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A lectin with antifungal and mitogenic activities from red cluster pepper (Capsicum frutescens) seeds.

A monomeric mannose/glucose-binding lectin, with a molecular mass of 29.5 kDa and an N-terminal sequence GQRELKL showing resemblance to that of the lectin-like oxidized low-density lipoprotein receptor from the rabbit, has been isolated from the seeds of red cluster pepper Capsium frutescens L. var. fasciculatum. The protocol involved anion exchange chromatography on diethylamino ethanol-cellulose and Q-Sepharose and fast protein liquid chromatography on Mono Q. Its hemagglutinating activity toward rabbit erythrocytes was inhibited by D: -mannose and glucose, specifically. The activity was stable from 0 to 40 degrees C, reached a maximum at pH 7 and 8, and was potentiated by Ca2+ and Mn2+ ions. The lectin showed strong mitogenic activity toward spleen cells isolated from BALB/c mice. The mitogenic activity, which reached a peak at a lectin concentration of 0.27 microM, was inhibited specifically by D(+)-mannose. The lectin was capable of inhibiting the germination of Aspergillus flavus and Fusarium moniliforme spores and hyphal growth in the two fungi.

Animals↗

The organization of mitochondrial atp6 gene region in male fertile and CMS lines of pepper (Capsicum annuum L.).

The mitochondrial atp6 gene in male fertile (N) and CMS (S) pepper has previously been compared and was found to be present in two copies (Kim et al. in J Kor Soc Hort Sci 42:121-127 2001). In the current study, these atp6 copies were amplified by an inverse PCR technique, and the coding region as well as the 5' and 3' flanking regions were sequenced. The atp6 copies in CMS pepper were detected as one intact gene and one pseudogene, truncated at the 3' coding region. When the atp6 genes in pepper were compared to other plant species, pepper, potato, and petunia all possessed a sequence of 12 identical amino acids at the 3' extended region, which was considered a hallmark of the Solanaceae family. Northern blot analysis showed differences in mRNA band patterns between CMS and restorer lines, indicating that atp6 gene is one of the candidates for CMS in pepper.

Amino Acid Sequence↗

Induction of some defense-related genes and oxidative burst is required for the establishment of systemic acquired resistance in Capsicum annuum.

The inoculation of primary pepper leaves with an avirulent strain of Xanthomonas campestris pv. vesicatoria induced systemic acquired resistance (SAR) in the non-inoculated, secondary leaves. This SAR response was accompanied by the systemic expression of the defense-related genes, a systemic microoxidative burst generating H2O2, and the systemic induction of both ion-leakage and callose deposition in the non-inoculated, secondary leaves. Some defense-related genes including those encoding PR-1, chitinase, osmotin, peroxidase, PR10, thionin, and SAR8.2 were markedly induced in the systemic leaves. The conspicuous systemic accumulation of H2O2 and the strong increase in peroxidase activity in the pepper leaves was suggested to play a role in the cell death process in the systemic micro-hypersensitive responses (HR), leading to the induction of the SAR. Treatment of the primary leaves with diphenylene iodinium (DPI), an inhibitor of oxidative burst, substantially reduced the induction of some of the defense-related genes, and lowered the activation of the oxidative bursts in the systemic leaves distant from the site of the avirulent pathogen inoculation and subsequently SAR. Overall, these results suggest that the induction of some defense-related genes as well as a rapid increase in oxidative burst is essential for establishing SAR in pepper plants.

Capsicum↗

Alteration of the expression of a plant defensin gene by exon shuffling in bell pepper (Capsicum annuum L.).

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.

3' Untranslated Regions↗

Pepper yellow mosaic virus, a new potyvirus in sweetpepper, Capsicum annuum.

A potyvirus was found causing yellow mosaic and veinal banding in sweetpepper in Central and Southeast Brazil. The sequence analysis of the 3' terminal region of the viral RNA revealed a coat protein of 278 amino acids, followed by 275 nucleotides in the 3'-untranslated region preceding a polyadenylated tail. The virus shared 77.4% coat protein amino acid identity with Pepper severe mosaic virus, the closest Potyvirus species. The 3'-untranslated region was highly divergent from other potyviruses. Based on these results, the virus found in sweetpepper plants could be considered as a new potyvirus. The name Pepper yellow mosaic virus (PepYMV) is suggested.

3' Untranslated Regions↗

EST and microarray analyses of pathogen-responsive genes in hot pepper (Capsicum annuum L.) non-host resistance against soybean pustule pathogen (Xanthomonas axonopodis pv. glycines).

Large-scale single-pass sequencing of cDNA libraries and microarray analysis have proven to be useful tools for discovering new genes and studying gene expression. As a first step in elucidating the defense mechanisms in hot pepper plants, a total of 8,525 expressed sequence tags (ESTs) were generated and analyzed in silico. The cDNA microarray analysis identified 613 hot pepper genes that were transcriptionally responsive to the non-host soybean pustule pathogen Xanthomonas axonopodis pv. glycines ( Xag). Several functional types of genes, including those involved in cell wall modification/biosynthesis, transport, signaling pathways and divergent defense reactions, were induced at the early stage of Xag infiltration. In contrast, genes encoding proteins that are involved in photosynthesis, carbohydrate metabolism and the synthesis of chloroplast biogenetic proteins were down-regulated at the late stage of Xag infiltration. These expression profiles share common features with the expression profiles elicited by other stresses, such as fungal challenge, wounding, cold, drought and high salinity. However, we also identified several novel transcription factors that may be specifically involved in the defense reaction of the hot pepper. We also found that the defense reaction of the hot pepper may involve the deactivation of gibberellin. Furthermore, many genes encoding proteins with unknown function were identified. Functional analysis of these genes may broaden our understanding of non-host resistance. This study is the first report of large-scale sequencing and non-host defense transcriptome analysis of the hot pepper plant species. (The sequence data in this paper have been submitted to the dbEST and GenBank database under the codes 10227604-10236595 and BM059564-BM068555, respectively. Additional information is available at http://plant.pdrc.re.kr/ks200201/pepper.html).

Capsicum↗

CAZFP1, Cys2/His2-type zinc-finger transcription factor gene functions as a pathogen-induced early-defense gene in Capsicum annuum.

A pepper zinc-finger protein gene, CAZFP1 , encoding the Cys2/His2-type zinc-finger transcription factor was isolated from pepper leaves inoculated with an avirulent strain Bv5-4a of Xanthomonas campestris pv. vesicatoria . The CAZFP1 protein is a nuclear targeting protein, which functions as a transcriptional regulator. The full-length CAZFP1 had no transcriptional activation activity, whereas the C-terminal region of CAZFP1 had transactivation activity. The CAZFP1 transcripts were constitutively expressed in the pepper stem, root, flower and red fruit, but were not detectable in the leaf and green fruit. The CAZFP1 transcripts accumulated earlier than the CAZFP1 (PR-1) gene in the incompatible interaction of the pepper leaves with X. campestris pv. vesicatoria . The CAZFP1 transcripts were significantly induced in the systemic, uninoculated leaf tissues early after inoculation with bacterial pathogens, but gradually declined thereafter. The CAZFP1 transcripts were localized, and confined to the phloem cells of the vascular bundle in the pepper leaf midrib in response to Colletotrichum. coccodes infection, ethylene and abscisic acid. The CAZFP1 gene was also induced much earlier by abiotic elicitors and environmental stresses, compared with the CAZFP1 gene. Overexpression of the CAZFP1 gene in the transgenic Arabidopsis plants enhanced not only the resistance against infection by Pseudomonas syringae pv. tomato, but also the drought tolerance. These results suggest that the CAZFP1 gene functions as an early-defense gene to enhance disease resistance and drought tolerance.

Amino Acid Sequence↗

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.

Amino Acid Sequence↗

Xanthophyll biosynthesis: molecular and functional characterization of carotenoid hydroxylases from pepper fruits (Capsicum annuum L.).

To dissect the mechanism by which carotenoid hydroxylases catalyze xanthophyll formation, we have cloned two pepper cDNAs encoding beta-cryptoxanthin and zeaxanthin biosynthetic enzymes. Using an in vitro system, we find that both enzymes are ferredoxin dependent and that their activity is strongly inhibited by iron chelators such as o-phenanthroline or 8-hydroxyquinoline. This suggests the transfer of a reducing equivalent from NADPH to the hydroxylase via ferredoxin and the involvement of an iron activated oxygen insertion process. Based on sequence analysis, the putative histidine clusters involved in the iron coordination were identified and their roles evaluated. Following site-directed mutagenesis of the identified histidine residues hydroxylase activity was totally inactivated. Collectively, our data indicate that carotenoid hydroxylases belong to a new class of diiron proteins structurally related to membrane fatty acid desaturases. Mechanistically, both types of enzymes exploit iron activated oxygen to break the C-H bond with concomitant formation of double bond or oxygen insertion. We propose that the same mechanism operates for beta-carotene ketolase and probably for other carotenoid oxygenases as well.

Amino Acid Sequence↗

Identification of a novel pathogen-induced gene encoding a leucine-rich repeat protein expressed in phloem cells of Capsicum annuum.

The CALRR1 gene, expressed in pepper leaves following infection by Xanthomonas campestris pv. vesicatoria, encodes a secreted leucine-rich repeat (LRR) with five tandem repeats of a 24-amino-acid LRR motif. Northern blot analyses revealed that CALRR1 is not constitutively expressed in pepper plants, but is strongly induced upon the infection by X. campestris pv. vesicatoria, Phytophthora capsici, Colletotrichum coccodes and Colletotrichum gloeosporioides on leaves. CALRR1 was not systemically induced in upper leaves by bacterial infection. The inoculation of bacterial live cells, and treatment with dead cells and culture filtrates of pathogenic or nonpathogenic bacteria triggered the accumulation of CALRR1 transcripts. Treatment with signaling molecules, including salicylic acid (SA), ethylene (ET), methyl jasmonate (MeJA), dl-beta-amino-n-butyric acid (BABA) and benzothiadiazole (BTH), did not activate the transcription of the CALRR1 gene, indicating that CALRR1 expression is not regulated by defense signaling pathways activated by these molecules. CALRR1 was induced by treatment with high salinity, abscisic acid (ABA) and wounding, but not by drought and cold stress. An in situ hybridization study showed that CALRR1 mRNA was localized in phloem tissues of leaves, stems, and green fruits of pepper plants during the pathogen infection and ABA exposure. The location characteristics and the spatio-temporal expression pattern of CALRR1 suggest that it may play a role in protecting phloem cells against biotic and abiotic stresses affecting phloem function.

Amino Acid Sequence↗

Identification of a short interspersed repetitive element in partially spliced transcripts of the bell pepper (Capsicum annuum) PAP gene: new evolutionary and regulatory aspects on plant tRNA-related SINEs.

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.

Animals↗

In vitro and in situ inhibition of carotenoid biosynthesis in Capsicum annuum by bleaching herbicides.

Pepper leaves treated with the herbicide J852 show an accumulation of phytoene and zeta-carotene, whereas treatment with norflurazon led to an accumulation of only phytoene. The effects of these herbicides were examined in vitro after the expression of carotenoid desaturases in Escherichia coli. Whereas norflurazon is a potent inhibitor of phytoene desaturase (PDS) (I(50) = 0.12 microM) but not of zeta-carotene desaturase (ZDS) (I(50) = 144 microM), J852 inhibits both PDS (I(50) = 23 microM) and ZDS (I(50) = 49 microM). The influence of PDS/ZDS inhibition on gene expression was examined by comparative RT-PCR. None of the examined genes, namely, encoding phytoene synthase, PDS, ZDS, or the terminal oxidase associated with phytoene desaturation, were induced upon herbicide treatment in pepper leaves or seedlings. This was unexpected because inhibition of carotene desaturation led to an up-regulation of the carotenoid biosynthetic capacity (higher amounts of accumulating precursors plus remaining colored carotenoids are present in treated tissues versus control).

Capsicum↗

Analysis of molecular species of glycolipids in fruit pastes of red bell pepper (Capsicum annuum L.) by high-performance liquid chromatography-mass spectrometry.

Five major glycolipid classes (acylated steryl glucoside, steryl glucoside, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and glucocerebroside) from fruit pastes of red bell pepper were separated by silica gel column chromatography. The molecular species of each glycolipid were separated and characterized by reversed-phase high-performance liquid chromatography coupled with on-line mass spectrometry using atmospheric pressure chemical ionization. The molecular species of steryl glucoside were beta-sitosteryl and campesteryl glucosides, and those of the acylated steryl glucoside were their fatty acid esters. The dilinolenoyl species was predominant in monogalactosyldiacylglycerol in addition to small amounts of another five molecular species, whereas digalactosyldiacylglycerol consisted of seven molecular species varying in their degree of unsaturation. The glucocerebroside class contained at least seven molecular species, which were characterized by proton nuclear magnetic resonance spectroscopy.

Capsicum↗