Table

Some Examples of Organisms in Which Carotenogenesis Is Nearly Fully Understood

Bacteria

Fungi

Rodobacter capsulatus and sphaeroides Neurospora sp.

Synechococcus sp. Myxococcus xanthus Thermus thermophilus Erwinia sp.

Phycomyces

Source: Adapted from Delgado-Vargas et al. (2000).2

carotenoid biosynthesis has been complicated because carotenoids are essential components and mutants could be very susceptible to stress conditions (e.g., photodamage), and under normal development conditions these mutants die. These problems are not observed with flower, fruit, or seed carotenoids, and a good collection of mutants has been generated with tomato fruit and maize seed.38 The mRNA for the e-cyclase of the 5-mutant of tomato CrtL-E gene is downregulated and transcriptional control is a major mechanism for lycopene accumulation during tomato fruit ripening. It has been observed that the 5-mutation does not affect the carotenoid composition or the CrtL-E mRNA level in leaves and flowers, suggesting the presence of multiple alleles for the e-cyclase. Moreover, plant PSY and PDS in wild types increase at the breaker stage during ripening, whereas CrtL-B and CrtL-E disappear at this stage. Thus, differential gene expression plays a major role in the accumulation of lycopene in tomato fruits by elevating the concentration of its biosynthetic enzymes and blocking the synthesis of enzymes that convert it to cyclic carotenoids. In the 5-mutant, CrtL-E mRNA increases at the breaker stage and remains elevated until the fruit fully ripens. Consequently, most of the lycopene is converted to 5-carotene.25 The importance of the cyclases in the carotenogenesis regulation was corroborated in the mRNA expression analysis of marigold (Tagetes erecta) e-cyclase. This enzyme is one of the most strongly induced in marigold flowers suggesting that its level controls the degree to which the intermediate lycopene is diverted into the branch of carotenoids having one e and one P ring.39

3. Molecular Biology of Carotenogenesis

Biochemical approaches have not been very successful in the study of carotenoid biosynthesis; membranal enzymes, such as those of carotenogenesis, are difficult to isolate and to work with. A molecular biology approach has permitted advances and, today, the main steps of the carotenogenesis pathway are almost completely described.

Great developments in the knowledge of the carotenoid pathway were obtained thanks to the complete elucidation of the gene clusters for several bacteria and fungi (Table 7.5). These clusters have a complete set of genes required for the biosynthesis of carotenoids and they are in tandem. Moreover, clusters are not present in eukaryotic cells or in cyanobacteria. Interestingly, the information obtained from microorganisms has permitted the discovery of many genes of cyanobacteria and higher plants. All these facts have permitted to get many genes or cDNA clones of carotenogenesis genes (Table 7.6). It has been suggested that carotenogenesis genes of eukaryotic organisms are present in only one copy, but some could have more than one copy, such as PSY and GGPP synthase of pepper.240

A few years ago, MVA synthesis was considered an important stage in the carotenoid biosynthesis, and it was suggested that HMGR was strongly regulated as occurs in animals. Certainly, HMGR appears in plants and other microorganisms, but it is involved in the biosynthesis of other isoprenoids. In fact, multiple copies of HMGR have been identified in Arabidopsis, Hevea, and Solanum.41 However, today, it is clear that plant carotenoid biosynthesis is carried out by the DXP pathway (Figures 7.4 and 7.5).

Various strategies have been utilized to identify the genes involved in carotenoid biosynthesis: use of heterologous probes, antibodies against a cDNA expression library, and transposon tagging, among others (Table 7.7).1042-51 After gene or cDNA isolation, it has been possible to identify specific coding regions involved in the active site of enzymes, possible regulatory stages in the pathway, and some mechanisms involved in regulation and accumulation of carotenoids. Interestingly, a gene coded in the mitochondria genome was identified in A thaliana (Table 7.7), suggesting that compart-mentalization is an important regulatory mechanism in isoprenoid biosynthesis.

The PSY sequence has been determined and it has been corroborated that the functional enzyme is a membrane integral protein, whereas a cytoplasmic soluble form is inactive. Thus, it is suggested that PSY activity is regulated by post-transcriptional mechanisms. In addition, the membranal active enzyme seems to involve reodox processes.48

With the introduction of plant transformation, it has been confirmed that iso-prenoid products share some of the pathway precursors, as was concluded with PSY1 transformed tobacco plants (Table 7.7). Recently, the first bacterial phytoene syn-thase was expressed and characterized. This enzyme has similar characteristics to those observed in plant phytoene synthases: it produces 15-cis-phytoene and all-trans-phytoene, depends on ATP and on Mg+2 or Mn+2, and the Michaelis constant (Km) for GGPP as a substrate is 41 \yM in Capsicum and 3 \yM in bacteria.52

In relation to PDS, two different enzymatic activities have been observed: (1) PDS in anoxygenic photosynthetic microbes catalyzes the conversion of phytoene to lycopene or neurosporene; and (2) plant PDS catalyzes the transformation of phytoene to Z-carotene.53 In the evaluation of the enzymatic activity, a dinucleotide binding site has been found in both PDS types; Norris et al.54 used Arabidopsis mutants to show that three genes are involved in the desaturation process, establishing that PDS, a-tocopherol, and plastoquinone are required. It has been proposed that plastoquinone/a-tocopherol is involved in electron transportation, whereas ubiquinone has been proposed for the anoxygenic microorganisms. On the other hand, PDS of Neurospora crassa was expressed in Escherichia coli; the enzyme shows significant similarities to all bacterial PDS. Nevertheless, the N. crassa enzyme is able to introduce up to five double bonds into phytoene, yielding 3,4-didehydrolycopene, whereas other PDS introduce only three or four double bonds. This enzyme is dependent on NAD but not on FAD as the others.55

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