| Leaf is the main organ of gas exchange between land plants and their environment.Although leaf development is largely genetically controlled,it is also affected by environment.The contents of different biomarkers(n-alkanes,fatty acids and a-cellulose)and their isotopic compositions(13C/12C,2H/1H and 18O/16O ratios)in a single leaf record the environmental conditions experienced by the leaf,such as temperature,humidity and water supply.The isotope composition of organic matter of leaves with reticulated veins can be used to reconstruct the environment experienced during the leaf life cycle under real world conditions(such as rainfall and soil evaporation).Up to now,most studies have focused on the response relationship between stable isotope of single biomarker in plant samples and environmental climate.In this paper,an improved method for systematic extraction of water,n-alkanes,fatty acids,and a-cellulose from<500 mg lotus leaf was proposed.Water,n-alkanes,fatty acids,and a-cellulose can be extracted from the same leaf fragment sequentially and in sufficient quantities for isotope characterisations.Specific work was as follows:(1)Firstly,leaves were cut and sealed in a pre-cooled centrifuge tube and water was extracted through a vacuum line.Secondly,leaves were extracted with a mixture of methylene chloride and methanol under ultrasonation,and n-hexane and ethyl acetate were respectively used to elute n-alkanes and fatty acids from silica gel column.Thirdly,fatty acids were then derived into fatty acid methyl ester(FAMEs)by BF3-CH3OH.Finally,α-cellulose was extracted from the residual solid under ultrasonication using a modified method by Zhou et al.(2)Two different methods were used to extract total lipids from lotus leaf slices.GC-MS was used to quantitatate n-alkanes by area normalization.The results showed that the reovery of ultrasonic extraction of n-alkanes from lotus leaf slices was higher.For the extraction of fatty acids,saponification method was improved to systematize the steps,and BF3-CH3OH was used for esterification.At the same time,the standard curve was used for quantitative analysis of mixed fatty acid methyl ester.The contents of hexadecanoic acid and octadecanoic acid in the systematic extraction saponification method were 0.225 mg/g and 0.183 mg/g,respectively.The characteristic peaks of a-cellulose extracted by Zhou’s method were all present in the infrared test results,XRD results showed that the crystallinity of a-cellulose was lower than that of standard a-cellulose.(3)Various biomarkers systematically extracted from leaf fragments were measured by GC/IRMS or EA/IRMS,and the spatial patterns of carbon,hydrogen and oxygen stable isotopes of different organic matter were studied according to the patterns before cutting of the original leaves.It was found that leaf water and a-cellulose in mature leaves were obviously enriched from center to edge.The δ18O gradient of a-cellulose in field mature leaves was only 1/4 of the gradient of leaf water,and the 82H of a-cellulose was only 1/8 of the gradient of leaf water,which means that oxygen isotope signal in leaf water was greatly diluted in a-cellulose.Compared with the δ13C center-to-edge enrichment trend of n-alkanes(C31,C33),the profile of δ13C in fatty acids(C16:0,C18:2)was opposite,which may be caused by the migration of 12C during the transport of organic matter or by the difference of CO2 concentration from the center to the edge of lotus leaf. |
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