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.     

The Popdc gene family in the rat: molecular cloning, characterization and expression analysis in the heart and cultured cardiomyocytes

Three Popeye domain-containing (Popdc 1-3) family-members are known in vertebrates. Their exact function is as yet unknown although involvement in cell adhesion has been suggested. We report herein sequencing of the rat Popdc 1-3 cDNAs that show high homology to other vertebrate orthologs and are expressed primarily in the heart and skeletal muscles. Popdc2 splice variants were identified, with Popdc2C showing a distinctive age-dependent decline. In isolated cardiomyocytes, Popdc genes were negatively regulated by serum, an effect that was reversed by EGFR-kinase inhibition, suggesting an EGFR-dependent modulation of Popdc gene expression.     

Genome-wide characterization of phenylalanine ammonia-lyase gene family in watermelon (Citrullus lanatus)

Phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid pathway, plays a critical role in plant growth, development, and adaptation. PAL enzymes are encoded by a gene family in plants. Here, we report a genome-wide search for PAL genes in watermelon. A total of 12 PAL genes, designated ClPAL1-12, are identified . Nine are arranged in tandem in two duplication blocks located on chromosomes 4 and 7, and the other three ClPAL genes are distributed as single copies on chromosomes 2, 3, and 8. Both the cDNA and protein sequences of ClPALs share an overall high identity with each other. A phylogenetic analysis places 11 of the ClPALs into a separate cucurbit subclade, whereas ClPAL2, which belongs to neither monocots nor dicots, may serve as an ancestral PAL in plants. In the cucurbit subclade, seven ClPALs form homologous pairs with their counterparts from cucumber. Expression profiling reveals that 11 of the ClPAL genes are expressed and show preferential expression in the stems and male and female flowers. Six of the 12 ClPALs are moderately or strongly expressed in the fruits, particularly in the pulp, suggesting the potential roles of PAL in the development of fruit color and flavor. A promoter motif analysis of the ClPAL genes implies redundant but distinctive cis-regulatory structures for stress responsiveness. Finally, duplication events during the evolution and expansion of the ClPAL gene family are discussed, and the relationships between the ClPAL genes and their cucumber orthologs are estimated.     

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.     

异叶天南星苯丙氨酸解氨酶基因的克隆与蛋白结构分析

以异叶天南星（Arisaema heterophyllum Blume）为材料,采用逆转录PCR（RT-PCR）法扩增其苯丙氨酸解氨酶（Phenylalanine ammonia-lyase,PAL）基因AhPAL,获得该基因的开放读码框（ORF）,并通过系统的生物信息学软件分析AhPAL的结构和理化性质。结果显示,AhPAL的ORF全长为2184 bp,编码727个氨基酸;AhPAL与郁金香（Tulipa fosteriana W.Irving）PAL的亲缘关系最近,序列相似性达88%。空间结构模型分析结果显示,AhPAL为同型四聚体,每个单体由3个结构域组成,其中MIO结构域含有PAL酶家族的保守序列和Ala-Ser-Gly三肽活性中心,是AhPAL酶活性的决定性结构域。利用荧光定量PCR方法检测3个AhPAL Unigene在根、块茎和叶中的表达情况,发现它们在根中表达量均最高,而在叶和块茎中表达较低。     

异叶天南星苯丙氨酸解氨酶基因的克隆与蛋白结构分析

以异叶天南星(Arisaema heterophyllum Blume)为材料,采用逆转录PCR(RT-PCR)法扩增其苯丙氨酸解氨酶(Phenylalanine ammonia-lyase,PAL)基因Ah PAL,获得该基因的开放读码框(ORF),并通过系统的生物信息学软件分析Ah PAL的结构和理化性质。结果显示,Ah PAL的ORF全长为2184 bp,编码727个氨基酸;Ah PAL与郁金香(Tulipa fosteriana W. Irving) PAL的亲缘关系最近,序列相似性达88%。空间结构模型分析结果显示,Ah PAL为同型四聚体,每个单体由3个结构域组成,其中MIO结构域含有PAL酶家族的保守序列和Ala-Ser-Gly三肽活性中心,是Ah PAL酶活性的决定性结构域。利用荧光定量PCR方法检测3个Ah PAL Unigene在根、块茎和叶中的表达情况,发现它们在根中表达量均最高,而在叶和块茎中表达较低。     

Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis

The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg^?1 and the k _cat/K _m was 1.9 × 10⁴ mM^?1 s^?1, exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.     

Genome-wide identification,characterization, and expression profiles of auxin responsive GH3 gene family in Salvia miltiorrhiza involved in MeJA treatment

GH3 (Gretchen Hagen 3) is a gene family involved in the response to auxin and plays a role in regulation of plant growth, development and stress responses. The GH3 gene family has been well investigated in genome wide in various plants containing Arabidopsis, rice, maize, etc. However, the study on the GH3 family and its roles involved in JA-signal pathway in Salvia miltiorrhiza is lacking. In this study, we performed a systematic identification of the SmGH3 gene family in genome wide and detected 11 members on 8 S. miltiorrhiza scaffolds. Phylogenetic analyses revealed that SmGH3 proteins could be clustered in two major categories with groups 1 and 2 of GH3 family of Arabidopsis. Diversified cis-elements in the promoter of SmGH3 were predicted as essential players in regulating SmGH3 expression patterns by using PlantCARE database. Gene structure and motif analyses indicated that most SmGH3 genes had relatively conserved exon/intron arrangements and motif compositions. RNA-seq analysis and RT-qPCR showed that 3 SmGH3s (SmGH3.2, SmGH3.6, and SmGH3.10) were up-regulated in S. miltiorrhiza treated by MeJA. Moreover, tissue-specific expression patterns of each SmGH3 genes in different tissues suggested that the various members of GH3 genes conducted their role in the different tissues of S. miltiorrhiza. These results would provide a comprehensive understanding of the GH3 gene family in S. miltiorrhiza and lay a foundation for exploration of their functional divergence and genetic manipulation.

     

Molecular cloning and functional characterization of a phenylalanine ammonia-lyase from liverwort Plagiochasma appendiculatum

Liverworts are rich in phenolic compounds, including flavonoids and the distinctive type of bisbibenzyls. The biosynthesis of both types of compounds is believed to involve the phenylpropanoid pathway. Phenylalanine ammonia-lyase (PAL) is thought to be the key enzyme in the biosynthesis of bisbibenzyls and flavonoids in liverworts. In this study, PAL (designated as PaPAL) was cloned and characterized from both the cDNA and genomic DNA of the liverwort Plagiochasma appendiculatum. The full-length cDNA sequence contains 2,202 bp and is predicted to encode a protein with 733 amino acids. Sequence alignment showed that PaPAL’s predicted amino acid sequence shares more than 70 % identity with PAL sequences reported in public databases. The recombinant protein was heterologously expressed in Escherichia coli and exhibited high PAL activity, catalyzing the conversion of l-phenylalanine to trans-cinnamic acid. However, the enzyme exhibited lower activity in catalyzing the formation of p-coumaric acid from l-tyrosine. Additionally, when the thallus of P. appendiculatum was treated with abiotic stress inducers methyl jasmonate and abscisic acid, PaPAL expression was enhanced, thereby augmenting bisbibenzyl formation. These results suggest that PaPAL plays a key role in the early steps of bisbibenzyl biosynthesis and that abiotic stress can stimulate the expression of PaPAL, resulting in the accumulation of bisbibenzyls in the plant.     

The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular cloning, characterization and expression

Summary The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the Al gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 by DNA fragment extending from –982 to –634 by upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 F1 gene and of several highly expressed plant genes.     

The SAUR gene family in coffee: genome-wide identification and gene expression analysis during somatic embryogenesis

Molecular Biology Reports - Small auxin-up RNA (SAUR) genes form a wide family supposedly involved in different physiological and developmental processes in plants such as leaf senescence, auxin...     

Hexose transporters of tomato: molecular cloning,expression analysis and functional characterization

A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries. Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members. The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa. The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers. The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily. Expression of the three genes in different organs of tomato was investigated by quantitative PCR. LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips. LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers. LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae. LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H⁺/hexose symporter. The K _m of the symporter for glucose is 45 M.