A Deficiency at the Gene Coding for ζ-Carotene Desaturase Characterizes the Sunflower non dormant-1 Mutant

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Abstract
The non dormant-1 (nd-1) mutant of sunflower (Helianthus annuus L.) is characterized by an albino and viviparous phenotype. Pigment analysis by spectrophotometer and HPLC demonstrated in nd-1 cotyledons the absence of β-carotene, lutein and violaxanthin. Additionally, we found a strong accumulation of ζ-carotene and, to a lesser extent, of phytofluene and cis-phytoene in nd-1 seedlings grown in very dim light (1 µmol m–2 s–1). These results suggested that ζ-carotene desaturation was impaired in the mutant plants. To understand the molecular basis of the nd-1 mutation, we cloned and characterized the ζ-carotene desaturase (Zds) gene from sunflower. A reconstructed full-length sequence (1,916 bp) of the Zds cDNA was obtained from homozygous Nd-1/Nd-1 wild-type plants. It contains a 1,761-bp CDS, 62 nucleotides of 5′-untranslated region (UTR), and 77 nucleotides of 3′-UTR. The predicted protein (64.9 kDa) consists of 587 amino acid residues with a putative transit sequence for plastid targeting in the N-terminal region and a typical amino oxidase domain that includes the flavin adenosine dinucleotide (FAD) binding motif. The phylogenetic analysis demonstrated that the sunflower Zds was clustered to marigold (Tagetes) Zds gene, for which it showed an overall aminoacidic identity of 96.6% and resulted strictly correlated with other Zds sequences of higher plants. Interestingly, RT-PCR analyses showed that nd-1 plants were unable to accumulate Zds transcripts. Sequence information from the Zds cDNA was used to design specific primers and to isolate the full-length exons/introns region of the gene. The sunflower Zds gene (HaZds) comprises 14 exons and 13 introns scattered in a ca. 5.0-kb region. Also, HaZds showed a high conservation of the distribution and size of the exons with rice Zds gene. Based on genomic Southern analysis, the nd-1 genome disclosed a large deficiency at the Zds locus.