RESUMEN

A cDNA, cakn, orthologous to the Arabidopsis KN gene, which is involved in vesicle fusion during cell plate formation, was isolated from bell pepper fruit. cakn seems to be monogenic and its expression mainly transcriptionally regulated. During fruit development, transcript and protein levels increase significantly in the early stages in which numerous cell divisions occur, but in the stages corresponding to fruit growth by cell enlargement, whereas the messengers are undetectable, proteins are still faintly present, suggesting a different stability rate for the two types of macromolecules.

Asunto(s)

Proteínas de Arabidopsis , Capsicum/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Plantas , Proteínas de la Membrana/genética , Plantas Medicinales , Secuencia de Aminoácidos , Ciclo Celular , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Qa-SNARE , Alineación de Secuencia , Transcripción Genética

RESUMEN

This work provides the first three-dimensional structure of a member of the plant annexin family and correlates these findings with biochemical properties of this protein. Annexin 24(Ca32) from Capsicum annuum was purified as a native protein from bell pepper and was also prepared by recombinant techniques. To overcome the problem of precipitation of the recombinant wild-type protein in crystallization trials, two mutants were designed. Whereas an N-terminal truncation mutant turned out to be an unstable protein, the N-terminal His-tagged annexin 24(Ca32) was crystallized, and the three-dimensional structure was determined by x-ray diffraction at 2. 8 A resolution. The structure refined to an R-factor of 0.216 adopts the typical annexin fold; the detailed structure, however, is different from non-plant annexins, especially in domains I and III and in the membrane binding loops on the convex side. Within the unit cell there are two molecules per asymmetric unit, which differ in conformation of the IAB-loop. Both conformers show Trp-35 on the surface. The loop-out conformation is stabilized by tight interactions of this tryptophan with residue side chains of a symmetry-related molecule and enforced by a bound sulfate. Characterization of this plant annexin using biophysical methods revealed calcium-dependent binding to phospholipid vesicles with preference for phosphatidylcholine over phosphatidylserine and magnesium-dependent phosphodiesterase activity in vitro as shown with adenosine triphosphate as the substrate. A comparative unfolding study of recombinant annexin 24(Ca32) wild type and of the His-tag fusion protein indicates higher stability of the latter. The effect of this N-terminal modification is also visible from CD spectra. Both proteins were subjected to a FURA-2-based calcium influx assay, which gave high influx rates for the wild-type but greatly reduced influx rates for the fusion protein. We therefore conclude that the N-terminal domain is indeed a major regulatory element modulating different annexin properties by allosteric mechanisms.

Asunto(s)

Anexinas/química , Capsicum/química , Proteínas de Plantas/química , Plantas Medicinales , Adenosina Trifosfatasas , Secuencia de Aminoácidos , Anexinas/genética , Calcio/farmacología , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Permeabilidad/efectos de los fármacos , Proteínas de Plantas/genética , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes/química , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Propiedades de Superficie

RESUMEN

Annexins interact in a calcium-dependent manner with membrane phospholipids. Although their exact function is not known, annexins have been proposed to be involved in a variety of cellular processes. To determine whether plant annexins are implicated in cell division, we have isolated cDNAs encoding annexin from TBY2 cells. Based on sequence analysis, these cDNAs fall into two families, differing mainly by deletions or insertions in their 5'- and 3'-untranslated regions. The two annexins Ntp32.1 and Ntp32.2 encoded by these cDNAs are homologous to p32 from bell pepper (Cap32.1): we propose that these Solanaceae annexins constitute a distinct type which we call Sp32 annexins. There are two genes (Ntan.1 and Ntan.2) derived from the separate progenitor species of Nicotiana tabacum and analysis of Southern blots is consistent with the presence of these two genes. We show that Sp32 transcript amounts are developmentally modulated in tobacco plants: RNA levels are highest in growing and dividing tissues. Sp32 annexin gene expression is also regulated in TBY2 cultured cells: transcripts and proteins are detected only in exponentially growing cells. In synchronized TBY2 cells, Sp32 annexin transcripts are expressed at the G2/M transition, in the M phase and at the G1/S transition. These results are the first evidence that the expression of plant annexins is modulated during the cell cycle. The Sp32 annexin proteins accumulate during the cell cycle and peak at the end of mitosis. Immunolocalization shows that the majority of Sp32 annexins is present in intercellular junctions, forming a ring structure under the plasma membrane. Since annexins are known to bind secretory vesicles during exocytosis, their localization at cell junctions suggests that annexins could be involved in cell wall maturation.

Asunto(s)

Anexinas/genética , Nicotiana/química , Proteínas de Plantas , Plantas Tóxicas , Secuencia de Aminoácidos , Anexinas/química , Ciclo Celular/genética , ADN Complementario/química , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Inmunohistoquímica , Datos de Secuencia Molecular , Semillas/química

RESUMEN

We have characterized two members of a gene family encoding defensin-type proteins, j1-1 and j1-2. The structures of these homologous genes are highly similar. Both genes contain an intervening sequence at the same position. Sequence analysis of the intron within the j1-2 gene revealed the existence of an additional exon (exon 2ji) which also encodes a defensin-type protein. It is very likely that this exon was derived by genomic shuffling from a gene, jx, belonging to another subfamily which remains to be characterized. Only transcripts resulting from the splicing of exons 1j2 and 2ji can be detected, indicating that the inserted exon has functionally replaced the original, leaving the latter as a partial pseudogene. This rearrangement did not alter the tissue specificity of expression of the gene j1-2. The corresponding transcripts, present at the stage of fruit set, accumulate progressively during the process of development. In contrast, gene j1-1 is expressed only at the later stages of ripening. Estimates of transcription rates show that in green fruits expression is mainly regulated at a post-transcriptional level, while transcriptional regulation is prominent during ripening.

Asunto(s)

Capsicum/genética , Defensinas , Exones/genética , Regulación del Desarrollo de la Expresión Génica , Reordenamiento Génico , Proteínas de Plantas/genética , Plantas Medicinales , Regiones no Traducidas 3'/genética , Empalme Alternativo/genética , Secuencia de Aminoácidos , Northern Blotting , Capsicum/crecimiento & desarrollo , Sondas de ADN , Frutas/genética , Intrones/genética , Datos de Secuencia Molecular , Mutagénesis Insercional/genética , Sistemas de Lectura Abierta/genética , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , ARN de Planta/análisis , ARN de Planta/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia

RESUMEN

In bell pepper, a gene encoding a major plastid-lipid associated protein is expressed as both partially and totally spliced transcripts (respectively PAP2 and PAP1). Although PAP is present as a single-copy gene in the bell pepper genome, Southern blots using PAP2 as a probe revealed multiple homologous copies. Analyses of the intronic sequence of PAP2 showed the existence of a 206bp short interspersed repetitive element (SINE) belonging to the Ts family of retrotransposons (Yoshioka et al., 1993). Comparison with PAP sequences in other Solanaceae species suggested that the structure of the gene is highly conserved: the two introns are inserted at the same position. However, the Ts insertion found in bell pepper is absent in tobacco and tomato. Studies using RT-PCR showed that in these latter species only totally spliced transcripts of PAP are present. On the other hand, RNA analyses of tobacco plants transformed with the bell pepper PAP revealed the presence of both totally and incompletely spliced transcripts. Altogether our results support the hypothesis that the Ts insertion into the first intron of PAP results in a splicing defect of the corresponding pre-mRNA. Based on the presence of peculiar, previously unidentified Ts elements, a possible horizontal transmission of Ts elements from animals to plants is discussed.

Asunto(s)

Capsicum/genética , Genes de Plantas , Plantas Medicinales , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Secuencia de Bases , Secuencia de Consenso , Cartilla de ADN/genética , ADN de Plantas/genética , Evolución Molecular , Genoma de Planta , Intrones , Datos de Secuencia Molecular , Proteínas Asociadas a Pancreatitis , Proteínas de Plantas/genética , Empalme del ARN , ARN de Planta/genética , ARN de Transferencia/genética , Retroelementos , Homología de Secuencia de Ácido Nucleico

RESUMEN

We have isolated a cDNA (PAP) corresponding to a single nuclear gene that encodes an approximately 30-kD major protein of bell pepper (Capsicum annuum L.) fruit chromoplasts. RNA and protein analyses revealed that, although at a low level, this gene is also expressed in every organ of the plant, the amount of the corresponding transcript and protein dramatically increasing in the latter stages of fruit development. Western-blot and immunocytochemical analyses of purified chloroplasts from leaves and fruits and of chromoplasts from red fruits showed that the encoded protein is the major component of plastoglobules and fibrils and is localized on the outer surface of these lipid structures. Analyses of PAP in plants belonging to different taxa revealed that it is expressed and highly conserved in both monocotyledonous and dicotyledonous plants. The presence of the protein in plastids not differentiating into chromoplasts indicates that PAP is expressed irrespective of the ontogeny of various plastid lines. In light of our results and since the encoded protein, identical to that previously named ChrB or fibrillin, is present in plastoglobules from several species and accumulates in the fibrils of bell pepper chromoplast, we propose to designate it as a plastid-lipid-associated protein.

Asunto(s)

Capsicum/genética , Genes de Plantas , Proteínas de Plantas/genética , Plantas Medicinales , Secuencia de Aminoácidos , Capsicum/fisiología , ADN Complementario , Inmunohistoquímica , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Fracciones Subcelulares/metabolismo

RESUMEN

We have isolated a 454-bp cDNA that encodes a novel fruit specific defensin from bell pepper (Capsicum annuum). The encoded 75-amino-acid polypeptide contains an N-terminal domain characteristic of a signal peptide and a 48-amino-acid mature domain named J1. The mature protein, from which the N-terminal amino acid sequence was determined, contains eight cysteines that from four intramolecular disulfide bridges, suggesting a monomeric form for J1. In healthy fruits J1 is undetectable at the green stage but high levels accumulate during ripening. In wound areas of the green fruit the accumulation of J1 dramatically increased, suggesting a role for J1 in the plant's defense response. Moreover, we have demonstrated that J1 possesses an antifungal activity. We have isolated and characterized the corresponding two homologous genes (j1-1 and j1-2) that exist in the bell pepper genome. Both genes are interrupted by the insertion, at the same position, of one intron of 853 bp for j1-1 and 4900 bp for j1-2. Northern blot and reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analyses revealed that j1-1 transcripts are present only in fruits, only in trace amounts in mature green fruits, and that they accumulate to high levels in fully ripe fruits, whereas no j1-2 transcripts were detected in the samples monitored.

Asunto(s)

Antifúngicos , Capsicum/genética , Defensinas , Genes de Plantas , Familia de Multigenes , Proteínas de Plantas/genética , Plantas Medicinales , Secuencia de Aminoácidos , Secuencia de Bases , Capsicum/metabolismo , Elementos Transponibles de ADN , ADN Complementario/genética , Expresión Génica , Intrones , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , Conformación Proteica , ARN Mensajero/análisis , ARN de Planta/análisis , Homología de Secuencia de Aminoácido , Distribución Tisular

RESUMEN

Alien are highly repeated plant transposable elements characterized by their small size (approx. 400 bp), high A + T content, target site specificity, potential to form stable secondary structures and possession of a conserved 28-bp terminal inverted repeat (TIR). Besides the TIR, they contain subterminal inverted repeat motifs (SIRM), as well as the 5'-CATGCAT domain which has been reported to be a cis-acting regulatory element of gene expression in some plant species. Although they were first identified in the intron of the bell pepper (Capsicum annuum) Sn-2 gene and in the promoter region of the potato starch phosphorylase-encoding gene, Alien arranged in tandem are present in the promoter of patatin class-II genes. PCR on the bell pepper genomic DNA using the Alien TIR consensus sequence as primer yielded DNA fragments of nearly 400 bp. These fragments have characteristics of transposable elements and contain numerous motifs reminiscent of Alien elements. Importantly, PCR on genomic DNA extracts from various monocotyledonous and dicotyledonous plants using the TIR consensus sequence as primer and subsequent hybridization with different Alien probes revealed that these elements are ubiquitously present and highly repeated in the genomes of higher plants.

Asunto(s)

Hidrolasas de Éster Carboxílico , Cotiledón/genética , Elementos Transponibles de ADN/genética , Genes de Plantas , Secuencia de Bases , Capsicum/genética , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Plantas Medicinales , Reacción en Cadena de la Polimerasa , Población , Regiones Promotoras Genéticas , Homología de Secuencia de Ácido Nucleico , Solanum tuberosum/genética , Especificidad de la Especie , Transcripción Genética

RESUMEN

Several lines of evidence indicate that annexins, as calcium-dependent phospholipid-binding proteins, are involved in a variety of plant cellular processes. We were interested in determining if annexins are implicated in the highly regulated fruit development of bell pepper. By differential screening of several cDNA libraries, we isolated a full-length cDNA of 1180 bp encoding an annexin. Northern blot analyses show a differential expression pattern of the transcripts during the early stages of development and during ripening. Immunoblots using antiserum raised against p33/p35 from maize reveal that cross-reactive polypeptides of about 30 kDa are present at each stage of fruit development in bell pepper. We partially purified the annexins from seedlings and green fruits. At least one annexin of 32 kDa is present in these plant tissues.

Asunto(s)

Anexinas/biosíntesis , Anexinas/química , Capsicum/fisiología , Regulación de la Expresión Génica de las Plantas , Plantas Medicinales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Capsicum/crecimiento & desarrollo , Capsicum/metabolismo , Secuencia de Consenso , ADN Complementario , Biblioteca de Genes , Medicago sativa/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Hojas de la Planta , Raíces de Plantas , ARN de Planta/análisis , Mapeo Restrictivo , Semillas , Homología de Secuencia de Aminoácido

RESUMEN

In Bidens pilosa (cv. radiata), a non-injurious stimulus induces a local and transient change in membrane potential, and an injurious stimulus induces a transmitted electrical signal described as the combination of an action potential and a slow wave. We have studied calmodulin gene expression after these stimuli. When the stimulus is non-injurious, calmodulin mRNA accumulation is only increased in the stimulated region. In contrast, when the stimulus is injurious, mRNA accumulation takes place in both wounded and distant, unwounded tissue. We propose that the slow wave plays a role in the long-distance transmission of a wound-induced information in plants.

Asunto(s)

Calmodulina/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Fenómenos Fisiológicos de las Plantas , Calor , Potenciales de la Membrana , Plantas/genética , ARN Mensajero/biosíntesis , ARN de Planta/biosíntesis , Agua

RESUMEN

In this study we have constructed a number of plants (cybrids), in which the nuclear genome of Nicotiana plumbaginifolia is combined with the plastome of Atropa belladonna, or the nuclear genome of N. tabacum with plastomes of Lycium barbarum, Scopolia carniolica, Physochlaine officinalis or Nolana paradoxa. Our biochemical and immunological analyses prove that in these cybrids the biogenesis of the chlorophyll a/b binding proteins (CAB) of the light harvesting complex II (LHCII) is altered. Besides normal sized CAB polypeptides of 27, 25.5 and 25 kDa, which become less abundant, the cybrids analyzed have additional polypeptides of 26, 24.5 and 24 kDa. Direct protein micro-sequencing showed that at least two truncated 26 kDa CAB polypeptides in plant cells containing a nucleus of N. plumbaginifolia and plastids of A. belladonna are encoded by the type 1 Lhcb genes. These polypeptides are 11-12 amino acids shorter at the N-terminus than the expected size. Based on the available data we conclude that the biogenesis of the LHCII in vivo may depend on plastome-encoded factor(s). These results suggest that plastome-encoded factors that cause specific protein degradation and/or abnormal processing might determine compartmental genetic incompatibility in plants.

Asunto(s)

Proteínas Portadoras/genética , Núcleo Celular/genética , Cloroplastos/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema II , Proteínas de Plantas , Plantas/genética , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Compartimento Celular , Quimera , Expresión Génica , Genes de Plantas , Variación Genética , Células Híbridas , Complejos de Proteína Captadores de Luz , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Plantas Tóxicas , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Nicotiana/genética

RESUMEN

Using a fruit-specific cDNA as a probe we isolated and sequenced the two corresponding homologous genes (Sn-1 and Sn-2) of the bell pepper (Capsicum annuum) genome. Both genes have a single intron and numerous unusual long inverted repeat sequences. The introns share 87% homology and Sn-2 contains one 450 bp additional sequence with structural features of a transposable element, which is highly repetitive in the bell pepper genome. Surprisingly, analysis in data banks showed that genes encoding the potato starch phosphorylase (EC 2.4.1.1) and patatin contain a similar element, named Alien, in their 5'-upstream region. Alien elements are characterized by a conserved 28 bp terminal inverted repeat (TIR), small size, high AT content, potential to form stable DNA secondary structures and they have probably been inserted in TA target sites. Interestingly, the TIR of the Alien elements shares high homology with sequences existing in the TIR of extrachromosomal linear pSKL DNA plasmid of Saccharomyces kluyveri. Northern blot analyses detected Sn-1 transcripts principally in the red fruit whereas no Sn-2 transcripts were detected in neither of the samples monitored. Western blot analyses detected a 16.8 kDa Sn protein principally in the ripe red fruit and wounded areas of green unripe fruit. A comparison of the deduced amino acid sequence of Sn-1 with protein sequences in data banks revealed a significant homology with proteins likely involved in the plant's disease resistance response. Analyses at the subcellular level showed that Sn-1 is localized in the membrane of vacuoles.

Asunto(s)

Elementos Transponibles de ADN , Proteínas de la Membrana/genética , Proteínas de Plantas/genética , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Intrones , Datos de Secuencia Molecular , Familia de Multigenes , Regiones Promotoras Genéticas , ARN Mensajero/genética , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico

RESUMEN

The ascorbate peroxidase (APX) system was studied in Capsicum annum during fruit ripening. A large increase in organelle APX activity was found during chloroplast-chromoplast transition whereas only a slight difference was detected in total fruit extracts. On native gels, four different isoforms were found in total fruit extracts but the patterns for red and green fruit were quite different. In isolated organelles, six isozymes were found and a comparison of the patterns showed significant differences. A cDNA encoding a cytosolic APX was cloned and sequenced. The corresponding transcript was shown to increase 3-4-fold during fruit ripening.

Asunto(s)

Capsicum/enzimología , Peroxidasas/metabolismo , Plantas Medicinales , Secuencia de Aminoácidos , Ascorbato Peroxidasas , Capsicum/crecimiento & desarrollo , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Frutas , Datos de Secuencia Molecular , Peroxidasas/genética , Homología de Secuencia de Aminoácido

RESUMEN

We have isolated cDNA and genomic clones corresponding to a gene highly and specifically expressed at the late stage of fruit-ripening in bell-pepper. Antibodies raised against the expressed protein allowed determination of the cellular localization of the gene product. The encoded protein is present only in chromoplasts from fully-ripe red fruits, as shown by Western analysis and import experiments. The corresponding mRNA accumulates to high levels during ripening at orange and red stages, but is not detected in yellow varieties impaired in the synthesis of ketocarotenoids. Several lines of evidence indicate that the encoded protein is an oxido-reductase involved in the synthesis of capsanthin and capsorubin.

Asunto(s)

Capsicum/genética , Capsicum/metabolismo , Cloroplastos/metabolismo , Expresión Génica , Genes de Plantas , Proteínas de Plantas/biosíntesis , Plantas Medicinales , Secuencia de Aminoácidos , Sitios de Unión , Western Blotting , Clonación Molecular , ADN Complementario , Flavina-Adenina Dinucleótido/metabolismo , Datos de Secuencia Molecular , NADP/metabolismo , Hojas de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , Transcripción Genética

RESUMEN

Screening of a lambda gt11 cDNA expression library of Euglena gracilis with antibodies directed against histones H2 from maize resulted in the isolation of a full-length cDNA for a histone H2A. The open-reading frame of 408 bp corresponded to a protein of 136 amino acid residues (14 kDa). Despite the presence of a poly(A) tail, which is typical of plant histone mRNA but not of animal histone mRNA, the size of the deduced protein and its percentage of homology were closer to animal histone H2As than to plant or lower eukaryotic histone H2A. Sequence alignment revealed that the Euglena H2A protein was characterized by a shorter C-terminus and a N-terminus which extended 10 residues past the animal H2A. In contrast to other organisms studied, the expression of the Euglena H2A gene appeared to be almost constant during an entire life-cycle and presented no cell-stage-specific expression during development. Similar results are obtained for another histone gene, H3, and for beta-tubulin. Regulation of gene expression at a post-transcriptional level seems to be a general feature of Euglena.

Asunto(s)

Euglena gracilis/genética , Histonas/genética , Secuencia de Aminoácidos , Animales , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Euglena gracilis/crecimiento & desarrollo , Euglena gracilis/metabolismo , Expresión Génica , Genoma , Histonas/biosíntesis , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido

Asunto(s)

Proteínas Portadoras/genética , Complejos de Proteína Captadores de Luz , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema II , Proteínas de Plantas , Zea mays/genética , ADN Complementario/análisis , Biblioteca de Genes , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido

RESUMEN

We have determined the nucleotide sequence of the plastid psbL gene from bell pepper. This gene has an ACG as a first codon. Isolation of RNA from pepper leaves and ripe fruits and subsequent sequencing of the psbL cDNA revealed that this ACG codon is post-transcriptionally edited into an AUG initiation codon in both leaves and fruits. These data indicate that the RNA editing machinery which exists in chloroplasts is still functional in chromoplasts from ripe fruits.

Asunto(s)

Genes de Plantas , Edición de ARN , Verduras/genética , Secuencia de Aminoácidos , Secuencia de Bases , Diferenciación Celular , Cloroplastos/fisiología , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Orgánulos/fisiología , ARN Mensajero/genética , Verduras/crecimiento & desarrollo

RESUMEN

Cysteine synthase (O-acetylserine sulfhydrylase) has been purified to homogeneity from bell pepper (Capsicum annuum) fruit chromoplasts. This enzyme consists of two subunits of 35 kDa. Immunocytochemical localization experiments confirmed the plastid location of this enzyme. A full-length cDNA was isolated from an expression library of C. annuum. The deduced peptide sequence revealed high similarity between the C. annuum cysteine synthase and its bacterial counterparts. In vitro transcription and translation of the cDNA and subsequent import experiments demonstrated that the encoded cysteine synthase is located in the plastids. The steady-state level of the cysteine synthase mRNA is almost constant in dark-grown hypocotyls, leaves, and fruits. However, a slight increase in this mRNA level was detected during fruit development (when the 25 S rRNA was taken as an internal standard). Similarly, the cysteine synthase activity in plastids was found to increase during fruit development and reaches the highest levels in the chromoplasts of red fruits. To address the physiological role of this phenomenon, we have shown that cysteine is engaged in the active metabolism of glutathione. Thus, in connection with the previous demonstration of an active tocopherol metabolism, it is concluded that differentiation of chloroplast to chromoplast in C. annuum involves an active synthesis of potential antioxidants or redox modulators.

Asunto(s)

Capsicum/enzimología , Cisteína Sintasa/genética , Cisteína Sintasa/metabolismo , Plantas Medicinales , Secuencia de Aminoácidos , Capsicum/genética , Capsicum/crecimiento & desarrollo , Cloroplastos/enzimología , Cromatografía , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular , Cisteína Sintasa/aislamiento & purificación , ADN/genética , ADN/metabolismo , Durapatita , Hidroxiapatitas , Datos de Secuencia Molecular , Peso Molecular , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética

RESUMEN

Cell-specific and light-regulated expression of the beta-glucuronidase (GUS) reporter gene from maize cab-m1 and rbcS-m3 promoter sequences was studied in maize leaf segments by using an in situ transient expression microprojectile bombardment assay. The cab-m1 gene is known to be strongly photoregulated and to be expressed almost exclusively in mesophyll cells (MC) but not in bundle sheath cells (BSC). Expression of GUS from a 1026-base-pair 5' promoter fragment of cab-m1 is very low in dark-grown leaves; GUS expression is increased about 10-fold upon illumination of dark-grown leaves. In illuminated leaves, the ratio of GUS expression in MC vs. BSC is about 10:1. The cab-m1 region between 868 and 1026 base pairs 5' to the translation start confers strong MC-preferred expression on the remainder of the chimeric gene in illuminated leaves, but a region between -39 and -359 from the translation start is required for photoregulated expression. Transcripts of rbcS-m3 are found in BSC but not in MC and are about double in BSC of greening dark-grown seedlings. In contrast to the behavior of the cab-m1-GUS construct, GUS expression driven by 2.1 kilobase pairs of the rbcS-m3 5' region was about twice as high in MC as in BSC of unilluminated dark-grown maize leaves. The number of BSC, but not MC, expressing GUS nearly doubled upon greening of bombarded etiolated leaves. These data suggest that the 5' region of rbcS-m3 used here could be responsible for most of the light-dependent increase in rbcS-m3 transcripts observed in BSC of greening leaves and that transcriptional or posttranscriptional mechanisms are responsible for the lack of rbcS-m3 transcripts in MC.

Asunto(s)

Regiones Promotoras Genéticas , Ribulosa-Bifosfato Carboxilasa/genética , TATA Box , Zea mays/genética , Secuencia de Bases , Oscuridad , Expresión Génica , Regulación de la Expresión Génica , Glucuronidasa/genética , Glucuronidasa/metabolismo , Luz , Datos de Secuencia Molecular , Virus del Mosaico/genética , Plásmidos , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Zea mays/citología , Zea mays/fisiología

RESUMEN

Geranylgeranyl pyrophosphate synthase is a key enzyme in plant terpenoid biosynthesis. Using specific antibodies, a cDNA encoding geranylgeranyl pyrophosphate synthase has been isolated from bell pepper (Capsicum annuum) ripening fruit. The cloned cDNA codes for a high molecular weight precursor of 369 amino acids which contains a transit peptide of approximately 60 amino acids. In-situ immunolocalization experiments have demonstrated that geranylgeranyl pyrophosphate synthase is located exclusively in the plastids. Expression of the cloned cDNA in E. coli has unambiguously demonstrated that the encoded polypeptide catalyzes the synthesis of geranylgeranyl pyrophosphate by the addition of isopentenyl pyrophosphate to an allylic pyrophosphate. Peptide sequence comparisons revealed significant similarity between the sequences of the C. annuum geranylgeranyl pyrophosphate synthase and those deduced from carotenoid biosynthesis (crtE) genes from photosynthetic and non-photosynthetic bacteria. In addition, four highly conserved regions, which are found in various prenyltransferases, were identified. Furthermore, evidence is provided suggesting that conserved and exposed carboxylates are directly involved in the catalytic mechanism. Finally, the expression of the geranylgeranyl pyrophosphate synthase gene is demonstrated to be strongly induced during the chloroplast to chromoplast transition which occurs in ripening fruits, and is correlated with an increase in enzyme activity.

Asunto(s)

ADN/genética , Dimetilaliltranstransferasa/genética , Plantas/enzimología , Plantas/genética , Secuencia de Aminoácidos , Capsicum/enzimología , Capsicum/genética , Clonación Molecular , Dimetilaliltranstransferasa/metabolismo , Escherichia coli/genética , Expresión Génica , Genes de Plantas , Datos de Secuencia Molecular , Plantas Medicinales , Homología de Secuencia de Aminoácido , Transcripción Genética