ENHANCING PLANT CAROTENOIDS VIA MANIPULATION OF SINK STRENGTH
Xiangjun Zhou & Li Libr
February, 2008

Carotenoids represent a large group of naturally occurring pigments found in many flowers, fruits, and roots. They provide essential phytonutrients, such as provitamin A, and confer many health benefits, such as reducing the incidence of certain diseases, including cancer, cardiovascular diseases, and age-related eye diseases. Therefore developing carotenoid enriched food may offer a sustainable avenue for improving human nutrition and health.

Engineering carotenoid biosynthesis
In past decades significant progress has been made in the dissection and genetic engineering of carotenoid biosynthesis in plants. Through a combination of molecular, genetic, biochemical, and genomic approaches, a nearly complete set of the genes and enzymes involved in carotenoid biosynthesis has been isolated and characterized¹. The availability of a large number of carotenogenic genes from various sources provides the necessary molecular tools for enhancing the carotenoid content and composition in food plants to benefit humans. One of the best known successful examples of improving crop nutritional value is Golden Rice. In "Golden Rice 2," ectopic expression of genes in a mini-carotenoid biosynthetic pathway results in the accumulation of up to 31 μg/g β-carotene in rice endosperm, a level adequate to provide most of the recommended dietary allowance of vitamin A for children consuming an average amount of rice daily². Manipulation of the biosynthetic genes that control the committed steps of carotenoid biosynthesis offers an effective approach to quantitative and qualitative alteration of carotenoids in food crops.

The orange cauliflower and the Or gene
Unlike Golden Rice, which was generated using a genetic engineering approach, the eye-appealing orange cauliflower (Fig. 1) arose from the spontaneous mutation of a single gene, designated as Or for Orange gene. The orange cauliflower was first discovered in a white cauliflower field in the early 1970s in Canada. Besides orange curd, the orange cauliflower mutant also exhibits visible orange coloration in the apical shoot and the stem pith. The orange color is due to the accumulation of high levels of primarily β-carotene.

Interestingly, mRNA from the expression of carotenoid and upstream isoprenoid biosynthetic genes is detected not only in the curd tissue of the mutant that accumulates β-carotene but also in the comparable unpigmented wild type tissue. The expression of carotenoid biosynthetic genes is not dramatically altered by the mutation; neither is the expression of upstream isoprenoid biosynthetic genes. Microscopic analysis showed that β-carotene accumulation in the Or mutant occurs in chromoplasts, predominantly as sheet structures. Furthermore, only one or two large chromoplasts are found in each affected cell in the apical meristem and curd meristem tissues of the Or mutant, suggesting limited plastid division in the Or mutation³. These results collectively imply that rather than regulating the capacity of biosynthesis, the Or gene may exert its effect on carotenoid accumulation through a novel mechanism.

The Or gene was isolated via a map-based cloning strategy and successfully verified by phenotypic complementation⁴. Sequence analysis revealed that the Or gene mutation is due to the insertion of a 4.7 kb copia-like LTR retrotransposon in the mutant allele, which results in a likely gain-of-function mutation that positively controls carotenoid accumulation. Three alternatively spliced Or mutant transcripts were produced following excision of the retrotransponson⁴. All of the spliced transcripts can read through, and they share the same start and stop codons as the wild type gene. Interestingly, overexpression of any one of the spliced transcripts does not cause the orange phenotype with associated carotenoid accumulation in transgenic plants, which suggests that Or-induced carotenoid accumulation may require the expression of more than one alternatively spliced transcript.

The most prominent feature of the OR protein is the presence of a cysteine-rich zinc finger domain in its C terminal. This domain is highly specific to DnaJ-like molecular chaperones, which participate in protein folding, assembly and disassembly, and translocation into organelles. Because OR lacks the typical N-terminal J domain that defines DnaJ-like molecular chaperones, OR is more likely a novel protein with a unique cellular function, and may exert its functional role in association with the molecular chaperone system. The OR protein is predicted to be targeted to the plastid. Indeed, the Orwt:GPF fusion protein was associated with non-colored plastids⁴.

The Or gene shares no sequence homology with the carotenoid biosynthetic genes and appears to exert no direct effect on the capacity of carotenoid biosynthesis. By contrast, analyses of the gene, the gene product, and the cytological effects of Or indicate that Or-induced carotenoid accumulation is associated with a metabolic process that triggers the differentiation of non-colored plastids into chromoplasts. Indeed, introduction of the Or transgene into white cauliflower results in the formation in the curd cells of large membranous chromoplasts containing increased levels of fusion protein β-carotene.

Or as a new molecular tool to enhance carotenoid accumulation
To examine the application of Or as a new genetic tool to enhance carotenoid content in a staple crop, the Or gene was transformed into potato plants under the control of a tuber-specific promoter. Remarkably, the Or transgenic potato plants produce tubers with a deep orange-yellow flesh. HPLC analysis showed that these transgenic tubers exhibit more than a 6-fold increase in total carotenoid content. The transgenic tubers contain not only increased levels of the violaxanthin and lutein that are normally present in nontransformed controls, but also accumulate significant levels of β-carotene. In addition, three other metabolic intermediates, phytoene, phytofluene, and ζ-carotene, which were not detected in the controls, also accumulated. The accumulation of these metabolic intermediates suggests a hindrance in desaturation in the carotenoid biosynthetic pathway. Such a hindrance may restrain the extent of Or-induced carotenoid accumulation in the transgenic potato tubers.

An examination of the cytological effects of the Or transgene by light microscopy revealed that expression of the Or transgene in the heterologous system leads to formation of chromoplasts with orange structures in transgenic potato tubers⁵. However, analysis of dark yellow-flesh potato cultivars with high levels of carotenoids revealed that they do not contain chromoplasts, demonstrating that high levels of carotenoid accumulation do not necessarily result in the formation of chromoplasts. Moreover, the Or transgene caused no dramatic changes in the transcript levels of endogenous carotenoid biosynthetic genes. Collectively, these results demonstrate that the induction of chromoplast formation is the cause of the Or-associated carotenoid accumulation.

Chromoplasts found in many flowers, fruits, and roots are characterized by high levels of carotenoids. Chromoplasts frequently derive from fully developed chloroplasts, as seen during the ripening process in tomato and pepper fruits. They also arise from other non-colored plastids, as in the case of carrot, squash, and the orange cauliflower. Chromoplasts have a unique mechanism for accumulating massive amounts of carotenoids: they generate carotenoid-lipoprotein structures, which create a chemical disequilibrium to effectively sequester the end products of biosynthesis. Such structures function as a deposition sink to store carotenoids, and also may prevent the end products of the carotenoid pathway from overloading chromoplast membranes, which are the site of carotenoid formation. A number of studies have demonstrated that biosynthesis of an appropriate sink structure correlates directly with increased carotenoid accumulation. Thus, chromoplasts serve as an effective metabolic sink to facilitate the sequestration and storage of carotenoids.

Alternative strategy for engineering carotenoids in low-pigmented crops
Carotenoid accumulation is not dependent solely upon the catalytic activities of carotenogenic enzymes, but also involves a network of other processes such as metabolite turnover and the stable storage of end products. The study of the Or mutant gene provides strong evidence showing that an increase in sink strength exerts a strong influence on carotenoid accumulation, and thus sink strength offers a novel approach for genetically engineering the carotenoid content in food crops.

Many important crops such as wheat, rice, barley, maize, potato, and cassava contain low levels of carotenoids in edible seeds or roots. These vitamin A-poor foods contribute to the prevalence of vitamin A deficiency in many parts of the world, because the population is dependent upon them as primary food sources. Despite low levels of carotenoid accumulation in these storage tissues, carotenoid biosynthetic enzymes are active. A number of possible reasons, such as low metabolic flux into the pathway, limited catalytic activity of particular enzymes in the pathway, or high turnover rate, could contribute to low levels of carotenoid accumulation. Moreover, lack of a suitable metabolic sink for effectively sequestering carotenoid end products could also be causative in some cases. For example, although white cauliflower curd accumulates negligible amounts of carotenoids, the genes involved in carotenoid biosynthesis are expressed at levels comparable to the orange cauliflower. The absence of a suitable sink structure restrains the accumulation of carotenoids. The Or gene mutation confers the formation of a storage sink structure, resulting in a dramatically increased accumulation of β-carotene, without alteration of the expression of carotenoid biosynthetic genes. Thus, it is likely that the introduction of an effective metabolic sink for carotenoid sequestration and deposition will facilitate the genetic engineering of carotenoid content in low-pigmented tissues of staple crops.

It should be noted that the extent of carotenoid enrichment in food crops via enhanced sink strength may be limited by a number of factors—most importantly, the maximal catalytic activity of the carotenoid biosynthetic pathway in particular crop tissues or organs. Thus, a concomitant increase in the sink capacity to effectively sequester and deposit carotenoids, along with the catalytic activity of this pathway to increase metabolic flux, would be a promising strategy to engineer increased carotenoid content in food crops to levels required for optimal human nutrition and health.

Xiangjun Zhou & Li Li
U.S. Department of Agriculture-Agricultural Research Service
Plant, Soil and Nutrition Laboratory
Department of Plant Breeding and Genetics, Cornell University
Ithaca, New York
ll37@cornell.edu