Histochemical Localization of Desiccation Induced Hydrogen ...

International Journal of Applied Environmental Sciences

ISSN 0973-6077 Volume 12, Number 8 (2017), pp. 1585-1603

© Research India Publications

http://www.ripublication.com

Histochemical Localization of Desiccation Induced

Hydrogen Peroxide Production and Its Relation to

H+ATPase in Dicranopteris linearis

Kavitha C H1* and Murugan K2

1Department of Botany, St Johns College, Anchal 2Plant Biochemistry and Molecular Biology Laboratory, Department of Botany,

University College, Trivandrum, 695 034, Kerala, India *Corresponding author

Abstract

Water scarcity is one of the major environmental stresses that induce a wide

array of responses like changes in osmotic potential, malondialdehyde (MDA)

level, ion homeostasis, osmolyte accumulation and pigment composition.

Research related to plant response to water stress is blooming due to the

changing climatic conditions in most parts of the earth. Water stress can vary

from a small decrease in water potential to the level of lethality in plants.

Currently, it has been proved that drought induced reaction is similar to

pathogen-induced responses and that could lead to deleterious tiring by the

ROS pathways normally noticed with the hypersensitive responses. The

subcellular localization of desiccation induced H2O2 synthesis and its relation

to H+ATPase of the desiccated fronds of Dicranopteris linearis was studied. Desiccation induced H2O2 accumulation was noticed initially on the plasma

membrane, cell wall and subsequently continued into the cytoplasm and cell

organelles like chloroplast, mitochondria. Inhibitor analysis reveals that

NADPH- dependent superoxide synthase and the cell wall peroxidases were

the possible sources for H2O2 production in the cell wall. Symptoms of visible

damages like vacuolation, lipid peroxidation were noticed with time span of

desiccation. These responses may not be directly connected with defense

1586 Kavitha C H and Murugan K

against oxidative stress, but may rather indicate changes in oxidative balance

within the cells that affect its metabolism and the homeostasis of the whole

cell, possibly leading into induction of programmed cell death. Desiccation

treatment also caused an inhibition of H+ATPase activity and that seems to be

a correlation between H2O2 production and H+ATPase activity.

Keywords: Dicranopteris linearis, desiccation, hydrogen peroxide, electron

microscopy, localization, H+ATPase.

1. INTRODUCTION

Cellular state represents the total active water level that regulates cells to mitigate

their habitats in a narrow manner.1Redox state is a part of the cellular reponse and

also a marker for accessing the physiological status of the cell. Intracellular plant

antioxidant expressions are closely related to the metabolic state of the cell and are

responding against the constantly changing environment. Similarly, the metabolic

pathways like photo respiratory, enzymatic and non-enzymatic defense and their

responsive-gene regulation and morpho- anatomical features play roles in regulating

the above state.2Abiotic stress includes drought, salinity, low temperature, UV-B and

others. Plant cell metabolism regulate via avoiding photodynamic or reductive

activation of molecular oxygen to produce ROS, particularly superoxide, H2O2 and

singlet oxygen.3Generally, in most cases, the formation of ROS is genetically

programmed, induced during the course of development and by environmental

fluctuations, and has complex downstream effects on both primary and secondary

metabolic compounds.4 Higher plant cells produce ROS, particularly superoxide and

H2O2 as secondary cascades in many events associated with plant growth and

development. Further, higher plants transmit information concerning changes in the

environment via the bursts of superoxide at the plasma membrane.

Situations which induce ROS synthesis in an uncontrolled manner leads to oxidative

stress, which is a harmful process enhance the oxidation of essential component of the

cell. The plant counteract via signals to make appropriate adjustments of gene

expression and cell structure in response to environmental and developmental cues.

Thus, ROS-antioxidant induction is not a simple signaling cassettes but a complex

relationship between metabolism and redox state in the plant system. H2O2 is

relatively stable, can diffuse freely throughout the cell, and hence may be able to

participate in HO formation at sites distant from its synthesis. Bestwick et al., 5

suggested that H2O2 accumulation may occur by a reduction in H2O2 scavenging and

an increase in H2O2 production involving coordinated changes in the enzymes like

peroxidase, superoxide dismutase, and catalase. H2O2 accumulation plays multiple

roles like membrane damage, a component of structural defense (lignin synthesis), a

signal molecule operating within the challenged cell, and also a defense compound.

Histochemical Localization of Desiccation Induced Hydrogen Peroxide… 1587

However, a little information on the localization of H2O2 production or accumulation

during plant-desiccation interactions was known especially among lower plant groups.

In this scenario, the present study aims to localize H2O2 in the desiccated fronds of the

forked fern and its relation with H+ATPase.

2. MATERIALS AND METHODS

2.1. Plant material

Dicranopteris linearis (Burm.f.) Underw. commonly known as forking fern belongs

to Gleicheniaceae and is widespread along the tropical and subtropical regions of the

earth .It grows horizontally at ground level with stalked compound fronds. It is found

dominating along the road cuttings in shaded or open areas where water availability is

scarce. The sporophyte is characteristic with dichotomously divided leaves and

creeping rhizome covered with septate, branched hairs.

2.2. Desiccation treatment protocol

Fresh D. linearis was fully hydrated and equilibrated in a controlled environment

chamber for 48 h at 20°C and a radiant flux intensity 75 µM /m2/ s. The samples were

desiccated in a desiccator over polyethylene glycol (PEG) in a controlled environment

chamber using the same light and temperature regimes as described above. The

species was subjected to five different desiccation regimes (a) 2 day (b) 4 day (c) 6

day (d) 8 day and (e) 10 day. Control plants were maintained in an optimal water

conditions in each case during the whole experimental period.

2.3. Histochemical localization of H2O2 by transmission electron microscopy

(TEM) using CeCl3 staining

Leaf sections were fixed with 2.5% glutaraldehyde in 50 mM pH 7.0 sodium

cacodylate buffer, first under vacuum and then at 4°C overnight for TEM analysis.

Subsequently, the samples were washed with buffer and stored at 4°C.The

localization of H2O2 accumulation by CeCl3 staining and subsequent pre-fixation was

carried by the protocol of Bestwick et al 5. Cerous ions (Ce3+) react with H2O2 results

in to electron dense cerium perhydroxide deposits 6.

H2O2+ CeCl3 → Ce(OH2)OOH

The CeCI3-treated and control sections were post-fixed in 1% osmium tetroxide,

dehydrated in ascending ethanol series and embedded in Epon LX 112 and

polymerized. Ultrathin sections were cut with a diamond knife on a Reichert Ultracut

microtome and mounted on copper slot grids (2 X 1 mm). Sections were observed


with transmission electron microscope at an accelerating voltage 60 kV. Intensity of

electron-dense was quantified using an image analyzer.

2.4. Inhibitor treatments

Twelve 2-month-old fronds were exposed to different regimes of desiccation 2 -10

days using polyethylene glygol (PEG). Leaves were collected at 0, 2 to 10 days and

sectioned. Sections were first vacuum infiltrated with buffer alone (50 mM Mops pH

7.2) or with buffer containing NADPH oxidase and peroxidase inhibitors, as

described by Bestwick et al. 5,for 30 min at room temperature. H2O2 accumulation

was analyzed from the leaf tissue.

2.5. Determination of H+ATPase activity

Plasma membrane H+ATPase was isolated from the leaf samples of Dicranopteris linearis under desiccation rehydration stress according to the protocol of Gallaghard

and Leonard7. The released inorganic phosphate (Pi) was determined according to the

method of Fiske and Subbarow 8. After termination the reactive solution was mixed

with 0.1 ml of 1-amino1-naphthol-sulphonic acid (0.125% in 15% NaHSO3 1%

Na2SO3). This mixture was shaken at 25oC for 30 min. The absorbance was recorded

at 750 nm. The protein content of the enzyme was estimated by Bradford method 9.

2.6. Statistical analysis

Results were presented as mean ± SD. All data were subjected to analysis of variance

performed on the window based Genestat package (6th ed., NAG Ltd., England).

3. RESULTS AND DISCUSSION

The control cells appeared highly vacuolated with maturity. Chloroplast was intact

showing grana with packed thylakoids and well defined starch grains were also

noticed. Mitochondria were normal (Fig.1). No H2O2 deposits were observed.


Fig.1. TEM (15000X) of unstained section of D.linearis showing intact chloroplast

(Control cells)

During the initial period of desiccation i.e.; 2 days, the cells showed many vesicles,

vacuolation and H2O2 deposition along the cell wall (Fig.2a&b). Endoplasmic

reticulum (ER)and mitochondria were intact.

Fig 2a&b. TEM images of leaf cells showing vacuolated cytoplasm with intact ER

and mitochondria.

Different levels of H2O2 deposits was noticed as electron-dense granules in various

parts of the cell wall. The morphology of ribosome, nuclei and endoplasmic

reticulum, does not appear to have been adversely affected by desiccation treatments

up to 6 days. Meanwhile, damaged membrane of organelles and plasmolysis with

separations of the plasma membrane from the cell wall was noticed in the cells. The

chloroplasts were flattened with scattered H2O2 (Fig.3).


Fig.3. TEM analysis of desiccated (6d) D.linearis frond cells showing chloroplasts

with marginal distortions. Black deposits of H2O2 along the chloroplast membrane.

Subsequent days of desiccation (8th and 10th days) displayed prominent deposits of

cerium perhydroxides along the membrane (Fig.4a and 4b).

Fig.4a and 4b. TEM images of 8 and 10 day desiccated leaf cells showing cerium per

hydroxides deposits along the membrane.

In addition, 8 day desiccated frond cells displayed massive irregular sized vacuoles.

Chloroplasts become oblong and more stretched. H2O2 could be seen prominently

along the intercellular region of the cells (Fig. 5). At higher desiccation periods, the

cells appeared highly vacuolated. Relatively large H2O2 deposits were observed in the

surface between cytoplasm and cell walls (Fig.6). Plastids were deformed with

irregular shape. Mitochondria also displayed irregularities (Fig.7).


Fig. 5, 6, 7. TEM images showing cell distortions and H2O2 deposits

Histo chemical localization of hydrogen peroxide (H2O2) using the cerium chloride

technique provided amble evidence of an enhanced cerium (IV) perhydroxide staining

along the cell wall and plasma membrane and subsequently into the cytoplasm.

Higher duration of exposure (8 to 10 d) showed noticeable increase in the

accumulation of H2O2. In the control, the staining of cell wall and plasma membrane

due to cerium (IV) perhydroxide deposits was negligible and fairly uniform (Fig.1).

The staining of these cell structures was noticeably more pronounced in the cells that

had been exposed to higher desiccation periods (Fig.8a and 8b).

Fig 8a and 8b. TEM images showing cerium (IV) perhydroxide deposits along the

plasma membrane and also between adjacent cell wall regions in the desiccated cells.

Figure 8a and 8b reveal the desiccation impact at higher magnitude, where dense

granular particles can be seen along the plasma membrane and also within adjacent

cell wall regions in the desiccated cells, but not in the control cell (Fig.1). Figures 2a

and 2b show the presence of cerium per hydroxide particles marginally in the

cytoplasm, which is filled with membranous vesicles, and showed signs of

degeneration.


Desiccation in the apoplast region of the cell can lead to the formation of diverse

short-lived ROS that accumulates along the cell wall and the plasma membrane

components 10. Visibility of CeCl3 precipitation in the membranes observed via the

TEM suggests the active H2O2 production by the cells as a consequence of desiccation

resulted oxidative stress in the cell wall.

To confirm that CeCl3 had penetrated into the cells and particularly the chloroplasts,

and that chloroplastic H2O2 production can be visualized with the CeCl3 staining, leaf

sections were infiltrated with methyl viologen that sets off ROS production in the

chloroplasts. In these leaves, H2O2 accumulation was visible in the chloroplast's

stroma of the cells. These fronds also displayed severe ultra-structural damages. The

appearance of methyl viologen-induced H2O2 in the cytoplasm, mitochondria, and

chloroplasts shows that CeCl3 obviously penetrates biological membranes and can be

used to detect intracellular H2O2 production in plant cells in a similar way as in

mammalian cells, where cerium-derived deposits have been detected inside

subcellular structures, including mitochondria 6.

To elucidate the possible sources of the H2O2 accumulation in desiccated leaves, the

fronds were exposed to desiccation for 10 d and infiltrated with inhibitors of possible

sources for radical production in the cell wall and subsequently with CeCl3, as

described in Bestwick et al 5. Inhibitor effects were analyzed from the cells. Results

from these inhibitor experiments presented in Table 1 show that precipitation of CeCl3

is H2O2-specific and that both cell wall peroxidases and the plasma membrane

NADPH oxidase are possible sources for the H2O2. Catalase removed the staining

almost completely, confirming that the precipitate detected is derived from H2O2.

DPI, an inhibitor of flavin containing oxidases 11, such as the plasma membrane

NADPH oxidase, reduced the percentage of H2O2 producing cells and abolished the

strong staining completely. KCN and NaN3 were used to inhibit peroxidases 5. KCN

was roughly as effective in inhibiting the desiccation induced H2O2 production as DPI,

whereas NaN3 abolished H2O2 accumulation detectable with the CeCl3 staining almost

completely.

Table.1. Effect of inhibitors on the cell wall and plasma membrane H2O2 localized in

8d desiccated fronds of D.linearis.

Intensity of CeCl3 (% of scored cells)

Treatment Strong Medium Marginal NIL

8d desiccated+ Buffer 10 24 30 28

8d desiccated+ CAT 0 2 1 97

8d desiccated+ DPI 0 1 38 55

8d desiccated+ KCN 0 14 27 50

8d desiccated+ NaN3 0 4 3 92


H+ATPase activity

The present study shows that desiccation causes a significant reduction in the activity

of plasma membrane associated H+ATPase in the cells (Table.2).

Table.2. Effect of desiccation stress on H+ATPase activity (U/mg protein) in

D.linearis

control 2 day

D

2day R 4day

D

4 day

R

6day D 6 day

R

8 day

D

8 day

R

10 day

D

10 day

R

H+AT

Pase

0.131±

0.09

0.128±

0.12

0.346±

0.12

0.25±

0.07

0.228±

0.02

0.104±

0.11

0.174±

0.13

0.098±

0.04

0.155±

0.09

0.057±

0.08

0.124±

0.17

Abbreviations: D-Desiccated; R- Desiccated and subsequently rehydrated. P < 0.01.

Interestingly a positive relationship between was seen H+ATPase and the elevated

production of H2O2 as indicated by cerium perhydroxide deposits. Penetration of

cerium chloride across tissues as a result of the reaction between CeCl3 and H2O2, is

likely to take more time and, therefore, this protocol may not be used to evaluate the

dynamic events related to H2O2 synthesis and their gradient across the plasma

membrane. However, it has been established as an ideal tool in assessing the

concentration differences in H2O2 over time 12, 5. H2O2 deposits along the membranes

correlated with adverse effects of desiccation on H+ATPase. The occurrence of

increased concentrations of H2O2, initially in the cell walls and along the cell

membrane, suggests that both the cell wall and the plasma membrane may be the

source of H2O2 13. Even though a direct link has yet to be proved, it seems to be that

elevated level of H2O2 leads alterations in membrane permeability, and also its

integral protein complexes. Ktitorova et al., 14 reported H2O2-mediated decrease in

hydraulic conductivity in roots of wheat in relation to salinity stress ,and the results

provided in the present analysis, showing a close relationship between H2O2 level and

H+ATPase activity in isolated the plasma membrane, substantiate this view. Prasad et

al., 15 showed a correlation between H2O2 concentration and chilling stress in corn

plants. Similarly, Frahry and Schopfer 16 also revealed that H2O2 content in the

soybean root could be induced significantly by exogenous NADH or NADPH. Thus,

the present results tempt to suggest that in addition to decrease of the H+ATPases, the

water- channel proteins, aquaporins, may be altered. Decrease of H+ATPase activity

may reduce cytoplasmic pH, and this may in turn lead to a decrease in root hydraulic

conductivity. Javot and Maurel17, revealed that the sensitivity of water-channel

activity to environmental parameters may affect the water uptake and the water

balance among plants. Zhang and Tyerman18, compared the activity of aquaporins

against chilling with the cell pressure probe in algae and also in the cortical cell of the

roots. Wan and Zwiazek19 reported that, mercurial compounds inhibits water transport


by reacting with sulphydryl groups of the proteins and there by block the channels.

This further leads to decrease of hydraulic conductivity and thereby water transport.

Elevated levels of H2O2 may interfere with the activity of H+ATPase and aquaporins.

Meanwhile, a direct correlation of the aquaporins activity (open/closed state) to H2O2

remains to be experimentally elucidated. The increased desiccation period may be

involved in the closing of aquaporins 20. Further, the decrease in the hydraulic

conductivity due to the increase in the water viscosity with desiccation, which may

inhibit water uptake. Cochard et al 21., ; Wan et al., 22 have proved that chilling

remarkably increased the resistance to water flow of Populus tremuloides seedlings,

and this feature may not be explained by the corresponding enhancement in the water

viscosity23. As noticed here, H2O2 caused a decrease in H+-ATPases activity in the

plasma membranes. Meanwhile, the relationship of this to water channel activity is

not yet clear, although the extrusion of protons from cells should be connected to the

uptake of nutrient ions. Zhang and Tyerman 18 have proved that the hydraulic pressure

conductivity was not altered by K+ channel blocker tetraethylammonium at doses that

usually block K+ channels. The ways in which desiccation inhibits H+ATPase are not

yet clear. H2O2 may influence ATP hydrolysis 24 and/or disulphide exchange of

oxidized glutathione with the reactive cysteine in V-ATPase. Studies in jack pine

seedlings revealed that the inhibition of the plasma membrane H+ATPase activity by

direct freeze and thaw was resulted by the thiol oxidation of plasma membrane

proteins25. Reduced glutathione level prevented lipid peroxidation through

glutathione-mediated ROS scavenging system. Simlarly, plasmodesmata can also

facilitate movement of water between adjacent cells, desiccation induced closure of

plasmodesmata may be another possibility for the observed reduction in H+ATPase

activity 26. Rapid accumulation of H2O2 initially at the plasma membrane may also be

relevant in this regard.

H2O2 formation in the present study appears to be an alternative to the desiccation-

induced O2· – based oxidative burst 28. Minibayeva and Beckett, 27 reported similar

trend in many lichens, thalloid liverworts and hornworts species. Runeckles and

Vaartnou 29 proved that the ozone derived O2.- signal disappeared in few minutes after

removing the plants from ozone source using Electron paramagnetic resonance

studies.

Schraudner et al., 30 have shown that O3 induces early bursts of active H2O2

production in the cell walls of the O3-sensitive tobacco cultivar Bel W3. The number

and distribution of these oxidative bursts correlated with the discrete sites of local cell

death and visible symptoms developed later. Similarly, Kettunen et al., 31 and Rao and

Davis, 32 reported O2·– formation similarly in ozone exposed Arabidopsis thaliana.

The oxidative burst is one of the earliest sign in plant-pathogen interactions. It has

been shown with H2O2-specific CeCl3 staining that the oxidative burst takes place

only in the immediate vicinity of the invading pathogen5.The subcellular location of


the desiccation induced H2O2 and O2·– synthesis, however, has not been documented

among ferns. In the present analysis, desiccation induced active H2O2 production in

the fronds of the fern in different subcellular locations was visualized. Initially, the

H2O2 accumulation was seen in the cell wall and plasma membrane and subsequently,

in the cytoplasm, chloroplast and mitochondria (Fig.2-8.) i.e., apoplastic phase

followed by symplasic regions. Continuing H2O2 accumulation in the fronds that can

be visualized with DAB (3,3-diaminobenzidine)- staining33 takes place only at the

locations that later develop macroscopically visible necrosis.

The apoplastic ROS induction may be via the plasma membrane NADPH oxidase

associated with an extracellular SOD and extracellular POXs, was addressed here

with inhibitor experiments (Table 1), which suggest that both are involved in the H2O2

production in the cell walls of desiccation exposed fronds. The plasma membrane

located NADPH oxidase is activated by biotic or elicitor challenge in various plant

species 34. The O2· – synthesized via NADPH oxidase has to dismutate rapidly to H2O2

either spontaneously or by the SOD activity 35.

Extracellular CuZn SOD is connected with the plasma membrane NADPH oxidase

and that may induce H2O2 production 36. Previously Kavitha and Murugan 39 proved

an increase in the total cellular AOX activity in desiccation exposed fern fronds at

different duration of desiccation stress. Furthermore, the increase in AOX activity was

parallel with periods of desiccation. It may be proposed that the enzyme activity

profile was related to the cellular damages.

Other possibility of apoplastic H2O2 synthesis is by the extracellular POXs. They are

involved in lignin biosynthesis and catalyze a reaction where H2O2 is produced at the

expense of NAD(P)H 37,38. In the fronds of the forked fern, the guaiacol peroxidase

activity was increased by desiccation 39 .The mode of increase was similar to that of

other enzyme activity and the increase was more pronounced in the 10th day after

desiccation. Further, DPI has a peroxidase inhibiting activity, especially at high

concentrations 40. In the French bean and rose cells, the increased concentration of

DPI reduced NADPH oxidase dependent H2O2 production compared to control 40.

Thus, the ability of DPI to partly inhibit peroxidases does not exclude the possibility

for involvement of NADPH oxidase as a source of H2O2 in the desiccated ferns. This

is also substantiated by two different locations for the apoplastic H2O2 accumulation

detectable with the CeCl3 staining; in Fig.3-8, CeCl3 precipitation is clearly visible on

the cell wall and also on the surface of the plasma membrane. Furthermore, the CeCl3

precipitates on the plasma membrane have a distinct spatial pattern which suggests the

presence of a single origin, presumably the oxidase protein complex, for each

precipitate. It must be proposed that the inhibitors used are not specific and that they

may have H2O2 scavenging properties as reported by Barcelo 41. KCN, NaN3 41 and

DPI 42 have an ability to scavenge H2O2 in addition to their inhibitor activity.

However, it has been shown in other systems, by combining localization, enzyme


activity and inhibitor studies that the two most likely candidates for stress-induced

H2O2 synthesis are plasma membrane NADPH oxidase and cell wall POXs 40

.Although the results from the inhibitor studies (Table 1) must be interpreted in

correlation with the histochemical localization of the H2O2 synthesized and with

enzyme assay. Tuomainen et al., 43 suggested that these parameters provide an

ambient system in dissecting the subcellular sites for ROS synthesis, thus leading the

way to more specific studies. Furthermore, the inhibitory effect of DPI on nitric oxide

(NO) synthesis and the interaction of NO and ROS production 44 substantiates the

present results.

In the present study, desiccation induced H2O2 production in the chloroplasts was

comparatively less may be probably due to the efficient H2O2 scavenging mechanisms

in the chloroplasts of these exposed plants. Chronic desiccation stress, results in

overexpression of SOD in the chloroplast reduced the leaf damage in tobacco45. In

tobacco, chloroplastic SOD activity may be the rate-limiting enzyme for ROS

scavenging and that was sufficient for detoxification of the H2O2 produced via SOD.

Methyl viologen-induced O2·– synthesis in the chloroplasts, on the other hand,

resulted in H2O2 accumulation that seemed to exceed the scavenging capacity in

chloroplasts, since increased CeCl3 precipitation was visible in the chloroplast stroma.

Many studies revealed that under high desiccation period, the cytoplasmic free radical

scavenging by antioxidant system was 46, 47 in safe guarding plants. It is well known

that peroxisomal catalase is a sink also for cytoplasmic H2O2, in addition to the H2O2

produced via photorespiration in peroxisomes 48. This scavenging mechanism was

crucial for defense against oxidative burst in the cell system. The H2O2 accumulation

in the cytoplasm, chloroplast and mitochondria suggest that cytoplasmic organelles

could be a sink for the desiccation induced H2O2 produced, but not for the apoplastic

H2O2. Intensity of H2O2 staining in chloroplast was marginal than in the surrounding

cytoplasm, which would be seen if catalase scavenges cytoplasmically synthesized

H2O2.

The mitochondrial H2O2 accumulation coincided with the symptoms of visible

damage formation. Naton et al., 49 reported similar changes with pathogen infected

parsley cells, where the mitochondria were swollen and their tubuli were damaged.

Subsequently, they reported the loss of mitochondrial membrane integrity, ROS

accumulation and the death of the infected cells. Jabs50, also suggested that in

mammals the mitochondria are the major source of ROS metabolism leading to

programmed cell death (PCD). The imbalance in mitochondria was through a

reduction in transmembrane potential followed by uncoupling of electron transport

from ATP synthesis. The latter causes O2.- production and subsequent bursting of

mitochondrial integrity.


The role of mitochondria in oxidative stress has not been extensively analyzed in

plants. Oxidative stress induced accumulation of heat shock proteins; especially the

mitochondrial HSP22 in tomato cell cultures was also reported 51. Similarly, Betula pendula the transcript levels of the mitochondrial phosphate translocator (Mpt1) were

significantly increased by ozone impact was reported 52. These reactions may not be

directly linked with defense against oxidative stress, but rather indicate changes in the

oxidative balance of the cell that affect mitochondrial metabolism and the

homeostasis of the cell. The relationship of ROSs: AOX machinery in plants to cell

organelle permeability transition, subsequent hypergeneration of O2- radicals and

following PCD, as in animal cells 53, remains to be elucidated. Michaeli and Galili 54

reported the degradation of organelles or specific organelle components via selective

autophagy in plant cells induced by ROSs. Pellinen et al., 55 reported ozone-induced

hydrogen peroxide production and its localization at subcellular level in Betula pendula leaf cells. Lee et al., 56 noticed rapid accumulation of H2O2 in cucumber roots

due to exposure to low temperature appears to mediate decreases in water transport.

Induction of an oxidative burst is a general feature of sensitive plants exposed to the

air pollutant ozone 57. Slesak et al., 58 stated the role of hydrogen peroxide in

regulation of plant metabolism and cellular signaling in response to environmental

stresses. Dat et al., 59 noticed changes in H2O2 homeostasis trigger an active cell death

process in tobacco cells. D’Haeze et al., 60 correlated reactive oxygen species and

ethylene and also their positive role in lateral root base nodulation of a semiaquatic

legume. Schutzendubel et al., 61 visualized cadmium-induced changes in

antioxidative systems, hydrogen peroxide content, and differentiation in scots pine

roots. Earlier reports of histochemical demonstration and localization of H2O2 in

organs of higher plants by tissue printing on nitrocellulose paper was carried by

Schopfer 62. Shao et al., 63 compared primary antioxidant free radical scavenging and

redox signaling pathways in higher plant cells. Dunand et al., 64 connected distribution

of superoxide and hydrogen peroxide in Arabidopsis root and their influence on root

development: possible interaction with peroxidases. Rubio et al., 65 localized

superoxide dismutases (SOD) and H2O2 in legume root nodules. All these data

substantiate the role of H2O2 accumulation as a signaling cascade to induce

antioxidant machinery and there by counter balance oxidative burst of cells. In the

present study, the forked fern produced enormous amount of H2O2 during increased

periods of desiccation and decreased H+ATPase activity.This further reduce water

transport between the cells.

4. CONCLUSION

The localized accumulation of H2O2 is consistent with the existence of a physically

close link between the putative receptor controlling recognition of the challenging

abiotic stress namely desiccation and activation of ROS production. Confirmation of

https://www.ncbi.nlm.nih.gov/pubmed/?term=Michaeli%20S%5BAuthor%5D&cauthor=true&cauthor_uid=24802874

https://www.ncbi.nlm.nih.gov/pubmed/?term=Galili%20G%5BAuthor%5D&cauthor=true&cauthor_uid=24802874


the molecular organization of the signaling cascade leading to ROS production will

require the identification of both the desiccation induced ligand and the plant’s

receptor, which are effective in generating drought resistant species.

5. ACKNOWLEDGEMENT

The authors hereby acknowledge the University Grant Commission regional office,

Bangalore for providing FDP status to the teacher fellow for completing the Ph.D.

work (Order No.F.No.FIP/12th plan/KLKE021 TF 06).

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