Structure and some characterization of the gene for phenylalanine ammonia‐lyase from rice plants

Abstract

A nearly full-length cDNA and a genomic clone were isolated that encoded the phenylalanine ammonia-lyase (PAL) of rice plants. and the complete nucleotide sequences were determined. The gene encodes a polypeptide of 701 amino acid residues. The deduced amino acid sequence is highly similar to that of PAL from Phaseolus vulgaris deduced from an incomplete cDNA fragment. The cloned gene spans 4412 bp and consists of two exons and one intron. The site of initiation of transcription was located -86 nucleotides (position 1) upstream from the translational initiation codon by the primer-extension method. Sequences analogous to TATA-box and GCbox were found in the 5'-upstream region from the transcriptional initiation site. Southern blot analysis showed that the PAL gene of rice plants exists as a small multi-gene family. Within this family, the genomic clone isolated in this study was shown to be responsive by light. This also indicated that this genomic sequence functions as a gene for phenylalanine ammonia-lyase in rice plants in vivo.Phenylalanine ammonia-lyase (PAL} catalyzes the first reaction in the general pathway of biosynthesis of polyphenolic compounds including lignin, cinnamate esters and flavonoids [11, and is one of the key enzymes in the metabolism of these compounds [l]. The activity of PAL increases dramatically in response to various stimuli, such as wounding [2, 31, infection [4, 51, growth regulators [2] and light [6]. It is caused by de novo synthesis of the enzyme due to the increase in the amount of translatable mRNA, suggesting that activation of the gene expression may be induced by these stimuli [7, 81. However, no reports have shown the whole genomic structure or complete amino acid sequence of PAL. Furthermore, our knowledge on the regulatory mechanisms of PAL activity is limited to dicotyledonous plants. As a first step to elucidate the molecular mechanisms for the regulation of expression of the gene for PAL, we isolated a nearly full-sized cDNA and relevant genomic DNA for PAL from rice plant and analyzed their primary structures. Using a probe specific to the isolated genomic clone for PAL, we demonstrated the accumulation of the transcripts from this gene during greening. This also showed that the isolated genomic DNA functions as a gene for PAL in vivo. Corre MATERIALS AND METHODS Plant materialsRice (Oryza sativa L. cv. Nipponbare) seeds were sterilized in a 2% (massivol.) solution of sodium hypochlorite, washed extensively in tap water, and allowed to germinate on sterile vermiculite in a green house at 25 "C. Twelve days after germination, primary leaves (about 5 cm in length) were harvested, immediately frozen in liquid nitrogen, and used for extraction of poly(A)-rich RNA and high-molecular-mass DNA. In some experiments, plants were allowed to germinate in the dark for seven days, then illuminated at 200 pmol . m ~ . s ~ for various periods of time with fluorescent lights. At each stage of greening, plants were harvested and used for the extraction of total cellular RNA. Prepar...

Transcription profiling identifies candidate genes for secondary cell wall formation and hydroxycinnamoyl-arabinoxylan biosynthesis in the rice internode

“…Monolignol synthesis and hydroxycinnamoylCoA synthesis were expected to be up-regulated in the wild-type whole internode and F71 pith parenchyma, respectively, and the expression profiles of genes in the phenylpropanoid pathway were determined. Although many researchers have studied genes in the phenylpropanoid pathway in rice, in many cases only a subset of these genes were targeted, and not all of the genes in this pathway have been identified (Kawasaki et al 2006; Lee et al 2007Lee et al , 2008Ma 2007;Minami et al 1989;Tobias and Chow 2005;Zhang et al 2006;Zhao et al 2004;Zhu et al 1995). The list of phenylpropanoid pathway genes in rice and the corresponding probe sets was based on all of these studies and a report on Arabidopsis genes by Raes et al (2003) (Table S4).…”

Nucleotide distribution in gymnosperm nuclear sequences suggests a model for GC-content change in land-plant nuclear genomes

“…CpG suppression is found to varying extents in different plant genomes (Hepburn et al 1987;Quigley et al 1989;Campbell and Gowri 1990;. Lack of suppression has been attributed to both protection against methylation (Bird 1986) as well as specific G-T mismatch repair (Hepburn et al 1987); for the purpose of our model, a distinction between these (Brinkmann et al 1987) and tobacco (Shih et al 1986), GapC from maize (Brinkmann et al 1987) and tobacco (Shih et al 1986), Lhcbl from rice (Lhcbl*l or 2120, Matsouka 1990) and tomato (Lhcbl*2 or Cab-lB, Pichersky et al 1985), Lhcb2 from rice (Lhcb2*l or 2123, Matsouka 1990) and tomato , Lhcb5 from barley (SCrensen et al 1992) and tomato , Pal from rice (Minami et al 1989) and tomato (EMBL M90692), Pcr from barley (Schultz et al 1989) and pea (Spano et al 1992a), and…”

Phenylalanine Ammonia-Lyase from Loblolly Pine

“…PAL is reported to be encoded by small multi-gene families in bean (4), rice (19), Arabidopsis (21), and parsley (16), based on evidence from Southern blot analysis of genomic DNA and isolation of genomic or cDNA clones. Southern blot analysis of pine genomic DNA, using a fragment of the pine xylem pal cDNA as probe, reveals bands consistent with a single pal gene corresponding to this cDNA.…”