Agricultural

Communication

Biosci. Biotech. Res. Comm. 9(3): 445-456 (2016)

Expression analysis of salt stress related expressed

sequence tags (ESTs) from

Aeluropus littoralis

quantitative real-time PCR

S. H. Hashemipetroudi

1,4

*, G. Nematzadeh

, G. Ahmadian

, A. Yamchi

and M. Kuhlmann

Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and

Natural Resources University, PO Box 578, Sari, Iran

National Institute of Genetics Engineering and Biotechnology (NIGEB), Tehran, Iran

Department of Plant Breeding and Biotechnology, Gorgan University of Agricultural Sciences & Natural

Resources, Gorgan, Iran

RG Abiotic Stress Genomics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben,

Germany

ABSTRACT

Aeluropus littoralis is a monocot halophyte grass and provide valuable genetic resources for understanding the molecular mecha-

nisms of stress-responsive genes, and improving tolerance to abiotic stresses in economically important crops. In an attempt to

identify salt stressed responsive genes, 154 isolated expressed sequence tags (EST) from A. littoralis were bioinformatically ana-

lyzed and functionally annotated. Of the 129 assembled unique transcripts, 111 (86%) and 18 (14%) comprised of singletons and

contigs, respectively. Among them, 58.9% could be assigned a putative identity, 20.9% with hypothetical or unknown functions

and 20.9% showed no match with existing sequences. Expression pattern of 41 selected ESTs were estimated by quantitative real-

time polymerase chain reaction (qPCR) in two different tissues. Expression pro ling were undertaken in control and three time

point of salt stress (6hrs, 24hrs and one week) followed by three time point of recovery condition (6hrs, 24hrs and one week). In the

root, the genes of SAMDC, ISB1 (6hrs), PP2C and SelO, HsfA1a, TFC D, Katanin, F-box were signi cantly up-regulated relative to

control while LecRLKs, ARP, HP3, PICKLE, Utp20, SYP81, CIPK20, HAK18, VDAC3, SND1, NAP1, ISB1 (6hrs and 24hrs), NUC2,

MUT, HP1 and PIP1;3 showed down-regulation in given conditions. In the case of leaf tissue, the genes of PP2C, SelO, Utp20,

SND1, PITP, LecRLKs, STPK, KCNK12, HsfA1a, HAK18, NUC2, ARP, HP3 and ARP were signi cantly up- or down-regulated.

Differential regulation of these genes were observed in root and tissue which con rm their role in salt stress tolerance. This func-

tionally annotated EST and gene expression pro ling provide initial insights into the transcriptome of A. littoralis.

KEY WORDS:

AELUROPUS LITTORALIS

, GENE EXPRESSION, SALT STRESS, RECOVERY CONDITION

445

ARTICLE INFORMATION:

*Corresponding Author: shr.hashemi@sanru.ac.ir

Received 1

Aug, 2016

Accepted after revision 5

Sep, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

446 EXPRESSION ANALYSIS OF

AELUROPUS LITTORALIS

ESTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Hashemipetroudi et al.

INTRODUCTION

The global climatic changes, such as prolonged drought,

temperature change and increasing salinity, cause to

a serious challenge for agricultural production world-

wide, affecting plant growth and yield. Drought and

salinity are becoming particularly widespread in many

regions, and may cause serious salinization of more than

50% of all arable lands by the year 2050. Therefore it

is important to secure food production for a growing

world population by increasing the yield of crop plants

while resources become more restricted (Yamaguchi and

Blumwald, 2005, Pitman and Läuchli, 2002 and Mittler

and Blumwald, 2010).

The discovery of novel stress-responsive genes, iden-

ti cation of new cis-and trans- acting elements that are

involved in stress adaptation provide an opportunity for

generating stress tolerance crops (Cushman and Bohnert,

2000, Patnaik and Khurana, 2001, Ben-Saad et al., 2012

and Hashemi et al., 2016).

The use of wild plant species or halophytic rela-

tives has been considered in plant breeding programs

for developing salt and drought tolerant crops Utilising

such approach, Aeluropus littoralis can serve as a halo-

phyte model for identi cation and isolation of the novel

adaptation genes. Aeluropus littoralis is a perennial

monocot grass with the small haploid genome of 349

Mb, using the C

mechanism for carbon  xation (Wang,

2004). Aeluropus littoralis grows in dry salty areas or

marshes (Saad et al., 2011) and can survive where the

water salinity is periodically high (Mesléard et al., 1993)

and tolerate up to 1100 mM sodium chloride (Barhoumi

et al., 2007). Therefore, A. littoralis serves as valuable

genetic resource for understanding the molecular mech-

anisms of stress-responses in monocots, and can poten-

tially be used for improving tolerance to abiotic stresses

in economically important crops (Saad et al., 2010).

The process of identifying new genes and character-

izing their functions generally is done at three molecular

biology levels viz: genomics, transcriptomics and prot-

eomics. Transcriptome-based gene discovery in response

to environmental stress offers insights into the roles of

the transcriptome in the regulation of physiological and

biological responses (Gracey, 2007). Because these meth-

ods strictly clarify changes in transcript level, a com-

plex multi-component process, such as salt and drought

stress, can be broken into their basic element (Umezawa

et al., 2002). Partial cDNA isolation often known as

expressed sequence tags (ESTs) is the rapid and cost-

effective Transcriptome-based gene discovery method

that has become an ef cient approach for identifying of

coding regions in a wide spectrum of organisms.

Various techniques such as differential display PCR

(DDPCR) (Hubank and Schatz, 1994), cDNA-ampli ed

fragment length polymorphism (AFLP) (Bachem et al.,

1996), suppression subtractive hybridization (SSH)

(Diatchenko et al., 1996), serial analysis of gene expres-

sion (SAGE) (Velculescu et al., 1995), massively parallel

signature sequencing (MPSS) and recently whole tran-

scriptome pro ling (RNA-Seq) (Brenner et al., 2000)

have been used for EST isolation. By use of these tech-

niques a large number of genes expressed during differ-

ent developmental, differentiation and growth stages or

in response to a variety of biotic and abiotic stresses has

already identi ed in plants (Priya et al., 2012). It is clear

now, the most biological processes, growth and develop-

mental programming are regulated by the precise control

of genetic expression (Agarwal et al., 2008). Genome-

wide analyses of mRNA level showed that the expression

level of genes may be changed (up or down-regulation)

in response to different condition (Rabbani et al., 2003),

in some case differential regulation of speci c genes and

pathways can lead to adaptation of crop genotypes to

different abiotic stress (Aglawe et al., 2012).

To gain insight into these processes, it is necessary

to study patterns of gene expression. Quantitative real-

time polymerase chain reaction (qPCR) analysis is one of

the most currently used approaches for measuring gene

expression level (Gutierrez et al., 2008). The sensitivity,

speci city and simplicity of this technique is incompa-

rable with other methods such as Northern and in situ

hybridization, RNase protection assays and semi-quan-

titative reverse transcription- polymerase chain reaction

(RT-PCR) (Bustin, 2000).

In our pervious study (Fatemi et al 2016.), 154 ESTs

(relative to salt and drought stresses) have been isolated

from A. littoralis by cDNA-AFLP and their sequences

were deposited in dbEST database (NCBI; www.ncbi.nlm.

nih.gov/dbEST). The primary goal of this investigation

was to annotate and assign putative functions of 154

isolated ESTs. Also, expression pattern of selected ESTs

was analyzed in two different tissues of A. littoralis at

salt stress and recovery condition.

MATERIAL AND METHODS

Aeluropus littoralis seeds were collected from Isfahan

province (Roddasht region) in Iran and the sterilized

seeds plated on full strength MS medium (Murashige

and Skoog, 1962) with vitamins, 3% sucrose and 0.7%

agar (pH 5.8). The cultures were incubated in germina-

tor at 25 ± 2 C with 16 h light/8 h dark photoperiod at

100 mol m

-2

-l

photon  ux density using cool-white

 uorescent light. Two weeks after germination, the seed-

lings were transferred to hydroponic culture containing

Hoagland’s solution (Hoagland and Arnon, 1950). The

30 day-old seedlings were stressed in 600 mM of sodium

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EXPRESSION ANALYSIS OF

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ESTS 447

Hashemipetroudi et al.

chloride at six passages (received 100 mM sodium chlo-

ride per 48 hrs up to 600 mM). At the end of the sixth

passage, salt stress samples were collected at 6hrs (S1),

24hrs (S2) and one week (S3) time point. In order to

plant recovery, the remained plants were transferred to a

sodium chloride-free Hoagland’s solution, and then were

collected after 6hrs (R1), 24hrs (R2) and one week (R3).

Leaf and root were sampled in parallel. Control sam-

ples were taken from unstressed plants at the start of

the experiment. All samples were immediately frozen in

liquid nitrogen and stored at -70°C for RNA extraction.

The EST sequences of Aeluropus littoralis were

retrieved from EST database at NCBI and were analyzed

using the BLASTN, tBLASTX and BLASTX algorithms

(Zhang et al., 2000). The database of gene ontology

(http.//www.geneontology.org) was used to investigate

the molecular function of each EST and its role in bio-

logical processes as well as its location in the cell. After

selection of candidate reference genes, the gene-speci c

primers were designed using the Primer 3 software

(Rozen and Skaletsky, 1999), and were synthesized by

Metabion GmbH (Martinsried, Germany). All designed

primers had 18-24 length, GC content ranging from 42%

to 61% and similar melting temperatures (55-64°C). The

amplicon length ranged from 60 to 282 bp. The primer

sequences and GenBank accession numbers of related

genes are presented in Table 1. The primer speci city

was evaluated by melt curve analysis, and size of the

amplicons was tested by end-point PCR on 3% agarose

gels.

Total RNA was extracted using TRIzol reagent (Inv-

itrogen Life Technologies, Karlsruhe, Germany) accord-

ing to the manufacturer’s instructions. The quality and

quantity of the extracted ribonucleic acid was checked

by measuring absorbance at 260/280 nm using a Nan-

oDrop spectrophotometer (Biochrom WPA Biowave II,

UK). Further, the purity and integrity of RNA was tested

by running on 1.2% agarose gel electrophoresis. Resid-

ual gDNA contaminating RNA extracts was removed

by DNase treatment (DNase I RNase-free, Thermo Sci-

enti c, USA). The qPCR with three rDNA-based primers

has recently been applied for DNA contamination assay

by using RNA as template, (Hashemi et al., 2016).

The cDNA was synthesized using the QuantiTect

reverse transcription kit (Qiagen) according to the man-

ufacturer’s instructions. In brief, 1 L (200 ng) of treated

RNA, 1 l of RT primer mix (blend of oligo-dT and ran-

dom primers), 1 µl Quantiscript Reverse Transcriptase

(contains RNase inhibitor), 4 l Quantiscript RT Buffer

5X (includes Mg

and dNTPs) and 13 l of RNase-free

water were added and incubated at 65°C for 20 min and

then followed by incubation at 95°C for 3 min for inacti-

vation of reverse transcriptase. The  nal cDNA reactions

were diluted 1:10, and stored at -20°C. Targets were

ampli ed by the Maxima SYBR Green/ROX qPCR Master

Mix (Thermo Scienti c) with two-step cycling in CFX96

real-time PCR instrument (Bio-Rad, USA) according to

the company’s suggestions. The reaction master mix

prepared by adding the following components: 1L of

cDNA (50 ng), 5 l of 2X SYBR Green Master Mix and

0.3 l of 10 M of each primers and 3.4 l of RNase-free

water. Thermal cycling were performed using two-step

cycling protocol according to the company’s procedures

as follow: 10 min initial activation step at 95° C followed

by 40 cycles of 95° C for 15 sec and 60° C for 1 min.

Data acquisition were performed during the anneal-

ing/extension step. After ampli cation, all PCR reac-

tions were subjected to a thermal melt with continuous

 uorescence measurement from 55°C to 95°C for dis-

sociation curve analysis. Curves were analyzed by CFX

Manager (Bio-Rad) with single threshold cycle and sub-

tracted curve  t method. At least one non-template con-

trol (NTC) was used for each primer pair master mix. The

threshold cycles (Ct) were automatically calculated for

all reactions in the plate using the CFX manager soft-

ware (Bio-Rad). All assays were carried out in three rep-

lications. The mean values for each assay were obtained,

and used for further analysis. The livak (2

–∆∆Ct

) method

(Livak and Schmittgen, 2001) were used for calculation

of relative gene expression ratio. RT2 Pro ler PCR Array

Data Analysis software (SABiosystems) was used to con-

struct vocalno plot and clusterogram.

RESULTS

BIOINFORMATIC ANALYSIS AND SELECTING

CANDIDATE ESTS

In our pervious study, 154 A. littoralis ESTs from four

different library were isolated by cDNA-AFLP method,

and used in this study. EST sequences were retrieved

through Entrez Gene -EST database- at the National

Centre for Biotechnology Information (NCBI). The four

libraries names were LIBEST_028119 (69 ESTs with

GenBank accession number of JK191110.1-JK191042.1),

LIBEST_027583 (25 ESTs with GenBank accession num-

ber of JK671243.1-JK671267.1), LIBEST_027584 (34

ESTs with GenBank accession number of JK671209.1-

JK671242.1), and LIBEST_027576 (26 ESTs with Gen-

Bank accession number of JK671176.1-JK671201.1).

All ESTs were evaluated by the CAP3 DNA sequence

assembly program. Assembling of the 154 ESTs produced

a total of 18 assembled contigs and 110 singletons. Of

the 129 assembled unique transcripts, 111 (86%) and 18

(14%) comprised of singletons and contigs, respectively.

A total of 129 non-redundant dataset were compared

to the GenBank non-redundant database using BLASTX

448 EXPRESSION ANALYSIS OF

AELUROPUS LITTORALIS

ESTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Hashemipetroudi et al.

to assign putative function. Of all aligned sequences,

58.9% could be assigned a putative identity, 20.9% with

hypothetical or unknown functions and 20.9% showed

no match with existing sequences. The size of most

ESTs (79%) ranged from 100 to 400 bp. Sequence simi-

larity searches using BLASTX were performed to com-

pare the selected ESTs to subset of non-redundant pro-

tein sequences (nr) of green plant (taxid: 33090), Oryza

sativa, Zea mays, Arabidopsis thaliana, Brachypodium

distachyon, Setaria italica and Sorghum bicolor with

several blast e-value cutoffs. We found that the A. litto-

ralis transcript set (129 ESTs) showed a greater number

of sequence similarity matches with Setaria italica and

Zea mays transcripts. Based on functional annotation,

41 ESTs were selected for relative expression analysis in

root and leaf tissues at different time-point of salt stress

and recovery condition. The primer features are listed in

Table 1.

DNA CONTAMINATION ASSAY

For DNA contamination assay, the RNA samples have

been tested by qPCR with. In our pervious study, a new

procedure for testing DNA contamination was explained

(Hashemi et al., 2016). To monitor residual gDNA con-

tamination in RNA sample, the total RNA samples were

examined by three rDNA-based primer pairs in qPCR.

Generally, observation of any band on the agarose gel

or melting curve peak in qPCR analysis were considered

as gDNA contamination. In this study, all RNA samples

were tested by this procedure, and DNase-free RNA was

used for cDNA synthesis.

PRIMER VALIDATION

The cDNA synthetized from control and treatment sam-

ples was also tested by qPCR. Pooled cDNA tissue sam-

ples containing equal amounts of the cDNA from con-

trol and treatment conditions were used to determine the

primer pairs annealing temperature and their speci c-

ity. Primers annealing temperature were adjusted to 55-

60°C, and their speci city was checked by melt curve

analysis and electrophoresis in 3% agarose gels. Single

sharp peak with no primer-dimer was used for relative

expression analysis. From 41 ESTs tested in the melt

curve analysis, three of the genes (C-NAD-MDH2, TTL,

SAP and PI/PC-TP) were not ampli ed and excluded

from further analysis. The sharp peak of 5PTASE11 was

only observed in root samples while no peak was detect-

able in leaf samples. The C

value of CHR11 in root time

point were higher than 35 and therefore excluded from

analysis. For RBPL39 and HP2 genes, sharp peak were

ampli ed only in leaf time points while unambiguous

TFC D peak was only observed in root tissue. The trend

of regulated genes in root and leaf tissues are presented

in Figure 1.

ROOT TIME POINT ANALYSIS

Distribution of C

values ampli ed from root samples

showed that in control and salt stressed samples, C

value

of most genes were lower than 25. Percent distribution

of C

values in C

range of <25 in different time-points

including control. Salt Stress: S1, S2, S3, Recovery con-

dition: R1, R2 and R3 were 54.63%, 59.26%, 50.00%,

53.70%, 6.48%, 5.56% and 59.26%, respectively. These

values indicated that the mRNA level of most genes in

control and salt-stressed samples were higher than in

the mRNA level of recovered samples (except R3). For

normalization of expression levels in Aeluropus litto-

ralis, different set of reference genes as well as their

optimal number were recommended for root and leaf

samples (Hashemi et al., 2016).

The three genes namely, RPS3, EF1A and UBQ were

used as normalizer in root samples. For identi cation

of genes with statistically signi cant gene expression

changes, a volcano plot were used. Volcano plots are

used to look at fold change and statistical signi cance,

simultaneously (Allison et al., 2006).

In this study, expression values of 31 genes across

six time point of root samples were compared to control

samples by volcano plots (Figure 2). Values above the

blue line and outside of the vertical lines were deter-

mined to statistically signi cant fold changes with 95%

con dence (=0.05). Genes with fold change higher

than 2 or lower than -2 and p-value < 0.05 are indicated

in blue in Figure 2.

Based on volcano plot analysis, seven genes includ-

ing ZF30, URM12, CAND1, SPIKE1, TBC1, HP1, MTL1

and 5PTASE had not signi cant difference relative to

control group. At time point S1, the expression level of

SAMDC (3.1) and ISB1 (3.3) were higher while LecRLKs

(-12.1), ARP (-18.2) and HP3 (-4.8) were downregulated

(p-value < 0.05). Despite of the expression level of PP2C

(4. 2) and GlyI (-7) were higher and lower than 2 and

-2, but their fold changes were not signi cant (value

inside parentheses is fold change). In S2 time point, two

gene of PP2C (5.7) and SelO (4.8) were signi cantly

upregulated while PICKLE (-4.5), Utp20 (-3.8), SYP81

(-3.6), CIPK20 (-4.1), HAK18 (-3.8) and LecRLKs (-18.9)

were signi cantly downregulated (p-value < 0.05). PP2C

(13.4) and SelO (11.9) and SAMDC (4.1) were upregu-

lated in S3 time point while LecRLKs (-6.2) were signi -

cantly downregulated.

Under recovery conditions at time point R1, the

expression level of HsfA1a (3.2), SelO (18.9), TFC D

(81.1), SAMDC (11.1), Katanin (9.5), F-box (4.3) were

increased relative to control while, the genes of Utp20

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EXPRESSION ANALYSIS OF

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Hashemipetroudi et al.

Table 1: showing the primer features

Gene

symbol

Accession

number

Name Function E-value Sequence

PIP3;1 JZ191087 Plasma membrane intrinsic

protein

water channel activity 3e-63 TGTCATGGGCGTCTCCAAGT

GCAGTAGACGAGCGCGAAGA

VDAC3 JZ191051 Voltage dependent anion

channel 3

regulation of anion

transmembrane transport

1e-13 TCCAGACCCAGCTGAAGCAC

GCCTGGTACACCAAGATCCTCA

SYP81 JZ191048 Syntaxin of plants 81 Vesicle traf cking protein

that functions in the

secretory pathway.

2e-32 CAGCATGGCGTGGCTCTTAT

AGCATCTTGAAAGCGCATGG

NAP1 JZ191072 Nucleosome assembly

protein1

modulate chromatin structure

by regulation of nucleosome

assembly/disassembly

0.29 CAGGGCTCCACAAATCCAAC

ACGACCTGCTGAGTGCAAGC

CAND1 JZ191057 Cullin-associated and

neddylation-dissociated

promotes the exchange of the

substrate-recognition F-box

subunit in SCF complexes

2e-06 TGGCAGTGACTACAGCATACGG

ACTGCGCACAGAGCGGTACT

SAMDC JZ191058 S-adenosylmethionine

decarboxylase

Essential for polyamine

homeostasis, and normal

plant embryogenesis, growth

and development.

5e-14 CCATCCATGGTCCTGCTTTC

GGGTTGAAGCCCATGACCTC

Katanin JZ191064 Katanin p80 WD40 microtubule severing 1e-61 TGATCCCTCCCTTCCCAGTT

CCTGAGCGAATGCGTAAACC

F-box JZ191080 F-box protein Unknown 1e-60 TGCCCATGAACCATTGTACG

GCCCTGCAGATCAGGTCAAC

SND1 JZ191081 Staphylococcal nuclease

domain-containing protein

1-like

posttranscriptional gene

silencing by RNA, response

to salt stress

1e-18 GCGGATCTGGCAGTATGGAG

ACCGCTGCCTGAACAGACTT

GTF3C5 JZ191082 General transcription factor

3C polypeptide 5-like

Involved in RNA polymerase

III-mediated transcription

2e-37 TTCCAAGTGGCCATCAGGTT

AAAGGGCTTCCTGCCTCTTG

ISB1 JZ191092 Importin subunit beta-1 protein transporter activity 1e-42 GCTCCAGCCAAATGTCAAGC

GGTCTTGGTCAACAGCTTCAGG

NUC2 JZ191093 Nucleolin 2-like Involved in pre-rRNA

processing and ribosome

assembly

7e-06 AAGTCCAGTGTTGCGGTTGC

CCGCATTTCTCTTCCCCTTC

GlyI JZ191094 Glyoxalase I carbohydrate metabolic

process

0.079 GTGGCATGGACTTGCTACGG

CCGTGGCATCACAGAGGATT

CIPK20 JZ191099 CBL-interacting protein

kinase 20

protein serine/threonine

kinase activity

2e-09 CAGGAGATGAGGCCAGCACT

CTGTTGCTGTTGCTGCTTGG

HAK18 JZ191100 High-af nity potassium

transporter

potassium ion

transmembrane transporter

activity

7e-37 GGCCAGACATTTCAGACCACA

AGCCCTGATGACCGTGTTTC

ZF30 JZ191101 Zinc  nger CCCH domain-

containing protein 30

regulation of transcription 3e-08 GCTCTTGTTGGCTCCCCTCT

TCACCATTTACGCCCCAATC

URM12 JZ191103 Ubiquitin-related modi er 12 involved intRNA

modi cation

4e-17 ACTGCGATTGGGAGCTGTGT

CGTGGAGATGAAGACCACCA

5PTASE11 Jk671224 Inositol polyphosphate

5-phosphatase

response to abscisic acid,

response to auxin, response

to jasmonic acid

6e-11 CACATGGAACATGAATGGCAAG

TGAACTCCTTGCTCCGAAAAGA

PITP Jk671260 Sec14p-like

phosphatidylinositol transfer

family protein

transporter activity 3e-55 GAAAGTAAAGATTGCGGAGAC

GGGTGCGAACTCTGAAAC

SPIKE1 Jk671264 DOCK family guanine

nucleotide exchange factor

vesicle-mediated transport 1e-24 TAAACAACACGGTGGCAGGTA

GCTCCCCATCAAATGTCCATA

TBC1 Jk671226 TBC1 domain family member

5 homolog B

act as a GTPase-activating

protein for Rab family

protein(s)

3e-18 CGGGATGGGAGCAACAAC

CACGGATAAGGGCACTGGT

450 EXPRESSION ANALYSIS OF

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TTL Jk671266 tubulin-tyrosine ligase cellular protein modi cation

process

1e-05 AAGAGGCAGTATCCTAATCAC

AAACTCATTCTGCCAATCTA

KCNK12 Jk671259 potassium channel subfamily

K, member 12

potassium ion

transmembrane transport

1.5 TCGGAATCTGCCCTGAATCT

TATGTATCCCGGTCCACCACT

LecRLKs JK671176 G-type lectin S-receptor-like

serine/threonine-protein

kinase

Protein kinase activity 5e-16 CGGCCGACAATGGGTGAAG

GGGCATGCCAACCTCCTGTAG

SAP JK671180 Putative senescence-

associated protein

- 2e-11 TGACACACCCCACACATACAA

GGTTTAGACCGTCGTGAGACAG

ARP JK671182 Auxin-repressed protein - 6e-08 GGAAGTTTTGGGCTGTCTTTA

ATTTCGATGTTGCCTACTCTCTA

HP1 JK671187 Hypothetical protein1 Similar to F-box family

protein

1.5 CCAACAACTCAGCTCCAA

GATGTGAAAATAAGCACGCTA

MTL1 JK671192 Mitochondrial translation

factor 1

Mitochondrial protein

translation and group II

intron splicing

6e-50 ATTTCGGCAAAAGGAATGGAG

GAAGCTTGATGAGGCGACAGA

HP2 JK671195 Hypothetical protein2 - 0.006 GTTTGGGCATTGGGTCCTCAAGT

CGAGCAACAGCAGCAAGAGCAC

MUT JK671196 Mutator-like transposase Transposable element gene 3e-18 GATGCCCATCTTGACAATAC

GCAGTGGGGAAGTTGATTT

HP3 JK671200 Hypothetical protein3 - 0.003 GGAAGTTTTGGGCTGTCTTTAC

AGATTTCGATGTTGCCTACTCTC

HsfA1a Jk671211 heat shock factor A1a transcription factor activity 4e-10 GCAGTGCCCAGTTGTCTT

TTGGGCCTGGTGTCATA

PP2C Jk671236 Protein phosphatase 2C protein serine/threonine

phosphatase activity

6e-34 TAATATGCAGGGGAGGAAA

CAGCGAGTACACCACCAA

C-NAD-

MDH2

Jk671223 Malate dehydrogenase Cytosolic-NAD-dependent

malate dehydrogenase 2

4e-20 AAAACGTCGTTCAAAGAG

GCCATAAGATCCGTCAG

5PTASE11 Jk671224 Inositol polyphosphate

5-phosphatase

response to abscisic acid,

response to auxin, response

to jasmonic acid

6e-11 CACATGGAACATGAATGGCAAG

TGAACTCCTTGCTCCGAAAAGA

HsfA1a Jk671211 heat shock factor A1a transcription factor activity 4e-10 GCAGTGCCCAGTTGTCTT

TTGGGCCTGGTGTCATA

PP2C Jk671236 Protein phosphatase 2C protein serine/threonine

phosphatase activity

6e-34 TAATATGCAGGGGAGGAAA

CAGCGAGTACACCACCAA

C-NAD-

MDH2

Jk671223 Malate dehydrogenase Cytosolic-NAD-dependent

malate dehydrogenase 2

4e-20 AAAACGTCGTTCAAAGAG

GCCATAAGATCCGTCAG

PI/PC-TP Jk671213 Putative

phosphatidylinositol/

phosphatidylcholine transfer

protein SFH8-like

phosphatidylinositol

transporter activity,

transporter activity

6e-13 TTGGCACATGCTTCCACATC

AGGACTGCCCCATCCATCAT

PICKLE Jk671232 CHD3-type chromatin-

remodeling factor

DNA helicase activity 1e-37 AGGGGTATGCTGAACTTGT

CACCTTCGCCTCAATAA

RBPL39 Jk671237 RNA-binding protein 39-like mRNA processing 3e-05 GGTGCCACTGGTCTGA

AAAGGGGAAGCTACAGGAG

SelO Jk671243 Selenoprotein O-like transferase activity 3e-13 TCAAGGGTAGCGGAAAGAC

GGATGCTGCTGCGTAGAAC

TFC D Jk671246 tubulin folding cofactor D GTPase activator activity 4e-25 TAAAAGATGCCGCAACATA

GAAGGTGGGGAGCAAG

CHR11 Jk671250 chromatin-remodeling

protein 11

ATP-dependent chromatin

remodeling, nucleosome

binding

7e-119 CGCTGTTTTCTCTTTGATT

CGCTTTTGCCCTATTCTA

Utp20 Jk671251 small subunit processome

component 20 homolog

rRNA processing, associates

with U3 snoRNA.

2e-60 -CTTTCAGTTGCGTTTAGATGT

CGCTTTCAGAAGTGATAAGG

STPK Jk671258 Serine/threonine-protein

kinase ULK4-like

protein kinase activity 1e-32 CATTTTCTGCCACTGTATCCT

ACTTTTACACAACCATGCTCC

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EXPRESSION ANALYSIS OF

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(-533.5), VDAC3 (-4.7), SND1 (-5.5), NAP1 (-10.5), ISB1

(-5.9), NUC2 (-11.7), HAK18 (-6.9), ARP (-6.3), MUT

(-5.9) showed downregulation in mRNA level. The genes

found to be upregulated at time point R2 were similar

to that of time point R1. HsfA1a (3.4), SelO (14.1), TFC

D (49.6), SAMDC (5.5), Katanin (7.6), F-box (3) showed

upregulation in R2 time point. A number of eleven genes

including Utp20 (-223.1), SYP81 (-5.7), SND1 (-15.4),

FIGURE 1: Trend of regulated genes during different time point of salt stress and recovery condition.

452 EXPRESSION ANALYSIS OF

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Hashemipetroudi et al.

FIGURE 2: Volcano plots of the fold changes and p-values distribution. A

total number of 31 genes were tested in the root salt-stressed and recov-

ered samples. The central vertical line represents a fold change of 1 (no

change) and two another vertical lines represent 2 fold upregulation or

downregulation cutoffs. The horizontal blue line represents the p-value

cutoff for identi cation of genes that are statistically signi cant. Value

inside parentheses is fold change.

NAP1 (-16.8), ISB1 (-3.7), NUC2 (-5), CIPK20 (-5.3),

HAK18 (-11.3), LecRLKs (-7.6), ARP (-3.3), HP1 (-4.1)

showed downregulation in R2 time point. In the R3 time

point, three genes of PIP1;3 (-9.9), ARP (-26.8) and HP3

(-4.8) only showed downregulation in mRNA level.

Visualization of gene expression differences among

different time point was done by RT2 Pro ler PCR

Array Data Analysis program (Figure 3 A). The result

of the cluster analysis is depicted in a clustergram that

shows all 31 genes analyzed and the magnitude each

is expressed in control and 6 different root time points

including group 1 (S1), group 2 (S2), group 3 (S3), group

4 (R1), group 5 (R2), group 6 (R3). Four major clusters of

genes were observed: cluster I and II contained upregu-

lated genes and cluster III and IV contained downregu-

lated genes.

LEAF TIME POINTS ANALYSIS

Distribution of Ct values in leaf samples showed that the

most of genes had Ct range of 25-30. Percent of Ct values

in range of 25-30 in different time-points including control,

S1, S2, S3, R1, R2 and R3 time-points were 57.41%, 61.11%,

42.59%, 48.15%, 50.00%, 45.37% and 54.63%, respectively.

Comparing of Ct values in root and leaf samples showed

that the mRNA level in root samples were higher than leaf

samples. The geometric mean of U2SURP and GTF Ct val-

ues were chosen as normalizer in leaf samples.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EXPRESSION ANALYSIS OF

AELUROPUS LITTORALIS

ESTS 453

Hashemipetroudi et al.

FIGURE 3: Clustergram of the magnitude of gene expression for 31 genes analyzed. Light green represents

minimal gene expression, and red indicated maximum gene expression as indicated by the legend.

454 EXPRESSION ANALYSIS OF

AELUROPUS LITTORALIS

ESTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Hashemipetroudi et al.

Similar to root analysis, expression values of 31

genes across 6 time point of leaf samples were com-

pared to control samples by volcano plots (Plots have

not shown). Based on volcano plot analysis, expres-

sion level of 18 genes including ZF30, URM12, TBC1,

HP1, MTL1, PICKLE, PIP1;3, VDAC3, SYP81, NAP1,

CAND1, SAMDC, Katanin, F-box, ISB1, GlyI, CIPK20,

HP2, MUT, RBPL39, SPIKE1 were unchanged relative

to control group in different leaf time point.

In S1 time point, PP2C (3.4) were upregulated while

NUC2 (-3.3), LecRLKs (-9.5), ARP (-9.9) and HP3 (-3.8)

and STPK (-6.7) were signi cantly downregulated

(p-value < 0.05). In S2 time point, SelO (8.9) were sig-

ni cantly upregulated while LecRLKs (-7.5), STPK

(-12.3) and KCNK12 (-4.7) were signi cantly downregu-

lated. In S3 time point, only upregulation of PP2C (3.4),

SelO (8.9) and Utp20 (-533.5) were observed among all

analyzed genes. PP2C (3.4), SND1 (3.1) and PITP (8.9)

showed upregulation in R1 time point while ARP (-7.5)

were signi cantly downregulated. In the R2 time point,

the expression level of PP2C (6.5), LecRLKs (3.5), STPK

(5.1) and KCNK12 (4.1) were signi cantly were increased

relative to control. Finally, HsfA1a (3.4), HAK18 (-6.9),

PITP (8.9) showed upregulation in R3 time point. The leaf

clustergram were presented in Figure 3 B. Three major

clusters of genes were observed. The most signi cantly

up or down-regulated genes allocated into cluster II. This

cluster represents genes reacting to recovery conditions.

DISCUSSION

The development of salt or drought-adopt crops either

through the use of the crops wild relatives as genetic

resources or domestication of naturally tolerant spe-

cies have been proposed as a strategy to face with the

environmental challenges. Halophytes as crops naturally

salt-tolerant species are now being promoted in agri-

culture, particularly to provide forage, medicinal plants,

aromatic plants (Flowers et al., 2010). Although, improv-

ing crop salt tolerance by genetic engineering is not

easy, halophyte germplasm can furnish “climate-ready”

genes for plant breeding program (Jaradat, 2010). Differ-

ent aspects of A. littoralis properties such as life style,

morphological, anatomical, ecological, physiological

and molecular characteristics have been investigated so

far (Hashemi et al., 2013, Hashemi-Petroudi et al., 2014).

In the present study, we have focused on gaining

insight on differential regulation of some responsive ESTs

in response to salt stress and recovery condition. Expres-

sion pattern of 41 selected ESTs were estimated by qPCR

in root and leaf tissue. In the root, the genes of SAMDC,

ISB1 (6hrs), PP2C and SelO, HsfA1a, TFC D, Katanin,

F-box were signi cantly up-regulated relative to con-

trol while LecRLKs, ARP, HP3, PICKLE, Utp20, SYP81,

CIPK20, HAK18, VDAC3, SND1, NAP1, ISB1 (6hrs and

24hrs), NUC2, MUT, HP1 and PIP1;3 showed down-reg-

ulation in given conditions. In the case of leaf tissue, the

genes of PP2C, SelO, Utp20, SND1, PITP, LecRLKs, STPK,

KCNK12, HsfA1a, HAK18, NUC2, ARP, HP3 and ARP

were signi cantly up- or down-regulated. Interestingly,

expression of some genes was induced by salt stress while

also signi cantly repressed by recovery condition. Here

interestingly the transcriptional regulator HsfA1a could

be found. HsfA1a was formerly described to be a main

component of the heat and drought stress response (Liu et

al., 2013, Wang et al., 2015).

In this study, the protein phosphatase 2C (PP2C)

were activated in salt stress and recovery condition. The

PP2Cs from various organisms have been implicated to

act as negative modulators of protein kinase pathways

involved in diverse environmental stress responses and

developmental processes (Xue et al., 2008). The SYP81

showed down regulation in given conditions. Syntax-

ins (with the exception of syntaxin 11) are transmem-

brane proteins which their functions respect to organ-

ism growth, physiology and development are not well

known (Teng et al., 2001). The coexpression analysis

showed that, the most signi cantly up or down-regu-

lated genes allocated into cluster II (leaf tissue). Genes

found in Cluster II are interesting candidates for physi-

ological reactions related to the recovery of the plant

after salt stress. This cluster represents genes reacting to

recovery conditions.

CONCLUSION

In this study we bioinformatically analyzed the 154 ESTs

from A. littoralis and the functionally annotation showed

that 58.9% of ESTs had a putative function, 20.9% were

hypothetical or unknown functions and 20.9% showed

no match with existing sequences. The qPCR expression

analysis of 41 selected ESTs showed different regula-

tion in leaf and root tissue. The gene expression pro l-

ing has done in this study will also provide insight into

the role of selected ESTs in different time points of salt

stress and recovery condition. Differential regulation of

these genes also point at their role in salt stress tolerance

in plant. These information facilitate understanding the

molecular mechanisms of stress related genes and could

be used as valuable starting point for further research

on these genes.

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