Metabolomic study of water hyacinth exposed to CuCI2, FeCI3, Na2HPO4 solution by GC m

.. .METABOLOMIC STUDY OF WATER HYACINTH EXPOSED TO CuCl2, FeCl3 AND Na2HPO4 SOLUTIONS BY GC- MS HUANG XULEI (M Sc., PEKING UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE... cultured in 0.3 mmol FeCl3, 1.2 mmol CuCl2 and 3.46 mmol Na2HPO4 solution for 15 days, respectively The metabolic profiles of roots, stems and leaves of water hyacinth were determined by GC- MS and further... study, water hyacinth was exposed to 0.3 mmol FeCl3, 1.2 mmol CuCl2 and 3.46 mmol Na2HPO4 concentrations for 15 days, respectively The metabolic profiles of roots, stems and leaves of water hyacinth

METABOLOMIC STUDY OF WATER HYACINTH

BY GC-MS

HUANG XULEI

NATIONAL UNIVERSITY OF SINGAPORE

2014

METABOLOMIC STUDY OF WATER HYACINTH

BY GC-MS

HUANG XULEI

(M Sc., PEKING UNIVERSITY)

A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE

2014

DECLARATION

I hereby declare that this thesis is my original work and it has been written by me

in its entirety, under the supervision of Prof Sam Li Fong Yau, Chemistry Department, National University of Singapore, between 12 August 2013 and 12 August 2014

I have duly acknowledged all the sources of information which have been used in the thesis

This thesis has also not been submitted for any degree in any university previously

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my supervisor Prof Li Fong Yau for

giving me the chance to join his group and for encouraging me to enter into the

wonderful world of analytical chemistry His integral view on research has made a

deep impression on me and has helped me out immensely by keeping me and my

research focused and on track I owe him lots of gratitude for having shown me the

ways of scientific research Besides of being an excellent supervisor, Prof Li was as

close as a relative and a good friend to all the students I am really glad that I have

come to get know Prof Li in my life

I would like to thank all the staffs and students in particular Dr.Liu Feng, Dr Gan

Pei Pei, Dr Li Ping Jing, Dr Guo Rui, Lin Xuanhao, Lai Linke, Feng Ting, Wu Ye

and Liang Xiaojian who were in the same lab with me Over the last year, I have

indeed enjoyed working with them They are so kind and ready to help me when

necessary We also discussed and shared some knowledge and information with each

other freely Best wishes to all of them

Finally, heartful thanks go to my family for their immense support along the way

TABLE OF CONTENTS

DECLARATION I ACKNOWLEDGEMENTS II TABLE OF CONTENTS III SUMMARY V LIST OF TABLES VI LIST OF FIGURES VII

1 INTRODUCTION 1

1.1 INTRODUCTION TO METABOLOMICS 1

1.2 PLANT METABOLOMICS 3

1.2.1 PLANT METABOLOMICS IN RESPONSE TO ABIOTIC STRESSES 5

1.3 HEAVY METAL ACCUMULATION IN WATER HYACINTH 14

1.4 BASIS AND SIGNIFICANCE OF DISSERTATION 15

2 MATERIALS AND METHODS 17

2.1 MATERIALS 17

2.2 INSTRUMENTS AND REAGENTS 17

2.3 SAMPLE EXTRACTION AND DERIVATIZATION 18

2.4 GC-MS ANALYSIS 18

2.5 DATA ANALYSIS 18

3 RESULTS AND DISCUSSION 20

3.1 GC RESULTS ANALYSIS 20

3.2 IDENTIFIED COMPOUNDS 25

3.3 PCA ANALYSIS 32

3.4 WEAKNESS AND PROSPECT OF THE STUDY 37

4 CONCLUSION 39

BIBLIOGRAPHY 41

SUMMARY

It is generally accepted that water hyacinth is capable of adsorbing excessive heavy metals, but the metal adsorption mechanism in the metabolic level is unknown In this study, the water hyacinth plants were cultured in 0.3 mmol FeCl3, 1.2 mmol CuCl2and 3.46 mmol Na2HPO4 solution for 15 days, respectively The metabolic profiles of roots, stems and leaves of water hyacinth were determined by GC-MS and further analyzed by PCA method Results showed that plants suffered severe damage under FeCl3 exposure but were tolerable to Na2HPO4 exposure Metabolites levels in stems and leaves increased but decreased in roots under CuCl2 exposure Leaves and stems

of the four differently treated plants could be distinctly separated in three-dimensional PCA, while roots could only be separated between control group and the treated groups individually by two-dimensional PCA Levels of D-glucopyranose, L-threonine, Butanoic acid and 9H-Purin-6-amine significantly increased in treated plants and acted as osmoprotectants This study provided an overall perspective of metabolites change in water hyacinth for mechanism of metal accumulation

LIST OF TABLES

Table 1 Advantages and disadvantages of NMR and MS for metabolomics study 3

Table 2 (a)Identified compounds in leaves of control and Na2HPO4, FeCl3, CuCl2

exposed samples in sequence of retention time (RT) and the corresponding peak

areas The peak areas were normalized to the mean response calculated for the

control of each measured batch (±standard error) 25

Table 2 (b)Identified compounds in stem of control and Na2HPO4, FeCl3, CuCl2

exposed samples in sequence of retention time (RT) and the corresponding peak

areas The peak areas were normalized to the mean response calculated for the

control of each measured batch (±standard error) 25

Table 2 (c) Identified compounds in root of control and Na2HPO4, FeCl3, CuCl2

exposed samples in sequence of retention time (RT) and the corresponding peak

areas The peak areas were normalized to the mean response calculated for the

control of each measured batch (±standard error) 25

LIST OF FIGURES

Figure 1 GC results of leaves pretreated by (a) wet grinding and (b) dry grinding

in the control group 20

Figure 2 GC results of stems pretreated by (a) wet grinding and (b) dry grinding

in the control group 21

Figure 3 GC results of roots pretreated by (a) wet grinding and (b) dry grinding

in the control group 22

Figure 4 GC results of leaf in the control group (a) before cultivation and (b)

after cultivation 23

Figure 5 GC results of (a) leaf, (b) stem and (c) root in the control group after

cultivation 24

Figure 6 Three-dimensional PCA of the metabolic profiles of (a)leaf, (b) stem

and (c) root in control and the exposed samples 33

Figure 7 Two-dimensional PCA of the metabolic profiles in roots of (a) control

vs Na2HPO4 treated group, (b) control vs FeCl3 treated group and (c) control

vs CuCl2 treated group 35

1 Introduction

1.1 Introduction to metabolomics

Metabolomics is a science for living systems (including cell, tissue and organism) research through examining metabolic responses or time-course variation of living systems when they are subjected to stimulation or disturbance Based on group indicators, the goal of metabolomics as a branch of systems biology is information modeling and system integration via high-throughput detection and data processing Metabolomics is another important research area in systems biology following after genomics, transcriptomics and proteomics The focus of metabolomics is metabolites variation of small molecules with molecular weight smaller than 1000 in metabolism, reflecting metabolic responses variation of cells or tissues subjected to outside stimuli

or genetic modification

The living organism is a dynamic system regulated by multi-factors integratively

In the biological information transport chain from genes to traits, organisms need to constantly adjust their own complex metabolic network to maintain normal dynamic balance within system or between system and external environment The existence of DNA, mRNA and protein provides a material basis for the biological processes, while metabolic substances and metabolic phenotype reflect a biological event that has happened The metabolic substances and metabolic phenotype are the comprehensive result of genotype and environment combination, and direct embodiment of physiological and biochemical function status in a biological system Therefore, as an important component of systems biology, metabolic groups can better reflect the system phenotype

The process of metabolomics analysis includes sample preparation, data collection, data analysis and interpretation The sample preparation is composed of sample extraction, pretreatment and compounds separation After extracted by water or organic solvent, samples are commonly pretreated using solid-phase microextraction, solid-phase extraction and affinity chromatographic methods Then compounds are separated via gas chromatography, liquid chromatography and capillary electrophoresis methods, etc Such separation and analysis methods as chromatography, mass spectrometry (MS), nuclear magnetic resonance (NMR), infrared spectroscopy, coulometric analysis, ultraviolet absorption, fluorescent scattering, radioactivity detection and light scattering and their combinations are all applied in the metabolomics study Among them, the NMR technology, especially the hydrogen spectrum (1H NMR), chromatography and MS become the main analysis tools, due to the universality of 1H NMR for hydrogen metabolites, and high resolution and high flux of chromatography, and universality, high sensitivity and specificity of MS Later period of Metabolomics research is to interpretate the biological significance of data based on data analysis and interpretation with the aid

of bioinformatics platform Constantly used methods by far include multiple regression, discriminant analysis, principal component analysis, hierarchical cluster analysis, factor analysis and canonical analysis, etc

NMR and MS based approaches in metabolomics have their own advantages and disadvantages, respectively (Table 1) The advantages of NMR involve high reproducibility, minimal sample preparation, short analysis time and low cost per sample, while MS has the advantages of high sensitivity, availability for targeted analysis, cheaper instrument cost Choosing whether NMR or MS for metabolomics study depends on the purpose of the study, the research object and the instrument

availability

Table 1 Advantages and disadvantages of NMR and MS for metabolomics study

Better for targeted analysis than NMR

Sample preparation Minimal sample preparation

Requires tissue extraction

Sample analysis time Fast The whole sample can be

analyzed in one measurement

Takes longer than NMR Requires different chromatography techniques for depending on type of metabolites analyzed

Instrument Cost More expensive and occupies

more space than MS

Cheaper and occupies less space than NMR

Sample Cost Low cost per sample High cost per sample

1.2 Plant metabolomics

In 1999, Nicholson team put forward the concept of metabonomics [1] So far they have done a lot of fruitful work in disease diagnosis and drug screening and so on ([2]; [3]; [4]) Fiehn [5] put forward the concept of metabolomics and correlated metabolites to biological gene function for the first time Afterwards, many plants

The application of metabolomics in plant research mainly included following aspects: (1) plant metabolites of certain species Such researches usually focused on a certain plant, selected a particular organ or tissue, analyzed the metabolites qualitatively and quantitatively, studied comprehensively on changes of metabolites types and contents in different periods or different parts, and further speculated the corresponding metabolic pathways and metabolic networks through these changes; (2) phenotype Metabolomics of different genotypes plants Such researches usually studied two or more than two plants (including normal controls and genetically modified plants), comparing and identifying different genotypes plants using metabolomics, as comparison of difference between the mutant or genetically modified plants and normal wild-type plants, or difference between tissue-cultured Metabolomics and the wild-type This category of studies played an important role in evaluation of the efficacy of genetic modification or tissue culture and screening of good varieties; (3) metabolomics of certain ecotypes plants Such researches usually chose the same type of plants under different ecological environment, and studied the effect of habitat on plant metabolites; (4) plant autoimmune response after external stimulation In such researches, changes of plant metabolites were induced by exogenous chemicals stimuli, physical or biological stimuli and were monitored and comprehensively analyzed by metabolomics method; (5) application in gene function research Metabolic products are the final products of gene expression and tiny changes in gene expression level may lead to massive changes of metabolites Previous determination of rise and fall of gene expression level through visible phenotypic change takes long time, and sometimes gene expression changes cannot cause phenotypic change, while the content of certain metabolites in plant body has already changed significantly Using of metabolomics method can judge the change of

gene expression level, so as to deduce the function of genes and their metabolic flux

A lot of research results have been made in the plant metabolomics research Fiehn

[5] analyzed the petiole vascular and leaf extract of Cucurbita maxima using GC-MS

and obtained more than 400 peaks By comparison with the mass spectrum database,

he preliminarily identified 90 compounds, and compared the differences on

metabolites in sugar and amino acid composition between petiole and leaf; Tiessen et

al (2002) [6] conducted Metabolomic analysis of Solanum tuberosum tuber using

high performance liquid chromatography (HPLC) They determined the quantity change of a series of substrates, intermediates, enzyme and products in the starch synthesis approach Then through comparison research between the wild and heterologous adenosine diphosphate glucose focal phosphorylase (AGPase) transgenic potatoes, they proposed a new regulating mechanism in the starch synthesis

approach; Maier, etc (1999) [7] studied the effect of Glomus intraradices on Nicotiana tabacum root metabolism, compared the metabolites difference between tobacco roots with and without Glomus intraradices To sum up, Metabolomics

technology is an ideal platform for plant metabolism study

1.2.1 Plant metabolomics in response to abiotic stresses

Recently many scientific research institutions carried out metabolomics studies on abiotic stress responses of plants Through qualitative and quantitative analysis of plant metabolites under stress environment via modern detection and analysis methods, the variation trend and rule of plant metabolites over time can be monitored The integration of various omics platforms such as genomics, proteomics and metabolomics is also a powerful toolkit [8] Combination of all these information

helps to study responses of biological systems to genes or environment changes as a whole For example, one can judge the level where metabolites change happens, helping people uncover the mysterious and complex mechanism of plant stress response These stress factors include water deficit, excessive high or low temperature, phosphorus and sulfur deficit, excessive salt and heavy metals and so on

(1) Drought stress

Water is one of the important factors that affect plant growth and development The harmful effect on plant due to less environmental moisture is called drought stress

In order to study the contribution of different wine grape (Vitis vinifera) fruit

organization to the wine quality and the influence of drought stress on wine quality, Grimplet (2009) [9] determined protein with specific differences in fruit (peel and fruit pulp) and tissue of wine grape planted under condition of enough moisture and dry environment Using two-dimensional gel electrophoresis (2-d PAGE) technology,

1047 proteins in fruits were detected, among which 90 were differentially expressed

in peel and fruit, while 695 proteins were detected in seeds, among which 163 proteins showed almost no difference in the seed and pee expression spectrum Drought stress changed abundance of about 7% skin protein, but showed little effect

on seed protein expression In the selected 32 small molecule metabolites to be determined, about 50% showed differences in the peel and seeds organizations, while under drought stress condition 7 compounds were affected in accumulation within grape fruits The metabolic fingerprinting results provided new inspiration for studying the effect of drought on the main compounds related to wine flavor and aroma in grape Deluc etc (2009) [10] studied the metabolomics and transcriptome of two different strains of grape Cabernet Sauvignon and Chardonnay under long-term

drought and seasonal drought Studies showed different metabolic pathway changes for two strains of grape in the response to drought stress For Cabernet Sauvignon, the glutamic acid and proline synthesis pathways and some important intermediate steps

in styrene acrylic acid synthesis pathway can be activated by drought stress, while for Chardonnay under drought stress, styrene acrylic acid, carotenoids and isoprenoid synthesis pathways were activated Both stress responses involved influence on abscisic acid metabolic pathway These metabolic products changes had a great influence on fruit and wine flavor

Mane etc (2009) [11] conducted metabolic profile analysis and biomass and yield

comparison of two genotypes Andean potato (Solanum tuberosum ssp Andigena Juz

& Buk Hawkes) Sullu and Ccompis Results showed that although the tuber yield of the two genotypes potatoes was not obviously affected, the aboveground biomass of Ccompis reduced and Sullu biomass was not affected Sucrose and and trehalose in regulatory molecules accumulated in Sullu blade, while in Ccompis blade, the oligosaccharide family way of cottonseed sugar was activated, and low level change

of sucrose and a small amount of stress-related trehalose change Proline and related gene expression level improved, and the expression amount of which in Sullu is 3 times more than Ccompis To sum up, the yield of two genotypes plants showed no obvious change under drought condition, but the biomass accumulation and metabolite changes were obviously different

(2) Temperature stress

Plant response to temperature in growth and development has three basic points: lowest temperature, optimum temperature and maximum temperature The harmful effect on plant caused by too low or too high temperature is called temperature stress

Shulaev et al (2008) [12] reviewed in detail plant metabolomics under temperature

stress The metabolic fingerprinting technology is used to explore response of

Arabidopsis thaliana plants (Arabidopsis) to temperature stress Kaplan et al (2004)

[13] studied metabolic fingerprint of Arabidopsis thaliana plant under high and low temperature environment using GC-MS technology and found a series of small molecule metabolites related to high temperature and low temperature or both The metabolite changes associated with low temperature were the most significant, but to our surprise, most of the metabolites produced under thermal stress would also be produced under cold stress, among which many metabolites were not considered to be related to the temperature stress in previous studies In subsequent research work ([14]), these metabolic fingerprint data were integrated in order to study the adaptive mechanism of Arabidopsis thaliana plants to low temperature Results showed that only part of the metabolites change were related to transcriptomics change, while the rest of metabolites change was not directly related to transcriptomics change It can be concluded through the above research that in the process of plant response to cold stress, the metabolites not directly related to transcriptomic changes played an important role in temperature response of Arabidopsis thaliana plants

Cook et al (2004) [15] compared metabolic fingerprint of Arabidopsis thaliana

plants with different cold resistance abilities and excessive expression (CBF) (C-repeat/dehydration responsive element-binding factor) using GC-MS technology Results showed that metabolism of Arabidopsis thaliana obviously changed in the process of cold stress responses, and that CBF pathway played an important role in the adaptation of low temperature environment in Arabidopsis

Morsy et al (2007) [16] studied the sugar metabolomics of cold stress and high salt

stress response for two genotypes rice with different cold resistance abilities Using

HPLC method, the authors quantitatively analyzed the soluble saccharide compounds

of cold resistance and cold non-resistance rice, and found that accumulation of soluble saccharide of the two genotypes rice under cold stress was different For cold resistant rice, galactose and raffinose accumulated under cold stress environment, while the content of these two kinds of sugar showed a downward trend in the other genotype rice The two genotypes rice also showed different saccharide metabolism characteristics under high salt stress environment

(3) Salt stress

The adverse effect on plant caused by too many soluble salts in the soil is called salt stress Metabolomics technology was used to identify metabolites change of

tomato (Solanum lycopersicum) under salt stress Johnson et al (2003) [17] selected

two tomato strains with different salt sensitivity Edkawy and Simge F1 for research, and found that the relative growth rate of Simge F1 under salt stress significantly decreased, while that of Edkawy was not affected Using Fourier transform infrared spectrum (FT-IR), the fresh tomato fruit extracts from control group and salt stress group were analyzed The obtained data was processed by PCA and discriminant function analysis (DFA), respectively PCA method could not distinguish the fruit difference between control group and high salt treatment group, while DFA method could distinguish between two different genotypes and fruits of different genotypes in control group and high salt treatment group Genetic algorithm (GA) model was used

to identify possible important functional groups in FT-IR spectrugram for salt stress response These functional groups included saturated nitrile compounds, unsaturated nitriles cyanide compounds and strong NH2 radicals peaks and other nitrogen compounds, etc

More detailed plant research in salt stress response was the time-course

metabolomics study by Kim et al (2007) [18] at Arabidopsis thaliana cells in high salt

culture The metabolic fingerprint metabolomics of Arabidopsis cells after treatment

by 100 mmol.L-1 NaCl for 0.5, 1, 2, 4, 12, 24, 48 and 72 h separately were determined using LC-MS and GC-MS The data was analyzed by PCA and self-organizing map Results showed that short-term metabolism change of plant cells in salt stress response included induction of methylation cycle which provided methyl, induction of hydroxyl methyl amine circulation which induced lignin synthesis, and 3-armour amino acid synthesis; while long-term metabolism changes included influence on glycolysis and sucrose metabolism, and co-reduction of methylation system

Cramer et al (2007) [19] compared the difference of grapevine (Vitis vinifera

‘Cabemet Sauvignon’) metabolites change between response to salt stress and drought

stress using GC-MS and anion exchange chromatography-ultraviolet detector method They found that in response to salt stress, the content of sugar, aspartic acid, succinic acid and fumaric acid in plant declined, while the content of proline, asparagine, malic acid and fructose etc was relatively higher Compared with response to salt stress, the content of glucose, malic acid and proline was higher in response to drought stress

Gong et al (2005) [20] also compared metabolites difference between salt-tolerant plants (Thellungiella halophila) and Arabidopsis thaliana by combination of GC-MS

metabolic fingerprint with biochip technology They found obvious differences between metabolites of the two species Compared with Arabidopsis thaliana, salt mustard maintained higher levels of metabolites whether in high salt environment or general environment Analysis of Arabidopsis thaliana showed that glucose, proline and possible polysaccharide significantly increased in Arabidopsis thaliana plants under 150 mmol.L-1 salt stress While in salt mustard, salt stress induced fairly

complex metabolic response Not only many metabolites levels were higher before salt stress, but also the content of many sugars, sugar alcohol, organic acid and phosphate after induction showed apparent change

(4) Sulfur and phosphorus stress

In addition to the above abiotic stresses, plants are also subjected to other environmental stresses, such as sulfur stress and phosphorus stress

Recently, many studies involved metabolomics research of plants subjected to

sulfur, and phosphorus stress Nikiforova et al ([21, 22]) analyzed Arabidopsis

thaliana plants under sulfur stress using GC-MS and LC-MS techniques They detected 134 known compounds and a series of unknown compounds in Arabidopsis thaliana related to sulfur stress, and made dynamic monitoring of these compounds, thus successfully rebuilt metabolic network of sulfur stress response in Arabidopsis thaliana Then, metabolic network data was consolidated with transcriptomics data of sulfur stress response in Arabidopsis thaliana, therefore the relationship between gene expression and metabolite changes under sulfur stress was obtained

The combination of Metabolomics and proteomics was also applied in the study of

leguminous plants under phosphorus stress Hernandez et al (2007) [23] made

metabolic profile analysis of roots of leguminous plant with sufficient phosphorus and insufficient phosphorus using GC-TOF-MS, and identified a series of metabolic products related to the phosphorus stress, many of which (including amino acids, polyols and sugars) increased in content in response to phosphorus stress

(5) Heavy metal stress

Several studies have investigated the metal stress responses in various plants As a

Kieffer et al [25] conducted research of proteome combined with metabolic profile analysis on Poplar (Poplar spp.) under cadmium stress Results showed that levels of

pigment and carbohydrates of Poplar changed in response to cadmium stress Under cadmium stress, the poplar showed growth inhibition and photosynthesis was also affected In the process of growth and development, photosynthesis products stored in the form of hexose or other complex carbohydrates in plants, thus adjusting osmotic pressure

Sun et al [26] elucidated the metabolic responses of Arabidopsis thaliana to

different cadmium concentrations (0, 5, 50 µM) for 2 weeks using GC-MS analysis Results showed that levels of carbohydrates, organic acids, amino acids, and other metabolites changed under cadmium stress Levels of Ala, b-ala, Pro, Ser, putrescine, Suc, 4-aminobutyric acid, glycerol, raffinose and trehalose increased in the treated plants compared to control group Concentrations of antioxidants such as alfa-tocopherol, campesterol, beta-sitosterol and isoflavone also significantly increased

Hediji et al [27] evaluated the long-term response of tomato plants to cadmium

exposure through 1H NMR, HPLC-PDA, and colorimetric methods The plants were

cultured in hydroponic conditions (0, 20, and 100 µM CdCl2) for 90 days Results showed that tomato plants adapted to 20 µm Cd concentration during long-term exposure and were perturbed physiologically leading to limited growth and fruit set abortion

Liu et al [28] studied the metabolites response of halophyte (Suaeda salsa)

exposed to 2, 10 and 50 µg L-1 cadmium concentration using NMR-based Metabolomics After cadmium exposures, the levels of amino acids, carbohydrates, intermediates of tricarboxylic acid cycle and osmolyte changed in the samples, indicating increased protein degradation and disturbances in the osmotic regulation and energy metabolism

Grid chara (Scenedesmus) plant is an important research object for study of metal stress and phenolic expression Dividing the genus plant S quadricauda into 3 groups, Jozef et al [29] conducted Metabolomics research after treatment of them using Cu2+, salicylic acid and combination of Cu2+ and salicylic acid, respectively Results showed that content of chlorophyll, soluble protein and phenolic compounds declined in the

Cu2+ treatment group; The alicylic acid treatment group showed opposite trend; In the

Cu2+ and salicylic acid treatment group, salicylic acid could not resist the downward trend of the three kinds of compounds under Cu2+ stress However, the concentration

of Cu2+ did not increased in plants body, possibly due to accumulation of benzoic acid which was related to salicylic acid

Brassica species plants have long been regarded as metal ion collector, and widely

used in the soil remediation, but the resistance mechanism for metal ions is unclear

Jahangir et al [30], studied the metabolites change of turnip (Brassica rapa)

subjected to metal ions stress The turnip was exposed to three kinds of metal ions

(Cu2+, Fe3+ and Mn2+) with four different concentrations (50, 100, 250 and 500 mmol.L-1) The plants sample after treatment was detected by 1H NMR and 2D-NMR, then the data was analyzed by PCA and partial least squares (PLS) Such primary metabolites as gluconic acid and hydroxy acid conjugate salt compounds, some carbohydrates and amino acids can be used to distinguish plants treated by different kinds of metal ions Results showed that the metabolites change of plants treated by

Cu2+ and Fe3+ was greater than those treated by Mn2+ Moreover, change of metabolic products was not only related to the species of metal ions, but also associated with the concentration of metal ions

1.3 Heavy metal accumulation in Water Hyacinth

Water hyacinth (Eichhornia crassipes) is a perennial, floating aquatic plant with

rapid-growing capability It is widely distributed in warm temperate, subtropical, and tropical regions around the world Various studies have shown that water hyacinth is capable of adsorbing excessive heavy metals Research by Misbahuddin and Fariduddin [31] showed that 81% of As with 400 ppb concentration was removed by the roots of living water hyacinth plants, and when stems and leaves of the water hyacinth plant were involved, 100% As was removed in six hours In Taiwan, Liao and Chang [32] demonstrated that water hyacinth removed large amounts of lead, copper, and zinc in a constructed wetland The results by Soltan and Rashed [33] showed that water hyacinth can survive in a mixture of heavy metal concentrations (Cd, Co, Cr, Cu, Mn, Ni, Pb and Zn) up to 3 mg.L-1 and in 100 mg.L-1 Pb solution

The uptake of metal ions from aqueous solutions by water hyacinth is a deprotonation reaction explained by a decrease in pH The mechanism was believed to

be founded on the chelation with carboxylic, amino acid and hydroxyl groups of macrocyclic molecules, such as ionophores existed in the mitochondria of water

hyacinth Haider et al (1983) suggested that the uptake of chemicals by water

hyacinth might happen through the cell membrane via osmosis and diffusion Field

studies by Ajmal et al [34] and Zaranyika and Ndapwadza [35] had shown that

metals accumulated in the leaves and roots of water hyacinth Cooly and Martin [36] found that Cu and Cd accumulated more in roots than least and petioles in leaves

Kelley et al [37] explained that carboxylic acids were responsible for chelating the

intracellular proportion of Eu(III) in the roots According to the study by Malik [38], almost all heavy metal ions accumulated in the roots rather than in the shoot system of

water hyacinth An exceptional research carried out by Zhu et al [39] suggested that

the metal Se was transported to the upper biomass of water hyacinth Metal accumulation was found in the sequence of roots > stems > leaves of water hyacinth, and there was a linear correlation between the external metal concentration and internal metal concentration [40] for Cr, Cu, Ni, and As [41] It was suggested that the capability of water hyacinth in storing most heavy metals enabled the plant to avoid toxicity of photosynthetic tissues caused by heavy metals

1.4 Basis and significance of dissertation

Modern industries have released large amounts of heavy metals into the environment Among various technologies for heavy metal removal, such as adsorption, reverse osmosis and electrochemical method etc., phytoremediation has been regarded as a promising method due to its low cost Water hyacinth has the capacity of adsorbing heavy metal during growth, thus is a promising plant for

heavy metal adsorption by water hyacinth, these studies all focused on reaction in parts of the plant such as roots [33], or certain features of the plant, such as reflectance [42], the complete metabolism response when water hyacinth is subjected

to heavy metal stress is unknown The application of metabolomics method on water hyacinth in response to heavy metal stress may provide incentive on the metabolites pathway of the water hyacinth for heavy metal adsorption from the perspective of the whole plant, thus building an solid scientific foundation for better application of water hyacinth in the phytoremediation

Previously, few studies on plant metabolomics in response to heavy metal stress involved comparison of metabolites change of different parts of plants This point deserved research as different parts of plant may play different roles in the metabolites change, subdivision of their functions could help us understand plant response to heavy metal stress as a whole Moreover, previous studies on plant metabolomics mostly described metabolites response to only one kind of stress, such as drought stress, metal stress or salt stress Few studies involved metabolites response to two or more kinds of stress The comparison between metabolites responses to different stresses may be significant, as it may help us better understand each stress response of the same plant, so as to understand those shared responses and stress-specific responses of the same plant

In this article, the water hyacinth plants were exposed to CuCl2, FeCl3 and

Na2HPO4 solutions Then we analyzed the metabolites change and differences of roots, stems and leaves of the plants using GC-MS method and PCA analysis The metabolism pathways of water hyacinth in response to different heavy metal stress were proposed

2 Materials and Methods

2.1 Materials

Water Hyacinth seedlings were all in the similar growth condition, purchased from the same batch in an ornamental fish shop in Clementi Station, Singapore They were washed by deionized water for several times before planting 32 water hyacinth seedlings were planted in 8 1mL glasses with 4 seedlings in each glass In the first week, all the plants were cultivated in deionized water which was changed for fresh water every three days After that, plants in 2 of 8 glasses were kept in deionized water continuingly as the control group In the rest 6 glasses, 2 glasses were replaced with 0.3 mmol FeCl3 solution, another 2 glasses replaced with 1.2 mmol CuCl2solution, the last 2 glasses replaced with 3.46 mmol Na2HPO4 solution All of the solutions were replaced with originally corresponding solutions every three days, respectively In the whole cultivation process, plants were placed in laboratory with temperature maintained around 23 oC and illumination time from 8:00 am to 18:00

pm every day 15 days later, the roots, stems and leaves of water hyacinth were cut into segments with length of 1 mm, then put in labelled centrifuge tubes and stored in the freezer with -20 oC

2.2 Instruments and Reagents

Instruments included 7980A GC-QQQ (Agilent Technologies) with HP-5 capillary gas chromatography column, drying machine, centrifuge Methanol, trichloromethane, pyridine and bis(trimethylsilyl)trifluoroacetamide (BSTFA) were all chromatographically pure purchased from Sigma, Singapore

2.3 Sample extraction and derivatization

Part of the plants samples (including leaves, stems and roots) were directly grinded without drying, others were dried before grinding Individual samples were extracted with 0.5 mL CHCl3 : CH3OH (3:1) solution, and then centrifuged at 161000 x g for 10 min The supernatant was lyophilized for derivatization Dried sample extracts were

reconstituted in 300 L pyridine 100 L BSTFA was added to each dried sample extract for derivatization for 120 minutes The obtained solution was agitated for 1 minute under room temperature and transferred to an amber vial for GC-MS analysis

2.4 GC-MS analysis

1.0 L aliquot of the BSTFA-derivatized sample was injected using the splitless mode into a GC/QqQ 7980A system equipped with a HP-5 capillary column The inlet temperature was set at 250 oC Helium was used as the carrier gas with a constant flow rate of 1.40 mL/min through the column The initial temperature was set at 100

o

C, 1 minute after injection the GC temperature was increased at a rate of 5 oC/min to

280oC and held for 5 minutes at 300 oC The transfer line temperature was set at 280