Lignosulfonate Improves Photostability and Bioactivity of Abscisic Acid under Ultraviolet Radiation

Article abstract of DOI:10.1021/acs.jafc.7b02002

Abscisic acid (ABA), as a commonly used plant growth regulator, is easy to be degraded and lose its bioactivity under sunshine. To select an eco-friendly and efficient photoprotectant for the improvement of photostability and bioactivity of ABA when exposed to ultraviolet (UV) light, we tested the effects of three biodegradable natural-derived high polymers, sodium lignosulfonates 3A [molecular weight (MW) > 50000, with degree of sulfonation (DS) of 0.48] and NA (20000 < MW < 50000, with DS of 0.7) and calcium lignosulfonate CASA (MW < 20000, with DS of 0.7), on the photodegradation of ABA. Lignosulfonates 3A, NA, and CASA showed significant photostabilizing capability on ABA. Lignosulfonate 3A showed preferable photostabilizing effects on ABA compared to CASA, while NA showed an intermediate effect. That indicated that lignosulfonate with a high MW and low DS had a stronger UV absorption and the hollow aggregate micelles formatted by lignosulfonate protect ABA from UV damage. Approximately 50% more ABA was kept when 280 mg/L ABA aqueous solution was irradiated by UV light for 2 h in the presence of 2000 mg/L lignosulfonate 3A. The bioactivity on wheat (JIMAI 22) seed germination was greatly kept by 3A in comparison to that of ABA alone. The 300 times diluent of 280 mg/L ABA plus 2000 mg/L 3A after 2 h of irradiation showed 20.8, 19.3, and 9.3% more inhibition on shoot growth, root growth, and root numbers of wheat seed, separately, in comparison to ABA diluent alone. We conclude that lignosulfonate 3A was an eco-friendly and efficient agent to keep ABA activity under UV radiation. This research could be used in UV-sensitive and water-soluble agrichemicals and to optimize the application times and dosages of ABA products.

Full text of DOI:10.1021/acs.jafc.7b02002

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Article
Lignosulfonate improves photostability and
bioactivity of abscisic acid under UV radiation
Fei Gao, Sha Yu, Qun Tao, Weiming Tan, Liusheng Duan, Zhaohu Li, and Haixin Cui
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02002 • Publication Date (Web): 29 Aug 2017
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Lignosulfonate improves photostability and bioactivity of abscisic acid under UV radiation
Fei Gaoa, b, Sha Yub, Qun Taob, Weiming Tanb, Liusheng Duanb*, Zhaohu Lib, Haixin Cuia
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a Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of
Agricultural Sciences, Beijing 100081, PR China
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b State Key Laboratory of Plant Physiology and biochemistry / Engineering Research Center of
Plant Growth Regulators, Ministry of Education / College of Agronomy and Biotechnology, China
Agricultural University, Beijing 100193, PR China
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*Corresponding author: Liusheng Duan (Tel: +86 10 62731301; Eꢀmail: duanlsh@cau.edu.cn)
This research was supported by Innovation and Development of New Plant Growth Regulator and
Bioꢀherbicide in the National Key Research and Development Program of China
(2017YFD0201300), Synergistic Key Technology of Chemical Pesticide and Product
Development in the National Key Research and Development Program of China
(2016YFD0200500), the Major National Scientific Research Program of China (2014CB932200),
and the Agricultural Science and Technology Innovation Program.
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Abstract: Abscisic acid (ABA), as a commonly used plant growth regulator, is easy to be
degraded and lose its bioactivity under sunshine. To select an ecoꢀfriendly and efficient
photoprotectant for the improvement of photostability and bioactivity of ABA when exposed to
UV light, we tested the effects of three biodegradable natural derived high polymers — sodium
lignosulfonates 3A (molecular weight (MW) > 50000, degree of sulfonation (DS) 0.48), NA
(20000 < MW < 50000, DS 0.7) and calcium lignosulfonate CASA (MW < 20000, DS 0.7) on the
photodegradation of ABA. Both 3A, NA and CASA showed significant photoꢀstabilizing
capability on ABA. 3A showed preferable photostabilizing effects on ABA than CASA, while NA
showed an intermediate effect. That indicated lignosulfonate with high molecular weight and low
degree of sulfonation had a stronger UV absorption and the hollow aggregates micelles formatted
by lignosulfonate protect ABA from UV damage. Approximately 50 % more ABA was kept when
280 mg/l ABA aqueous solution was irradiated by UV light for 2 hours in the presence of 2000
mg/l lignosulfonate 3A. The bioactivity on wheat (JIMAI 22) seed germination was greatly kept
by 3A, comparing to that of ABA alone. The 300 times diluent of 280 mg/l ABA plus 2000 mg/l
3A after 2 hours irradiation showed 20.8 %, 19.3 % and 9.3% more inhibition on shoot growth,
root growth and root numbers of wheat seed, separately, comparing to ABA diluent alone. We
conclude that lignosulfonate 3A was an ecoꢀfriendly and efficient agent to keep ABA activity
under UV radiation. This research could be used in UVꢀsensitive and water soluble agrichemicals
and to optimize the application times and dosages of ABA products.
Keywords: abscisic acid (ABA); UV light; photodegradation; lignosulfonate; shoot and root
growth
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1 Introduction
Most of bioꢀagrichemicals are susceptible to UV damage in the field and their halfꢀlives were
very short, for example, 6 hours for avermectin (1) and 1 hour for spinosad (2). Therefore,
bioꢀagrichemicals requires more applications and high amount to ensure their effectiveness (3).
Abscisic acid (ABA) is an effective bioꢀplantꢀgrowthꢀregulator, which could prolong florescence
(4-7), promote differentiation of flower bud (8-10), enhance the growth of root and shoot (11-13),
raise fruit coloring rate and quality (14-19), and improve resistance to drought stress (20-25). ABA
also had curative effects on inflammation, type Ⅱ diabetes and tumor of animals and humans
(26-28). ABA is a potential product that would be widely applied both in agriculture and medical
care. However, due to UV light of sunshine, ABA is readily losing its bioactivity by isomerizing to
inactive transꢀABA (Figure 1) (29-31). The halfꢀlife of ABA is only 24 min (32). This increases
economic cost for ABA field application.
Many efforts have been made to keep the bioactivity of ABA and reduce its photosensitivity
(33-36), such as replaced the double bond or hydrogen in ꢁ2 (the location of double bond) of ABA
side chain to benzene ring and cyclopropyl or fluorine to relieve cis–trans isomerization. However,
the bioactivities of these new compounds were much less than that of ABA. Our previous study
found benzophenoneꢀtype UV absorbers (BPs) UVꢀ531 (2ꢀHydroxyꢀ4ꢀnꢀoctoxyꢀbenzophenone)
and BPꢀ4 (2ꢀHydroxyꢀ4ꢀmethoxybenzophenoneꢀ5ꢀsulfonic acid) greatly improved the stability and
bioactivity of ABA under UV radiation (37). However, potential endocrine disrupting effects of
these UV absorbers were reported (38-43). They have been revealed to act in a similar way to
estrogen and/or antiandrogen (38, 39), exert mutagenic effects on Salmonella (40), generate
oxidative stress in Carassius auratus (41), suppress immunity of the immune system (42) and
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induce sister chromatid exchanges or chromosomal aberrations in Chinese hamster ovarian cells
(43). What’s worse, BPs contained in personal care products, industrial and agricultural products
might pollute the aquatic environment and be bioaccumulated through food chains, as residues
have been detected in water, sediments, human breast milk, urine and blood (38). Though the toxic
of BPꢀ4 is rather low (39), the potential impacts cannot be excluded. Potential environmental
impacts of BPs have led to an increase concern over their usage in commercial products and their
presence in the environment. Therefore, it is better to develop ecoꢀfriendly photoprotectants to
alleviate the photosensitivity of ABA in application.
Lignosulfonate is sulfonated, fragmented lignin from the waste liquor of sulfite pulp mills
(44-48) (Figure 2). It is highly crossꢀlinked polymer formed from different phenylꢀpropanoid units
and keeps approximately the original chemical structure of native lignin except for the
introduction of a great number of sulfonic acid groups on the αꢀcarbons of lignin side chains (49).
The sulfonic acid groups make it easy to be soluble in the water. Due to hydrophilic groups
(sulphonic, phenylic hydroxyl, carboxylic and alcoholic hydroxyl) and hydrophobic groups
(aromatic, aliphatic and carbon chains), lignosulfonate showed surface activities (44, 46, 47, 50).
Due to the existence of phenolic hydroxyl group, carbonyl and other chromophores, some
researchers used lignin and lignosulfonate as sun blockers added into the emulsions of cosmetics
(51), or as wall material of microcapsule for protecting the agrichemicals and microbial pesticide
from UV damage and controlling actives release (52-54). The physicoꢀchemical properties of three
commercialized lignosulfonates 3A, CASA and NA were shown in Table 1. 3A has low degree of
sulfonation and high molecular weight which might make it has more αꢀOꢀ4 ether bonds and
conjugated systems separately. CASA has low sulfonation degree and molecular weight which
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might make it have fewer αꢀOꢀ4 ether bonds and conjugated systems. NA has high sulfonation
degree and intermediate value of molecular weight which might make it have fewer αꢀOꢀ4 ether
bonds and intermediate levels of conjugated systems.
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We hypothesized that applying lignosulfonate could reduce photosensitivity of ABA. The
objective of this study was therefore to test the hypothesis and to further select an efficient
photoprotectant. The dynamics of ABA degradation at different doses of selected lignosulfonate
and afterwards bioactivity of ABA plus lignosulfonate on plant growth were also analyzed. The
bioactivity of lignosulfonate itself at different doses on plant growth was analyzed as well.
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2 Materials and methods
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2.1 Experimental design and managements
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Experiment 1 was designed for comparing the stabilizing effects of three lignosulfonates on
ABA degradation under UV light. Three lignosulfonates were calcium lignosulfonate Borresperse
CAꢀSA, abbreviated as CASA; sodium lignosulfonate Borresperse NA, abbreviated as NA; and
modified and purified sodium lignosulfonate 3A, abbreviated as 3A. Solid contents all above were
greater than 93 % and main properties were listed in Table 1. All lignosulfonates were purchased
from Borregaard LignoTech. These lignosulfonates at the dose of 0 mg/l and 500 mg/l were added
into 5 mg/l ABA (pure content above 98 %, purchased from J&K Scientific Ltd.) aqueous solution.
Quartz test tubes (16 mm×13 mm×150 mm, obtained from Nanjing Xujiang Electromechanical
Plant) with 10 ml corresponding ABA solution prepared above were exposed to UV light in
photochemical reactor (XPAꢀ7, obtained from Nanjing Xujiang Electromechanical Plant). Test
tube of each treatment covered with aluminum foil was set as dark control. Mercury lamp (300 W)
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equipped with 365 nm light filter (both obtained from Nanjing Xujiang Electromechanical Plant)
was applied as UV radiation source (S1), around which the test tubes could orbit. The actual
irradiance is the energy of mercury lamp multiplying the transmittance of UV filter. The actual
irradiance of UVA was detected as 9974 ꢂW/cm2, UVB as 2730 ꢂW/cm2. And the actual
irradiance ratio of UVA to UVB was 3.7. Solutions in the tubes were stirred in order to achieve
uniform irradiation. At intervals of 1 min, 0.5 ml solution of each treatment under irradiation was
sampled and directly injected into high performance liquid chromatography (HPLC) after high
speed centrifugation. Dark control of each treatment was determined after irradiation ended. Total
radiation time lasted for 8 min and the experiments were repeated three times.
Experiment 2 was for further optimizing the dosage of lignosulfonates added into ABA
application. The lignosulfonate 3A was added into ABA aqueous solution (280 mg/l). The dose of
3A was 0 mg/l, 500 mg/l, 1000 mg/l, 2000 mg/l, 4000 mg/l and 5000 mg/l respectively. Referring
to the method of experiment 1, the solutions were exposed to UV light for 8 h. At intervals of 1
hour, 20 ꢂl solution of each treatment under irradiation was sampled and injected into HPLC after
diluted by 100 times and filtered by strainer (0.22 ꢂm). Dark control of each treatment was
determined after irradiation ended. The experiments were repeated three times.
Experiment 3 was for testing the remaining bioactivity of ABA in the presence of selected
lignosulfonate after irradiation. In order to compare the bioactivity of ABA with and without 3A
after irradiation, ABA were diluted to lower concentrations, as preꢀbioassay of ABA above 2 mg/l
showed badly suppression on wheat (JIMAI 22) seed germination and growth, which resulted in a
difficulty to compare the differences among treatments. Therefore, ABA (280 mg/l) with and
without 3A (2000 mg/l) were exposed to UV light for two hours and then diluted 300 times to
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prepare 25 ml solution each and added with 20 ꢂl Tweenꢀ20 for bioactivity assay. Petri dish (Ф =
90 mm) placed with two layers of filter paper was immersed by 4 ml each prepared solution and
then seeded by twenty wheat seeds disinfected with 10 % hydrogen peroxide solution for 10 min.
Five dishes were applied for each treatment as repeats. Artificial climate chest (PRXꢀ450C,
obtained from Ningbo Saifu Experimental Instrument Co., Ltd.) was used to incubate wheat seeds
of all treatments in darkness at the temperature of 25 °C and humidity of 85 %. After three days’
incubation, 10 seeds were chosen randomly in each dish to measure the shoot length, root length
and root numbers of each treatment.
Experiment 4 was for examining the bioactivity of lignosulfonate 3A at different dosages on
wheat seed germination. 25 ml lignosulfonate solution at the dose of 0 mg/l, 1.3 mg/l, 6.7 mg/l,
33.3 mg/l, 500 mg/l, 1000 mg/l, 2000 mg/l and 5000 mg/l were prepared separately for bioactivity
assay referring the methods of experiment 3. After three days’ incubation, 10 seeds were chosen
randomly in each dish to measure the shoot length, root length and root numbers of each
treatment.
Experiment 5 was for determination of critical aggregation concentration (CAC). CAC was
determined with Platinum ring (ϕ = 20.5 mm × 0.6 mm) on automatic interfacial tension meter
(JK99BM, obtained from Shanghai Zhongchen Digital Technic Apparatus Co., Ltd) at 20 °C.
Surface tensions of lignosulfonate 3A at a series of concentration were determined and the turning
point on the curve of γꢀc plot was CAC. Surface tension of water was 73.7 mN/m.
Experiment 6 was for the measurements of particle size distribution of lignosulfonate 3A at a
series of concentration by dynamic light scattering (DLS). The measurements were carried out by
ZETASIZER Nano series (Nano ꢀ ZS90, obtained from Malvern Instruments) with a scattering
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angle of 90° at 25 ± 0.01 °C. The light source is a standard laser with a power of 4 mW and a
wavelength of 633 nm. The range of the particle size measurement was from 0.3 nm to 5000 nm.
Accurate concentrations of lignosulfonate 3A were prepared in ultrapure water and then
equilibrated for 24 h before data collection. 4 ml of each prepared solution was added into
polystyrene disposable cuvette (12 mm × 12 mm × 45 mm). Each measurement was repeated three
times, and the average result was accepted as the final hydrodynamic diameters whenever all the
values fluctuated within reasonable experimental errors.
Experiment 7 was for investigating the morphology of lignosulfonate 3A at the concentrations
of CAC. The experiment was carried out by scanning electron microscopy (SEMꢀSU8010,
obtained from Hitachi) with an accelerating voltage of 5 kV and a working distance of 8 mm. The
analysis was done in a high vacuum, with Everhart–Thornley secondary electron (SE) detector. To
prepare samples for SEM investigation, lignosulfonate 3A at 2 g/l was dissolved in ultrapure water
and equilibrated for 24 h. Then the sample was added dropwise onto the surface of a polished
silicon substrate and naturally dried at room temperature overnight being protected from dust in
the air. The sample was thereafter coated with platinum (thickness = 5 nm) by a sputter coater
(EM ACE600, obtained from Leica Vienna, Austria) before capturing SEM images. Three parallel
experiments were carried out to exclude accidental errors.
Experiments 1 to 4 were carried out at Engineering Research Centre of Plant Growth Regulators,
Ministry of Education in China Agricultural University in 2015, Beijing, China. Experiments 5 to
7 were carried out at Institute of Environment and Sustainable Development in Agriculture,
Chinese Academy of Agricultural Sciences in 2017, Beijing, China. Experiments 1 to 3 were
focused on the photodegradation and afterwards bioactivity of ABA plus lignosulfonates.
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Experiments 4 to 7 were focused on the property of lignosulfonates.
2.2 Measurements
HPLC equipped with Agilent Eclipse Plus C18 column (4.6 mm×5 ꢂm, 250 mm) (Agilent 1200,
obtained from Agilent Technologies) was used to determine ABA concentrations. Methanol and
water (60/40, V/V) contained 0.2 % acetic acid inside were applied as mobile phase and operated
at the speed of 1 ml/min, in which temperature was set at 30 °C. UV detector wavelength was set
at 260 nm. The injection volume of samples was 20 ꢂl. ABA concentrations in samples were
calculated by external standard method according to a series of ABA standard solutions.
Full wavelength scan ranging from 190 nm to 400 nm were carried out to determine absorptions
of accurate amount of lignosulfonates 3A, CASA, NA, UV absorber BPꢀ4
(2ꢀHydroxyꢀ4ꢀmethoxybenzophenoneꢀ5ꢀsulfonic acid) and ABA in aqueous solution separately by
UV/Vis spectrophotometer (UVꢀ4802, obtained from Unico Instruments Co. Ltd). Absorptivity per
unit mass was calculated from absorbance according to LambertꢀBeer’s Law.
UV intensity of this research and natural sunshine were detected by UVA and UVB
radiationmeters (obtained from Photoelectric Instrument Factory of Beijing Normal University).
2.3 Data analysis
The bioactivity differences of ABA with ABA plus lignosulfonate 3A after being exposed to UV
radiation were analyzed by OneꢀWay ANOVA using LSD test at significant level of 0.05.
Bioactivity differences of a series of lignosulfonate 3A concentrations compared to pure water
were analyzed by the same statistical method as well.
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3 Results
3.1 Photodegradation of ABA in the presence of lignosulfonates
Three types of lignosulfonates, CASA, NA and 3A significantly stabilized ABA under UV
radiation (Figure 3). After 8 mins’ UV radiation, only 72 % ABA remained in ABA alone
treatment. However, all the treatments of ABA plus lignosulfonate kept at least 89 % ABA. 3A had
more significant effects on stabilizing ABA (degradation slope ꢀ0.005) than CASA (degradation
slope ꢀ0.055). Intermediate values of remained effective ABA were achieved in adding NA
(degradation slope ꢀ0.014).
3.2 Degradation dynamics of ABA adding with different dosages of lignosulfonate 3A
With adding 3A at the dose of 2 g/l to 5 g/l, more than 92 percent effective ABA remained after
two hour’s radiation, more than 80 percent after 4 hours, and more than 67 percent after 8 hours
(Figure 4). Meanwhile, there was only 23 percent ABA remained in ABA alone treatment after 8
hours radiation. Lower level but significant values of remained effective ABA were also achieved
in adding 3A with doses of 0.5 g/l and 1 g/l, in which the latter showed better stabilizing effects.
3A at 4 g/l and 5 g/l showed no significant difference on stabilizing ABA. Though 3A at 2 g/l
showed significant less effect on stabilizing ABA at longer duration of radiation than at 4 g/l and 5
g/l, it showed nearly the same stabilizing capability in the first 4 hours’ radiation. Thus, 3A at 2g/l
would be more convenient for further tests.
3.3 Remaining bioactivity of ABA in the presence of lignosulfonate 3A after irradiation
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Shoot and root length of wheat were significantly suppressed by the diluents of 280 mg/l ABA
alone or the diluents of 280 mg/l ABA plus 2000 mg/l 3A, compared to the control of pure water
(Figure 5a and 5b). Shoot and root length in ABA plus 3A treatment were 20.8 % and 19.3 %
shorter than that of ABA alone respectively, and there was no significant difference with the
treatments of unꢀdegraded ABA (Figure 5a and 5b). Root numbers in treatment of ABA plus 3A
were 9.3 % fewer than that in ABA alone. Root numbers in ABA alone showed no significant
difference with that of pure water treatment (Figure 5c). The bioactivity of unꢀdegraded ABA with
or without 3A showed no significant difference.
3.4 Impacts of lignosulfonate 3A on wheat seed germination
3A showed no significant bioactivity on wheat seed germination at the doses ranged from 1.3
mg/l to 2000 mg/l, compared to that of pure water treatment, while significant differences were
found among various lignosulfonate concentrations (Figure 6a, 6b and 6c). However, 3A at the
dose of 5000 mg/l suppressed 27 % shoot growth and 32 % root growth comparing to pure water
treatment (Figure 6a and 6b). The root numbers in all treatments showed no significant difference
with that of pure water control (Figure 6c).
3.5 Critical aggregation concentration of lignosulfonate 3A
According to the relationship between γ value and concentration (Figure 7), surface tension of
lignosulfonate 3A decreased sharply when the concentration was lower than 2 g/l. However, as the
concentration continued to increase, surface tension reduced slowly. So we defined concentration
at 2 g/l as a critical aggregation concentration of lignosulfonate 3A in aqueous solution.
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3.6 Dynamic aggregation behavior of lignosulfonate 3A in aqueous solution
From the intensity distribution of particle diameter for lignosulfonate 3A in aqueous solution
(Figure 8), there was a single peak at 0.5 g/l and 1g/l respectively, however, bimodal peaks
presented at 2 g/l. The smallest particle at one peak top was about 250 nm and the largest at
another peak top was about 5.5 ꢂm. The intensity of particle size distribution of 3A in aqueous
solution decreased with increasing concentration of 3A, but the intensity and the size of 3A
aggregates increase. These results indicate that assemble of 3A is a gradually process.
3.7 Morphologic characterization of lignosulfonate 3A
From the SEM photograph (Figure 9a and 9b), aggregated structures of lignosulfonate 3A at the
concentration of 2 g/l were observed. The aggregated structures were within the diameters of
about 500 nm to about 5µm and the larger ones appeared to be hollow, as deep grey color in the
middle of the aggregated structures was observed (Figure 9b). Solid aggregated structures would
show the same pale white color with the exterior, but if the structures are hollow, the middle
would be collapsed under the strong voltage of SEM. This resulted in deep grey color in the center
of the aggregated structures.
4 Discussions and conclusions
By adding lignosulfonates, ABA degradation under UV light was significant reduced.
Lignosulfonate greatly kept the bioactivity of ABA after exposed to UV radiation. Lignosulfonate
itself had no significant bioactivity. Adding lignosulfonate kept 67 percent ABA remained after 8
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hours exposing to UV irradiation, while only 23 percent ABA remained in ABA alone control. The
efficiency of ABA was increased nearly 3 times by adding lignosulfonate.
It is well known that UV bands of field sunshine mainly range from 290 nm to 400 nm (55). At
this range, ABA has some absorption in UVB (290 nm ~ 320 nm) but very little in UVA (320 nm ~
400 nm) (56) (Figure 10). Thus, UVB takes a great role on ABA bioactivity reduction in the field.
Lignosulfonates absorb UV radiation at most of UV region due to many chromophores it
contained. And the complex structure of lignosulfonate, which is widely accepted as a kind of
threeꢀdimensional crossꢀlinked biopolymer (57), can effectively dissipate the energy associated
with the absorption of UV light internally thereby preventing transfer to other proximate actives.
Though absorption of lignosulfonate per unit mass is not greater than that of ABA (Figure 10),
especially, in UVB region, increased dosage of lignosulfonate could show great UVB absorption.
Our experiments proved that lignosulfonate at proper high mass dosage strongly competed UVB
absorption, compared to that of ABA. Besides, the UV radiation applied in this research mainly
ranged from UVB to UVA (S1). UVB intensity applied in this study (2730 ꢂW/cm2) was much
higher than under natural sunshine (263 ꢂW/cm2). Therefore, laboratory results in this study that
ABA photodegradation could be greatly reduced by competitive UV absorption of lignosulfonate
can be applicable in field situation.
Lignosulfonate with different molecular weight and sulfonation degree showed different
stabilizing effects on ABA degradation under UV radiation. Lignosulfonate with higher molecular
weight may have more aryl ring structural units (58), which strengthens and shifts the UV
absorption of lignosulfonate to longer wavelength and thereby absorbs more UV damage from
ABA around. Lignosulfonate with lower sulfonation degree may have more αꢀOꢀ4 ether bonds not
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being replaced by sulfonic acid groups (57), which also enhances and induces red shift and finally
help improve UV protection effects. 3A with low degree of sulfonation and high molecular weight,
thus, showed most preferable photostabilizing effect on ABA degradation.
Absorptivity of lignosulfonate per unit mass is much lower than that of UV absorbers such as
2ꢀHydroxyꢀ4ꢀmethoxybenzophenoneꢀ5ꢀsulfonic acid (BPꢀ4) (Figure 10), therefore, theoretically,
much higher dosages of lignosulfonate should be applied to stabilize ABA comparing to that of
UV absorber. According to our measurements in this study and in the former researches (37), we
found 3A at 500 mg/l showed no significant difference with that of BPꢀ4 at 200 mg/l on stabilizing
ABA under UV radiation (Figure 11). However, adding 2000 mg/l 3A had a much higher
stabilizing effect on ABA than adding BPꢀ4. In consideration of potential impacts on human health
and the environment (38-43), caution is needed to increase the dosage of BPꢀ4 applied on ABA in
order to improve its stabilizing effects. Lignosulfonate, however, is sulfonated natural lignin,
proved safety to human beings (49, 59) and degradable in the environment (60). Lignosulfonate in
aqueous solution is quite stable even under strong and short wavelength UV radiations (61-63).
Properly increasing dosages of lignosulfonate was proved little impacts on plant growth by our
experiments. Thus, lignosulfonate is more efficient and ecoꢀfriendly to stabilizing ABA under UV
radiation even applied at high dosage.
Lignosulfonate 3A at 2 g/l (CAC) showed similar photostabilizing effects with that of 3A at
higher concentration (4 g/l or 5 g/l). There is no significantly increased effect on ABA
photostabilizing by further increasing lignosulfonate 3A above CAC, which indicates there might
be other factors that also protect ABA from UV damage except for competitive UV absorption.
One factor might be the micelles that lignosulfonate formed. It is well known that micelles were
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formed by surfactants above critical micelle concentration. A typical micelle is an aggregate with
the hydrophilic “head” regions in contact with surrounding solvent, sequestering the hydrophobic
singleꢀtail regions in the micelle center. Though lignosulfonate has amphiphilic activity and could
form micelles, the lignosulfonate molecules cannot aggregate into hollow micelles by their
hydrophobic sites as conventional linear molecule surfactants, as the hydrophobic base and
hydrophilic groups in lignosulfonate molecules are not totally separated from one another and the
hydrophilic groups are scattered in the whole molecule (Figure 12) (64). According to our research
and other studies (46, 64), hollow aggregates are the main form of the lignosulfonate at the
dosages of above CAC in solution. Moreover, sulfonic groups and some phenolic hydroxyl groups
constructed the surface of lignosulfonate aggregates. And the core of the aggregates is formed by
hydrophobic chains, which is loose and contains many weakly ionized groups such as carboxyls
and phenolic hydroxyl groups (Figure 12) (46). ABA has hydroxyl, conjugated carboxyl and
ketene groups and is slightly dissolved in water (3 g/l) and strongly dissolves in methanol, acetone
and ethyl acetate (above 90 g/l) (65), thus it showed relative weak polarity which makes it tend to
presence in the core of lignosulfonate aggregates through hydrogenꢀbonding and intermolecular
van der Waals forces (Figure 12). This explains the similar stabilizing effects of lignosulfonate 3A
at dosages above CAC. Therefore, we concluded that the formation of hollow aggregates might be
the other important factor that helped lignosulfonate protect ABA from UV damage. CAC found
in this study can be applied as one of the reference indexes for adding lignosulfonate into ABA.
Absorbance of agrichemicals by plant stems and leaves mainly takes place in the first four
hours after their application. According to our former measurement in natural conditions of winter
time, the applied ABA (540 mg/l) lost more than 50 % within 4 hours and even worse during other
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seasons due to much higher UV radiation. By adding lignosulfonate, the loss of ABA bioactivity
can be reduced to less than 20 % and even much lower. Although our previous studies (37) proved
UV absorber BPꢀ4 had a good photostabilizing effect on ABA, however, the simple small
molecule of BPꢀ4 might loss its stabilizing capability on ABA due to the rain. Lignosulfonate has
amphiphilic groups and plant leave surface is always hydrophobic or hydrophilic, which could
help ABA well adhere on the leaves and thereby increases its effects on ABA stabilization.
Therefore, the usage of surfactant could be thereafter reduced and the limitation of using BPꢀ4 can
be relieved.
In the future studies, appropriate time and dosage of lignosulfonate added into ABA application
are still required, especially, by more experiments at field conditions. In the field, the bioactivity
of ABA plus lignosulfonate in the sunny day might be different from that of in the cloudy due to
different degrees of degradation, so the effect and suitable doses of lignosulfonate should be
testified at both situations in the future. The effects of ABA plus lignosulfonate on growth,
development and yields of plant in relation to optimal applying time and dosage should be
determined at field condition. Moreover, the effects of new ABA products with adding
lignosulfonate on prolonging florescence, promoting fruit coloring rate and improving resistance
to drought stress is necessary to be explored, especially under natural sunshine.
In conclusion, lignosulfonate greatly reduced ABA photodegradation. Lignosulfonate 3A
addition significantly prevented ABA from UV damage and thereafter greatly kept ABA
bioactivity after exposed to UV radiation. Besides, lignosulfonate showed no significant impacts
on plant growth at suitable dosage. Consequently, lignosulfonate could be a safe and high
efficiency agent to photostabilize ABA. The usage of surfactants could also be reduced due to the
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surface activities of lignosulfonate. And waste liquor of sulfite pulp mills can be reduced and
reused. Pretty low cost would be made by applying lignosulfonate to prepare novel photostable
formulations of ABA or alternatively to be applied tankꢀmixed in the field, compare to the
application of UV absorbers. Thereafter, the ABA efficiency would be significantly promoted and
crop yield and quality would be improved. This research could be widely used in the application
of UVꢀsensitive and waterꢀsoluble agrichemicals and help to reduce environmental impact of
applying plant growth regulators.
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Acknowledgment
We thank Professor Lizhen Zhang, College of Resources and Environmental Sciences, China
Agricultural University, for technical improvement of the manuscript.
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Figure captions
Figure 1 Structure and photoisomerization of ABA to transꢀABA.
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Figure 2 Sulfonation process of lignin in sulfite pulping. Lignin in the group sites of the structure
represents for propagated lignin structure. This figure was modified from Lauten et al (57).
Figure 3 Photodegradation dynamics of ABA in the presence of varieties of lignosulfonate. ABA
was applied at 5 mg/l. Lignosulfonate 3A, CASA and NA were all applied at 500 mg/l. Error bars
are s. e. (standard error) of three replicates.
Figure 4 Dynamics of ABA (280 mg/l) photodegradation in aqueous solution affected by adding
different doses of lignosulfonate 3A. ABA was applied at 280 mg/l. Lignosulfonate 3A was
applied at 500 mg/l, 1000 mg/l, 2000 mg/l, 4000 mg/l and 5000 mg/l. lingo. is the abbreviation for
lignosulfonate 3A. Error bars are s. e. of three replicates.
Figure 5 Bioactivity of ABA in the presence of lignosulfonate 3A after irradiation on the growth
of wheat (JIMAI 22). ABA (280 mg/l) with or without 3A (2000 mg/l) were exposed to UV
radiation for 2 h and then diluted for 300 times to compare their bioactivity on wheat shoot and
root growth. Error bars are s. e. of five replicates. Within each figure, treatments with same letter
are not significantly different at a=0.05. unꢀdeg. is the abbreviation for unꢀdegraded; pꢀdeg. is the
abbreviation for partiallyꢀdegraded.
Figure 6 Impacts of lignosulfonate 3A on the growth of wheat (JIMAI 22). 3A is applied at 1.3
mg/l, 6.7 mg/l, 33.3 mg/l, 500 mg/l, 1000 mg/l, 2000 mg/l and 5000 mg/l. Error bars are s. e. of
five replicates. Within each figure, treatments with same letter are not significantly different at
a=0.05.
Figure 7 Surface tension of lignosulfonate 3A as a function of 3A concentration at 25 °C. CAC is
the abbreviation for critical aggregation concentration. γ represents for surface tension.
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Figure 8 Influence of concentration on the particles size distribution of lignosulfonate 3A at
25 °C.
Figure 9 SEM micrography of lignosulfonate 3A on polished silicon substrate. Lignosulfonate
was applied at the concentration of 2 g/l. a and b represent for micrograph captured at different
magnification.
Figure 10 Absorption spectra per unit mass of ABA, lignosulfonate 3A, CASA and NA, and UV
absorber BPꢀ4 in aqueous solution. Gray and light gray shadows indicate UVB (290ꢀ320 nm) and
UVA (320ꢀ400 nm) region in natural UV radiation.
Figure 11 Comparison effects of lignosulfonate 3A and UV absorber BPꢀ4 on stabilizing ABA
under UV radiation. ABA was applied at 280 mg/l. Error bars are s. e. of three replicates.
Figure 12 The formation of lignosulfonate aggregate and its interaction with ABA.
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Tables
Table 1 Physicoꢀchemical properties of three commercialized lignosulfonates
Name Molecular weight Na (%) Ca (%) Sulfonation degree
3A
NA
> 50000
20000 ~ 50000
< 20000
8.5
9
0.02
0.3
5
0.48
0.7
CASA
< 0.1
0.7
565
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 38
566
567
Figure graphics
568
569
Figure 1
ACS Paragon Plus Environment

Page 27 of 38
Journal of Agricultural and Food Chemistry
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571
572
Figure 2
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 38
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Figure 3
ACS Paragon Plus Environment

Page 29 of 38
Journal of Agricultural and Food Chemistry
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Figure 4
ACS Paragon Plus Environment