Establishing And Applying Of A Position-Specific Oxygen Isotope Composition Measuring Procedure For Higher Plants-Derived Lignin

Lignin is a three-dimensional,amorphous polymer in plants,mainly composed of three structural monomers of p-coumaryl alcohol,coniferyl alcohol and sinapyl alcohol(referred to as H-type,G-type and S-type structural monomers).The content of lignin is second only to cellulose,which is an important carrier of ecological,climate,environmental and physiological metabolic information.However,there is currently no effective method to access the 18O/16O ratios of individual oxygens in lignin structural(monomeric)units so that this abundant material is inaccessible for climate,environmental,physiological,botanical and metabolic studies.The main purpose of this PhD work is to break this technical bottleneck by developing a novel wet-chemistrybased method to measure the 18O/16O of the three oxygens(O-3,O-4 and O-5)attached to the aromatic ring and those in the side chains of the structural units.Four aspects of lignin oxygen isotopes at natural abundance levels have been addressed:(1)How to access the 18O/16O ratios of individual oxygens within the structural units of lignin,i.e.,position-specific oxygen isotope analysis(PSIA-18O)of lignin.This was achieved by depolymerizing the lignin to monomeric units(bearing no oxygen in their side chains)using highly selective hydrogenolysis catalyzed by W2C/AC(tungsten semicarbide supported by activated carbon)followed by simultaneous 18O/16O ratio measurement of all individual structural units(CSIA-18O:compound-specific oxygen isotope analysis)with GC/Py/IRMS(gas chromatography-pyrolysis-isotope ratio mass spectrometry).18O/16O for O-4 was obtained directly from the measurement of 18O/16O of H-type structural units,while those for O-3 and O-5 can be calculated following isotope mass balance calculation between the H,G and S-types structural units.(2)The intramolecular isotope profile for oxygen(IMIP-O)promised by the new technique.This work analyzed the PSIA-18O and CSIA-18O profiles of the structural units of lignin extracted from the leaves(photosynthetic tissues)and stems/branches(non-photosynthetic tissues)from a cohort of woody and herbaceous plant species grown in close vicinity at the Xi’an Botanic Garden(CHINA).The analysis led to the discovery of an extraordinary isotopic order for the three major structural units(H,G and S types)of lignin in herbaceous plants:δ18OH>δ18OG>δ18Os,and an exactly opposite order:δ18OH<δ18OG<δ18OS for the lignin in woody plants.(3)The carbon isotope compositions of structural units released with the catalyzed hydrogenolysis(CSIA-13C),and a CSIA-13C signal for the released lignin structural units for which the PSIA-18O signal is also obtained will be a valuable added dimension of constraint.Consequently,lignin structural units from a total of 36 plant leaf and stem(branch)samples were obtained and analyzed forδ13C by GC/C/IRMS(gas chromatography/combustion/isotope ratio mass spectrometry).A carbon isotopic order amongst the three structural units similar to that for oxygen isotopes emerged:δ13CH>δ13CG>δ13Cs.Again,a thorough investigation of the histories of the carbons in the three(H,G and S)structural units led to the conclusion that such an isotopic order is the result of continuous addition of a 13C-highly depleted methyl groups to the aromatic ring during the synthesis of G and S-type units from H-type units.Additionally,the CSIA-13C data of lignin structural units,already allowed for a reliable assignment of their source plants to specific photosynthetic carbon assimilation mode,even though,no clear isotopic pattern is discernable between the δ13C values of the lignin structural units and the life forms of their source plants,possibly due to a limited sample set.For example,the δ13C values of the H units ranged from-8.9‰ in Aristida brevissima(a C4 plant)to-39.4‰ in the leaves of Bambusa multiplex(a C3 plant),the δ13C values of the G units ranged from-16.8‰in the leaves of Lolium perenne(a C4 plant)to-44.4‰ in the leaves of Cercis chinensis(a C3 plant),while those for the S units spanned from-31.3‰ in the leaves of Ligustrum sinense(a C4 plant)to-46.2‰ in the stem of Rumex acetosa(a C3 plant).(4)The δ13C values of lignin precursors from plants were investigated.Removal of the unambiguity on the sources of the precursors(L-phenylalanine and L-phenylalanine)for the synthesis of lignin monomers in woody and herbaceous plants stands to benefit the interpretation of the PSIA-18O and CSIA13C of the lignin structural units as such precursors can originate from both leaves and stems.Unfortunately,no effective method for measuring the δ13C of Lphenylalanine and L-phenylalanine in plants leaves and stems is readily available.Effort was made,consequently,to establish a GC/C/IRMS-based method to measure the δ13C of free amino acids(FAAs)derivatized with methyl chloroformate(MCF),following,in sequence,an ultrasonication-assisted ethanol extraction(for plant leaves),or KCl-assisted water extraction(for soils)step and a strong cation ion-exchange cleanup step.As a demonstration of its effectiveness,the method was applied to measure the FAAs δ13C values in the leaves of 12 naturally grown plants(in the Qingling Mountains).Data from this pilot set of samples showed that a)the δ13C values varied largely from species to species:the lowest value-47.1‰ was found in the threonine of the leaves of Ilex asprella,while the highest value-21.1‰ was found in the threonine of the leaves of Tadehagi triquetrum the δ13C values of individual FAAs in the same plant species also varied greatly:lower values were found in valine and leucine while higher values were found in tyrosine.