Differential expression of two cinnamate 4-hydroxylase genes in 'Valencia' orange (Citrus sinensis Osbeck)

Two different full-length cDNAs for cinnamate 4-hydroxylase (C4H1 and C4H2) were isolated from Citrus sinensis Osbeck cv. Valencia libraries. C4H1 (1708 bp) and C4H2 (1871 bp) share only 65% identity on nucleotide and 66% identity on the amino acid level, respectively. C4H1 is most homologous to a cinnamate 4-hydroxylase sequence from French bean (Phaseolus vulgaris), but codes for a unique N-terminus. C4H2 shows highest similarity to a poplar (Populus kitakamiensis) sequence, but also shows a unique N-terminus. The two genes are expressed differentially in orange flavedo, C4H2 is constitutive, C4H1 is wound-induced. In competitive RT-PCR, the mRNA for both genes in wounded and untreated tissue was quantified. C4H1 is strongly wound-inducible from `not detectable' to about 35 fg mRNA per 50 ng total RNA 8 h after wounding. The first detectable C4H1 mRNA was found 4 h after wounding. After reaching peak levels 4 h later the levels slightly declined, but stayed elevated until the end of the experiment (48 h). C4H2 is expressed 3-10 times higher than wound-induced C4H1 even in the control sample; wounding transiently increases the level of expression another 2-3 times. The existence of different N-termini and their effects on the possible role of both genes in phenylpropanoid pathways is discussed.