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Original Article

Effect of proline on Triticum aestivum (wheat) under thedrought conditions of salinity

Manisha Jain a, Elsa Mini Jos a, Deepika Arora b,Y.V.R. Kameshwar Sharma c,*aDepartment of Biological Sciences (III), Sri Venkateswara College, University of Delhi, Delhi 110021, IndiabDepartment of Botany (II), Sri Venkateswara College, University of Delhi, Delhi 110021, IndiacDepartment of Biochemistry, Sri Venkateswara College, University of Delhi, Delhi 110021, India

a r t i c l e i n f o

Article history:

Received 12 February 2013

Accepted 25 March 2013

Available online 18 July 2013

Keywords:

Osmolyte

Proline

Salt tolerance

Triticum aestivum

Stress

* Corresponding author. Tel.: þ91 (0) 11 2411E-mail address: sharmabiochem@gmail.c

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.05.002

a b s t r a c t

Background: The increasing salinity is a major problem in agriculture around the world. Salt

stress results in inhibition or reduction in plant growth which affects productivity. Also,

most of the crop plants like wheat are glycophytes, which are sensitive to even low salt

concentrations.

Hence it is important to find out the role played in plant salt tolerance by compatible

osmolytes such as proline. Proline appears to be the preferred organic osmoticumin many

plants. The proposed functions of proline under stress conditions include osmotic ad-

justments, protection of enzymes and membrane, as a hydroxyl radical scavenger, as well

as acting as a reserve of energy and nitrogen for utilization during exposure to salinity.

Method: Wheat (Triticumaestivum) seedlings were grown in salt solutions ranging from 0.5M

to 5MNaCl concentration for 7 days. The tissueswerehomogenized in 3%sulphosalicylic acid

toobtain theextract.Theextractwasmade toreactwithninhydrin inacidic conditions to form

a red chromophore which was read at 520 nm to determine the proline concentration.

Result: Experimental findings confirm the accumulation of proline under salt stress. There

is a linear correlation between the given salt stress, and the proline accumulated implying

the role of proline as a compatible osmolyte.

Conclusion: The development of salt tolerant crop varieties would have a huge impact in the

agricultural economy. Salt tolerant genotypeswould reducepressure onwater resources and

low quality water could be used for irrigation. Engineering crops to accumulate compatible

osmolytes like proline could be a nature friendly method of adapting plants to stress.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction creating a water stress in plants. Secondly, they cause se-

Wheat is an important food crop worldwide. High salt con-

centrations decrease the osmotic potential of soil solution

8590, þ91 9910374426 (moom (Y.V.R. Kameshwar S2013, JPR Solutions; Publi

vere ion toxicity, since Naþ is not readily sequestered into

vacuoles as in halophytes. Finally, the interactions of salts

with mineral nutrition may result in nutrient imbalances

bile); fax: þ91 (0) 11 24118535.harma).shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 5 0 6e5 0 9 507

and deficiencies.1 The consequence of all these ultimately

leads to inhibition of growth and development, reduction

in photosynthesis, respiration, and protein synthesis and

disturbs nucleic acid metabolism in wheat.2e5 Plants are

exposed to many types of environmental stress. Among

these stresses, osmotic stress, in particular, due to drought

and salinity is the vital problem that limits plant growth and

crop productivity in agriculture.6 Salt acts as a toxic sub-

stance that restricts plant growth the most. It is estimated

that salinity affects at least 20% of world’s arable land and

more than 40% of irrigated land to various degrees.7 Hence

there is an increasing need for salt tolerance in plants. So we

need to find out the prominent role in plant salt tolerance by

organic compounds such as proline.8 Based on their capacity

to grow on high salt medium, plants are traditionally clas-

sified as glycophytes or halophytes. Most plants, including

the majority of crop species, are glycophytes and cannot

tolerate high salinity. For glycophytes, salinity imposes ionic

stress, osmotic stress, and secondary stresses such as

nutritional disorders and oxidative stress. Sodium toxicity

represents the major ionic stress associated with high

salinity.7 For cells that successfully adapt to cellular dis-

turbances, especially water stress, three generalizations

have emerged. First, during short-term water loss cells often

restore volume with inorganic ions as osmolytes while

up-regulating stress (“heat-shock”) proteins,9e11 possibly

indicating disturbances in protein structures. Second, under

long-term water stress, organic osmolytes replace ions for

volume regulation, while stress proteins decline. High levels

of inorganic ions appear to be incompatible with long-term

normal protein function, as perhaps are stress proteins,

which may provide no protection against osmotic stress.12,13

Third, these solutes are limited to a few chemical types.14

Compatible osmolytes are potent osmoprotectants that

play a role in counteracting the effects of osmotic stress.

Osmolyte compatibility is proposed to result from the

absence of osmolyte interactions with substrates and co-

factors, and the non-perturbing or favorable effects of

osmolytes on macromolecular solvent interactions. The

compatible solutes may be classified into two categories:

one is nitrogen-containing compounds such as proline and

other amino acids, quaternary ammonium compounds and

polyamines and the other is hydroxy compounds, such as

sucrose, polyhydric alcohols and oligosaccharides. Proline

(Pro) is one of the most common compatible osmolytes in

water-stressed plants.6 Proline accumulation in dehydrated

plant tissues was first reported by Kemble and Mac Pherson

(1954) in wilted ryegrass.15 The accumulation of Pro has

been observed not only in plants but also in eubacteria,

marine invertebrates, protozoa, and algae.6 While several

amino acids are known to accumulate in response to os-

motic stress, proline apparently has a specific protective role

in the adaptation of plant cells to water deprivation and

appears to be the preferred organic osmoticum in many

plants.16,17 It helps in osmotic adjustment and protection of

plasma membrane integrity and acts as a sink of energy or a

reducing power, as a source of carbon and nitrogen, and/or

as a hydroxyl radical scavenger. Salinity stress may increase

activities of proline biosynthetic enzymes and/or inhibit

proline dehydrogenase (ProDH) activity.18

2. Applications

Studying salt stress is an important means to the under-

standing of plant ion homeostasis and osmo-balance. Salt

stress research, benefits agriculture as soil salinity signifi-

cantly limits plant productivity on agricultural lands.19 It is

evident from the literature that, properties of osmolytes

are becoming increasingly useful in molecular biology, agri-

culture, biotechnology and medicine.20,21 Transfer of genes

for osmolyte production from salt tolerant into salt-intolerant

species is being used to adapt plants for saline and drought

conditions in agriculture.22 A variety of other stresses

viz; oxidative, protein perturbing, etc. can also occur along

with water stress, and many osmolytes probably have

unique properties that protect cells from these disturbances,

either through cytoprotective metabolic reactions such as

anti-oxidation or stabilization of macromolecules through

wateresolute or soluteemacromolecule interactions.21

Among known compatible solutes, proline is the most

widely distributed osmolyte.17 Proline, which increases pro-

portionately faster than other amino acids in plants under

water stress, has been suggested as an evaluating parameter

for irrigation scheduling and for selecting drought-resistant

varieties.23 Stabilizers are used to prevent aggregation of IgG

molecules during manufacture and storage. Proline is used in

amino acid infusion material. A 3-h-intravenous infusion of

an amino acid mixture containing L-proline in healthy male

volunteers did not result in increased glucose release from the

kidneys24; implying that increased blood levels of glucose are

not anticipated following L-proline stabilized IVIG infusion.

From the literature, the present study intricacies to eluci-

date the role of osmolyte, accumulation of proline in wheat

under the drought conditions of sodium chloride to regulate

salt stress.

3. Materials and methods

3.1. Chemicals

Acid Ninhydrin, 3% Aqueous Sulphosalicyclic Acid, Glacial

Acetic Acid, Benzene, Proline and Sodium Chloride were used

of analytical reagent of standard company. Colorimeter

(Systronics, India) was used for measuring the absorbance to

detect the proline contents.

3.2. Methodology

3.2.1. Extraction of plant materialPlant material Triticum aestivum was treated with different

concentrations of sodium chloride ranging from 0.5 to 5.0 M

and the one without the treatment was considered to be

control. Plant tissue (0.5 g) was extracted by homogenizing in

3% sulphosalicyclic acid (10 mL) in the ratio of 1:20. It was

filtered through Whatmann Paper No.1.

3.2.2. Proline estimationTo the filtered extract, acetic acid and acid ninhydrin (Warm

1.25 g ninhydrin in 30 mL glacial acetic acid and 20 mL 6 M

a b

Fig. 1 e (a) and (b) Proline accumulation at various concentrations of NaCl.

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 5 0 6e5 0 9508

phosphoric acid) were added in the ratio 1:1 and then boiled

for 1 h. Reaction was terminated by placing in ice bath after

which 4 mL of benzene was added. Benzene layer was sepa-

rated and warmed to room temperature. The absorbance

values were determined at 520 nm.23,25

Standard curve was prepared using pure proline and used

for the detection of proline in the experimental conditions.

4. Results and discussion

Proline accumulation is one of the common characteristics in

many monocotyledons under saline conditions.26 It is well

documented that the accumulation of proline is a response of

plants to increased noxious elements.27 Among these, sodium

ion is known as the most prominent one.8

Very high accumulation of cellular proline (above 100% of

the total amino acid pool under stress as compared to just 5%

under the normal condition) has been earlier reported in

many higher plants species due to increased synthesis and

decreased degradation under the stress conditions such as

water, salt, drought and heavy metal.28

Seedlings of T. aestivum (wheat) was subjected to drought

conditions of salinity with different concentrations of NaCl

(0.5e5 M). Sample which was treated with 1.0 M NaCl showed

high accumulation of proline with 65 times of more than that

of the control, whereas at low saline conditions of 0.5MNaCl it

showed only 31.42% of proline. On increasing the saline con-

ditions it was found to be 84.28% and 98.57% at salt concen-

trations of 2.5 M and 5 M, respectively (Fig. 1).

Above the concentration of 1 M NaCl the decline of proline

accumulation at higher values might be some interference of

other amino acids with the colorimetric reading.

The standard plot was prepared using pure proline which

shows the amount of accumulation of proline under various

drought conditions of NaCl.

5. Summary and conclusion

From the above result we can conclude that there is accu-

mulation of proline in the plant under induced drought

conditions of salinity. The accumulation is greater at higher

concentration of sodium chloride. The expected linear in-

crease in colorimetric absorbance reading at 520 nmmay have

been affected by other interfering materials. Nevertheless, it

has been seen that proline is accumulated under water stress

and may have a role in protecting the plant, and helping in its

recovery when replenished with water at a later time.

Conflicts of interest

All authors have none to declare.

Acknowledgment

Authors are highly thankful to DBT for financial support and

Principal, Dr. P. Hemalatha Reddy for providing lab facilities to

work.

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