Cloning and characterization of a maize cDNA encoding phytoene desaturase, an enzyme of the carotenoid biosynthetic pathway

Abstract

To study regulation of the plastid-localized maize carotenoid biosynthetic pathway, a cDNA encoding phytoene desaturase (PDS) was isolated and characterized. The DNA sequence of the maize Pds cDNA was determined and compared with available dicot Pds genes. The deduced PDS protein, estimated at 64.1 kDa (unprocessed), had a dinucleotide binding domain and conserved regions characteristic of other carotene desaturases. Alignment of available PDS sequences from distantly related organisms suggests that Pds has potential as a phylogenetic tool. By use of heterologous complementation in Escherichia coli, maize PDS was shown to catalyze two desaturation steps converting phytoene to zeta-carotene. RFLP (restriction fragment length polymorphism) mapping was used to place Pds on chromosome 1S near viviparous5 (vp5), and RT-PCR (reverse-transcriptase polymerase chain reaction) analysis indicated reduced Pds transcript in vp5 mutant relative to normal endosperm. Other phytoene-accumulating mutant endosperms, vp2 and white3 (w3), showed no difference in Pds transcript accumulation as compared with normal endosperm counterparts. RT-PCR analysis of Pds transcript accumulation in developing endosperm showed Pds was constitutively expressed. Therefore, endosperm carotenogensis is not regulated by increasing the level of Pds transcripts.

The Maize Phytoene Synthase Gene Family: Overlapping Roles for Carotenogenesis in Endosperm, Photomorphogenesis, and Thermal Stress Tolerance

“…There remain open questions about the specific roles of these three genes that encode a key enzyme operating at the pathway entry point; the six downstream steps all appear to involve single-copy genes in maize (Li et al, 1996;Matthews et al, 2003;Li et al, 2007;Harjes et al, 2008). Whereas some species contain a single PSY gene, the PSY gene duplication in the grasses could potentially provide grasses with a fine tune control of carotenoid biosynthesis for different regulatory scenarios.…”

“…In assembly of the biosynthetic metabolons, the nuclear-encoded pathway enzymes are recruited to membrane locations that may differ from one plastid type to another. Because many of the pathway enzymes are encoded by single copy genes (Li et al, 1996;Matthews et al, 2003), specificity of targeting must be controlled by factors beyond the transit sequence. Optimization of metabolic engineering/breeding of carotenoids is predicated on elucidating the mechanisms that control recruitment of pathway enzymes to metabolons on different plastid membranes in various plastid types.…”

Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis

“…First-strand cDNA synthesis was performed in a 20-µL-reaction volume consisting of 3 µg of total RNA, oligodT [12][13][14][15][16][17][18] and SuperScript TM reverse transcriptase (GIBCO BRL). The reaction was allowed to proceed at 42 ºC for 50 min before being terminated by treatment at 70 ºC for 15 min.…”

“…The PDS gene was first cloned from cyanobacterium [7]. Homologous PDS genes were then cloned from soybean [8], pepper [9], tomato [10,11], Arabidopsis [12] and maize [13]. These homologous proteins share about 60% identity and 75% similarity to the cyanobacterium PDS in amino acid sequences.…”

Timing and Biosynthetic Potential for Carotenoid Accumulation in Genetically Diverse Germplasm of Maize

“…For the carotenoid biosynthetic pathway, we identified two CrtISO (for carotene isomerase) genes in maize on chromosomes 2 and 4, adding to previously isolated pathway genes for PDS (for phytoene desaturase), ZDS (for zetacarotene desaturase), LCYE (for lycopene e-cyclase), and LCYB (for lycopene b-cyclase), all of which are single copy in maize and other grasses (Buckner et al, 1990;Li et al, 1996Li et al, , 2008aLi et al, , 2008bMatthews et al, 2003;Singh et al, 2003;Harjes et al, 2008;Supplemental Table S1). In contrast to two maize CrtISO genes, only one was found in rice, sorghum, and Arabidopsis (Arabidopsis thaliana; Supplemental Table S1; Supplemental Fig.…”