Despite recent progress in our understanding of carotenogenesis in plants, the mechanisms that govern overall carotenoid accumulation remain largely unknown. The Orange (Or) gene mutation in cauliflower (Brassica oleracea var botrytis) confers the accumulation of high levels of β-carotene in various tissues normally devoid of carotenoids. Using positional cloning, we isolated the gene representing Or and verified it by functional complementation in wild-type cauliflower. Or encodes a plastid-associated protein containing a DnaJ Cys-rich domain. The Or gene mutation is due to the insertion of a long terminal repeat retrotransposon in the Or allele. Or appears to be plant specific and is highly conserved among divergent plant species. Analyses of the gene, the gene product, and the cytological effects of the Or transgene suggest that the functional role of Or is associated with a cellular process that triggers the differentiation of proplastids or other noncolored plastids into chromoplasts for carotenoid accumulation. Moreover, we demonstrate that Or can be used as a novel genetic tool to induce carotenoid accumulation in a major staple food crop. We show here that controlling the formation of chromoplasts is an important mechanism by which carotenoid accumulation is regulated in plants.

SummaryThe Or gene of cauli¯ower (Brassica oleracea var. botrytis) causes many tissues of the plant to accumulate carotenoids and turn orange, which is suggestive of a perturbation of the normal regulation of carotenogenesis. A series of experiments to explore the cellular basis of the carotenoid accumulation induced by the Or gene was completed. The Or gene causes obvious carotenoid accumulation in weakly or unpigmented tissues such as the curd, pith, leaf bases and shoot meristems, and cryptically in some cells of other organs, including the roots and developing fruits. The dominant carotenoid accumulated is b-carotene, which can reach levels that are several hundred-fold higher than those in comparable wildtype tissues. The b-carotene accumulates in plastids mainly as a component of massive, highly ordered sheets. The Or gene does not affect carotenoid composition of leaves, nor does it alter color and chromoplast appearance in¯ower petals. Interestingly, mRNA from carotenogenic and other isoprenoid biosynthetic genes upstream of the carotenoid pathway was detected both in orange tissues of the mutant, and in comparable unpigmented wild-type tissues. Thus the unpigmented wild-type tissues are likely to be competent to synthesize carotenoids, but this process is suppressed by an unidenti®ed mechanism. Our results suggest that the Or gene may induce carotenoid accumulation by initiating the synthesis of a carotenoid deposition sink in the form of the large carotenoid-sequestering sheets.

Transgenic plants have facilitated our understanding of the functional roles of genes and the metabolic processes affected in plants. Recently, the Or gene was isolated from an orange cauliflower mutant and it was shown that the Or gene could serve as a novel genetic tool to enrich carotenoid content in transgenic potato tubers. An in-depth characterization of these Or transgenic lines is presented here. It was found that the Or transgene may facilitate the identification of potential rate-limiting step(s) of the carotenoid biosynthetic pathway. The Or transgenic tubers accumulated not only increased levels of carotenoids that normally are present in the controls, but also three additional metabolite intermediates of phytoene, phytofluene, and zeta-carotene, indicating that the desaturation steps became limiting following the expression of the Or transgene. Moreover, we observed that long-term cold storage greatly enhanced carotenoid content in the Or transgenic tubers to a level of 10-fold over controls. Expression of the Or transgene in the transgenic plants caused no dramatic changes in the transcript levels of the endogenous carotenoid biosynthetic genes, which is in agreement with the Or gene not directly controlling carotenoid biosynthesis. Microscope analysis revealed that the Or transgene conferred the formation of chromoplasts containing carotenoid sequestering structures in a heterologous system. Such structures were not observed in tubers of potato cultivars that accumulate high levels of carotenoids. Collectively, these results provide direct evidence demonstrating that the Or gene indeed controls chromoplast differentiation and that regulation of chromoplast formation can have a profound effect on carotenoid accumulation in plants.

SUMMARYFull appreciation of the roles of the plant cuticle in numerous aspects of physiology and development requires a comprehensive understanding of its biosynthesis and deposition; however, much is still not known about cuticle structure, trafficking and assembly. To date, assessment of cuticle organization has been dominated by 2D imaging, using histochemical stains in conjunction with light and fluorescence microscopy. This strategy, while providing valuable information, has limitations because it attempts to describe a complex 3D structure in 2D. An imaging technique that could accurately resolve 3D architecture would provide valuable additions to the growing body of information on cuticle molecular biology and biochemistry. We present a novel application of 3D confocal scanning laser microscopy for visualizing the architecture, deposition patterns and micro-structure of plant cuticles, using the fluorescent stain auramine O. We demonstrate the utility of this technique by contrasting the fruit cuticle of wild-type tomato (Solanum lycopersicum cv. M82) with those of cutin-deficient mutants. We also introduce 3D cuticle modeling based on reconstruction of serial optical sections, and describe its use in identification of several previously unreported features of the tomato fruit cuticle.

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The Or mutation in cauliflower (Brassica oleracea L. var. botrytis) leads to abnormal accumulations of beta-carotene in orange chromoplasts, in tissues in which leucoplasts are characteristic of wild-type plants. Or chromoplasts were investigated by light microscopy of fresh materials and electron microscopy of glutaraldehyde- and potassium permanganate-fixed materials. Carotenoid inclusions in Or chromoplasts resemble those found in carrot root chromoplasts in their optical activity and angular shape. Electron microscopy revealed that the inclusions are made up of parallel, membrane-bound compartments. These stacks of membranes are variously rolled and folded into three-dimensional objects. We classify Or chromoplasts as "membranous" chromoplasts. The Or mutation also limits plastid replication so that a single chromoplast constitutes the plastidome in most of the affected cells. There are one to two chromoplasts in each cell of a shoot apex. The ability of differentiated chromoplasts to divide in the apical meristems of Or mutant plants resembles the ability of proplastids to maintain plastid continuity from cell to cell in meristems of Arabidopsis thaliana mutants in which plastid replication is drastically limited. The findings are used to discuss the number of levels of regulation involved in plastid replication.

We report the biomechanics and anatomy of fruit wall peels (before and after cellulase/pectinase treatment) from two Lycopersicon esculentum cultivars (i.e., Inbred 10 and Sweet 100 cherry tomatoes). Samples were tested before and after enzyme treatment in uniaxial tension to determine their rate of creep, plastic and instantaneous elastic strains, breaking stress (strength), and work of fracture. The fruit peels of both cultivars exhibited pronounced viscoelastic and strain-hardening behavior, but differed significantly in their rheological behavior and magnitudes of material properties, e.g., Inbred 10 peels crept less rapidly and accumulated more plastic strains (but less rapidly), were stiffer and stronger, and had a larger work of fracture than Sweet 100 peels. The cuticular membrane (CM) also differed; e.g., Sweet 100 CM strain-softened at forces that caused Inbred 10 to strain-harden. The mechanical behavior of peels and their CM correlated with anatomical differences. The Inbred 10 CM develops in subepidermal cell layers, whereas the Sweet 100 CM is poorly developed below the epidermis. Based on these and other observations, we posit that strain-hardening involves the realignment of CM fibrillar elements and that this phenomenon is less pronounced for Sweet 100 because fewer cell walls contribute to its CM compared to Inbred 10.