丙酮和二甲基亚砜法测定植物叶绿素和类胡萝卜素的方法学比较

Methodological Comparison of Chlorophyll and Carotenoids Contents of Plant Species Measured by DMSO and Acetone-extraction Methods

丙酮和二甲基亚砜作为提取剂测定植物光合色素含量是最为普遍的两种方法,但是对于二者之间的可比性问题探讨很少。本文基于对19中木本植物叶片和树枝内叶绿素含量的测定结果发现如下结果:(1)二甲基亚砜测定叶绿素a、叶绿素b和叶绿素总量的结果均显著高于丙酮测定结果(而对叶绿素a/b比值的影响较小),与此相反,丙酮对类胡萝卜素提取测定结果要远高于二甲基亚砜的测定结果。(2)两种方法对叶绿素和类胡萝卜素测定结果之间均存在显著线性相关,可以应用相关线性拟合方程进行不同方法测定结果之间的转换,以减少叶绿素含量、类胡萝卜素含量比较过程中产生的测定方法误差。(3)不同单位表示方法,对两种方法测定结果的相关性存在影响,使用鲜重单位表示的相关程度普遍高于以表面积为单位的相关程度。(4)两种方法对树枝和叶片测定结果的影响存在差异:对于叶绿素含量低的树枝,两种方法测定结果的差异较小,而对于叶绿素含量高的叶片,二甲基亚砜测定结果则远高于丙酮的测定结果,可能原因在于丙酮对于叶绿素的提取效果远低于二甲基亚砜。(5)以二甲基亚砜为浸提试剂,通过不同精确度仪器(0.1和1nm)检测发现尽管不同仪器之间相关关系达到0.99以上,但二者相比精确度为0.1nm检测叶绿素a、总叶绿素浓度高出15%～33%,而叶绿素b浓度低4%左右。这些研究发现为不同方法之间的比较提供了依据。

Chlorophyll and carotenoids measurement by dimethyl sulfoxide （DMSO） and acetone extractions are two typical and popular methods used in plant ecophysiological research. However, few studies on the compara- bility between these two methods have been carried out. To answer this question, the contents of pigments in leaves and young branches of 19 woody species were measured in this study and we found the following conclu- sions. Firstly, chlorophyll a （Chla）, Chlb and total chlorophyll contents measured by the DMSO method were much higher than those measured by the acetone method, however, the result of Chla/b ratio was slightly affect- ed. Contrarily, carotenoids contents measured by the acetone method were much higher than those measured by DMSO method. Direct comparison of these data will make serious bias; Secondly, both chlorophyll data and ca- rotenoids data measured by these two methods were significantly linearly correlated. Thus, it is possible to use these linear relations to make data correction in comparative studies; Thirdly, unit differences also affect the data correlations of these two methods. When using the unit of fresh mass, the correlation was much higher than that unit of surface area; Fourthly, the influences of these two methods on the data precision differed between leaves and branches. In the case of branches （low chlorophylls） , two methods give slightly difference. However, the difference was much significant in the case of leaves （ high chlorophylls）. The possible reason may attribute to the extracting efficiency of DMSO is much higher than acetone. Finally, spectrophotometer precision （0.1 and 1 nm） were strongly affected the result of different pigments measured by DMSO method. The 0.1 nm spectropho- tometer produced 15 % - 33 % higher concentration of Chla, Chla ＋ b and carotenoids, but a 4% lower concentration of Chlb although all the correlation coefficient R2 of two equipments were over 0.99. Our above methodo- logical findings may provide a basis for pigments comparison in physiological field.