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American Journal of Plant Sciences, 2014, 5, 3396-3407
Published Online November 2014 in SciRes. http://www.scirp.org/journal/ajps
http://dx.doi.org/10.4236/ajps.2014.522355
How to cite this paper: Rajashekar, C.B., Fu, J. and Giri, A. (2014) Exogenous Vitamin K3 and Peroxides Can Alleviate Hypox-
ia in Bean Seedlings (Phaseolus vulgaris L.). American Journal of Plant Sciences, 5, 3396-3407.
http://dx.doi.org/10.4236/ajps.2014.522355
Exogenous Vitamin K3 and Peroxides Can
Alleviate Hypoxia in Bean Seedlings
(Phaseolus vulgaris L.)
C. B. Rajashekar1*, Jinmin Fu2, Anju Giri1
1Department of Horticulture, Forestry and Recreation Resource, Kansas State University, Manhattan, USA
2Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden,
Chinese Academy of Sciences, Wuhan, China
Email: *crajashe@ksu.edu
Received 3 October 2014; revised 17 October 2014; accepted 6 November 2014
Academic Editor: Thomas M K Elmore-Meegan, Dept Public Health Research, International Community for
Relief of Starvation and Suffering; Dept International Health, College of Medicine, Tampere University, Finland
Copyright © 2014 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/
Abstract
Oxygen limiting conditions are a common occurrence in root zones of most crop plants and can
adversely affect nearly all aspects of plant growth and development including its survival. The ob-
jective of this study was to determine the effectiveness of a novel redox cycling agent, vitamin K3,
and various peroxides including hydrogen peroxide, calcium peroxide and magnesium peroxide in
alleviating the effects of hypoxia in bean seedlings grown in nutrient culture. All the anti-hypoxic
agents including vitamin K3 had a positive impact on the overall growth of bean seedlings under
hypoxic conditions, but their responses were variable depending on the concentration. With re-
gard to shoot growth, vitamin K3 (5 μM) increased the leaf area significantly, by more than 58%
over the hypoxic control plants and produced the highest stem fresh weight similar to calcium
peroxide (20 μM) and magnesium peroxide (10 μM). In addition, the use of vitamin K3 resulted in
the highest accumulation of chlorophyll (chla + chlb) in the leaves, an increase of nearly two-fold
over the hypoxic control plants. Furthermore under hypoxia, calcium peroxide (20 μM) and mag-
nesium peroxide (10 μM) produced the highest leaf biomass (FW) followed by vitamin K3. Vitamin
K3 (1 μM) also favored root growth in bean seedlings under hypoxia; it produced the largest in-
crease in root length and root biomass (DW) similar to calcium peroxide and magnesium peroxide.
Based on the overall shoot and root growth response of bean seedlings to various anti-hypoxic
substances under hypoxic conditions, calcium peroxide, magnesium peroxide and vitamin K3 per-
formed better than hydrogen peroxide. These findings show that vitamin K3 and peroxide salts are
*
Corresponding author.
C. B. Rajashekar et al.
3397
effective in alleviating hypoxic stress in bean seedlings and also, further highlight their potential
for dealing with hypoxia in wide ranging situations.
Keywords
Hypoxia, Vitamin K3, Menadione, Peroxides, Beans
1. Introduction
Oxygen limitation to the plants especially to the roots is a common occurrence in crop plants. It is a chronic
problem in heavy, poorly drained soils but it can occur in most regions, regardless of soil type, during a transient
flooding after a heavy rainfall or prolonged flooding in river basins and coastal areas. Furthermore, in recent
decades with the growing threat of global climate change, the risk of more severe and frequent flooding has been
dramatically increasing, affecting food production and food security [1]. In the US among all the abiotic stresses,
too much or too little water is by far the major reason for crop losses [2].
Flooding can adversely affect seed germination, seedling establishment, impair growth and development of
plants leading to serious reduction in crop productivity and even crop losses (see review by [3]-[7]). Severity of
these effects however depends on crop species and the length of their exposure to hypoxia. However, most crop
plants including beans are typically sensitive to hypoxia [8] [9]. Moreover, roots are especially sensitive to oxy-
gen deficiency which can accrue rapidly in waterlogged soils as diffusion of air in saturated soils is relatively
low.
The major metabolic consequence of hypoxia is a shift in respiration from a high-energy yielding aerobic
pathway to low-energy yielding fermentative glycolysis. Low-energy status in plants is expected to have a broad
range of negative consequences on just about all the plant functions including diminished photosynthetic activity
leading to reduced growth and biomass accumulation [10]. In addition, in response to hypoxia, plants exhibit a
number of physiological and morphological changes which may help plants to deal with oxygen deficiency and
are thought to promote plant adaptation to hypoxia. These include changes in carbohydrate metabolism [4], in-
duction of ethylene, lowering of cellular pH, development of aerenchyma [11] and activation of a number of
hypoxia-responsive genes [1] [12].
Although there have been a number of efforts to reclaim land affected by long-term flooding or polluted by
harmful toxic substances by using oxidizing agents, very little attention has been focused on alleviating the ad-
verse effects of oxygen limitation in crop plants. For soil bioremediation, many oxygen-rich peroxides which
have a strong oxidizing capacity, including hydrogen peroxide, calcium peroxide and magnesium peroxide, have
been studied extensively for their potential use in reclaiming these soils [13] [14]. In the case of crop plants,
oxygen-rich peroxides have been used, mainly as a seed treatment, to provide oxygen and to improve crop per-
formance in wetland rice, and to improve germination of seeds [15]-[18]. In addition, use of hydrogen peroxide
has been found to improve crop performance of avocado in poorly drained soils [19]. However, very little is
known as to the relative effectiveness of these oxygen-rich compounds used in improving germination or plant
performance under hypoxic conditions.
In addition to evaluating the relative effectiveness of some of the commonly used peroxides, we report here a
novel redox cycling agent, vitamin K3, as an anti-hypoxic substance that can alleviate hypoxia in bean seedlings.
Vitamin K consists of many structural analogs and belongs to a family of napthaquinones which not only play
an important role in human health but also in maintaining redox homeostasis in plants and microbes. The two
well-known naturally occurring forms of this vitamin are vitamin K1, also known as phylloquinone, found in
green plants and involved in electron transport in photosystem I of photosynthesis, and the other is the vitamin
K2, also known as menaquinone, produced by certain bacteria including the anaerobic bacteria in human gut.
The synthetic and commonly used water soluble analog of this vitamin is vitamin K3 (menadione) which is used
as redox mediator or to generate reactive oxygen species [20] [21]. Menadione plays a complex role in plants; it
is known, in addition to its ability to produce reactive oxygen species, to induce resistance against biotic stresses
in plants [22], to induce chilling tolerance in plants [23] and to overcome the adverse effects of hypoxia in ani-
mal tissues [24].
C. B. Rajashekar et al.
3398
Potentially, these anti-hypoxic compounds can be used in poorly drained heavy soils which are prone to oxy-
gen deficiency and during transient flooding following heavy rains to mitigate oxygen deficiency in root zone of
crop plants. In addition, these compounds would be a valuable tool in the emerging hydroponic industry, bio-
reactors and liquid cell cultures where these additives can substitute the current cumbersome and expensive ap-
proaches used to provide aeration. The main objective of this study was to compare the effectiveness vitamin K3
with oxygen-rich peroxides including hydrogen peroxide, calcium peroxide and magnesium peroxide in alle-
viating the adverse effects of hypoxia on the growth and development of bean seedlings.
2. Materials and Methods
2.1. Plant Culture and Treatments
Bean seeds (Phaselous vulagris L. cv. Tendergreen) were purchased from Chesmore seed company (St. Joseph,
MO) and were planted in wet calcareous sand contained in plastic pots (9 cm × 9 cm) and germinated at 30˚C/
25˚C (day/night) in a growth chamber. After emergence of true leaves, the seedlings were transplanted into
200-mL glass bottles, each containing 200 mL of Hoagland solution. The seedling was held in upright position
by a rubber stopper in such a way that the roots were completely submerged in Hoagland solution while the
shoots were exposed to the air with the seedling stem passing through an 8 mm hole in the center of the rubber
stopper. The stopper was cut across radially from the hole so that the stem can be placed in the hole. Each bottle
contained one seedling and the stopper was sealed using Parafilm so as to prevent air leaking into the bottles.
The bottles were tightly wrapped with aluminum foil to keep the roots in the dark.
Seedlings were grown in a growth chamber with day/night temperature of 30˚C/25˚C, and a 8-h photoperiod
under a light intensity (photo synthetically active radiation) of 400 μmol∙m−2∙s−1. Bean seedlings were grown in
nutrient medium for 15 d to induce hypoxia. This was confirmed by measuring dissolved oxygen in the medium
before transferring the seedlings to the medium and after 15 d of plant growth by using a YSI ProODO oxygen
meter (YSI Inc., Yellow Springs, OH). After calibrating the instrument, the probe was immersed the nutrient
solution to record the dissolved in oxygen levels in triplicate samples. The dissolved oxygen level dropped from
7.57 ± 0.02 to 1.86 ± 0.59 mg/L after 15 d of plant growth indicating the hypoxic conditions in the nutrient me-
dium. The treatments included Hoagland solution containing freshly prepared hydrogen peroxide at 10 and 20
mM, calcium peroxide at 10 and 20 μM, magnesium peroxide at 10 and 20 μM or vitamin K3 (menadione so-
dium bisulfate) at 1 and 5 μM (Sigma Chemical Co., St. Louis) while the control consisted of Hoagland solution.
The experiments were conducted on a completely randomized design with 4 replications.
2.2. Plant Growth Measurements
After 15 d of plant growth in nutrient culture, plants were harvested and leaves, stem and roots were separated.
Fresh weights of leaves and stem were measured immediately. To determine dry weights, samples were initially
subjected to 105˚C for 30 min and subsequently dried in an oven at 85˚C until constant weight. Roots were
washed thoroughly with tap water and blotted gently with paper towels. Leaf area, root surface area and root
length were determined by using a digital image analyzer (Monochrome AgVision System, Decagon Devices,
Inc., Pullman) following the procedure of Harris and Campbell [25]. Leaf chlorophyll content was determined
by extracting leaf samples (0.05 g) with dimethyl sulfoxide for 48 h and the absorbance of the extract was
measured at 635 and 645 nm using a spectrophotometer (Spectronic Instruments, Inc., New York).
2.3. Statistical Analyses
Data on plant growth were analyzed by analysis of variance using the General Linear Models (GLM) Procedure
of Statistical Analysis System (SAS Institute, Cary, NC). Treatment means were separated using the Least Sig-
nificant Difference (LSD) test at a 0.05 probability level.
3. Results
Growth characteristics of bean seedlings were analyzed in response to various anti-hypoxic agents in seedlings
grown in the liquid medium for 15 d. Bean plants were sensitive to hypoxia, and growing plants for 15 days in
the nutrient medium reduced its dissolved oxygen level by more than 75%. The seedlings subjected to hypoxia
C. B. Rajashekar et al.
3399
showed reduced overall growth compared to those grown in the nutrient medium containing oxygen-rich perox-
ides and vitamin K3. All the major shoot and root growth characteristics measured in control plants were signif-
icantly lower than those in plants grown in the medium containing anti-hypoxic agents. Although all the anti-
hypoxic agents had a positive impact on overall growth of been seedlings, the responses were variable and va-
ried depending on the anti-hypoxic agents and their concentrations in the nutrient medium.
Total leaf area of bean seedlings treated with all the peroxides and vitamin K3 increased significantly with the
exception calcium peroxide at 10 μM (Figure 1). However, the largest increase in leaf area was with calcium
peroxide at 20 μM, magnesium peroxide at 10 μM and vitamin K3 at 5 μM, producing a leaf area more than 58%
of that in the control plants. Similarly, the specific leaf area/plant was higher in plants treated with anti-hypoxic
agents than in the control, reflecting an overall positive relationship between leaf area and leaf fresh weight
(Table 1). Leaf fresh weight of seedlings increased with all the anti-hypoxic treatments except hydrogen perox-
ide and magnesium peroxide both at 20 μM. However, the largest increase in leaf fresh weight resulted from the
treatment of seedlings with calcium peroxide (20 μM) and magnesium peroxide (10 μM) followed by vitamin K3
(5 μM) which, respectively, produced 37% and 35% more leaf fresh weight than the control plants. Furthermore,
treating bean seedlings with vitamin K3 (5 μM) produced the highest stem fresh weight similar to calcium pe-
roxide (20 μM) and magnesium peroxide (10 μM, Table 1). Overall, these three treatments consistently showed
better shoot growth responses in bean seedlings than did other anti-hypoxic treatments as evidenced by their
similar responses in relation to their stem fresh weight, leaf expansion, leaf fresh weight and shoot biomass (FW)
(Table 1, Figure 1, Figure 2 and Figure 3). However, it should be noted that these changes in shoot growth
characteristics were more pronounced on the fresh weight basis than on the dry weight basis. It is interesting to
note that hydrogen peroxide applied at both concentrations (10 and 20 mM) showed the least positive impact on
many of the shoot growth characteristics compared to other anti-hypoxic agents (at concentrations evoking the
best response). These shoot growth characteristics of bean plants included leaf area, leaf fresh weight, stem fresh
weight and shoot biomass.
Leaf chlorophyll content (chla + chlb) increased significantly in bean seedlings grown in the nutrient medium
containing anti-hypoxic agents compared to the hypoxic control (Table 2). Largest increase in leaf chlorophyll
(chla) content was observed in plants grown in the medium containing vitamin K3 (5 μM), which was more than
80% over that in the hypoxic control plants. Similar pattern was also noted in the chlorophyll (chla + chlb) con-
tent. Bean seedlings treated with vitamin K3 (5 μM) produced the highest amount of chlorophyll (chla + chlb),
representing a 96% increase over the control seedlings. The sharp increase in chlorophyll content is consistent
with better overall shoot growth observed in seedlings treated with vitamin K3.
After growing bean seedlings in nutrient solutions under hypoxic conditions and with anti-hypoxic treatments
for 15 d, their root growth characteristics were also analyzed. The total root length of bean seedlings grown in
Figure 1. Leaf area of bean seedlings after growing them for 15 d in nu-
trient solution containing oxygen-rich peroxides and vitamin K3 at various
concentrations. Control consisted of seedlings grown in the nutrient solu-
tion with hypoxic conditions. Bars with same letters are not significantly
different. Data represent means (n = 4) separated by LSD at p < 0.05.
C. B. Rajashekar et al.
3400
Figure 2. Shoot biomass (FW basis) accumulation in bean seedlings after
growing them for 15 d in nutrient solution containing oxygen-rich perox-
ides and vitamin K3 at various concentrations. Control consisted of seedl-
ings grown in the nutrient solution with hypoxic conditions. Bars with
same letters are not significantly different. Data represent means (n = 4)
separated by LSD at p < 0.05.
Figure 3. Photos showing bean seedling growth in nutrient solution with
hypoxic conditions (control) and in nutrient solution containing hydrogen
peroxide (10 mM), calcium peroxide (20 μM), magnesium peroxide (10
μM) and vitamin K3 (5 μM).
C. B. Rajashekar et al.
3401
Table 1. Leaf and stem growth characteristics of bean seedlings after growing them for 15 d in nutrient solution containing
oxygen-rich peroxides and vitamin K3. Control consisted of seedlings grown in the nutrient solution with hypoxic condi-
tions. Increase in shoot biomass accumulation (FW, %) due to treatments was calculated over that of the control. Data
represent means (n = 4) and the means were separated by LSD at p < 0.05.
Treatments Leaf weight
g/plant Specific leaf area
cm2∙g−1 FW Stem FW
g/plant Stem DW
g/plant Shoot FW
g/plant
Shoot FW
% increase over
control
Control 2.83 f 32.52 e 2.28 b 0.21 b 5.06 -
H2O2-10 mM 3.15 de 34.44 cd 2.68 c 0.26 bcd 5.83 15.21
H2O2-20 mM 2.89 f 36.74 ab 2.43 de 0.23 cd 5.32 5.13
CaO2-10 μM 3.25 d 33.17 de 2.58 cd 0.27 abcd 5.83 15.21
CaO2-20 μM 4.24 a 34.36 cd 3.00 ab 0.33 a 7.24 43.08
MgO2-10 μM 4.24 a 35.43 bc 3.02 ab 0.31 ab 7.26 43.47
MgO2-20 μM 3.01 ef 36.11 ab 2.37 de 0.22 cd 5.47 8.1
Vit K3-1 μM 3.63 c 34.25 cd 2.79 bc 0.28 abc 6.42 26.87
Vit K3-5 μM 3.89 b 37.28 a 3.08 a 0.26 abcd 6.97 37.74
Table 2. Chlorophyll a and chlorophyll b content of leaves of bean seedlings after growing
them for 15 d in nutrient solution containing oxygen-rich peroxides and vitamin K3. Control
consisted of seedlings grown in the nutrient solution with hypoxic conditions. Increase (%) in
chlorophyll (chla + chlb) due to treatments was calculated over that of the control. Data
represent means (n = 4) and the means were separated by LSD at p < 0.05.
Treatments Chla
mg∙g−1 FW Chlb
mg∙g−1 FW Chla + Chlb
mg∙g−1 FW Chla + Chlb
% increase over control
Control 0.68 f 0.07 g 0.75 e -
H2O2-10 mM 0.82 e 0.09 f 0.91 d 21.33
H2O2-20 mM 1.02 d 0.12 cd 1.14 c 52.00
CaO2-10 μM 1.07 cd 0.11 de 1.18 c 57.33
CaO2-20 μM 1.16 b 0.13 bc 1.29 b 72.00
MgO2-10 μM 1.11 bc 0.11 e 1.22 bc 62.67
MgO2-20 μM 1.14 b 0.12 c 1.27 b 68.00
Vit K3-1 μM 1.16 b 0.13 b 1.29 b 72.00
Vit K3-5 μM 1.32 a 0.14 a 1.47 a 96.00
the nutrient medium containing peroxides and vitamin K3 was significantly larger than the control (Figure 4).
Among the treated plants, calcium peroxide (10 μM), magnesium peroxide (10 μM) and vitamin K3 (1 μM)
produced the largest root length followed by other anti-hypoxic treatments whose responses were very similar.
Similarly, all the anti-hypoxic agents had a positive impact on root surface area with calcium peroxide (20 μM)
and magnesium peroxide (10 μM) showing the largest increase followed by vitamin K3 (1 μM). However, root
dry matter accumulation was not affected by most of the anti-hypoxic treatments except calcium peroxide (20
μM) and magnesium peroxide (10 μM) which produced about 81% and 76% more root dry matter (on the DW
basis), respectively, than did the hypoxic control. The results show that, by and large, calcium peroxide and
C. B. Rajashekar et al.
3402
Figure 4. Root growth characteristics including root length, root surface
area and root biomass (DW) in bean seedlings after growing them for 15
d in nutrient solution containing oxygen-rich peroxides and vitamin K3 at
various concentrations. Control consisted of seedlings grown in the nu-
trient solution with hypoxic conditions. Bars with same letters are not
significantly different. Data represent means (n = 4) separated by LSD at
p < 0.05.
C. B. Rajashekar et al.
3403
magnesium peroxide had a overall positive impact on root growth parameters including root length, root surface
area and root biomass. This was followed by vitamin K3 which produced significant increase both in root length
and root surface area but not in the root biomass (Figure 4).
The total biomass accumulation of bean seedlings (DW basis) in response to various treatments followed the
same trend as their shoot biomass accumulation (Figure 5). The highest biomass accumulation was observed in
seedlings that were treated with calcium peroxide (20 μM) and the lowest with magnesium peroxide (20 μM).
Seedlings treated with vitamin K3 at both concentrations (1 and 5 μM) produced higher biomass accumulation
than did the control plants.
The results on root-shoot ratio was variable with regard to the various hypoxic treatments. Bean seedlings treated
with calcium peroxide (10 μM) had no effect on root-shoot ratio compared to the hypoxic control (Figure 6).
Figure 5. Total biomass (DW basis) accumulation in bean seedlings after
growing them for 15 d in nutrient solution containing oxygen-rich perox-
ides and vitamin K3 at various concentrations. Control consisted of seedl-
ings grown in the nutrient solution with hypoxic conditions. Bars with
same letters are not significantly different. Data represent means (n = 4)
separated by LSD at p < 0.05.
Figure 6. Root-shoot ratio (DW basis) of bean seedlings after growing
them for 15 d in nutrient solution containing oxygen-rich peroxides and
vitamin K3 at various concentrations. Control consisted of seedlings
grown in the nutrient solution with hypoxic conditions. Bars with same
letters are not significantly different. Data represent means (n = 4) sepa-
rated by LSD at p < 0.05.
C. B. Rajashekar et al.
3404
Other treatments increased the root-shoot ratio, and the only exception being vitamin K3 (5 μM) which had a
lower ratio than the control. Although hydrogen peroxide treatment (20 mM) did not increase the root growth as
much as the other treatments, it did produce the highest root-shoot ratio. This may be due to the fact that hydro-
gen peroxide (20 mM) was not able to produce as much as shoot biomass as other anti-hypoxic agents.
Although vitamin K3 has a positive impact both on shoot and root growth characteristics of bean seedlings
grown under hypoxic conditions, its concentration seems to play a key role in their responses. Table 3 summa-
rizes the positive impact of vitamin K3 at 2 different concentrations on a number of shoot and root growth para-
meters of bean seedlings grown in non-aerated nutrient medium. The shoot growth was favored at higher con-
centration of vitamin K3 (5 μM) while lower concentration (1 μM) had a strong positive impact on the root
growth in seedlings.
4. Discussion
All the anti-hypoxic agents showed a positive effect on both shoot and root growth in bean seedlings compared
to the hypoxic control. Compared to the anti-hypoxic treatments, bean seedlings under hypoxia produced lowest
shoot and root biomass accumulation, leaf area, chlorophyll content, root length and root surface area. Many
studies have characterized the adverse impact of hypoxia on plant growth and development. Consistent with our
observations in bean seedlings, hypoxia typically has been shown to reduce biomass of shoots and roots [26].
Gravatt and Kirby [27] compared the growth responses of hypoxia intolerant and tolerant seedlings of hardwood
woody species to flooding and found that oxygen deficiency resulted in reduced net photosynthesis, total chlo-
rophyll contents, and relative growth rates of stems, roots and the whole plants in all the species. When bean
seedlings were subjected to hypoxia, vitamin K3 and the peroxides, especially calcium peroxide and magnesium
peroxide showed positive results on the growth. Based on both shoot and root growth analyses, vitamin K3 pro-
motes growth in bean seedlings under oxygen limiting conditions. Vitamin K3 (5 μM) produced the largest leaf
area and stem fresh weight in bean seedling relative to other anti-hypoxic agents. Also largest increases were
also observed with vitamin K3 treatment (depending on the concentration) in stem dry weight, root length and
root biomass (DW), similar to the traditional oxygen-rich peroxide salts. Furthermore, bean seedlings under hy-
poxic conditions accumulated more chlorophyll (chla + chlb) in the presence of vitamin K3 than any other anti-
hypoxic agents tested in this study. Previous studies have shown that vitamin K3 (menadione) improved growth
(biomass accumulation) of maize seedlings and increased their survival of during chilling stress [23], induced
resistance in plants against pathogens [22] and reduced the effects of hypoxia in animal tissues [24].
It is interesting note that shoot and root growth responses were consistently very different based on the con-
centration of vitamin K3. Typically, shoot growth, characterized by almost all the leaf and stem growth characte-
ristics measured [leaf area, specific leaf area, stem biomass (FW), shoot biomass (FW) and leaf chlorophyll
content], were favored at higher concentration of vitamin K3 (5 μM) while in contrast, root growth [characte-
rized by root length, root surface area and root biomass (DW) and root-shoot ratio (DW)] was promoted at lower
concentration of vitamin K3 (1 μM). It is worth noting that suppression of root growth at higher concentration of
vitamin K3 is striking, for example, the root biomass decreased drastically by more than 2.5 fold by increasing
its concentration from 1 to 5 μM. However, this reduction in root growth was perhaps compensated by increase
in shoot growth, as evidenced by the nearly identical total biomass accumulations when plants were treated with
Table 3. Increase (%) in shoot and root growth parameters in bean seedlings over control caused by different concentra-
tions of vitamin K3. The seedlings were grown in nutrient solution containing vitamin K3 for 15 d. Control consisted of
seedlings grown in nutrient solution without aeration.
Percent increase over control
Treatments
Shoots Roots
Leaf area Chlorophyll
a + b Biomass
(DW) Total length Total
surface area Biomass
(DW)
Vit K3-1 μM 38.64 72 30.61 57.33 46.26 47.54
Vit K3-5 μM 64.12 96 30.61 34.78 26.1 17.32
C. B. Rajashekar et al.
3405
either concentrations of vitamin K3. This may suggest a reallocation of biomass in bean seedlings to shoots and
roots with the variable vitamin K3 concentrations. Nevertheless, it should be recognized that vitamin K3 at much
higher concentration can produce adverse effects on plant growth because of its ability to generate reactive oxy-
gen species [21]. However on the other hand, it should also be noted that plant tissues can prevent the oxidative
stress by inducing a number of antioxidants enzymes [23]. For example, vitamin K3 is known to produce supe-
roxide anion which, in the presence of superoxide dismutase, undergoes dismutation to form hydrogen peroxide
and molecular oxygen [20]. Further, hydrogen peroxide can be decomposed to molecular oxygen and water by
catalase in the cells. Thus, vitamin K3 can facilitate the availability of oxygen within the cells that naturally con-
tain these antioxidant enzymes, producing very little changes in the dissolved oxygen levels in the external me-
dium. The assumption is that vitamin K3 can generate oxygen in the tissue containing these antioxidant enzymes,
thus uptake of vitamin K3 by roots appears to be critical. However, it is not known as to how rapidly this anti-
hypoxic agent is taken up by the roots of bean seedlings.
There was also a concentration-dependent response of bean seedlings to calcium peroxide and magnesium
peroxide under hypoxic conditions. However, the concentration dependence in this case was different from that
observed with vitamin K3 in that calcium peroxide at 20 μM and magnesium peroxide peroxide at 10 μM pro-
duced better overall growth of bean seedlings including both shoot and root growth in bean seedlings grown
under hypoxic conditions than other peroxides tested. These treatments consistently produced higher shoot bio-
mass and favorable various other shoot growth parameters such as leaf fresh weight, leaf area, leaf dry weight,
stem fresh weight and stem dry weight. Similarly, root growth was also favored by this treatment, including root
biomass, root area and root length. However, higher concentration of magnesium peroxide (20 μM) had the least
favorable effect on various shoot and root growth parameters of bean seedlings except for the leaf chlorophyll
content. It had markedly more negative impact on root growth compared to shoot growth. For example, at higher
concentration of magnesium peroxide (20 μM), the reduction in shoot biomass (FW) was about 25% while it
was about 76% for root biomass (DW) compared to that at a lower concentration of magnesium peroxide (10
μM).
Results also show that calcium and magnesium peroxides tend to alleviate hypoxia better than hydrogen pe-
roxide based on the plant responses with regard to the various growth parameters in bean seedlings. Based on
the average responses (average of two concentrations for each peroxide treatment), hydrogen peroxide treatment
produced less shoot growth, characterized by leaf fresh weight, leaf area, leaf dry weight, stem fresh and dry
weights, shoot biomass (both on dry and fresh weight basis), and total biomass (on dry weight basis) than did
either calcium peroxide or magnesium peroxide. Similar response was also observed with regard to various root
growth characteristics. However, hydrogen peroxide has been traditionally used as a seed treatment to reduce the
adverse effects of hypoxia in plants and in soil bioremediation [14] [15] [19]. Previous studies have shown that
peroxide salts are more stable and may release oxygen more slowly than hydrogen peroxide [14] [28]. Thus,
these peroxide salts may be more suitable to sustain plants against hypoxia over longer periods of time and for
bioremediation because of their ability to release oxygen more slowly than hydrogen peroxide [14] [29].
5. Conclusion
In summary, vitamin K3, a redox cycling agent, was able to offset the adverse effects of hypoxia in bean seedl-
ings. It produced better shoot and root growth in bean seedlings under hypoxia than hydrogen peroxide did, but
was comparable to traditional calcium peroxide or magnesium peroxide. The shoot and root growth characteris-
tics included leaf area, shoot biomass, root length and root biomass. The largest increase in leaf chlorophyll
content in bean seedlings was due to vitamin K3; the chlorophyll (chla + chlb) concentration nearly doubled un-
der hypoxic conditions compared to the control. Thus, the peroxide salts and vitamin K3 produced better overall
growth in bean seedlings under hypoxic condition than hydrogen peroxide did. The results suggest that these an-
ti-hypoxic agents including vitamin K3 have a potential use in addressing the problems associated with hypoxia
in a wide ranging situations such as poorly drained soils, flooded soils, bodies of water, aquaculture, bioreactors
and liquid tissue culture.
Acknowledgements
We thank Kansas Agricultural Experiment Station for supporting this study and this is a contribution by the
KAES.
C. B. Rajashekar et al.
3406
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