Cloning and properties of a rice gene encoding phenylalanine ammonia-lyase

Abstract

Phenylalanine ammonia-lyase (PAL) genomic sequences were isolated from a rice (Oryza sativa L.) genomic library using a PCR-amplified rice PAL DNA fragment as a probe. There is a small family of PAL genes in the rice genome. The nucleotide sequence of one PAL gene, ZB8, was determined. The ZB8 gene is 4660 bp in length and consists of two exons and one intron. It encodes a polypeptide of 710 amino acids. The transcription start site was 137 bp upstream from the translation initiation site. Rice PAL transcripts accumulated to a high level in stems, with lower levels in roots and leaves. Wounding of leaf tissues induced ZB8 PAL transcripts to a high level. In rice suspension-cultured cells treated with fungal cell wall elicitors, the ZB8 PAL transcript increased within 30 min and reached maximum levels in 1-2 h. The transcription of the ZB8 gene was investigated by fusing its promoter to the reporter gene beta-glucuronidase (GUS) and transforming the construct into rice and tobacco plants, as well as rice suspension-cultured cells. High levels of GUS activity were observed in stems, moderate levels in roots and low levels in leaves of transgenic rice and tobacco plants. Histochemical analysis indicated that in transgenic rice the promoter was active in root apical tips, lateral root initiation sites, and vascular and epidermal tissues of stems and roots. In rice flowers, high GUS activity was observed in floral shoots, receptacles, anthers and filaments, occasionally GUS activity was also detected in lemma and awn tissues. In tobacco flowers, high GUS activity was detected in the pink part of petals. Consistent with the activity of endogenous PAL transcripts, wounding of rice and tobacco leaf tissues induced GUS activity from low basal levels. Tobacco mosaic virus (TMV) infection of tobacco leaves induced GUS activity to a high level. Fungal cell wall elicitors strongly induced GUS activity and GUS transcripts to high levels in transgenic rice suspension-cultured cells. We demonstrated that the promoter of ZB8 gene is both developmentally regulated and stress-inducible.