Phytoene accumulation

A second class of herbicides primarily affects ( -carotene desaturase. These herbicides are apparent feedback inhibitors of PD as well. This class of compounds includes dihydropyrones like LS 80707 [90936-96-2] (56) and 6-methylpyridines (57,58). The third class consists of the ben2oylcyclohexane-diones, eg, 2-(4-chloro-2-nitroben2oyl)-5,5-dimethyl-cyclohexane-I,3-dione. This class of atypical bleaching herbicides induces phytoene accumulation when appHed either pre- or post-emergence. However, it does not inhibit phytoene desaturase activity in vitro (59). Amitrole also has been considered a bleaching herbicide, though its main mode of action is inhibition of amino acid synthesis. 

The carotenoid pathway may also be regulated by feedback inhibition from the end products. Inhibition of lycopene cyclisation in leaves of tomato causes increase in the expression of Pds and Psy-1 (Giuliano et al, 1993 Corona et al, 1996). This hypothesis is supported by other studies using carotenoid biosynthesis inhibitors where treated photosynthetic tissues accumulated higher concentrations of carotenoids than untreated tissues (reviewed by Bramley, 1993). The mechanism of this regulation is unknown. A contrary view, however, comes from studies on the phytoene-accumulating immutans mutant of Arabidopsis, where there is no feedback inhibition of phytoene desaturase gene expression (Wetzel and Rodermel, 1998). 

Rice Psy cDNA from daffodil CaMV 35S Phytoene accumulation in endosperm Burkhardt et al., 1997... 

Fig. 2. Effect of homogentisate (HGA) on phytoene accumulation in the third leaves of early watergrass 6 days after treatment. Error bars are SE of the means. Re Mx>duced with the permission from ref. 12. Copyright Blackwell Publishing Ltd.

Isomerisation of 15-c/5-phytoene to the all-/ra x configuration must occur during the desaturation steps, since most desaturated carotenes are in the all-trans form. The CRTI type desaturases appear to be able to carry out this isomerisation themselves (Fraser et al, 1992 Bartley etal, 1999), but mutants of PDS/ZDS-type organisms accumulate cis isomers of unsaturated carotenes, suggesting the presence of a separate isomerase (Clough and Pattenden, 1983 Ernst and Sandmann, 1988). Three recent publications have reported the cloning of a carotene isomerase (CrtlSO) from tomato (Isaacson et al, 2002), Arabidopsis (Park et al, 2002) and Synechocystis 6803 (Breitenbach... 

POTRYKUS I (1997) Transgenic rice Oryza sativa) endosperm expressing daffodil Narcissuspseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis , Plant J, 11, 1071-78. 

Over-expression of bacterial phytoene synthase led to only modest increases in pigment accumulation (except in the case of chloroplast-contaiifing tissues). Attention turned to CrtI, one gene that might control flux through the entire four desaturation steps from phytoene to lycopene (discussed in Section 5.3.2.4). Only a modest increase in carotenoid content in tomatoes and a variety of changes in carotenoid composition including more P-carotene, accompanied by an overall decrease in total carotenoid content (no lycopene increase), resulted when CrtI was over-expressed under control of CaMV 35S. Apparently, the bacterial desaturase... 

Plant phytoene synthase (Psy) has been used in a variety of transgenics. As noted above, P yl over-expression under a strong constitutive promoter caused a decrease in carotenoid accumulation, probably due to transcription silencing. Similarly, over-expression of the gene sequence backward (antisense) also silenced activity. In another approach to over-expression of tomato 1 in fruits, a synthetic alternative in which the third position of each codon was changed in order to avoid transcriptional silencing was successful in conditioning an increase in carotenoid accumulation. 

In rice endosperm that accumulated phytoene by virtue of daffodil Psy transgenesis, Burkhart et al. reported the failure of daffodil Pds over-expression in rice endosperm to condition accumulation of -carotene, even though an increase in PDS antigen was detected. ... 

The stability of phytoene desaturase and lycopene cyclase transcripts also influenced accumulation of carotenoids. Efforts in directed evolution of carotenogenic enzymes have also continued. Alternate approaches using systematic and combinatorial gene knockout targets have allowed for enhancement of carotenoid production in the absence of a priori assumptions of regulatory mechanisms. 

Linden, H. et al.. Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenoids, Z. Naturforsch. 46c, 1045, 1991. 

Wong, l.C. et al., QTL and candidate genes phytoene synthase and zetacarotene desaturase associated with the accumulation of carotenoids in maize, Theor. Appl. Genetics 108, 349, 2004. 

Kinetic studies of the incorporation of the " C-labelled precursors mevalonic acid, isopentenyl pyrophosphate, and phytoene into C40 carotenes by Halobac-terium cutirubrum cell-free preparations" produced results consistent with the pathways outlined in Schemes 2 and 3. Only the trans-isomers seemed to be involved. A mutant strain, " PGl, of the green alga Scenedesmus obliquus accumulates phytoene (135), phytofluene (136), and -carotene (137) in place of the... 

The amount of carotenoids formed by biosynthesis was quantified by the accumulation of the colourless carotenoid phytoene in the presence of the inhibitor, norflurazon. When applied, substantial amounts of this... 

In the photosynthetic bacteria Rhodomicrobium vannielii, which normally contains acyclic carotenoids with tertiary hydroxy- and methoxy-groups at C-1 and C-T, phytoene only accumulated when diphenylamine was present, but the occurrence of hydroxy-derivatives of phytofluene, 7,8,11,12-tetrahydrolycopene, neurosporene, and lycopene in the presence of the inhibitor indicated that hydroxylation could take place at any level of desaturation although only the more desaturated half of the molecule was so substituted. 

---