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Acetyl Bromide Soluble Lignin (ABSL) Assay for Total Lignin Quantification from Plant Biomass
采用乙酰溴可溶性木质素(ABSL)测定法定量植物生物量中的总木质素   

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Abstract

Lignin is the second most abundant biopolymer on Earth, providing plants with mechanical support in secondary cell walls and defense against abiotic and biotic stresses. However, lignin also acts as a barrier to biomass saccharification for biofuel generation (Carroll and Somerville, 2009; Zhao and Dixon, 2011; Wang et al., 2013). For these reasons, studying the properties of lignin is of great interest to researchers in agriculture and bioenergy fields. This protocol describes the acetyl bromide method of total lignin extraction and quantification, which is favored among other methods for its high recovery, consistency, and insensitivity to different tissue types (Johnson et al., 1961; Chang et al., 2008; Moreira-Vilar et al., 2014; Kapp et al., 2015). In brief, acetyl bromide digestion causes the formation of acetyl derivatives on free hydroxyl groups and bromide substitution of α-carbon hydroxyl groups on the lignin backbone to cause a complete solubilization of lignin, which can be quantified using known extinction coefficients and absorbance at 280 nm (Moreira-Vilar et al., 2014).

Keywords: Lignin(木质素), Biochemical measurement(生化测定), Plant biomass(植物生物量), Acetyl bromide(乙酰溴), Plant cell walls(植物细胞壁)

Background

The acetyl bromide method for quantification of lignin from plant biomass has been used to accurately measure total lignin content for decades (Johnson et al., 1961). Recently, this method has gained support as an optimal procedure for lignin quantification, as opposed to the alternative thioglycolic acid and Klason lignin methods (Moreira-Vilar et al., 2014). Comparison of these three methods has empirically shown that the acetyl bromide method consistently results in the highest recovery of lignin, and is insensitive to tissue type, extent of lignification, and lignin composition (Moreira-Vilar et al., 2014). In our previous work (Kapp et al., 2015), we adapted the scale of the acetyl bromide assay to facilitate a rapid, small-scale determination of lignin that uses a small amount of alcohol insoluble residue (AIR) derived from Brachypodium distachyon, based on a protocol described in the ‘Microscale Method for Cuvettes’ method detailed by Chang et al. (2008). The protocol described below can be performed with standard laboratory equipment and requires 5-9 days total after harvesting plant material, which can be derived from a variety of tissues or developmental stages.

Materials and Reagents

  1. Personal protective equipment (PPE; these should be worn at all times when dealing with concentrated acids and alkali. see Note 1)
    1.  Safety glasses
    2.  Lab coat
    3.  Gloves
  2. Pipette tips  
  3. Pipette set
  4. Ice bucket
  5. 2 ml Sarstedt tubes (SARSTEDT, catalog number: 72.694.007 )
  6. Plant material or cell wall preparation of choice
  7. Liquid nitrogen
  8. Acetone, ≥ 99.5% (EMD Millipore, catalog number: AX0120 )
  9. Lugol’s Iodine staining solution (Sigma-Aldrich, catalog number: 32922 )
  10. 1.5 N sodium hydroxide (NaOH, strong base), ≥ 97% (Fisher Scientific, catalog number: BP359-500 )
  11. 0.5 M hydroxylamine hydrochloride (strong reducing agent; store in Drierite container, make fresh in water), 98% (Sigma-Aldrich, catalog number: 255580 )
  12. Chloroform, ≥ 99.5% (Sigma-Aldrich, catalog number: C2432 )
  13. Methanol, ≥ 99.8% (Fisher Scientific, catalog number: A412P )
  14. Deionized water
  15. Ethanol, 100%, 200 proof (Decon Labs, catalog number: V1001 )
  16. Acetyl bromide (strong acid, violently reacts with water; dilute in acetic acid), 99% (Sigma-Aldrich, catalog number: 135968 )
  17. Glacial acetic acid, ≥ 99.7% (EMD Millipore, catalog number: AX0073 )
  18. DMSO (Dimethyl sulfoxide), ≥ 99.9% (Sigma-Aldrich, catalog number: 276855 )
  19. Chloroform-methanol mixture (see Recipes)
  20. 70% ethanol (see Recipes)
  21. 25% acetyl bromide (see Recipes)
  22. 90% DMSO (see Recipes)

Equipment

  1. 80 °C freezer
  2. Wiley Mini-Mill (Thomas Scientific, catalog number: 3383L10 )
  3. Cryogenic-compatible containers
  4. CryoMill ball mill along with all sizes of steel balls, grinding jars, safety valves, and Autofill (Retsch, catalog numbers: 20.749.0001 ; 02.480.0002 )
  5. Microcentrifuge capable of spinning at 10,000 x g (e.g., Eppendorf, catalog number: 5424 )
  6. Chemical hood
  7. Platform rocker (VWR, catalog number: 12620-906 )
  8. 10 ml glass graduated cylinder (Kimble Chase Life Science and Research Products, catalog number: 20024D-10 )
  9. NanoDrop 2000C spectrophotometer, or other UV-Vis (Thermo Fisher Scientific, Thermo ScientificTM, model: ND-2000C-PC )
  10. Quartz cuvette, 0.7 ml volume (Sigma-Aldrich, catalog number: Z600199 )
  11. Pipette  
  12. Water bath or incubator capable of reaching 70 °C (e.g., VWR, catalog number: 89032-216 )
  13. Analytical balance (Mettler Toledo, Excellence Series )
  14. Vortex mixer (VWR, catalog number: 58816-123 )
  15. Tissue lyophilizer (4.5 Liter Freeze Dry Freeze Dryer) (Labconco, catalog number: 7750020 )
  16. Solid cap with PTFE Liner 15 mm, for 7 ml glass screw-cap vial (Sigma-Aldrich, catalog number: 27152 )
  17. Optional: Glass beads (Kimble Chase Life Science and Research Products, catalog number: 13500-4 )
  18. Optional: Reacti-Therm Heating and Stirring Module (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: TS-18823 )

Procedure

  1. Plant material preparation (requires 2-4 d after harvesting of tissue)
    1. After harvesting approximately 5 g (fresh weight) of desired tissue for lignin content determination, flash freeze in liquid nitrogen and store at -80 °C for at least 1 h until use.
    2. Lyophilize tissue until completely dry; duration of lyophilization often depends on tissue density, but commonly takes at least 24 h and up to 72 h for complete drying.
    3. Grind freeze-dried tissue to pass through a 60-mesh screen using a Wiley Mini-Mill. Wiley Mini-Mills are equipped to grind dried samples ranging from long, thin Arabidopsis stems (several mm thick) to thicker woody tissues (~1 cm).
    4. Transfer coarsely-ground samples to cryogenic-compatible containers with steel balls (leave 1/3 of the container empty for air space) and mill using a Retsch CryoMill ball mill with a pre-cooling cryo setting at 5 Hz for ~3 min, followed by grinding at 5-10 min at 30 Hz. If the sample is not a homogenously fine powder after this initial grinding, another round of cryogenic milling should be performed. It is essential to obtain a fine powder with high surface area to facilitate the complete digestion of lignin present in the AIR (Figure 1). This tissue will be used to prepare alcohol insoluble residue (AIR).


      Figure 1. Representation of insufficiently and sufficiently ground dry plant material

  2. Alcohol insoluble residue (AIR) preparation (requires 2-3 d)
    1. Weigh roughly 70 mg of finely ground tissue into a 2 ml Sarstedt tube (see Note 2).
    2. Add 1.5 ml of 70% ethanol, vortex thoroughly, centrifuge at ≥ 10,000 x g for 10 min to pellet residue, remove as much supernatant as possible without disturbing the pellet.
    3. Wash with 1.5 ml 1:1 chloroform:methanol, vortex to resuspend pellet, then centrifuge and remove supernatant as described in the previous step.
    4. Add 1.5 ml acetone, vortex, and then centrifuge and remove supernatant as previously described. Allow material suspended in residual acetone to air dry in a chemical hood overnight with the tube cap removed, or dry under a stream of air at 35 °C on a Reacti-Therm apparatus until completely dry (see Note 3). The remaining material is AIR.
    5. To destarch AIR, add 1.5 ml 90% DMSO to the pellet, vortex thoroughly, and allow to shake overnight on a platform rocker at a speed of at least 50 rpm speed at the highest angle to facilitate mixing. The following day, centrifuge and remove supernatant as previously described (see Note 4).
    6. Wash once in 1.5 ml 90% DMSO, vortex, centrifuge, remove supernatant as described above.
    7. Wash six times in 1.5 ml 70% ethanol; vortex, then centrifuge and remove supernatant each time as previously described.
    8. Add 1.5 ml acetone, vortex, and then centrifuge and remove supernatant as previously described. Allow material suspended in residual acetone to air dry in a chemical hood overnight, or dry under a stream of air at 35 °C on a Reacti-Therm apparatus (see Note 3). The remaining material is de-starched AIR.
    9. Verify the absence of starch by staining a small portion of de-starched AIR with Lugol’s Iodine solution and washing with water once. For staining, use at least 2 mg AIR for ease of visualizing stained starch. Add 500 µl Lugol’s Iodine solution to AIR and vortex, incubate at room temperature for 5 min, centrifuge and remove supernatant as previously described, wash with 1 ml water, centrifuge and remove supernatant as previously described, and observe color of AIR. If starch is present, the AIR will appear dark in color and the destarching protocol should be repeated (see Note 5; Figure 2).


      Figure 2. Image of non-destarched and destarched AIR following staining with Lugol’s Iodine solution

  3. Acetyl bromide soluble lignin determination (requires 1-2 d)
    1. Weigh out approximately 5 mg of destarched AIR and put into a 7 ml glass screw-cap vial; record the exact mass. Small aggregates of particulate AIR below ~0.5 mm in diameter will not affect lignin solubilization as small aggregates should be permeable to the 25% acetyl bromide due to cryo-milling and will likely disintegrate during digestion. For each biological sample, perform technical replicates in triplicate.
    2. In a chemical hood, prepare 25% acetyl bromide by diluting in glacial acetic acid (see Note 6). Be sure to wear appropriate PPE and use a glass graduated cylinder for measuring and only glass containers for all steps involving acetyl bromide.
    3. To each glass tube containing destarched AIR, gently add 1 ml of 25% acetyl bromide, swirl gently to mix AIR into 25% acetyl bromide. Also add 1 ml of 25% acetyl bromide to an empty glass tube, which will serve as a blank.
    4. Put all samples at 70 °C for 1 h with gentle swirling every 10 min (see Note 7).
      Note: If using a water bath to fit the large 7 ml tubes, make sure to use a glass secondary container, such as a tall glass beaker, to prevent the tubes from tipping over.
    5. Immediately put samples on ice to cool. While samples are on ice, add 5 ml of glacial acetic acid and vortex thoroughly in the chemical hood.
    6. Allow any residual AIR to settle to the bottom of the glass vials for at least 1 h to overnight at room temperature. The reaction is complete and stable after the addition of glacial acetic acid and the absorbance corresponding to ABSL values will not be significantly affected; waiting for any residue to settle is important because suspended particles may interfere with the accuracy of the spectrophotometer (see Note 8).
    7. Once settled, gently transfer 300 µl from the top of each acetyl bromide solution to a quartz cuvette while avoiding resuspending any residual AIR. Add 400 µl 1.5 N NaOH, then 300 µl 0.5 M freshly made hydroxylamine hydrochloride. Pipette solutions gently into the cuvette until fully mixed together after addition of the hydroxylamine hydrochloride (pipette up and down ~15-20 times for thorough mixing, look for complete miscibility of the mixed solutions), and measure absorption at 280 nm against a blank on a spectrophotometer immediately after mixing is complete. The blank consists of the blank acetyl bromide digestion sample mixed with 1.5 N NaOH and hydroxylamine hydrochloride in the same ratio as previously described. Due to the utilization of a single quartz cuvette, each sample must be prepared in the cuvette immediately before reading A280. If the A280 measurement exceeds 1.000, it is recommended to dilute the mixture with acetic acid to ensure accurate readings and to best observe the resulting curve for any aberrant characteristics (see Figure 3 for expected spectrum characteristics). Be sure to account for this dilution factor in addition to the other dilutions from the 1 ml digestion during calculation of percentage of acetyl bromide soluble lignin (see Note 9).


      Figure 3. Representative absorbance spectrum of ABSL reading measured against a blank. A280 absorbance is due to the presence of ABSL, and A600 is depicted to show that there is minimal residual AIR particulate matter present in the sample during absorbance reading.

    8. Between samples, wash the inside of the cuvette with glacial acetic acid and wipe the outside of the cuvette clean with 70% ethanol.
    9. Use Beer’s Law to calculate the percentage of acetyl bromide soluble lignin with the proper extinction coefficient (see Table 1; see Note 10).

Data analysis

  1. The following formula can be used to determine the mass percentage of AIR that is ABSL, and can be converted to µg mg-1 AIR:

    Where,
    A280 = Absorbance at 280 nm (Blank corrected),
    ε = extinction coefficient (g-1 L cm-1),
    L = spectrophotometer path length (cm),
    D = dilution factor from digested AIR,
    m = mass of de-starched AIR (mg).
  2. The extinction coefficient has been determined for numerous organisms and tissues in several studies to account for a lack of lignin standards within the cell wall field (Table 1). Additional values for softwoods and hardwoods can be found in Johnson et al. (1961), but are in the range of 23.3-23.6 g-1 L cm-1 (see Note 11).

    Table 1. List of previously determined extinction coefficients for the acetyl bromide soluble lignin method of lignin quantification


  3. After calculation of percent ABSL, apply a two-tailed t-test for comparing two samples or One-Way ANOVA for comparing multiple samples.

Notes

  1. Always wear PPE when dealing with concentrated acids and bases such as acetyl bromide, glacial acetic acid, and sodium hydroxide.
  2. A 3 mm glass bead can be added to each tube before preparing AIR to facilitate pellet resuspension and mixing after centrifugation. This will speed up the process considerably, extract lipids more efficiently, and maintain high yields of AIR, especially when dealing with a large number of samples.
  3. All steps of AIR preparation are performed at room temperature, with the option of drying at 35 °C under a stream of air at steps B4 and B8. Use of a Reacti-Therm module is not required, but will dry samples faster and more evenly due to the supply of gentle airflow and heat. Drying in the Reacti-Therm module usually takes < 1 h, and the pellet will appear cracked when it is completely dry. This method, as opposed to allowing samples to dry in the chemical hood overnight, accounts for the differences in estimated time given for each sub-heading of the Procedure section.
  4. Other destarching protocols are available, such as the enzymatic method used by Hatfield et al. (2009). However, in our experience (Kapp et al., 2015), using 90% DMSO appeared to be the more effective technique.
  5. Lugol’s Iodine staining is recommended to verify the presence or absence of starch due to the facile nature of staining and noticeable darker coloration of AIR containing starch after staining. Lugol’s Iodine staining is commonly used to stain starch granules in intact plant tissues, with sensitivity of detection largely limited to visual restraints (Ovecka et al., 2012). Removal of starch is necessary for accurate determination of lignin content because the ABSL quantification method quantifies lignin content with respect to the mass of cell wall material (AIR). If AIR contains starch, starch will contribute to the overall weight of the AIR in the acetyl bromide digestion and result in a lower and variable weight of cell wall material in each sample, thus diminishing the accuracy of lignin determination.
  6. When diluting acetyl bromide to 25% in acetic acid in step 2 of Procedure C (Acetyl bromide soluble lignin determination), be sure to measure fuming acetyl bromide into glass containers such as a glass graduated cylinder or using a glass Pasteur pipette. Using a micropipette will result in damage to the micropipette and the need for repair.
  7. It has been reported that excessive duration of the acetyl bromide digestion or increased temperature of the reaction causes xylan degradation (Hatfield et al., 1999). Xylans are present in various amounts in all lignified plant tissues; xylan degradation produces furfural derivatives, which absorb in the range of 270-280 nm and can influence absorbance at 280 nm during ABSL measurements. Hatfield et al. (1999) recommend lowering the digestion temperature to 50 °C and extending the incubation time two-fold if xylan degradation is suspected of producing variable results. Occasionally, researchers have included perchloric acid in their acetyl bromide digestion, but Hatfield et al. (1999) recommend excluding perchloric acid as this also increases the propensity for xylan degradation. 
  8. We recommend allowing remaining AIR to settle to the bottom of the tube for 12-24 h after addition of 5 ml glacial acetic acid to sample to prevent residual AIR colloids from interfering with A280 measurements. When comparing measurements of A280 after 1 h of settling to after 24 h of settling of the same sample, A280 values at 24 h vary from 0.4-4.4% of the A280 measurement recorded at 1 h, indicating that the A280 readings are consistent following digestion and mixing of glacial acetic acid.
  9. Dilution factor should be calculated from with respect to the volume of the 25% acetyl bromide digestion of 1 ml. For a sample yielding A280 readings in the range of 0-1.000 without the need for dilution, the following dilutions should be accounted for when using the protocol as read above: a 6x dilution upon addition of 5 ml of glacial acetic acid following digestion, and a (10/3)-fold dilution during mixing of 300 µl digested AIR material, with 400 µl 1.5 N NaOH, and 300 µl hydroxylamine hydrochloride, to produce a total dilution factor of 20.
  10. The method described above produced highly consistent results between different biological samples of Arabidopsis basal stem tissue (Xiao et al., 2017) and Brachypodium distachyon (Kapp et al., 2015). The higher variation in the study performed by Kapp et al. (2015) is likely derived from the heterogeneity of tissues samples, as percentage ABSL was determined for the entirety of aerial tissue from 2-12 week-old Brachypodium distachyon plants, with variation in leaf:stem:tiller biomass ratios. Determination of ABSL content in the basal three internodes of Arabidopsis stems yielded a standard deviation ranging from 3.7-8% ABSL of the mean across three biological replicates, with standard deviation between technical replicates within a single biological replicate ranging from 0.07-1.12% ABSL, indicating highly precise results (Xiao et al., 2017).
  11. For determination of ABSL content in Arabidopsis stem tissue using this method in Xiao et al. (2017), we chose to use the extinction coefficient determined by Chang et al. (2008) because this extinction coefficient was determined using a method most similar to the protocol above on Arabidopsis thaliana (Col-0) stem tissues, and was experimentally determined across various Arabidopsis thaliana ecotypes, allowing our values to be readily compared to other studies using this method in the future.

Recipes

  1. Chloroform-methanol mixture
    Chloroform:methanol (1:1, v/v)
  2. 70% ethanol
    100% ethanol (200 proof):double distilled water (7:3, v/v)
  3. 25% acetyl bromide
    Acetyl bromide:glacial acetic acid (1:3, v/v)
  4. 90% DMSO
    Double distilled Water:DMSO (1:9, v/v)

Note: The final volume of each mixture required by the procedure is dependent on the number of samples.

Acknowledgments

This work was supported as part of The Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences under Award # DE-SC0001090. Thanks to the authors of Foster et al. (2010) for guidelines and tips on AIR preparation, and the authors of Chang et al. (2008) for developing the ABSL method across different scales and providing excellent insight and expertise on the subject matter. The authors have no conflicts of interest in submitting this work.

References

  1. Carroll, A. and Somerville, C. (2009). Cellulosic biofuels. Annu Rev Plant Biol 60: 165-182.
  2. Chang, X. F., Chandra, R., Berleth, T. and Beatson, R. P. (2008). Rapid, microscale, acetyl bromide-based method for high-throughput determination of lignin content in Arabidopsis thaliana. J Agric Food Chem 56(16): 6825-6834.
  3. Foster, C. E., Martin, T. M. and Pauly, M. (2010). Comprehensive compositional analysis of plant cell walls (Lignocellulosic biomass) part I: lignin. J Vis Exp(37).
  4. Fukushima, R. S. and Hatfield, R. D. (2004). Comparison of the acetyl bromide spectrophotometric method with other analytical lignin methods for determining lignin concentration in forage samples. J Agric Food Chem 52(12): 3713-3720.
  5. Hatfield, R. D., Grabber, J., Ralph, J., and Brei, K. (1999). Using the acetyl bromide assay to determine lignin concentrations in herbaceous plants: some cautionary notes. J Agric Food Chem, 47, 628-632.
  6. Hatfield, R. D., Marita, J. M., Frost, K., Grabber, J., Ralph, J., Lu, F. and Kim, H. (2009). Grass lignin acylation: p-coumaroyl transferase activity and cell wall characteristics of C3 and C4 grasses. Planta 229(6): 1253-1267.
  7. Johnson, D. B., Moore, W. E., and Zank, L. C. (1961). The spectrophotometric determination of lignin in small wood samples. Tappi, 44, 793-798.
  8. Kapp, N., Barnes, W. J., Richard, T. L. and Anderson, C. T. (2015). Imaging with the fluorogenic dye Basic Fuchsin reveals subcellular patterning and ecotype variation of lignification in Brachypodium distachyon. J Exp Bot 66(14): 4295-4304.
  9. Moreira-Vilar, F. C., Siqueira-Soares, Rde. C., Finger-Teixeira, A., de Oliveira, D. M., Ferro, A. P., da Rocha, G. J., Ferrarese, Mde. L., dos Santos, W. D., and Ferrarese-Filho, O. (2014). The acetyl bromide method is faster, simpler and presents best recovery of lignin in different herbaceous tissues than klason and thioglycolic acid methods. PloS One, 9(10), e110000. doi:10.1371/journal.pone.0110000.
  10. Ovecka, M., Bahaji, A., Munoz, F. J., Almagro, G., Ezquer, I., Baroja-Fernandez, E., Li, J. and Pozueta-Romero, J. (2012). A sensitive method for confocal fluorescence microscopic visualization of starch granules in iodine stained samples. Plant Signal Behav 7(9): 1146-1150.
  11. Wang, Y., Chantreau, M., Sibout, R. and Hawkins, S. (2013). Plant cell wall lignification and monolignol metabolism. Front Plant Sci 4: 220.
  12. Xiao, C., Barnes, W. J., Zamil, M. S., Yi, H., Puri, V. M. and Anderson, C. T. (2016). Activation tagging of Arabidopsis POLYGALACTURONASE INVOLVED IN EXPANSION2 promotes hypocotyl elongation, leaf expansion, stem lignification, mechanical stiffening, and lodging. Plant J.
  13. Zhao, Q. and Dixon, R. A. (2011). Transcriptional networks for lignin biosynthesis: more complex than we thought? Trends Plant Sci 16(4): 227-233.

简介

木质素是地球上第二大丰富的生物聚合物,为植物在二次细胞壁提供机械支持,防止非生物和生物胁迫。然而,木质素还可以作为生物燃料生成的糖化的障碍(Carroll和Somerville,2009; Zhao和Dixon,2011; Wang等人,2013)。由于这些原因,研究木质素的性质对于农业和生物能源领域的研究人员非常感兴趣。该方案描述了总木素提取和定量的乙酰溴方法,其对于其不同组织类型的高回收率,一致性和不敏感性的其它方法是有利的(Johnson等人,1961; Chang < em等人,2008; Moreira-Vilar等人,2014; Kapp等人,2015)。简言之,乙酰溴消化导致游离羟基上的乙酰基衍生物形成和木质素骨架上的α-碳羟基的溴取代引起木质素的完全溶解,其可以使用已知的消光系数和280nm处的吸光度进行定量(Moreira-Vilar等人,2014)。

背景 用于从植物生物量定量木质素的乙酰溴方法已被用于精确测量几十年的总木质素含量(Johnson等,1961)。最近,与替代的巯基乙酸和Klason木质素方法(Moreira-Vilar等人,2014))相比,该方法获得了木质素定量的最佳方法的支持。这三种方法的比较经验证明,乙酰溴方法一直导致木质素回收率最高,对组织类型,木质化程度和木质素组成不敏感(Moreira-Vilar等人)。 ,2014)。在我们以前的工作(Kapp等人,2015)中,我们调整了乙酰溴测定的规模,以促进使用少量不溶于醇的残留物快速,小规模测定木质素基于由Chang等人详细描述的“Microscale Method for Cuvettes”方法中描述的方案。下面描述的方案可以用标准实验室设备进行,并且在收获植物材料之后总共需要5-9天,其可以从各种组织或发育阶段得到。

关键字:木质素, 生化测定, 植物生物量, 乙酰溴, 植物细胞壁

材料和试剂

  1. 个人防护装备(PPE;处理浓酸和碱时应始终佩戴),见注1)
    1.  安全眼镜
    2. 实验室外套
    3.  手套
  2. 移液器提示
  3. 移液器套件
  4. 冰桶
  5. 2毫升Sarstedt管(SARSTEDT,目录号:72.694.007)
  6. 选择植物材料或细胞壁制备
  7. 液氮
  8. 丙酮≥99.5%(EMD Millipore,目录号:AX0120)
  9. Lugol碘染色溶液(Sigma-Aldrich,目录号:32922)
  10. 1.5N氢氧化钠(NaOH,强碱)≥97%(Fisher Scientific,目录号:BP359-500)
  11. 0.5M盐酸羟胺(强还原剂;储存在Drierite容器中,在水中新鲜),98%(Sigma-Aldrich,目录号:255580)
  12. 氯仿,≥99.5%(Sigma-Aldrich,目录号:C2432)
  13. 甲醇≥99.8%(Fisher Scientific,目录号:A412P)
  14. 去离子水
  15. 乙醇,100%,200证明(Decon Labs,目录号:V1001)
  16. 乙酰溴(强酸,与水剧烈反应;在乙酸中稀释),99%(Sigma-Aldrich,目录号:135968)
  17. 冰醋酸≥99.7%(EMD Millipore,目录号:AX0073)
  18. DMSO(二甲基亚砜),≥99.9%(Sigma-Aldrich,目录号:276855)
  19. 氯仿 - 甲醇混合物(参见食谱)
  20. 70%乙醇(见食谱)
  21. 25%乙酰溴(见配方)
  22. 90%DMSO(参见食谱)

设备

  1. 80°C冷冻机
  2. Wiley Mini-Mill(Thomas Scientific,目录号:3383L10)
  3. 低温兼容容器
  4. CryoMill球磨机以及各种规格的钢球,研磨罐,安全阀和自动填充(Retsch,目录号:20.749.0001; 02.480.0002)
  5. 能够以10,000 x g(例如,Eppendorf,目录号:5424)旋转的微量离心机
  6. 化学罩
  7. 平台摇杆(VWR,目录号:12620-906)
  8. 10毫升玻璃量筒(Kimble Chase生命科学研究产品,目录号:20024D-10)
  9. NanoDrop 2000C分光光度计,或其他UV-Vis(Thermo Fisher Scientific,Thermo Scientific,型号:ND-2000C-PC)
  10. 石英比色皿,0.7ml体积(Sigma-Aldrich,目录号:Z600199)
  11. 移液器
  12. 能够达到70℃的水浴或孵化器(例如,VWR,目录号:89032-216)
  13. 分析天平(Mettler Toledo,卓越系列)
  14. 涡街搅拌机(VWR,目录号:58816-123)
  15. 组织式冷冻干燥机(4.5升冷冻干燥冷冻干燥机)(Labconco,目录号:7750020)
  16. 带PTFE衬里的固体盖15毫米,用于7毫升玻璃螺旋瓶小瓶(Sigma-Aldrich,目录号:27152)
  17. 可选:玻璃珠(Kimble Chase Life Science and Research Products,catalog number:13500-4)
  18. 可选:Reacti-Therm加热和搅拌模块(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:TS-18823)

程序

  1. 植物材料制备(收集组织后2-4 d)
    1. 收获大约5g(鲜重)所需的组织用于木质素含量测定后,在液氮中闪蒸冷冻,并在-80℃下储存至少1小时直到使用。
    2. 冻干组织直到完全干燥;冷冻干燥的持续时间通常取决于组织密度,但通常需要至少24小时和高达72小时才能完全干燥。
    3. 使用Wiley Mini-Mill研磨冷冻干燥的组织以通过60目筛网。 Wiley Mini-Mills装备了从长而薄的拟南芥茎(几毫米厚)到较厚的木质组织(〜1厘米)的干燥样品。
    4. 将粗磨样品转移到具有钢球的低温相容容器中(将空间中的空间的1/3留在空气中),并使用Retsch CryoMill球磨机在5Hz下预冷却冷冻设定约3分钟,然后通过在30Hz下5-10分钟研磨。如果样品在初始研磨后不是均匀的细粉末,则应进行另一轮低温研磨。获得具有高表面积的细粉末是有必要的,以促进AIR中存在的木质素的完全消化(图1)。该组织将用于制备不溶于醇的残留物(AIR)。


      图1.表示不足够充分地干燥植物材料

  2. 酒精不溶性残留物(AIR)制剂(需2-3 d)
    1. 将大约70毫克精细磨碎的组织称重到2毫升Sarstedt管中(见注2)。
    2. 加入1.5 ml的70%乙醇,充分涡旋,≥10,000×g离心10分钟以沉淀残留物,尽可能多地除去上清液,而不会干扰沉淀物。
    3. 用1.5ml 1:1氯仿:甲醇洗涤,涡旋重悬沉淀,然后如上一步骤所述离心并除去上清液。
    4. 加入1.5ml丙酮,旋转,然后如前所述离心并除去上清液。将悬浮在残留丙酮中的材料在化学品罩中空气干燥,取出管帽,或在35℃的Reacti-Therm装置下在空气流下干燥直到完全干燥(见注3)。剩余的材料是AIR。
    5. 为了去除空气,向沉淀物中加入1.5ml 90%DMSO,彻底涡旋,并以最高角度以至少50rpm的速度在平台摇臂上摇动过夜以促进混合。第二天,如前所述离心分离上清液(见附注4)。
    6. 在1.5ml 90%DMSO中洗涤一次,旋转,离心,如上所述除去上清液。
    7. 在1.5ml 70%乙醇中洗涤6次;旋转,然后如前所述每次离心并除去上清液。
    8. 加入1.5ml丙酮,旋转,然后如前所述离心并除去上清液。将悬浮在残留丙酮中的材料在化学品罩中空气干燥过夜,或在35℃的空气流下在Reacti-Therm装置上干燥(见注3)。剩余的材料是去淀粉的AIR。
    9. 通过用Lugol碘溶液染色一小部分去淀粉的AIR并用水洗涤一次来验证淀粉的不存在。对于染色,使用至少2毫克的AIR可以方便地染色淀粉。向AIR中加入500μlLugol碘溶液涡旋,室温孵育5 min,如前所述离心分离上清液,用1 ml水洗涤,离心并除去上清液,并观察AIR的颜色。如果存在淀粉,则AIR将显示为暗色,并且应重复脱粒方案(见附注5;图2)。


      图2.使用Lugol碘溶液染色的非破坏性和破坏性AIR的图像

  3. 乙酰溴可溶性木质素测定(需要1-2 d)
    1. 称量大约5毫克的破坏的AIR,并放入7毫升的玻璃螺旋瓶小瓶中;记录精确质量。直径小于0.5毫米的颗粒物空气的小颗粒不会影响木质素的溶解,因为小的聚集体由于冷冻研磨而对透明的25%乙酰溴是可渗透的,并且在消化过程中可能会崩解。对于每个生物样品,进行一式三份的技术重复。
    2. 在化学防护罩中,通过在冰醋酸中稀释制备25%乙酰溴(见附注6)。确保佩戴适当的PPE,并使用玻璃量筒测量和仅玻璃容器,用于涉及乙酰溴的所有步骤。
    3. 向每个含有脱气AIR的玻璃管中轻轻加入1ml 25%乙酰溴,轻轻旋转,将AIR混合至25%乙酰溴。还加入1毫升25%乙酰溴到一个空的玻璃管,这将作为一个空白。
    4. 将所有样品在70℃下放置1小时,每10分钟轻轻旋转(见注7)。
      注意:如果使用水浴装配大型7 ml管,请确保使用玻璃二次容器,如高玻璃烧杯,以防止管子翻倒。
    5. 立即将样品放在冰上冷却。当样品在冰上时,加入5ml冰醋酸,并在化学罩中彻底涡旋
    6. 允许任何残留的AIR在室温下沉降至玻璃小瓶的底部至少1小时至过夜。加入冰醋酸后反应完全稳定,对ABSL值的吸光度不受影响;等待任何残留物沉淀是重要的,因为悬浮颗粒可能会干扰分光光度计的准确性(见注8)。
    7. 一旦沉降,轻轻地将300μl从每个乙酰溴溶液的顶部转移到石英比色杯,同时避免重新悬浮任何残留的AIR。加入400μl1.5N NaOH,然后加入300μl0.5M新鲜的盐酸羟胺。在加入羟胺盐酸盐(移液管上下〜15-20次后彻底混合,寻找混合溶液的完全混溶性),轻轻取出溶液,直到完全混合在一起,并测量280nm处的空白对混合后立即分光光度计完成。空白由与前述所述相同比例的与1.5N NaOH和盐酸羟胺混合的空白乙酰溴消化样品组成。由于使用单个石英比色杯,每个样品必须在阅读A 280之前立即在比色皿中制备。如果A 280 测量超过1.000,建议用乙酸稀释混合物,以确保准确的读数,并最佳地观察任何异常特征的结果曲线(参见图3的预期光谱特性)。在计算乙酰溴可溶性木质素的百分比时,除了从1ml消化的其他稀释度外,请务必考虑此稀释因子(见附注9)。


      图3.以空白测量的ABSL读数的代表性吸收光谱。吸光度是由于ABSL的存在而导致的,A 600 是描绘为显示在吸光度读数期间样品中存在的残留AIR颗粒物最少。

    8. 在样品之间,用冰醋酸洗涤比色皿的内部,并用70%乙醇擦拭比色杯的外部。
    9. 使用啤酒定律计算具有适当消光系数的乙酰溴可溶性木质素的百分比(见表1;见附注10)。

数据分析

  1. 以下公式可用于确定ABS的质量百分比,可以转换为μgmg -1 AIR:

    哪里,
    A 280 = 280nm处的吸光度(空白校正),
    ε=消光系数(g -1 L cm -1 ),
    L =分光光度计路径长度(cm),
    D =消化的AIR的稀释因子,
    m =去冰空气的质量(mg)。
  2. 在几项研究中已经确定了许多生物和组织的消光系数,以解决细胞壁区域内缺乏木质素标准(表1)。约翰逊等人可以找到软木和硬木的附加价值。 (1961),但是在23.3-23.6g Lcm -1的范围内(参见附注11)。

    表1.木质素定量的乙酰溴可溶性木质素方法的先前确定的消光系数清单


  3. 在计算百分比ABSL后,应用双尾测试用于比较两个样本或单因素方差分析以比较多个样本。

笔记

  1. 处理浓缩酸和碱如乙酰溴,冰醋酸和氢氧化钠时,始终佩戴PPE。
  2. 在制备AIR之前,可以向每个管中加入3mm玻璃珠,以便离心后使颗粒再悬浮和混合。这将大大加快这一过程,更有效地提取脂质,并保持高产量的AIR,特别是在处理大量样品时。
  3. AIR制备的所有步骤在室温下进行,可以在步骤B4和B8的空气流下在35℃下干燥。不需要使用Reacti-Therm模块,但是由于提供温和的气流和热量,会使样品更快,更均匀地干燥样品。在Reacti-Therm模块中干燥通常, 1小时,当它完全干燥时,颗粒会出现裂纹。这种方法,而不是让样品在化学品通风橱中干燥过夜,这说明了程序部分每个子标题给出的估计时间差异。
  4. 还有其他的解决方案是可用的,例如Hatfield等人使用的酶法。 (2009)。然而,根据我们的经验(Kapp等人,2015),使用90%的DMSO似乎是更有效的技术。
  5. 推荐使用Lugol碘染色,以鉴别淀粉的存在与否,因为染色后含有AIR的淀粉的染色容易,显色较暗。 Lugol的碘染色通常用于染色完整植物组织中的淀粉颗粒,检测灵敏度主要限于视觉限制(Ovecka等,2012)。由于ABSL定量方法相对于细胞壁材料质量(AIR)量化木质素含量,因此淀粉的去除对于精确测定木质素含量是必需的。如果AIR含有淀粉,淀粉将有助于在乙酰溴消化中AIR的总重量,并导致每个样品中细胞壁材料的重量和重量可变,从而降低木质素测定的准确性。
  6. 当程序C(乙酰溴可溶性木质素测定)的步骤2中将乙酰溴稀释至25%时,一定要测量发烟乙酰溴到玻璃容器如玻璃量筒或使用玻璃巴斯德吸管。使用微量移液器会导致微量移液器受损,需要修理。
  7. 据报道,乙酰溴消化过程持续时间过长或反应温度升高会导致木聚糖降解(Hatfield等,1999)。木聚糖以各种量存在于所有木质化植物组织中;木聚糖降解产生糠醛衍生物,其在270-280nm的范围内吸收,并且可以在ABSL测量期间影响280nm处的吸光度。 Hatfield等人。 (1999)建议将消化温度降低至50°C,如果怀疑木聚糖降解产生可变结果,则将孵化时间延长两倍。偶尔,研究人员已经将高氯酸包括在乙酰溴消化中,但是哈特菲尔德等人。 (1999)建议排除高氯酸,因为这也增加了木聚糖降解的倾向
  8. 我们建议允许剩余的AIR在加入5毫升冰醋酸后,将样品中的残留AIR定位到管底部12-24小时,以防止残留的AIR胶体干扰A 280 测量。当将沉降1小时后的A 280 的测量值与相同样品的沉降24小时进行比较时,24小时的A 280值在0.4-4.4%在1小时记录的 280 测量,表明在消化和混合冰醋酸之后,A 280读数是一致的。
  9. 稀释因子应根据25毫升乙酰溴消化1毫升的体积计算。对于在0-1.000范围内产生A 280 读数的样品,无需稀释,当使用上述方案时,应考虑以下稀释度:加入5ml的6x稀释液的消化后的冰醋酸和在混合300μl消化的AIR材料,400μl1.5N NaOH和300μl盐酸羟胺的混合期间的(10/3)倍稀释,以产生20的总稀释倍数。 />
  10. 上述方法在拟南芥基础茎组织的不同生物样品(Xiao等人,2017年)和不同生物样品之间产生了高度一致的结果(
  11. 用于在肖氏等人中使用该方法测定拟南芥茎组织中的ABSL含量。 (2017),我们选择使用由Chang等人确定的消光系数。 (2008),因为这种消光系数是使用与上述在拟南芥(Col-0)茎组织上的方案最相似的方法测定的,并且通过各种拟南芥(Arabidopsis thaliana)进行实验测定>生态型,允许我们的价值观与以后使用这种方法的其他研究相比较。

食谱

  1. 氯仿 - 甲醇混合物
    氯仿:甲醇(1:1,v/v)
  2. 70%乙醇
    100%乙醇(200证明):双蒸水(7:3,v/v)
  3. 25%乙酰溴
    乙酰溴:冰醋酸(1:3,v/v)
  4. 90%DMSO
    双蒸水:DMSO(1:9,v/v)

注意:程序所需的每种混合物的最终体积取决于样品数量。

致谢

这项工作是由美国能源部科学,基础能源科学办公室资助的能源前沿研究中心的木质纤维素结构与形成中心的一部分得到支持,授予#DE-SC0001090。感谢福斯特等人的作者。 (2010年),关于AIR准备的指导和提示,以及Chang等人的作者。 (2008年),用于开发不同规模的ABSL方法,并在主题上提供卓越的洞察力和专业知识。作者在提交这项工作时没有兴趣。

参考文献

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引用:Barnes, W. J. and Anderson, C. T. (2017). Acetyl Bromide Soluble Lignin (ABSL) Assay for Total Lignin Quantification from Plant Biomass. Bio-protocol 7(5): e2149. DOI: 10.21769/BioProtoc.2149.
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