Salinity-induced expression of pyrrolline-5-carboxylate synthetase determine salinity tolerance in Brassica spp

ORIGINAL PAPER

Salinity-induced expression of pyrrolline-5-carboxylate synthetasedetermine salinity tolerance in Brassica spp

K. Chakraborty • R. K. Sairam • R. C. Bhattacharya

Received: 8 April 2011 / Revised: 26 March 2012 / Accepted: 29 March 2012 / Published online: 20 April 2012

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2012

Abstract The objective of the present study was to assess

the role of salinity-induced expression of pyrrolline

5-carboxylate synthetase (P5CS), P5CS activity, and pro-

line accumulation on salinity tolerance in Brassica geno-

types. A pot culture experiment was conducted with four

Brassica genotypes viz. CS 52, CS 54, Varuna, (B. juncea)

and T 9 (B. campestris) under control and two salinity

levels, i.e., 1.65, 4.50 and 6.76 dS m-1. Proline contents

increased with increasing levels of salinity, and the highest

content were recorded at post-flowering stage in CS 52 and

CS 54. Activity of P5CS recorded at flowering stage was

highest at higher level of salinity, with CS 52 and CS 54

recording highest activity. Gene expression of P5CS, which

regulates the synthesis of proline, was higher in CS 52 and

CS 54 under salt stress than Varuna and T 9. Comparison

of partial nucleotide as well as amino acid sequence

showed conserved domains, and inter and intra generic

relatedness of these genes. The study suggests that salinity-

induced expression of P5CS, pyrrolline-phosphate synthe-

tase activity and proline accumulation may serve as one of

the mechanism of salinity stress tolerance in Brassica

genotypes.

Keywords Brassica � Compatible solutes � Gene

expression � Osmolytes � Proline � Pyrrolline 5-carboxylae

synthetase � Salinity stress

Abbreviations

ECe Electrical conductivity of extract

MSI Membrane stability index

P5CS Pyrrolline 5-carboxylae synthetase

RWC Relative water content

Introduction

Soil salinity is a global problem affecting productivity on

about 80 million hectares of global arable land, especially

in regions with hot and dry climate, including Asia, Africa,

Australia, parts of North and South America, and Medi-

terranean Europe. The ability of a plant to cope with

salinity stress is an important determinant of crop distri-

bution and productivity in many areas. It is, therefore, very

important to understand the mechanism that confers toler-

ance to salinity stress (Gilbert et al. 1998). Salinity exerts

undesirable effects through osmotic inhibition as a result of

salts present in soil solution, which reduces the plant’s

ability to take up water, and thus leads to slower growth,

and ionic toxicity caused by excessive amount of sodium

and chloride ions entering through transpiration stream,

which eventually injures cells in the leaf tissues and may

further reduce growth (Munns et al. 2006). Recent studies

have shown existence of a salt overly sensitive (SOS)

pathway for the exclusion and sequestration of Na? ions

mainly consisting of SOS1, SOS2, SOS3, and NHX1 in

Arabidopsis thaliana and rice (Martınez-Atienza et al.

Communicated by M. Stobiecki.

K. Chakraborty � R. K. Sairam (&)

Division of Plant Physiology, Indian Agriculture Research

Institute, New Delhi 110 012, India

e-mail: [email protected]

R. C. Bhattacharya

National Research Center on Plant Biotechnology, Indian

Agriculture Research Institute, New Delhi 110 012, India

123

Acta Physiol Plant (2012) 34:1935–1941

DOI 10.1007/s11738-012-0994-y

2007; Chinnusamy et al. 2005). Recently, Chakraborty

et al. (2012) have shown existence of a similar mechanism

in Brassica spp.

Accumulation under salinity stress of osmolytes/com-

patible solutes like, proline, sugar, sugar alcohol, glycine

betaine etc. help in maintenance of osmoregulation as well

as protection of macromolecules (Yancey 2005). Some

osmolytes are essential elemental ions, such as K?, but the

majority are organic solutes. Compatible solute accumu-

lation as a response to osmotic stress is a ubiquitous pro-

cess in organisms as diverse as bacteria to plants and

animals. A major category of organic osmotic solutes

consists of simple sugars (mainly fructose and glucose),

sugar alcohols (glycerol and methylated inositols), and

complex sugars (trehalose, raffinose, and fructans) (Bohn-

ert and Jensen 1996). Others include quaternary amino acid

derivatives (proline, glycine betaine, b-alanine-betaine,

proline-betaine), tertiary amines 1,4,5,6-tetrahydro-2-

mehyl-4-carboxyl pyrimidine), and sulfonium compounds

(choline osulfate, dimethyl sulfonium propironate) (Nuccio

et al. 1999).

It is known for a long time that the concentration of

proline increases up to 100 times the normal level under

stress, which makes up to 80 % of the total amino acid

pool, in a large number of plant species. Abiotic stresses,

such as, cold (Hura et al. 2004), heat (Song et al. 2005), salt

(Kholova et al. 2009, Sairam et al. 2002, 2005), drought

(Zhu et al. 2005, Mohammadkhani and Heidari 2008), and

heavy metal (Handique and Handique 2009; Siripornadulsil

et al. 2002) causes significant increase in the proline con-

centration in a variety of plant species. Goudarzi and

Pakniyat (2009) reported that salinity induced proline

content in wheat could be used to screen tolerant and

susceptible genotype. The increase in proline content under

salt stress has been correlated with the increased activity of

D-pyrroline-5-carboxylate reductase (Madan et al. 1995)

and with the low activity of proline oxidase and proline

dehydrogenase (Girija et al. 2002). Proline is synthesized

from L-glutamate by the catalytic action of enzyme

D-pyrroline-5-carboxylate synthetase (P5CS). A positive

correlation between stress-induced expression of P5CS and

proline content has been reported in Opuntia streptacantha

(Silva-Ortega et al. 2008), and wheat and barley (Dong

et al. 2010). Transgenic tobacco, cotton, and potato plants

over-expression of P5CS genes have been reported to

increase the levels of proline and consequently growth,

biomass production, and under drought and salinity stress

conditions (Yamada et al. 2005; Parida et al. 2008; Hmida-

Sayari et al. 2005).

The objective of the present study was to investigate the

role of salinity-induced gene expression of P5CS, P5CS

activity and proline content on salinity tolerance in dif-

ferentially tolerant and susceptible Brassica genotypes.

Materials and methods

Plant material and growth conditions

An experiment was conducted in the net house of the

Division of Plant Physiology, Indian Agricultural Research

Institute, New Delhi, India during the winter season of

2009–2010 with four cultivars of Brassica viz. CS 52, CS

54, Varuna (B. juncea), and T 9 (B. campestris). Sowing

was done in 30 cm earthen pots filled with clay loam soil

and farm yard manure in 3:1 ratio. Prior to pot filling, the

pots were lined with 400 gauge polythene sheets to avoid

leakage of the solution from pots. Nitrogen, phosphorus,

and potash fertilizers were applied at the rate of

60:60:40 kg ha-1, respectively. The fertilizer dose was

calculated considering 3.24 9 106 kg of soil per hectare.

Salinity treatment was given to each pot having approxi-

mately 10 kg of air-dried soil in terms of 2.5 l of water (S0)

or 50 (S1), and 100 mM NaCl (S2) solutions. The electrical

conductivity of extract (ECe), which is mean of four esti-

mations made at three stages, viz., 1 month after sowing,

anthesis, and after harvest was 1.65, 4.50, and 6.76 dS m-1

for control (S0) and saline (S1 and S2) treatments. Both

control and salt-stressed plants were kept under well-irri-

gated condition, and re-irrigated as and when the soil water

status went below 75–80 % of field capacity.

The plant samples were recorded at (a) vegetative/pre-

flowering (25 DAS), (b) flowering, and (c) post-flowering

(25 DAA) stages. Leaf samples were taken from the third

fully matured leaf from the top in triplicate from three pots.

Sample from each replicate were analyzed twice, thus each

value is mean of six estimations.

For gene expression studies, the leaf samples were

collected at flowering stage from plants subjected to vari-

ous salinity treatments for 24 h only, and total RNA

extraction was done immediately after sample collection.

Proline estimation

Free proline content in the leaves was determined follow-

ing the method of Bates et al. (1973). Leaf samples (0.5 g)

were homogenized in 5 mL of sulfo-salycylic acid (3%)

using mortar and pestle. The material was filtered through

Whatman # 2 paper. Two millilitres of extract was taken in

test tube and to it 2 mL of glacial acetic acid and 2 mL of

ninhydrin reagent were added. The reaction mixture was

boiled in water bath at 100 �C for 30 min. After cooling

the reaction mixture, 6 mL of toluene was added and then

transferred to a separating funnel. After thorough mixing,

the chromophore containing toluene was separated and

absorbance read at 520 nm in UV-visible spectrophotom-

eter (Model: Specord Bio-200, AnalytikJena, Germany). A

blank and standards using L-proline were also run along

1936 Acta Physiol Plant (2012) 34:1935–1941

123

with the samples. Concentration of proline was estimated

by referring to a standard curve of proline.

D-pyrroline-5-carboxylate synthetase activity

For extraction of enzyme, 0.5 g of leaf tissue was grounded

with liquid nitrogen and 10 mL of extraction buffer con-

taining 50 mM Tris–HCl (pH 7.2), 10 mM MgCl2, 0.6 M

KCl, 3 mM EDTA, 1 mM DTT, 5% PVPP, and 1 mM

b-marcapto ethanol. After grinding, the homogenate was

filtered through four layers of muslin cloth. The filtrate

then centrifuged at 10,000g for 20 min at 4 �C. The

supernatant was taken as enzyme extract.

D-Pyrroline-5-carboxylate synthetase activity was

assayed by recoding the decrease in optical density due to

NADPH at 340 nm (Garcia-Rios et al. 1997). One millilitre

reaction mixture contained 100 mM Tris–HCl (pH 7.2),

25 mM MgCl2, 75 mM sodium glutamate, 5 mM ATP,

0.4 mM NADPH, and distilled water to make the volume

up to 1 mL. The reaction was started by addition of

enzyme extract. The reaction velocity was measured as the

rate of consumption of NADPH, monitored as decrease in

absorption at 340 nm as a function of time. One unit of

enzyme activity was defined as 0.01 changes in absorbance

per minute and specific activity as units per minute per mg

of protein.

Gene expression study by RT-PCR

The nucleotide sequences for P5CS were obtained from

National Centre for Biotechnology Information (http://

www.ncbi.nlm.nih.gov/). The Basic Local Alignment

Search Tool (Altschul et al. 1997; http://www.ncbi.nlm.

nih.gov/BLAST/) was used to identify the homologs of

candidate genes. For RT-PCR expression analysis and

cloning of cDNAs, the following oligonucleotide primers

were designed manually, and oligo quality (to avoid primer

dimmer, self dimer etc.), GC% and Tm were analyzed by

using Oligoanalyzer 3.0 tool (http://www.idtdna.com/

analyzer/Applications/OligoAnalyzer/, Intergrated DNA

Technologies, Coralville, IA 52241, USA).

Gene expression of P5CS was studied in leaf tissue.

Leaf samples were harvested from control and treated

plants and total RNA was extracted using RNAeasy kit

(Qiagen Inc., Chatsworth CA 91311, USA, Cat No:

749040) according to the manufacturer’s instruction. DNA

contamination was removed from the RNA samples using

DNase I (Qiagen Science, MA, USA). One micrograms of

total RNA was reverse transcribed using gene specific

primers and Qiagen one step RT-PCR kit. PCR conditions

were standardized using gene-specific primers for tubulin.

Linear amplification for semi-quantitative RT-PCR was

obtained with 27 cycles. Reactions were conducted using

My Genie 32 Thermal Block PCR (Bioneer, Korea) under

the following conditions: initial PCR activation step:

15 min at 95 �C, reverse transcription: 30 min at 50 �C,

denaturation: 1 min at 94 �C, annealing: 1 min at 60 �C for

P5CS and Tubulin, extension: 2 min at 72 �C, 27 cycles,

final extension: 10 min at 72 �C. The amplification prod-

ucts were electrophoresed on 1.2 % agarose gel at 120

volts in TBE buffer (0.4 M Tris–borate, 0.001 M EDTA,

pH 8.0) using known concentration DNA ladders. Gels

were stained with ethidium bromide and visualized on Uvi

Pro Gel Documentation system (Uvitec, England, UK).

The purified cDNA for each gene were cloned into

pTz57R/T vectors and transformed in Escherichia coli

(strain DH5a) cells. DH5a cells transformed with recom-

binant plasmid were selected based on antibiotic resistance

as well as a-complementation method. Ampicillin-resistant

putative recombinants were selected for further analysis.

Plasmid were isolated from the confirmed colonies and

restriction analysis was carried out by using KpnI and

HindIII enzymes flanking the cloning site of vector

pTz57R/T, to confirm the presence of cloned insert cDNA.

E. coli cells containing desired recombinant plasmid were

given to Xcelris Labs Limited, Bodakdev, Ahmadabad,

India, for sequencing the cloned insert cDNA.

The data collected on different parameters were sub-

jected to statistical analysis following the procedure

described by Cochran and Cox (1957). The critical dif-

ference (CD) was worked out where variance ratio was

found significant for treatment effect. The treatment effects

were tested at 5 % probability level for their significance.

Name Sequence Length (bases) GC % Tm (�C) Product size (bp)

BnP5CS-F CTA TCT TAC ACA AGG TGA TCA CTG 24 41.7 60.3 625

BnP5CS-R GTG CTT GCA TTG TGG ATA ACA G 22 45.5 61.8

Tubulin-F CAG CAA TAC AGT GCC TTG AGT G 19 57.9 60.0 360

Tubulin-R CCT GTG TAC CAA TGA AGG AAA GCC 24 50.0 62.2

Oligo concentration 1.0 lM, Na? concentration 50 mM

Acta Physiol Plant (2012) 34:1935–1941 1937

123

Results and discussion

The results obtained in the present study, conducted with three

B. juncea genotypes, i.e., CS 52 and CS 54 (tolerant), Varuna

(susceptible), and one B. campestris genotype T 9 (suscepti-

ble) revealed differential response to salinity stress. All the

varieties recorded a decline in relative water content (RWC),

membrane stability index (MSI), and yield. However, the

declines in RWC, MSI, and yield were significantly greater in

susceptible genotypes Varuna and T 9 when compared with

tolerant genotype CS 52 and CS 54, which could maintain

higher RWC, MSI, and seed yield even at 6.76 dS m-1

salinity level (data not presented). Salt stress has been found to

reduce both RWC and fresh weight significantly in different

Brassica genotypes; however, the degree of reductions was

different in different species (Siddiqui et al. 2008). Thomas

(1997) reported that cell-membrane stability is affected by

dehydration, oxidative stress, and Na-injury. Ashraf et al.

(1999) reported that the reduction in seed yield may also be

due to decreasing assimilates production associated with

decreased plant size and biomass.

Proline content increased in all the genotypes under

salinity stress, and also with age, i.e., compared to vege-

tative stage, proline accumulation was higher at flowering

and post flowering stages both in control and salt-stressed

plants (Fig. 1). CS 54 showed 160.62 and 97.65 %

increases, respectively, at flowering and post flowering

stages. However, at vegetative stage highest increase over

control plants was observed in CS 52 (75.25 %). Concen-

tration of proline increases in a large variety of plants under

stress, up to 100 times the normal level, which makes up to

80 % of the total amino acid pool under salt stress (Sairam

et al. 2002, 2005; Sumithra et al. 2006). Zhu et al. (2005)

have also reported increase in proline accumulation with

age. However, the magnitudes of increases were more in

the comparatively tolerant genotypes CS 52 and CS 54 than

susceptible genotypes Varuna and T 9. Proline content also

increased in the susceptible genotypes under salt stress,

which can be explained as one of the early response of all

plants towards any abiotic stress. Salinity-induced proline

accumulation has been reported in Indian mustard (Ahmad

2009), potato (Hmida-Sayari et al. 2005), and wheat (Khan

et al. 2009). Greater proline accumulation has been

reported in salinity tolerant maize hybrids (Kholova et al.

2009) and wheat genotypes (Sairam et al. 2002, 2005) than

the susceptible ones.

D-Pyrroline-5-carboxylate synthetase (P5CS) activity

recorded at flowering stage increased significantly in the

tolerant genotypes CS 52 and CS 54 (Fig. 2). The increases

were 152.11 and 205.63 % in CS 52 and 90.14 and

166.19 % in CS 54 under S1 and S2 treatments, respec-

tively, compared to control. However, only a small increase

in enzyme activity was observed in both Varuna and T 9

under salinity stress. P5CS, which is the key enzyme cat-

alyzing the synthesis of proline showed pattern similar to

proline content, as the activity was much higher in tolerant

cultivars CS 52 and CS 54 under salinity stress than Varuna

and T 9, which could be the reason for higher accumulation

of proline in these genotypes. Hong et al. (2000) have

reported relationship between P5CS activity and proline

content in Vigna aconitifolia. Salinity induced differential

increase in the proline content and activities of D-pyrro-

line-5-carboxylate synthetase have also been reported in

cotton (Parida et al. 2008).

In case of D-pyrrolline-5-carboxylate synthetase (P5CS)

gene, RT-PCR was performed with gene-specific primers

obtained from B. napus and expected amplicon size of

625 bp was obtained in all the four genotypes under all the

three treatments. There was little expression in control

plants of the four genotypes; however, there was progres-

sive increase in gene expression in tolerant genotypes (CS

52 and CS 54) with increase in salinity level. In case of

Varuna and T 9, there was very little increase in mRNA

expression with the increasing level of salinity treatment

(Fig. 3). The b-tubulin expression was almost constant in

all the genotypes, and did not change under control and

salinity conditions (Fig. 4).

Increase in proline content and activity of P5CS in the

salt-stressed plants of the tolerant cultivars CS 52 and CS

54 were associated with increase in the gene expression of

0

10

20

30

40

50

60

70

80

90

1.65 4.5 6.76 1.65 4.5 6.76 1.65 4.5 6.76POST FLOWERING STAGEFLOWERING STAGEVEGETATIVE STAGE

Pro

line

Co

nte

nt

(mg

g-1

DW

)

CS-52 CS-54 Varuna T9

Salinity levels (dS m )-1

-1

Fig. 1 Effect of different

salinity levels on proline content

at different growth stages in

Brassica genotypes. LSD

(P B 0.05). Vertical bars show

±SE of mean (n = 6)

1938 Acta Physiol Plant (2012) 34:1935–1941

123

P5CS. Less expression of P5CS under salt stress in sus-

ceptible genotypes like Varuna and T 9 could be the reason

for the observed lower activity of P5CS and less accumu-

lation of proline in these genotypes. Increase in P5CS gene

expression and proline content under salinity stress have

also been reported in cactus pear (Silva-Ortega et al. 2008)

and transgenic potato (Hmida-Sayari et al. 2005). The

expression of b-tubulin did not vary under control or

salinity stress conditions in all the genotypes. b-Tubulin is

a component of heterodimeric protein composed of two

closely related 55 kDa proteins called a and b-tubulin. The

sequences of these genes are highly conserved throughout

the eukaryotic kingdom. The expression of tubulin is not

much affected by environmental conditions, and therefore,

b-tubulin was used as an internal control.

Partial nucleotide sequences of 587, 590, 592, and 593 bp

were obtained in case of CS 52, CS 54, Varuna and T 9,

respectively. The partial nucleotide and deduced amino acid

sequences of P5CS were compared with A. thaliana and

B. napus using BLAST tool and CLUSTAL W (1.83) mul-

tiple alignment (Fig. 5a, b). All the four genotypes showed

approximately 90 and 97 % similarity with A. thaliana and

B. napus, respectively, while among the four genotypes, the

similarity was more than 98 %. This indicates that the RT-

PCR transcribed portion of the P5CS gene is highly

conserved in these genotypes. Conserved domains were

identified using ‘PROSITE’ (release 20.61) and the partial

amino acid sequence of P5CS showed one conserved

domain: PROA, gamma-glutamyl phosphate reductase sig-

nature (PS 01223) (amino acid residues 144–165 in CS 52,

CS 54, Varuna and T 9). Observed differences in secondary

structure, such as shorter length of the a-helix at the 30 end of

the sequences in B. juncea genotypes, viz., CS 52 and CS 54,

compared to longer in B. campestris T 9, and existence of

random coiled structure at the end after the last a-helix in CS

52 and CS 54, where Varuna has a b-turn in that position

could be having a bearing on more efficient enzyme protein

in tolerant genotypes than the susceptible ones.

From the foregoing discussion it is clear that tolerant

genotypes CS 52 and CS 54, which were able to maintain

higher RWC and MSI, and showed fewer declines in yield

under salinity stress, also accumulated significantly higher

concentration of proline than Varuna and T 9. Further the

proline accumulation was correlated with salinity induced

greater gene expression of P5CS and activity of pyrrolline-5-

carboxylate synthetase in these tolerant genotypes. Unlike

other compatible solutes, proline plays a major role via

osmotic adjustment and protection of macromolecules

against salinity stress in Brassica spp. It can thus be con-

cluded that tolerant genotypes CS 52 and CS 54 have inbuilt

mechanism in the form of greater gene expression and

activity of P5CS, whose product proline provides osmotol-

erance in the form of retention of moisture (higher RWC) and

MSI, resulting in more yield stability (less reduction).

0

0.5

1

1.5

2

2.5

6.764.51.65

Pyr

rolli

ne

carb

oxy

late

syn

thet

ase

ac

tivi

ty

(un

its

min

-1m

g-1

pro

tein

)

CS-52 CS-54 Varuna T9

Salinity levels (dS m-1)

Fig. 2 Effect of different salinity levels on pyrrolline-5-carboxylate

synthetase activity at flowering stage in Brassica genotypes. LSD

(P B 0.05). Vertical bars show ±SE of mean (n = 6)

Fig. 3 Gene expression of P5CS in leaves of Brassica genotypes

under different salinity levels. Tubulin was used as internal standard

(M marker, 1 control, T1 50 mM NaCl, T2 100 mM NaCl)

Fig. 4 Predicted secondary

structure of deduced protein of

P5CS of Brassica genotypes

(color code: red a-helix, 10;

yellow b-sheets, 10; blue b-turn,

13; grey random coil, 7)

Acta Physiol Plant (2012) 34:1935–1941 1939

123

Fig. 5 Clustal W (1.83) multiple sequence alignment and comparison

of partial coding (a) and deduced protein (b) sequences of P5CS in

leaf tissues in B. juncea genotypes CS 52 and CS 54 (tolerant),

Varuna and B. campestris genotype T9 with A. thaliana (GeneBank

Acc. Nos. NM115419.4 and NP191120.2) and B. napus (GeneBank

Acc. Nos. AF314812.1 and AAK01361.1) (Asterisk shows conserved

nucleotides; dark/bold letters show nucleotide polymorphisms)

1940 Acta Physiol Plant (2012) 34:1935–1941

123

Author contribution K. Chakraborty was the senior

research fellow working on the project, R.K. Sairam and

R.C. Bhattacharya were the PI and Co-PI of the project.

Acknowledgments K. Chakraborty gratefully acknowledges the

Council of Scientific and Industrial Research, New Delhi, India for

the award of senior research fellowship during the course of the study.

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