The Simultaneous Repression of CCR and CAD - Essay Example

?INTRODUCTION Numerous plant-based secondary metabolites have already been isolated. The functions of most of these biomolecules have also been well-defined. Saponins enhance nutrient absorption. Cocaine is used for protection against foraging insects. Lignins, on the other hand, contribute in the maintenance of the stable structure of secondary plant cellular walls. Aside from determining the functions of these bimolecules, the pathways in which these compounds are involved in are also being elucidated by researchers. The discovery of such complex information may allow a deeper understanding of the physiological processes that plants go through. Later on, understanding pathways may be used in the laboratory synthesis of these compounds. Moreover, the induction, as well as repression, of their production in plants through anthropogenic intervention to promote optimal plant growth and/or fruit bearing may be conducted. This is important as many secondary metabolites are already used by humans. In the case of lignins, genes CCR and CAD have been identified as the encoders for the last and specific steps for monolignol biosynthesis, the first part of lignin pathway. Because of the complex nature of biochemical production, regulation of a certain pathway may affect other pathways as well. Such effects may be great enough to cause changes in phenotype. In the case of monolignol biosynthesis in tobacco, the silencing of both CCR and CAD genes resulted not only to a reduction of lignin production but to a decrease in plant size as well. The recent study by Thevenin et al. (2011) looked into the effects of silencing monolignol-specific CAD and CCR genes in Arabidopsis thaliana. The plant contains two CCR genes, but only one, CCR 1, is specific for lignification. The mutant lines for this gene, irx4, ccr1s and ccr1g, are dwarves, have a reduced amount of lignin (25-35%), and have a modified set of phenolic metabolites. On the other hand, A. thaliana contains nine CAD genes, two of which, CAD C and CAD D, are involved in lignin biosynthesis. CAD C and CAD D double mutants, unlike CCR 1 mutants, has a normal size and 40% amount of lignin. The effects on leaf and flower morphology, lignin structure and content, amount of sugar, and other metabolites were observed. RESULTS Identification and Phenotyping of the ccc Triple Mutant After crossing ccr 1 g mutant with cad c and cad d double mutant, and producing ccc mutant, the absence of CCR 1, CAD C, and CAD D expression in ccc mutants was verified using RT-PCR. 1. Leaf Morphology The growth of wild type, ccc, cad c, cad d, and ccr 1 g from plantlets to senescence were observed at greenhouse conditions. At 30 days, the absence of CCR 1 caused the leaves to change its morphology from rosette to pointed and rolled. The decrease in leaf size was also noted most noticeably among ccc plantlets (5-fold shorter), and less prominently in ccr 1 g (3-fold shorter. On the other hand, the absence of both cad c and cad d expression did not cause any decrease in leaf size. 2. Flower Morphology More changes were observed in the flowers. Similar to leaves, the mature floral stem is smaller in ccc than in ccr 1 g mutants. However, when compared to the wild type, the triple mutant senesced later, despite its first inflorescence being prematurely shriveled. In addition, male flowers are sterile. Despite possessing normal pollens, the anthers were unable to release them. As a result, more than 50% of ccc mutants were not able to undergo seed germination. The ccc triple mutant possesses non-dehiscent anthers Initial flower development and stamen filament elongation were similar in ccc and wild-type A. thaliana. However, ccc anthers, despite containing pollens whose sizes germinating capability (through Alexander and aniline blue staining) were similar to that of wild-type, did not dehisce as what normally happens. Probably, the absence of lignified secondary thickening observed among ccc plants may have caused the non-dehiscent of anthers. The ccr 1 g plants, on the other hand, have few amounts of the said secondary thickening. The ccc mutant presents cytological modifications in the floral stem Looking closer into floral stems, ccc, cad c, and cad d mutants had red coloration on the xylem vessels and interfascicular fibers in their basal area. On the other hand, ccr 1 g and wild-type A. thaliana had no coloration. Moreover, the absence of CCR 1 caused the xylem vessels to collapse, with ccc stems having the greatest extent of collapse. Under UV light, the characteristic blue autofluorescence of lignins was drastically reduced in its fibers and xylem vessels as compared to that of wild-type A. thaliana. Through Wiesner staining, however, ccc cross section were stained at a greater extent than cad c and cad d, but reduced in ccr 1 g. Lignin content and structure are affected to various extents in the different lines The lignin content of all mutant lines were the same. However, the lignin content of extract-free, cell wall residues (CWR) of the different A. thaliana lines used for this study were different. CWR of ccc mutants was less compared to the other lines. In comparing Klason lignin (KL) and acido-soluble lignin (ASL) among the mutant lines, ccc had less KL and more ASL. In measuring H, G and S thioethylated monomers involved in B-O-4 bonds among cell lines, compared to the control and ccr 1 g samples, the thioacidolysis monomers released from coniferaldehyde end-groups were recovered in higher relative amounts from the lignins of the cad c cad d and ccc mutants. Ferulic acid was released at a greater amount in all mutant cell lines. The sugar composition is modified in the floral stem cell wall In measuring arabinose, xylose, glucose, and uronic acid in extract-free stems upon acid hydrolysis, lesser amount of sugars were obtained from ccc. On the other hand, cad c and cad mutants have more uronic acid than wild-type A. thaliana. In addition, glucose was reduced in ccr 1 g and ccc mutants. However, this is compensated by high levels of xylose and uronic acid in ccr 1 g, and arabinose plus glucuronic acid in ccc. The hypolignified and dwarf stems of the ccc mutant accumulate both soluble phenolics and auxin Even at plantlet stage, flavones glycosides were reduced in ccc. Sinapoyl esters were similarly reduced in ccr 1 g and ccc mutants. In contrast to the other plant lines, ccr 1 g and ccc had increased levels of feruloyl malate. On the other hand, flavonol gylcosides and sinapoyl malate were increased in ccc. Expression of genes involved in phenolic compound biosynthesis is modified in the ccc mutant In ccc, the expression of REF1 and FSH1 were decreased, while that of CCR 2 and CAD 1 were increased. In addition, CADB1 expression were induced. Despite this, CADB1 was not able to restore CAD effect on lignin biosynthesis. Those of PAL1, PAL2, CHS, COMT1, and CADG remained unchanged. CONCLUSION By observing the effects on leaf and flower morphology, it is evident that the CAD has a supplementary effects on the action CCR 1. Through the results of Wiesner staining, it was determined that A. thaliana triple mutants caused the accumulation of coniferaldehyde end-groups in lignin and not the lignin itself. Probably, it is the absence of CAD gene expressions that caused coniferaldehyde accumulation. ccc mutants have less biomass and more soluble components than the other plant lines. In addition, because ccc mutants have more ASL than KL, its structure is weaker than the other lines. This probably causes the dwarfism observed among these plants. The decrease of H, G and S thioethylated monomers in ccc mutants means that either lignins involved in B-O-4 bonds are less, or that the ccc lignin units are involved in links highly resistant to thioacidolysis. The changes in sugar composition may indicate a decrease in the amount of cellulose in plant cell walls. This explains why glucose was depleted in ccr 1 g and ccc mutants. However, even xyloglucan remained unchanged or even decreased. The high flavonoid levels in ccc perturbed auxin transport along the plants. This causes the dwarfism observed in this study, as well as in other previous researches. The understanding of lignin biosynthesis is important in the advancement of what is known about the lignocellulose-to-bioethanol process. Since lignification interferes with this process, it is important to determine means by which lignin production is decreased without affecting plant growth and development. Read More