OCE 2001 USF Coastal Ecology Virtual Field Trip Report

OCE2001.650U21 Introduction to OceanographyCoastal Ecology Virtual Field Trip Assignment (70 points)
Start by watching the Coastal Ecology Field Trip videos, which can be found at the link below:
http://usfweb.usf.edu/courseresources/ms/oce2001/videos/coastal-ecology/index.html
15 pts
1) Mangroves are salt tolerant trees. List the three types of mangroves found in Tampa Bay that are
discussed in the videos, and describe the anatomical differences mentioned in the videos. Dig deeperusing the review article provided (Parida and Jha 2010), discuss physiological adaptations in mangroves
for water uptake and water conservation. Finally, discuss the three salt-eliminating mechanisms
mangroves use to live in saltwater, as listed in the article.
10 pts
2) Mangroves and seagrasses provide similar benefits to the ecosystem. Discuss at least three benefits
of mangroves and seagrasses.
15 pts
3) Discuss the difference in camouflage techniques of needlefish and toadfish discussed in the video.
What does the camouflage technique used by each fish tell you about where it lives? Looking at the
cartoons below, what does the anatomy of each fish tell you about how it swims and how it feeds? Be
sure to answer these questions for both fish, needlefish and toadfish.
10 pts
4) Below is a figure depicting the life history of a gag (species of grouper). Their life history is complex,
involving many different habitats, including inshore seagrasses as nursery habitat. Dig deeper- using the
article provided (Switzer et al. 2012), discuss characteristics of the habitat that the authors found
juvenile gags prefer (i.e. salinity and vegetation type and coverage). How are these habitats beneficial to
gags and other juvenile fishes? How could the loss or decline of seagrasses impact local fisheries?
10 pts
5) Provide two characteristics that are different between the mangrove sediment core and the lowenergy beach sediment core. Describe two other differences in these two habitats from observing the
videos in each location. Which one is a better habitat for most juvenile organisms? Why?
10 pts
6) Seagrass coverage in Tampa Bay was dangerously low, down by approximately 80%, a few decades
ago. Name two factors that contributed to this decline. Describe three changes in Tampa Bay that have
helped to restore seagrass habitats. What is the current state of seagrass coverage in Tampa Bay? There
are a variety of reputable references on the subject so look here for the answers— websites by Tampa
Bay Watch, Florida Fish and Wildlife, NOAA, and USGS.
Trees (2010) 24:199–217
DOI 10.1007/s00468-010-0417-x
REVIEW
Salt tolerance mechanisms in mangroves: a review
Asish Kumar Parida • Bhavanath Jha
Received: 15 April 2009 / Revised: 19 December 2009 / Accepted: 20 January 2010 / Published online: 11 February 2010
Ó Springer-Verlag 2010
Abstract Mangroves are woody plants which form the
dominant vegetation in tidal, saline wetlands along tropical
and subtropical coasts. The current knowledge concerning
the most striking feature of mangroves i.e., their unique
ability to tolerate high salinity is summarized in the present
review. In this review, we shall discuss recent studies that
have focused on morphological, anatomical, physiological,
biochemical, molecular and genetic attributes associated
with the response to salinity, some of which presumably
function to mediate salt tolerance in the mangroves. Here
we shall also review the major advances recently made at
both the genetic and the genomic levels in mangroves.
Salinity tolerance in mangroves depends on a range of
adaptations, including ion compartmentation, osmoregulation, selective transport and uptake of ions, maintenance of
a balance between the supply of ions to the shoot, and
capacity to accommodate the salt influx. The tolerance of
mangroves to a high saline environment is also tightly
linked to the regulation of gene expression. By integrating
the information from mangroves and performing comparisons among species of mangroves and non-mangroves, we
could give a general picture of salt tolerance mechanisms
of mangroves, thus providing a new avenue for development of salt tolerance in crop plants through effective
breeding strategies and genetic engineering techniques.
Communicated by U. Lüttge.
A. K. Parida (&)
B. Jha
Discipline of Marine Biotechnology and Ecology, Central Salt
and Marine Chemicals Research Institute, Council of Scientific
and Industrial Research (CSIR), G. B. Marg, Bhavnagar 364002,
Gujarat, India
e-mail: asishparida@csmcri.org
Keywords Antioxidant
Compatible solutes

Mangrove
Na?/H? antiporter
Salt tolerance

Salt secretor
Salt exclusion
Propagules
Viviparous
Introduction
True mangroves and mangrove associates
Mangroves are constituent plants of tropical intertidal
forest community. They include woody trees and shrubs,
which flourish in the zone between land and sea along the
tropical coastline of the globe. They fall into two groups
according to their habitats in nature: true mangroves and
mangrove associates. True mangroves occur only in mangrove habitat and their existence is rare elsewhere such as
Rhizophora, Kandelia, Ceriops, Bruguiera, Avicennia,
Xylocarpus, Aegiceras, Sonneratia, Laguncularia, Lumnitzera, Nypha etc. Mangrove associates are non-exclusive
mangrove species occurring in the landward margin of
mangal and often non-mangal habitats such as rainforest,
salt marsh or lowland freshwater swamps (e.g. Excoecaria,
Camptostemon, Pemphis, Osbornia, Pelliciera, Aegialitis,
Acrostichum, Scyphiphora, Heritiera etc.). Heritiera fomes
is a true mangrove with tolerance to high-saline conditions
(Santisuk 1983; Naskar and Mandal 1999), but the status of
H. littoralis as mangrove is controversial. The species H.
littoralis has been described by most of the authors as fresh
water loving back mangal or mangrove associate (Kartawinata et al. 1979; Mukherjee et al. 2003), whereas a few
authors have described it as true mangrove (Das et al.
1994; Tam et al. 1997; Parani et al. 1998). Mangroves are
taxonomically diverse. True mangroves include about 54
species in 20 genera belonging to 16 families (Hogarth
1999). Mangroves grow in the intertidal zone between land
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and sea. They are frequently inundated by tide leading to
waterlogging and fluctuation in salinity (Naidoo et al.
2002; Sengupta and Chaudhuri 2002; Paliyavuth et al.
2004; Jagtap and Nagle 2007). Like other marine organisms, they are exposed to the air and thus to the risk of
desiccation and overheating; on the other hand they face
waterlogging and salinity (Naidoo et al. 2002; Sengupta
and Chaudhuri 2002; Paliyavuth et al. 2004; Jagtap and
Nagle 2007). Under high temperature conditions in tropics
the above problems become worse. Firstly, at low tide,
overheating and desiccation is greater, and secondly,
through evapotranspiration, any water that remains may
become even more highly saline than that of the open sea.
At high tide, the warmth of water lowers the oxygen in
water (Hogarth 1999). High salinity makes it more difficult
for mangroves to extract water from the soil, even though
the soils on which mangroves grow are usually waterlogged. Consequently, many mangrove species have morphological characteristics and high water-use efficiencies
characteristic of terrestrial xerophytes (Clough et al. 1982;
Ball 1988a; Clough and Sim 1989).
Economic and ecological importance of mangroves
The mangrove forests have enormous economic potentiality and utilitarian value at the ecosystem as well as at the
component levels (Robertson and Alongi 1992; UNEPWCMC 2006). Mangroves have been playing a significant
role in the economy of tropical societies for thousands of
years, providing a wide variety of goods and services
including wood production, support for commercial and
subsistence fisheries, salt production and shoreline protection and coastal erosion control (Hamilton and Murphy
1988; Krauss et al. 2008). Mangrove trees are good sources
of firewood for local communities; their wood makes a
superior kind of charcoal and is a source of tannins, resins,
medicines, etc. (Hamilton and Murphy 1988; Baconguis
and Mauricio 1991). Many of the animals found within the
mangal are harvested for food: fish, crabs and prawns in
particular. Therefore, mangrove trees are very important
for conserving and maintaining mangrove ecosystems.
Mangrove ecosystems constitute a refuge, feeding ground
and nursery for species of cyanobacteria, red algae,
manglicolous fungi, invertebrates, fish and shrimp
(Martosubroto and Naamin 1977; Sheridan 1991; Turner
1992; Sasekumar et al. 1992; Hyde and Lee 1995; De Graaf
and Xuan 1999). Reptiles, including crocodiles, alligators,
lizards, snakes and sea turtles also live in many mangroves.
Mangroves provide important nesting sites for land birds,
shorebirds and waterfowl, and they are home to a number
of threatened species including spoonbills, large snowy
egrets, scarlet ibis, fish hawks, royal terns, West Indian
whistling-ducks, and Storm’s Storks (Staus 1998).
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A variety of mammals make their homes in the mangal by
association with the mangroves. Some of the noteworthy
species present include dolphins, crab-eating monkeys,
proboscis monkeys, fishing cats, mangrove monkeys and
otters in India (Gopal and Krishnamurthy 1993), flying fox
in northern Australia (Loughland 1998) and capuchin in
Brazil (Fernandes 1991).
Exploitation of mangroves
Finally, mangroves are currently threatened by many different human activities. Exploitation is often taken beyond
the level of natural replacement. Inland irrigation schemes
divert river water from coastal regions and mangroves
suffer from the resulting increase in salinity. Pollution
takes its toll. Deliberate clearance for the development of
aqua-culture takes place without consideration of what may
be lost. Mangroves are likely to be one of the first ecosystems to be affected by global changes because of their
location at the interface between land and sea. It has been
predicted that as sea level rises, accompanying the rapid
climate changes (Pernetta 1993; Field 1995; Ellison and
Farnsworth 1997; Das et al. 2002; Kim et al. 2005; Jagtap
and Nagle 2007; Gilman et al. 2008), mangroves tend to
retreat landwards. And, of course, any significant rise in sea
level resulting from global warming will mean encroachment on the relatively narrow zone within which mangroves flourish (Pernetta 1993; Das et al. 2002; Kim et al.
2005; Jagtap and Nagle 2007; Gilman et al. 2008).
Destruction of mangrove habitats means a loss of the
mangrove resource: as mangroves decline, so too do timber
and charcoal production and fisheries, and the livelihood of
the people who depend on them. This seems almost too
obvious to need pointing out. The productivity and diversity of mangroves as well as their considerable social and
economic values therefore make them of great interest to
biologists and ecologists to understand their significance.
Mangroves and salt tolerance
Salt tolerance is the ability of plants to grow and complete
their life cycle on a substrate that contains high concentrations of soluble salt. Plants that can survive on high
concentrations of salt in the rhizosphere and grow well are
called halophytes. Depending on their salt-tolerating
capacity, halophytes are either obligate, and characterized
by low morphological and taxonomical diversity with relative growth rates increasing up to 50% seawater or facultative and found in less saline habitats along the border
between saline and non-saline upland and characterized by
broader physiological diversity which enables them to cope
with saline and non saline conditions. Mangroves are facultative halophytes tolerant to both high and fluctuating
Trees (2010) 24:199–217
salinity. Some authors have also categorized mangroves
under obligate halophytes (Downton 1982; Clough 1984).
Several mangrove tree species reach an optimum growth at
salinities of 5–25% of standard seawater (Downton 1982;
Clough 1984; Ball 1988a; Burchett et al. 1989; Ball and
Pidsley 1995). However, the range of salinity in which the
plant is able to survive varies according to the species (Ball
1988a); in several species growth may be affected by either
absence of or excess of NaCl in the substrate (Downton
1982; Clough 1984; Burchett et al. 1989; Pezeshki et al.
1990; Ball and Pidsley 1995). Because mangroves successfully live in high salinity environments it is advantageous to use them to study the mechanisms by which plants
respond and adapt to these environments. Mangrove
research has advanced considerably in the last few years,
and the time seems right for an attempt to present our
current understanding of the mechanisms of salt tolerance
in mangroves. Although voluminous works are available
on salt tolerance mechanism taking the model plant Arabidopsis thaliana (Apse et al. 1999; Nanjo et al. 1999;
Quesada et al. 2000; Shi et al. 2000; Liu et al. 2000; Zhu
2000; Elphick et al. 2001), facultative model halophyte
Mesembryanthemum crystallinum (Ratajczak et al. 1994;
Low et al. 1996; Su et al. 2001; Golldack and Deitz 2001;
Agarie et al. 2007), the information on morphological,
anatomical, physiological, biochemical and molecular
basis of salt tolerance mechanisms in mangroves which are
facultative halophytes and potential stress adaptor
(Downton 1982; Clough 1984) will throw new light and
give a new dimension to salt stress research. Understanding
the mechanisms of salt tolerance in mangroves and identification of salt tolerant genes from mangroves will lead to
effective means to breed or genetically engineer salt tolerant crops.
Mechanisms of salt tolerance in mangroves
The ecological success of the mangroves under harsh
conditions is explained by several morphological, anatomical, physiological, biochemical and molecular features. Mangroves develop a plethora of mechanisms to
cope with salt stress that are discussed under following
headings.
Morphological and anatomical features
Mangroves grow in a soil that is more or less waterlogged
and in water whose salinity fluctuates and may be as high
as that of the open sea (Naidoo et al. 1997). Mangrove trees
adapt to survive such uncompromising surroundings with
several morphological features such as salt-excreting
leaves, and viviparous water dispersed propagules. In
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response to salinity where water economy is stringent,
leaves tend to be smaller and thicker in mangroves (Ball
1988a). A small leaf design enhances cooling as small
leaves lose more heat by convection than do large ones.
There are interspecific differences in salt tolerance among
the mangroves (Ball and Pidsley 1995). Ball and Pidsley
(1995) examined the effects of soil salinity on the growth
of two closely related species, Sonneratia alba and S.
lanceolata in relation to their differential distributions
along naturally seasonal salinity gradients. They showed
that there were interspecific differences in salt tolerance
which were founded on the inherent growth characteristics
of the two species. In fact, these species showed a neat
trade-off between growth and salt tolerance with S.
lanceolata growing in salinities of up to 50% that of seawater while S. alba can grow in 100% seawater. Both the
species, however, showed optimal growth in culture at 5%
seawater. Nevertheless, at optimal salinity the growth of
more salt tolerant species S. alba, measured as biomass,
height and leaf area, is less than half that of the less salt
tolerant species S. lanceolata, indicating that S. lanceolata
will be the successful competitor even at a salinity that is
optimal for both the species. On the other hand, S.
lanceolata could not grow or compete in high salinities. In
this species pair, a species can apparently opt for salt tolerance, or for rapid growth and competitive ability under
low salinity conditions, not both (Ball and Pidsley 1995).
The root-to-shoot ratio becomes relatively high and
increases with increasing concentration of salinity in Avicennia and Aegiceras (Ball 1988b; Saintilan 1997). In
addition, vivipary is also a characteristic that might have
some role to tolerate high salinity in mangroves (Zheng
et al. 1999). Vivipary is the condition found in some species of mangroves in which the sexually produced embryo
of the seed continues its development without dormancy
into a seedling, while still attached to the mother plant
(Elmqvist and Cox 1996). Vivipary is known in the four
genera of mangroves such as Bruguiera, Kandelia, Rhizophora and Ceriops of Rhizophoraceae (Tomlinson 1986).
The seedling develops without dormancy largely by elongation of the hypocotyl to produce a cigar-shaped seedling
known as propagule, which remains conspicuously pendulous on the mother tree for several months (Tomlinson
and Cox 2000). The viviparous condition is so strongly
associated with mangroves that it is suggested to have
adaptive significance in the intertidal environment (Tomlinson 1986). It was assumed that vivipary in mangroves
may be an adaptive characteristic permitting avoidance of
high salinity at germination (Henkel 1979). Some evidence
has proved that developing viviparous propagules (i.e.
hypocotyls) in Rhizophoraceae retain lower salt concentrations than in other organs of mother trees especially the
leaves, but it increases gradually during the process of
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propagule maturation (Wang et al. 2002). This lowered salt
concentration in developing propagules, whatever its origin, is interpreted as a mechanism to ‘‘protect’’ the embryo
from the deleterious effects of high salt concentrations until
maturity (Hogarth 1999). Nevertheless, a few authors have
argued that the adaptation of developing propagules
(hypocotyls) to salt originates when it is still attached to the
mother tree by continuously absorbing salt from the mother
tree (Lin 1988). After following the element concentrations
on a dry weight basis, Zheng et al. (1999) concluded that
the development of propagules was not a salt accumulation
process, but a desalinating process, because salt concentration on a dry weight basis declined with propagule
development. If it were truly a desalinating process, how
can the propagule keep the osmotic balance and by what
means can the propagule cope with the salt stress and
osmotic stress after the propagule settles down in the hyposaline environment? Little research has dealt with the
changes after a propagule leaves the mother plant (Tomlinson and Cox 2000). Wang et al. (2002) reported that no
direct correlation was found between salt tolerance and
vivipary. All features mentioned above reveal higher
competence of salt tolerance of mangroves in terms of
morphology.
Mangroves develop diverse mechanisms associated with
anatomic characteristics in order to tolerate high salinity.
Mangrove species usually have salt resistant-associated
anatomic structures. Many species of mangrove possess
salt glands in their leaves. All species in genus Aegiceras,
Avicennia, Acanthus and Aegialitis have typical salt gland
structures, and species in Laguncularia and Conocarpus
have analogous structure to salt glands (Tomlinson 1986).
In L. racemosa and C. erectus ‘‘tiny bumps’’ or ‘‘pimples’’
like structures are found on the petioles of the leaves that
somewhat resemble salt glands (Tomlinson 1986). However, the salt secretory ability of these structures has not
been precisely demonstrated. Biebl and Kinzel (1965)
describe three morphologically different structures in the
leaves of L. racemosa, the smallest of which sit in deep,
irregular epidermal depressions and extrude chains of salt
crystals. However, Kemis (1984) raised specimens of
Conocarpus and observed them to secrete clear, sweettasting nectar. His observations and electron microscope
studies led him to conclude that the ‘‘pimples’’ are, in fact,
‘‘extra floral nectarines (EFN)’’. Kemis (1984) reported
that these extra-floral nectaries (EFN) may serve an adaptive role and used mutualistically to attract ants to defend
the tree from herbivorous pests. Kemis and Lersten (1984)
follow up the study of Conocarpus with a similar electron
microscope study of Laguncularia and conclude that, the
pimples lack a salt excretory function, but the morphology
is so different that there is ‘‘no evidence of an opening’’.
No evidence of an opening indicates that these pimples
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secrete neither salt nor nectar. They suggest a theory
regarding the differences in the L. racemosa and C. erectus
petiolar ‘‘pimples.’’ They suggest that the apparently nonfunctional nature of the pimples on Laguncularia is a
consequence of a reversion from functionality to vestigiality. It is suspected that the mythical salt glands in
Conocarpus are really the sugar glands become vestigial
remnant in Laguncularia. Saenger (2002) pointed out that
in L. racemosa, the formation of these salt glands analogous structures or pimples is found only in the presence of
salt implying a secretory function (2002). Saenger (2002)
reported that these salt glands analogous structures may be
interpreted as hydathodes, salt glands or nectaries. Leaves
of Aegiceras and Avicennia have deposits of salt crystals as
a result of salt leak from the salt glands. The lower leaf
surface of Avicennia is densely covered with hairs, which
raise the secreted droplets of salt water away from the leaf
surface, preventing the osmotic withdrawal of water from
the leaf tissues (Osborne and Berjak 1997). In species of
Rhizophora, Sonneretia, Avicennia and Xylocarpus, salt is
also deposited in the bark of stem and roots (Scholander
1968). Another typical characteristic of mangrove is the
induction of leaf succulence by thickening of leaves with
increasing water content (Suarez and Sobrado 2000). For
example, Laguncularia racemosa can increase leaf thickness and water content when stressed with high salinity
(Sobrado 2005), by which absorbed salt was diluted and
salt-induced damage was reduced to some extent. Under
the condition of high salinity, there is increase of leaf
thickness from the youngest to the oldest leaves along
shoot in L. racemosa (Werner and Stelzer 1990). Saltinduced succulence can lower the resistance to CO2 uptake
and this increases the photosynthetic rate by increasing the
internal leaf surface for gas exchange. The waxed epidermis in the leaves is also a protective trait in some mangroves, which contributes to low transpiration of mangrove
species relative to that of other plants without this trait
(Werner and Stelzer 1990). Several species deposit salt in
senescent leaves, which are then shed (Zheng et al. 1999).
This helps in removing salt from the metabolic tissues.
There is also change in leaf anatomy i.e. development of
kranz anatomy and dimorphism of chloroplast, reduction in
number of stomata per leaf area and wide opening of stomata which is a typical feature of succulence (Werner and
Stelzer 1990; Parida et al. 2004a).
In Rhizophora mangle salt-induced increase in succulence is predominantly based on the considerable expansion of the hyalinous hypodermal cells below the upper
epidermis (Werner and Stelzer 1990). The high Na? and
Cl- concentrations in hypodermal and endodermal vacuoles of Rhizophora are the result of cellular metabolism.
These vacuoles represent storage pools, supplied and/or
unloaded by membrane activity. Apparently, the outer
Trees (2010) 24:199–217
hypodermis and the endodermis represent ion regulating
steps or two sheaths of the ultrafilter, respectively. The
hypodermal cells layers of Rhizophora form a tight ring of
cells with relatively long radial cell walls. Their vacuoles
represent more than 40% of the total root volume and they
contain relatively high Na? and Cl- concentrations. The
vacuoles of these cells act as salt traps, protecting
the subsequent cell layers (Werner and Stelzer 1990). The
processes of root elongation and suberization of cell walls
are two possible other ways to avoid an invasion of these
cells by salt. Root elongation maintains the capacity for
accumulating ions by providing new cells (Stelzer et al.
1988); suberization seals the ion-invaded cells and blocks
the apoplasmic pathway (Peterson 1988; Taura et al. 1988).
The mangrove species have several morphological and
anatomical features which are quite similar to high wateruse efficiency characteristics of terrestrial xerophytes
(Clough et al. 1982; Ball 1988a; Clough and Sim 1989).
These characteristics enable the mangroves to grow in a
‘‘physiologically’’ dry (or) saline environment without
apparent adverse effects of severe water stress. The water
conserving function of mangroves is very much similar to
xerophytes (Ball 1988a). Most of the mangrove genera
have thick-walled epidermis with waxy cuticle and sunken
stomata. The waxy cuticles are covered by a tomentum of
various shaped hairs, like tricellular peltale hairs in Avicennia sp., stellate hairs in Hibiscus tiliaceous and stellate
scales in Heritiera sp. (Miller et al. 1975). This tomentumlike outgrowth reduced water loss via the stomata and the
salt gland. Sunken stomata beneath the epidermis are
prominent in Avicennia sp., Bruguiera spp., Ceriops spp.,
Lumnitzera spp. and Rhizophora spp. (Miller et al. 1975).
Sclerenchymatous cells are distributed throughout the
mangrove leaves including the epidermis (Sidhu 1975).
The presence of large-celled water-storing hypodermis
with strongly developed palisade mesophylls with small
intercellular spaces in the mangrove leaves indicate their
xerophytic characters (Sidhu 1975). The isobilateral leaves
of Aegialitis rotundifolia have no hypodermal tissue and
spongy mesophyll; on the other hand the isobilateral leaves
of Ceriops tagal have enlarged spongy mesophyll cells
both in upper and lower hypodermis (Saenger 1982). The
isobilateral leaves of Sonneratia caseolaris and Lumnitzera
racemosa also have enlarged water-storing hypodermal
cells. The dorsiventral leaves of Acanthus ilicifolius, B.
gymnorrhiza, Excoecaria agallocha and xylocarpus spp.
have several layers of hypodermal cells (Saenger 1982).
Rhizophora spp. also have marked differentiation of
hypodermis with small tannin cells and large colorless
water-storing cells. The leaves of most of the mangroves
are succulent due to the well-developed large-celled water
storing hypodermis and strongly developed palisade
mesophyll tissues with small intercellular spaces (Saenger
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1982). Several layered hypodermal aqueous tissues are
present in most of the mangroves, e.g., Avicennia sp.,
Hibiscus spp., Acrostichum spp. and single-layered hypodermal cells have also been reported in Bruguiera spp.,
Ceriops spp., Cynometra spp., Accanthus spp. and Excoecaria spp. (Saenger 1982). Large undifferentiated mesophyll cells have formed a central aqueous tissue in
Sonneratia sp. and Lumnitzera sp. (Saenger 1982). In
Aviccenia spp., Bruguiera spp. and Ceriops spp., the ends
of the vascular bundles are surrounded by irregular groups
of tracheids (Saenger 1982). The walls of the tracheids bear
spiral reticulated or pitted thickenings and the water storage function has been attributed to them (Saenger 1982).
The stone cells and sclereides have been reported from
Avicennia spp., Rhizophora spp., Sonneratia spp., Bruguiera spp., and Xylocarpus spp. (Saenger 1982). These cells
give toughness and rigidity to leaves. The mucilaginous
cells have been reported from Sonneratia spp. and Rhizophora spp. and these mucilaginous cells may be involved in
reducing damages from wilting by conserving water (Ball
1988a). The water use efficiency of glycophytes is comparatively less than that of mangroves.
Physiological and biochemical mechanisms
Some of the most salt-tolerant mangrove species grow in
an environment where tidal influence is minimized but
evaporation of water from the soil surface is high. In these
areas the concentration of salt in the soil rises to such an
extent that it becomes hypersaline (more salty than seawater, Lovelock and Feller 2003). Seawater contain 35 g/l
of salt (3.5%), 483 mM Na? and 558 mM Cl-) with an
osmotic potential of -2.5 MPa (Scholander 1968). Mangroves, therefore, have to maintain continuous water
uptake, and regulate ion uptake and compartmentation
against a strong external salt gradient (Ball 1996). To
maintain water uptake, mangroves not only have to restrict
water loss by means of conservative morphological and
physiological adaptations such as thickening of leaves
giving rise to greater retention times for leaf nitrogen and
conservative water use efficiency (Ball 1996; Lovelock and
Feller 2003) but also they need to maintain sufficiently low
water potentials. Agricultural crops under well-saturated
conditions generally have water potentials of approximately -1.0 MPa. However, as the osmotic potential of
seawater is approximately -2.5 MPa (Sperry et al. 1988),
mangrove leaf water potentials have to range between -2.5
and -6.0 MPa (Scholander et al. 1966; Aziz and Khan
2001a; Sobrado and Ewe 2006). Salinity stress causes low
stomatal conductance, which decreases the rate of CO2
accumulation and uptake, rate of transpiration and increase
in xylem tension (Ball and Farquhar 1984; Aziz and Khan
2001a; Parida et al. 2004a). The low transpiration rates and
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slow water uptake are common features for all mangroves
(Scholander et al. 1966). However, increase in transpiration
in response to increase in salinity has also been reported in
B. gymnorrhiza (Takemura et al. 2000) and in both Avicennia alba and Rhizophora apiculata (Becker et al. 1997).
This is suggested to be the aim at increasing internal salt
concentration and eventually balance the increased external
salt concentration. As water salinity increases, some species of mangroves simply become increasingly conservative in their water use, thus achieving greater tolerance
(Ball and Passioura 1995). Because mangrove roots
exclude salts when they extract water from soil, soil salts
could become very concentrated, creating strong osmotic
gradients (Passioura et al. 1992). Zimmermann et al. (1994)
reported that the xylem vessels of the roots and stems of
mangrove (Rhizophora mangle) contain high molecular
weight viscous, mucilage made up of acid polysaccharides
(mucopolysaccharides). These mucopolysaccharides in the
xylem sap are apparently involved in water transport in the
xylem conduit of R. mangle (Zimmermann et al. 1994).
Accumulation of mucilage in xylem vessels is an important
strategy of mangrove trees to save water on its tortuous
pathway to the uppermost crown (Zimmermann et al. 1994,
2002). The viscous, polymeric substances in the xylem sap
limit flow rate and decrease transpiration (Zimmermann
et al. 1994, 2002). This, combined with high water-use
efficiency, slows the rate of water uptake and prevents salts
from accumulating in the soil surrounding the roots. This
helps the mangroves conserve water and regulate internal
salt concentrations (Ball and Passioura 1995; Ball 1996).
Mangroves experience hypersaline conditions as a result
of evaporation (which raises the salinity level) and fluctuation of salinity caused by tide (Lovelock and Feller 2003).
High salinity and salinity variation pose a problem for
mangroves (Lovelock and Feller 2003). To cope with this
uncompromising situation, a number of physiological and
biochemical mechanisms are adopted by mangroves,
namely, salt excretion (Hanagata et al. 1999), salt accumulation (Popp 1984a, b; Kura-Hotta et al. 2001; Mimura
et al. 2003), salt secretion (Sobrado and Greaves 2000),
accumulation of compatible solutes (Ashihara et al. 1997)
and induction of antioxidative enzymes (Takemura et al.
2000; Parida et al. 2004b) which are discussed under the
following headings.
Salt secretion, salt exclusion and salt accumulation
Depending on their salt eliminating mechanism mangroves
and their associates have been classified into three groups:
(1) salt excluders, (2) salt secretors and (3) salt accumulators (Table 1). The salt-excluding mangrove species (e.g.
Rhizophora spp., Ceriops spp., Bruguiera spp., Lumnitzera
spp., Excoecaria spp.) eliminate excess salt by an ultrafiltration mechanism occurring at the root cell membranes of
cortical cells (Scholander 1968; Zheng et al. 1999;
Takemura et al. 2000; Aziz and Khan 2001a; Khan and
Aziz 2001; Wang et al. 2002). Though the exact mechanism of ultrafiltration is not well characterized, it is
understood that the process is a physical one. Negative
hydrostatic pressure developed in plants by transpiration is
enough to overcome negative osmotic pressure in the
environment of the roots. Water is therefore drawn in,
unwanted ions and other substances are excluded. Rhizophora mangle is not provided with salt glands, but it keeps
the xylem sap essentially free of NaCl by ultrafiltration at
the membranes of root cells (Scholander et al. 1966;
Scholander 1968). The concentration of xylem sap is about
one-tenth than that of seawater (Lawton et al. 1981). Furthermore, in B. gymnorrhiza, hypocotyls function as an
additional filter to retain salt from the shoot (Lawton et al.
1981).They maintain ionic balance through K?/Na?
exchange at the xylem parenchyma cells in the basal part of
Table 1 Mechanism of salt adaptation and their known distribution in some mangrove species
Species
Exclude Secrete Accumulate References
Acanthus
Aegialitis
?
?
Hogarth (1999); Ye et al. (2005); Nguyen et al. (2007)
?
Naidoo and von Willert (1995); Hogarth (1999)
Aegiceras
?
?
Avicennia
?
?
Bruguiera
?
Ceriops
?
Excoecaria
?
Laguncularia
Naidoo and von Willert (1995); Mishra and Das (2003); Ye et al. (2005)
?
?
Sobrado (2002); Ye et al. (2005); Suarez and Medina (2006); Griffiths et al. (2008)
Takemura et al. (2000); Kura-Hotta et al. (2001); Li et al. (2008); Miyama and Tada (2008)
Hogarth (1999); Zheng et al. (1999); Aziz and Khan (2001b)
Tomlinson (1986); Hogarth (1999)
Hogarth (1999); Sobrado (2004)
?
Osbornia
?
?
Tomlinson (1986); Hogarth (1999)
Rhizophora
?
?
Clough (1984); Werner and Stelzer (1990); Hogarth (1999)
Sonneratia
?
Xylocarpus
123
?
?
Tomlinson (1986); Hogarth (1999); Yasumoto et al. 1999
?
Hogarth (1999); Paliyavuth et al. (2004)
Trees (2010) 24:199–217
the plants and vacuolar K?/Na? exchange in transpiring
leaves and circulation of exchanged ions within the plants.
The ultrafiltration process and K?/Na? exchange contribute to keeping the xylem sap concentration of salt-treated
plants low with mean rates of net ion transport into the
shoot being even lower due to recirculation of exchanged
ions (Mallery and Teas 1984; Werner and Stelzer 1990).
Salt secretors regulate internal salt levels by secreting
excess salt through foliar glands and are represented by
Acanthus spp., Avicennia marina, Av. officinalis, Av. alba,
Aegiceras corniculatum and Aegialitis spp. (Meher-Homji
1988; Selvam 2003). Salt stimulation to secretion was a
common feature in salt-secreting mangrove species (Ball
1988a; Drennan et al. 1992; Sobrado 2002). Salt secretion
occurs through glands and it is facilitated by efficient leaf
turnover for salt shedding (Aziz and Khan 2001b). Dschida
et al. (1992) identified an energy-dependent process in salt
excretion, achieved by plasma membrane H?-ATPase in
Avicennia germinans. The salt tolerance of three saltsecreting mangrove species, i.e. Acanthus ilicifolius, Aegiceras corniculatum and A. marina was compared by Ye
et al. (2005). They reported that all of the three species
exhibited increases in salt secretion with increases in
salinity. The capacity to secrete salts at any given salinity
was different between species, following an order of A.
marina [ A. corniculatum [ A. ilicifolius, similar to the
case of seedling growth. The salt tolerance of the three saltsecreting mangrove species was in the descending order of
A. marina [ A. corniculatum [ A. ilicifolius. This indicated that salt secretion from leaves was related to salt
tolerance (Ye et al. 2005). There are species, which can
employ more than one mechanism to protect against
adverse effects of salinity (Table 1). Aegiceras and Avicennia, which are provided with salt glands also, exclude
90–97% salt through the process of ultrafiltration (Ball
1988b). Salt-secreting ability is absent in glycophytes.
Salt accumulators accumulate high concentration of
salts in their cells and tissues and avoid salt damage by
efficient sequestering of ions to the vacuoles in the leaf,
translocation outside the leaf, possible cuticular transpiration and efficient leaf turnover to salt shedding (Tomlinson
1986; Aziz and Khan 2001b). Species of Lumnitzera and
Excoecaria accumulate salts in leaf vacuoles and become
succulent. Salt concentrations in the sap may also be
reduced by transferring the salts into senescent leaves or by
storing them in the bark or roots (Tomascik et al. 1997;
Perry et al. 2008). Regulation of K? uptake and/or prevention of Na? entry, efflux of Na? from the cell, and
utilization of Na? for osmotic adjustment are strategies
commonly used by plants to maintain desirable K?/Na?
ratios in the cytosol. Osmotic homeostasis is established
either by Na? compartmentation into the vacuole or by
biosynthesis and accumulation of compatible solutes. A
205
high K?/Na? ratio in the cytosol is essential for normal
cellular function of plants. Na? competes with K uptake
through Na?–K? co-transporters, and may also block the
K?-specific transporters of root cells under salinity (Zhu
2003). This results in toxic levels of sodium as well as
insufficient K? concentration for enzymatic reactions and
osmotic adjustment. Under salinity, sodium gains entry
into root cell cytosol through cation channels or transporters (selective and nonselective) or into the root xylem
stream via an apoplastic pathway depending on the plant
species (Chinnusamy et al. 2005). Silica deposition and
polymerization of silicates in the endodermis and rhizodermis block Na? influx through the apoplastic pathway in
the root (Yeo et al. 1999). Restriction of sodium influx
either into the root cells or into the xylem stream is one
way of maintaining the optimum cytosolic K?/Na? ratio of
plants under high salinity. In saline conditions, cellular
potassium level can be maintained by activity or expression
of potassium-specific transporters. In Mesembryanthemum
crystallinum L., high affinity K? transporter-K? uptake
genes are up-regulated under NaCl stress (Su et al. 2002).
Sodium efflux from root cells prevents accumulation of
toxic levels of Na? in the cytosol and transport of Na? to
the shoot. Molecular genetic analysis in Arabidopsis have
led to the identification of a plasma membrane Na?/H?
antiporter, SOS1 (Salt Overly Sensitive 1), which plays a
crucial role in sodium extrusion from root epidermal cells
under salinity (Chinnusamy et al. 2005). Sodium efflux by
SOS1 is also vital for salt tolerance of meristem cells such
as growing root-tips and shoot apex as these cells do not
have large vacuoles for sodium compartmentation (Shi
et al. 2002). The expression of SOS1 is ubiquitous, but
stronger in epidermal cells surrounding the root-tip, as well
as parenchyma cells bordering the xylem. Thus, SOS1
functions as a Na?/H? antiporter on the plasma membrane
and plays a crucial role in sodium efflux from root cells and
the long distance Na? transport from root to shoot (Shi
et al. 2002). Sodium efflux through SOS1 under salinity is
regulated by SOS3–SOS2 kinase complex (Chinnusamy
et al. 2005).
Vacuolar sequestration of Na? is an important and costeffective strategy for osmotic adjustment that also reduces
the Na? concentration in the cytosol. Na? sequestration
into the vacuole depends on expression and activity of
Na?/H? antiporters as well as V-type H?- ATPase and H?PPase. These phosphatases generate the necessary proton
gradient required for activity of Na?/H? antiporters. Salt
accumulation in mangroves occurs with the sequestration
of Na? and Cl- into the vacuoles of the hypodermal
storage tissue of the leaves (Werner and Stelzer 1990; Aziz
and Khan 2001a; Kura-Hotta et al. 2001; Mimura et al.
2003). Cram et al. (2002) reported two subsequent phases
of salt accumulation in leaves of Bruguiera cylindrica,
123
206
Avicennia rumphiana and A. marina. The first phase is the
rapid increase in leaf salt concentration, as it grows from
bud to maturity followed by a slower but continuous
change in salt content via changes in ion concentration and/
or in increased leaf thickness. Increased accumulation of
Na? is generally coupled with reduced Ca?? and Mg??
uptake (Greenway and Munns 1980; Delphine et al. 1998)
and sometimes with decline in carbon assimilation (Parida
et al. 2004a). It was reported that Na? and Cl- ion levels
increase in root and shoot tissues of Kandelia candel and
B. gymnorrhiza with increasing NaCl stress but, B. gymnorrhiza shows a rapid Na? accumulation upon the initiation of salt stress and leaves contain 90% more Na? and
40% more Cl- than K. candel (Li et al. 2008). The X-ray
microanalysis of leaf mesophyll cells shows evidence of
distinct vacuolar compartmentation of Na? in K. candel but
Cl- in B. gymnorrhiza seedlings subjected to 100 mM
NaCl. Moreover, Na? within cell wall, cytoplasm, vacuole
and chloroplast remains 23–72% lower in stressed B.
gymnorrhiza as compared to K. candel. It was concluded
that B. gymnorrhiza exhibit effective salt exclusion from
chloroplasts although increasing salt stress causes a rapid
and higher build up of Na? and Cl- in the leaves (Li et al.
2008). Net photosynthetic rate (PN) declines with increasing salinity in both the species, and the most marked
reduction occurred after exposure of mangrove seedlings to
a severe salinity, 400 mM NaCl. However, the inhibitory
effects of severe stress varied with species: PN decreased
by 80% in K. candel whereas in B. gymnorrhiza the decline
was 60%. The lesser reduction in photosynthesis in
B. gymnorrhiza could be a consequence of salt exclusion
from mesophyll chloroplasts (Li et al. 2008). Compartmentalizing NaCl into the vacuole is likely to depend on
Na?/H? antiporter systems (Garbarino and DuPont 1988;
Tanaka et al. 2000; Fu et al. 2005), H?-coupled Cl- antiport (Schumaker and Sze 1987) or ion channels (Pantoja
et al. 1989; Maathuis and Prins 1990). Ion compartmentation in the vacuole would limit excessive salt accumulation in the symplast, thus protecting salt-sensitive
enzymes in the cytoplasm and chloroplasts. Hence, the
ability to maintain lower Na? and Cl- in the symplast may
be an underlying determinant of the tolerance of mangroves (Li et al. 2008).
Accumulation of compatible solutes and osmolytes
One of the important biochemical mechanisms by which
mangroves counter the high osmolarity of salt is accumulation of compatible solutes (Takemura et al. 2000; Parida
et al. 2004c). To accommodate the ionic balance in the
vacuoles, cytoplasm accumulates low-molecular-mass
compounds termed compatible solutes because they do not
interfere with normal biochemical reactions; rather they
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Trees (2010) 24:199–217
replace water in biochemical reaction (Hasegawa et al.
2000; Ashihara et al. 2003). With accumulation proportional to the change of external osmolarity within speciesspecific limits, protection of structures and osmotic balance
supporting continued water influx (or reduced efflux) are
accepted functions of compatible solutes (Parida et al.
2005). Hibino et al. (2001) reported that three mangroves,
B. gymnorrhiza, K. candel, and R. stylosa, accumulate
exclusively pinitol as compatible solute. Mannitol is the
compatible solute for intact plants of Sonneratia alba under
salinity (Yasumoto et al. 1999). Although, pinitol and
mannitol are the most common compatible solutes of a
number of mangrove species, proline (in Xylocarpus species), methylated quaternary ammonium compounds (in
Avicennia eucalyptifolia, A. marina, Acanthus ilicifolius,
Heritiera littoralis and Hibiscus tiliaceus), and carbohydrates (in Acanthus ilicifolius, Heritiera littoralis and
Hibiscus tiliaceus) are found to be dominant osmoregulating compounds (Popp et al. 1985). Glycinebetaine, the
most common compatible solute, which offers protection to
photosynthetic machinery, is found in some mangroves like
A. marina (Ashihara et al. 1997; Hibino et al. 2001).
Although glycinebetaine accumulates as compatible solute
in mangroves and many species of non-mangrove halophytes such as Suaeda maritima, Atriplex nummularia and
Salicornia europoea but is absent from many crop species
(e.g. rice) and the model species in plant transformation,
tobacco (Flowers and Colmer 2008). There has therefore
been considerable work on engineering the production of
glycinebetaine in crop species that do not produce it naturally. Compatible solutes of Rhizophora stylosa and B.
gymnorrhiza are O-methylmucoinositol (Ashihara et al.
2003). Mannitol is a compatible solute in Sonneratia alba
and Lumnitzera racemosa (Ashihara et al. 2003). Many
mangrove species e.g. Sonneratia alba, A. marina also
accumulate inorganic ions and use them as osmolytes to
maintain osmotic and water potential (Yasumoto et al.
1999; Suarez and Medina 2006). Salt tolerance of B.
gymnorrhiza might be attributed to their ability to accumulate high concentrations of Na? and Cl- (Miyama and
Tada 2008). The uptake of additional Na? and Cl- by this
mangrove is used as osmolytes and to maintain K?
homeostasis under salt stress (Miyama and Tada 2008).
A summary of specific compatible solutes in mangrove
species is given in Table 2. The increased synthesis of
compatible solutes is achieved by modulating genes
encoding enzymes of the osmolyte biosynthetic pathway.
For instance, upregulation of pyrroline-5-carboxylate synthetase (P5CS) gene that is involved in proline biosynthesis
leads proline accumulation during salt stress in A. marina
(Mehta et al. 2005). A significant upregulation of AcP5CS
gene was also observed in A. corniculatum during
salinity stress. The upregulation of Betaine-aldehyde
Trees (2010) 24:199–217
207
Table 2 Compatible solutes in mangroves and mangrove associates
Compatible solutes
Mangrove species
References
Pinitol
Kandelia candel, Rhizophora stylosa, Bruguiera gymnorrhiza,
Avicennia marina
Hibino et al. (2001)
Ceriops tagal
Popp et al. (1985)
Mannitol
Kandelia candel, Rhizophora stylosa, Bruguiera gymnorrhiza
Hibino et al. (2001)
Proline
Glycinebetaine
Sonneratia alba
Yasumoto et al. (1999); Ashihara et al. (2003)
Lumnitzera racemosa
Ashihara et al. (2003)
Kandelia candel, Rhizophora stylosa, Bruguiera gymnorrhiza
Hibino et al. (2001)
Bruguiera parviflora
Aegiceras corniculatum
Parida et al. (2002)
Fu et al. (2005)
Bruguiera sexangula, Avicennia alba, Xylocarpus granatum
Datta and Ghosh (2003)
Acanthus ilicifolius, Hibiscus tiliaceus
Datta and Ghosh (2003)
Avicennia marina
Datta and Ghosh (2003); Hibino et al. (2001)
Ceriops roxburghiana
Rajesh et al. (1999)
Ceriops tagal
Aziz and Khan (2001b)
Avicennia marina
Hibino et al. (2001); Ashihara et al. (1997); Popp
et al. (1985)
Ceriops roxburghiana
Rajesh et al. (1999)
Hibiscus tiliaceus
Popp et al. (1985)
O-methylmucoinositol
Rhizophora stylosa, Bruguiera gymnorrhiza
Ashihara et al. (2003)
Starch/polysaccharide
Aspartic acid
Aegiceras corniculatum
Aegiceras corniculatum, Acanthus ilicifolius
Parida et al. (2004c)
Datta and Ghosh (2003)
Sterol
Ceriops roxburghiana
Suarez and Medina (2006)
dehydrogenase (BADH) gene that is involved in glycinebetaine synthesis results in the accumulation of glycinebetaine in A. marina under salt stress (Hibino et al. 2001).
Waditee et al. (2002) reported that under high salinity
conditions, the betaine/proline transporters (AmT1, -2, and
-3) also involved in the accumulation of betaine by
increasing the mRNA levels as well as post-translational
activation in A. marina.
Induction of antioxidative enzymes
Mangroves inhabiting the intertidal zones suffer from
diverse stresses such as high salinity, hypoxia, ultraviolet
radiation, nutrition deficiency and so on. These primary
stresses may lead to secondary oxidative stress, resulting in
accumulation of reactive oxygen species (ROS) such as
superoxide (O.2 ), hydrogen peroxide (H2O2), hydroxyl
radical and singlet oxygen (1O2) (Parida et al. 2004b). These
cytotoxic reactive oxygen species can seriously disrupt
normal metabolism through oxidative damage to lipids,
protein and nucleic acids (Parida et al. 2004b). Mangroves
with high levels of antioxidants, either constitutive or
induced, have been reported to have greater resistance to
this oxidative damage (Cheeseman et al. 1997; Takemura
et al. 2000; Parida et al. 2004b; Jithesh et al. 2006). The
activities of the antioxidative enzymes such as catalase
(CAT), ascorbate peroxidase (APX), guaiacol peroxidase
(POD), glutathione reductase (GR), and superoxide dismutase increase under high salinity and a correlation of
these enzyme levels and salt tolerance exists in mangroves
(Parida et al. 2004b; Takemura et al. 2000). There are
several reports of upregulation of antioxidative enzymes
and their corresponding genes in mangroves under salinity.
In B. gymnorrhiza, the activities of the antioxidant
enzymes, superoxide dismutase (SOD) and catalase, show
an immediate increase after the plants are transferred from
water to high salinity (Takemura et al. 2000). In B. parviflora, salt treatment preferentially enhances the content of
H2O2 as well as the activity of ascorbate peroxidase (APX),
guaiacol peroxidase (GPX), glutathione reductase (GR),
and superoxide dismutase (SOD), whereas it induces the
decrease of catalase (CAT) activity (Parida et al. 2004b). It
has also been reported that in the salt-secreting mangrove
Aegiceras corniculatum, there is a linear increase of salt
secretion of leaf with increase in period of salt treatment
and a concomitant decrease in antioxidative enzymes such
as catalase, ascorbate peroxidase and guaiacol peroxidase
(Mishra and Das 2003).
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208
Molecular mechanism of salt tolerance in mangroves
The mechanism of salt tolerance of mangroves can be
partially explained by morphological, anatomical, physiological and biochemical studies. However, these are
insufficient to clarify the salt tolerance mechanism.
Recently, some progress has been achieved in understanding the mechanism of salt tolerance in mangroves at a
molecular level. Several salt stress-associated genes from
mangroves have been evaluated for their contribution to
salt tolerance in laboratory studies (Table 3). Table 4 also
lists some relevant studies in mangroves at the genomic
levels. These results indicated that the tolerance of mangroves to a high-saline environment is indeed tightly linked
to the regulation of gene expression. Some molecular
biological studies of mangroves are discussed below in
order to illustrate this point.
A molecular biological study of salt tolerance was first
reported in a mangrove B. gymnorrhiza by Sugihara et al.
(2000) and they suggested that an oxygen evolving
enhancer protein 1 (OEE1) has an important role in the salt
tolerance of this mangrove. They reported that when young
plants of B. gymnorrhiza are transferred from freshwater to
a medium with a seawater salt level (500 mM NaCl), the
intensity of a 33 kDa protein with pI 5.2 increases in the
leaf extract as observed in 2-D gel electrophoresis. The Nterminal amino acids sequence of this protein has a significant homology with the mature region of the oxygen
evolving enhancer protein 1 (OEE1) precursor. The
deduced amino acid sequence consists of 322 amino acids
and is 87% identical to that of Nicotiana tobacum. The
expression of OEE1 was analyzed together with other OEE
subunits (OEE2 and OEE3) and D1 protein of photosystem
II. The transcript levels of all the three OEEs were
enhanced by NaCl treatment, but the significant increase of
D1 protein was not observed in B. gymnorrhiza. Sugihara
et al. (2000) suggested that not only the OEE1 but also the
OEE2 and OEE3 play an important role in the maintenance
of PSII activity under NaCl stress conditions. In particular,
OEE1 is essential for oxygen evolving activity and PSII
stability. The expression of OEE1 is also considered to be
the rate-limiting step in the assembly of the PSII subunit
(Mizobuchi and Yamamoto 1989). Therefore, it is considered that the recovery or turnover of OEE1 is one of the
mechanisms to maintain the capacity of PSII under NaCl
stress.
Currently there are also ongoing genomic studies of B.
gymonorrhiza. The first B. gymonorrhiza expressed
sequence tag (EST) library, which collected 14,842 ESTs
from leaves and roots after high salinity or hormone
treatments were established by Miyama et al. (2006).
Clustering and assembling of these sequences resulted
in 6,943 unique genes. The EST collection of
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Trees (2010) 24:199–217
B. gymonorrhiza includes genes that share significant
similarities with salt stress inducible genes. These genes
can be categorized into five major categories such as (1)
osmolyte biosynthesis, (2) reactive oxygen scavenger, (3)
chaperones, (4) transporters and (5) signaling components.
The unique gene collections obtained from the assembly of
those ESTs were later used in microarray experiments to
monitor transcript profiling in leaves and roots of saltstressed B. gymonorrhiza (Miyama and Hanagata 2007).
Totally, 228 genes displayed transcript levels fivefold
higher than in controls, while 61 genes were downregulated
to one-fifth of control levels. Among these remarkably
differentially expressed genes, only 32.5% upregulated and
3.3% downregulated genes were co-regulated in upper and
lower leaves, as well as in roots (Miyama and Hanagata
2007). The rest showed tissue-specific expression patterns
(Miyama and Hanagata 2007). Differing from EST analysis, differentially expressed genes in microarray analysis
fell into six categories of gene expression patterns (Miyama and Hanagata 2007).
The transcriptional response of B. gymnorrhiza to high
salinity (500 mM NaCl) and hyperosmotic stress (1 M
sorbitol) was investigated by microarray analysis (Miyama
and Tada 2008). Statistical analysis of microarray revealed
that 865 of 11,997 genes showed significant differential
expression under salt and osmotic stress. Comparison of
gene ontology (GO) categories of differentially expressed
genes under the stress conditions revealed that the adaptation of B. gymnorrhiza to salt stress was accompanied by
the upregulation of genes categorized for cell communication, signal transduction, lipid metabolic process, photosynthesis, multicellular organismal development and
transport, and by downregulation of genes categorized for
catabolic process. Hierarchical clustering of the 865 genes
showed that expression profiles under salt stress were distinctly different from those under osmotic stress. B. gymnorrhiza maintains its leaf water potential and recovered
from its photosynthesis rate that declined temporarily
under salt stress, but not under osmotic stress. These results
demonstrated a fundamental difference between the
responses to salt and osmotic stress (Miyama and Tada
2008). The salt-responding genes that showed no sequence
similarity to the public database entries were also detected
in B. gymnorrhiza (Miyama and Tada 2008). Therefore, it
is possible that these unknown salt-responding genes may
contribute to the salt adaptation of B. gymnorrhiza in a
unique and specific manner.
The differential display method was applied to transcripts extracted from leaves of B. gymnorrhiza treated
with 500 mM NaCl for 0, 6 h, 3 and 28 days to identify
genes that are differentially expressed in response to salt
stress (Banzai et al. 2002). The expression of 12 transcripts,
whose corresponding cDNA fragments differentially
Trees (2010) 24:199–217
209
Table 3 Salt inducible genes reported in mangroves
Mangrove
species
Genes
Characteristic feature(s)
References
Delta 1-pyrroline-5-carboxylate synthetase, a key enzyme of proline synthesis pathway
Accumulation of transcript of this gene under high salinity tended to accompany
recruitment of proline in Aegiceras corniculatum
Fu et al. (2005)
PIP1
PIP1 aquaporin—This gene was upregulated by salt stress
Fu et al. (2005)
PIP2
PIP2 aquaporin—This gene was upregulated by salt stress
Fu et al. (2005)
NHA
Na?/H? antiporter—This gene was upregulated by salt stress
Fu et al. (2005)
BADH
Betaine-2-aldehyde dehydrogenase—High salinity induced increase of transcript level and Hibino et al.
such an increase was accompanied by accumulation of betaine. Although activity of this (2001)
enzyme decreased with an increase in salinity, the extent of decrease is less than its
homologue in E. coli and spinach
Sod1
Cytosolic Cu/Zn superoxide dismutase—Overexpression of this gene in indica Rice var
Pusa Basmati-1 confers abiotic stress tolerance
Prashanth et al.
(2008)
Sod1
Cu/Zn superoxide dismutase—High salinity did not lead to transcriptional change but
osmotic stress decreased transcript level of this gene. Under oxidative stress, its
transcription was transiently upregulated
Jithesh et al.
(2006)
Cat1
Catalase—It was upregulated by saline or oxidative stress but downregulated by osmotic
stress
Jithesh et al.
(2006)
Fer1
Ferritin 1—It was transcriptionally Upregulated by saline or oxidative stress but didn’t
change under osmotic stress
Jithesh et al.
(2006)
AmT1; AmT2
Betaine/Proline transporter—Transgenic E. coli with such genes could accumulate
Waditee et al.
(2002)
Aegiceras
P5CS
corniculatum
Avicennia
marina
AmT3 [partial] Betaine under salt stress; In Avicennia marina, salt stress induced transcription of such
genes in root and leaf
Bruguiera
gymnorrhiza
OEE1
OEE1 is one component of PS II and high salinity induced accumulation of its transcript and Sugihara et al.
protein
(2000)
DLDH
Dihydrolipoamide dehydrogenase—Upregulated when treated with 500 mM NaCl for 1 day Banzai et al.
(2002)
LAS
Lipoic acid synthase—Being upregulated when treated with 500 mM NaCl for 1 day
Banzai et al.
(2002)
Unnamed gene Fructose-6-phosphate, 2-kinase/fructose-2, 6-bisphophatase—Transcription of this gene
Banzai et al.
increased after 6 h of salt stress. It was supposed to act in osmotic regulation process by (2002, 2003)
controlling the content of Fru-2, 6-P2
Cytosolic
Cytosolic Cu/Zn superoxide dismutase—High salinity, mannitol and ABA induced
accumulation
Cu/Zn SOD
Of its transcripts in leaves; Transcript was induced by high salinity in young and mature
leaves rather than in old leaves
Takemura et al.
(2002)
Cytosolic CAT Catalase——No significant change occurred in the expression of this gene during the
Takemura et al.
(partial)
treatment with NaCl, mannitol and ABA, but CEPA (2-chloroethylphosphonic acid) can (2002)
increase its transcript level
Bruguiera
sexangula
Kandelia
candel
CCTa
CCTa a subunit of CCT complex——Transgenic E. coli with one domain of this subunit Yamada et al.
displayed enhanced tolerance to high salinity
(2002a)
Mangrin
Partially homologous to gene encoding Allene Oxide Cyclase (AOC)——It was
Yamada et al.
upregulated by high salinity and its overexpression enhanced salt tolerance of transgenic (2002b)
yeast and tobacco cell
SIGKC1;
SIGKC2
Cytosolic low molecular mass heat shock protein (sHSPs)—sHSPs act as molecular
Huang et al.
chaperones to prevent thermal aggregation of proteins by binding non-native intermediates (2003)
SIGKC3
ADP-ribosylation factor (ARF)—a ubiquitous, highly conserved 21 kDa GTP-binding
protein; The ARF proteins are thought to function as regulators of membrane traffic
Unknown function
SIGKC4;
SIGKC5
Huang et al.
(2003)
Huang et al.
(2003)
The names of various genes have been by and large reproduced here as per the original publications of the authors
123
210
Trees (2010) 24:199–217
Table 4 Genomic studies in mangroves
Mangrove species Description
Aegiceras
corniculatum
References
A leaf SSH library was constructed. By sequencing the whole SSH library 577 ESTs were found that are Fu et al. (2005)
up-regulated by high salinity. Fourteen catagories were assigned for this EST collection and those
categories of ‘‘protein synthesis’’, ‘‘defense’’, ‘‘transport’’, ‘‘ion homeostasis’’, ‘‘protein destination’’
and ‘‘signal transduction’’ were remarkable
Avicennia marina A leaf cDNA library was constructed from 500 mM NaCl-treated seedlings. Random sequencing
Mehta et al.
generated 1602 ESTs which were grouped into 13 categories; Among these, 7% were homologous with (2005)
stress-responsive genes
Bruguiera
gymnorrhiza
Large-scale sequencing of ESTs collected from high salinity or hormone treated leaves and roots.
Assembly of 14,842 high quality sequences generated 6,943 unique genes and 62.5% of such EST
collection matched known proteins in Blast searching. Totally 129 statistically-confident genes were
grouped into 4 clusters depending on their EST frequency and each group has specific pattern of
transcript profiling under high salinity
Miyama et al.
(2006)
Salt-responsive transcript profilings of 7,029 unique genes in leaf and root tissues were monitored using Miyama and
microarray technique. Clustering generated at least six categories of transcript accumulating under
Hanagata
saline condition; Some genes displayed similar salt-responsive patterns to those in other plants,
(2007)
indicating shared mechanisms in Bruguiera gymnorrhiza and other glycophytes. Distinct expression
patterns of other genes suggested existence of specific mechanisms in this species
Differentially expressed candidates genes were identified through differentially display technique from Banzai et al.
leaves stressed with 500 mM NaCl for 0 h, 6 h, 3 days, and 28 days. Totally 89 clones were identified (2002)
as differentially expressed candidates; nine out of these candidates were verified by Northern Blot and
classed into three groups depending on their salt-induced patterns of transcript accumulation
Bruguiera
cylindrica
126 salt tolerant cDNAs were identified and isolated from the root using suppression subtractive
Wong et al.
hybridization (SSH) and bacterial functional screening. Sequencing of 51 subtracted cDNA clones that (2007)
were differentially expressed in the root of B. cylindrica exposed to 342 mM NaCl revealed 10 tentative
unique genes (TUGs) with putative functions in protein synthesis, storage and destination, metabolism,
intracellular trafficking and other functions; and nine unknown proteins. The 75 cDNA sequences of B.
cylindrica that conferred salinity tolerance to Escherichia coli consisted of 29 TUGs with putative
functions in transportation, metabolism and other functions; and 33 with unknown functions. Both
approaches yielded 42 unique sequences that have not been reported elsewhere to be stress related
Acanthus
ebracteatus
A leaf cDNA library was constructed from seawater-growing seedlings. Random sequencing generated Nguyen et al.
521 readable sequences and 67% of them matched function-known genes by homolog searching; among (2006)
which, 18% were predicted to function in stress response, 23.9% in metabolism, 7.3% in regulation of
transcription and 2.7% in others
Sesuvium
portulacastrum
In an attempt to isolate and identify the target genes relevant to salt tolerance in the mangrove associate Zeng et al.
(Sesuvium portulacastrum L.), a subtracted cDNA library was constructed via suppressive subtractive (2006)
hybridization (SSH). Screening of this subtracted cDNA library revealed five clones involved in salttolerance pathways. Among the clones isolated, P66, P175, and P233 are novel because no significant
similarity was obtained upon alignment with the GenBank database. Clone P89 demonstrated high
homology with NADPH of Arabidopsis thaliana, whereas clone P152 was highly homologous with the
gene encoding late embryogenesis abundant (LEA) protein of A. thaliana
appeared on differential display, was then analyzed by
Northern hybridization. Nine of them were affirmed to be
upregulated or induced by salt treatment. The nine transcripts were divided into three groups based on their
expression patterns. RNA transcripts to BG7, BG50, BG51,
BG55 and BG67, whose levels were temporarily raised at
6 h, were assigned to Group I. Transcripts to BG60 and
BG70, whose levels increased at 6 h, and remained at a
high level for at least 28 days were assigned to Group II.
Transcripts to BG56 and BG64 were assigned to Group III
as their levels increased at 3 days, but then decreased
(Banzai et al. 2002). By screening a cDNA library, five
full-length cDNAs were cloned and putative protein
products were identified for three of them by homology
123
with known protein. The RNA transcript bg51 encodes a
protein with homology to fructose-6-phosphate, 2-kinase/
fructose-2,6-bisphophatase (F6P, 2-K/F26BPase), the
bifunctional enzyme catalyzing both synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P2) which is
an important regulator in the carbohydrate metabolism.
The protein encoded by bg55 has homology with a protein
of A. thaliana whose function is unknown. Transcripts
bg56 and bg64 are encoded by proteins having very significant homology with dihydrolipoamide dehydrogenase
(DLDH) and lipoic acid synthase (LAS), respectively. The
results suggest that these RNA transcripts and their
respective encoding proteins have some role in salt tolerance in B. gymnorrhiza (Banzai et al. 2002). The protein
Trees (2010) 24:199–217
encoded by bg70, Group II transcript, is a novel protein
with no homology with any proteins registered in the databases so far (Banzai et al. 2002). Further analysis of such
Group II genes will reveal how B. gymnorrhiza adapts to
high salinity.
In order to identify key genes in the regulation of salt
tolerance in the mangrove plant B. gymnorrhiza, cDNA
expression libraries were constructed from salt-treated
roots and leaves using the host organism Agrobacterium
tumefaciens (Ezawa and Tada 2009). Functional screening
of the Agrobacterium libraries identified 44 putative salt
tolerance genes in B. gymnorrhiza. A cDNA clone which is
homologous to an unknown cDNA from the mangrove
plant K. candel and the cyc02 gene from Catharanthus
roseus conferred the highest level of salt tolerance to A.
tumefaciens, which indicates that it plays a major role in
the regulation of salt tolerance in mangrove plants. Several
of the genes that were identified have not previously been
implicated in plant salt tolerance (Ezawa and Tada 2009).
Transgenic Arabidopsis plants expressing Bg70 and cyc02
homolog exhibited increased tolerance to NaCl. These
results demonstrate that Agrobacterium functional screening is an effective supplemental method to pre-screen
genes involved in salt tolerance (Ezawa and Tada 2009).
Screening for salinity tolerant genes was done in the root
of the mangrove plant Bruguiera cylindrica by using suppression subtractive hybridization (SSH) and bacterial
functional screening (Wong et al. 2007). One hundred
twenty-six salinity tolerant cDNAs were identified and
isolated from the root of B. cylindrica by both approaches
(Wong et al. 2007). Sequencing of 51 subtracted cDNA
clones that were differentially expressed in the root of B.
cylindrica exposed to 342 mM NaCl revealed 10 tentative
unique genes (TUGs) with putative functions in protein
synthesis, storage and destination, metabolism, intracellular trafficking and other functions; and 9 unknown proteins.
Bacterial functional screening revealed that the 75 cDNA
sequences of B. cylindrica that conferred salinity tolerance
to Escherichia coli consisted of 29 TUGs with putative
functions in transportation, metabolism and other functions; and 33 with unknown functions. Both approaches
yielded 42 unique sequences that have not been reported
elsewhere to be stress related might have some role in salt
tolerance in B. cylindrical (Wong et al. 2007). These
unique genes may be valuable gene candidates for further
analyses to unravel the mystery of salinity tolerance in
mangrove plants.
In order to reveal the molecular mechanism of salt tolerance in mangroves gene expression pattern was identified
in Kandelia candel (Huang et al. 2003). Ten cDNAs of
genes were isolated and identified from K. candel by representational difference analysis of cDNA (cDNA RDA)
under different NaCI concentrations (Huang et al. 2003).
211
Of five genes expressed preferentially under salt condition,
two were unknown; three were two kinds of cytosolic lowmolecular mass heat-shock proteins (sHSPs) and ADPribosylation factor (ARF), respectively. The upregulation
of sHSPs and ARF has some role in osmotic equilibrium by
which the K. candel plant can resist salt damage. The
expressions of other five genes were repressed under NaCI
stress, two encoded cyclophilins; three were tonoplast
intrinsic protein, early light-induced protein and 60S
ribosomal protein, respectively.
Betaine-aldehyde dehydrogenase (BADH) gene that is
involved in glycinebetaine synthesis in A. marina was first
identified and cloned by Hibino et al. (2001). BADH was
upregulated under salt stress, and this tendency was consistent with the accumulation of glycinebetaine in A.
marina. Three other genes that encode A. marina betaine
and/or proline transporters 1 (AmT1), 2 (AmT2) and 3
(AmT3), respectively, were also isolated by Waditee et al.
(2002). The mRNA levels of the three transporters were
measured, and it was observed that the levels of mRNAs
for AmT1, AmT2 and AmT3, were constitutively low and
almost the same in both leaf and root when A. marina was
grown without salt stress. Upon the salt stress (400 mM
NaCl), the levels of mRNAs in all three transporters,
AmT1, -2 and -3, increased in both leaf and root. However,
the increases in leaf were more pronounced than those in
root for all three transporters. Salt stress induced the
highest level of mRNA for AmT1 followed by AmT2 and
AmT3, respectively. The accumulation levels of mRNA for
AmT1, -2 and -3 increased with increasing concentration of
NaCl, especially in leaves (Waditee et al. 2002). Since
betaine synthesis occurs in chloroplasts upon the salt stress,
it is reasonable that the synthesis of betaine transporters is
also induced upon the salt stress because the synthesized
betaine must be transported to the other plant organs where
betaine synthesis activity is low (Takabe et al. 1997).
Therefore, under high-salinity conditions, the betaine/proline transporters (AmT1, -2 and -3) appear to be involved
in the accumulation of betaine by increasing the mRNA
levels as well as post-translational activation in betaineaccumulating A. marina (Waditee et al. 2002).
In order to characterize the genes that contribute to
combating salinity stress, the cDNA library of A. marina
genes was constructed by Mehta et al. (2005). Random
EST sequencing of 1,841 clones produced 1,602 quality
reads. These clones were classified into functional categories, and BLAST comparisons revealed that 113 clones
were homologous to genes earlier implicated in stress
responses. Of the ESTs analyzed, 30% showed homology
to previously uncharacterized genes in the public plant
databases. Of this 30%, 52 clones were selected for reverse
Northern analysis: 26 were shown to be upregulated and 5
shown to be downregulated under NaCl stress (Mehta et al.
123
212
2005). Expression patterns for the ‘unknown’ genes provide a starting point for the isolation of salt tolerance
candidate genes, and further functional analysis will elucidate their role in salt tolerance. ESTs showing homology
to stress-tolerant genes reported in literature represent the
sixth most abundant category. Dehydrins (34 clones/12
genes) predominate in this class and include late-embryogenesis-related proteins and desiccation or droughtinduced proteins. Other stress-induced genes present in this
category include heat shock proteins, thioredoxin, osmotin
and genes for osmolyte production such as BADH and
pyrroline-5-carboxylate synthase. The analyzed EST pool
contained transcripts coding for enzymes involved in the
oxidative stress response such as catalase, superoxide dismutase, peroxidases, glutathione S-transferase and epoxide
hydrolase. In addition, genes reported to be induced by
heavy metal stress, such as metallothioneins, aluminuminduced protein(s), truncated copper-binding protein
CUTA and divalent cation-tolerant gene, and those induced
by anaerobic stress, such as alcohol dehydrogenases and
submergence-induced genes, were also found in the ESTs
sequenced. Transcripts coding for proteins involved in
membrane transport and processing play an integral role in
the response to water deficit such as that imposed by salt
stress and include aquaporins, proline/glycine betaine
transporter and Na?/H? antiporter. In addition, protein
factors involved in the regulation of signal transduction
events, such as receptors, protein and lipid kinases, calmodulins and protein phosphatases, which may have a role
in stress signaling pathways, have been categorized separately. Genes for a variety of transcription factors that
contain typical DNA binding motifs, such as MYB, bZIP,
ERF/AP2, have been demonstrated to be stress-inducible
(Zhu 2002). Transcription factors containing similar
domains are present in the A. marina ESTs and may have a
role in regulating the response to salt stress (Mehta et al.
2005). At the genomic level, a large number of ESTs have
been collected from NaCl stressed seedlings of A. marina
through techniques of differential display or random
sequencing of cDNA library clones (Mehta et al. 2005).
Some genes in such EST collections, e.g., those coding for
dehydrin and polypeptide hormone phytosulphokine, were
continuously upregulated after 48 h of salt-stress (Mehta
et al. 2005). Their transcript abundance returned to the
normal level if salt stress was prolonged to 12–24 weeks,
indicating adaptation of A. marina after long-term stress
(Mehta et al. 2005).
Jithesh et al. (2006) studied the expression of antioxidant genes such as Cu–Zn SOD (Sod1), catalase (Cat1) and
ferritin (Fer1) in response to salt, iron, hydrogen peroxide,
mannitol and light stress by mRNA expression analysis in
A. marina. In response to NaCl stress Cat1, Fer1 showed
short-term induction while Sod1 transcript was found to be
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Trees (2010) 24:199–217
unaltered. Sod1, Cat1 and Fer1 mRNA levels were induced
by iron, light stress and by direct H2O2 stress treatment,
thus confirming their role in oxidative stress response.
Transcripts that showed enhanced expression during salt
stress was cloned in the leaves of a salt-tolerant mangrove
species, Aegiceras corniculatum using suppressive subtractive hybridization (SSH, Fu et al. 2005). cDNAs of
freshwater germinated and irrigated seedling were used as
driver and cDNAs of 6 h salt-stress seedling were used as
tester. By sequencing the whole SSH library, 577 ESTs
were found. Among which, 30 had no significant homology
to any previously identified genes and 527 of the remaining
547 ESTs represent singletons. Real-time quantitative RTPCR analysis of four transcripts’ expression pattern
showed that all of their transcripts were up-regulated during 24 h after salt shock. Their sequences showed high
homology to the delta-1-pyrroline-5-carboxylate synthetase, Na?/H? antiporter and plasma membrane intrinsic
proteins, respectively.
Another salt secreting mangrove species Acanthus ebracteatus has also been of concern recently. A. ebracteatus
was chosen as a source for the generation of ESTs to
isolate genes involved in salt tolerance of this mangrove
plant (Nguyen et al. 2006). In this study, they isolated and
sequenced 864 randomly selected cDNA clones from the
primary cDNA library of A. ebracteatus. Sequence analyses demonstrated that 349 of these ESTs showed significant homology to functional proteins, of which 18% are
particularly interesting as they correspond to genes
involved in stress response. Some of these clones,
including putative mannitol dehydrogenase, plastidic
aldolase, secretory peroxidase, ascorbate peroxidase, and
vacuolar H?-ATPase, may be related to osmotic homeostasis, ionic homeostasis, and detoxification (Nguyen et al.
2006). Hundred seven salinity tolerance candidate genes
have been identified and isolated from the mangrove plant,
A. ebracteatus by using bacterial functional assay (Nguyen
et al. 2007). Sequence analysis of these putative salinity
tolerant cDNA candidates revealed that 65% of them have
not been reported to be stress related and may have great
potential for the elucidation of unique salinity tolerant
mechanisms in mangrove. Among the genes identified
were also genes that had previously been linked to stress
response including salinity tolerance (Nguyen et al. 2007).
Even though many of these mangrove genes or gene
products were reported in stress tolerance of higher plants,
the mangrove proteins may display unique properties and
function. For example, the mangrove allene oxide cyclise
(AOC) homolog or ‘‘mangrin’’ (Yamada et al. 2002b),
containing an usual sequence of 70 amino acids that is
essential to salt tolerant phenotype, is not found in Lycopersicon and Arabidopsis AOC homologs. The successful
identification and isolation of salt-tolerant candidates from
Trees (2010) 24:199–217
A. ebracteatus provide a basis for further function elucidation, in addition to providing useful information for
plant breeding and genetic engineering of crop plants with
greater salinity tolerance that allow agriculture on saline
soil.
Conclusion and future prospects
Salinity is a major abiotic stress that greatly affects
plant growth and crop production globally. Sodium ions
in saline soil are toxic to plants because of their adverse
effects on potassium nutrition, cytosolic enzyme activities, photosynthesis, and metabolism. Mangroves tolerate
high salinity by rejecting potentially harmful salts. Some
species of mangroves actively excrete those salts leaking into the plant by means of specialized salt glands in
their leaves and some species excrete salt by ultra filtration at the root cell membranes of cortical cells. Salt
accumulators avoid the toxic effects of salt by compartmentation of Na? and Cl- ions into the vacuoles by
Na?/H? antiporter system. Accumulation of compatible
solutes and induction of antioxidative enzymes are other
mechanisms of salt tolerance in mangroves. Mangroves
also provide a reservoir for some of the best known,
and at times, novel genes and proteins, involved in
tolerance to salinity stress, that are likely at work in
other plants. The salt tolerance genes listed in this
review most likely represent only the tip of the iceberg,
and continuous efforts to isolate and identify novel
useful genes and promoters from mangroves are necessary; DNA microarray technology in particular is likely
to become a powerful tool for this purpose. Eventually,
the largest challenge will be to combine these genes and
promoters in a systematic and logical way in order to
maximize plant salinity tolerance. When realized,
genetic engineering of crop and industrial plant for
salinity tolerance using genes isolated from mangroves
will be a vitally important tool in the quest to alleviate
the earth’s future problems concerning food, energy, and
the environment.
Acknowledgments The financial support received from Council of
Scientific and Industrial Research (CSIR), India (project no. NWP020) is gratefully acknowledged.
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