Occurrence of guaiacyl/p-hydroxyphenyl lignin in Arabidopsis thaliana T87 cells

Abstract

Abstruct Arabidopsis thaliana is the most widely used model plant in the area of plant biosciences, and the cell line T87 is one of the most commonly used lines in Arabidopsis cell cultures. The characteristics of lignins in cultured cells often differ from those of lignins in intact plants. To date, nothing is known about the lignins of T87 cells. In this study, we characterized lignins of T87 cells using six analytical methods; phloroglucinol staining, thioacidolysis, nitrobenzene oxidation, alkaline hydrolysis, and Klason and acetyl bromide methods. These analyses clearly showed that lignins of T87 cells are composed of guaiacyl and p-hydroxyphenyl units, and are quite different from lignin found in the Arabidopsis inflorescence stem, which is composed of guaiacyl and syringyl units. Materials and methods T87 cells and plant materialsT87 suspension-cultured cells were cultivated in 200 ml mJPL3 liquid medium according to Ogawa et al. (2008b) and harvested at 1-21 days after subculturing. The harvested T87 suspensioncultured cells were collected onto filter paper (Advantec Co., Ltd.) in a Buchner funnel. After removal of the medium, the collected cells were washed with 50 ml MilliQ water, dewatered by suction filtration for 30 s, weighed, and then stored in liquid nitrogen until use. In addition, T87 cells were maintained on mJPL3 agar medium and subcultured every 14 days. A. thaliana Columbia plants were cultivated under a 16-h light/8-h dark regime as reported previously (Nakatsubo et al. 2008a(Nakatsubo et al. , 2008b. Sample preparation for lignin analysesHarvested T87 suspension-cultured cells and A. thaliana inflorescence stems (60 days old, 30 cm in height) were individually freeze-dried, powdered with a pestle and mortar, and extracted with 95% methanol at 60°C until the color of the powdered cells changed from green to pale yellow. Then, the powders were further extracted successively with hexane and distilled water and freeze dried. Hereafter, these samples are referred to as extracts-free powders in this paper. Then, the extracts-free powders were dried in an oven at 60°C to constant weight. The lignins in the dried extract-free powders were then analyzed by thioacidolysis, nitrobenzene oxidation, alkaline hydrolysis, and Klason and acetyl bromide methods. Chemicals InstrumentationCells were observed under an Olympus BX51 microscope (Olympus Co., Ltd), and images were obtained using a digital camera (Olympus DP71, Olympus Co., Ltd.) connected to the microscope.GC-MS was performed using a Shimadzu QP-5050A GC-MS system (Shimadzu Co., Ltd.). The GC-MS conditions were as follows: Shimadzu Hicap CBP10-M25-0.25 column (25 mϫ 0.22 mm); carrier gas, helium; injection temperature, 230°C; oven temperature, 40°C at tϭ0 to 2 min, then to 230°C at 40°C min Ϫ1; ionization, electron-impact mode (70 eV). Absorbance was measured with a UV-1600PC UV-visible spectrophotometer (Shimadzu Co., Ltd.). Phloroglucinol-hydrochloric acid stainingThe phloroglucinol-hydrochloric acid reaction was carried out according to the...

Retraction: Characterization of lignocellulose of Erianthus ravennae in relation to enzymatic saccharification efficiency

“…Inner part and outer part: the inner and outer parts of 1st internode, respectively. only of β-O-4 substructure which is obtained by the thioacidolysis but also of other substructures, each organ was analysed by the microscale nitrobenzene oxidation method (Yamamura et al 2010(Yamamura et al , 2011. The GC-MS analysis of the nitrobenzene oxidation products showed that lignin in each organ consisted of guaiacyl, syringyl, and p-hydroxyphenyl units (Table 2), consistent with the result of thioacidolysis.…”

“…The aromatic substitution patterns of the β-O-4 substructure and also other substructures were analysed by alkaline nitrobenzene oxidation (Yamamura et al 2010(Yamamura et al , 2011.…”

“…Cell-wall ester-linked ferulic and p-coumaric acids were analysed as previously described (Yamamura et al 2011).…”

Characterization of Jatropha curcas lignins

“…To determine the lignin aromatic components not only of β-O-4 substructure which is obtained by the thioacidolysis but also of other substructures, each organ of J. curcas was analyzed by the microscale nitrobenzene oxidation method (Yamamura et al 2010(Yamamura et al , 2011. GC-MS analysis of the nitrobenzene oxidation products indicated that the molar ratios of syringyl to guaiacyl moieties (S/V), as measured by that of syringaldehyde to vanillin, varied among organs (Table 1).…”

“…Secondary xylem of a one-year-old stem of the plant was also analyzed by the thioglycolic acid method. First, each sample was subjected to thioacidolysis (Nakatsubo et al 2008;Yamamura et al 2011) to confirm the presence of lignin by identifying phenyltrithioethylpropane compounds (Figure 2), which are derived speci cally from β-O-4 substructures of lignin polymers. The detection of the diagnostic monomers clearly indicated the presence of lignin in all analyzed samples (Figure 3).…”