulgare) aleurone layers are subjected to heat shock there is a selective degradation of the normally stable mRNAs encoding secreted proteins. Messages for nonsecreted proteins are not degraded. The synthesis of heat shock proteins is not required for this selective message degradation. Our hypothesis explaining this phenomenon is that a component of the early steps in the synthesis of secreted proteins is damaged by heat shock, resulting in a selective halt in translation on secretory mRNAs, which may in turn lead to degradation of those messages. The first committed step in the synthesis of secreted proteins is the binding of the nascent signal sequence to the signal recognition particle. We have obtained cDNA clones and antibodies for the barley 54-kDa subunit of the signal recognition particle. In cell fractionation experiments, more signal recognition particle was bound to the endoplasmic reticulum membranes and less was in the free particle fraction following a heat shock. The results suggest that heat shock inhibits the release of the signal recognition particle from the endoplasmic reticulum. This would, in turn, inhibit the resumption of translation and may be the underlying cause of the secretory message degradation.

Using empirical data to model transgene dispersal.

One element of the current public debate about genetically modified crops is that gene flow from transgenic cultivars into surrounding weed populations will lead to more problematic weeds, particularly for traits such as herbicide resistance. Evolutionary biologists can inform this debate by providing accurate estimates of gene flow potential and subsequent ecological performance of resulting hybrids. We develop a model for gene flow incorporating exponential distance and directional effects to be applied to windpollinated species. This model is applied to previously published data on gene flow in experimental plots of Agrostis stolonifera L. (creeping bentgrass), which assessed gene flow from transgenic plants resistant to the herbicide glufosinate to surrounding non-transgenic plants. Our results show that although pollen dispersal can be limited in some sites, it may be extensive in others, depending on local conditions such as exposure to wind. Thus, hybridization under field conditions is likely to occur. Given the nature of the herbicide resistance trait, we regard this trait as unlikely to persist in the absence of herbicide, and suggest that the ecological consequences of such gene flow are likely to be minimal.

Mutualistic fungal endophytes express a proteinase that is homologous to proteases suspected to be important in fungal pathogenicity.

Many cultivated and wild grass species are hosts to mutualistic fungal endophytes. These associations are ecologically and agronomically significant, yet little is known regarding the physiological aspects of the interaction. In the Poa ampla/Acremonium typhinum interaction, a fungal serine proteinase, At1, is surprisingly abundant and may constitute 1 to 2% of the total leaf-sheath protein. Sequence analysis of cDNA and genomic clones indicates that proteinase At1 is a member of the eukaryotic subtilisin-like protease family. It is homologous to proteases suspected to be virulence factors in fungal pathogens of insects, nematodes, and other fungi. Gel blot analysis of RNA extracted from infected leaf-sheath tissue indicates that the proteinase At1 transcript level is extremely high. RNA gel blots and immunoblots of purified enzymes indicate that similar proteinases are produced by Epichloë festucae and Acremonium lolii, the fungal endophytes infecting Festuca rubra subsp. rubra and Lolium perenne, respectively. Fungal expression of proteinase At1-like enzymes may be a general feature of endophyte infection.

Chloroplast biogenesis. Identification of chlorophyllide a (E458f674) as a divinyl chlorophyllide a.

The chemical identification of chlorophyllide (E458F674) (Belanger, F. C., and Rebeiz, C. A. (1980) Plant Sci. Lett. 18, 343-350) has been confirmed by chemical derivatization coupled to spectrofluorometric, spectrophotometric, and chromatographic analysis. Chlorophyllide (E458F674) and its demetallated analog were converted by catalytic hydrogenation into mesochlorophyllide a and mesopheophorbide a. Furthermore, methyl chlorophyllide (E458F674) was converted by partial hydrogenation into a mixture of monovinyl chlorophyllide a isomers and the latter into mesochlorophyllide a by further hydrogenation. On the other hand, chemical oxidation of methyl chlorophyllide (E458F674) converted it into methyl divinyl protochlorophyllide. Chlorophyllide (E458F674) was detected in several plant species and is proposed to be an important intermediate of the chlorophyll a biosynthetic pathway.

Chloroplast biogenesis: detection of a magnesium protoporphyrin diester pool in plants.

The discovery of a novel metalloporphyrin pool in etiolated cucumber cotyledons and in dark-grown Euglena gracilis is described. The novel pool exhibited the chromatographic properties of a fully esterified metalloporphyrin, devoid of free carboxylic groups, and the spectrophotometric and spectrofluorometric properties of a magnesium protoporphyrin. Demetalation and hydrolysis indicated that the tetrapyrrole moiety of the metalloporphyrin was a protoporphyrin diester. High-pressure liquid chromatography of the fully esterified metalloporphyrin pool and gas chromatographic/mass spectroscopic analysis of the saponified alcohol fraction revealed that the latter was made up of three major long-chain alcohols. None of those alcohols was identifiable, however, with known isoprenoids such as geraniol, farnesol, or phytol. Similar analysis of the saponified alcohol fraction of the protochlorophyllide ester pool likewise revealed the presence of three major long-chain alcohols none of which was identifiable with known isoprenoid alcohols or with the alcohols of the novel metalloporphyrin pool. On the basis of the above observations, the novel metalloporphyrin pool was tentatively identified as a magnesium protoporphyrin diester pool. It is suggested that this pool is a metabolic intermediate of the fully esterified branch of the chlorophyll biosynthetic pathway [Rebeiz, C. A., Smith, B. B., Matthesis, I. R., Cohen, C. E., & McCarthy, S. A. (1978) in Chloroplast Development (Akoyunoglou, G., & Argyroudi-Akoyunoglou, J. H., Eds.) pp 56-76, Elsevier/North-Holland Bio-Medical Press, Amsterdam] and is probably the precursor of the protochlorophyllide ester pool in plants.

Heat shock proteins are not required for the degradation of alpha-amylase mRNA and the delamellation of endoplasmic reticulum in heat-stressed barley aleurone cells.

When barley (Hordeum vulgare) aleurone layers are heat shocked, the synthesis and secretion of alpha-amylase and other secretory proteins is arrested and the synthesis of heat shock proteins (hsps) is induced. alpha-Amylase mRNA, normally a very stable mRNA, is actively degraded during heat shock. In addition, endoplasmic reticulum (ER) is delamellated during heat shock, possibly causing the destabilization of the mRNA for the secreted alpha-amylase. To ascertain whether or not hsps play any role in the destabilization of alpha-amylase mRNA or in the delamellation process of ER, heat shocked cells were treated with the transcription inhibitor cordycepin, which effectively inhibits the synthesis of hsps yet does not affect alpha-amylase synthesis after this enzyme has been fully induced by gibberellic acid (12 hours). In the absence of hsp expression, heat shock still causes the destabilization of alpha-amylase mRNA and the delamellation of ER. Alternatively, the synthesis of hsps may be induced in the absence of temperature increase by incubating cells in the presence of arsenite. Arsenite-induced expression of some hsps in the absence of increased temperature does not result in the destabilization of alpha-amylase mRNA or in the delamellation of ER. If cordycepin or cycloheximide are used to inhibit hsp synthesis during heat shock, the tissue recovers from heat shock with normal recovery kinetics. Although hsps have been implicated in the establishment of thermotolerance, our observations indicate that hsps do not play a role in the other heat shock-induced changes observable in aleurone cells. Furthermore, if the synthesis of hsp mRNA is inhibited during heat shock (by cordycepin) hsp mRNAs are synthesized later, during recovery, indicating that there is a stable inducer of hsp synthesis in aleurone tissues.

Heat shock causes destabilization of specific mRNAs and destruction of endoplasmic reticulum in barley aleurone cells.

In response to a phytohormone, gibberellic acid, the aleurone layers of barley seeds synthesize and secrete alpha-amylases, which are coded by a set of stable mRNAs. When aleurone layers are subjected to heat shock treatment, the synthesis of alpha-amylase is suppressed while heat shock proteins are induced. The suppression of alpha-amylase synthesis is not the result of translational control as reported in several other systems. Rather, the sequences of alpha-amylase mRNA are rapidly degraded during heat shock as shown by in vitro translation and dot blot hybridization with a cDNA probe. Upon recovery from heat shock, the tissue resumes the synthesis of alpha-amylase in 2-4 hr. However, in the presence of a transcription inhibitor, cordycepin, the resumption of synthesis of alpha-amylase does not take place, indicating that new transcription of alpha-amylase genes is necessary for this recovery process. The degradation of alpha-amylase mRNAs correlates with the rapid destruction of endoplasmic reticulum as observed by electron microscopy, a phenomenon that has not been reported previously as a heat shock response. Since alpha-amylase mRNA is associated with the endoplasmic reticulum via membrane-bound polyribosomes, we suggest that the destruction of the endoplasmic reticulum during heat shock causes the destabilization and the eventual degradation of alpha-amylase mRNA.

Arabidopsis thaliana expresses three divergent Srp54 genes.

The Arabidopsis thaliana Srp54 gene family was determined to consist of three genes, all of which were cloned and sequenced. In addition, cDNAs corresponding to two of the genes were obtained. To our knowledge this is the first description of multiple Srp54 genes within an organism. In contrast to the situation in mammals, where there are only three amino acid differences between the mouse and canine sequences, there was significant amino acid sequence diversity among the genes, particularly in the methionine-rich region of the protein, which is the region responsible for binding to the 7S RNA of the signal recognition particle and to the signal sequence of newly synthesized proteins. The amino acid sequences of the GTP-binding domains of the three clones were 86% identical, whereas the methionine-rich domains were only 65% identical. RNA gel blots of various tissues and developmental stages hybridized with gene-specific probes revealed that all three genes were expressed in all the tissues investigated. There were, however, quantitative differences in expression levels.

Isolation and characterization of an Arabidopsis thaliana gene for the 54 kDa subunit of the signal recognition particle.

The first step in the routing of newly synthesized proteins into the secretory pathway is the binding of the nascent signal sequence to the signal recognition particle. The mammalian signal recognition particle is a complex consisting of 6 proteins and a single 7S RNA molecule. Signal recognition particle-like complexes have been described from wheat and maize but none of the protein components have yet been described from any plant species. Here we report the cloning and characterization of an Arabidopsis thaliana gene encoding the 54 kDa protein subunit of the signal recognition particle. This is the first report of a SRP-54 sequence for any plant species and the first genomic sequence for any multicellular organism.

Characterization of a multifunctional methyltransferase from the orchid Vanilla planifolia.

The final enzymatic step in the synthesis of the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) is believed to be methylation of 3,4-dihydroxybenzaldehyde. We have isolated and functionally characterized a cDNA that encodes a multifunctional methyltransferase from Vanilla planifolia tissue cultures that can catalyze the conversion of 3,4-dihydroxybenzaldehyde to vanillin, although 3,4-dihydroxybenzaldehyde is not the preferred substrate. The higher catalytic efficiency of the purified recombinant enzyme with the substrates caffeoyl aldehyde and 5-OH-coniferaldehyde, and its tissue distribution, suggest this methyltransferase may primarily function in lignin biosynthesis. However, since the enzyme characterized here does have 3,4-dihydroxybenzaldehyde-O-methyltransferase activity, it may be useful in engineering strategies for the synthesis of natural vanillin from alternate sources.