Supporting Information

Molecular Mechanisms of Early Flowering in Tomato

Induced by Manganese Ferrite (MnFe2O4) Nanomaterials

Le Yueab, Yan Fengab, Chuanxin Mac, Chuanxi Wangab, Feiran Chenab, Xuesong

Caoab, Jing Wangab, Jason C. Whited, Zhenyu Wang*ab, Baoshan Xinge

a Institute of Environmental Processes and Pollution Control and School of Environment and

Civil Engineering, Jiangnan University, Wuxi 214122, China.

b Jiangsu Engineering Laboratory for Biomass Energy and Carbon Reduction Technology,

Wuxi 214122, China.

c Key Laboratory for City Cluster Environmental Safety and Green Development of the Ministry

of Education, Institute of Environmental and Ecological Engineering, Guangdong University

of Technology, Guangzhou 510006, China.

d The Connecticut Agricultural Experiment Station, New Haven, Connecticut 06504,

United States

e Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts

01003, United States.

*Corresponding author:

Tel.: +86 0510 85911123; Fax: +86 0510 85197773

E-mail address: wang0628@jiangnan.edu.cn (Dr. Zhenyu Wang)

Text. S1 Optimum concentration selected to explore the promoting mechanisms of
tomato flowering
Fig. S4 shows the growth of tomato plants after spraying MnFe2O4 NMs at different
concentrations and different times. MnFe2O4 NMs significantly promoted flowering
time as the dose increased (Fig. S4a). After foliar spraying 10 and 50 mg L-1 MnFe2O4
NMs, the flower formation time was significantly reduced by 13 days and 15 days as
compared to control, respectively. At harvest, the fruit number of all tomato plants upon
1, 10 and 50 mg L-1 MnFe2O4 NMs treatment was significantly increased compared of
the control (Fig. S4b), and the sugar content in the fruits was 1.4, 2.0 and 1.2 times that
of the control, respectively (Fig. S4c). Therefore, 10 mg L-1 was chosen as the
concentration for subsequent experiments due to the best growth effects. Also, the net
photosynthetic rate of tomato leaves after 4 and 7 times of consecutive applications at
different concentrations was analyzed (Fig. S4d). After spraying 4 times, the net
photosynthetic rate of all treatments increased significantly, and the effects of 10 mg
L-1 and 50 mg L-1 were the greatest. However, only 1 mg L-1 had the better net
photosynthetic rate than control after 7 times of spraying. This might be because that
the increased continuous foliar spraying caused stomatal blockage. Fig. S4e further
verified that the stomatal conductance of tomato leaves increased significantly by 26.6%
after spraying 1 time but decreased significantly by 38.9% after spraying 7 times. In
addition, the sucrose content in leaves increased by 7.5% and 8.8% after spraying 10
mg L-1 MnFe2O4 NMs for 1 and 4 times, respectively (Fig. S4f). Therefore, 10 mg L-1
MnFe2O4 NMs spraying for 4 times was selected for further investigation.
Text. S2 HPLC-MS/MS operating parameters used in this study
After the plant sample extraction, 10 μL samples were injected onto a Acquity UPLC
HSS T3 (2.1×100 mm, 1.8μm) using an 18-min linear gradient at a flow rate of 0.35
mL/min. The eluents in negative/positive mode were eluent A (0.1% v/v formic acid in
water) and eluent B (0.1% formic acid in acetonitrile). The solvent gradient was set as
follows: 5% B, 0 min; 5% B, 1.5 min; 100% B, 14.0 min; 100% B, 15.5 min; 5% B, 16
min; 5% B, 18 min. Nitrogen was used as sheath gas (35 L / min) and aux gas (15 L /
min). The spray voltage was set as 3 kV (-3 kV for negative mode and 3 kV for positive
mode) and capillary temperature was 320 ℃. The resolution of full MS was set as 70000
and scan range was acquired between 70 to 1050 m/z in full MS-dd MS2 mode. The
following dd-MS2 product ion spectra were collected at resolution of 17500, isolation
window of 1.5 m/z, and collision energy of nce: 20, 40, 60. Compounds were identified
based on their retention time and mass spectrum values in the mzCloud and mzVault
library.
Fig. S1. Zeta potential, hydrodynamic diameter, and TEM image (a), size distribution
(b), and VSM (d) of MnFe2O4 NMs
Fig. S2. Fe and Mn content (a, b), single particle content (c) in tomato leaves, and the
number of NM particles in leaf sap at 0 h (d), 12 h (e), and 72 h (f). The significant
differences between CK and NMs are marked with “*” (p < 0.05).
Fig. S3. Magnetic attraction of chloroplast mixture (left) and chloroplast-NM mixture
(right).
 Fig. S4. Flowering time (a), fruit number (b), sugar content (c), net photosynthetic
rate (d), stomatal conductance (e), sucrose content (f) of tomato upon treatment with
different doses of MnFe2O4 NMs. Bars with different letters are significantly
different, and values are mean ± SD (standard deviation). The significant differences
between CK and NMs are marked with “*” (p < 0.05).
 Fig. S5. The relative expression of Ferredoxin, PsaA, and PsbA (a), and SPS in
tomato leaves after spraying MnFe2O4 NMs. The significant differences between CK
and NMs are marked with “*” (p < 0.05).
Fig. S6. Phenotype of tomato fruits (a), fruit number (b), fruit fresh weight (c). Bars
with different letters are significantly different, and values are mean ± SD (standard
deviation).
Fig. S7. The companion field experiment to verify the greenhouse effect.
Fig. S8. Partial least squares-discriminate analysis (PLS-DA) score plots of metabolic
profiles of tomato fruits between control and MnFe2O4 NMs.
Fig. S9. Volcano plot of metabolites in control and tomato fruits
upon MnFe2O4 NMs (CK/NMs).
Fig. S10. Sugar-acid ratio (a), and ascorbic acid content (b) in fruit upon MnFe2O4
NMs. The significant differences between CK and NMs are marked with “*” (p <
0.05).
Fig. S11. Relative expression of JA synthesis genes (a), SA synthesis genes (b), and
ethylene synthesis genes (c) in tomato fruits after MnFe2O4 NM application. The
significant differences between CK and NMs are marked with “*” (p < 0.05).
Fig. S12. The auto fluorescence of chlorophyll observed by fluorescence microscopy
in tomato fruit upon control (a) and MnFe2O4 NMs (b), fluorescence intensity of
control and NMs (c).
Fig. S13. Mass of Fe and Mn (a), and P content (b) in tomato fruits upon MnFe2O4
NMs. The significant differences between CK and NMs are marked with “*” (p <
0.05).
Fig. S14 Raw data of the NM determination in tomato fruits by SP-ICP-MS
Table. S1. Fresh/dry weight and root parameters of tomato plants after MnFe2O4 NM
application. The significant differences between CK and NMs are marked with “*” (p
< 0.05).
Table. S2. Differentially expressed metabolites of tomato fruits in CK and upon
MnFe2O4 NMs
Table. S3. Fe and Mn content (mg/g) in fruit in CK and upon MnFe2O4 NMs.
Table S4. Measures of sensitivity, detection limits, and R2 for element detection by
ICP-MS.
Table. S5. Instrumental parameters for SP-ICP-MS data acquisition.
Table. S6. Primer sets list for this study
Gene Primer Sequence (5' to 3')
Actin Forward ATGATAACTCGACGGATCGC
Reverse CTTGGATGTGGTAGCCGT
SPS Forward CGGTGGATGGCAAAACG
Reverse GGCAATCGGCCTCTGGT
Ferredoxin Forward TTCCTCACTCACAATGGCAAC
Reverse CCAGCTCTGTAGTTTTACCTT
PsaA Forward GATTTCTCATAGTTGGTGCTGCTG
Reverse TACAAACCAAAACTGTGAAAGCCT
PsbA Forward CCAAGGTTAGCACGGTTGAT
Reverse CGTAGCCGCTCATGGTTATT
SFT Forward CACCGATATTCCAGCTACCA
Reverse TGTTTGCCGACCTAATTGTC
LeSUT1 Forward TTCCATAGCTGCTGGTGTTC
Reverse TACCAGAAATGGGTCCACAA
LeSUT2 Forward CCTACAGCGTCCCTTTCTCT
Reverse GGATACAACCATCTGAGGTACAA
LIN5 Forward TGAGACTCTGAATGCTTGGAG
Reverse CCATACACACACTTTGCCTTC
LIN7 Forward TGGCAATTAACGACGAGGCACA
Reverse CCCCTTTTACCATAGTTCCTTTCTCCT
GA20ox2 Forward GTGATCCGATTGCAGCTAACGG
Reverse ACGGATGTGCAAGTGAGATAAG
GA20ox3 Forward CTAGTGTTACTAGAGAACTACA
Reverse TGTCAACCCCATGGTTAACCAC
SIGAST1 Forward GCACGTACCGGTGTTCAAAGAC
Reverse CAGTTATTGTAGCAAGGGCAAC
SHT1 Forward AACATCCCTGGAGGAACTTG
Reverse GCATTTGAATTCCACCTTCA
SHT2 Forward TCAACTACGGAACAGCCAAG
Reverse TCAGGTTCAATGTTGTCGGT
SHT3 Forward GTGCTATGAGGTTCGGGATT
Reverse TGGTTGAGTCCATTGGTGTT
LOX13 Forward GGACCACAGATGAAGAACCATTAC
Reverse CTCTGTTCTTCAAGTTCGGATCAT
AOS Forward CGAAACTCCCAGCCCAAAAAG
Reverse ACCTGGTGGCATGTTCGTTC
AOC Forward TTCTACTTCGGCGATTACGGTC
Reverse GGTTAAGTACGCTCCCTGAACG
OPR3 Forward CTTTGAGGAACGCGTATCAGG
Reverse TGACACGAGATCAGCATCACC
PAL5 Forward AACAGCAACATTACCCCGTGTT
Reverse GCAATGTATGACAACGGGACAA
ICS Forward TCATTAGACGATTGGCGTGCTA
Reverse GCTGTTGCATCAAATCGGATT
ACS2 Forward TATGGAGAGTTATTATAAACGATGTTA
Reverse CTAAGTACATAGACCAGTTGTCAATAC
ACS4 Forward ATTCACTAGAGGACTTGAAGAAATAG
Reverse CAAGCTTTATAACTTTATTTGATTGTA
ACO2 Forward AAATTTTGGGACTAAAGTAAGTAACTA
Reverse TAAATTTAGGGTAAACTTGTTTGTTAT
ACO4 Forward GTTACTTGACTTACTCTGTGAAAATCT
Reverse ATATAACTTCATGTAATCATCAAACAC