Carotenoid accumulation and characterization of cDNAs encoding phytoene synthase and phytoene desaturase in garlic (Allium sativum).

Phytoene synthase (PSY) and phytoene desaturase (PDS), which catalyze the first and second steps of the carotenoid biosynthetic pathway, respectively, are key enzymes for the accumulation of carotenoids in many plants. We isolated 2 partial cDNAs encoding PSY (AsPSY-1 and AsPSY-2) and a partial cDNA encoding PDS (AsPDS) from Allium sativum. They shared high sequence identity and conserved motifs with other orthologous genes. Quantitative real-time PCR analysis was used to determine the expression levels of AsPSY1, AsPSY2, and AsPDS in the bulbils, scapes, leaves, stems, bulbs, and roots of garlic. High-performance liquid chromatography demonstrated that carotenoids were not biosynthesized in the underground organs (roots and bulbs), but were very abundant in the photosynthetic organs (leaves) of A. sativum. A significantly higher amount of β-carotene (73.44 μg·g(-1)) was detected in the leaves of A. sativum than in the other organs.

Tuan PA ，Kim JK ，Kim HH ，Lee SY ，Park NI ，Park SU ... -  《-》

Expression of the genes encoding the early carotenoid biosynthetic enzymes in Capsicum annuum.

We have shown that both geranylgeranyl pyrophosphate synthase and phytoene synthase from pepper and tomato chromoplasts are very similar in terms of enzymic activity and immunological properties. This enabled us to clone cDNAs specific for phytoene synthase from ripening tomato and pepper fruits and to compare their amino acid sequences with those of bacterial phytoene synthase and yeast squalene synthase. Comparison of the nucleotide sequence of the 3'-untranslated region of the pepper phytoene synthase cDNA suggests that this region was subjected to complex recombination events. RNA gel blot hybridizations revealed that induction of phytoene synthase gene expression is much stronger in tomato than in pepper fruits. In contrast with tomato, 2 phytoene synthase transcripts of different size are present in pepper leaves and fruits. Comparison of the expression pattern of the genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase and phytoene desaturase revealed that these genes are not co-regulated during pepper fruit ripening.

Römer S ，Hugueney P ，Bouvier F ，Camara B ，Kuntz M ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Isolation and characterization of phytoene desaturase cDNA involved in the beta-carotene biosynthetic pathway in Dunaliella salina.

The green alga Dunaliella salina is one of the best and most important biological sources of beta-carotene; however, to date the molecular basis of the beta-carotene biosynthesis process in D. salina is still unresolved. The dehydrogenation of phytoene is the second step in the carotenoids biosynthetic pathway, and the phytoene-related desaturases are the key enzymes in the beta-carotene biosynthetic pathway. A phytoene desaturase (Pds) cDNA with a 1752 bp open reading frame was cloned by RT-PCR and RACE-PCR methods on the basis of a modified switching mechanism at 5' end of the RNA transcript (SMART) technology from D. salina. The predicted protein sequence displays a high identity (up to 65%) with phytoene desaturases of higher plants and cyanobacteria. The highest amino acid sequence identity (91%) is shared with the phytoene desaturase sequence of Dunaliella bardawil, and a dinucleotide-binding motif lies in the N-terminal. The phylogenetic analysis shows that D. salina Pds is closer to higher plants and cyanobacteria than bacterial and fungi. These results together demonstrated the cloned Pds cDNA of D. salina is a Pds-type gene, and it is postulated that in D. salina the first two dehydrogenations, by which phytoene is converted into zeta-carotene, are carried out by this putative phytoene desaturase.

Zhu YH ，Jiang JG ，Yan Y ，Chen XW ... -  《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》

Expression of carotenoid biosynthesis genes during carrot root development.

Carotenogenesis has been extensively studied in fruits and flower petals. Transcriptional regulation is thought to be the major factor in carotenoid accumulation in these organs. However, little is known about regulation in root organs. The root carotenoid content of carrot germplasm varies widely. The present study was conducted to investigate transcriptional regulation of carotenoid biosynthesis genes in relation to carotenoid accumulation during early carrot root development and up to 3 months after sowing. HPLC carotenoid content analysis and quantitative RT-PCR were compared to quantify the expression of eight genes encoding carotenoid biosynthesis enzymes during the development of white, yellow, orange, and red carrot roots. The genes chosen encode phytoene synthase (PSY1 and PSY2), phytoene desaturase (PDS), zeta-carotene desaturase (ZDS1 and ZDS2), lycopene epsilon-cyclase (LCYE), lycopene beta-cyclase (LCYB1), and zeaxanthin epoxidase (ZEP). All eight genes were expressed in the white cultivar even though it did not contain carotenoids. By contrast with fruit maturation, the expression of carotenogenic genes began during the early stages of development and then progressively increased for most of these genes during root development as the total carotenoid level increased in coloured carrots. The high expression of genes encoding LCYE and ZDS noted in yellow and red cultivars, respectively, might be consistent with the accumulation of lutein and lycopene, respectively. The results showed that the accumulation of total carotenoids during development and the accumulation of major carotenoids in the red and yellow cultivars might partially be explained by the transcriptional level of genes directing the carotenoid biosynthesis pathway.

Clotault J ，Peltier D ，Berruyer R ，Thomas M ，Briard M ，Geoffriau E ... -  《-》

Functional analysis of the early steps of carotenoid biosynthesis in tobacco.

Carotenoids contribute to energy transduction in the light harvesting complexes and serve in protection from excess light fluence. Because of the importance of carotenoids, the genes encoding enzymes of carotenoid biosynthesis in higher plants are potential targets for herbicides. To obtain further insight into tobacco carotenoid biosynthesis and to investigate and prioritize potential herbicide targets in the pathway, the effects of changed phytoene synthase (PSY) and phytoene desaturase (PDS) gene expression were studied in transgenic tobacco (Nicotiana tabacum Petit Havana SR1) plants. Genes for both enzymes were cloned from tobacco, and surprisingly two functional PSY genes were found. Transgenic tobacco plants constitutively expressing these genes in both sense and antisense orientations were examined regarding phenotype, carotenoid content and transcript levels of carotene biosynthesis genes. Overexpression of either psy gene resulted in severe phenotypic effects including dwarfism, altered leaf morphology, and pigmentation. A correlation among phenotype, transcript level, and metabolic profile was demonstrated by comparison of hemizygous and homozygous plants from the same transformation event. Antisense expression of PSY and PDS also caused lethal phenotypes. Transcript levels of other carotene biosynthesis genes remained unaltered in the transgenic mutant. Phytoene accumulated in plants expressing antisense RNA to pds. However, elevated levels of phytoene were detected suggesting an increase in metabolic flux into this pathway.

Busch M ，Seuter A ，Hain R 《-》