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.

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.

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.

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.

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.

Chromoplast Biogenesis in Cucumis sativus Corollas (Rapid Effect of Gibberellin A3 on the Accumulation of a Chromoplast-Specific Carotenoid-Associated Protein).

The development of cucumber (Cucumis sativus L.) corollas is accompanied by the accumulation of chromoplasts. In mature corollas, chromoplasts, but no chloroplasts, were detected by electron microscopy. Chlorophyll was also undetectable in corollas at anthesis. The contents of carotenoids and a carotenoid-associated, chromoplast-specific, 35-kD protein in corollas increased in parallel with flower development, peaking concomitantly at anthesis. The involvement of phytohormones and light in the regulation of their expression was studied. When gibberellin A3 (GA3) was added to an in vitro bud culture system, accumulation of both carotenoids and the 35-kD protein was markedly enhanced. The specific up-regulation of the 35-kD protein was very rapid: after only 2 h of culture, increased levels were detected in GA3-treated versus untreated corollas. During this period, corolla fresh weight and total protein and carotenoid contents remained unchanged. Inclusion of abscisic acid in the culture medium counteracted the effect of GA3. Accumulation of the 35-kD protein was also enhanced when flower buds on plants were sprayed with GA3 or etiolated.