ABSTRACT
Vitamin E has been found to be associated with an important antioxidant property in mammals and plants. In photosynthetic organisms, the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD; E.C. 1.13.11.27) plays an important role in the vitamin E biosynthetic pathway. The full-length cDNA encoding HPPD was isolated from Lactuca sativa L. by rapid amplification of cDNA ends (RACE). The cDNA, designated as LsHPPD, was 1743 base pairs (bp) long containing an open reading frame (ORF) of 1338 bp encoding a protein of 446 amino acids. Sequence analysis indicated that LsHPPD shared high identity with HPPD from Medicago truncatula L. Real-time fluorescent quantitative PCR (qPCR) analysis revealed that LsHPPD was preferentially expressed in mature leaves compared with other tissues and that the LsHPPD expression was sensitive to high light and drought stress treatments. Transient expression of LsHPPD via agroinfiltration resulted in 12-fold increase in LsHPPD mRNA expression level and 4-fold enhancement in α-tocopherol content compared with the negative control. A decrease in chlorophyll content and inhibition of photosystem II were observed during stress treatments and agroinfiltration.
Subject(s)
4-Hydroxyphenylpyruvate Dioxygenase/genetics , 4-Hydroxyphenylpyruvate Dioxygenase/metabolism , Lactuca/enzymology , Lactuca/genetics , 4-Hydroxyphenylpyruvate Dioxygenase/isolation & purification , 4-Hydroxyphenylpyruvate Dioxygenase/radiation effects , Amino Acid Sequence , Base Sequence , Chlorophyll/metabolism , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Dehydration/metabolism , Gene Expression Regulation, Plant , Lactuca/radiation effects , Light/adverse effects , Molecular Sequence Data , Phylogeny , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Leaves/radiation effects , Plant Roots/genetics , Plant Roots/metabolism , Plant Roots/radiation effects , Plant Stems/genetics , Plant Stems/metabolism , Plant Stems/radiation effects , RNA, Plant/genetics , Real-Time Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNA , Stress, Physiological , Vitamin E/biosynthesis , Vitamin E/chemistryABSTRACT
4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria-Bertani medium supplemented with l-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific beta-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium.