The essential requirements for enzyme assays are described and frequently occurring errors and pitfalls as well as their avoidance are discussed. The main factors, which must be considered for assaying enzymes, are temperature, pH, ionic strength and the proper concentrations of the essential components like substrates and enzymes. Standardization of these parameters would be desirable, but the diversity of the features of different enzymes prevents unification of assay conditions. Nevertheless, many enzymes, especially those from mammalian sources, possess a pH optimum near the physiological pH of 7.5, and the body temperature of about 37 °C can serve as assay temperature, although because of experimental reasons frequently 25 °C is preferred. But in many cases the particular features of the individual enzyme dictate special assay conditions, which can deviate considerably from recommended conditions.In addition, exact values for the concentrations of assay components such as substrates and enzymes cannot be given, unless general rules depending on the relative degree of saturation can be stated. Rules for performing the enzyme assay, appropriate handling, methodical aspects, preparation of assay mixtures and blanks, choice of the assay time, are discussed and suggestions to avoid frequent and trivial errors are given. Particularities of more complex enzyme assays, including reversible reactions and coupled tests are considered.Finally the treatment of experimental data to estimate the enzyme activity is described.
The procedure for determining the initial enzyme velocity and its transformation into defined enzyme units as well as suggestions for documentation of the results are presented. Previous article in issue.
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BackgroundLignin is a phenolic heteropolymer in secondary cell walls that plays a major role in the development of plants and their defense against pathogens. The biosynthesis of monolignols, which represent the main component of lignin involves many enzymes. The cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis as it catalyzes the final step in the synthesis of monolignols. The CAD gene family has been studied in Arabidopsis thaliana, Oryza sativa and partially in Populus. This is the first comprehensive study on the CAD gene family in woody plants including genome organization, gene structure, phylogeny across land plant lineages, and expression profiling in Populus.
ResultsThe phylogenetic analyses showed that CAD genes fall into three main classes (clades), one of which is represented by CAD sequences from gymnosperms and angiosperms. The other two clades are represented by sequences only from angiosperms.
All Populus CAD genes, except PoptrCAD 4 are distributed in Class II and Class III. CAD genes associated with xylem development ( PoptrCAD 4 and PoptrCAD 10) belong to Class I and Class II.
Most of the CAD genes are physically distributed on duplicated blocks and are still in conserved locations on the homeologous duplicated blocks. Promoter analysis of CAD genes revealed several motifs involved in gene expression modulation under various biological and physiological processes. The CAD genes showed different expression patterns in poplar with only two genes preferentially expressed in xylem tissues during lignin biosynthesis. ConclusionThe phylogeny of CAD genes suggests that the radiation of this gene family may have occurred in the early ancestry of angiosperms. Gene distribution on the chromosomes of Populus showed that both large scale and tandem duplications contributed significantly to the CAD gene family expansion. The duplication of several CAD genes seems to be associated with a genome duplication event that happened in the ancestor of Salicaceae. Phylogenetic analyses associated with expression profiling and results from previous studies suggest that CAD genes involved in wood development belong to Class I and Class II.
The other CAD genes from Class II and Class III may function in plant tissues under biotic stresses. The conservation of most duplicated CAD genes, the differential distribution of motifs in their promoter regions, and the divergence of their expression profiles in various tissues of Populus plants indicate that genes in the CAD family have evolved tissue-specialized expression profiles and may have divergent functions. Lignin is a phenolic heteropolymer that provides plant cells with structural rigidity, a barrier against insects and other pestilent species, and hydrophobicity –. Its role in hydrophobicity helps xylem cells facilitate the conduction of water and minerals throughout the plant. Lignin is the second most abundant plant molecule on earth next to cellulose and comprises approximately 35% of the dry matter of wood in some tree species. The composition of lignin consists of various phenylpropanoids, predominantly the monolignols p-coumaryl, coniferyl, and sinapyl alcohols. Lignin varies in content and composition between gymnosperms and angiosperms.
In gymnosperms, lignin contains guaiacyl subunits (G units) and p-hydroxyphenyl units (H units) polymerized from coniferyl alcohol and from p-coumaryl alcohol respectively. Lignin in angiosperms comprises, in addition to G-units and some H-units , syringyl units (or S-units) polymerized from sinapyl alcohol. However, there are exceptions found within each group and variation in lignin composition can even occur between cell types within the same plant.The monolignol biosynthetic pathway involves many intermediates and enzymes. The first step in the process consists of a deamination of phenylalanine by the phenylalanine ammonia-lyase (PAL) that produces cinnamic acid. Cinnamic acid is then hydroxylated by the enzyme cinnamate-4-hydroxylase (C4H) producing p-coumaric acid , which is in turn activated by 4-coumarate:CoA ligase (4CL) to produce p-coumaroyl-CoA ,.
This product is processed by cinnamoyl-CoA reductase (CCR) to coniferaldehyde, which in turn is converted to coniferyl alcohol by the action of CAD. P-coumaroyl-CoA can also be transformed to p-coumaroyl-CoA shikimate by the action of hydroxycinamoyl transferase (HCT). P-coumaroyl-CoA shikimate proceeds through a series of transformations into caffeoyl shikimate, caffeoyl-CoA, feruloyl CoA, and coniferaldehyde by the action of the enzymes p-coumarate 3-hydrolase (C3H), HCT, caffeoyl-CoA O-methyltransferase (CCOMT), and cinnamoyl CoA reductase (CCR), respectively. Coniferaldehyde can be transformed to coniferyl alcohol by the action of CAD or lead to 5-Hydroxy- coniferaldehyde and sinapyl aldehyde under the action of ferulate 5-hydrolase (F5H) and caffeic/5-hydroxyferulic acid O-methyltransferase (COMT).
The sinapyl alcohol is produced either from sinapyl aldehyde by CAD or from coniferyl alcohol by F5H and COMT. It has also been reported that the synthesis of sinapyl alcohol can be catalyzed by sinapyl alcohol dehydrogenase (SAD). However, recent studies did not find any detectable sinapyl alcohol dehydrogenase activity in Arabidopsis and Oryza indicating that the same CAD gene products can synthesize both coniferyl and sinapyl alcohols.Because of its economic importance and biological role in various developmental and defense processes, the function of lignin biosynthesis related genes has been well studied in various plants ,. Down-regulation of genes involved in the early steps of the monolignol synthesis pathway can lead to a reduction in lignin biosynthesis. However, altered expression of CAD genes in various plants resulted in only slight variations in lignin content –.
This is mainly due to the incorporation of other phenolic products that compensate for monolignols in lignin as well as the compensation by other members of the CAD gene family. A significant reduction of lignin was detected in natural CAD mutants in Pinus (5%) and the bm2, bm3, and bm4 mutants in maize (20%) ,. The gene underlying the bm1 mutant in maize is not a CAD gene, however, and may encode a regulator of several CAD genes. Down-regulating the expression of CAD genes in Nicotiana tabacum, Populus, and Pinus showed no gross morphological variations but CAD deficient plants were enriched in coniferyl aldehyde and sinapyl aldehyde ,. The accumulation of the aldehyde molecules is responsible for the red-brown color in the stems of natural and induced CAD mutants in Populus, Zea, Oryza, and Pinus ,. A recent study in Arabidopsis showed that double mutants in the two major CAD genes associated with lignin biosynthesis ( AtCADC and AtCADD named AtCAD4 and AtCAD5) present prostrate stems because of the weakness of the vasculature.
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A reduction in the size and the diameter of the stems was also observed in the double mutant plants. Beside its role in plant development, CAD also seems to play a key role in plant defense against abiotic and biotic stresses ,.CAD proteins are encoded by a gene family in plants ,.
Complete sets of CAD genes and CAD-like genes have been previously identified in the genomes of model species ( Arabidopsis, Oryza, and Populus) and partially from expressed sequences of non-model plants. In Arabidopsis, CAD exists as a multigene family consisting of nine genes ( AtCAD1 to AtCAD9) ,. Although all nine have been classified as CAD genes based on their predicted protein sequences, only CAD-C ( AtCAD5) and CAD-D ( AtCAD4) have been shown to have major roles in lignin synthesis in Arabidopsis ,. AtCAD7 and AtCAD8 may also be involved to some extent in lignin biosynthesis. AtCAD2, AtCAD3, AtCAD6, and AtCAD9 appear to encode mannitol dehydrogenases. A double mutation of AtCAD2 and AtCAD6 led to an over-expression of AtCAD1 ( AtCAD7) suggesting a compensation between some CAD genes. In Oryza, 12 CAD genes have been reported.Phylogenetic analysis of the predicted amino acid sequences of CAD genes in Arabidopsis has shown that CAD is organized into three classes with gymnosperm sequences clustering in a separate group.
On the contrary, another study showed that CAD genes were distributed in two classes both containing monocot and eudicot genes. The contradictory results obtained in these two studies were obtained using a limited set of genes and were not conclusive.In this study we retrieved and compared CAD sequences from a wide variety of plants, making full use of the available plant genome sequences ( Arabidopsis, Oryza, Populus, Medicago, and Vitis) as well as expressed sequence databases for species of basal angiosperms, gymnosperms, and mosses. This dataset was used to analyze the phylogeny of the CAD gene family. We also analyzed the organization, the structure, and the expression of CAD genes in Populus. This provided insight into the evolution of their structure and function as well as mechanisms that contributed to gene duplications. CAD gene family organizationIn model species for which the genome is completely sequenced, 71 CAD genes have been identified to date (see Additional file ): 9 in Arabidopsis , 12 in Oryza , 15 in Populus (this study), 18 in Vitis (this study), and 17 in Medicago (this study).
Furthermore, we identified 54 more CAD genes in 31 other species, which include a variety of eudicots, monocots, basal angiosperms, and gymnosperms. Additional file includes the list of these CAD gene names based on the standard established by the International Populus Genome Consortium (IPGC) with the names of species (Poptr for Populus trichocarpa for example), the protein name (CAD), and a designation of family and clade memberships derived from this study. Additional file also provides the accession number and database source for each gene.Analysis of the physical gene distribution in the Arabidopsis and Populus genomes showed that most CAD genes were located on duplicated blocks. In Arabidopsis only one gene ( AtCAD5) is not located on duplicated chromosomal blocks.
Almost all of the genes are still in conserved positions within the duplicated blocks. In Populus, we found 14 of the 15 CAD genes distributed on duplicated regions. The Populus CAD genes were distributed on seven chromosomes with chromosomes I, IX, and XVI having three or more genes each (Fig. PoptrCAD9 was located on a scaffold not yet assigned to a chromosome (see Additional file ). Homologous pairs from the nine duplicated genes ( PoptrCAD6, PoptrCAD11, PoptrCAD3, PoptrCAD4, PoptrCAD15, PoptrCAD16.