Phytoene Synthase Enzyme Research Articles

Co-chaperoning of chlorophyll and carotenoid biosynthesis by ORANGE family proteins in plants

Chlorophylls and carotenoids are essential photosynthetic pigments. Plants spatiotemporally coordinate the needs of chlorophylls and carotenoids for optimal photosynthesis and fitness in response to diverse environmental and developmental cues. However, how the biosynthesis pathways of these two pigments are coordinated, particularly at posttranslational level to allow rapid control, remains largely unknown. Here, we report that the highly conserved ORANGE (OR) family proteins coordinate both pathways via posttranslationally mediating the first committed enzyme in each pathway. We demonstrate that OR family proteins physically interact with magnesium chelatase subunit I (CHLI) in chlorophyll biosynthesis pathway in addition to phytoene synthase (PSY) in carotenoid biosynthesis pathway and concurrently stabilize CHLI and PSY enzymes. We show that loss of OR genes hinders both chlorophyll and carotenoid biosynthesis, limits light-harvesting complex assembly, and impairs thylakoid grana stacking in chloroplasts. Overexpression of OR safeguards photosynthetic pigment biosynthesis and enhances thermotolerance in both Arabidopsis and tomato plants. Our findings establish a novel mechanism by which plants coordinate chlorophyll and carotenoid biosynthesis and provide a potential genetic target to generate climate-resilient crops.

Differential interaction of Or proteins with the PSY enzymes in saffron

Colored apocarotenoids accumulate at high concentrations in few plant species, where display a role in attraction of pollinators and seed dispersers. Among these apocarotenoids, crocins accumulate at high concentrations in the stigma of saffron and are responsible for the organoleptic and medicinal properties of this spice. Phytoene synthase and Orange protein are key for carotenoid biosynthesis and accumulation. We previously isolated four phytoene synthase genes from saffron with differential roles in carotenoid and apocarotenoid biosynthesis. However, the implications of Orange genes in the regulation of apocarotenoid accumulation are unknown. Here, we have identified two Orange genes from saffron, with different expression patterns. CsOr-a was mainly expressed in vegetative tissues and was induced by light and repressed by heat stress. Both CsOr-a and CsOr-b were expressed in stigmas but showed a different profile during the development of this tissue. The interactions of CsOr-a and CsOr-b were tested with all the four phytoene synthase proteins from saffron and with CsCCD2. None interactions were detected with CCD2 neither with the phytoene synthase 2, involved in apocarotenoid biosynthesis in saffron. The obtained results provide evidence of different mechanisms regulating the phytoene synthase enzymes in saffron by Orange for carotenoid and apocarotenoid accumulation in saffron.

Energy conversion processes and related gene expression in a sunflower mutant with altered salicylic acid metabolism

Salicylic acid (SA) is involved in several responses associated with plant development and defence against biotic and abiotic stress, but its role on photosynthetic regulation is still under debate. This work investigated energy conversion processes and related gene expression in the brachytic mutant of sunflower lingering hope (linho). This mutant was characterized by a higher ratio between the free SA form and its conjugate form SA O-β-D-glucoside (SAG) compared to wild type (WT), without significant changes in the endogenous level of abscisic acid and hydrogen peroxide. The mutant showed an inhibition of photosynthesis due to a combination of both stomatal and non-stomatal limitations, although the latter seemed to play a major role. The reduced carboxylation efficiency was associated with a down-regulation of the gene expression for both the large and small subunits of Rubisco and the Rubisco activase enzyme. Moreover, linho showed an alteration of photosystem II (PSII) functionality, with reduced PSII photochemistry, increased PSII excitation pressure and decreased thermal energy dissipation of excessive light energy. These responses were associated with a lower photosynthetic pigments concentration and a reduced expression of genes encoding for light-harvesting chlorophyll a/b binding proteins (i.e. HaLhcA), chlorophyll binding subunits of PSII proteins (i.e. HaPsbS and HaPsbX), phytoene synthase enzyme and a different expression level for genes related to PSII repair cycle, such as HaPsbA and HaPsbD. The concomitant stimulation of respiratory metabolism, suggests that linho activated a coordinate modulation of chloroplast and mitochondria activities to compensate the energy imbalance and regulate energy conversion processes.

Molecular analysis of the expression of a crtB transgene and the endogenous psy2-y 1 and psy2-y 2 genes of cassava and their effect on root carotenoid content.

A conventional breeding program was established to transfer the bacterial phytoene synthase transgene-crtB-from a transgenic, white-rooted cassava to yellow-rooted cassava plants carrying the endogenous phytoene synthase alleles named psy2-y 1 and/or psy2-y 2. Combining endogenous phytoene synthase enzymes (PSYs) with CRTB in a single cassava plant would allow the molecular dissection of individual allele contributions to carotenoid synthesis and/or accumulation in cassava roots. The simultaneous expression of the crtB transgene and psy2-y 2 in individuals planted in the field coincided with higher total, HPLC-quantified carotenoid content in roots, although the variability among replications (plants) precluded the detection of statistically significant differences. Nevertheless, the highest total carotenoid content in roots within a family coincided with one individual of the F1 progeny carrying both psy2-y 2 and crtB genes. The results also indicated the presence of at least one more key gene-different from psy or crtB-which too is necessary for the synthesis and/or accumulation of Pro-Vitamin A carotenoids in cassava roots.

A Metagenomics Analysis on B-Carotene Synthesis in Neurospora Crassa

We have studied insilico on evolutionary uniqueness of phytoene synthase, which is one of the regulatory enzymes of ?-carotene synthesis in Neurospora crassa. This study reveals multiple sequence alignments showed high sequences with similarity within a species of bacteria, fungi and higher plants. This results designate interestingly between species of bacteria-fungi, fungi-plant, and among the species of bacteria-fungi-plant, showed tremendously less sequence with similarity, except bacteria-plant (high sequence with similarity) respectively. In Phylogenetics tree analysis showed within species of bacteria, fungi and plant 91%, 92% and 99% homology. Whereas in between species of bacteria-fungi, bacteria-plant, fungi-plant, and among the species bacteria-fungi-plant showed 99%, 96%, 100%, and 91%-99% homology respectively. N. crassa phytoene synthase enzyme encode (Isoprenoid Biosynthesis enzymes, Class 1) protein size 610aa, Cyanobacteria phytoene encode (Isoprenoid Biosynthesis enzymes, Class 1) protein size 310aa, and Oryza sativa Indica phytoene synthase 1 (chloroplast), (Isoprenoid Biosynthesis enzymes, Class 1) encode protein size 421aa (e- value 0.0, 0.0 and 0.0; identity 100%, 100% and 100%; Max.score:1238, 644 and 870) respectively. We studied insilico on basis of an evolutionary Endosymbiotic theory; a bacterium is the ancestors to eukaryotes. Int J Appl Sci Biotechnol, Vol 3(3): 490-503

Functional Analysis of the Phycomyces carRA Gene Encoding the Enzymes Phytoene Synthase and Lycopene Cyclase

Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.

Increases in cell elongation, plastid compartment size and phytoene synthase activity underlie the phenotype of the high pigment-1 mutant of tomato.

A characteristic trait of the high pigment-1 ( hp-1) mutant phenotype of tomato ( Lycopersicon esculentum Mill.) is increased pigmentation resulting in darker green leaves and a deeper red fruit. In order to determine the basis for changes in pigmentation in this mutant, cellular and plastid development was analysed during leaf and fruit development, as well as the expression of carotenogenic genes and phytoene synthase enzyme activity. The hp-1 mutation dramatically increases the periclinal elongation of leaf palisade mesophyll cells, which results in increased leaf thickness. In addition, in both palisade and spongy mesophyll cells, the total plan area of chloroplasts per cell is increased compared to the wild type. These two perturbations in leaf development are the primary cause of the darker green hp-1 leaf. In the hp-1 tomato fruit, the total chromoplast area per cell in the pericarp cells of the ripe fruit is also increased. In addition, although expression of phytoene synthase and desaturase is not changed in hp-1 compared to the wild type, the activity of phytoene synthase in ripe fruit is 1.9-fold higher, indicating translational or post-translational control of carotenoid gene expression. The increased plastid compartment size in leaf and fruit cells of hp-1 is novel and provides evidence that the normally tightly controlled relationship between cell expansion and the replication and expansion of plastids can be perturbed and thus could be targeted by genetic manipulation.

Cloning and expression in Escherichia coli of the gene coding for phytoene synthase from the cyanobacterium Synechocystis sp. PCC6803

The gene coding for the carotenoid biosynthesis enzyme phytoene synthase ( pys) has been cloned from the unicellular cyanobacterium Synechocystis sp. PCC6803. The gene has been functionally expressed in Escherichia coli, where it directs the biosynthesis of phytoene from geranylgeranyl pyrophosphate (GGPP). Analysis of the sequence of the Synechocystis pys protein deduced from the gene sequence shows that it is highly homologous to the tomato and Synechococcus phytoene synthases and shows conserved domains with other bacterial phytoene synthase enzymes. The pys gene starts 60 nucleotides downstream of the pds gene (which codes for phytoene desaturase). However, it seems to be transcribed mainly from its own promoters, because insertions that disrupt the pds gene do not affect significantly the expression of the pys gene.

Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression

A tomato phytoene synthase gene, Psy1, has recently been isolated as the clone GTOM5 and shown by sequence identity to be the gene from which the major fruit-ripening cDNA clone TOM5 was derived. Sequence analysis of transcripts from two allelic yellow-fruited tomato mutants, mapped to chromosome 3, has shown the lack of carotenoids in fruit of these mutants to be due to the production of aberrant TOM5 transcripts which are unlikely to encode a functional phytoene synthase enzyme. In one mutant (yellow flesh) the aberrant transcript contained a sequence that, by its strong hybridization to a wide size range of genomic fragments, appeared to be repeated many times within the genome. Southern and PCR analysis of the phytoene synthase genes in the mutant revealed restriction fragment length polymorphisms, suggesting that the production of altered mRNAs was associated with specific genomic rearrangements. Constitutive over-expression of a TOM5 cDNA clone in transgenic mutant plants restored synthesis of the carotenoid lycopene in ripening fruit and also led to unscheduled pigment production in other cell types. In some mutant plants transformed with the TOM5 cDNA construct, inhibition of carotenoid production in immature green fruit, leaves and flowers was observed, due to the phenomenon of co-suppression, indicating that different insertion events with the same gene construct can lead to overexpression or co-suppression in transgenic plants. Green organs of these plants were susceptible to photobleaching, due to the lack of carotenoids. These results suggest the existence of separate Psy genes for carotenoid synthesis in green organs.