Halophytes for food security in dry lands

HALOPHYTES FOR
FOOD SECURITY IN
DRY LANDS


HALOPHYTES FOR
FOOD SECURITY IN
DRY LANDS
Edited by

MUHAMMAD AJMAL KHAN
MUNIR OZTURK
BILQUEES GUL
MUHAMMAD ZAHEER AHMED

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FOREWORD BY
SHEIKHA ABDULLA AL MISNAD

Water scarcity is one of the defining issues for the future of
Gulf Cooperation Council (GCC) countries. With the rapid pace
of urban development and population growth in this region, the
demand for water will only increase. Desalination of water for
agricultural and domestic use is not without substantial financial cost and grave environmental implications. Both food and
water security are key for Qatar’s future and for its development
plans. Innovative solutions are urgently needed.
Through academic programs and research initiatives, Qatar
University has been contributing to the multi-faceted issue of
sustainable development, with special emphasis on the roles of
education, science, and technology. In November 2012, Qatar
University (QU) and the Qatar National Food Security Program
hosted the International Conference on Food Security in Dry
Lands. Based on the conviction that high-quality scientific
research is essential for finding sustainable development solutions in dry lands, QU created a Centre for Sustainable
Development to address water and food security and wider
environmental management issues and to link research with
human, social, and economic developments in Qatari society. In
May 2014, Qatar Shell Professorial Chair in Sustainable
Development organized another conference on Halophytes for
Food Security in Dry Lands with the participation of scientists
from all over the world. This book was born out of the ideas
and discussions at that conference and the pressing need for
creative and context-appropriate solutions.
One such innovative idea is the use of vast resources of ground
saline water or seawater for the production of economically
important crops from the indigenous Qatari plants distributed in
coastal and inland sabkha salt marshes and deserts. Halophytes
are a group of plants that are naturally equipped with the
mechanisms to survive under highly saline and arid conditions

and produce high biomass. This high productivity could be used
as fodder, forage, biofuel, turf, medicine, edible and essential oils,
and biodiesel. The scientific community has made limited but
steady progress in developing these salt-tolerant plant species as
cash-crops, and attempts are ongoing to enhance research and
implementation in farming and landscaping. Throughout the

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FOREWORD BY SHEIKHA ABDULLA AL MISNAD

Arabian Peninsula, promising results have been seen with certain
halophytic species. This area therefore holds exciting potential
explored throughout the conference papers.
Many of the participants of the May 2014 Halophytes for
Food Security in Dry Lands conference have contributed to this
volume and to enriching knowledge about halophyte productivity in the harsh Qatari environment. The editors have already
produced four volumes on the Sabkha Ecosystem in regions of
the world, including the Arabian Peninsula and adjacent countries. This volume is a continuation of those efforts. Importantly,
the conference was followed-up with promising collaborations
and research funding proposals around developing nonconventional crops that can alleviate some of the chronic food and
water security issues in the region.
The professional contributions that have gone into the production of this volume are immense, and I encourage students
and scientists to make use of this rich resource in the search for
innovative and much-needed models to achieve food security
in dry lands of this region and the rest of the world.
Sheikha Abdulla Al Misnad, Ph.D.

President, Qatar University,
Doha, Qatar


FOREWORD BY
EIMAN AL-MUSTAFAWI
Since one of the tenets that Qatar’s National Vision 2030
(QNV2030) resets on is advancing sustainable development,
there has been an urgent need for new interdisciplinary
approaches for food and water security enhancement.
To serve the needs of Qatar, the College of Arts and Sciences
at Qatar University launched the Center for Sustainable
Development to produce with our partners to make an interdisciplinary contribution towards promoting sustainable development in Qatar, and the Gulf region, with a focus on food security,
given its importance both for current and future generations.
Qatar is a water-scarce country where per capita availability of
water is amongst the lowest in the world. The population of Qatar
has grown rapidly (as of 2015) to over 2 million, compared with a
few hundred thousand over the last two decades. Most food is
imported and the source of fresh water is through desalinating
seawater into fresh water. This desalination process requires a
huge amount of energy that substantially increases CO2 emissions, which contribute to the challenge of global warming.
An innovative focus of our food security program has been to
examine the possibility of developing coastal salt deserts into
man-made ecosystems for agricultural productivity, with the
food supply requirements of the growing human population in
mind. It is encouraging that studies undertaken in this arid
region have revealed that various medicinal/aromatic plants can
be cultivated easily on slightly saline-alkaline soils using seawater
irrigation. Many salt-tolerant plant taxa found in nature can be
domesticated to provide better economic returns. Whilst initial

results are encouraging, what is needed is vision, planning,
and the involvement of scientific and agricultural authorities and
politicians.
The Qatar Shell Professorial Chair in Sustainable Development,
housed in the College of Arts and Sciences, organized an
International Conference on Halophytes for Food Security in
Dry Lands from May 12À13, 2014, Doha, at which distinguished
scientists, participants, and contributors from all over the world
were present. The theme of this conference was very timely: no
longer do we merely try to understand the importance of
halophytes for sustainable development, but we have also started

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FOREWORD BY EIMAN AL-MUSTAFAWI

to understand the tremendous importance of sabkha for the conservation of halophyte biodiversity. Halophytes hold significant
potential to counteract adverse environmental impacts, such as
climate change, marine discharge waters, ecosystem restoration,
and the enhancement of primary productivity. It is for these
reasons that this important volume includes all aspects of
halophyte biology spanning from ecosystem to molecular levels.
This information can be useful in making crop plants for
food consumption salt-tolerant. This volume also contributes
to our understanding of the economic significance of halophytes
for food security in dry regions.
It is on this hopeful note that I offer my thanks to the editors

and the authors for their contributions to the scientific community, given their recommendations and suggestions for future
research. Overall, I am hopeful that if halophytes are properly
utilized, it could be a blessing for dry lands and food security.
Dr. Eiman Al-Mustafawi
Dean, College of Arts and Science,
Qatar University, Doha, Qatar


LIST OF CONTRIBUTORS
Chedly Abdelly Laboratoire des Plantes Extreˆmophiles, Centre
de Biotechnologie de Borj Ce´dria, Hammam Lif, Tunisia
Muhammad Zaheer Ahmed Institute of Sustainable Halophyte
Utilization, University of Karachi, Karachi, Pakistan; Gene
Research Center, University of Tsukuba, Tsukuba City, Ibaraki,
Japan
A.J. Al Dakheel International Center for Biosaline, Dubai, UAE
Volkan Altay Biology Department, Science and Arts Faculty,
Mustafa Kemal University, Antakya-Hatay, Turkey
Ernaz Altunda˘g Biology Department, Science and Arts Faculty,
Duzce University, Duzce, Turkey
Jorge Batlle-Sales Department of Vegetal Biology, University of
Valencia, Valencia, Spain
Laila Bouqbis Polydisciplinary Faculty, Ibn Zohr University,
Taroudant, Morocco
Franc¸ois Bouteau Institut des Energies de Demain, Universite´
Paris Diderot, Sorbonne Paris Cite´, Paris, France
Meryem Brakez Laboratory of Plant Biotechnologies, Faculty of
Sciences, Ibn Zohr University, Agadir, Morocco
Zahra Brakez Laboratory of Cell Biology & Molecular Genetics,
Faculty of Sciences, Ibn Zohr University, Agadir, Morocco

Siegmar-W. Breckle Department of Ecology, University of
Bielefeld, Bielefeld, Germany
Cylphine Bresdin Environmental Research Laboratory of the
University of Arizona, Tucson, AZ, USA
J. Jed Brown Center for Sustainable Development, College of
Arts and Sciences, Qatar University, Doha, Qatar
Isabel Cac¸ador Marine and Environmental Sciences Centre,
Faculty of Sciences of the University of Lisbon, Lisbon, Portugal
John Cheeseman Department of Plant Biology, University of
Illinois at Urbana-Champaign, Urbana, IL, USA

xvii


xviii

LIST OF CONTRIBUTORS

¨sener-Godt UNESCO Man and the Biosphere
Miguel Clu
Programme, Division of Ecological and Earth Sciences, Paris,
France
Salma Daoud Laboratory of Plant Biotechnologies, Faculty of
Sciences, Ibn Zohr University, Agadir, Morocco
Joann Diray-Arce Department of Microbiology and Molecular
Biology, Brigham Young University, Provo, UT, USA
Richard Doyle School of Land and Food, University of
Tasmania, Hobart, TAS, Australia
Bernardo Duarte Marine and Environmental Sciences Centre,
Faculty of Sciences of the University of Lisbon, Lisbon, Portugal

Hassan M. El Shaer Desert Research Center, Mataria, Cairo,
Egypt
Khalid Elbrik Faculty of Sciences, Ibn Zohr University, Agadir,
Morocco
Marı´a Ferrandis Department of Vegetal Biology, University of
Valencia, Valencia, Spain
Angelo Maria Gioffre` Department of Plant and Environmental
Sciences, Faculty of Science, University of Copenhagen, Ta˚strup,
Denmark
Edward P. Glenn Environmental Research Laboratory of the
University of Arizona, Tucson, AZ, USA
¨cel Institute of Environmental Sciences, Near East
Salih Gu
University, Lefko¸sa, Northern Cyprus
Bilquees Gul Institute of Sustainable Halophyte Utilization,
University of Karachi, Karachi, Pakistan
Ibtissem Ben Hamad Laboratoire des Plantes Extreˆmophiles,
Centre de Biotechnologie de Borj Ce´dria, Hammam Lif, Tunisia;
Institut des Energies de Demain, Universite´ Paris Diderot,
Sorbonne Paris Cite´, Paris, France
Karim Ben Hamed Laboratoire des Plantes Extreˆmophiles,
Centre de Biotechnologie de Borj Ce´dria, Hammam Lif, Tunisia
Abdul Hameed Institute of Sustainable Halophyte Utilization,
University of Karachi, Karachi, Pakistan
Marcus Hardie School of Land and Food, University of
Tasmania, Hobart, TAS, Australia


LIST OF CONTRIBUTORS

Gabriel Haros The Punda Zoie Company Pty Ltd, Melbourne,
VIC, Australia
Moulay Che´rif Harrouni Hassan II Agronomic and Veterinary
Institute, Agadir, Morocco
A.K.M. Nazrul Islam Ecology Laboratory, Department of
Botany, University of Dhaka, Dhaka, Bangladesh
Sven-Erik Jacobsen Department of Plant and Environmental
Sciences, Faculty of Science, University of Copenhagen, Ta˚strup,
Denmark
M. Ajmal Khan Institute of Sustainable Halophyte Utilization,
University of Karachi, Karachi, Pakistan; Centre for Sustainable
Development, College of Arts and Sciences, Qatar University,
Doha, Qatar
Peter Lane School of Land and Food, University of Tasmania,
Hobart, TAS, Australia
Joa˜o Carlos Marques Marine and Environmental Sciences
Centre, Faculty of Sciences and Technology, University of
Coimbra, Coimbra, Portugal
David G. Masters School of Animal Biology, The University of
Western Australia, Crawley, WA, Australia; CSIRO Agriculture,
Wembley, WA, Australia
Adele Muscolo Department of
University, Reggio Calabria, Italy

Agriculture,

Mediterranea

Brent Nielsen Department of Microbiology and Molecular
Biology, Brigham Young University, Provo, UT, USA

Hayley C. Norman CSIRO Agriculture, Wembley, WA, Australia
Suresh Panta School of Land and Food, University of Tasmania,
Hobart, TAS, Australia
Maria Rosaria Panuccio Department of
Mediterranea University, Reggio Calabria, Italy

Agriculture,

Juan Bautista Peris Department of Vegetal Biology, University
of Valencia, Valencia, Spain
Sergey Shabala School of Land and Food, University of
Tasmania, Hobart, TAS, Australia
Noomene Sleimi UR-MaNE, Faculte´ des Sciences de Bizerte,
Universite´ de Carthage, Tunisia

xix


xx

LIST OF CONTRIBUTORS

Naima Tachbibi Laboratory of Plant Biotechnologies, Faculty of
Sciences, Ibn Zohr University, Agadir, Morocco
Marı´a Rosa Ca´rdenas Tomazˇicˇ UNESCO Man and the
Biosphere Programme, Division of Ecological and Earth
Sciences, Paris, France
Kazuo N. Watanabe Gene Research Center, University of
Tsukuba, Tsukuba City, Ibaraki, Japan
¨ ztu

¨nir O
¨rk Botany Department, Science Faculty, Ege
Mu
University, Bornova-Izmir, Turkey


INTRODUCTION
The world population has been increasing steadily and has
reached seven billion whilst registering an increase of one billion during the last decade. One-sixth of the world population
inhabits arid or/and semi-arid regions where the per capita
availability of water is among the lowest in the world. Water
availability has remained constant globally, however, its utilization has increased many fold due to the increase in population.
Activities of humans to survive in these conditions could lead
to global warming, for example, through huge expenditure of
energy in the desalination of seawater for domestic purposes in
the Arabian Gulf region.
Gulf Cooperation Council countries suffer from severe water
scarcity and their natural resources are not sufficient for domestic usage. Therefore, using this scarce precious water for agriculture is not possible. This area is going through a period of
unprecedented development and consequently the population
is rising and annual water production through desalination is
also increasing rapidly. Qatar is striving hard to ensure food and
water security, as envisaged in Qatar National Vision 2030. Food
security cannot be achieved through conventional agriculture
but requires “out of the box” solutions. Halophytes are a group
of plants that are naturally equipped with the mechanisms to
survive under highly saline and arid conditions and produce
high biomass. This high productivity could be used as fodder,
forage, medicine, edible oil, and in some cases as food for
humans. An “International Conference on Halophytes for Food
Security in Dry Lands” was organized by the College of Arts and

Sciences Qatar University from May 12À13, 2014 to address the
issue of food security for Qatar and adjacent regions. The
themes of the conference were: (i) halophyte ethno-botany, traditional uses, nontraditional crop development, (ii) halophyte
research (ecology, bio-geography, eco-physiology, biochemistry,
genetics, molecular biology; chemistry; fodder value; animal
nutrition, pharmaceuticals and cosmetics, etc.) and education,
(iii) food and water security, environment management, conservation and global changes, (iv) stakeholders (farmers, donors,
investors, landowners, agro-industry), projects, pilot forms, network, etc. and (v) social, economic, human, and cultural aspects
of scientific research.

xxi


xxii

INTRODUCTION

This book addresses aspects of food security (particularly
biomass production under saline conditions) that cover the
themes of the conference. It also contains the communication
of innovative ideas, such as research into halophyte farming
with economic sustainability, as well as salt-tolerant plant utilization as a possible alternative to salt-sensitive crops. It is
hoped that the information provided will not only advance
vegetation science, but that it will truly generate more interdisciplinarily, networking, and awareness, and inspire farmers,
and agricultural and landscaping stakeholders, to seriously
engage in halophyte cash crop production in coastal and inland
saline areas, especially those with an arid climate.
M. Ajmal Khan, Munir Ozturk, Bilquees Gul,
and Muhammad Zaheer Ahmed

1
CHARACTERIZATION AND
FUNCTION OF SODIUM
EXCHANGER GENES IN
AELUROPUS LAGOPOIDES
UNDER NaCL STRESS
Muhammad Zaheer Ahmed1,2, Bilquees Gul1,
M. Ajmal Khan1,3 and Kazuo N. Watanabe2
1

Institute of Sustainable Halophyte Utilization, University of Karachi, Karachi,
Pakistan 2Gene Research Center, University of Tsukuba, Tsukuba City,
Ibaraki, Japan 3Centre for Sustainable Development, College of Arts and
Sciences, Qatar University, Doha, Qatar

1.1

Introduction

Soil salinization is the key issue in irrigated arid and semi-arid
areas that have substantial impact on plant productivity. To cope
with salinity, plants have developed several adaptive mechanisms
including altered growth pattern, osmotic adjustment, and ion
homeostasis (Flowers and Colmer, 2008). These complex traits are
extensively reported in both salt-sensitive (glycophytes) and saltresistant (halophytes) plants (Zhu, 2001; Tester and Davenport,
2003). Moreover, recent molecular studies indicate that halophytes have better ability to alter the expression of genes linked
with a wide array of plant processes which support them in surviving in saline areas (Maathuis and Amtmann, 1999; Zhu, 2001).
In this scenario, there is a need to enhance knowledge about the
multi-genic response of halophytes in NaCl to improve the salt

tolerance of conventional crops.
Halophytes can reduce Na1 toxicity in cytoplasm, minimize
water deficit, manage essential mineral deficiency and reactive
species damage when grown under salinity-affected soil in various ways (Blumwald, 2000; Chen et al., 2007; Cosentino et al.,
M.A. Khan, M. Ozturk, B. Gul, & M.Z. Ahmed (Eds): Halophytes for Food Security in Dry Lands.
DOI: />© 2016 Elsevier Inc. All rights reserved.

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2

Chapter 1 CHARACTERIZATION AND FUNCTION OF SODIUM EXCHANGER GENES

2010). The Na1 partitioning between the below- and aboveground biomass of plants is an important aspect for salinity
resistance (Flowers and Colmer, 2008). Grasses accumulate
lower amounts of Na1 in shoots compared to dicots (Marcum,
2008). Plants exclude Na1 from root to soil solution, regulate
its loading in vascular tissues, compartmentalize in the vacuole/apoplast and excrete it from above ground epidermal bladder cells to reduce its negative effect on metabolic processes
(Tester and Davenport, 2003; Flowers and Colmer, 2008;
Shabala, 2013).
The movement of Na1 into the vacuoles or toward apoplast
is enabled by the action of tonoplast and plasma membranebound Na1/H1 antiporters, respectively, that use the electrochemical gradient of H1 generated by H1 ATPases and
H1 PPase. Knowledge is available about the sequence of
Na1/H1 antiporters (NHX) (Apse et al., 1999; Shi et al., 2000;
Tao et al., 2002; Zhang et al., 2008), expression and function of
NHX genes when plants are exposed to salinity (Gaxiola et al.,
1999; Oh et al., 2009). Some reports highlight the improvement
in the salt tolerance of many crop plants by overexpressing
NHX genes (He et al., 2005; Xu et al., 2010).

Poaceae is the most economically important plant family
because 70% of all crops are salt-sensitive grasses. About 3.6
billion ha from 5.2 billion ha of the world’s agricultural land is
already salt-affected and not suitable for conventional crop
farming. In contrast, the demand for food is continuously
increasing and we expect to need to feed around nine billion
by the end of 2050 (Millar and Roots, 2012). However, extensive efforts are underway to improve the salinity tolerance of
conventional crops either through breeding or modern molecular techniques, but still no crop can tolerate half the level of
salinity of seawater. In such a scenario, a major breakthrough
in crop breeding for salinity tolerance is needed. Regulation of
the number, size, and shape of the salt-excreting structure—
trichome could be one such possibility. About 15% of
halophytic grasses excrete Na1 and Cl2 through bicellular
microhairs, which are present on the leaf surface (Adams
et al., 1998). Aeluropus lagopoides (Linn.) Trin. Ex Thw. is a
salt-excreting, salinity- (1000 mmol L21 NaCl; Gulzar et al.,
2003) and drought-tolerant (Mohsenzadeh et al., 2006) grass.
Therefore, it could be used as a model plant to improve the
salinity tolerance of crops like rice, wheat, and maize (Flowers
and Colmer, 2008). Detailed ecological and physiological studies on A. lagopoides have been carried out (Waghmode and
Joshi, 1982; Sher et al., 1994; Abarsaji, 2000; Gulzar et al.,


Chapter 1 CHARACTERIZATION AND FUNCTION OF SODIUM EXCHANGER GENES

2003). However, information related to the function of its Na1
transport genes in salinity is lacking. Therefore, the goals of
this study were: (i) to isolate the cDNA sequences of VNHX
and PMNHX from A. lagopoides; (ii) to observe the change in
the expression of both genes under saline condition; and (iii)

to explore the role of both genes in the salt tolerance of A.
lagopoides.

1.2
1.2.1

Materials and Methods
Plant Material

Tillers of A. lagopoides were collected from a population
located in coastal areas of Karachi, Pakistan and used for the
growth of new seedlings.

1.2.2

Isolation of the cDNA and Sequence
Analysis of VNHX and PMNHX

One-month-old plants were treated with half-strength
Hoagland culture solution containing 400 mmol L21 NaCl for 2
days. Total RNA was extracted using an RNAqueous Kit
(Ambion). The first strand of cDNA was synthesized from 1 µg
RNA (DNA free) with the help of protocol provided with cDNA
Takara RNA-PCR Kit (AMV; Ver 3.0). Polymerase chain reaction
(PCR) were performed using with a pair of primers: (P1: 50 TTC
ATC TAC CTG CTC CCG CCC ATC AT30 ; P2: 50 CCA CAG AAG
AAC ACG GTT AGA ATA CC30 ) for VNHX and (P3: 50 TTC ATC
TAC CTG CTC CCG CCS ATC AT30 ; P4: 50 CCA CAG AAG AAC
ACG GTT AGA ATR CC30 ) for PMNHX, which were designed
based on the conserved regions of previously reported Na1/H1

antiporter from other plants. PCR product was cloned through
TA cloning kit (Takara) and pGEM-T vector. After cloning, plasmid was extracted and used for sequencing. The sequencing of
50 and 30 un-translated regions of VNHX was performed using
P5: 50 GTT GTG AAT GAT GCC ACG TC30 ; P6: 50 GAG AGC AGG
AGA TCC CAA TC30 ; P7: 50 CCA CAG AAG AAC ACG GTT AGA ATA
CC30 and M13-primer: 50 GTT TTC CCA GTC ACG AC30 . After
amplification of the 30 and 50 regions, fragments were
sequenced and assembled to provide the full-length cDNA of
VNHX. The analysis of the VNHX and PMNHX sequences was
performed by DNA-Dynamo software and NCBI program.

3


4

Chapter 1 CHARACTERIZATION AND FUNCTION OF SODIUM EXCHANGER GENES

1.2.3

Growth Conditions and Harvest

Tillers of A. lagopoides were potted in plastic pots (26 cm
high 3 20 cm diameter) in prewashed field collected sand culture and sub-irrigated with half-strength Hoagland nutrient
solution (Hoagland and Arnon, 1950) to establish for 1 month.
Equal-sized plantlets were treated with different concentrations
(0, 150, 300, and 600 mmol L21) of NaCl. The concentration of
test solution was maintained every alternate day by distilled
water to compensate for evaporation; whereas all test solutions
were completely replaced after every fifth day.

Growth parameters (length of shoot and leaf, number of total
and senesced leaves) were recorded initially and at the end of
the experiment. Each plantlet was carefully removed from the
soil after 15 days of experiment and washed thoroughly. Roots
and shoots were washed and separated from each other before
treating with liquid N2. All samples were stored at 280 C.

1.2.4

Quantification of Gene Expression by
qRT-PCR

For quantitative real-time PCR (qRT-PCR), a pair of primers
were designed for PMNHX (PMN-F: 50 TAT CGA ATG GTG CTC
GGA AGA30 ; PMN-R: 50 AGC CCA GCC ACA GTA CCG ATA30 ) and
for VNHX (VNHX-F: 50 GCA GGT CCT CAA TCA GGA TG30 ;
VNHX-R: 50 ACT CCA AGG AAG GTG CTT GA30 ) by using the
gene sequence information of A. lagopoides. Expression of Actin
gene was used to normalize the data. The quantitative expression data of both genes was recorded on a Light CyclerCarousel-based System (ROCHE), while the analysis of data was
performed by software 4.0. All standard curves had R2 $ 0.99.

1.2.5

Measurement of Na 1 in Plant Sap

The press sap method was used (Cuin et al., 2009) to determine
the soluble fraction of Na1 in leaves and roots of NaCl-treated
plants. Sap was mixed thoroughly before preparing dilutions and
used for the determination of Na1 on atomic absorption spectrometer (AA-700; Perkin Elmer, Santa Clara, CA, USA).

1.2.6

Secretion of Na 1

Fully expanded young leaves of three plantlets were tagged
from each NaCl treatment (0, 150, 300, and 600 mmol L21). All
tagged leaves were prewashed 72 h before the final data


Chapter 1 CHARACTERIZATION AND FUNCTION OF SODIUM EXCHANGER GENES

collection. Leaves were rinsed with 2 mL deionized water and
collected in Eppendorf tubes and the rate of Na1 excretion was
determined by atomic absorption spectrometry. The area of
rinsed leaves was calculated by Image-J software version 1.45
( and data were expressed in µmol
Na1 cm22 per day.

1.2.7

Malondialdehyde Content

Malondialdehyde (MDA) content was determined in leaf
samples as an indicator of lipid peroxidation (Heath and Packer,
1986). An extinction coefficient of 155 mM21 cm21 was used to
calculate the MDA content in the supernatant while absorbance
was recorded at 532 and 600 nm wavelengths. The result of
MDA was expressed as µg mg21 FW.

1.2.8

Statistical Analyses

Statistical analysis was done by SPSS version 11.0 for
Windows (SPSS, 2001). Two-way analysis of variance (ANOVA)
was used to test for a significant (P,0.05) effect of NaCl on
growth, MDA, Na1 concentration, and expression data. A posthoc Bonferroni test was used to test for significant differences
between means. Correlation analysis was performed between
different parameters of A. lagopoides through SPSS. Graphs
were constructed with the help of SigmaPlot (11.0).

1.3
1.3.1

Results
Molecular Characterization of VNHX and
PMNHX

The full-length cDNA of VNHX contained 2353 bp including
a putative poly (A) addition signal site in the end of sequence.
Whereas, the un-translated region (UTR) of 50 and 30 consisted
of 337 and 393 bp respectively, the open reading frame (ORF) of
1623 bp encoded a protein of 540 amino acids with a theoretical
molecular mass of 59.36 kDa (Figure 1.1A). The cDNA sequence
of VNHX has been deposited at GenBank with the name
AlaNHX under accession number GU199336.1. Sequence
homology revealed a high degree of homology sequences of
AlNHX (VNHX) and putative vacuolar Na1/H1 antiporter of
other higher plants.

5


6

Chapter 1 CHARACTERIZATION AND FUNCTION OF SODIUM EXCHANGER GENES

Figure 1.1 Information from two isolated genes from Aeluropus lagopoides. (A) The cDNA and deduced amino
acid sequence of VNHX (AlaNHX), and (B) cDNA sequence of PMNHX.


Chapter 1 CHARACTERIZATION AND FUNCTION OF SODIUM EXCHANGER GENES

The “expressed sequence tag” (EST) of PMNHX contained
204 bp and showed a high degree of homology with previously
reported Na1/H1 antiporter located on the plasma membrane
of higher plants (Figure 1.1B). The cDNA sequence of PMNHX
has been deposited at GenBank under accession number
GW796824.1.

1.3.2

Growth

The number of leaves and plant height decreased significantly (P,0.01) with the increases in salinity. In addition, a substantial (P,0.0001) increase in leaf senescence was observed at
300 and 600 mmol L21 NaCl (Table 1.1; Figure 1.2).

1.3.3

Peroxidation of Lipid Membrane

MDA content was unchanged at up to 300 mmol L21 NaCl
treatment, whereas around a 40% increase was found when
plants were treated with 600 mmol L21 NaCl compared to nonsaline controls (Figure 1.1).

Table 1.1 Results of One-Way
ANOVA Showed the Effect of NaCl
on Different Parameters of
Aeluropus lagopoides
Parameters

df

Mean Square

F significance

Height of shoot
# of leaves
# of yellow leaves
MDA
Na1—secretion
Leaf—Na1
Leaf—VNHX
Leaf—PMNHX
Root—Na1
Root—VNHX
Root—PMNHX

3

3
3
3
3
3
3
3
3
3
3

1480.183
1318.175
32.458
2025.764
102.231
321064.380
55926.904
83948.736
407017.080
838020.333
9805.678

15.911Ã
8.901Ã
86.555ÃÃÃ
138.764ÃÃÃ
3.032Ã
158.165ÃÃÃ
95.712ÃÃÃ

76.645ÃÃ
293.535ÃÃÃ
67.553ÃÃ
22.467ÃÃ

Ã

P,0.05; Ã Ã P,0.001; ÃÃ Ã P,0.0001.
Values of correlation are provided with degree of significance.

7


a

60

70

a

60
b

50

50

40

40
b

30
20

c
c

10

30
d

20

c

10

0

0

21

21

14

b
a

7
0

a
ab

a

7

b

a
0

14

c

0
150

300

Increase in height
of shoot plant–1 (%)

70

Number of senescent
leaves plant–1

Increase in number
of leaves plant–1 (%)

Chapter 1 CHARACTERIZATION AND FUNCTION OF SODIUM EXCHANGER GENES

MDA
(µmol g–1 FW)

8

600

NaCl (mmol L–1)

Figure 1.2 Change in the growth and biochemical parameters [filled circles:
increase in number of leaves plant21; empty circles: increase in height of shoot
plant21; filled squares: malondialdehyde (MDA) content; empty squares: number
of senescent leaves plant21] of Aeluropus lagopoides treated with different
NaCl concentrations (0À600 mmol L21) for 15 days (n 5 3). Values with at least
one Bonferroni letter the same were not significantly different at P,0.05.

1.3.4

Flux in Na 1

Na1 content increased significantly (P,0.0001) in both
leaves and roots of A. lagopoides under NaCl treatment
(Table 1.1; Figures 1.3 and 1.4). Moreover, this increase was
approximately tenfold higher in plants treated with
600 mmol L21 NaCl than in nonsaline controls (Figures 1.3 and
1.4). In general, the amount of Na1 was similar in both parts of
plants except at 300 mmol L21 NaCl where roots accumulated a
higher amount of Na1 than leaves (Figures 1.3 and 1.4).

1.3.5

Secretion of Na 1

Sodium excretion from the leaf surface increased significantly (P,0.01) with increase in NaCl concentrations up to
300 mmol L21 NaCl, however, no difference was noted in the
Na1 secretion rate of plants exposed to 300 and 600 mmol L21
NaCl (Figure 1.5).


Chapter 1 CHARACTERIZATION AND FUNCTION OF SODIUM EXCHANGER GENES

600

800
450
600
300
400
150

Gene expression actin–1

Na+ in leaf tissue (mmol L–1)

1000

200
0
0

0

150
NaCl (mmol

300

600

L–1)

Figure 1.3 Bars represent the concentration of Na1 in leaf of Aeluropus
lagopoides treated with different NaCl concentrations (0À600 mmol L21) for 15
days (n 5 3). Change in the expression of genes in leaves was shown by line
graph (square and circle symbols were used for VNHX and PMNHX gene,
respectively). Values with at least one Bonferroni letter the same were not
significantly different at P,0.05.

Na+ in root tissue (mmol L–1)

1200
800

1000
800

600

600
400
400
200

Gene expression actin–1

1400

1000

200
0

0
0

150
300
NaCl (mmol L–1)

600

Figure 1.4 Bars represent the concentration of Na1 in roots of Aeluropus
lagopoides treated with different NaCl concentrations (0À600 mmol L21) for 15
days (n 5 3). Change in the expression of genes in roots was shown by line
graph (square and circle symbols were used for the VNHX and PMNHX genes,
respectively). Values with at least one Bonferroni letter the same were not
significantly different at P,0.05.

9


Chapter 1 CHARACTERIZATION AND FUNCTION OF SODIUM EXCHANGER GENES

c
b

c
a a

0.9

800
b

a
0.6

600
400
a

0.3
a

200

Rate of Na+ secretion
(µmol cm–2 d–1)

Leaf
Root

1000
Na+ in tissue (mmol L–1)

10

a

a

a

0.0

0
0

150
300

NaCl (mmol L–1)

600

Figure 1.5 Bars represent the difference in the concentration of Na1 between
leaves and roots of Aeluropus lagopoides plants treated with different NaCl
concentrations (0À600 mmol L21) for 15 days (n 5 3). Bonferroni letters were
used to show significant difference (P,0.05) between means of leaves and
roots within each salinity level. Salt secretion rate was showed by line graph
and Bonferroni letters were used to compare values among salinity
concentrations.

1.3.6

Gene Expression

The expression of AlaNHX (VNHX) gene was significantly
up-regulated in both leaves (P,0.0001; Table 1.1; Figure 1.3)
and roots (P,0.001; Table 1.1; Figure 1.4) of plants when treated
with NaCl. However, higher gene expression was observed in
roots than leaves, especially in plants treated with 300 and
600 mmol L21 NaCl (Figures 1.3 and 1.4). The expression of
AlaNHX gene was similar at 300 and 600 mmol L21 NaCl, where
it was approximately tenfold (root) and fourfold (leaf) greater
than the respective nonsaline controls (Figures 1.3 and 1.4).
A negative correlation (r2 5 0.79; P,0.05) was found between
the expression of AlaNHX and PMNHX genes in leaves
(Table 1.2). Expression of PMNHX gene increased significantly
(P,0.001) under NaCl treatment (Table 1.1; Figures 1.3 and 1.4).
PMNHX gene showed approximately threefold higher expression in leaves than roots (Figures 1.3 and 1.4). Gene expression

did not change in leaves under salinity except at 150 mmol L21
NaCl where substantial up-regulation was found (Figure 1.3). In
contrast to leaves, the maximum expression of PMNHX gene
was found in roots treated with 600 mM NaCl (Figure 1.4).


Table 1.2 Pearson Correlation Analysis Between Changes in
Different Parameters of Aeluropus lagopoides Under NaCl
Parameters

Leaf—
Na1

Leaf—
VNHX

Leaf—
PMNHX

Root— Root—
Na1
VNHX

Root—
PMNHX

Na1—
Secretion

MDA Total

leaves

Yellow
Leaves

Leaf—Na1
(Significance)
Leaf—VNHX
(Significance)
Leaf—PMNHX
(Significance)
Root—Na1
(Significance)
Root—VNHX
(Significance)
Root—PMNHX
(Significance)
Na1—Secretion
(Significance)
MDA
(Significance)
Total leaves
(Significance)
Yellow leaves
(Significance)

À

0.84

0.96

0.95

0.94

0.76

0.78

0.90

ÃÃ

ÃÃ

ÃÃ

Ã

Ã

0.84

À

2 0.40
ns
2 0.79

0.88

0.89

0.75

0.82

Ã

ÃÃ

ÃÃ

Ã

Ã

2 0.44
ns
À

2 0.48
ns
0.98

2 0.32
ns
0.85

2 0.60
ns
0.80

ÃÃ

ÃÃ

Ã

0.98

À

0.80

0.71

Ã

Ã

Ã

ÃÃ

ÃÃ

2 0.40
ns

0.96

2 0.79

À

0.88

ÃÃ

ÃÃ

0.95

0.89

ÃÃ

ÃÃ

2 0.44
ns
2 0.48
ns
2 0.32
ns
2 0.60
ns
2 0.26
ns

0.71

Ã

0.94

0.75

ÃÃ

Ã

0.76

0.82

Ã

Ã

0.78
2 0.86

0.55
ns
2 0.93
ÃÃ

Ã

0.90

0.77

ÃÃ

Ã

2 0.41
ns

Ã

ÃÃ

P,0.05; ÃÃ P,0.001; ns, nonsignificant.
Values of correlation are provided with degree of significance.

ÃÃ

0.85

0.80

ÃÃ

Ã

À

0.80

0.71

0.77

Ã

Ã

Ã

0.60
ns
2 0.85

0.56
ns
2 0.88

0.91

ÃÃ

ÃÃ

ÃÃ

2 0.74

Ã

0.78

0.78

0.94

Ã

Ã

ÃÃ

2 0.86

ÃÃ

0.77

0.55
ns
2 0.26
ns
0.60
ns
0.56
ns
0.91

Ã

ÃÃ

Ã

ÃÃ

À

0.57
ns
À

Ã

2 0.72

0.64
ns
0.90

0.57
ns
2 0.72
Ã

0.64
ns

2 0.93

ÃÃ

Ã

0.71

2 0.41
ns
0.78

Ã

2 0.85

ÃÃ

2 0.88

ÃÃ

2 0.74

2 0.63
ns
2 0.63 À
ns
0.90
2 0.81

ÃÃ

0.77

ÃÃ

Ã

Ã

0.78
Ã

0.94

ÃÃ

2 0.81

Ã

À


12

Chapter 1 CHARACTERIZATION AND FUNCTION OF SODIUM EXCHANGER GENES

1.4

Discussion

Survival of salt-excreting grasses under saline conditions
depends on the extent of Na1 accumulation in cytoplasm which
is the function of increase in the ability of Na1 exclusion, excretion and sequestration into vacuoles (Ahmed et al., 2013).
Sodium/hydrogen antiporter genes are considered to play an
important role in controlling Na1 flux, cytoplasmic pH and cell
volume (Mahnensmith and Aronson, 1985). To better understand
salt-tolerance mechanisms in A. lagopoides that survives successfully under highly saline conditions we cloned and characterized the cDNA of salt stress-related genes (PMNHX and VNHX
(AlaNHX)). A full-length cDNA was isolated from A. lagopoides
grown under saline conditions which was 2353 bp long including
the predicted ORF of 1623 bp long (338À1960 bp of full-length
cDNA) which encodes protein consisting of 540 amino acids.
Comparison of both cDNA sequences with other proteins indicates that AlaNHX shares a higher identity with AlNHX isolated
from Aeluropus littoralis (Zhang et al., 2008). Similarly, the EST of
PMNHX had shown greater homology with the SOS1 gene of
Phragmites australis (Takahashi et al., 2009). These data allowed
us to classify PMNHX and AlaNHX as new members of the
plasma membrane and vacuole Na1/H1 antiporter family and to
suggest that they might be involved in Na1 regulation.
Growth of grasses was reduced when exposed to salinity, even
if they survived in higher NaCl concentrations (Gulzar et al., 2003;
Barhoumi et al., 2007; Flowers and Colmer, 2008). Similarly, A.
lagopoides has the ability to survive in up to 1000 mmol L21 NaCl
but nonsaline conditions appear to be optimal for the production
of plant biomass (Gulzar et al., 2003). The negative correlation
between total number of leaves and Na1 content (r2 5 20.86:
P,0.001; Table 1.2), but a greater positive correlation between
Na1 and leaf senescence (r2 5 0.90: P,0.001; Table 1.2) was found.
A decreasing trend in the shoot length, leaf elongation, and leaf

emergence in higher salinities could be attributed to minimal Na1
accumulation in shoots (Torrecillas et al., 2003). A delay in the
emergence of new leaves and accelerated shedding of mature
leaves at 600 mmol L21 NaCl could be related to the specific ionic
toxicity, particularly Na1 and Cl2 (Rudmik, 1983). Halophytic
grasses usually employ mature leaf shedding and decreasing leaf
elongation rates that could help to reduce the Na1 transport
towards young and active plant tissues, but at the cost of reduced
biomass (Munns, 2002; Flowers and Colmer, 2008). In contrast, a
rapid growth reduction in 600 mmol L21 NaCl-treated plants is
due to oxidative stress (Sobhanian et al., 2010) indicated by higher