Cloning and in silico analysis of a cinnamyl alcohol dehydrogenase gene in Pennisetum purpureum

Lignin is a major constituent of plant cell walls and indispensable to the normal growth of a plant. However, the presence of lignin complicates the structure of the plant cell walls and negatively influences pulping industry, lignocellulose utilization as well as forage properties. Cinnamyl alcohol dehydrogenase (CAD), a key enzyme involved in lignin biosynthesis, catalyses the last step in monolignol synthesis and has a major role in genetic regulation of lignin production. In the present study, a 1 342-bp cDNA fragment of CAD gene, named PpCAD, was isolated from Pennisetum purpureum using strategies of homologous clone and rapid amplification of cDNA end. It was translated into an intact protein sequence including 366 amino acid residues by ORF Finder. The genomic full-length DNA of PpCAD was a 3 738-bp sequence containing four exons and three introns, among which the 114-bp exon was considered to be a conserved region compared with other CADs. Basic bioinformatic analysis presumed that the PpCAD was a nonsecretory and hydrophobic protein with five possible transmembrane helices. The phylogenetic analysis indicated that the PpCAD belonged to the class of bona fide CADs involved in lignin synthesis and it showed a high similarity (nearly 90%) with CAD protein sequences of Sorghum bicolor, Panicum virgatum and Zea mays in Gramineae. Furthere, PpCAD amino acid sequence was demonstrated to have some conserved motifs such as Zn-binding site, Zn-catalytic centre and NADP(H) binding domain after aligning with other bona fide CADs. Three-dimensional homology modelling of PpCAD showed that the protein had some exclusive features of bona fide CADs.     

Expression patterns of a cinnamyl alcohol dehydrogenase gene involved in lignin biosynthesis and environmental stress in Ginkgo biloba

The cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis as it catalyzes the final step in the synthesis of monolignols. A cDNA sequence encoding the CAD gene was isolated from the leaves of Ginkgo biloba L, designated as GbCAD1. The full-length cDNA of GbCAD1 was 1,494?bp containing a 1,074?bp open reading frame encoding a polypeptide of 357 amino acids with a calculated molecular mass of 38.7?kDa and an isoelectric point of 5.74. Comparative and bioinformatic analyses revealed that GbCAD1 showed extensive homology with CADs from other gymnosperm species. Southern blot analysis indicated that GbCAD1 belonged to a multi-gene family. Phylogenetic tree analysis revealed that GbCAD1 shared the same ancestor in evolution with other CADs and had a further relationship with other gymnosperm species. GbCAD1 was an enzyme being pH-dependent and temperature-sensitive, and showing a selected catalyzing. Tissue expression pattern analysis showed that GbCAD1 was constitutively expressed in stems and roots, especially in the parts of the pest and disease infection, with the lower expression being found in two- to four-year-old stem. Further analysis showed the change in lignin content had some linear correlation with the expression level of GbCAD1 mRNA in different tissues. The increased expression of GbCAD1 was detected when the seedling were treated with exogenous abscisic acid, salicylic acid, ethephon, ultraviolet and wounding. These results indicate that the GbCAD1 gene may play a role in the resistance mechanism to biotic and abiotic stresses as well as in tissue-specific developmental lignification.     

A Genomewide Analysis of the Cinnamyl Alcohol Dehydrogenase Family in Sorghum [Sorghum bicolor (L.) Moench] Identifies SbCAD2 as the Brown midrib6 Gene

The content and composition of the plant cell wall polymer lignin affect plant fitness, carbon sequestration potential, and agro-industrial processing. These characteristics, are heavily influenced by the supply of hydroxycinnamyl alcohol precursors synthesized by the enzyme cinnamyl alcohol dehydrogenase (CAD). In angiosperms, CAD is encoded by a multigene family consisting of members thought to have distinct roles in different stages of plant development. Due to the high sequence similarity among CAD genes, it has been challenging to identify and study the role of the individual genes without a genome sequence. Analysis of the recently released sorghum genome revealed the existence of 14 CAD-like genes at seven genomic locations. Comparisons with maize and rice revealed subtle differences in gene number, arrangement, and expression patterns. Sorghum CAD2 is the predominant CAD involved in lignification based on the phylogenetic relationship with CADs from other species and genetic evidence showing that a set of three allelic brown midrib (bmr) lignin mutants contained mutations in this gene. The impact of the mutations on the structure of the protein was assessed using molecular modeling based on X-ray crystallography data of the closely related Arabidopsis CAD5. The modeling revealed unique changes in structure consistent with the observed phenotypes of the mutants.     

Purification, Characterization, and Cloning of Cinnamyl Alcohol Dehydrogenase in Loblolly Pine (Pinus taeda L.)

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1. 195) has been purified to homogeneity from differentiating xylem tissue and developing seeds of loblolly pine (Pinus taeda L.). The enzyme is a dimer with a native molecular weight of 82,000 and a subunit molecular weight of 44,000, and is the only form of CAD involved in lignification in differentiating xylem. High levels of loblolly pine CAD enzyme were found in nonlignifying seed tissue. Characterization of the enzyme from both seeds and xylem demonstrated that the enzyme is the same in both tissues. The enzyme has a high affinity for coniferaldehyde (K_m = 1.7 micromolar) compared with sinapaldehyde (K_m in excess of 100 micromolar). Kinetic data strongly suggest that coniferin is a noncompetitive inhibitor of CAD enzyme activity. Protein sequences were obtained for the N-terminus (28 amino acids) and for two other peptides. Degenerate oligonucleotide primers based on the protein sequences were used to amplify by polymerase chain reaction a 1050 base pair DNA fragment from xylem cDNA. Nucleotide sequence from the cloned DNA fragment coded for the N-terminal protein sequence and an internal peptide of CAD. The N-terminal protein sequence has little similarity with the λCAD4 clone isolated from bean (MH Walter, J Grima-Pettenati, C Grand, AM Boudet, CJ Lamb [1988] Proc Natl Acad Sci USA 86:5546-5550), which has homology with malic enzyme.     

Molecular cloning and functional analysis of nine cinnamyl alcohol dehydrogenase family members in Populus tomentosa

Main conclusion

Nine CAD/CAD-like genes in P. tomentosa were classified into four classes based on expression patterns, phylogenetic analysis and biochemical properties with modification for the previous claim of SAD. Cinnamyl alcohol dehydrogenase (CAD) functions in monolignol biosynthesis and plays a critical role in wood development and defense. In this study, we isolated and cloned nine CAD/CAD-like genes in the Populus tomentosa genome. We investigated differential expression using microarray chips and found that PtoCAD1 was highly expressed in bud, root and vascular tissues (xylem and phloem) with the greatest expression in the root. Differential expression in tissues was demonstrated for PtoCAD3, PtoCAD6 and PtoCAD9. Biochemical analysis of purified PtoCADs in vitro indicated PtoCAD1, PtoCAD2 and PtoCAD8 had detectable activity against both coniferaldehyde and sinapaldehyde. PtoCAD1 used both substrates with high efficiency. PtoCAD2 showed no specific requirement for sinapaldehyde in spite of its high identity with so-called PtrSAD (sinapyl alcohol dehydrogenase). In addition, the enzymatic activity of PtoCAD1 and PtoCAD2 was affected by temperature. We classified these nine CAD/CAD-like genes into four classes: class I included PtoCAD1, which was a bone fide CAD with the highest activity; class II included PtoCAD2, -5, -7, -8, which might function in monolignol biosynthesis and defense; class III genes included PtoCAD3, -6, -9, which have a distinct expression pattern; class IV included PtoCAD12, which has a distinct structure. These data suggest divergence of the PtoCADs and its homologs, related to their functions. We propose genes in class II are a subset of CAD genes that evolved before angiosperms appeared. These results suggest CAD/CAD-like genes in classes I and II play a role in monolignol biosynthesis and contribute to our knowledge of lignin biosynthesis in P. tomentosa.     

LtuCAD1 Is a Cinnamyl Alcohol Dehydrogenase Ortholog Involved in Lignin Biosynthesis in Liriodendron tulipifera L., a Basal Angiosperm Timber Species

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis and catalyzes the final step in the synthesis of monolignols. Seven CAD homologs (LtuCAD1 to LtuCAD7) have been previously identified from a basal angiosperm species Liriodendron tulipifera L., which is an important timber tree species with significant ecological and economic values. The phylogenetic analysis indicates that LtuCAD1 is the only Liriodendron CAD grouped with the bona fide CADs, the primary CAD genes involved in lignification. In this study, the predicted protein sequence of LtuCAD1 was found to have conserved domains and the same key determinant site with the bona fide CADs in other plant species. Additionally, LtuCAD1 had the highest expression level in xylem as revealed by quantitative RT-PCR analysis. The expression of beta-glucuronidase (GUS) driven by the LtuCAD1 promoter was largely localized in vascular tissues in Arabidopsis. In stem cross sections, GUS staining was found exclusively in xylem and phloem. When expressed in the Arabidopsis cad4 cad5 double mutant, LtuCAD1 was able to restore the total lignin content and decrease the S/G lignin ratio. Our data indicate that LtuCAD1 is a CAD ortholog involved in lignin biosynthesis in Liriodendron.     

The Evolution of the Conserved ATPase Domain (CAD): Reconstructing the History of an Ancient Protein Module

The AAA proteins (ATPases Associated with a variety of cellular Activities) are found in eubacterial, archaebacterial, and eukaryotic species and participate in a large number of cellular processes, including protein degradation, vesicle fusion, cell cycle control, and cellular secretory processes. The AAA proteins are characterized by the presence of a 230 to 250-amino acid ATPase domain referred to as the Conserved ATPase Domain or CAD. Phylogenetic analysis of 133 CAD sequences from 38 species reveal that AAA CADs are organized into discrete groups that are related not only in structure but in cellular function. Evolutionary analyses also indicate that the CAD was present in the last common ancestor of eubacteria, archaebacteria, and eukaryotes. The eubacterial CADs are found in metalloproteases, while CAD-containing proteins in the archaebacterial and eukaryotic lineages appear to have diversified by a series of gene duplication events that lead to the establishment of different functional AAA proteins, including proteasomal regulatory, NSF/Sec, and Pas proteins. The phylogeny of the CADs provides the basis for establishing the patterns of evolutionary change that characterize the AAA proteins. Received: 28 January 1997 / Accepted: 8 May 1997     

A novel activation mechanism of caspase-activated DNase from Drosophila melanogaster

Caspase-activated DNase (CAD) is an enzyme that cleaves chromosomal DNA in apoptotic cells. Here, we identified a DNase in Drosophila Schneider cells that can be activated by caspase 3, and purified it as a complex of two subunits (p32 and p20). Using primers based on the amino acid sequence of the purified proteins, a cDNA coding for Drosophila CAD (dCAD) was cloned. The polypeptide encoded by the cDNA contained 450 amino acids with a calculated M(r) of 52,057, and showed significant homology with human and mouse CAD (22% identity). Mammalian CADs carry a nuclear localization signal at the C terminus. In contrast, dCAD lacked the corresponding sequence, and the purified dCAD did not cause DNA fragmentation in nuclei in a cell-free system. When dCAD was co-expressed in COS cells with Drosophila inhibitor of CAD (dICAD), a 52-kDa dCAD was produced as a heterotetrameric complex with dICAD. When the complex was treated with human caspase 3 or Drosophila caspase (drICE), the dICAD was cleaved, and released from dCAD. In addition, dCAD was also cleaved by these caspases, and behaved as a (p32)(2)(p20)(2) complex in gel filtration. When a Drosophila neuronal cell line was induced to apoptosis by treatment with a kinase inhibitor, both dCAD and dICAD were cleaved. These results indicated that unlike mammalian CAD, Drosophila CAD must be cleaved by caspases to be activated.     

Switchgrass, Big Bluestem, and Indiangrass Monocultures and Their Two- and Three-Way Mixtures for Bioenergy in the Northern Great Plains

High yielding, native warm-season grasses could be used as renewable bioenergy feedstocks. The objectives of this study were to determine the effect of warm season grass monocultures and mixtures on yield and chemical characteristics of harvested biomass and to evaluate the effect of initial seeding mixture on botanical composition over time. Switchgrass (Panicum virgatum L.), indiangrass [Sorghastrum nutans (L.) Nash], and big bluestem (Andropogon gerardii Vitman) were planted as monocultures and in all possible two- and three-way mixtures at three USA locations (Brookings and Pierre, SD and Morris, MN) during May 2002. Biomass at each location was harvested after a killing frost once annually from 2003 to 2005. Total biomass yield significantly increased with year at all locations. Switchgrass monocultures or mixtures containing switchgrass generally out-yielded big bluestem or indiangrass in monocultures or the binary mixture. Cellulose and hemicellulose concentrations were higher in 2004 and 2005 compared with 2003. Switchgrass or mixtures containing switchgrass tended to have less cellulose than either big bluestem or indiangrass. Results were more variable for total N, lignin, and ash. Switchgrass was the dominant component of all mixtures in which it was present while big bluestem was dominant when mixed with indiangrass. Indiangrass was maintained only in monocultures and declined over years when grown in mixtures at all locations. Our results indicated if biomass yield in the northern Great Plains is a primary objective, switchgrass should be a component of binary or tertiary mixtures that also contain big bluestem and/or indiangrass.     

Unusual NADPH conformation in the crystal structure of a cinnamyl alcohol dehydrogenase from Helicobacter pylori in complex with NADP(H) and substrate docking analysis

Cinnamyl alcohol dehydrogenase is a zinc- and NADPH-dependent dehydrogenase catalyzing the reversible conversion of p-hydroxycinnamaldehydes to their corresponding hydroxycinnamyl alcohols. A CAD homolog from Helicobacter pylori (HpCAD) possesses broad substrate specificities like the plant CADs and additionally a dismutation activity converting benzaldehyde to benzyl alcohol and benzoic acid. We have determined the crystal structure of HpCAD complexed with NADP(H) at 2.18 Å resolution to get a better understanding of this class of CAD outside of plants. The structure of HpCAD is highly homologous to the sinapyl alcohol dehydrogenase and the plant CAD with well-conserved residues involved in catalysis and zinc binding. However, the NADP(H) binding mode of the HpCAD has been found to be significantly different from those of plant CADs.Structured summary of protein interactionsHpCAD and HpCAD bind by x-ray crystallography (View interaction)     

Review and Model-Based Analysis of Factors Influencing Soil Carbon Sequestration Beneath Switchgrass (Panicum virgatum)

A multi-compartment model was developed to summarize existing data and predict soil carbon sequestration beneath switchgrass (Panicum virgatum) in the southeastern USA. Soil carbon sequestration is an important part of sustainable switchgrass production for bioenergy because soil organic matter promotes water retention, nutrient supply, and soil properties that minimize erosion. A literature review was undertaken for the purpose of model parameterization. A sensitivity analysis of the model indicated that predictions of soil carbon sequestration were affected most by changes in aboveground biomass production, the ratio of belowground-to-aboveground biomass production, and mean annual temperature. Simulations indicated that the annual rate of soil carbon sequestration approached steady state after a decade of switchgrass growth while predicted mineral soil carbon stocks were still increasing. A model-based experiment was performed to predict rates of soil carbon sequestration at different levels of nitrogen fertilization and initial soil carbon stocks (to a 30-cm depth). At a mean annual temperature of 13°C, the predicted rate of soil carbon sequestration varied from ?28 to 114?g?C?m^?2?year^?1 (after 30?years) and was greater than zero in 11 of 12 simulations that varied initial surface soil carbon stocks from 1 to 5?kg?C?m^?2 and nitrogen fertilization from 0 to 18?g?N?m^?2?year^?1. The modeling indicated that more research is needed on the process of biomass allocation and on nitrogen loss from mature plantations, respectively, to improve our understanding of carbon and nitrogen dynamics in switchgrass agriculture.     

Switchgrass Biomass and Nitrogen Yield with Over-Seeded Cool-season Forages in the Southern Great Plains

In dry climates with long, hot summers and freezing winters, such as that of the southern Great Plains of North America, switchgrass (Panicum virgatum L.) has proven potential as a cellulosic bioenergy feedstock. This trial looked at dry matter (DM) and N yield dynamics of switchgrass overseeded with cool-season legumes and rye (Secale cereale L.), compared to switchgrass fertilized with 0, 56 and 112 kg N ha^-1 yr^-1 at an infertile and a fertile location. Optimal N fertilizer rate on switchgrass was 56 kg N ha^-1 at the infertile location. Legume yield was greater in the first season after planting, compared to subsequent years where annual legumes were allowed to reseed and alfalfa (Medicago sativa L.) was allowed to grow. This suggests that the reseeding model for annual legumes will not work in switchgrass swards grown for biomass unless soil seed banks are built up for more than one year, and that overseeding with alfalfa may have to be repeated in subsequent years to build up plant populations. Overseeding rye and legumes generally did not suppress or enhance switchgrass biomass production compared to unfertilized switchgrass. However, cumulative spring and fall biomass yields were generally greater due to winter and spring legume production, which could be beneficial for grazing or soil conservation systems, but not necessarily for once-yearly late autumn harvest biofuel production systems.