#METABOLOMICS WORKBENCH Tatiana_20240129_130504 DATATRACK_ID:4619 STUDY_ID:ST003064 ANALYSIS_ID:AN005019 PROJECT_ID:PR001910
VERSION             	1
CREATED_ON             	January 31, 2024, 1:10 pm
#PROJECT
PR:PROJECT_TITLE                 	Metabolic responses of Amaranthus caudatus roots and leaves to zinc stress
PR:PROJECT_TYPE                  	GCMS-based untargeted and targeted analysis
PR:PROJECT_SUMMARY               	During the last decades pollution with heavy metals became an important stress
PR:PROJECT_SUMMARY               	factor. Plants are characterized by significant biochemical plasticity and can
PR:PROJECT_SUMMARY               	adjust their metabolism to ensure survival under changing environmental
PR:PROJECT_SUMMARY               	conditions. In the most straightforward way these metabolic shifts can be
PR:PROJECT_SUMMARY               	addressed by the untargeted mass spectrometry-based metabolomics approach.
PR:PROJECT_SUMMARY               	However, so far this methodology was only minimally employed in studies of
PR:PROJECT_SUMMARY               	Zn-induced metabolic shifts in plants. Moreover, the genus Amaranthus is still
PR:PROJECT_SUMMARY               	not addressed in this respect. Therefore, here we propose, to the best of our
PR:PROJECT_SUMMARY               	knowledge, the first gas chromatography-mass spectrometry (GC-MS)-based
PR:PROJECT_SUMMARY               	metabolomics study of Zn2+-induced stress responses in Amaranthus caudatus
PR:PROJECT_SUMMARY               	plants. The GC-MS-based study was performed with root and leaf aqueous
PR:PROJECT_SUMMARY               	methanolic extracts after their lyophylization and sequential derivatization
PR:PROJECT_SUMMARY               	with methoxylamine hydrochloride and N-trimethylsilyl-N-methyl
PR:PROJECT_SUMMARY               	trifluoroacetamide. Thereby, 419 derivatives were detected, of which 144 could
PR:PROJECT_SUMMARY               	be putatively annotated. The metabolic shifts in seven-week old A.caudatus
PR:PROJECT_SUMMARY               	plants in response to a seven-day treatment with 300 µmol/L ZnSO4·7H2O in
PR:PROJECT_SUMMARY               	nutrient solution were organ-specific and more pronounced in roots. The most of
PR:PROJECT_SUMMARY               	the responsive metabolites were up-regulated and dominated with sugars and sugar
PR:PROJECT_SUMMARY               	acids. These effects could be attributed to the involvement of these metabolites
PR:PROJECT_SUMMARY               	in osmoregulation, ROS scavenging and complexation of Zn2+ ions. Galactose was
PR:PROJECT_SUMMARY               	the most Zn2+-responsive root sugar that indicated its possible role in the
PR:PROJECT_SUMMARY               	binding of Zn2+ ions to the root cell walls. A 59-fold up-regulation of gluconic
PR:PROJECT_SUMMARY               	acid in roots clearly indicated its involvement in chelation of Zn2+. A high
PR:PROJECT_SUMMARY               	Zn2+–induced up-regulation of salicylic acid in roots and shoots suggested a
PR:PROJECT_SUMMARY               	key role of this hormone in the activation of Zn2+ stress tolerance mechanisms.
PR:PROJECT_SUMMARY               	Thus, our study provides the first insight in the general trends in Zn-induced
PR:PROJECT_SUMMARY               	biochemical rearrangements and main adaptive metabolic shifts in A. caudatus
PR:PROJECT_SUMMARY               	plants.
PR:INSTITUTE                     	K.A. Timiryazev Institute of Plant Physiology RAS
PR:LABORATORY                    	Laboratory of Analytical Biochemistry and Biotechnology
PR:LAST_NAME                     	Frolov
PR:FIRST_NAME                    	Andrej
PR:ADDRESS                       	Botanicheskaya st. 35., Moskow, 127276, Russian Federation
PR:EMAIL                         	frolov@ifr.moscow
PR:PHONE                         	+79046097095
PR:FUNDING_SOURCE                	Russian Scientific Foundation (grant # 21-74-30003), Ministry of Science and
PR:FUNDING_SOURCE                	Higher Education of the Russian Federation (theme # 122042700043-9)
#STUDY
ST:STUDY_TITLE                   	Metabolic responses of Amaranthus caudatus roots and leaves to zinc stress
ST:STUDY_TYPE                    	GCMS-based untargeted and targeted analysis
ST:STUDY_SUMMARY                 	During the last decades pollution with heavy metals became an important stress
ST:STUDY_SUMMARY                 	factor. Plants are characterized by significant biochemical plasticity and can
ST:STUDY_SUMMARY                 	adjust their metabolism to ensure survival under changing environmental
ST:STUDY_SUMMARY                 	conditions. In the most straightforward way these metabolic shifts can be
ST:STUDY_SUMMARY                 	addressed by the untargeted mass spectrometry-based metabolomics approach.
ST:STUDY_SUMMARY                 	However, so far this methodology was only minimally employed in studies of
ST:STUDY_SUMMARY                 	Zn-induced metabolic shifts in plants. Moreover, the genus Amaranthus is still
ST:STUDY_SUMMARY                 	not addressed in this respect. Therefore, here we propose, to the best of our
ST:STUDY_SUMMARY                 	knowledge, the first gas chromatography-mass spectrometry (GC-MS)-based
ST:STUDY_SUMMARY                 	metabolomics study of Zn2+-induced stress responses in Amaranthus caudatus
ST:STUDY_SUMMARY                 	plants. The GC-MS-based study was performed with root and leaf aqueous
ST:STUDY_SUMMARY                 	methanolic extracts after their lyophylization and sequential derivatization
ST:STUDY_SUMMARY                 	with methoxylamine hydrochloride and N-trimethylsilyl-N-methyl
ST:STUDY_SUMMARY                 	trifluoroacetamide. Thereby, 419 derivatives were detected, of which 144 could
ST:STUDY_SUMMARY                 	be putatively annotated. The metabolic shifts in seven-week old A.caudatus
ST:STUDY_SUMMARY                 	plants in response to a seven-day treatment with 300 µmol/L ZnSO4·7H2O in
ST:STUDY_SUMMARY                 	nutrient solution were organ-specific and more pronounced in roots. The most of
ST:STUDY_SUMMARY                 	the responsive metabolites were up-regulated and dominated with sugars and sugar
ST:STUDY_SUMMARY                 	acids. These effects could be attributed to the involvement of these metabolites
ST:STUDY_SUMMARY                 	in osmoregulation, ROS scavenging and complexation of Zn2+ ions. Galactose was
ST:STUDY_SUMMARY                 	the most Zn2+-responsive root sugar that indicated its possible role in the
ST:STUDY_SUMMARY                 	binding of Zn2+ ions to the root cell walls. A 59-fold up-regulation of gluconic
ST:STUDY_SUMMARY                 	acid in roots clearly indicated its involvement in chelation of Zn2+. A high
ST:STUDY_SUMMARY                 	Zn2+–induced up-regulation of salicylic acid in roots and shoots suggested a
ST:STUDY_SUMMARY                 	key role of this hormone in the activation of Zn2+ stress tolerance mechanisms.
ST:STUDY_SUMMARY                 	Thus, our study provides the first insight in the general trends in Zn-induced
ST:STUDY_SUMMARY                 	biochemical rearrangements and main adaptive metabolic shifts in A. caudatus
ST:STUDY_SUMMARY                 	plants.
ST:INSTITUTE                     	K.A. Timiryazev Institute of Plant Physiology RAS, Moscow, Russia
ST:LABORATORY                    	Laboratory of Analytical Biochemistry and Biotechnology
ST:LAST_NAME                     	Frolov
ST:FIRST_NAME                    	Andrej
ST:ADDRESS                       	Botanicheskaya st. 35., Moskow, 127276, Russian Federation
ST:EMAIL                         	frolov@ifr.moscow
ST:PHONE                         	+79046097095
#SUBJECT
SU:SUBJECT_TYPE                  	Plant
SU:SUBJECT_SPECIES               	Amaranthus caudatus L.
SU:TAXONOMY_ID                   	3567
SU:GENDER                        	Not applicable
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	LY_cont_1	Plant_organs:Young_leaves | Treatment:Control	RAW_FILE_NAME=LY_cont_1.CDF
SUBJECT_SAMPLE_FACTORS           	-	LY_cont_2	Plant_organs:Young_leaves | Treatment:Control	RAW_FILE_NAME=LY_cont_2.CDF
SUBJECT_SAMPLE_FACTORS           	-	LY_cont_3	Plant_organs:Young_leaves | Treatment:Control	RAW_FILE_NAME=LY_cont_3.CDF
SUBJECT_SAMPLE_FACTORS           	-	LY_Zn_1	Plant_organs:Young_leaves | Treatment:Zn	RAW_FILE_NAME=LY_Zn_1.CDF
SUBJECT_SAMPLE_FACTORS           	-	LY_Zn_2	Plant_organs:Young_leaves | Treatment:Zn	RAW_FILE_NAME=LY_Zn_2.CDF
SUBJECT_SAMPLE_FACTORS           	-	LY_Zn_3	Plant_organs:Young_leaves | Treatment:Zn	RAW_FILE_NAME=LY_Zn_3.CDF
SUBJECT_SAMPLE_FACTORS           	-	LM_cont_1	Plant_organs:Mature_leaves | Treatment:Control	RAW_FILE_NAME=LM_cont_1.CDF
SUBJECT_SAMPLE_FACTORS           	-	LM_cont_2	Plant_organs:Mature_leaves | Treatment:Control	RAW_FILE_NAME=LM_cont_2.CDF
SUBJECT_SAMPLE_FACTORS           	-	LM_cont_3	Plant_organs:Mature_leaves | Treatment:Control	RAW_FILE_NAME=LM_cont_3.CDF
SUBJECT_SAMPLE_FACTORS           	-	LM_Zn_1	Plant_organs:Mature_leaves | Treatment:Zn	RAW_FILE_NAME=LM_Zn_1.CDF
SUBJECT_SAMPLE_FACTORS           	-	LM_Zn_2	Plant_organs:Mature_leaves | Treatment:Zn	RAW_FILE_NAME=LM_Zn_2.CDF
SUBJECT_SAMPLE_FACTORS           	-	LM_Zn_3	Plant_organs:Mature_leaves | Treatment:Zn	RAW_FILE_NAME=LM_Zn_3.CDF
SUBJECT_SAMPLE_FACTORS           	-	R_cont_1	Plant_organs:Roots | Treatment:Control	RAW_FILE_NAME=R_cont_1.CDF
SUBJECT_SAMPLE_FACTORS           	-	R_cont_2	Plant_organs:Roots | Treatment:Control	RAW_FILE_NAME=R_cont_2.CDF
SUBJECT_SAMPLE_FACTORS           	-	R_cont_3	Plant_organs:Roots | Treatment:Control	RAW_FILE_NAME=R_cont_3.CDF
SUBJECT_SAMPLE_FACTORS           	-	R_Zn_1	Plant_organs:Roots | Treatment:Zn	RAW_FILE_NAME=R_Zn_1.CDF
SUBJECT_SAMPLE_FACTORS           	-	R_Zn_2	Plant_organs:Roots | Treatment:Zn	RAW_FILE_NAME=R_Zn_2.CDF
SUBJECT_SAMPLE_FACTORS           	-	R_Zn_3	Plant_organs:Roots | Treatment:Zn	RAW_FILE_NAME=R_Zn_3.CDF
#COLLECTION
CO:COLLECTION_SUMMARY            	Amaranthus caudatus L., variety Karwa dauta plants were used in the study. After
CO:COLLECTION_SUMMARY            	two weeks culturing in the hydroponic system (nutrient solution (in mmol/L) was
CO:COLLECTION_SUMMARY            	as follows: Ca(NO3)2·4H2O - 3.81; KNO3 - 6.44; MgSO4·7H2O - 0.81; KH2PO4 -
CO:COLLECTION_SUMMARY            	1.83; NH4NO3 - 0.87; Fe-EDTA - 0.09; H3BO3 - 0.047; MnSO4·5H2O - 0.007;
CO:COLLECTION_SUMMARY            	ZnSO4·7H2O - 0.0007; CuSO4·5H2O - 0.0008; (NH4)2MoO4 - 0.0005), the vessels
CO:COLLECTION_SUMMARY            	with six-week-old plants (experimental group) were subjected to Zn2+ stress for
CO:COLLECTION_SUMMARY            	one week which was accomplished by supplementation of 300 µmol/L ZnSO4·7H2O in
CO:COLLECTION_SUMMARY            	the nutrient solution. Control plants remained untreated. Roots, young and
CO:COLLECTION_SUMMARY            	mature leaves of seven-week-old Zn-treated and control plants were collected
CO:COLLECTION_SUMMARY            	separately. Approximately 10 and 20 mg of ground dry leaf and root material,
CO:COLLECTION_SUMMARY            	respectively, were extracted with 1 mL methanol. After vortexing (3000 g, 30 s)
CO:COLLECTION_SUMMARY            	and centrifugation (12000 g, 4 °C, 10 min) of the suspensions, the resulted
CO:COLLECTION_SUMMARY            	supernatants were collected. The plant material residues were additionally
CO:COLLECTION_SUMMARY            	supplemented with 0.1 mL of deionized water. After a following vortex and
CO:COLLECTION_SUMMARY            	centrifugation cycle, the obtained supernatants were combined with the first
CO:COLLECTION_SUMMARY            	portions. The total extract volume was 1090 μL. Aliquots (30 μL) of the
CO:COLLECTION_SUMMARY            	resulted aq. methanolic extracts were freeze-dried under reduced pressure with
CO:COLLECTION_SUMMARY            	Labconco CentriVap centrifugal concentrator. The residues were sequentially
CO:COLLECTION_SUMMARY            	derivatized with methoxyamine hydrochloride in pyridine, and
CO:COLLECTION_SUMMARY            	N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) according to the
CO:COLLECTION_SUMMARY            	established procedure (Leonova et al., 2020,
CO:COLLECTION_SUMMARY            	http://dx.doi.org/10.3390/ijms21020567)
CO:SAMPLE_TYPE                   	Plant leaves and roots
#TREATMENT
TR:TREATMENT_SUMMARY             	Amaranthus caudatus L., variety Karwa dauta plants were used in the study. After
TR:TREATMENT_SUMMARY             	two weeks culturing in the hydroponic system (nutrient solution (in mmol/L) was
TR:TREATMENT_SUMMARY             	as follows: Ca(NO3)2·4H2O - 3.81; KNO3 - 6.44; MgSO4·7H2O - 0.81; KH2PO4 -
TR:TREATMENT_SUMMARY             	1.83; NH4NO3 - 0.87; Fe-EDTA - 0.09; H3BO3 - 0.047; MnSO4·5H2O - 0.007;
TR:TREATMENT_SUMMARY             	ZnSO4·7H2O - 0.0007; CuSO4·5H2O - 0.0008; (NH4)2MoO4 - 0.0005), the vessels
TR:TREATMENT_SUMMARY             	with six-week-old plants (experimental group) were subjected to Zn2+ stress for
TR:TREATMENT_SUMMARY             	one week which was accomplished by supplementation of 300 µmol/L ZnSO4·7H2O in
TR:TREATMENT_SUMMARY             	the nutrient solution. Control plants remained untreated. Roots, young and
TR:TREATMENT_SUMMARY             	mature leaves of seven-week-old Zn-treated and control plants were collected
TR:TREATMENT_SUMMARY             	separately.
TR:TREATMENT_PROTOCOL_COMMENTS   	6 sample groups: LY_cont - young leaves of control plants; LY_Zn - young leaves
TR:TREATMENT_PROTOCOL_COMMENTS   	of Zn-treated plants; LM_cont - mature leaves of control plants; LM_Zn - mature
TR:TREATMENT_PROTOCOL_COMMENTS   	leaves of Zn-treated plants; R_cont - roots of control plants; R_Zn - roots of
TR:TREATMENT_PROTOCOL_COMMENTS   	Zn-treated plants.
TR:TREATMENT                     	Heavy metal stress
TR:TREATMENT_COMPOUND            	ZnSO4·7H2O
TR:TREATMENT_ROUTE               	supplementation in the nutrient solution
TR:TREATMENT_DOSE                	300 µmol/L
TR:TREATMENT_DOSEDURATION        	1 week
TR:PLANT_PLOT_DESIGN             	total 27 plants in nine vessels
TR:PLANT_LIGHT_PERIOD            	16 : 8 day/night regimen
TR:PLANT_HUMIDITY                	70-75% relative humidity
TR:PLANT_TEMP                    	day/night temperatures of 24/18° C
TR:PLANT_WATERING_REGIME         	plant were culturing in the hydroponic system
TR:PLANT_NUTRITIONAL_REGIME      	nutrient solution in mmol/L as follows: Ca(NO3)2·4H2O - 3.81; KNO3 - 6.44;
TR:PLANT_NUTRITIONAL_REGIME      	MgSO4·7H2O - 0.81; KH2PO4 - 1.83; NH4NO3 - 0.87; Fe-EDTA - 0.09; H3BO3 - 0.047;
TR:PLANT_NUTRITIONAL_REGIME      	MnSO4·5H2O - 0.007; ZnSO4·7H2O - 0.0007; CuSO4·5H2O - 0.0008; (NH4)2MoO4 -
TR:PLANT_NUTRITIONAL_REGIME      	0.0005
TR:PLANT_GROWTH_STAGE            	vegetative stage
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Approximately 10 and 20 mg of ground dry leaf and root material, respectively,
SP:SAMPLEPREP_SUMMARY            	were extracted with 1 mL methanol. After vortexing (3000 g, 30 s) and
SP:SAMPLEPREP_SUMMARY            	centrifugation (12000 g, 4 °C, 10 min) of the suspensions, the resulted
SP:SAMPLEPREP_SUMMARY            	supernatants were collected. The plant material residues were additionally
SP:SAMPLEPREP_SUMMARY            	supplemented with 0.1 mL of deionized water. After a following vortex and
SP:SAMPLEPREP_SUMMARY            	centrifugation cycle, the obtained supernatants were combined with the first
SP:SAMPLEPREP_SUMMARY            	portions. The total extract volume was 1090 μL. Aliquots (30 μL) of the
SP:SAMPLEPREP_SUMMARY            	resulted aq. methanolic extracts were freeze-dried under reduced pressure with
SP:SAMPLEPREP_SUMMARY            	Labconco CentriVap centrifugal concentrator. The residues were sequentially
SP:SAMPLEPREP_SUMMARY            	derivatized with methoxyamine hydrochloride in pyridine, and
SP:SAMPLEPREP_SUMMARY            	N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) according to the
SP:SAMPLEPREP_SUMMARY            	established procedure (Leonova et al., 2020,
SP:SAMPLEPREP_SUMMARY            	http://dx.doi.org/10.3390/ijms21020567).
SP:PROCESSING_STORAGE_CONDITIONS 	4℃
SP:EXTRACT_STORAGE               	-20℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	The samples (1μL) were injected with CTC GC PAL Liquid Injector (Shimadzu
CH:CHROMATOGRAPHY_SUMMARY        	Deutschland GmbH, Duisburg, Germany) into GC2010 gas chromatograph coupled
CH:CHROMATOGRAPHY_SUMMARY        	online to a quadrupole mass selective detector Shimadzu GCMS QP201 operating
CH:CHROMATOGRAPHY_SUMMARY        	under the instrumental settings summarized in PR2.pdf
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Shimadzu GC-2010
CH:COLUMN_NAME                   	Phenomenex ZB-5MS (30 m × 0.25 mm, 0.25 μm)
CH:SOLVENT_A                     	-
CH:SOLVENT_B                     	-
CH:SOLVENT_C                     	-
CH:FLOW_GRADIENT                 	-
CH:FLOW_RATE                     	1 mL/min
CH:COLUMN_TEMPERATURE            	1 min at 40°C, ramp 15°C/min to 70°C, 1 min at 70°C, ramp 6°C/min to
CH:COLUMN_TEMPERATURE            	320°C, 12 min at 320°C
CH:INJECTION_TEMPERATURE         	250
CH:SAMPLE_INJECTION              	1μm
CH:ANALYTICAL_TIME               	5.5-55 min
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Shimadzu QP2010 Plus
MS:INSTRUMENT_TYPE               	Single quadrupole
MS:MS_TYPE                       	EI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Targeted GC-MS analysis The samples (1μL) were injected with CTC GC PAL Liquid
MS:MS_COMMENTS                   	Injector (Shimadzu Deutschland GmbH, Duisburg, Germany) into GC2010 gas
MS:MS_COMMENTS                   	chromatograph coupled online to a quadrupole mass selective detector Shimadzu
MS:MS_COMMENTS                   	GCMS QP201. The GC-MS instrumental settings are summarized in PR2.pdf. The
MS:MS_COMMENTS                   	quality of the acquired chromatograms was assessed by verification of the
MS:MS_COMMENTS                   	baseline regularity, background MS noise, the symmetry, width and height of
MS:MS_COMMENTS                   	chromatographic peaks. To obtain qualitative information about the Zn-related
MS:MS_COMMENTS                   	dynamics of individual metabolites, the chromatograms were processed by AMDIS
MS:MS_COMMENTS                   	software (www.amdis.net/) to accomplish deconvolution of mass spectra, peak
MS:MS_COMMENTS                   	picking, calculation of Kovach retention indices (RI) and annotation of
MS:MS_COMMENTS                   	analytes. The analytes annotated in the experimental samples were quantified by
MS:MS_COMMENTS                   	integration of the corresponding extracted ion chromatograms (XIC, m/z ± 0.5
MS:MS_COMMENTS                   	Da) for representative intense signals at specific retention times. This analyte
MS:MS_COMMENTS                   	quantification procedure was accomplished with XcaliburTM (version 2.0.7),
MS:MS_COMMENTS                   	LCquanTM (version 2.5.6, TermoFisher Scientific Inc., Bremen, Germany) and
MS:MS_COMMENTS                   	MSDial (http://prime.psc.riken.jp/compms/msdial/main.html) softwares which
MS:MS_COMMENTS                   	perform alignment of chromatograms by retention times of analytes and the
MS:MS_COMMENTS                   	integration of analyte peak areas. Metabolite identification and targeted
MS:MS_COMMENTS                   	absolute quantitative analysis relied on external standardization with 29
MS:MS_COMMENTS                   	authentic standards (oxalic acid, malonic acid, succinic acid, tartaric acid,
MS:MS_COMMENTS                   	malic acid, aconitic acid, citric acid, fumaric acid, benzoic acid, ascorbic
MS:MS_COMMENTS                   	acid, erythronic acid, glycerol, arabinose, glucose, galactose, myo-inositol,
MS:MS_COMMENTS                   	sucrose, urea, Ala, Trp, Ile, Leu, Asn, Asp, Glu, Pro, Val, Ser, Thr) prepared
MS:MS_COMMENTS                   	as a total mix serially diluted in the range from 0.2 pmol/μL to 200 pmol/μL.
MS:MS_COMMENTS                   	Among these, only 21 compounds were confirmed in leaves and roots of control and
MS:MS_COMMENTS                   	Zn2+-treated A. caudatus plants (Result table).
MS:ION_SOURCE_TEMPERATURE        	240
MS:IONIZATION                    	EI
MS:IONIZATION_ENERGY             	70eV
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	μmol/g DW
MS_METABOLITE_DATA_START
Samples	LY_cont_1	LY_cont_2	LY_cont_3	LY_Zn_1	LY_Zn_2	LY_Zn_2	LM_cont_1	LM_cont_2	LM_cont_3	LM_Zn_1	LM_Zn_3	R_cont_1	R_cont_2	R_cont_3	R_Zn_1	R_Zn_2	R_Zn_3
Factors	Plant_organs:Young_leaves | Treatment:Control	Plant_organs:Young_leaves | Treatment:Control	Plant_organs:Young_leaves | Treatment:Control	Plant_organs:Young_leaves | Treatment:Zn	Plant_organs:Young_leaves | Treatment:Zn	Plant_organs:Young_leaves | Treatment:Zn	Plant_organs:Mature_leaves | Treatment:Control	Plant_organs:Mature_leaves | Treatment:Control	Plant_organs:Mature_leaves | Treatment:Control	Plant_organs:Mature_leaves | Treatment:Zn	Plant_organs:Mature_leaves | Treatment:Zn	Plant_organs:Roots | Treatment:Control	Plant_organs:Roots | Treatment:Control	Plant_organs:Roots | Treatment:Control	Plant_organs:Roots | Treatment:Zn	Plant_organs:Roots | Treatment:Zn	Plant_organs:Roots | Treatment:Zn
Oxalic acid	5.13	19.16	4.65	12.24	11.23	3.38	20.97	26.45	21.82	27.64	1.53	11.49	12.68	11.21	0.58	6.02	0.61
Malonic acid	0.48	0.66	8.17	0.98	0.74	0.30	0.86	1.01	0.66	0.78	1.01	0.53	0.41	0.50	0.83	1.08	1.01
Succinic acid	4.14	4.18	4.48	2.84	2.49	1.36	4.70	5.37	4.37	2.44	3.33	2.74	2.54	2.77	1.53	1.78	1.78
Fumaric acid	0.40	0.47	0.45	0.46	0.40	0.30	0.58	0.64	0.52	0.53	0.77	0.79	0.75	0.81	0.74	0.83	0.76
Malic acid	2.20	1.98	2.31	3.54	1.87	1.58	2.81	2.90	2.39	6.09	7.57	4.62	4.70	4.89	4.98	6.04	5.83
Pyroglutamic acid	16.40	9.86	4.27	33.01	18.86	23.42	6.37	2.87	3.10	3.96	10.71	17.48	19.51	21.35	21.83	15.41	15.13
Citric acid	0.25	0.32	6.13	0.58	0.20	0.23	0.49	0.15	1.95	0.24	0.56	0.13	0.12	0.11	0.27	0.38	0.36
Aconitic acid	0.82	1.37	1.90	1.47	1.12	0.77	1.94	3.41	3.28	2.02	2.62	0.07	0.03	0.04	0.25	0.03	0.03
Erythronic acid	2.39	2.51	2.73	3.66	2.99	2.24	2.69	3.20	2.82	4.50	6.23	1.05	0.97	1.12	1.86	2.02	2.03
Benzoic acid	7.78	7.83	7.59	4.02	5.51	4.18	9.80	13.58	11.68	8.43	9.19	6.83	6.01	12.19	9.50	10.02	9.64
Alanine	10.23	4.14	9.47	4.57	4.08	2.86	5.87	3.85	3.42	2.33	3.64	1.29	1.42	1.40	3.10	3.32	2.41
Valine	0.82	0.50	0.46	0.39	0.44	0.26	0.74	0.46	0.37	0.18	0.17	0.49	0.50	0.54	1.45	0.84	0.88
Isoleucine	0.32	0.14	0.26	0.12	0.11	0.09	0.26	0.06	0.08	0.06	0.11	0.27	0.35	0.38	1.07	0.44	0.44
Proline	1.32		0.32				1.46	0.65			1.10	0.76	1.51	1.07	2.17	2.18	2.13
Urea	10.34						6.42				1.53	0.54	3.01	1.04	6.32	2.66	2.62
Glycerol	1.01	0.90	1.07	0.44	0.66	2.44	1.08	0.56	1.06	1.62	1.44	2.82	2.79	3.09	4.10	4.32	4.25
Arabinose	0.23	0.20	0.19	0.23	0.15	0.16	0.16	0.22	0.16	0.27	0.37	0.06	0.05	0.06	0.11	0.14	0.12
Galactose	0.04	0.04	0.06	0.12	0.11	0.07	0.06	0.04	0.02	0.12	0.17	0.05	0.04	0.05	0.37	0.40	0.38
Glucose	0.24	0.21	0.24	1.04	0.70	0.55	0.28	0.30	0.22	0.64	0.84	0.96	0.88	1.04	3.01	3.52	3.49
Myo-inositol	0.35	0.24	0.34	0.69	0.67	0.41	0.13	0.18	0.15	0.18	0.32	0.36	0.34	0.39	0.66	0.82	0.74
Sucrose	2.04	1.83	3.24	29.73	12.72	11.93	3.97	4.75	4.00	8.27	11.39	7.74	8.05	8.42	7.74	9.39	9.01
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	KEGG ID	PubChem ID	RI	quantitated m/z
Oxalic acid	C00209	971	1145	190
Malonic acid	C04025	867	1211	233
Succinic acid	C00042	1110	1316	247
Fumaric acid	C00122	444972	1352	245
Malic acid	C00149	222656	1487	233
Pyroglutamic acid	C01879	7405	1516	156
Citric acid	C00158	311	1814	273
Aconitic acid	C00417	643757	1747	229
Erythronic acid	C21593	2781043	1540	292
Benzoic acid	C00180	243	1253	179
Alanine	C00041	5950	1113	116
Valine	C00183	6287	1217	144
Isoleucine	C00407	6306	1292	158
Proline	C00148	145742	1577	142
Urea	C00086	1176	1249	189
Glycerol	C00116	753	1276	205
Arabinose	C00259	439195	1758	307
Galactose	C00984	439357	1979	319
Glucose	C00031	5793	1991	319
Myo-inositol	C00137	892	2065	305
Sucrose	C00089	5988	2457	361
METABOLITES_END
#END