Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.
Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.
维管束植物依靠充当植物组织和外部环境之间的防水屏障外层。这些亲脂性的细胞壁相关结构限制病原体感染和调节的气体,水和溶解的物质的被动转运进出植物组织1。这种障碍是植物表皮,一个synapomorphic结构独特的植物2,以及不同的含软木脂扩散壁垒。角质层是由表皮细胞经由果胶层上的细胞壁3-5的外侧合成并结合到它们的亲油性层。它包住高等植物的主天线器官,用作植物组织和环境之间的一个重要的接口。
角质,角质层的结构基体,和软木脂与溶剂萃取的蜡2,4相关联的两个不溶甘油脂聚酯。这些聚合物升ipids是由饱和和不饱和脂肪酸衍生物的且都是结构上和功能上类似。然而,它们是可区分由在化学组成和沉积点特性的差异。
软木脂是一种脂族聚酯位于形成次生壁一定的外部和内部组织的细胞壁内。 Suberized组织包括根periderms,块茎和树皮,根内皮层,种皮层和愈合伤口2。不像角质,所述木栓质聚酯通常含有醇,饱和的和单不饱和二羧酸,和相当大的比例非常长链的单体的(C≥20)。
角质是在维管植物6中最丰富的脂类聚酯,和由甘油和C 16 -C 18交换的脂肪酸衍生物,如羟基和羟基取代的环氧脂肪酸4。而角质聚合物的组合物跨越维管种类而异,最主要主单体为10,16二羟基16:0,18羟基-9,10-环氧18:0,和9,10,18三羟基18:0脂肪酸。有趣的是,拟南芥属叶和茎角质主要由18:2的二羧酸7,8。
植物表皮也厚度呈现出相当大的变化,从几纳米到几微米9。由于角质层隔离是费力和耗时的步骤,特别是对于非常薄的叶子表皮诸如拟南芥 8,绕过角质层分离方法已经开发和验证7,8。在这里,我们描述了一个详细的协议来研究角质的拟南芥叶片由甲醇钠(甲醇钠)催化的解聚和随后的气相色谱/质谱(GC / MS)分析该单体组合物。该协议提供了一个强大的方法测定共mposition在全脱脂组织的植物脂肪聚酯,并已被改编自先前报告协议7,10,11。整个组织样品是第一匀浆,彻底去脂,除去溶剂萃取的脂质包括表皮和epicuticular蜡,膜脂,和三酰基甘油。细胞壁富集残基,然后由甲醇钠催化的甲醇分解解聚成其组成甲酯单体。脂肪酸甲基酯被提取在酸化时,并衍生以得到其相应的三甲基甲硅烷基或乙酰基的衍生物。衍生的残基是高度挥发性的,并且可以从在适当温度下用气相色谱柱洗脱而不会在GC / MS分析改变其结构构象。
不像其他的生物聚合物如DNA和蛋白质,植物脂质聚酯不是从模板制成。相反,他们的组合物依赖于存在于使这些细胞外聚合物组织中的酶的特异性。这样,化学组成的分析成分是关键的了解脂质聚酯组合物。
化学方法裂解的酯键包括皂化,氢解,酸催化transmethylation,和碱催化transmethylation 2。他们每个人都有优点和缺点。皂化产生游离脂肪酸羟基酸,可以进行二次反应。氢解用氢化铝锂(的LiAlH 4)16已被用于角质分析7。氢解降低官能碳为醇和原始结构需要由deuteriolysis锂铝氘(LiAlD 4)推断。该这种方法的缺点是高清晰度的GC / MS的要求来比较获得使它们的结构分配的脂肪多元醇的deuteriation的程度。酸催化的酯交换用甲醇三氟化硼(BF 3)已被常用于角质和软木脂depolymerizations 8,17,18,但该试剂具有有限的货架寿命并可能引入由于副反应15假象。硫酸甲醇也产生的单体,但具有较大的2-羟基脂肪酸的比例,这大概是不正确的脂质聚酯组分甲酯,相比其他方法10。
在这个协议中所描述的的NaOMe催化酯交换法产生由羟基甲硅烷基化衍生的脂肪酸甲酯,用于识别提供特性质谱,或通过乙酰化,以提供羟基更稳定的衍生物FO- [R量化。这种技术的一个缺点是水解竞争与酯交换当水存在于反应。水发生反应的NaOMe(催化剂),并产生氢氧化钠,这反过来水解脂肪酸甲酯,得到游离酸(图2D)。这是一种不希望的副反应,因为两个峰值将存在对于每个脂肪酸:甲基酯和TMSI酯衍生物,因此复杂的分析。用无水试剂并加入乙酸甲酯作为助溶剂与皂化竞争因此关键步骤,以防止水解(图2D)。
1和26%甘油4之间的角质和木栓质含有。但是,此单体不会被在这个协议中所描述的实验条件下进行检测。甘油是高度亲水性的并且,不同于脂肪酸甲酯单体,将在水性溶剂洗涤步骤被消除。这种限制也是一个pplies其他角质解聚方法,但甘油可以在使用酶方法酯交换后得到的水层来确定。可替代地,它可以使用较温和的条件(,0.05M的的NaOMe 当量)定量无需进一步水萃取,以检测所有的单体,包括甘油19,20。虽然对于甘油量化的目的是有用的,在温和的条件通常给角质的不完全解聚和木栓质。
如果耦合到一个火焰离子化检测器(FID)的GC一个是可用的,所有的重复可在该仪器进行定量目的被分析,有代表性的样品的峰已确定的GC / MS后。或者,在GC / FID痕迹单体可以识别,如果他们的保留指数是已知的。火焰离子化检测器具有特别高的灵敏度和宽范围相称,这是主要和次要样品组分定量临界在单次运行。此外,它是坚固和易于维护和操作15。
所描述的协议允许的可靠和可重复的分离,鉴定和定量植物脂质聚酯单体,允许其在不同的一种或多种脂质聚酯单体的组合物的突变体的化学特性。该过程是可伸缩的,它可以很容易地适用于处理小型和大批量的各种植物材料,包括根,种子,叶,茎和花。来自许多物种的脂质聚酯单体的质谱数据已经公布,例如 ,21-26和构成宝贵资源适应这个协议对其他组织和/或物种时识别未知单体。这种方法适用于生物合成,调节和高等植物分布脂质聚酯的调查。
The authors have nothing to disclose.
This work was supported by a Natural Sciences and Engineering Research (NSERC)-USRA grant to S.J., and by an NSERC-Discovery Grant to I.M. We thank Richard Bourgault, Meghan Rains, and Amanda Fluke for technical assistance. Seeds of att1-1 and att1-2 mutants were kindly provided by Dr. Jian-Min Zhou, Institute of Genetics and Developmental Biology, Beijing, China.
Chemicals | |||
2-propanol | Fisher Scientific | BPA451-4 | Solvent for delipidation |
Anhydrous sodium sulfate | Fisher Scientific | S421500 | |
Acetic anhydride | Sigma Aldrich | 320102 | Derivatization agent |
BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide) | Sigma-Aldrich | 15222 | Derivatization agent |
Butylated hydroxytoluene (BHT) | Sigma-Aldrich | 101162 | Antioxidant |
Calcium chloride, anhydrous | Fisher Scientific | C614-3 | Desiccation agent |
Calcium suflate, anhydrous (DRIERITE- 8 MESH with indicator) | Acros Organics | 219090020 | Desiccation agent |
Chloroform (Trichloromethane) | Fisher Scientific | C6074 | Organic solvent |
Glacial acetic acid | Fisher Scientific | BP2401212 | Acidification agent |
Helium carrier gas, compressed | Air Liquide | ALPHAGAZ1-UN1046 | Carrier gas, GC/MS |
Heptane | Fisher Scientific | H3501 | Organic solvent |
Hexanes | Fisher Scientific | H3024 | Organic solvent |
Methanol | Fisher Scientific | A4124 | Organic solvent, transmethylation reactive |
Methyl acetate | Sigma-Aldrich | 296996 | Organic solvent |
Methyl heptadecanoate | Sigma-Aldrich | H4515 | Internal standard (1mg/mL stock) |
Methylene dichloride (Dichloromethane) | Fisher Scientific | D374 | Organic solvent |
Nitrogen, compressed | Air Liquide | ALPHAGAZ1-UN1044 | Carrier gas, GC-FID |
Pentadecanolactone | Fluka | 76530 | Internal standard (1 mg/mL stock) |
Pyridine | Sigma-Aldrich | 270970 | Co-solvent for derivatization |
Sodium chloride | Fisher Scientific | BP358212 | Saline solution |
Sodium methoxide (25wt.%) | Sigma-Aldrich | 156256 | Nucleophile |
Toluene | FIsher Scientific | T2904 | Organic solvent |
Plant Growth Supplies | |||
Pro-Mix PGX | Premier Tech Horticulture Ltd | Pro-Mix PGX is recommended to grow Arabidopsis plants (Eddy, R. and Hahn, D.T., 2012,http://docs.lib.purdue.edu/pmag/2) Purdue Methods for Arabidopsis Growth. |
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PermaNest Humidity Dome | Grower's Solution, LLC, Cookeville, TN | GD2211-24 | |
Perma-Nest Plant Trays (22x11in) | Grower's Solution, LLC, Cookeville, TN | N/A | |
Square greenhouse pots, 3.5 inch | Grower's Solution, LLC, Cookeville, TN | P86 | |
General Purpose Plant Fertilizer, Plant-Prod 20-20-20 | Premier Tech Home and Garden In., Brantford, ON | N/A | |
Glassware | |||
13 x 100 mm glass test tube with Teflon-faced screw cap | Kimble Chase Life Science and Research Products LLC | 45066A-13100 | |
16 x 125 mm glass test tube with Teflon-faced screw cap | Kimble Chase Life Science and Research Products LLC | 45066A-16125 | |
20 x 125 mm glass test tubes with Teflon-faced screw cap | Kimble Chase Life Science and Research Products LLC | 45066A-20125 | |
GC vial caps | National Scientific | C400051A | |
GC vial microinserts | National Scientific | C4011631 | |
GC vials | National Scientific | C40001 | |
Disposable pasteur pipets | Fisher Scientific | 1367820B | |
Flasks | Fisher Scientific | ||
Equipment | |||
Allegra X15R centrifuge | Beckman Coulter | ||
Analytic balance | Fisher Scientific | ||
Belly dancer | A shaker can be used for this purpose if Belly Dancer not available | ||
DB-5 Capillary GC column | J&W Scientific, CA, USA; | 30 m x 0.25 mm x 0.25 μm film thickness | |
Desiccator | |||
Isotemp 202 water bath | Fisher Scientific | ||
ISQ LT single quadupole mass spectrometer | Thermo Scientific | ||
Heat block | Fisher Scientific | ||
Nitrogen evaporator | |||
Polytron homogenizer | Birkmann | ||
Trace 1300 gas chromatograph | Thermo Scientific | ||
Two-stage regulator | Air Liquide | Q1-318B-580 | |
Vacuum desiccator | Fisher Scientific | ||
Vortex mixer | Fisher Scientific |