The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana

Phenylpropanoid derivatives are a complex class of secondary metabolites that have many important roles in plants during normal growth and in responses to environmental stress. Phenylalanine ammonialyase (PAL) catalyzes the first step in the biosynthesis of phenylpropanoids, and is usually encoded by a multi-gene family. Genomic clones for three Arabidopsis thaliana PAL genes containing the entire protein-coding region and upstream and downstream sequences have been obtained and completely sequenced. Two A. thaliana PAL genes (PAL1 and PAL2) are structurally similar to PAL genes that have been cloned from other plant species, with a single intron at a conserved position, and a long highly conserved second exon. Previously identified promoter motifs plus several additional sequence motifs were found in the promoter regions of PAL1 and PAL2. Expression of PAL1 and PAL2 is both qualitatively and quantitatively similar in different plant organs and under various inductive conditions. A third A. thaliana PAL gene, PAL3, differs significantly from PAL1 and PAL2 and other sequenced plant PAL genes. PAL3 contains an additional intron, and its deduced amino acid sequence is less homologous to other PAL proteins. The PAL3 promoter region lacks several sequence motifs conserved between A. thaliana PAL1 and PAL2, as well as motifs described in other genes involved in phenylpropanoid metabolism. A. thaliana PAL3 was expressed at very low levels under the conditions examined.     

Promoter Activity of Phenylalanine Ammonia-Lyase Gene of Pharbitis nil in Arabidopsis

Promoter activity of phenylalanine ammonia-lyase (PAL) gene of Pharbitis nil was examined by introducing a PAL:GUS construct into Arabidopsis. GUS staining was observed in vascular bundles of hypocotyl and cotyledons, endodermal cells of the primary root, hydathodes, stigma and pollens of mature flower, abscission zones of petals and sepals and inner layer of seed coat. Light induced GUS expression in cotyledons and the upper part of hypocotyl in which anthocyanin was accumulated. Wounding also induced GUS expression. Endogenous PAL activity increased earlier than the GUS activity directed by the PAL promoter.     

鸭梨PAL基因的反义遗传转化及表达分析

苯丙氨酸解氨酶(phenylalanin ammonia-lyase,PAL,EC4.3.1.5)是植物通过苯丙烷代谢途径合成木质素的关键酶和限速酶,其通过影响木质素的合成而与果实中石细胞的分化、发育及果实品质密切相关。为了降低鸭梨中苯丙氨酸解氨酶的含量,该研究利用反义PAL基因遗传转化鸭梨、降低鸭梨内源PAL基因的表达。结果表明:(1)采用RT-PCR技术,利用根据Gen Bank中西洋梨PAL基因序列设计特异性引物,扩增得到496 bp的鸭梨PAL基因片段。(2)将扩增片段反向插入载体p BI121的MCS区域,构建植物PAL基因反义表达载体p BI121-As PAL。接着采用电转化法将反义表达载体转入农杆菌EHA105中,并制备出农杆菌工程菌液。(3)利用农杆菌介导法对鸭梨组培苗叶片外植体进行遗传转化,得到23株转基因鸭梨苗。PCR检测证实PAL反义基因片段转入鸭梨中,实时定量PCR检测表明转基因鸭梨苗体内PAL基因表达量均有所降低,为非转基因苗的65%~75%。该研究结果表明利用反义RNA技术获得了抑制内源性PAL基因表达的转基因鸭梨植株,为改善鸭梨果实品质、改良品种奠定了基础。     

Control of phenylalanine ammonia-lyase gene promoters from pea by UV radiation

The gene fusion system was used to study UV light-control of PS PAL1 and PS PAL2 genes encoding phenylalanine ammonia-lyase of pea. The induction of pea PAL promoters was analysed in transgenic tobacco plants. Binary plasmids (derivatives of pBI101.2 vector) containing 5′ regulatory fragments of PS PAL1 and PS PAL2 linked to reporter genes (GUS,LUC) were constructed. The analyses were performed with the use of single constructs (containing one variant of PS PAL promoter and one reporter gene) and dual constructs (containing both PS PAL1 and PS PAL2 promoters connected with different reporter genes). The use of dual constructs enabled the evaluation of both PS PAL promoters activity in the same plant. The analyses of in vitro grown plants have shown that both PAL promoters are strongly induced in leaves subjected to UV radiation. In some cases, the UV-stimulated expression exceeded the exposed areas. This phenomenon was observed more often in the leaves of plants containing the PS PAL1::GUS than PS PAL2::GUS construct. Removal of boxes 2, 4, 5 from PS PAL1 promoter and deletion of its 5′ end region (-339 to -1394) decreases the level of gene expression but does not eliminate its responsiveness to UV.     

Tissue-specific expression of the PAL3 promoter requires the interaction of two conserved cis sequences

The bean PAL2 and PAL3 promoters confer expression in overlapping sets of tissue types in transgenic tobacco. The PAL3 promoter contains motifs that resemble two AC cis elements which are required for tissue-specific expression of the PAL2 promoter. The functions of these motifs in the PAL3 promoter were determined by analysis of mutated PAL3 promoter-GUS constructs in transgenic tobacco. This revealed that the AC motifs are necessary for tissue-specific expression of the PAL3 promoter. Therefore, a key role is indicated for AC elements, which are Myb-protein binding sites, in regulating tissue-specific expression of the bean PAL gene family.     

Upregulation of the promoter activity of the carrot (Daucus carota) phenylalanine ammonia-lyase gene (DcPAL3) is caused by new members of the transcriptional regulatory proteins, DcERF1 and DcERF2, which bind to the GCC-box homolog and act as an activator to the DcPAL3 promoter

The phenylalanine ammonia-lyase (PAL) gene, DcPAL3, was expressed during the synthesis of anthocyanin in suspension-cultured cells of carrot (Daucus carota). There were two putative cis-elements in the DcPAL3 promoter region: the box-L and GCC-box homologs. Both of these are committed to the upregulation of promoter activity. Although box-L is known as the conserved cis-element present in the promoter region of most PAL genes of many plant species targeted by the R2R3-MYB protein, among PAL genes, the GCC-box homolog is unique to the promoter region of the DcPAL3 gene. We have isolated two proteins belonging to the ethylene-responsive element-binding factor (ERF) family, DcERF1 and DcERF2, from two different cDNA libraries prepared from anthocyanin-synthesizing cells of different cultured cell lines of carrot. The methodology employed was yeast one-hybrid screening with the GCC-box homolog as a bait. Both DcERF1 and DcERF2 bound to the GCC-box homolog sequence in vitro. Transient expression analysis showed that, in carrot protoplasts, DcERF1 was able bind to the GCC-box homolog and act as an activator of the DcPAL3 promoter. In contrast, DcERF2 itself had no ability to activate DcPAL3 promoter activity, possibly because transiently expressed DcERF2 may not be exported into the nucleus. These results suggest that DcERF1 and DcERF2 may function in different ways in committing to the upregulation of the DcPAL3 promoter activity in anthocyanin-synthesizing cells of carrot.     

BBX16, a B‐box protein,positively regulates light‐induced anthocyanin accumulation by activating MYB10 in red pear

The red coloration of pear (Pyrus pyrifolia) results from anthocyanin accumulation in the fruit peel. Light is required for anthocyanin biosynthesis in pear. A pear homolog of Arabidopsis thaliana BBX22, PpBBX16, was differentially expressed after fruits were removed from bags and may be involved in anthocyanin biosynthesis. Here, the expression and function of PpBBX16 were analysed. PpBBX16's expression was highly induced by white‐light irradiation, as was anthocyanin accumulation. PpBBX16's ectopic expression in Arabidopsis increased anthocyanin biosynthesis in the hypocotyls and tops of flower stalks. PpBBX16 was localized in the nucleus and showed trans‐activity in yeast cells. Although PpBBX16 could not directly bind to the promoter of PpMYB10 or PpCHS in yeast one‐hybrid assays, the complex of PpBBX16/PpHY5 strongly trans‐activated anthocyanin pathway genes in tobacco. PpBBX16's overexpression in pear calli enhanced the red coloration during light treatments. Additionally, PpBBX16's transient overexpression in pear peel increased anthocyanin accumulation, while virus‐induced gene silencing of PpBBX16 decreased anthocyanin accumulation. The expression patterns of pear BBX family members were analysed, and six additional BBX genes, which were differentially expressed during light‐induced anthocyanin biosynthesis, were identified. Thus, PpBBX16 is a positive regulator of light‐induced anthocyanin accumulation, but it could not directly induce the expression of the anthocyanin biosynthesis‐related genes by itself but needed PpHY5 to gain full function. Our work uncovered regulatory modes for PpBBX16 and suggested the potential functions of other pear BBX genes in the regulation of anthocyanin accumulation, thereby providing target genes for further studies on anthocyanin biosynthesis.     

Increased anthocyanin accumulation in aster flowers at elevated temperatures due to magnesium treatment

Temperature is one of the main external factors affecting anthocyanin accumulation in plant tissues: low temperatures cause an increase and elevated temperatures cause a decrease in anthocyanin concentration. Several metals have been shown to increase the half-life time of anthocyanins, by forming complexes with them. We studied the combined effect of elevated temperatures and increased metal concentrations on the accumulation of anthocyanins in aster 'Sungal' flowers. It has been found that magnesium treatment of aster plants or detached flower buds, partially prevents colour fading at elevated temperatures. Anthocyanin concentration of aster 'Sungal' flowers grown at 29°C/21°C day/night, respectively, was about half that of flowers grown at 17°C/9°C. The activity of phenylalanine ammonia-lyase (PAL) and chalcone isomerase (CHI) decreased as the temperature increased. Treatment of both whole plants and detached flower buds grown at elevated temperatures in the presence of magnesium salts, increased flower anthocyanin concentration by up to 80%. Measurement of magnesium following these treatments revealed an increased level of the metal in the petals, suggesting a direct effect. Magnesium treatment does not seem to cause increased synthesis of anthocyanin through a stress-related reaction, since the activities of both PAL and CHI did not increase due to this treatment. The results of this study show that increasing magnesium levels in aster petals prevents the deleterious effect of elevated temperatures on anthocyanin accumulation, thus enhancing flower colour.     

Phenylalanine Ammonia-Lyase Genes Involved in Anthocyanin Synthesis and the Regulation of its Expression in Suspension Cultured Carrot Cells

D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed. Received 10 June 2000/ Accepted in revised form 1 July 2000     

RNAi-mediated suppression of the phenylalanine ammonia-lyase gene in Salvia miltiorrhiza causes abnormal phenotypes and a reduction in rosmarinic acid biosynthesis

Medicinal Salvia miltiorrhiza contains two main groups of active pharmaceutical ingredients: lipid-soluble tanshinones and water-soluble phenolic acids, including rosmarinic acid and salvianolic acid B. Phenylalanine ammonia-lyase (PAL) catalyzes the first step in the phenylpropanoid pathway and is assumed to be closely related to the accumulation of rosmarinic acid and its derivatives. We selected a 217-bp fragment, located at the 3′ end of the coding region of PAL1, to establish an RNA interference construct that was introduced into S. miltiorrhiza via Agrobacterium tumefaciens-mediated transformation. PAL-suppressed plants exhibited several unusual phenotypes such as stunted growth, delayed root formation, altered leaves, and reduced lignin deposition. The total phenolic content was decreased by 20–70% in PAL-suppressed lines, and was accompanied by lower PAL activity. Down-regulation of PAL also affected the expression of C4H, 4CL2, and TAT, which are related genes in the rosmarinic acid pathway. Moreover, rosmarinic acid and salvianolic acid B were markedly reduced in PAL-suppressed lines, as detected by HPLC analysis. Our results indicate that PAL is very important for the synthesis of major water-soluble pharmaceutical ingredients within S. miltiorrhiza.