Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala

Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.Keyword: Abiotic stress, Cinnamoyl CoA reductase, Developing seedlings, Leucaena leucocephala     

in Silico mutagenesis and docking studies of active site residues suggest altered substrate specificity and possible physiological role of Cinnamoyl CoA Reductase 1 (Ll-CCRH1)

Cinnamoyl CoA reductase (CCR) carries out the first committed step in monolignol biosynthesis and acts as a first regulatory point in lignin formation. CCR shows multiple substrate specificity towards various cinnamoyl CoA esters. Here, in Silico mutagenesis studies of active site residues of Ll-CCRH1 were carried out. Homology modeling based modeled 3D structure of Ll-CCRH1 was used as template for in Silico mutant preparations. Docking simulations of Ll-CCRH1 mutants with CoA esters by AutoDock Vina tools showed altered substrate specificity as compared to wild type. The study evidences that conformational changes, and change in geometry or architecture of active site pocket occurred following mutations. The altered substrate specificity for active site mutants suggests the possible physiological role of CCR either in lignin formation or in defense system in plants.

Abbreviations

Ll-CCRH1 - Leucaena leucocephala cinnamoyl CoA reductase 1, OPLS - Optimized Potentials for Liquid Simulations, RMSD - Root Mean Square Deviation.     

Characterization of a cinnamoyl-CoA reductase that is associated with stem development in wheat

Cinnamoyl-CoA reductase (CCR) is responsible for the CoA ester to aldehyde conversion in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of lignin biosynthesis and its biological function, a cDNA encoding CCR was identified from wheat (Triticum aestivum L.), and designated as Ta-CCR1. Phylogenetic analysis indicated that Ta-CCR1 grouped together with other monocot CCR sequences while it diverged from Ta-CCR2. DNA gel-blot and mapping analyses demonstrated that Ta-CCR1 is present as a single copy gene in the wheat genome. Recombinant Ta-CCR1 protein converted feruloyl CoA, 5-OH-feruloyl CoA, sinapoyl CoA, and caffeoyl CoA, but feruloyl-CoA was the best substrate, suggesting the preferential biosynthesis of G-type lignin. RNA gel-blot analysis indicated that Ta-CCR1 was highly expressed in stem, with lower expression in leaves, and undetectable expression in roots. CCR enzyme activity was increased progressively along with the lignin biosynthesis and stem maturity. During stem development, Ta-CCR1 mRNA levels remained high at elongation, heading, and milky stages in the wheat H4564 cultivar, while they declined dramatically at the heading and milky stages in stems of the C6001 cultivar. Ta-CCR1 mRNA expression paralleled extractable CCR enzyme activity in these two cultivars. Furthermore, high Ta-CCR1 mRNA levels and high CCR enzyme activity in wheat stem were correlated with a higher Klason lignin content and greater stem mechanical strength in the H4564 cultivar. This suggests that Ta-CCR1 and its related CCR enzyme may be involved in the regulation of lignin biosynthesis during stem maturity and then contributes to stem strength support in wheat.     

Kinetic and inhibition studies of cinnamoyl-CoA reductase 1 from Arabidopsis thaliana.

Cinnamoyl coenzyme A reductase (CCR, EC 1.2.1.44), one of the key enzymes in the biosynthesis of lignin monomers, catalyzes the NADPH-dependent reduction of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. AtCCR1, one of the two distinct isoforms isolated from Arabidopsis thaliana, was shown to be involved in lignin biosynthesis during development. Here, we report on the purification of the recombinant AtCCR1 protein expressed in Escherichia coli and the subsequent determination of its kinetic properties (K(m) and k(cat)/K(m) values) towards its main substrates i.e. feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA esters. In addition, the potential inhibitory effect of five substrate-like analogs possessing an N-acetylcysteamine thioester group was tested on CCR activity using either feruloyl-CoA or sinapoyl-CoA as substrates. The K(i) values were in the range of 4.4-502 microM and the type of inhibition was found to be either uncompetitive or noncompetitive. Interestingly, for compounds 3 and 5, the type of inhibition was found to be different depending on the substrate used to monitor the enzyme activity. The best inhibitors were those possessing the feruloyl (compound 3) and sinapoyl (compound 5) aromatic moiety (4.1 and 7.1 microM) while the enzyme activity was monitored using the corresponding substrates.     

Biochemical characterization of a cinnamoyl-CoA reductase from wheat

Cinnamoyl-CoA reductase (CCR) is responsible for the CoA ester-->aldehyde conversion in monolignol biosynthesis, which can divert phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of lignin biosynthesis in wheat (Triticum aestivum L.), a cDNA encoding CCR was isolated and named Ta-CCR2. DNA hybridization analyses demonstrated that the Ta-CCR2 gene exists in three copies in the wheat genome. RNA blot hybridization indicated that Ta-CCR2 was expressed most abundantly in root and stem tissues that were in the process of lignification. The secondary and three-dimensional structures of Ta-CCR2 were analyzed by molecular modeling. Recombinant Ta-CCR2 protein purified from E. coli converted feruloyl CoA, 5-OH-feruloyl CoA, sinapoyl CoA and caffeoyl CoA with almost similar efficiency, suggesting that it is involved in both G and S lignin synthesis. Ta-CCR2 had a very low V max value for 4-coumaroyl CoA, which may serve as a mechanism to control metabolic flux to H lignin in vivo . Furthermore, the reaction mechanism of Ta-CCR2 was analyzed in relation to its possible three-dimensional structure. The activity of Ta-CCR2 in relation to lignin biosynthesis is discussed.     

Down-regulation of the<Emphasis Type="Italic"> AtCCR1</Emphasis> gene in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>: effects on phenotype,lignins and cell wall degradability

Cinnamoyl CoA reductase (CCR; EC 1.2.1.44) is the first enzyme specific to the biosynthetic pathway leading to monolignols. Arabidopsis thaliana (L.) Heynh. plants transformed with a vector containing a full-length AtCCR1 cDNA in an antisense orientation were obtained and characterized. The most severely down-regulated homozygous plants showed drastic alterations to their phenotypical features. These plants had a 50% decrease in lignin content accompanied by changes in lignin composition and structure, with incorporation of ferulic acid into the cell wall. Microscopic analyses coupled with immunolabelling revealed a decrease in lignin deposition in normally lignified tissues and a dramatic loosening of the secondary cell wall of interfascicular fibers and vessels. Evaluation of in vitro digestibility demonstrated an increase in the enzymatic degradability of these transgenic lines. In addition, culture conditions were shown to play a substantial role in lignin level and structure in the wild type and in the effects of AtCCR1 repression efficiency.     

Redirection of the phenylpropanoid pathway to feruloyl malate in <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants deficient for cinnamoyl-CoA reductase 1

Cinnamoyl-CoA reductase 1 (CCR1, gene At1g15950) is the main CCR isoform implied in the constitutive lignification of Arabidopsis thaliana. In this work, we have identified and characterized two new knockout mutants for CCR1. Both have a dwarf phenotype and a delayed senescence. At complete maturity, their inflorescence stems display a 25–35% decreased lignin level, some alterations in lignin structure with a higher frequency of resistant interunit bonds and a higher content in cell wall-bound ferulic esters. Ferulic acid-coniferyl alcohol ether dimers were found for the first time in dicot cell walls and in similar levels in wild-type and mutant plants. The expression of CCR2, a CCR gene usually involved in plant defense, was increased in the mutants and could account for the biosynthesis of lignins in the CCR1-knockout plants. Mutant plantlets have three to four-times less sinapoyl malate (SM) than controls and accumulate some feruloyl malate. The same compositional changes occurred in the rosette leaves of greenhouse-grown plants. By contrast and relative to the control, their stems accumulated unusually high levels of both SM and feruloyl malate as well as more kaempferol glycosides. These findings suggest that, in their hypolignified stems, the mutant plants would avoid the feruloyl-CoA accumulation by its redirection to cell wall-bound ferulate esters, to feruloyl malate and to SM. The formation of feruloyl malate to an extent far exceeding the levels reported so far indicates that ferulic acid is a potential substrate for the enzymes involved in SM biosynthesis and emphasizes the remarkable plasticity of Arabidopsis phenylpropanoid metabolism.     

Sequence analysis, in silico modeling and docking studies of Caffeoyl CoA-O-methyltransferase of Populus trichopora

Caffeoyl coenzyme A-O-methyltransferases (CCoAOMTs) which are characterized under class I plant OMTs, methylates CoA thioesters, with an in vitro kinetic preference for caffeoyl CoA. CCoAOMTs exhibit association with lignin biosynthesis by showing a prime role in the synthesis of guaiacyl lignin and providing the substrates for synthesis of syringyl lignin. The sequence analysis of CCoAOMT from Populus trichopora exhibits 58 nucleotide substitutions, where transitions overcome transversions. Validation of homology models of both CCoAOMT1 and 2 isoforms reveals that 92.4% and 96% residues are falling in the most favorable region respectively in the Ramachandran plot, indicating CCoAOMT2 as the more satisfactory model, and the overall quality factor of both isoforms is 98.174. The structural architecture analysis is showing very good packing of residues similar to protein crystal structures data. The active site residues and substrate-product interactions showed that CCoAOMT2 possesses more affinity toward caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapoyl CoA than CCoAOMT1, therefore it exist in a more active conformation. The affinity of CCoAOMT2 with feruloyl CoA is highest among all the affinities of both CCoAOMT isoforms with their substrates and products. This information has potential implications to understand the mechanism of CCoAOMT related enzymatic reactions in Populus trichopora, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.     

Reassessment of effects on lignification and vascular development in the irx4 Arabidopsis mutant

The Arabidopsis thaliana irregular xylem4 (irx4) cinnamoyl-CoA reductase 1 (CCR1) mutant was reassessed for its purported exclusive rate-limiting or key effects on lignification. Analyses of gross growth characteristics and stem cross-section anatomy, from seedling emergence to senescence, revealed that stunted irx4 mutant lines were developmentally delayed, which in turn indirectly but predictably led to modest reductions (ca. 10-15%) in overall lignin amounts. Such developmental changes are not generally observed in suppression of other monolignol pathway forming enzymes (e.g., 4-coumarate CoA ligase) even when accompanied by significant reductions in lignin amounts. With the greatly arrested development of the irx4 mutant, formation of the lignin-derived syringyl moieties was also predictably delayed (by about 1-2 weeks), although at maturation the final guaiacyl:syringyl ratios were essentially identical to wild-type. No evidence was obtained for so-called abnormal lignin precursors being incorporated into the lignin, as shown by solid-state 13C NMR spectroscopic analysis in contrast to a claim to the contrary [Jones, L., Ennos, A.R., Turner, S.R., 2001. Cloning and characterization of irregular xylem4 (irx4): a severely lignin-deficient mutant of Arabidopsis. Plant J. 26, 205-216]. A previous claim of an "abnormal" lignin present in stunted CCR downregulated tobacco was also not substantiated, with only trace differences being noted in the presumed cell-wall constituent levels. More importantly, a linear correlation between total lignin amounts and lignin-derived fragmentation products was observed at all stages of Arabidopsis growth/development in both wild-type and irx4 mutant lines, regardless of lignin content, i.e., in harmony with an exquisitely controlled and predictable macromolecular assembly process. Recombinant CCR1 displayed fairly broad substrate versatility for all phenylpropanoid CoA substrates, with both feruloyl and 5-hydroxyferuloyl CoA being the best substrates. Taken together, these data indicate that other CCR isoforms are apparently capable of generating monolignol-derived lignified elements in irx4 when CCR1 is impaired, i.e., indicative of a functionally redundant CCR metabolic network operative in Arabidopsis. Other dwarfed phenotypes have also been observed following downregulation/disruption of unrelated metabolic processes but which also involve CoA ester metabolism, i.e., with hydroxymethylglutaryl CoA reductases in Arabidopsis and a bacterial enoyl CoA hydratase/lyase overexpressed in tobacco. Although the reasons for dwarfing in each case are unknown, a common mechanism for the various pleiotropic effects is proposed through perturbation of CoASH pool levels. Finally, this study demonstrates the need for progressive analyses over the lifespan of an organism, rather than at a single time point which cannot reveal the progressive developmental changes occurring.     

Clarification of cinnamoyl co-enzyme A reductase catalysis in monolignol biosynthesis of Aspen

Cinnamoyl co-enzyme A reductase (CCR), one of the key enzymes involved in the biosynthesis of monolignols, has been thought to catalyze the conversion of several cinnamoyl-CoA esters to their respective cinnamaldehydes. However, it is unclear which cinnamoyl-CoA ester is metabolized for monolignol biosynthesis. A xylem-specific CCR cDNA was cloned from aspen (Populus tremuloides) developing xylem tissue. The recombinant CCR protein was produced through an Escherichia coli expression system and purified to electrophoretic homogeneity. The biochemical properties of CCR were characterized through direct structural corroboration and quantitative analysis of the reaction products using a liquid chromatography-mass spectrometry system. The enzyme kinetics demonstrated that CCR selectively catalyzed the reduction of feruloyl-CoA from a mixture of five cinnamoyl CoA esters. Furthermore, feruloyl-CoA showed a strong competitive inhibition of the CCR catalysis of other cinnamoyl CoA esters. Importantly, when CCR was coupled with caffeoyl-CoA O-methyltransferase (CCoAOMT) to catalyze the substrate caffeoyl-CoA ester, coniferaldehyde was formed, suggesting that CCoAOMT and CCR are neighboring enzymes. However, the in vitro results also revealed that the reactions mediated by these two neighboring enzymes require different pH environments, indicating that compartmentalization is probably needed for CCR and CCoAOMT to function properly in vivo. Eight CCR homologous genes were identified in the P. trichocarpa genome and their expression profiling suggests that they may function differentially.     

Phylogenetic analysis, homology modelling, molecular dynamics and docking studies of caffeoyl–CoA-O- methyl transferase (CCoAOMT 1 and 2) isoforms isolated from subabul (Leucaena leucocephala)

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) is an important enzyme that participates in lignin biosynthesis especially in the formation of cell wall ferulic esters of plants. It plays a pivotal role in the methylation of the 3-hydroxyl group of caffeoyl CoA. Two cDNA clones that code CCoAOMT were isolated earlier from subabul and in the present study; 3D models of CCoAOMT1 and CCoAOMT2 enzymes were built using the MODELLER7v7 software to find out the substrate binding sites. These two proteins differed only in two amino acids and may have little or no functional redundancy. Refined models of the proteins were obtained after energy minimization and molecular dynamics in a solvated water layer. The models were further assessed by PROCHECK, WHATCHECK, Verify_3D and ERRAT programs and the results indicated that these models are reliable for further active site and docking analysis. The refined models showed that the two proteins have 9 and 10 α-helices, 6 and 7 β-sheets respectively. The models were used for docking the substrates CoA, SAM, SAH, caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapyl CoA which showed that CoA and caffeoyl CoA are binding with high affinity with the enzymes in the presence and absence of SAM. It appears therefore that caffeoyl CoA is the substrate for both the isoenzymes. The results also indicated that CoA and caffeoyl CoA are binding with higher affinity to CCoAOMT2 than CCoAOMT1. Therefore, CCoAOMT2 conformation is thought to be the active form that exists in subabul. Docking studies indicated that conserved active site residues Met58, Thr60, Val63, Glu82, Gly84, Ser90, Asp160, Asp162, Thr169, Asn191 and Arg203 in CCoAOMT1 and CCoAOMT2 enzymes create the positive charge to balance the negatively charged caffeoyl CoA and play an important role in maintaining a functional conformation and are directly involved in donor-substrate binding.     

In situ analysis of lignins in transgenic tobacco reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular and subcellular levels

Using tobacco transgenic lines altered in the monolignol biosynthetic pathway and which differ in their lignin profiles we have evaluated lignin deposition at the cellular and subcellular levels using several microanalytical techniques. Surprisingly, whereas a Cinnamoyl CoA reductase (CCR) down-regulated line with a strong decrease in lignin content exhibited an overall reduction in lignin deposition in the walls of the different xylem cell types, this reduction was selectively targeted to the fibers in a double transformant (down-regulated for both CCR and Cinnamyl alcohol dehydrogenase (CAD)) displaying a similar degree of global lignin content decrease. Fiber and vessel secondary walls of the transgenic tobacco line homozygous for the ccr antisense gene (CCR.H) down-regulated plants were dramatically destructured, particularly in the S2 sublayer, whereas the deposition of lignins in the S1 sublayer was not significantly modified. In contrast, cell wall organization was slightly altered in xylem cells of the double transformant. The relative distribution of non-condensed and condensed units in lignin, evaluated microscopically with specific antibodies, was differentially affected in the transgenics studied and, in a general way, a drop in non-condensed lignin units (beta- 0-4 interunit linkages) was associated with a loss of cohesion and extensive disorganization of the secondary wall. These results demonstrate that lignification is tightly and independently regulated in individual cell types and cell wall sublayers. They also show that down-regulation of specific genes may induce targeted changes in lignin structure and in spatial deposition patterns of the polymer.     

The Arabidopsis cinnamoyl CoA reductase irx4 mutant has a delayed but coherent (normal) program of lignification

Previous studies have indicated that the Arabidopsis thalianairregular xylem 4 (irx4) mutant is severely lignin-deficient, forming abnormal lignin from aberrant monomers. Studies of lignin structure in dwarfed cinnamoyl CoA reductase (CCR)-downregulated tobacco were also previously reported to incorporate feruloyl tyramine derivatives. The lignin in the Arabidopsis irx4 mutant was re-investigated at 6 weeks and at maturation (9 weeks). Application of (1)H, (13)C, 2D Heteronuclear Multiple Quantum Coherence and 2D Heteronuclear Multiple Bond Coherence spectroscopic analyses to the lignin-enriched isolates from both Arabidopsis wild-type (Ler) and the CCR-irx4 mutant at both developmental stages revealed that only typical guaiacyl/syringyl lignins were formed. For the irx4 mutant, the syringyl content at 6 weeks growth was lower, in accordance with a delayed but coherent program of lignification. At maturation, however, the syringyl/guaiacyl ratio of the irx4 mutant approached that of wild-type. There was no evidence for feruloyl tyramines, or homologues thereof, accumulating as a chemical signature in lignins resulting from CCR mutation. Nor were there any noticeable increases in other phenolic components, such as hydroxycinnamic acids. These findings were further confirmed by application of thioacidolysis, alkaline nitrobenzene oxidation and acetyl bromide analyses. Moreover, in the case of CCR downregulation in tobacco, there were no NMR spectroscopic correlations that demonstrated feruloyl tyramines being incorporated into the lignin biopolymers. This study thus found no evidence that abnormal lignin formation occurs when CCR activity is modulated.