Biotechnological
Communication
Biosci. Biotech. Res. Comm. 9(4): 672-679 (2016)
Unravelling desiccation and rehydration tolerance
mechanism in the fern,
Adiantum latifolium
Lubaina AS
1
, Brijithlal ND
2
and K Murugan
3
1
Department of Botany, Christian College, Kattakada, Thiruvananthapuram, Kerala
2
Department of Botany, Bishop Moore College, Mavelikkara, Kerala - 690110
3
Plant Biochemistry and Molecular Biology Lab, Department of Botany, University College, Trivandrum
695 034, Kerala
ABSTRACT
Desiccation-tolerance (DT), the ability to lose virtually all free intracellular water and then recover normal function
upon rehydration, is one of the most remarkable features of ferns. The phenomenon of desiccation tolerance in pteri-
dophytes is well known and many species can with stand drying with water content to 10–20% of their dry weight
and return to normal metabolism and growth following rehydration. The time required to recover from desiccation
increases with duration of desiccation. Hence the present investigation was carried to reveal the biochemical adapta-
tions against desiccation and also subsequent rehydration in Adiantum latifolium Lam.a fern. Experimental design
for desiccation periods was1 D (24 h), 2 D (48 h) and 3 D (72 h) in a desiccator contain PEG. Desiccation caused an
enhanced production of reactive oxygen species (ROS) and increased lipid peroxidation. The analytical data were sub-
stantiated by histochemical localization. Total phenolic content increased with desiccation. Fractionation of phenols
revealed a pool of phenolic acids. Further, high induced level of polyphenol oxidase and phenylalanine ammonia
lyase enzyme activates provide tolerance to desiccation. Further studies are planned to analyze the molecular mecha-
nism involved in providing desiccation tolerance in the fern.
KEY WORDS: DESICCATION-TOLERANCE; REHYDRATION; REACTIVE OXYGEN SPECIES; LIPID PEROXIDATION
672
ARTICLE INFORMATION:
*Corresponding Author: harimurukan@gmail.com
Received 5
th
Nov, 2016
Accepted after revision 20
th
Dec, 2016
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007
Thomson Reuters ISI ESC and Crossref Indexed Journal
NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006
© A Society of Science and Nature Publication, 2016. All rights
reserved.
Online Contents Available at: http//www.bbrc.in/
Lubaina, Brijithlal and Murugan
INTRODUCTION
Pteridophytes, the seedless vascular plants character-
ized with independent heteromorphic alternation of
generation and primitive vasculature. These form the
conspicuous ora of tropical plant world. The pterido-
phytes consist of ferns and fern allies; of these, the ferns
are predominantly distributed in a wide range of habi-
tats. Out of 1250 species of pteridophytes occurring in
India, 173 species have been found to be used as food,
avor, dye, medicine, bio-fertilizers, oil, ber and
bio-gas production (Manickam and Irudayaraj, 1992).
Besides sugar, starch, proteins and amino acids, ferns
contain alkaloids, glycosides, avonoids, terpenoids,
sterols, phenols, sesquiterpenes as potential raw materi-
als used in various industries.In addition to medicinal
properties they play an important role in the ecological
niches of forest ecosystem i.e., integral part of biogeo-
chemical cycling of minerals; also they help as ecologi-
cal indicators of pollution, (Srivastava and Paul, 2016;
Bergenon and Pellerin, 2014).
Among the vascular land plants there are some 60
to70 species of ferns and fern allies exhibit some degree
of vegetative desiccation tolerance. Oxidative stress is
a major hazard of desiccation which inturn leads to the
formation of ROS (Reactive Oxygen Species). Most of
the studies in ferns indicate that enzyme conservation
may be important in the physiological reactivation pro-
cess; de novo synthesis of some enzymes during rehy-
dration also contributes to full physiological recovery
in these plants. Through the years of evolution and cli-
matic changes, this plant group showed a resistance and
persistence in maintaining its diversity in the earth. The
stress tolerance capacity of these plants is the reason for
its established biodiversity in the earth (Pereira et al.,
2014; Scoffoni et al., 2014; Maia et al., 2014)
The primary response of an organism to an oxida-
tive stress condition includes the activation of antioxi-
dant enzymes, and the use of water-soluble antioxidant
compounds and lipid-soluble antioxidant molecules.
Antioxidant enzymes include catalase (CAT) and ascor-
bate peroxidase (AP), which are able to reduce H
2
O
2
to
water, and peroxiredoxins (PRXs), expressed mainly in
the organelles and involved in detoxi cation of H
2
O
2
and alkyl hydroperoxides. Antioxidant mechanisms are
present in all living organisms, and have been studied
with particular emphasis in host–pathogen interactions
and to clarify how organisms cope with extreme envi-
ronmental conditions. Rabara et al., (2015) reported that
when water uptake and loss cannot keep balance by pri-
mary adaptive responses, different drought mechanisms
through abscisic acid and other signaling pathways, may
be exploited to avoid and/or tolerate dehydration plants.
Similarly, various genes that function as stress sensors
in signaling transduction pathways, which comprise a
network of protein-protein reactions, transcription fac-
tors (TFs) and promoters, are activated in Arabidopsis
and other plants (Liu et al., 2016).
In this scenario, the present study was undertaken to
evaluate the stress tolerance mechanism in terms of des-
iccation tolerant capacity of a fern Adiantum latifolium
Lam. Commonly known as the maidenhair fern.
MATERIAL AND METHODS
PLANT MATERIAL
The plant material Adiantumlatifolium collected from
the KattakadaTaluk of Trivandrum district, Kerala, was
used for the present study. It was identi ed using the
standard ora of Manickam and Irudayaraj (1992). The
identi cation of the fern was further con rmed by her-
baria of University of Calicut and a voucher specimen
was kept intheDepartment herbarium (CCB 047).
DESICCATION TREATMENT
The fresh and mature sporophyte of Adiantaum latifo-
liumcleaned carefully and the samples were desiccated
in a desiccators over polyethylene glycol (PEG) in a con-
trolled environment chamber (Fig.2 a & b) . The selected
plants were subjected to three different desiccation
regimes like 1D (24 h), 2D (48h) and 3D (72h) (Fig.3 a, b
& c). After desiccation a set of desiccated samples were
subjected to rehydration for 30 min. Thus the samples
were divided into two groups namely desiccated and
desiccated subsequently rehydrated. Control plants were
maintained in an optimal water conditions in each case
during the whole experimental period.
ESTIMATION OF RELATIVE WATER CONTENT
(RWC)
The water status at each desiccated stage was calculated
as relative water content (RWC).
(FW-Fresh Weight, DW - Dry Weight)
QUANTIFICATION OF PHENOL
Total phenol content was estimated by the method of
Mayr et al. (1995).
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Lubaina, Brijithlal and Murugan
REVERSE PHASE HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (RP-HPLC) OF PHENOLIC
ACIDS
Quantitative fractionation of various phenolic acids in
the samples was studied by RP-HPLC analysis. Phenolic
acids extracted from fresh and desiccated tissues in
aqueous methanol were used for the study.
PREPARATION OF THE SAMPLE
1g leaf tissue was re uxed in boiling 80% methanol for
10min. The tissue was homogenized, ltered through
cheesecloth and centrifuged at 15000 rpm for 10 min.
The resultant supernatant was made up to 5 ml with
80% methanol and used for RP-HPLC analysis.
PROCEDURE OF RP-HPLC
A modi ed method of Beta et al. (1999) was followed
for HPLC analysis. An HPLC system (Waters Associates)
equipped with a 7725 Rheodyne injector and Waters
510 HPLC pump, 486 tunable absorbance detector and
Millennium 2010 software data module were used for
the study. An HPLC column of 4.6x250 mm id reverse
phase (RP) C8 was used for the fractionation of phe-
nolic acids. Potassium hydrogen phosphate and acetoni-
trile in a ratio of 75:25 was used as the mobile phase
for the isocratic elution. An elution period of 20 min
with a ow rate of 0.8 ml min
-1
was given.10µl of the
sample was injected and the absorbance at 254 nm was
recorded. Standard phenolic acids such as gallic, vanil-
lic, p-hydroxybenzoic, ferulic, chlorogenic, sinapic, par-
acoumarate and cinnamic acids were injected in to the
column separately. Comparing with the retention time
of the standard identi ed phenolic acids in the sample.
Height of the peaks was taken for quanti cation. Con-
centration of the standard and height of the standard
peak were taken as the standard parameters.
QUANTIFICATION OF HYDROGEN
PEROXIDE (H
2
O
2
)
H
2
O
2
concentration of the experimental tissues was
estimated as per the procedure of Bellincampi et al.
(2000).
LOCALIZATION OF HYDROGEN
PEROXIDE (H
2
O
2
)
Histochemical localization of H
2
O
2
was done by staining
the tissues with Tetramethylbenzidine (TMB) reagent, as
per the method of RosBarcelo (1998).
QUANTIFICATION OF LIPID PEROXIDATION
(LPX)
The level of lipid peroxidation in the cells was measured
in terms of malondialdehyde (MDA) content determined
by the thiobarbituric acid (TBA) reaction as described by
Zhang and Kirkham (1996).
ISOLATION AND ASSAY OF PHENYLALANINE
AMMONIA LYASE (PAL)
AND POLYPHENOL
OXIDASE (PPO)
Isolation of PAL was made by the method of Morrison
et al. (1994) and the activity of PAL was estimated by the
method of Whetten and Sederoff (1992).Isolation and
the assayof PPO determined as per the method of Mayr
et al.(1965).
STATISTICAL ANALYSES
The values were mean of six independent analysis ±
SD and signi cance of the differences in all parameters
tested was determined by two-way analysis of variance
(ANOVA).
RESULTS AND DISCUSSION
Pteridophytes gain water in their cells both through
external (ectohydric) capillary movement and internal
(endohydric) transport. When fully hydrated, their water
content is very high, attained several times more than
their dry mass. The relative water content of control and
76 h desiccated fern were 80% and 31% respectively
(Table 1). Unlike other plants, water content of ferns is
highly related to environmental conditions and weakly
regulated by their internal and morphological structures.
Alpert and Oechel (1987) found that those species that
occurred in microsites with lower water availability were
able to attain maximum net photosynthetic gain at lower
Table 1: In uence of desiccation
(24, 48 and 72h) stress on the levels
of relative water content (RWC) % in
Adiantum latifolium. Values are mean
±SD of six independent replications
Stages of desiccation RWC (%)
Control 80±0.23
24hour desiccated 62±0.81
48 hour desiccated 48±0.19
72hour desiccated 31±0.34
674 DESICCATION TOLERANCE IN THE FERN BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Lubaina, Brijithlal and Murugan
water content and to recover better from prolonged des-
iccation than those taxa in less xeric microsites.
The total phenolic content in the tissues varied
among the experimental ferns. After desiccation (72h),
the total phenols increased by 5 fold; following rehydra-
tion it was decreased substantially than the value found
in desiccated phase (Table 2). The level of phenols may
be supported by the activity pro le of phenylalanine
ammonia lyase (PAL) during desiccation. The waxing
and waning pattern of phenols was further investigated
by fractionating the phenols by reverse phase high per-
formance liquid chromatography (RP-HPLC). The gures
1a and b represent the HPLC chromatogram of phenolic
extracts from leaf tissues of A. latifolium with control
and desiccated conditions.
Phenolic acids such as cinnamic acid, caffeic acid,
ferulic acid, sinapic acid, coumaric acid, hydroxy ben-
zoic acid, chlorogenic acid, gallic acid and vanillic acid
were used as standards for detecting the compounds.
It is evident from the gures 1a and b that phenolic
extracts of the samples, contain the peaks of most of
the standards, indicating the functional compartmen-
tation of phenolic acids during desiccation when com-
pared with the control. The major phenolic acid noticed
in the plant under control and desiccated condition was
represented in the table 3. Interestingly, the desiccated
fern shows signi cant pro le of phenolic acids such
as sinapicacid(4105.56g/g), ferullate (5136.73g/g),
pholorogucinol (3006.56g/g),cinnamate (617.413 g/g),
gallate(529.21 g/g), vanillate (617.413 g/g),catechol
(705.615g/g) and hydroxyl benzoic acid(3301.59g/g).
The antioxidant activity of phenolic acids is related to
the acid moiety and the number and relative positions of
hydroxyl groups on the aromatic ring structure. Hydrox-
ycinnamic acids are more effective antioxidants than
hydroxybenzoic acids due to increased possibilities for
delocalization of the phenoxyl radical. Hydroxylation
in the 2- and 4-positions or in the 3-, 4- and 5-posi-
tions confers the greatest antioxidant activity. Adjacent
hydroxyl groups, as found in protocatechuic acid, are
less favourable for antioxidant activity than those meta-
orientated with respect to each other.
Higher plants respond to various stresses by activat-
ing secondary metabolic pathways such as phenyl pro-
panoid pathway (Zadworna and Zenkteler, 2006) which
plays an important role in plant desiccation. Browning
Table 2: Hydrogen peroxide (H2O2), lipid peroxidation (LPX) and total phenolic content in Adiantum
latifolium subjected to desiccation (24, 48 and 72hours) and rehydration. Values are mean ±SD of three
independent replications
Name of the
assay
Control 24D 24R 48D 48R 72D 72R
H
2
O
2
(µmolg
-1
DW) 3.9 ±0.53 5.64±0.89 4.85±0.24 11.03±0.71 7.54±0.59 16.04±0.53
9.35±0.82
LPX (µmolg
-1
DW) 3.41±0.23 6.87±0.15 7.65±0.54 8.82±0.48 6.73±0.35 12.91±0.49
7.19±0.98
Total Phenol
(mg g
-1
tissue)
3.2 ±0.21 5.4 ±0.35 4.1±0.42 11.6 ±0.98 6.2±0.87 15.9 ±0.76
10.2±0.57
D-Desiccated R-Rehydrated
Table 3: Phenolic acid pro le of the control and desiccated A. latifolium.
Control Desiccated
Retention
time(minutes)
Amount
(μg/g tissue)
Retention
time(minutes)
Amount
(μg/g tissue)
Catechol 3.66 640.03 3.656 705.615
Cinnamic Acid 3.663 560.03 3.656 617.413
Gallate 3.663 480.02 3.656 529.29
Vanillate 3.663 560.027 3.656 617.413
Sinapic Acid 4.284 4043.30 4.296 4105.56
Ferullate 4.310 4877.08 4.602 5136.73
Hydroxybenzoic Acid 5.284 2339.99 5.350 3301.589
Pholorogucinol 4.284 2557.85 4.296 3006.56
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS DESICCATION TOLERANCE IN THE FERN 675
Lubaina, Brijithlal and Murugan
of plant tissue probably connected with phenolic accu-
mulation, is a mor phological symptom of drought stress
( Hossain et al., 2013a and Moharramnejad et al., 2015 ).
The total phenolic content in the desiccated fronds
showed signi cant increase. It is possible to suggest
that negative impact of desiccation stress was reduced
by total phenols. Increasing evidences indicate that
phenols enhanced water stress tolerance via the induc-
tion of antioxidant defence systems in the plants (Jiang
and Zhang, 2004). Moreover, Oliver and Bewley (1997)
stated that vascular plants, even belonging to res-
urrection, could survive desiccation, only when they are
dehydrated slowly. Longer rapid desiccation could cause
even irreparable damages as suggested by Proctor et al.,
(2007a) would allow insuf cient time for cytological
osmotic adjustment, which is critical for plant survival
during water stress. In all the fronds, after rehydration
following desiccation, phenolic levels decreased, but not
below control values. It seems that rehydration was too
quick to put in motion the cascade of protection mecha-
nisms in them (Nadernejad et al., 2013; Solti et al., 2014)
H
2
O
2
, the reactive oxygen species (ROS) is derived
from the O
2
•−
by dismutation reaction. In the present
study H
2
O
2
was quanti ed in the fronds of control plants
and experimentals. Table 2 demonstrates the H
2
O
2
con-
tent accumulated in the desiccated and rehydrated tis-
sues of A. latifolium. From the data it is clear that, the
desiccated tissues showed higher deposition of H
2
O
2
than
the control. The assay data of H
2
O
2
in the desiccated tis-
sues corroborates with the deep blue colour deposits in
the histochemical analysis (Fig.2 a, b, c and d). Apart
from the synthesis of H
2
O
2
in cell system as a normal
metabolic process, the over production of H
2
O
2
in the
tissues of desiccated plants is possibly a manifestation
of oxidative stress. It can be postulated that H
2
O
2
play
a central role in plant as a signaling cascade or toler-
ance to stress (Bhattacharjee 2013; Sathiyaraj et al.,
2014).
Irrespective of the comparison regarding the depo-
sition of ROS-H
2
O
2
between desiccated and control the
higher accumulation of ROS remains as a physiological
impact of the pteridophytes against desiccation stress.
The level of H
2
O
2
formation in the pteridophytes can be
considered as an indicator of magnitude of abiotic stress
in the species. Thus the results clearly indicates physi-
ological impairedness of plants, due to this oxidative
burst caused by the higher level of H
2
O
2
production and
it also highlights the reaction response of the pterido-
phytes against abiotic stress. The role of abiotic stress
response in the formation of H
2
O
2
in plant tissue has
FIGURE 1. RP-HPLC chromatograms showing the peaks of phenolic acids in control and
desiccated Adiantumlatifolium respectively.
676 DESICCATION TOLERANCE IN THE FERN BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Lubaina, Brijithlal and Murugan
FIGURE 2. Histochemicallocalization of H
2
O
2
(a) Contorl (b) 24 hour desiccated (c) 48 hour
desiccated (d) 72 hour desiccated A. atifolium leaves.
Table 4: Phenylalanine ammonia lyase (PAL) and poly phenol oxidase (PPO) content in Adiantum
latifolium subjected to desiccation (24, 48 and 72h) and rehydration. Values are mean ±SD of three
independent replications
Name of the
assay
Control 24D 24R 48D 48R 72D 72R
PAL (Umg-1 protein) 37±0.62 59±1.23 48±1.01 78±1.48 54±0.98 96±0.89 63±1.16
PPO (Umg-1 protein) 3.25±0.14 10.79±0.98 6.89±0.36 6.81±0.28 3.77±0.13 2.48±0.11 1.23±0.05
been conclusively proved by several studies (Pukacka
and Ratajczak, 2006).
The present data conclusively proved the excess gen-
eration of H
2
O
2
in the species as a desiccation effect. As a
constitutive metabolite of cell wall for lignin formation,
the presence of H
2
O
2
on cell system of plants is a com-
mon physiological feature. No lignin content and the
tendency of plant cell to accumulate H
2
O
2
in the exces-
sive level, requires further analysis at biochemical level,
for detecting the role of the marker enzyme involved in
the process (Wang et al., 2014a).
Accumulation of H
2
O
2
concomitant with lipid peroxi-
dation resulted in signi cant decrease in cell membrane
stability. It has been suggested that decrease in cell
membrane stability re ects the extent of lipid peroxida-
tion caused by ROS. Dat et al., (1998) reported that by
heat acclimation pre-treatment, accumulation of H
2
O
2
in plants can be reduced, compared with those without
heat acclimation pre-treatment, which may be indicative
of an enhanced antioxidant potential in leaf tissues and
would contribute to enhance thermo tolerance. Desic-
cation stress injury may involve different mechanisms,
depending on environmental conditions, before or dur-
ing the dehydration stress. Genetic differences may also
be important for elevated levels of H
2
O
2
contributing to
stress injury in plants.
Effect of desiccation stress on plant tissues were
determined in terms of membrane lipid peroxidation, i.e.,
estimated as the content of thiobarbituric acid-reactive
substances (TBARS). Increase in membrane lipid peroxi-
dation was seen in all the desiccated fronds but at a slow
pace right from 24 hour onwards, when compared with
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS DESICCATION TOLERANCE IN THE FERN 677
Lubaina, Brijithlal and Murugan
control plants (Table 2). Within the experimental period
of 72 hour, it increased to a maximum of 12.91 µmolg
-1
DW. Thus, desiccation appeared to cause some damage
in membrane con guration, but this was reversed and or
repaired on rehydration to control values suggesting the
active phase of antioxidant machinery in scavenging the
ROS in the system.
Sairam et al., (2002) reported that, osmotic stress
causes severe damage to the membrane integrity of
various tissue types in different plant species. Moreover,
membrane integrity is frequently correlated with plant
tolerance to stress such as heat, salt, drought and other
osmotic stress. Lipid peroxidation is an effective indica-
tor of cellular oxidative damage, and was estimated by
the levels of TBARS. The observed increase in TBARS
concentration in stressed plants, might indicate lipid
peroxidation of cell membrane components, caused by
ROS generated by the oxidative stress (Sairam et al.,
2002), i.e., LPX data strongly corroborates with the level
of H
2
O
2
content in the species (Zhou et al., 2014)
The key enzyme of phenyl propanoid metabolism
known as phenylalanine ammonia lyase (PAL) was iso-
lated from the control, desiccated and rehydrated fronds
of A. latifolium and assayed spectrophotometrically in
order to ascertain their role in secondary metabolite ini-
tiation. Being the initial enzyme of PPM, the presence of
PAL, in plant tissue becomes an indispensable part for
the synthesis of phytoalexins, lignin and phenolics thus
involved in the defence response of plant cells (Morrison
et al., 1994). Thus, PAL has been generally recognized as
a marker of environmental stress in different plant spe-
cies (MacDonald and D’ Cunha, 2007).
Effects of desiccation on PAL activity in A.latifolium
was shown in Table 4.A sig ni cant increase in PAL
activity was observed in the fern at all tested desicca-
tion periods, and the highest activity of 96mg
-1
pro-
teins observed at the 72 h desiccation period compared
to the control. In few studies, PAL activity was induced
by various biotic and abiotic stresses, such as wounding,
chilling, heavy metal and infection by viruses, bacteria
or fungi (MacDonald and D’Cunha, 2007). The responses
of PAL activity subjected to desiccation could be a pro-
tective response to the cellular damage provoked by des-
iccation stress (John et al., 1971). The results suggest
that changes in activity of enzyme strongly depend on
desiccation periods (Huang et al., 2015)
D-DESICCATED R-REHYDRATED
PPOs are copper containing metalloenzymes that cata-
lyze the oxidation of hydroxy phenols to their quinone
derivatives. Polyphenol oxidase (PPO) showed varied
expression in the desiccated fern compared to the con-
trol. PPO activity decreased from the 1
st
to 3
rd
day after
desiccation i.e., 10.79±0.98 and 2.48±0.11respectively
(Table 4). Initially the activity of endogenous polyphenol
oxidase was higher in desiccated and rehydrated samples
followed by a decrease in subsequent days of desiccation
and rehydration (Table 4). Sofo et al., (2005) reported
that a decrease in PPO activity following abiotic stress
was associated with improved antioxidant capacity. The
suppression of PPO increased the drought tolerance in
tomato plants (Thipyapong et al., 2004a).
CONCLUSION
The results obtained in this study demonstrate that des-
iccation treatment from 24 to 72 h duration, in Adian-
tum latifolium, generated an oxidative stress condition,
and morphological and biochemical alterations. The
activation of different biochemical mechanisms helps to
explain the high tolerance to desiccation of this species.
In this context, in vitro rehydration experiments dem-
onstrated the rapid capacity of the species to recover
from oxidative stress, and provide the functional basis
that helps to explain, at least in part, the position of this
species at the highest tropical zones.
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