Floral fragrance. New inroads into an old commodity.
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Magnoliopsida↗
Biomedical subjects
A Vainstein
Publications and source records attributed to A Vainstein.
Carotenoid sequestration in plants: the role of carotenoid-associated proteins.
In plants, carotenoid accumulation and sequestration take place within chloroplasts and chromoplasts. In the chloroplast, practically all carotenoids are associated with chlorophyll-binding proteins, whereas chromoplasts have developed a unique mechanism to sequester carotenoids within specific lipoprotein structures. Recent research into the existence of a group of homologous genes that encode carotenoid-associated proteins that aid in the generation of carotenoid-lipoprotein structures in chromoplasts, offers a new framework for elucidating the carotenoid sequestration mechanism.
Journal Article↗
Molecular mechanisms underlying carotenogenesis in the chromoplast: multilevel regulation of carotenoid-associated genes
In nature, carotenoid function and mode of action are highly determined by the neighboring protein and lipid molecules. Therefore an understanding of the proteins' involvement in carotenoid sequestration would be of great help in elucidating carotenoid function in vivo. Based on a study of the expression of chromoplast-specific carotenoid-associated genes from cucumber corolla (CHRC and CHRD), a working model is presented wherein two major regulatory factors control carotenoid sequestration within the chromoplasts: (i) floral tissue-specific transcriptional regulators of chromoplasto- genesis; and (ii) post-transcriptional regulators related to the amount/type of sequestered carotenoids. This model is supported by the major role transcriptional regulation was found to play in the temporal and spatial expression of the CHRC gene, and by the fact that phytohormones such as gibberellic acid (GA3), abscisic acid and ethylene also acted as transcriptional regulators of CHRC expression. The primary response to GA3 was localized within the CHRC promoter to a 290 bp fragment. Furthermore, we demonstrated strong down-regulation of CHRC expression at post-transcriptional and translational/post-translational levels resulting from inhibition of carotenoid biosynthesis, thus revealing a close link between carotenoid biosynthetic and sequestration machineries.
Journal Article↗
Genetic engineering for cut-flower improvement.
The application of modern biotechnological approaches to cut flowers has clearly become instrumental and rewarding for the floriculture industry. In recent years, several gene-transfer procedures have been developed for some of the major commercial cut flowers. Using Agrobactrium or microprojectile bombardment, several basic protocols are now available. However, despite the great progress and interest in gene transfer to these crops, their transformation is routine in only a limited number of laboratories, and its application is still considered to be an "art form". This review summarizes the reported gene-transfer procedures for the main cut-flower crops, with an emphasis on the unique factors of each method and the recent progress in introducing new traits of horticultural interest into these species.
Journal Article↗
CHRC, encoding a chromoplast-specific carotenoid-associated protein, is an early gibberellic acid-responsive gene.
CHRC, a corolla-specific carotenoid-associated protein, is a major component of carotenoid-lipoprotein complexes in Cucumis sativus chromoplasts. Using an in vitro flower bud culture system that mimics in vivo flower development, CHRC mRNA levels in corollas were shown to be specifically up-regulated by gibberellic acid. The response to gibberellic acid was very rapid (within 20 min) and insensitive to protein synthesis inhibition by cycloheximide. Abscisic acid, known to antagonize gibberellin in many developmental systems, strongly down-regulated CHRC mRNA levels. The gibberellin synthesis inhibitor paclobutrazol exhibited a similar negative effect on CHRC expression. Inclusion of exogenous gibberellic acid into the in vitro bud culture system with the paclobutrazol not only prevented the CHRC mRNA down-regulation, it up-regulated transcript accumulation to the level of gibberellic acid-treated corollas. CHRC mRNA accumulation in response to gibberellic acid displayed a dose-dependent increase up to 10(-4) M gibberellic acid. The up-regulation could be detected with as little as 10(-7) M gibberellic acid. Based on these data, we suggest that CHRC is the first structural gene identified to date whose expression is regulated by gibberellic acid in a primary fashion. The critical role of the rapid response of CHRC to gibberellic acid in aiding carotenoid sequestration while preserving chromoplast structural organization is discussed.
Carrier Proteins↗
Isolation and regulation of accumulation of a minor chromoplast-specific protein from cucumber corollas.
The differentiation of chloroplasts to chromoplasts in cucumber (Cucumis sativus L.) corollas parallels flower development. Chromoplast biogenesis involves chlorophyll degradation, carotenoid accumulation, and the appearance of a new set of proteins. To study factors involved in chromoplast biogenesis in floral tissues, a minor (in abundance) protein of about 14 kD, CHRD (chromoplast protein D), was isolated from cucumber corolla chromoplasts. Immunological characterization revealed that the protein is chromoplast-specific and that its steady-state level in corollas increases in parallel to flower development. The protein was not detected in cucumber leaves or fruits. Immunological analysis of corollas and fruits from variety of other plants also did not reveal cross-reactivity with the CHRD protein antisera. Using an in vitro bud culture system, we analyzed the effect of phytohormones on CHRD expression. Gibberellic acid rapidly enhanced, whereas paclobutrazol down-regulated, the steady-state level of CHRD. Ethylene also down-regulated the protein's steady-state level. It is suggested that hormonal control of chromoplastogenesis is tightly regulated at the tissue/organ level and that mainly developmental signals control carotenoid accumulation in nonphotosynthetic tissues.
Blotting, Western↗
Molecular cloning of a carotenoid-associated protein from Cucumis sativus corollas: homologous genes involved in carotenoid sequestration in chromoplasts.
Chromoplasts are carotenoid-accumulating plastids found in the corollas and fruits of many higher plants. In most cases, the pigment in these plastids is accumulated with the aid of carotenoid-associated proteins located within unique structures. This paper reports the isolation and characterization of the cDNA (CHRC) from Cucumis sativus corollas which encodes the chromoplast-specific carotenoid-associated protein CHRC. The transit peptide cleavage site was determined and, using a chloroplast uptake system, it is shown that CHRC can be post-translationally targeted to these plastids where it is peripherally associated with thylakoids. Analysis of CHRC transcript level in Cucumis sativus revealed its temporal and tissue-specific regulation: the transcript was detected only in corollas, where its level increased in parallel to flower development, peaking just before anthesis. CHRC shares significant homology (59%) with the gene coding for fibrillin-a protein in Capsicum annuum red fruits whose function is essentially identical to that of CHRC. A CHRC fragment including the potential active site of the protein was used as a probe in Northern blot analyses of floral and fruit tissues from various plants containing chromoplasts of different types: CHRC homologs of similar sizes were revealed in all cases. The existence of a group of homologous genes coding for chromoplast-specific proteins which aid in the sequestration of carotenoids within specific structures is proposed.
Amino Acid Sequence↗
Expression of plant genes in transfected mammalian cells: accumulation of recombinant preLHCIIb proteins within cytoplasmic inclusion bodies.
Transfection of the monkey COS-7 cells with an expression vector bearing the Lemma gibba LHCIIb AB30 or AB19 genes led to the synthesis of the LHCIIb polypeptide precursors (preLHCIIb). This was inferred mainly from Western blot analysis which has revealed the appearance of a single immunoprecipitation band following the use of three different preparations of anti-LHCIIb antibodies. Synthesis of the precursor polypeptides, not the mature processed LHCIIb protein, was evident from the molecular weight of the newly synthesized protein, inferred from its position in the gel. Expression of the AB30 and AB19 genes was also evident from the appearance of specific transcripts only in transfected cells. Immunofluorescence observations revealed the appearance of distinct fluorescent spots in about 1-2% of the transfected cells. The same was observed following immunogold staining and electron microscopy studies, which revealed a specific association of gold particles with amorphous structures only in transfected cells. The preLHCIIb synthesized by transfected COS-7 cells was insoluble in a variety of detergents and could be solubilized only by 8 M urea or 0.1 N NaOH. These properties are characteristic of proteins accumulating within inclusion bodies of prokaryotes.
Animals↗
Correlation of some published amino acid sequences for photosystem I polypeptides to a 17 kDa LHCI pigment-protein and to subunits III and IV of the core complex.
Photosystem I (PSI) in barley consists of at least 11 polypeptides of which three have apparent sizes of 15-19 kDa. Two of these polypeptides (subunits III and IV) are constituents of the core complex (CCI), the third is a component of the light-harvesting complex (LHCI). After fractionation of PSI into its CCI and LHCI components, each of the polypeptides has been isolated and its N-terminal region sequenced. We conclude that the gene sequence published for subunit IV of spinach [(1988) FEBS Lett. 237, 108-112] is not that of subunit IV but rather that of the 17 kDA LHCIc pigment protein. We confirm that the published sequence for subunit III [(1988) Curr. Genet. 14, 511-518] is indeed that of subunit III; seemingly conflicting identifications, based on apparent sizes on SDS-PAGE, of which polypeptides are subunits III and IV are probably explained by subunit III's electrophoretic migration rate being dependent on the solvent.
Amino Acid Sequence↗
Light-harvesting pigment-proteins of photosystem I in maize. Subunit composition and biogenesis.
Three different pigment-binding proteins of the light-harvesting complex (LHC I) of maize photosystem I (PS I) have been isolated. Absorption and fluorescence excitation spectral analyses showed that each pigment-protein can transfer absorbed energy from its carotenoid and/or chlorophyll b components to chlorophyll alpha. Their apoproteins with apparent sizes of 24 (LHC Ia), 21 (LHC Ib), and 17 (LHC Ic) kDa have been purified to homogeneity. Differences in their pigment and amino acid compositions and in their reactions with antibodies demonstrate that the two smaller pigment-proteins are not proteolytically derived from the largest one. LHC Ib's apoprotein is particularly enriched in cysteine residues. None of the three apoproteins cross-reacted with antibodies raised against the major light-harvesting chlorophyll a/b-protein of photosystem II (LHC IIb) or against the PS I core complex (CC I) subunits. Studies of the biogenesis of PS I during greening of etiolated plants showed that all of the CC I subunits accumulated to a detectable level prior to the appearance of the 17-kDa subunit of LHC I, the accumulation of which preceded those of the 24- and 21-kDa subunits of LHC I. In addition, subunit VI of CC I is shown to be differentially expressed in mesophyll and bundle sheath cells; a slightly larger form of it accumulates in mesophyll than in bundle sheath thylakoids during plastid development.
Apoproteins↗
Expression of the Major Light-Harvesting Chlorophyll a/b-Protein and Its Import into Thylakoids of Mesophyll and Bundle Sheath Chloroplasts of Maize.
Distribution of the major light-harvesting chlorophyll a/b-protein (LHCII) and its mRNA within bundle sheath and mesophyll cells of maize (Zea mays L.) was studied using in situ immunolocalization and hybridization, respectively. In situ hybridization with specific LHCII RNA probes from maize and Lemna gibba definitively shows the presence of high levels of mRNA for LHCII in both bundle sheath cells and mesophyll cells. In situ immuno-localization studies, using an LHCII monoclonal antibody, demonstrate the presence of LHCII polypeptides in chloroplasts of both cell types. The polypeptide composition of LHCII and the amount of LHCII in bundle sheath cells are different from those in mesophyll cells. Both mesophyll and bundle sheath chloroplasts can take up, import and process the in vitro transcribed and translated LHCII precursor protein from L. gibba. Although bundle sheath chloroplasts incorporate LHCII into the pigmented light-harvesting complex, the efficiency is lower than that in mesophyll chloroplasts.
Journal Article↗
A new method for reconstitution of highly fusogenic Sendai virus envelopes.
A new way for reconstituting highly fusogenic Sendai virus envelopes is described. As opposed to previously described methods, in the present one the detergent (Triton X-100) is removed by direct addition of SM-2 Bio-beads to the detergent solubilized mixture of the viral phospholipids and glycoproteins, thus avoiding the long dialysis step. The vesicles obtained in the present work resemble, in their composition, size and features, envelopes of intact Sendai virus particles. The present method allows the enclosure of low and high molecular weight material within the reconstituted viral envelopes.
DNA, Viral↗
Fusion-mediated injection of SV40-DNA. Introduction of SV40-DNA into tissue culture cells by the use of DNA-loaded reconstituted Sendai virus envelopes.
Sendai virus envelopes can be solubilized by non-ionic detergents such as Triton X-100. Removal of the detergent from a supernatant containing the solubilized viral envelope glycoproteins results in the formation of reconstituted fusogenic viral envelopes. When SV40-DNA is added to the reconstitution system, it is trapped within the viral envelope. Incubation of SV40-DNA-loaded Sendai virus envelopes with permissive cells (CV1 and TC7 cells) resulted in fusion-mediated injection of the trapped DNA, as was demonstrated by the ability of the injected cells to synthesize SV40-T-antigen. Quantitative estimation revealed that up to 20% of the injected cells were able to synthesize T-antigen. Loaded viral envelopes were able to inject SV40-DNA and to promote synthesis of T-antigen also in cells which are resistant to infection by intact SV40 viruses, such as F1' 1-4 cells. In addition, it is shown that reconstituted envelopes of Sendai virus are able to transfer membrane fragments from SV40 receptor-positive into SV40 receptor-negative cells, such as F1' 1-4 cells. After implantation of SV40 receptors, the F1' 1-4 cells became susceptible to infection by intact SV40 viruses.
Animals↗
Fusogenic reconstituted Sendai virus envelopes as a vehicle for introducing DNA into viable mammalian cells.
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Animals↗
The Evergreen gene is essential for flower initiation in carnation.
One of the leading cut-flower crops in the world, the greenhouse carnation (Dianthus caryophyllus), has been subjected to intense breeding efforts for the past few hundred years. As an ornamental crop, flowering and flower architecture are major breeding targets that are constantly in demand. In an ongoing breeding program aimed at improving these characteristics, two mutants heterozygous for a mutation in a gene termed evergreen (e) were identified. In these mutants, spike-like clusters of bracteoles subtend each flower. Genetic analysis of the mutants confirmed the semidominant nature of this nuclear mutation and that the two original mutants were allelic at the evergreen locus. In homozygous mutant plants, a more severe phenotype was observed. Flower formation was completely blocked and spikelike clusters of bracteoles did not subtend any flowers. Morphological characterization of mutant plants revealed that vegetative growth and inflorescence structure are not affected by the mutant allele. In plants heterozygous for the evergreen mutation, fertility, petal and pistil length, calyx diameter, and stamen number were not affected. However, flowers from these heterozygous plants had a reduced number of petals, suggesting an intriguing link between evergreen and the double flower (d) gene that determines petal number in carnation. The control by evergreen of bracteole formation, floral meristem initiation, and petal number in carnation is discussed in comparison to the recessive leafy (lfy) and floricaula (flo) mutants of Arabidopsis and Antirrhinum, respectively.
Genes, Plant↗