Uptake of humic substances by
wheat plants: preferentialaccumulation in lipid fraction
Natalia Kulikova, E. Tsvetkova, G. Badun,
M. Chernysheva, V. Korobkov, Z. Tyasto,
I. Perminova
Lomonosov Moscow State University, Moscow, Russia
Zelinskii Institute of Organic Chemistry, Moscow, Russia
www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Probable mechanisms of HS
physiological activity
HS physiological activity
Membrane activity
Hormone-like activity

Redox activity
Biosynthesis¸ Do humic substances enter the plants?
¸ What is the distribution of HS in plants?
www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

The goal¸ to study uptake anddistribution of HS in wheat
seedlings

www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Preparation of labeled HS
Isolation fromlabeled substrate

Introduction
14С or 15N
labeledsubstances

Synthesis fromprecursors

Polymerization
14
C- or 15Nphenols, proteins,sugars etc.
in the presenceof enzymes

Composting,
HS isolation

Precipitation,
HS isolation

14C or 15N analogues of humic substances
www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Preparation of 3H-HS
Tritium thermal activation method
Tritium

Atomictritium

Tungsten wire,2000К

Н 2 → 2 3Н3
Н + RH → R• + 3НHR• + 3Н → R3Н
3Target,77К

Dialysis,HS isolation

3Н humic substances

www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

SEC analysis of 3H-HS
UV-detection

Labeled HS are identicalto parent

Parent HS
Labeled HSV
UV-detection

Radioactivity

Tritium is uniformly
distributed in HS

UV-detection
Radioactivitydetection

Vwww.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Studied humic substances¸ Coal humic acids¸ Peat humic and fulvic acid¸ Aquatic fulvic acidElemental analysis
Size-exclusion chromatography
13C NMR analysis

Labeling with tritium

Estimation of hydrophobicity(i.e. determination of KTW)

Bioassay experiments

www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Bioassay design
Target object: wheat Triticum aestivum L.
HS concentration: 50 mg/lGrowing: Knopp nutrition solution

Extraction of proteins,lipids, sugars

Soaking: distilled water25°С, 72 h

5 daysEquilibrium
concentration of HS

www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Kinetics of HS uptake by plants
Coal HA

HS accumulation, mg/(kg h)
16

R2=0.96

12Michaelis-Mentenequation

8V=40

Vmax[C]
Km + [C]010203040
Equilibrium concentration of HS, mg/lV[C]Vmax

is rate of nutrient uptake (amount/unit time)

Kmis the Michaelis constant (in units of concentration)www.humus.ru

50

is nutrient concentration in the solution
is the maximum rate of nutrient uptake due to saturation of thetransporter mechanism

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Kinetics of HS uptake by plants
8Peat FA

R2=0.98

68

Coal HMA

R2=0.9664

422HS, mg/l

0010203040

R2=0.91

40050

620

41030

4030

5050

HS, mg/l0102030¸ Kinetics of HS uptake by plants can be describedusing Michaelis-Menten equation¸ Uptake of HS by plants is hypothesized to be
carrier-mediated (transporter-limited)www.humus.ru

40

Coal 1 HMA0020202HS, mg/l
1010R2=0.97

Peat HA 8300HS, mg/l

0Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

4050

HS uptake vs. HS properties
Vmax, mg/(kg h)
60Km, mg/l
504040302020100005Mp, kD

1015-4-3-2lg(Ktw)¸ The higher hydrophobicity, the less Km¸ The higher molecular weight of HS, the higher Vmax

www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia-10

HS in lipid fraction of plants
HS content in lipid fraction, % of accumulated
Coal HA
100

Drying
Lipid extraction

80

Aquatic FA60
40200

¸ Mainly hydrophobic fragments of HS are
accumulated by plants?
www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Identifying of individual compounds
in HS in plants
Fourier Transform Ion Cyclotron Resonance
Mass Spectrometry
Intensity, a.u.

Extraction

m/z
150

250

350

450

Identifying of individual compounds

HSwww.humus.ru

Extraction

Gas chromatography

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Identifying of individual compounds
in HS in plants Identified compounds

HS3H-C H O
18 32 23Н-Linoleic acid C18:2

3H-C H O
14 24 23Н-Product of lipids decomposition•Acetic acid C2:0
•Propionic acid C3:0
•Butyric acids C4:0
•Valeric acids C5:0
•Caproic acids C6:0
•Palmitic acid C16:0•Oleic acid C18:1

Volatile fatty acids¸ HS can be involved in lipid metabolism of plants
www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Uptake of HS by plants: furtherissues to solve
HS content in fraction, % of accumulated

140120¸
Sum of HS determined in proteins, lipids, and100 sugars exceeded 100% of HS accumulated
by plants
8060
40
200
Proteins

www.humus.ru

LipidsSugarsHumic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

TotalUptake of HS by plants: further
issues to solve
Thin layer chromatography andlipid fraction¸ Sumtritiumof HSautoradiography
determined in ofproteins,lipids, andsugars exceeded 100% of HS accumulated
by plants
Carotenoids?
Neutral pigments?
¸ Lipids are a heterogeneous
mixture ofdifferent compounds

Neutral lipids

www.humus.ru

Polar lipids

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Uptake of HS by plants: furtherissues to solve¸ Sum of HS determined in proteins, lipids, andsugars exceeded 100% of HS accumulated
by plants¸ Lipids are a heterogeneous mixture ofdifferent compounds¸ Further study of distribution of HS in plants
is needed

www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia

Thanks for yourattention!
Ďakujem!

www.humus.ru

Humic Substances in Ecosystems 8, September 13-17, Šoporňa, Slovakia