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Biomedical subjects

K Apel

Publications and source records attributed to K Apel.

At least 37 records · Page 2Linked to original sources
PubMed1996

Identification of two MADS box genes that are expressed in the apical meristem of the long-day plant Sinapis alba in transition to flowering.

To understand the molecular mechanisms involved in the transition from vegetative to reproductive growth, the photoperiodic-controlled induction of flowering in the long-day plant Sinapis alba was used to screen for regulatory genes which are expressed upon induction in the apical meristem. By using the conserved MADS box-encoding region of the organ identity gene AGAMOUS, the genes SaMADS A and SaMADS B were identified which are expressed in transition stages of mustard. RNA blot analysis has confirmed that the transcript levels of both genes are drastically increased shortly after the induction of flowering and that both genes are expressed earlier than the known MADS box genes. In situ hybridization studies have shown that the expression of the genes is restricted to the apical meristem of induced plants during early phases of reproductive development. The expression of SaMADS A is first detectable in the central zone of the meristem, a region where the earliest changes of an evoked meristem could be detected by classical physiological methods, suggesting that SaMADS A may have an important function during the transition to flowering.

Amino Acid Sequence↗
PubMed1996

Distinct roles for light-dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in higher plants.

Light-dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear-encoded plastid-localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR-encoding genes, PorA and PorB, that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A-mediated far-red high irradiance response. Wild-type seedlings grown in continuous far-red light (cFR) display the morphology of white light (WL)-grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR-induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide-F655, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR-grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR-grown transgenic Arabidopsis wild-type seedlings restores Chl accumulation and WL-induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light-dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide-F655.

Arabidopsis↗
PubMed1995

Two routes of chlorophyllide synthesis that are differentially regulated by light in barley (Hordeum vulgare L.).

NADPH-protochlorophyllide oxidoreductase (POR; EC 1.6.99.1) catalyzes the only known light-dependent step in chlorophyll synthesis of higher plants, the reduction of protochlorophyllide (Pchlide) to chlorophyllide. In barley, two distinct immunoreactive POR proteins were identified. In contrast to the light-sensitive POR enzyme studied thus far (POR-A), levels of the second POR protein remained constant in seedlings during the transition from dark growth to the light and in green plants. The existence of a second POR-related protein was verified by isolating and sequencing cDNAs that encode a second POR polypeptide (POR-B) with an amino acid sequence identity of 75% to the POR-A. In the presence of NADPH and Pchlide, the in vitro-synthesized POR-A and POR-B proteins could be reconstituted to ternary enzymatically active complexes that reduced Pchlide to chlorophyllide only after illumination. Even though the in vitro activities of the two enzymes were similar, the expression of their genes during the light-induced transformation of etiolated to green seedlings was distinct. While the POR-A mRNA rapidly declined during illumination of dark-grown seedlings and soon disappeared, POR-B mRNA remained at an approximately constant level in dark-grown and green seedlings. Thus these results suggest that chlorophyll synthesis is controlled by two light-dependent POR enzymes, one that is active only transiently in etiolated seedlings at the beginning of illumination and the other that also operates in green plants.

Amino Acid Sequence↗
PubMed1995

Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana.

We compared the organ specificity and the strength of different constitutive (CaMV-35S, CaMV-35Somega, Arabidopsis ubiquitin UBQ1, and barley leaf thionin BTH6 promoter) and one inducible promoter (soybean heat-shock promoter Gmhsp17.3) in stably transformed Arabidopsis thaliana plants. For this purpose we constructed a set of plant expression vectors equipped with the different promoters. Using the uidA reporter gene we could show that the CaMV-35S promoter has the highest expression level which was enhanced two- to threefold by the addition of a translational enhancer (TMV omega element) without altering the organ specificity of the promoter. The barley leaf thionin promoter was almost inactive in the majority of lines whereas the ubiquitin promoter exhibited an intermediate strength. The heat-shock promoter was inducible up to 18-fold but absolute levels were lower than in the case of the ubiquitin promoter. Conclusive quantitative results for different organs and developmental stages were obtained by the analysis of 24 stably transformed lines per promoter construct.

Antimicrobial Cationic Peptides↗
PubMed1995

Isolation and classification of chlorophyll-deficient xantha mutants of Arabidopsis thaliana.

Mutant lines of Arabidopsis thaliana that are either blocked at various steps of the biosynthetic pathway of chlorophyll (Chl) or that are disturbed in one of the subsequent steps leading to the assembly of an active photosynthetic membrane were isolated by screening for Chl-deficient xantha (xan) mutants. Only mutants that segregated in a 3:1 ratio, that contained the same carotenoid spectrum as etiolated wild-type seedlings and less than 2% of the Chl of wild-type control seedlings, and whose Chl content was not affected by the addition of sucrose to the growth medium were selected for a more detailed analysis. As a final test for the classification of the selected mutants, light-grown xan mutants were vacuum-infiltrated and incubated with the common precursor of tetrapyrroles, delta-aminolevulinic acid (ALA), in the dark. Two major groups of mutants could be distinguished. Some of the mutants were blocked at various steps of the Chl pathway between ALA and protochlorophyllide (Pchlide) and did not accumulate the latter in the dark. The other mutants accumulated Pchlide in the dark regardless of whether exogenous ALA was added. This latter group could be subdivided into mutants with a biochemical lesion in a recently discovered second light-dependent Pchlide reduction step that occurs in green plants and mutants that have blocks in the assembly of Chl protein complexes. In the present work a total of seven different loci could be defined genetically in Arabidopsis that affect the synthesis of Chl and its integration into the growing photosynthetic membrane.

Arabidopsis↗
PubMed1995

Enzymatic product formation impairs both the chloroplast receptor-binding function as well as translocation competence of the NADPH: protochlorophyllide oxidoreductase, a nuclear-encoded plastid precursor protein.

The key enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR, EC 1.6.99.1), is a nuclear-encoded plastid protein. Its posttranslational transport into plastids of barley depends on the intraplastidic availability of one of its substrates, protochlorophyllide (PChlide). The precursor of POR (pPOR), synthesized from a corresponding full-length barley cDNA clone by coupling in vitro transcription and translation, is enzymatically active and converts PChlide to chlorophyllide (Chlide) in a light- and NADPH-dependent manner. Chlorophyllide formed catalytically remains tightly but noncovalently bound to the precursor protein and stabilizes a transport-incompetent conformation of pPOR. As shown by in vitro processing experiments, the chloroplast transit peptide in the Chlide-pPOR complex appears to be masked and thus is unable to physically interact with the outer plastid envelope membrane. In contrast, the chloroplast transit peptide in the naked pPOR (without its substrates and its product attached to it) and in the pPOR-substrate complexes, such as pPOR-PChlide or pPOR-PChlide-NADPH, seems to react independently of the mature region of the polypeptide, and thus is able to bind to the plastid envelope. When envelope-bound pPOR-PChlide-NADPH complexes were exposed to light during a short preincubation, the enzymatically produced Chlide slowed down the actual translocation step, giving rise to the sequential appearance of two partially processed translocation intermediates. However, ongoing translocation induced by feeding the chloroplasts delta-aminolevulinic acid, a precursor of PChlide, was able to override these two early blocks in translocation, suggesting that the plastid import machinery has a substantial capacity to denature a tightly folded, envelope-bound precursor protein. Together, our results show that pPOR with Chlide attached to it is impaired both in the ATP-dependent step of binding to a receptor protein component of the outer chloroplast envelope membrane, as well as in the PChlide-dependent step of precursor translocation.

Biological Transport, Active↗
PubMed1995

Identification of NADPH:protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana.

Illumination releases the arrest in chlorophyll (Chl) biosynthesis in etiolated angiosperm seedlings through the enzymatic photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the first light-dependent step in chloroplast biogenesis. NADPH: Pchlide oxidoreductase (POR, EC 1.3.1.33), a nuclear-encoded plastid-localized enzyme, mediates this unique photoreduction. Paradoxically, light also triggers a drastic decrease in the amounts of POR activity and protein before the Chl accumulation rate reaches its maximum during greening. While investigating this seeming contradiction, we identified two distinct Arabidopsis thaliana genes encoding POR, in contrast to previous reports of only one gene in angiosperms. The genes, designated PorA and PorB, by analogy to the principal members of the phytochrome photoreceptor gene family, display dramatically different patterns of light and developmental regulation. PorA mRNA disappears within the first 4 h of greening, whereas PorB mRNA persists even after 16 h of illumination, mirroring the behavior of two distinct POR protein species. Experiments designed to help define the functions of POR A and POR B demonstrate exclusive expression of PorA in young seedlings and of PorB both in seedlings and in adult plants. Accordingly, we propose the existence of a branched light-dependent Chl biosynthesis pathway in which POR A performs a specialized function restricted to the initial stages of greening and POR B maintains Chl levels throughout angiosperm development.

Amino Acid Sequence↗
PubMed1995

An Arabidopsis thaliana thionin gene is inducible via a signal transduction pathway different from that for pathogenesis-related proteins.

Two cDNAs encoding thionin preproteins have been isolated from Arabidopsis thaliana. The corresponding genes have been designated Thi2.1 and Thi2.2. Southern blot analysis suggests that A. thaliana most probably contains single genes for both thionins. Thi2.2 transcripts have a low basal level in seedlings and show circadian variation. Thi2.2 transcripts were also detected in rosette leaves. No potent elicitors have been found for the Thi2.2 gene. Transcripts of the Thi2.1 gene are not detectable in seedlings but are present in rosette leaves and at a very high level in flowers and in siliques. The expression of the Thi2.1 gene is highly inducible in seedlings by pathogens, silver nitrate, and methyl jasmonate, but not by salicylate, indicating that the gene is induced by a signal transduction pathway that is at least partly different from that for the pathogenesis-related proteins.

Amino Acid Sequence↗
PubMed1995

Substrate-dependent transport of the NADPH:protochlorophyllide oxidoreductase into isolated plastids.

The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translocation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with delta-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seedlings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurnal fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a novel regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide.

Biological Transport↗
PubMed1995

A light-induced protease from barley plastids degrades NADPH:protochlorophyllide oxidoreductase complexed with chlorophyllide.

The NADPH:protochlorophyllide oxidoreductase precursor protein (pPorA) of barley (Hordeum vulgare L. cv. Carina), synthesized from a full-length cDNA clone by coupling in vitro transcription and translation, is a catalytically active protein. It converts protochlorophyllide to chlorophyllide in a light- and NADPH-dependent manner. At least the pigment product of catalysis remains tightly bound to the precursor protein. The chlorophyllide-pPorA complex differs markedly from the protochlorophyllide-pPorA complex with respect to sensitivity to attack by a light-induced, nucleus-encoded, and energy-dependent protease activity of barley plastids. The pPorA-chlorophyllide complex is rapidly degraded, in contrast to pPorA-protochlorophyllide complexes containing or lacking NADPH, which are both resistant to protease treatment. Unexpectedly, pPorA devoid of its substrates or products was less sensitive to proteolysis than the pPorA-chlorophyllide complex, suggesting that both substrate binding and product formation during catalysis had caused differential changes in protein conformation.

Adenosine Triphosphate↗
PubMed1994

Organ-specific expression of highly divergent thionin variants that are distinct from the seed-specific crambin in the crucifer Crambe abyssinica.

Most thionins of higher plants are toxic to various bacteria, fungi, and animal and plant cells. The only known exception is the seed-specific thionin, crambin, of the crucifer Crambe abyssinica. Crambin has no net charge, is very hydrophobic and exhibits no toxicity. In the present work, the organization of the crambin precursor polypeptide was deduced from cD-NA sequences. The precursor shows a domain structure similar to that of the preproprotein of other thionins, which contains a signal peptide, a thionin domain and a C-terminal amino acid extension. Unlike the thionin precursors studied thus far, both the thionin domain and the C-terminal amino acid extension of the crambin precursor have no net charge and are hydrophobic, thus facilitating their interaction, by analogy to that proposed for the corresponding domains of other thionin precursors that have positive and negative charges. The existence of a large number of novel and highly variable thionin variants in Crambe abyssinica has been deduced from cDNA sequences that were amplified by the polymerase chain reaction (PCR) from RNA of seeds, leaves and cotyledons. While the deduced amino acid sequences of the thionin domains of most of these thionin precursor molecules are highly divergent, the two other domains are conserved. Most of the predicted thionin variants are positively charged. The presence of positively charged residues in the thionin domains consistently correlates with the presence of a negatively charged residue in the C-terminal amino acid extension of the various thionin precursors. The different thionin variants are encoded by distinct sets of genes and are expressed in an organ-specific manner.

Amino Acid Sequence↗
PubMed1994

JIP60, a methyl jasmonate-induced ribosome-inactivating protein involved in plant stress reactions.

Plant tissues treated with the naturally occurring cyclopentanone compound methyl jasmonate or exposed to stress causing in planta jasmonate accumulation express distinctive proteins and, concomitantly, reduce the synthesis of most preexisting proteins. One of the recently identified jasmonate-induced proteins, designated JIP60, in barley is a ribosome-inactivating protein that cleaves polysomes of both animal and plant origin into their ribosomal subunits. By attacking foreign and self ribosomes, respectively, JIP60 appears to be both a defense protein and a potent regulator of protein synthesis in stressed plant tissues.

Acetates↗
PubMed1994

A light- and temperature-entrained circadian clock controls expression of transcripts encoding nuclear proteins with homology to RNA-binding proteins in meristematic tissue.

To investigate physiological processes generated by endogenous circadian rhythms on the molecular level, we have identified clock-controlled genes in the long-day plant Sinapis alba L. A cDNA library was differentially screened using cDNA probes representing transcripts expressed at either the middle of the light period or the middle of the dark period. Two closely related groups of transcripts, Sagrp1 and Sagrp2, controlled by a circadian rhythm have been isolated. RNA blot analysis verified that transcript levels oscillate in plants grown in light/dark cycles with maxima between 'Zeitgeber' time (zt)8 and zt12 (8-12 h after onset of illumination) and minima around zt20. Steady-state mRNA levels continue to oscillate in plants shifted from light/dark cycles to constant light. No synchronous mRNA oscillations are found in plants grown from seed in constant light at constant temperature, suggesting that the clock has to be entrained initially. In contrast, when plants grown in constant light are exposed to rhythmic temperature shifts oscillations of steady-state Sagrp mRNA levels are induced, indicating that temperature acts as an alternative external stimulus (zeitgeber) other than light to entrain the oscillator. In situ hybridization reveals that both transcript groups are expressed predominantly in meristematic and growing tissue. Strong expression is observed in the leaf primordia of the shoot apex, the procambial strands, cambium and in all cell layers of young leaves around zt12. In contrast, little or no signal is found on tissue sections isolated at zt20. This indicates that the oscillator(s) regulating Sagrp transcript fluctuations operate(s) synchronously in different organs. For both transcript groups cDNAs were isolated corresponding to unspliced pre-mRNAs or to transcripts generated by the use of a second 5' splice site. The cDNAs corresponding to the fully spliced transcripts contain open reading frames for polypeptides of 16 kDa, each containing a putative N-terminal RNA recognition motif and a C-terminal region rich in glycine. The predicted proteins show strong homology to an ABA-inducible glycine-rich protein from maize embryos and to the mammalian RNA-binding protein A1 of the heterogeneous nuclear ribonucleoprotein complex involved in pre-mRNA splicing. The SaGRP protein fluctuates with a very low amplitude over light/dark cycles. Immunogold labeling demonstrates the presence of the SaGRP protein within the nucleus of the investigated meristematic cells of young leaves.

Amino Acid Sequence↗
PubMed1994

Circadian oscillations of a transcript encoding a germin-like protein that is associated with cell walls in young leaves of the long-day plant Sinapis alba L.

As part of an attempt to analyze rhythmic phenomena in the long-day plant Sinapis alba L. at the molecular level, we have searched for mRNAs whose concentration varies as a function of time of day. Differential screening of a cDNA library established from mRNAs expressed at the end of the daily light phase with probes representing transcripts expressed predominantly in the morning or evening has identified one major transcript. The cDNA, Saglp, encodes a predicted 22-kD protein with an N-terminal signal sequence. The protein shows homology to germin, a protein expressed in wheat embryos after onset of germination. The Saglp mRNA level undergoes circadian oscillations in light/dark cycles with maxima between 8 and 12 PM (zeitgeber time [zt]12-zt16) and minima around 8 PM (zt0). In plants grown from seed in constant light, transcript levels are constitutive. In constant light regular temperature shifts function as an alternative "zeitgeber" to initiate Saglp transcript oscillations. At the cellular level, Saglp transcripts are expressed in the epidermis and spongy parenchyma of young leaves, and in distinct regions of the epidermis and the cortex in stems and petioles. Strong signals are observed in these tissues around zt12, whereas little expression is found around zt20, suggesting that the underlying oscillatory mechanism(s) operate(s) synchronously in different plant organs. The SaGLP steady-state protein concentration remains constant over light/dark cycles. Immunogold labeling shows that the SaGLP protein is associated with primary cell walls.

Amino Acid Sequence↗
PubMed1994

The proteins encoded by two tapetum-specific transcripts, Sa tap35 and Sa tap44, from Sinapis alba L. are localized in the exine cell wall layer of developing microspores.

By differential screening of a copy DNA (cDNA) library from flowering Sinapis alba L. apices against cDNAs from vegetative apices, two cDNA clones were isolated representing transcripts that are expressed transiently at an early stage of tapetum development. The Sa tap35 cDNA encodes a polypeptide with a predicted molecular weight of 12.7 kDa and an isoelectric point of 10.4. The sa tap44 cDNA codes for a putative 12.4-kDa polypeptide with an isoelectric point of 7.5. The deduced amino-acid sequences display 76% sequence identity and contain an N-terminal stretch of hydrophobic amino acids which has characteristics of secretory signal sequences. In-vitro transcription of the cDNAs and translation of the resulting RNAs in the presence of canine pancreatic microsomes demonstrates that the two proteins are translocated into the microsomes and that the putative preproteins are proteolytically processed to the mature forms. By immunoelectron microscopy the Sa TAP35 and Sa TAP44 proteins were detected at the developing peritapetal membrane between the tapetal cytoplasm and the adjacent middle layer of the anther wall. Furthermore, labelling was observed within the locule in association with globules resembling pro-Ubisch bodies which appeared at the tetrad stage. During the early vacuolate stage of microspore development the young exine was strongly labelled. The exine and the peritapetal membrane both are composed of sporopollenin, and the pro-Ubisch bodies are thought to contain sporopollenin precursors. Thus, Sa TAP35 and Sa TAP44 might be involved in sporopollenin formation and/or deposition.

ATP-Binding Cassette Transporters↗
PubMed1993

cDNA-derived identification of novel thionin precursors in Viscum album that contain highly divergent thionin domains but conserved signal and acidic polypeptide domains.

The existence of new thionin variants in Viscum album has been deduced from cDNA sequences. Unlike the viscotoxins and related thionins previously found in different members of the Viscaceae, these novel thionins contain eight rather than six cysteine residues. In this respect they resemble thionins described previously from various cereals and from Pyrularia pubera, which also contain eight cysteine residues at identical positions. All of the new thionins of V. album are encoded as higher-molecular-weight precursors consisting of a signal peptide, a thionin domain and an acidic polypeptide domain. While the deduced amino acid sequences of the thionin domains of different precursor molecules are highly divergent, the two other domains are conserved among all of the variants and are distinct from the corresponding domains of thionin precursors of other plant species.

Amino Acid Sequence↗
PubMed1993

Light-independent and light-dependent protochlorophyllide-reducing activities and two distinct NADPH-protochlorophyllide oxidoreductase polypeptides in mountain pine (Pinus mugo).

Lower plants and gymnosperms synthesize chlorophyll and develop photosynthetically competent chloroplasts even when grown in the dark. In cell-free extracts of pine (Pinus mugo, Turra, ssp. mugo) seedlings, light-independent and light-dependent protochlorophyllide-reducing activities are present. Two distinct NADPH-protochlorophyllide-oxidoreductase (POR) polypeptides can be detected immunologically with an antiserum raised against the POR of barley. The subcellular localization and amounts of the two POR polypeptides are differentially affected by light: one of them is predominantly present in prolamellar bodies of etiochloroplasts and its abundance rapidly declines once the pine seedlings are exposed to light; the other is found in thylakoid membranes and its amount does not change during illumination of dark-grown seedlings. Two types of cDNA sequences are identified that encode two distinct POR polypeptides in pine. The relevance of these POR polypeptides for the two chlorophyll biosynthetic pathways active in gymnosperms is discussed.

Amino Acid Sequence↗