Regulation of biosynthesis and cellular localization of Sp32 annexins in tobacco BY2 cells.

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.

A ubiquitous plant housekeeping gene, PAP, encodes a major protein component of bell pepper chromoplasts.

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.

Fruit-specific expression of a defensin-type gene family in bell pepper. Upregulation during ripening and upon wounding.

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.

Characterization and gene expression of an annexin during fruit development in Capsicum annuum.

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.

Characterization of a family of genes encoding a fruit-specific wound-stimulated protein of bell pepper (Capsicum annuum): identification of a new family of transposable elements.

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.

Changes in ascorbate peroxidase activities during fruit ripening in Capsicum annum.

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.

A chromoplast-specific protein in Capsicum annuum: characterization and expression of the corresponding gene.

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.

Structure and expression of a cDNA encoding a histone H2A from Euglena gracilis.

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.