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May 2019

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Cell Wall Compositional Analysis of Rice Culms
水稻茎杆的细胞壁组成分析   

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Abstract

The plant cell wall is a complicated network that is mainly constituted of polysaccharides, such as cellulose, hemicellulose and pectin. Many noncellulosic polysaccharides are further acetylated, which confers these polymers flexible physicochemical properties. Due to the significance of cell wall in plant growth and development, the analytic platform has been the focus for a long time. Here, we use internodes/culms, an important organ to provide mechanical support for rice plants, as an experimental sample to explore the method for cell wall composition analysis. The method includes preparation of cell wall residues, sequential extraction of polysaccharides, and measurement of cellulose. The procedure for acetate examination is also described. This method is applicable to determine the composition of individual cell wall polymers and the modifier acetates, and is suitable to identify cell wall relevant mutants based on the advantages in high throughput, precision and repeatability.

Keywords: Xylan (木聚糖), Pectin (果胶), Cellulose (纤维素), Acetylation (乙酰化), Cell wall (细胞壁), Rice (水稻)

Background

The plant cell wall represents one of the most complicated cellular structure in nature and is essential for plant growth and adaptations to environments. Besides presenting multiple polysaccharide components and phenolic compounds, acetylation is a prevalent modification on most cell-wall polymers, which alters the physicochemical properties and increases the complexity of cell wall structure. Establishment of the effective analytic platform for cell wall composition is always a challenging task. The previous analytic method often uses alkali to extract cell wall residues, but removes acetate. Recent works have revealed that acetate patterns on xylan determine the folding of this polymer and impact the binding to cellulose or lignin, indicating its importance in cell wall formation and plant growth control (Grantham et al., 2017; Kang et al., 2019; Zhang et al., 2019). The method that can simultaneously examine the composition of diverse cell-wall polysaccharides and their acetyl modifications needs to be developed. It becomes realizable as solvent dimethyl sulfoxide has been found extracting xylan without trimming acetyl esters (Goncalves et al., 2008). Rice culms are representative for cell wall composition methodology analysis because this organ is rich of secondary wall-bearing fiber cells and also contains multiple cell types. In addition to abundant materials, acetylation level varies on different wall polymers and during the culm development. By using rice culms as analytic samples, we developed a protocol for cell wall composition and acetyl modification analyses with some changes from the previous method (Foster et al., 2010). This protocol offers a widely used way to examine the composition of diverse cell wall polymers and determine the acetate content in different rice varieties and other crops.

Materials and Reagents

  1. 96-well flat bottom assay plate (Greiner bio-one, catalog number: 655180)
  2. UV capable 96-well flat bottom assay plate (Corning, catalog number: 3635)
  3. Glass bottle
  4. Eppendorf tubes (1.5 ml) (Eppendorf, catalog number: 0030120.086)
  5. Sarstedt tubes 2 ml (Sarstedt, D-51588)
  6. 50 ml plastic centrifuge tube (Corning CentriStar)
  7. Glass microfiber filters (Whatman, catalog number: 1820-025)
  8. Rice mature plants
  9. Endopolygalacturonase M2 (Megazyme, catalog number: PGALUSP, 4 °C)
  10. Pectin methyl esterase (Sigma-Aldrich, catalog number: P5400-1KU, −20 °C)
  11. α-amylase (Megazyme, catalog number: E-BLAAM, 4 °C)
  12. ddH2O
  13. Acetatic Acid Assay Kit (Megazyme, catalog number: K-ACET, 4 °C)
  14. Acetone
  15. DMSO (Sigma-Aldrich, catalog number: D5879)
  16. 70% (v/v) aqueous ethanol
  17. Chloroform/methanol (1:1, v/v) solution
  18. Updegraff reagent (Acetic acid: nitric acid: water, 8:1:2 v/v)
  19. 72% Sulfuric acid (Prepared with concentrated Sulfuric acid GR)
  20. 1 mg/ml glucose stock (Prepared from D-(+)-glucose) (Sigma-Aldrich, catalog number: G8270, −20 °C)
  21. Anthrone reagent (2 mg/ml Anthrone in concentrated sulfuric acid) (Sigma-Aldrich, catalog number: 319899)
  22. Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: T6508)
  23. Ammonium formate (Aldrich, catalog number: 516961)
  24. 11% peracetic acid solution (prepared from 35% peracetic) (Aladdin, catalog number: P112625)
  25. Ethanol: methanol: water solution (7:2:1, adjust the pH to 3.0 with HCOOH)
  26. 1% ammonium oxalate (Sigma-Aldrich, catalog number: 09898)
  27. 37% hydrogen chloride
  28. MES/Tris buffer (pH 8.1-8.3) (see Recipes)
  29. 2 M trifluoroacetic acid (see Recipes)
  30. 1 N sodium hydroxide (see Recipes)
  31. 1 N hydrogen chloride (see Recipes)
  32. 50 mM ammonium formate (pH 4.5) (see Recipes)

Equipment

  1. Freeze dryer (Beijing Songyuanhuaxing Technology Develop Co. Ltd., model: LGJ-12)
  2. Ball mill (QIAGEN, TissueLyser II, catalog number: 85300)
  3. (Optional) Vortex shaker 
  4. Basket centrifuge (Eppendorf, model: 5810R)
  5. Centrifuge (Eppendorf, model: 5430) (to fit Eppendorf 1.5 ml tubes)
  6. Thermomixer comfort (Eppendorf)
  7. Dri-Block heaters (Techne, model: DB200/3)
  8. Microplate reader (PerkinElmer, Enspire)
  9. Concentrator (Eppendorf, concentrator plus)
  10. (Optional) Drying oven 
  11. (Optional) Shaking incubator
  12. Sieves (Mesh size of 0.15 mm)
  13. pH meter (Mettler Toledo)
  14. Semi-micro scales (dual resolutions starting at 0.01 mg)

Procedure

  1. Preparation of destarched alcohol-insoluble cell-wall residues (AIR)
    1. Pool the whole 2nd internodes (numbered from the top down) of 5-20 rice mature plants.
    2. Freeze the fresh samples in liquid nitrogen and then lyophilize them in a freeze dryer (The rice internodes were lyophilized for 48 h to ensure complete dryness).
    3. Grind tissues to a particle size no more than 0.15 mm using ball mill and sieve through mesh with a size of 0.15 mm.
    4. Weigh approximately 1 g of the ground plant biomass into a 50 ml plastic centrifuge tube.
    5. Add 30 ml of 70% (v/v) aqueous ethanol, mix thoroughly using a vortex mixer and leave in a thermomixer comfort set at 37 °C and 200 rpm for 12 h.
    6. Centrifuge at 1,500 x g for 10 min in a basket centrifuge and discard the supernatant.
    7. Repeat Steps A5-A6 once.
    8. Add 30 ml of the chloroform/methanol (1:1 v/v) solution, mix thoroughly using a vortex mixer and leave in a shaking incubator for 30 min at 37 °C and 200 rpm.
    9. Centrifuge at 1,500 x g for 10 min at room temperature and discard the supernatant.
    10. Repeat Steps A8-A9 twice.
    11. Add 15 ml of acetone, shake the tube to re-suspend the pellet. 
    12. Centrifuge at 1,500 x g for 10 min and discard the supernatant.
    13. Repeat Steps A11-A12 twice.
    14. Let the biomass samples dry in a drying oven at 40 °C without shaking for approximately 16 h.
    15. Treat the residues with 100 U α-amylase in 40 ml MES/Tris buffer (pH 8.1) at 97 °C for 35 min, then 60 °C for 1 h.
    16. Centrifuge at 1,500 x g for 10 min and discard the supernatant.
    17. Wash the pellet with 30 ml ddH2O three times and with 15 ml acetone twice, with centrifugation (1,500 x g for 10 min) and supernatant removal after each wash.
    18. Let the biomass samples dry in an oven at 40 °C for approximately 16 h to get destarched AIR.

  2. Analysis of the crystalline cellulose content
    1. Weigh 2 mg AIR material in five replicates into 2 ml Sarstedt tubes.
    2. Add 250 µl of 2 M trifluoroacetic acid (TFA) to each sample and make ensure no material is splashed up onto the tube walls.
    3. Cap tightly and incubate for 90 min at 121 °C in Dri-Block heaters.
    4. Cool the heating blocks and samples on ice.
    5. Centrifuge at 11,000 x g for 10 min, then transfer the supernatant to a new Sarstedt tube for optional noncellulosic polysaccharides composition analysis (Foster et al., 2010) and keep the pellet for crystalline cellulose assay.
    6. Add 1 ml of Updegraff reagent (Acetic acid: itric acid: water, 8:1:2 v/v) to the pellets left over from the Step B5, cap tightly and vortex.
    7. Heat in Dri-Block heaters at 100 °C for 30 min.
    8. Cool samples on ice. 
    9. Centrifuge samples at 11,000 x g for 10 min. 
    10. Discard supernatant ensuring no pellet material is discarded.
    11. Wash once with 1 ml water and four times with 1 ml acetone, centrifuge and discard supernatant as done above.
    12. Air dry the pellet in the Dri-Block heaters at 35 °C.
    13. Add 175 µl 72% Sulfuric acid and incubate at room temperature for 60 min.
    14. Add 825 µl ddH2O, vortex and centrifuge samples at 11,000 x g for 5 min.
    15. Analyze the glucose content of the supernatant using the anthrone assay in a 96-well flat bottom assay plate.
    16. Add 10 µl of sample and 90 µl of ddH2O for a total volume of 100 µl in each sample well.
    17. Prepare standards using 1 mg/ml glucose stock (stock at -20 °C). Make 0, 2, 4, 6, 8, and 10 µg standards by pipetting 0, 2, 4, 6, 8, and 10 µl into the appropriate well, adding 100, 98, 96, 94, 92, and 90 µl ddH2O accordingly.
    18. Add 200 µl freshly prepared Anthrone Regent.
    19. Heat the plate for 30 min at 80 °C with shaking in a thermomixer comfort. Cool to room temperature. 
    20. Shake the plate thoroughly in a thermomixer comfort and then read absorption at 625 nm within 1 h.

  3. Extraction of pectin from cell wall residues 
    1. Weigh about 6 mg destarched AIR for 5 replicates into 1.5 ml Eppendorf tubes. 
    2. Add 1 ml of 50 mM ammonium formate (pH 4.5) buffer, then add 2 U of endopolygalacturonase M2 and 0.04 U of pectin methyl esterase. Mix and incubate at 37 °C for 16 h with shaking at 300 rpm in a thermomixer comfort. 
    3. Centrifuge at 3,000 x g for 10 min and transfer the pectin-rich supernatants to a new tube. Keep the remains as pectin-free samples.
    4. Freeze the supernatants using liquid nitrogen and then lyophilize them in a freeze dryer, finally get the gelatinous pectin.

  4. Isolation of the acetyl-xylan from AIR
    1. Weigh about 400 mg of destarched AIR into a 50 ml centrifuge tube. 
    2. Add 30 ml 1% ammonium oxalate and incubate at 85 °C for 2 h. Centrifuge at 1,500 x g for 15 min in a basket centrifuge. Discard the supernatant to remove pectin.
    3. Add 30 ml 11% peracetic acid solution, incubate in a water bath at 85 °C for 30 min for delignification.
    4. Centrifuge at 1,500 x g for 10 min. Discard the supernatant and wash the pellets with 30 ml ddH2O three times and with 15 ml acetone once, through centrifugation (1,500 x g for 10 min) and supernatant removal.
    5. Let the pellets dry in an oven set at 40 °C for approximately 16 h.
    6. Add 30 ml DMSO and then incubate at 70 °C for 12 h to conduct extraction, centrifuge at 1,500 x g for 10 min, transfer the supernatant to a new glass bottle. 
    7. Repeat Step D6 with 12 ml DMSO, and combine the supernatants containing the extractives.
    8. Filter the extracts through glass microfiber filters.
    9. Precipitate the extracts with 5 volume of ethanol: methanol: water solution (7:2:1, pH 3.0) at 4 °C for 12 h. 
    10. Centrifuge at 1,500 x g for 15 min. Discard the supernatant and wash the pellets four times each with 10 ml anhydrous ethanol with centrifugation (1,500 x g for 10 min) and supernatant removal after each wash.
    11. Dry the pellets under vacuum in a concentrator at room temperature.

  5. Determining the content of acetyl esters
    1. Weigh about 1 mg destarched AIRs/pectin/xylan for 5 replicates into 1.5 ml Eppendorf tubes. 
    2. Add 100 µl 1 N sodium hydroxide to tubes and shake for 1 h at 28 °C and 200 rpm to release the bound acetate. 
    3. Add 100 µl of 1 N hydrogen chloride to neutralize samples. 
    4. Centrifuge at 15,000 x g for 10 min, and transfer 10 µl supernatant aliquot to a UV capable 96-well flat bottom assay plate and immediately quantify released acetic acids according to the instruction of Acetic Acid Assay Kit.
    5. Add 94 µl of ddH2O to each sample well in the UV capable 96-well flat bottom assay plate.
    6. Add 42 µl of freshly prepared mixture of kit-supplied Solutions 1 and 2 (2.5:1, 30 µl + 12 µl each), mix and incubate at 25 °C for 3 min with shaking at 300 rpm in a thermomixer comfort.
    7. Read the absorption at 340 nm (A0).
    8. Add 12 µl of freshly diluted kit-supplied solution 3 (1:10, v/v, in ddH2O), mix and incubate at 25 °C for 4 min with shaking at 300 rpm in a thermomixer comfort.
    9. Read the absorption at 340 nm (A1).
    10. Add 12 µl of freshly diluted kit-supplied solution 4 (1:10, v/v, in ddH2O), mix and incubate at 25 °C for 12 min with shaking at 300 rpm in a thermomixer comfort.
    11. Read the absorption at 340 nm (A2).
    12. At the same time, it is necessary to make a blank control and a standard curve in parallel. To make a standard curve, add 5, 10, 15, 30, 50 µl acetic acid standard (solution 5) equal to 0.5, 1, 1.5, 3, 5 µg, and adjust the volume of ddH2O in Step E5 to 99, 94, 89, 74, 54 µl, respectively. 

Data analysis

  1. Generation of standard curve and calculation of cellulose content
    To calculate cellulose amount, the standard curve needs to be plotted out first (The absorbance of glucose standards versus the concentration of glucose standards). Then calculate cellulose content (µg glucose/mg AIR) of sample with standard curve.
  2. For acetylation content analysis: Δacetic acid = (A2 - A0)sample - (A1 - A0)2sample/(A2 - A0)sample - [(A2-A0)blank - (A1 - A0)2blank/(A2 - A0)blank].
    Locate the sample value in the standard curve, and get acetic acid content in the sample aliquot. Calculate sample contents by multiplying with a factor of (100 + 100)/10/weight (µg/mg AIR).
  3. For statistical analysis, typically use at least five biological replicates from a pool of internodes. Results are reported as means and standard deviation and statistical significance is assessed by Student’s t-test or one-way ANOVA followed by Tukey’s multiple comparison test.

Notes

  1. If a freeze-dryer is not available, it could be alternative to prepare AIR directly from frozen tissue that is homogenized with a mortar and pestle immersed in liquid nitrogen or a ball mill pre-immersed in liquid nitrogen.
  2. For acetylation analysis, the acetic acid released should be assayed as soon as possible, do not leave too long before plate reading.

Recipes

  1. MES/Tris buffer (pH 8.1)
    1. Add 488 mg MES and 355 mg Tris in 40 ml double distilled water 
    2. Adjust the pH to 8.1 with sodium hydroxide and finally dilute to 50 ml
  2. 2 M trifluoroacetic acid
    Dilute 15.31 ml TFA with ddH2O to a final volume of 100 ml
  3. 1 N sodium hydroxide
    Add 2 g of sodium hydroxide to 50 ml of ddH2O
  4. 1 N hydrogen chloride
    Mix 4 ml 37% hydrogen chloride with 44 ml ddH2
  5. 50 mM ammonium formate (pH 4.5)
    1. Add 0.15 g of ammonium formate to 50 ml ddH2O
    2. Adjust the pH to 4.5 with formic acid

Acknowledgments

This protocol was adapted from previous work (Zhang et al., 2019). This work was supported by the National Natural Science Foundation of China (grant No. 31530051, 31571247, and 91735303), the Youth Innovation Promotion Association, Chinese Academy of Sciences (grant No. 2016094). This protocol is based on the following previously published reports: Updegraff (1969), Harholt et al. (2006), Goncalves et al. (2008), Li et al. (2009), Foster et al. (2010), Bromley et al. (2013), de Souza et al. (2014), Zhang et al. (2017).

Competing interests

The authors declare no conflict of interest.

References

  1. Bromley, J. R., Busse-Wicher, M., Tryfona, T., Mortimer, J. C., Zhang, Z., Brown, D. M. and Dupree, P. (2013). GUX1 and GUX2 glucuronyltransferases decorate distinct domains of glucuronoxylan with different substitution patterns. Plant J 74(3): 423-434.
  2. de Souza, A., Hull, P. A., Gille, S. and Pauly, M. (2014). Identification and functional characterization of the distinct plant pectin esterases PAE8 and PAE9 and their deletion mutants. Planta 240(5): 1123-1138.
  3. Goncalves, V. M., Evtuguin, D. V. and Domingues, M. R. (2008). Structural characterization of the acetylated heteroxylan from the natural hybrid Paulownia elongata/Paulownia fortunei. Carbohydr Res 343(2): 256-266
  4. Foster, C.E., Martin, T.M., Pauly, M. (2010). Comprehensive compositional analysis of plant cell walls (Lignocellulosic biomass): Part II. Carbohydrates. J Vis Exp 37: e1837.
  5. Grantham, N. J., Wurman-Rodrich, J., Terrett, O. M., Lyczakowski, J. J., Stott, K., Iuga, D., Simmons, T. J., Durand-Tardif, M., Brown, S. P., Dupree, R., Busse-Wicher, M. and Dupree, P. (2017). An even pattern of xylan substitution is critical for interaction with cellulose in plant cell walls. Nat Plants 3(11): 859-865.
  6. Harholt, J., Jensen, J. K., Sorensen, S. O., Orfila, C., Pauly, M. and Scheller, H. V. (2006). ARABINAN DEFICIENT 1 is a putative arabinosyltransferase involved in biosynthesis of pectic arabinan in Arabidopsis. Plant Physiol 140(1): 49-58.
  7. Kang, X., Kirui, A., Dickwella Widanage, M. C., Mentink-Vigier, F., Cosgrove, D. J. and Wang, T. (2019). Lignin-polysaccharide interactions in plant secondary cell walls revealed by solid-state NMR. Nat Commun 10(1): 347.
  8. Li, M., Xiong, G., Li, R., Cui, J., Tang, D., Zhang, B., Pauly, M., Cheng, Z. and Zhou, Y. (2009). Rice cellulose synthase-like D4 is essential for normal cell-wall biosynthesis and plant growth. Plant J 60(6): 1055-1069. 
  9. Updegraff, D. M. (1969). Semimicro determination of cellulose in biological materials. Anal Biochem 32(3): 420-424.
  10. Zhang, B., Zhang, L., Li, F., Zhang, D., Liu, X., Wang, H., Xu, Z., Chu, C. and Zhou, Y. (2017). Control of secondary cell wall patterning involves xylan deacetylation by a GDSL esterase. Nat Plants 3: 17017.
  11. Zhang, L., Gao, C., Mentink-Vigier, F., Tang, L., Zhang, D., Wang, S., Cao, S., Xu, Z., Liu, X., Wang, T., Zhou, Y. and Zhang, B. (2019). Arabinosyl deacetylase modulates the Arabinoxylan acetylation profile and secondary wall formation. Plant Cell 31(5): 1113-1126.

简介

植物细胞壁是一个复杂的网络,主要由多糖组成,例如纤维素,半纤维素和果胶。许多非纤维素多糖被进一步乙酰化,赋予这些聚合物灵活的物理化学特性。由于细胞壁在植物生长和发育中的重要性,因此分析平台长期以来一直是人们关注的焦点。在这里,我们使用节间/茎秆(一种为水稻植物提供机械支撑的重要器官)作为实验样品,探索细胞壁组成分析的方法。该方法包括细胞壁残留物的制备,多糖的顺序提取以及纤维素的测量。还描述了乙酸盐检查的程序。该方法适用于确定单个细胞壁聚合物和改性剂乙酸酯的组成,并且基于高通量,精确度和可重复性的优点,适合于鉴定与细胞壁相关的突变体。
【背景】 植物细胞壁代表自然界中最复杂的细胞结构之一,对于植物生长和适应环境至关重要。除了提供多种多糖成分和酚类化合物外,乙酰化是大多数细胞壁聚合物的普遍修饰,可改变其理化性质并增加细胞壁结构的复杂性。建立有效的细胞壁组成分析平台始终是一项艰巨的任务。先前的分析方法通常使用碱提取细胞壁残留物,但除去乙酸盐。最近的研究表明,木聚糖上的乙酸酯图案决定了该聚合物的折叠并影响了与纤维素或木质素的结合,表明其在细胞壁形成和植物生长控制中的重要性(Grantham et al。,2017; Kang et al。,2019; Zhang et al。,2019)。需要开发一种能够同时检查多种细胞壁多糖的组成及其乙酰基修饰的方法。由于已发现溶剂二甲基亚砜可提取木聚糖而不需修整乙酰基酯,因此它变得可实现(Goncalves等,2008)。稻秆代表细胞壁成分方法学分析的代表,因为该器官富含次生壁纤维细胞,并且还包含多种细胞类型。除丰富的材料外,乙酰化水平在不同的壁聚合物上和茎秆发育过程中也不同。通过使用稻秆作为分析样品,我们开发了一种用于细胞壁组成和乙酰基修饰分析的方案,与以前的方法相比有所变化(Foster et al。,2010)。该协议提供了一种广泛使用的方法来检查各种细胞壁聚合物的组成并确定不同水稻品种和其他农作物中的乙酸盐含量。

关键字:木聚糖, 果胶, 纤维素, 乙酰化, 细胞壁, 水稻

材料和试剂

  1. 96孔平底测定板(Greiner bio-one,目录号:655180)
  2. 具有紫外线功能的96孔平底测定板(Corning,目录号:3635)
  3. 玻璃瓶
  4. Eppendorf管(1.5 ml)(Eppendorf,目录号:0030120.086)
  5. Sarstedt管2毫升(Sarstedt,D-51588)
  6. 50 ml塑料离心管(Corning CentriStar)
  7. 玻璃超细纤维过滤器(Whatman,目录号:1820-025)
  8. 水稻成熟植物
  9. 内聚半乳糖醛酸内切酶M2(Megazyme,目录号:PGALUSP,4°C)
  10. 果胶甲基酯酶(Sigma-Aldrich,目录号:P5400-1KU,-20°C)
  11. α-淀粉酶(Megazyme,目录号:E-BLAAM,4°C)
  12. ddH 2 O
  13. 乙酸测定试剂盒(Megazyme,目录号:K-ACET,4°C)
  14. 丙酮
  15. DMSO(Sigma-Aldrich,目录号:D5879)
  16. 70%(v / v)乙醇水溶液
  17. 氯仿/甲醇(1:1,v / v)溶液
  18. 上接枝试剂(乙酸:硝酸:水,8:1:2 v / v)
  19. 72%硫酸(用浓硫酸GR制备)
  20. 1 mg / ml葡萄糖原液(由D-(+)-葡萄糖制备)(Sigma-Aldrich,目录号:G8270,−20°C)
  21. 蒽酮试剂(浓硫酸中的2 mg / ml蒽酮)(Sigma-Aldrich,目录号:319899)
  22. 三氟乙酸(TFA)(Sigma-Aldrich,目录号:T6508)
  23. 甲酸铵(Aldrich,目录号:516961)
  24. 11%过氧乙酸溶液(由35%过氧乙酸制备)(阿拉丁,目录号:P112625)
  25. 乙醇:甲醇:水溶液(7:2:1,用HCOOH调节pH值至3.0)
  26. 1%草酸铵(Sigma-Aldrich,目录号:09898)
  27. 37%氯化氢
  28. MES / Tris缓冲液(pH 8.1-8.3)(请参阅食谱)
  29. 2 M三氟乙酸(请参阅食谱)
  30. 1 N氢氧化钠(请参阅食谱)
  31. 1 N氯化氢(请参阅食谱)
  32. 50 mM甲酸铵(pH 4.5)(请参阅食谱)

设备

  1. 冷冻干燥机(北京松原华兴科技发展有限公司,型号:LGJ-12)
  2. 球磨机(QIAGEN,TissueLyser II,目录号:85300)
  3. (可选)涡旋振动筛
  4. 篮式离心机(Eppendorf,型号:5810R)
  5. 离心机(Eppendorf,型号:5430)(适合Eppendorf 1.5 ml管)
  6. 温控器舒适度(Eppendorf)
  7. Dri-Block加热器(技术,型号:DB200 / 3)
  8. 酶标仪(PerkinElmer,Enspire)
  9. 集中器(Eppendorf,集中器加)
  10. (可选)干燥箱
  11. (可选)摇动培养箱
  12. 筛子(筛孔尺寸0.15毫米)
  13. pH计(梅特勒-托利多)
  14. 半微米秤(双分辨率起始于0.01 mg)

程序

  1. 脱醇的不溶性细胞壁残留物(AIR)的制备
    1. 汇集5-20个水稻成熟植物的整个第二节间(从上到下编号)。
    2. 将新鲜样品在液氮中冷冻,然后在冷冻干燥机中冻干(将稻节间冻干48小时以确保完全干燥)。
    3. 使用球磨机将组织研磨至不超过0.15毫米的粒度,并通过0.15毫米大小的筛网过筛。
    4. 在50 ml塑料离心管中称取约1 g地面植物生物量。
    5. 加入30 ml的70%(v / v)乙醇水溶液,使用涡旋混合器充分混合,然后在37°C和200 rpm的温度混合器中放置12小时。
    6. 在篮式离心机中以1,500 x g 离心10分钟,并弃去上清液。
    7. 重复步骤A5-A6。
    8. 加入30 ml的氯仿/甲醇(1:1 v / v)溶液,使用涡旋混合器充分混合,然后在37°C和200 rpm的振动培养箱中放置30分钟。
    9. 在室温下以1,500 x g 离心10分钟,弃去上清液。
    10. 重复步骤A8-A9两次。
    11. 加入15毫升的丙酮,摇动试管以重新悬浮沉淀物。
    12. 以1,500 x g 离心10分钟,弃去上清液。
    13. 重复步骤A11-A12两次。
    14. 让生物质样品在40°C的干燥箱中干燥,不要摇动约16 h。
    15. 在97°C下用40 ml MES / Tris缓冲液(pH 8.1)中的100 Uα-淀粉酶处理残留物35分钟,然后在60°C下处理1 h。
    16. 以1,500 x g 离心10分钟,弃去上清液。
    17. 用30 ml ddH 2 O洗涤沉淀3次,用15 ml丙酮洗涤2次,离心(每次1,500 x g 10分钟),每次洗涤后除去上清液。
    18. 让生物质样品在40°C的烤箱中干燥约16小时,以去除空气中的淀粉。

  2. 结晶纤维素含量的分析
    1. 称量2 mg AIR材料,一式五份,放入2 ml Sarstedt管中。
    2. 向每个样品中加入250 µl 2 M三氟乙酸(TFA),并确保没有材料溅到管壁上。
    3. 盖紧盖子,在Dri-Block加热器中于121°C孵育90分钟。
    4. 在冰上冷却加热块和样品。
    5. 以11,000 xem 离心10分钟,然后将上清液转移至新的Sarstedt管中以进行可选的非纤维素多糖成分分析(Foster et al。,2010),并保持沉淀结晶纤维素测定。
    6. 向步骤B5剩下的沉淀物中加入1 ml Updegraff试剂(乙酸:itric酸:水,8:1:2 v / v),盖紧瓶盖并涡旋。
    7. 在Dri-Block加热器中于100°C加热30分钟。
    8. 在冰上冷却样品。
    9. 将样品以11,000 x g 的速度离心10分钟。
    10. 丢弃上清液,以确保不丢弃任何沉淀物。
    11. 用1 ml水洗涤一次,并用1 ml丙酮洗涤四次,离心并按照上述方法弃去上清液。
    12. 在35°C的Dri-Block加热器中风干沉淀。
    13. 加入175 µl 72%硫酸,在室温下孵育60分钟。
    14. 加入825 µl ddH 2 O,涡旋并以11,000 x g 的速度离心5分钟。
    15. 在96孔平底测定板中使用蒽酮测定法分析上清液中的葡萄糖含量。
    16. 加入10 µl样品和90 µl ddH 2 O,每个样品孔中的总体积为100 µl。
    17. 使用1 mg / ml葡萄糖储备液(-20°C的储备液)准备标准液。通过将0、2、4、6、8和10 µl移入适当的孔中,加入100、98、96、94、92和90 µl ddH,制成0、2、4、6、8和10 µg标准液 2 O。
    18. 加入200 µl新鲜制备的蒽酮摄政剂。
    19. 将板在80°C的温度下加热30分钟,同时在热混合器中摇晃。冷却至室温。
    20. 在恒温混合器中彻底摇动平板,然后在1小时内读取625 nm处的吸收。

  3. 从细胞壁残留物中提取果胶
    1. 称重约6 mg脱淀粉的AIR,重复5次,放入1.5 ml Eppendorf管中。
    2. 加入1 ml 50 mM甲酸铵(pH 4.5)缓冲液,然后加入2 U内聚半乳糖醛酸酶M2和0.04 U果胶甲基酯酶。混合并在37°C下孵育16小时,然后在300 rpm的温度下摇动,并置于舒适的温度混合器中。
    3. 于3,000 x g 离心10分钟,然后将富含果胶的上清液转移至新试管中。将残留物保留为无果胶样品。
    4. 用液氮冷冻上清液,然后在冷冻干燥机中冻干,最后得到凝胶状果胶。

  4. 从AIR分离乙酰木聚糖
    1. 称量约400 mg脱去淀粉的AIR到50 ml离心管中。
    2. 加入30 ml 1%草酸铵,在85°C下孵育2小时。在篮式离心机中以1,500 x g 离心15分钟。丢弃上清液以除去果胶。
    3. 加入30 ml 11%过氧乙酸溶液,在85°C的水浴中孵育30分钟以进行脱木素作用。
    4. 以1,500 x g 离心10分钟。弃去上清液,并通过离心(1,500 x g 10分钟)并用30 ml ddH 2 O洗涤3次,并用15 ml丙酮洗涤一次,沉淀。
    5. 让颗粒在设定为40°C的烤箱中干燥约16小时。
    6. 加入30 ml DMSO,然后在70°C下孵育12 h以进行提取,以1,500 x g 离心10分钟,然后将上清液转移至新的玻璃瓶中。
    7. 用12 ml DMSO重复步骤D6,并合并含有提取物的上清液。
    8. 通过玻璃微纤维过滤器过滤提取物。
    9. 在4°C下用5体积的乙醇:甲醇:水溶液(7:2:1,pH 3.0)沉淀提取物12小时。
    10. 以1,500 x g 离心15分钟。弃去上清液,并用10 ml无水乙醇离心4次(每次1,500 x g 10分钟)洗涤沉淀四次,每次洗涤后除去上清液。
    11. 在室温下在浓缩器中真空干燥沉淀。

  5. 测定乙酰基酯的含量
    1. 称重约1 mg脱淀粉的AIRs /果胶/木聚糖,重复5次,放入1.5 ml Eppendorf管中。
    2. 向试管中加入100 µl 1 N氢氧化钠,并在28°C和200 rpm下摇动1 h,以释放结合的乙酸盐。
    3. 加入100微升的1 N氯化氢以中和样品。
    4. 以15,000 x g 的速度离心10分钟,然后将10 µl上清液等分试样转移至具有UV功能的96孔平底测定板中,并根据乙酸测定试剂盒的说明立即定量释放的乙酸。
    5. 向具有UV功能的96孔平底测定板的每个样品孔中加入94 µl ddH 2 O。
    6. 加入42 µl由试剂盒提供的溶液1和2(2.5:1、30 µl + 12 µl各自)制成的新鲜制备的混合物,混合并在25°C下温育3分钟,并在300 rpm的温度下摇动,并置于热混合器中。
    7. 读取340 nm(A0)处的吸收。
    8. 加入12 µl新鲜稀释的试剂盒提供的溶液3(1:10,v / v,在ddH 2 O中),混合并在25°C下孵育4分钟,并在300 rpm下摇动。温控器的舒适性。
    9. 读取340 nm(A1)处的吸收。
    10. 加入12 µl新鲜稀释的试剂盒提供的溶液4(1:10,v / v,在ddH 2 O中),混合并在25°C下孵育12分钟,并在300 rpm下摇动。温控器的舒适性。
    11. 读取340 nm(A2)处的吸收。
    12. 同时,必须同时进行空白对照和标准曲线。要绘制标准曲线,请添加等于,等于0.5、1、1.5、3、5 µg的5、10、15、30、50 µl乙酸标准溶液(溶液5),并调节ddH 2 O,分别为99、94、89、74、54 µl。

数据分析

  1. 标准曲线的生成和纤维素含量的计算
    要计算纤维素含量,首先需要绘制标准曲线(葡萄糖标准品的吸光度与葡萄糖标准品的浓度)。然后用标准曲线计算样品的纤维素含量(微克葡萄糖/毫克空气)。
  2. 用于乙酰化含量分析:Δ乙酸=(A2-A0)样品-(A1-A0) 2 样品 /(A2-A0) sample -[(A2-A0)空白-(A1-A0) 2 空白 /(A2-A0) 空白]。
    在标准曲线中找到样品值,并获得等分样品中的乙酸含量。通过乘以(100 + 100)/ 10 /重量(µg / mg AIR)的系数来计算样品含量。
  3. 对于统计分析,通常使用节点间池中的至少五个生物学副本。结果以均值报告,标准差和统计显着性通过学生的 t 检验或单向ANOVA进行评估,然后再进行Tukey的多重比较检验。

笔记

  1. 如果没有冷冻干燥机,则可以选择直接用冷冻的组织制备AIR,冷冻后的组织用研钵和研杵浸入液氮中匀浆,或者将球磨机预先浸入液氮中。
  2. 为了进行乙酰化分析,应尽快测定释放出的乙酸,不要在板读数前放置太久。

菜谱

  1. MES / Tris缓冲液(pH 8.1)
    1. 在40毫升双蒸馏水中加入488毫克MES和355毫克Tris。
    2. 用氢氧化钠调节pH至8.1,最后稀释至50 ml
  2. 2 M三氟乙酸
    用ddH 2 O稀释15.31 ml TFA,使最终体积为100 ml
  3. 1 N氢氧化钠
    向50 ml的ddH 2 O中添加2 g氢氧化钠
  4. 1 N氯化氢
    将4 ml 37%氯化氢与44 ml ddH 2 O
  5. 50 mM甲酸铵(pH 4.5)
    1. 将0.15克甲酸铵加到50毫升ddH 2 O中
    2. 用甲酸将pH调节至4.5

致谢

该协议改编自以前的工作(Zhang et al。,2019)。这项工作得到了中国国家自然科学基金(批准号31530051、31571247和91735303),中国科学院青年创新促进会的支持(批准号2016094)。该协议基于以下先前已发布的报告:Updegraff(1969),Harholt 等人()(2006),Goncalves 等人()(2008),Li 等人((2009),福斯特(emoster)等人()(2010),布罗姆利(emrom)等人()(2013),德苏扎(em Souza)等人(2014),张 et al。(2017)。

利益争夺

作者宣称没有利益冲突。

参考文献

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引用:Zhang, L., Zhang, B. and Zhou, Y. (2019). Cell Wall Compositional Analysis of Rice Culms. Bio-protocol 9(20): e3398. DOI: 10.21769/BioProtoc.3398.
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