EFFECT OF PLANT DERIVED SMOKE SOLUTION ON

PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION

OF MAIZE (Zea mays L.) UNDER SALT STRESS

by

MUHAMMAD ABDUL WAHEED SHAH

DEPARTMENT OF BOTANY

KOHAT UNIVERSITY OF SCIENCE & TECHNOLOGY, 26000

KOHAT

2019

EFFECT OF PLANT-DERIVED SMOKE SOLUTION ON PHYSIOLOGICAL

AND BIOCHEMICAL CHARACTERIZATION OF MAIZE (Zea mays L.) UNDER

SALT STRESS

Thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY (PhD)

In

Botany

By

MUHAMMAD ABDUL WAHEED SHAH

(Registration No. BO 420092001)

Department of Botany

Kohat University of Science & Technology, Kohat-26000

Khyber Pakhtunkhwa, Pakistan

(2019)

ACKNOWLEDGMENTS

This thesis becomes a reality with the kind support of many individuals. I would like to

extend my sincere thanks to all of them.Foremost, I want to offer this endeavor to our Almighty

Allah for the wisdom he bestowed upon me, the strength, peace of my mind and good health for

continuing this work.I would like to express my gratitude towards my family for the

encouragement that helped me in completion of this work. I am thankful to my parents, wife

and my child who helped me in successful completion of this research work.

I am highly indebted to the faculty of Botany department for their guidance and constant

supervision as well as for providing necessary information regarding this research and also for

their support in completing this endeavor. I would like to express my special gratitude and

thanks to my supervisor Dr. Shafiq ur Rehman and Co-supervisor Dr. Muhammad Jamil and Dr

Muhammad Daud khan for imparting their knowledge and expertise in this study.

My thanks and appreciations also go to my colleagues PhD scholar who has willingly

helped me out with their abilities.

MUHAMMAD ABDUL WAHEED SHAH

Dedication

Every challenging work needs self effort as well as

guidance of elders especially those who were very close to

our heart.

My humble efforts I dedicate to my sweet and lovely

Father and Mother

Whose efforts, love, encouragement and prays of day and

night make me able to get such success and honor,

Along with all hard working and respected

Teachers.

TABLE OF CONTENTS

S.NO Page

No

Acknowledgments

Abstract

CHAPTER 1 INTRODUCTION

1-16

1.1 Fire as a germination cue

1

1.2 Smoke, as a regulator

1

1.3 Role of Smoke as a germination cue

1

1.4 Mechanism of action (compound in smoke)

3

1.5 Effect of smoke on seedling vigour and plant growth

4

1.6 Effect of smoke on some other biological processes

4

1.7 Applications of smoke technology

4

1.8 Effects of salinity on crops yield

5

1.9 Plant growth under salt stress

5

1.10 Osmotic adjustment

5

1.11 Specific ion toxicity

6

1.12 Physiological and biochemical processes

7

1.13 Ion analysis

8

1.14 Chlorophyll and Total carotenoids

9

1.15 Protein and nitrogen contents

10

1.16 Proline induces the expression of salt-stress

11

1.17 Reactive Oxygen Species (ROS) and antioxidants

12

CHAPTER 2 Materials and methods

17-22

2.1 Seeds collection

17

2.2 Collection of plant

17

2.3 Preparation of smoke solutions

17

2.4 Dilutions of concentrated smoke solutions 18

2.5 Experiements designed

18

2.6 Surface sterilization

18

2.7 Priming of seeds

18

2.8 Germination experiment

18

2.9 Germination test

18

2.10 Root shoot length

18

2.11 Root shoot fresh weight

18

2.12 Root shoot dry weight

19

2.13 Sand pot experiment

19

2.14 Biochemical analysis

19

2.15 Determination of ions

19

2.16 Determination of photosynthetic pigment

19

2.17 Determination of nitrogen and protein content 20

2.18 Proline assay

20

2.19 Quantification of MDA contents and ROS (H2O2)

21

2.20 Total soluble proteins and antioxidants status

21

2.21 Statistical analysis 22

CHAPTER 3 RESULTS 23-39

3.1 Effect of plant-derived smoke solutions on seed germination

23

3.2 Effect of plant-derived smoke solutions on seedling growth

24

3.3 Effect of plant-derived smoke on root and shoot fresh and dry

weighs

25

3.4 Effect of plant-derived smoke water on different ions contents

Na+, K+ and Ca+2

27

3.5 Effect of plant-derived smoke water on chlorophyll and total

caroteniods

29

3.6 Effect of plant-derived smoke water on total nitrogen and protein

contents

31

3.7 Proline contents

32

3.8 Soluble protein contents

32

3.9 H2O2 concentration

33

3.10 APX activity of Maize (Azam variety) under salt stress

34

3.11 POD activity of Maize under salt stress

34

3.12 CAT activity of Maize under salt stress

35

3.13 Thiobarbituric acid reactive substances activity

36

3.14 Correlations

37

CHAPTER 4 DISCUSSION 40-47

CHAPTER 5 REFERENCES

48-90

LIST OF FIGURES

S.No Topic Page No

1 Chemical structure of 3-methyl-2H-furo [2, 3-c] pyran-2-

one.

2

2 Structural comparison of Karrikins with Strigolactone.

(Source: Nelson et al., 2009)

4

3 Plant (Cymbopogonjawarancusa) used for preparation of

smoke solutions

17

4 Chimny used for the preparation of plant derived smoke

17

5 Effect of smoke solutions (1:100, 1:200, 1:300, 1:400, 1:500)

ongermination % in smoke primed seeds under normal and

saline (50,100,150,200 and 250 mM) conditions after 36

hours

23

6 Effect of Plant-derived smoke solutions (1:100,

1:200,1:300,1:400 and 1:500 dilutions) on root and shoot

length of smoke primed maize seeds under normal and saline

(50, 100, 150, 200 and 250mM) conditions

24

7 Effect of smoke solutions (1:100, 1:200,1:300,1:400 and

1:500) on Root and Shoot fresh and dry weight in smoke

primed maize seeds under normal and saline (50, 100, 150,

200 and 250 mM) condition

25

8 Effect of smoke solutions (1:100,and 1:400 ) on (Na+, K+

and Ca+2) concentrations in smoke primed seeds grown

under normal and saline ( 100 150, 200 and 250 mM)

conditions

27

9 Effect of smoke solutions (1:100, and 1:400 ) on chla, b and

total carotenoids in smoke primed seeds under normal and

saline 100,150,200 and 150 mM conditions

30

10 Effect of smoke solutions (1:100 and1:400 ) on nitrogen and

protein content in smoke primed seeds under normal and

saline ( 100,150,200 and 250 mM) conditions

31

11 Effect of smoke solutions (1:100 and1:400 ) on prolin

content in smoke primed seeds under normal and saline (

100,150,200 and 250 mM) conditions

32

12 Effect of Plant-derived smoke solutions (1:100 1:400

dilutions) on soluble protein of smoke primed maize seeds

32

under normal and saline ( 100, 150, 200 and 250Mm

conditions

13 Effect of Plant-derived smoke solutions (1:100 and1:400

dilutions) on H2O2 concentration of smoke primed maize

seeds under normal and saline ( 100, 150, 200 and 250mM)

conditions

33

14 Effect of Plant-derived smoke solutions (1:100 and1:400

dilutions) on APX activity of smoke primed maize seeds

under normal and saline ( 100, 150, 200 and 250mM)

conditions

34

15 Effect of Plant-derived smoke solutions (1:100 and 1:400

dilutions) on POD activity of smoke primed maize seeds

under normal and saline ( 100, 150, 200 and 250mM)

conditions

35

16 Effect of Plant-derived smoke solutions (1:100 and1:400

dilutions) on CAT activity of smoke primed maize seeds

under normal and saline ( 100, 150, 200 and 250mM)

conditions

35

17 Effect of Plant-derived smoke solutions (1:100 and1:400

dilutions) on MDA activity of smoke primed maize seeds

under normal and saline ( 100, 150, 200 and 250mM)

conditions

-

36

LIST OF ABBREVIATIONS

GER Germination

RL Root length

SL Shoot length

RF Root fresh weight

SF Shoot fresh weight

RD Root dry weight

SD Shoot dry weight

ROS Reactive oxygen species

SOD Superoxide dismutase

POD Peroxidase

APX Ascorbate peroxidase

CAT Catalase

MDA Malondialdehyde

MG/G Microgram per gram

MM/G Milimol per gram

H2O2 Hydrogen peroxide

EDTA Ethylene diamine tetraacitic acid

NBT Nitro blue tetrazolium chloride

TCA Trichloroacitic acid

TBARS Thiobarbituric acid reactive species

H2SO4 Sulphoric acid

G Gram

Abstract

In nature, biotic and abiotic stresses impose negative effects on all life forms. Being a

sessile life habitat, plant always exposed to these stresses. Plant when subjected to stress

condition, always resulted in a reduced growth. Among the abiotic stresses, salinity is a

major cause often hindering plant growth which inturn curtails the crops yield. To

lessen the impacts of salinity i.e. alleviation for the betterment of

plant growth and crop yield is the matter of interest in the current

research scenario. Plant-derived smoke obtaining from burning vegetation have been

using in agricultural and horticultural since long. Being a cheap and environment friendly

source, smoke play an important role in the enhancement of crops yield. Plant derived

smoke possesses alleviation potential for number of stresses. The present research work

was conducted to investigate the effect of Cymbopogon jawaracusa smoke extracts

(1:100, 1:200,1:300,1:400 and 1:500 dilutions) on physiological and biochemical aspects

of maize (Zea mays L.) under different concentrations of NaCl (50,100, 150, 200 and 250

mM). To investigate the alleviation potential of smoke, seeds were primed for 24 h with

smoke solution and then subjected to salt stress. Obtained results showed that seed

germination percentage was improved up to 93% with smoke as compared to control

(70%). Similarly, seedling vigor in term of root and shoot length, fresh and dry weights

were also significantly increased in plants raised from seeds primed with smoke extracts.

Priming of seeds with smoke solution alleviated the adverse effects of salt stress. Ions

analysis indicated that priming with smoke solution increased the level of potassium and

calcium while reduced the level of sodium in the exposed plants. In addition, the levels of

photosynthetic pigments, total nitrogen and protein contents were also improved with the

application of seed priming techniques by using plant smoke solution as compared to salt

stressed plants. Salt stress increased the level of Reactive Oxygen Species (ROS) as well

as antioxidants in the exposed maize seedlings. Seed priming with smoke solution

showed alleviation of stress and the level of ROS as well as antioxidants remained

lowered although the seedlings were exposed to salt stress. Findings of this study showed

that smoke solution has the potential to alleviate the toxic effects of saline condition as

well as can increase the productivity in plants.

Key words: Smoke, Salt stress, Cymbopogon, Zea mays, Germination, Growth, Biomass

1

INTRODUCTION

1.1 Fire as a germination cue

Fire, is an environmental force, that can influence vegetation in different parts

around the globe and is considered to be an important characteristic in species of different

areas [1,3], Australian Kwongon [4], African savanna [5], fynbos [6,7], Californian

chaparral [8], and habitats related to grassland [9]. Besides the burning of vegetation, fire

can also affect various other aspects like growth, developmental process, germination of

seeds, biomasses, flowering production, dispersal of seeds and increase the rate of

mortality in plants. Plants thus have been adapted to the different factors related to fire

induced impacts. These adaptations have also been evolved in seed due to which it can

respond to chemical as well as physical germination cues associated with fire [10,11].

There are two theories regarding the breaking of seed dormancy in seeds of post fire

environment [12]. One theory states that this cue might be due to the loss/removal of

allelopathic compounds and toxins produced by nearby plants and microorganisms.

Second theory suggested a direct impact of fire on vegetation itself. It is also suggested

that direct effect of fire has more prominent role in cueing seed germination in different

species of plants [13,18]. Fire is also found to provide some chemical stimuli in the form

of ammonia and ethylene, nitrogen dioxide [19], ash [20], charcoal [21, 22] and nutrient

elements [23].

1.2 Smoke, as a growth regulator

Smoke produced due to wildfire is act as growth regulator. Since 1990s, it has

been proved that smoke enhances germination of seeds. It can be used in agriculture and

horticulture industries for high yield of vigorous crops [24]. Various cues have been

suggested and observed in post-fire environmental condition [25]. Smoke has been found

to promote seed germination in over 1200 species belong to 80 genera [26]. Smoke and

its solution can enhance germination of seeds in different species related to fire-prone

habitat [27]. Smoke motivates various metabolic processes through interacting with

endogenous plant hormones. Solution of smoke is prepared by burning parts of vegetation

and bubble the smoke in to bottle with water [28]. Flowering [29] seed embryo and

radical emergence in germinating seeds [30].

1.3 Role of smoke as a germination cue

Seed germination plays important role and ensuring the development of new plants

from seeds during quiescent dormant period. In plant life cycle, smoke obtained from

plant has been reported to promote germination of different species worldwide [31]. More

2

over burnt cellulose can also motivate seed germination [32]. Attempts has been made by

Australian researchers and South African scientists who isolated some important

compounds acting as germination stimulant e.g. (3-methyl-2H-furo [2, 3-c] pyran-2-one)

[33]. It is also referred as ‘butenolide’.

Figure 1.1 Chemical structure of 3-methyl-2H-furo [2, 3-c] pyran-2-one [35].

The discovery of more similar compounds in plant derived smoke water,

butenolide has been considered as a class of chemical compounds known as Karrikins,

after ‘Karrik’ the indigenously applied Nyungar word used for plant derived smoke [34].

Karrikins is a potent stimulator and enhancing seed germination at very low level [35].

Recently it has been observed that the Karrikins found in smoke water enhancing

germination of seed of Arabidopsis thaliana by using pathway which required light and

synthesis of gibberellic acid [36].

Plant derived smoke and its aqueous extracts activates the dormant seeds to

germinate in the threatened fynbos species [37]. It has been found that smoke activate

germination of different species from a region of fire prone environments all over the

world including Californian Chapparal [38-40] Australian Kwongon [41-48] and

Lanthana camara verbena species of tropical (forests) of India [49]. Smoke is a useful

stimulant that activates germination of about 1200 species worldwide in over 80 genera

[50]. It has recently been investigated that in the matorral of central Chile, smoke and

heat increases the emergence of exotic and native plants [51]. Smoke was also found to

stimulate species span of different genera of angiosperms and gymnosperms as well as

plant range from phenerophytes to geophytes [52]. In Mediterranean fire prone

ecosystems, wide range of species from different regions of earth suggested that

activation of germination by plant derived smoke or aqueous smoke solution is common

[53]. Due to stimulatory role of smoke, it has been observed that once the seeds treated

with smoke or smoke concentration, it was impossible to eradicate the germination cue by

soaking the seeds in water. This miraculous properties of smoke has allowed it to be used

as a useful seed pre-treatment [54]. The inhibitory effects of high concentrations of smoke

3

solutions assumed to be reversible, contrary to the maintenance of the germination cue

[55].

1.4 Mechanism of action (compounds in smoke)

It has been studied that smoke and aqueous smoke extracts obtained from different

plant species work like hormone. These hormones or growth stimulatory compounds

interact with plant growth regulators such as auxins, ethylene, cytokinins, gibberellins,

and abscisic acid in different kinds of seeds [56]. The compounds of butenolides found in

aqueous smoke extracts are recommended to increase germination, seedling growth and

number of nuclei ratio in quiescent Lycopersicun estulentum seeds [57]. In seeds of Avena

fatua, Butenolide is also found to stimulate germination by enhancing number of nuclei at

(Gs/ G2) phases of cell division. It also increases the ratio of (G2 and G1) before

coleorhizae protrusion [58] and suggesting that butenolide plays a key role in initiating

DNA replication and has hormone like actions in cell division as well as cell elongation

[59]. It has been studied that in various species smoke extracts show hormone like

responses and interacts with cytokinins, auxins and GA, abscisic acid ethylene and

abscisic acid in different seeds [60].

It was concluded that dormancy of seeds in quiescent embryos occur due to slow

cell cycle in G0 or G1 phases [61] while it is nearly stop in Gs or G2 phases of cell division

in other embryos. In dormant seeds of Avena fatua, synthesis of DNA was found to be

restricted in G2 phase. Butenolides present in plant derived smoke and aqueous smoke

extracts promotes both the percentage and germination of nuclei number in quiescent

seeds of tomato [62] indicating it possible role in the initiation of DNA replication, cell

division and elongation [63].

Daws et al. [64] reported that butenolide can substitute the hormone by motivating

germination of parasitic plant species i.e. Orobanche and Striga. This suggests that

butenolide function just like strigolactones which suggesting the structure and activity

similarity with strigolactones. By using synthetic analogues of strigol, which shows that

the lactone-enol ether plays main role in the biological action of these compounds [65,

66].

4

Figure 1.2 Structural comparisons of Karrikins with Strigolactone. [36]

1.5 Effect of smoke on seedling vigor and plant growth

Smoke solution promotes seed germination and inhibits seed dormancy. It has been

described by some researcher that the effect of smoke and its concentration expand after

the post germination actions and resulted in the improvement of seedling vigor [67,68]. In

some arable weeds, Butenolide have significant effects on seedling growth [69], woody

Acacia species [70,71], Eragrostis tef (Zucc.) Trotter [72], Vegetables such as tomatoes,

okra, and beans [73], commercial maize cultivar [74] and rice [75]. Both butenolide and

smoke water treated plants produce tomato fruits in huge number as compare to untreated

control [76]. The results of different authors have shown that butenolide compound and

smoke solutions plays regulatory role in seed germination, development and growth.

Hence, the physiological functions in plants are in part controlled by plant growth

regulators (PGRs) or phytoharmons, which shows that compounds in plant derived smoke

extracts relate with endogenous plant growth regulators in some way [77].

1.6 Effect of smoke on some other biological processes

Smoke extracts activates number of biological processes such as rooting [78] and

somatic embryogenesis [79]. Plant derived smoke has also been found to alleviate ZnCl2

and HgCl2 stress during germination of tomato seeds [80]. Smoke was also found to

alleviate B and Na stress during maize, wheat, millet and sorghum seed germination [81].

1.7 Applications of smoke technology

Plant derived smoke and smoke solutions can potentially be used for a variety of

application related to seed technology [82]. It can be used in pre-treatment of seed, weed

5

control, agriculture, re-vegetation of ruined areas [83,84] and antiestablishment of

communities of soil dwelling organisms i.e. fungi and bacteria etc. [85].

1.8 Effects of salinity on crops yield

Salinity is a global problem which affects large cropped lands of the world

resulting in significant reductions in crop yield [86]. Salt stress affects biochemistry and

physiology of plants which decreases plant dry matter causing yield loss in various crops

[87] as well as changed morphological traits of the plants [88]. Salinity cause serious

problem in the world that cause reduction in crops yield [89]. According to an estimate,

one third part of the agriculture area has been damaged by salt stress and it is gradually

growing annualy. In Asia, 21.5 million hectares of agriculture is thought to be affected by

salt stress [90]. In Pakistan 40,000 hectares of arable land is wasted every year because of

salinity [91]. Salinity is considered a major factor in reduction of crop yield [92]. Plants

show impaired growth when grown under salt stress due to specific ionic effect. Salt

causes osmotic effect, nutrient disturbance and harm plants due to higher concentration of

ROS as well as changes occur in endogenous hormones [93].

The NaCl toxicity due to NaCl causes problem to crop that overall reduces crop

production [94]. Na and Cl ion severely harm the plant cells through both ionic and

osmotic stress [95]. Several reports in the literature revealed that salinity causes many

adverse effects on the morphology, anatomy and physiology of pearl millet [96].

However, improvement in salt tolerance of crop plants remains elusive. Salinity affects

almost every aspect of physiology and biochemistry of plants [97] at both whole plant and

cellular levels [98]. Exposure of plants to a stressful environment during various

developmental stages appears to induce various physiological and developmental changes

[99].

1.9 Plant growth under salt stress

In plant, growth reduction occurs due to salt stress because plant may have

subjected to four different kinds of stresses i.e. water stress, ionic toxicity, imbalance in

the nutrients level and production of Reactive oxygen species [100].

1.10 Osmotic adjustment

Abiotic stress due to salt diminishes the ability of plants to absorb water that

ultimately results in stunted growth. Salinity causes drought stress at cellular level. In

plants, the development of young leaves requires high water potential, but salinity in the

xylem lowering the potential which in turn reduces the growth of young leaves [101]. The

effect of water stress reduces the growth of root and leaf more than that of direct impacts

6

of salinity [102]. It has been observed that in the growing cells, the levels of Na+ and Cl-

remained below toxic concentrations. For instance, in wheat it was 120 mM

concentration, in which the Na+ level was 20 mM found in leaves of the growing tissues

and 10 mM in the zone of fast growing area while Cl– was found about 50 mM in the

wheat [103]. It was also observed that the concentrations of Na+ and Cl– was 40 mM in

the growing tissues and is not correlated with tissues of maize exposed to 80 mM of NaCl

[104]. In barley plant mesophyll and epidermis cells, the concentration of Na+ was 38

and 49 mM, respectively when exposed to 100 mM of NaCl concentration for 24 h [105].

Severity of the stress causes reduction in plant growth in the leaves and stems due to

increased level of osmotic stress, however, roots may continue to grow and elongate

[106]. It was also investigated that water relation in shoot shows that there are some

hormonal signals that produced in the roots due to osmosis effects NaCl which regulate

the cell elongation [107].

1.11 Specific ion toxicity

In salt stress, ions have phytotoxic properties and can cause toxicity to wide range

of crops through irrigation water. These toxic ions include Na+, Cl– and SO+4 and can

decrease crop yield. Most crops and woody everlasting plants are sensitive to salt but not

all crops have same influences [108]. The accumulation of salt in the old leaves for longer

time results in the accumulation of Na+ and Cl– and ultimately causes cell death. It is

because plant cannot cope with the compartmentalizing of salts cell vacuoles. In

cytoplasm NaCl rapidly build up the concentrations which slower the enzyme activities in

the cells. Toxic ions accumulated in the walls of plant cells and cell subjected to

dehydration but no effect was observed in varieties of maize having varying degree of salt

tolerance [109,110]. Plants tolerate the salt stress through mechanism of reducing its

higher level in the plant cell or through minimize the level of cytoplasmic salts. Na+ in the

root cytosol is in the range of 10 - 30 mM [111] while in leaves its concentration was

found below 100 mM [112].

Cl– toxicity shows that roots must exclude most of the Na+ and Cl– dissolved in

the soil solution, or the salt in the shoot will gradually build up with the passage of time

to toxic levels. Plants transpire about 50 times more water than they retain in their leaves

[113]. Similarly, Husain et al. [114] used two wheat genotypes seeds exposed to different

concentration of Na+ and have different ability to tolerate salt stress in which some were

found with the quality of reducing cell injury in the leaves with enhanced yield. It was

also investigated that high level of Na+ in the cell can damage chlorophyll in older leaves

7

which dry up and fall earlier than leaves with low Na+ level. In saline soil at moderate

salinity the low-Na+ concentrations can improve the crop yield up to 20%. This indicates

that traits other than Na+, exclusion are important at high salinity, where the osmotic

effect of the NaCl outweighs its salt-specific effect on growth and yield. Na+ increment

inside plants had toxic effects on seed germination, mainly by affecting the plant water

relations or through displacement of Ca2+ by Na+ from critical cell wall binding sites,

which could disrupt cell wall synthesis and hence inhibit plant growth [115]. It was

observed that level of Cl- was found 80mM which may alter the morphology as well as

stomata shows less response hence, reduction occurred in the leaf thickness [116].

Similarly, Marschner [117] observed that extreme leaf burn due to toxic level of chloride

leads to the early leaf drop; as a result the whole plant became dries up.

1.12 Physiological and biochemical processes

Salinity affects various physiological and biochemical processes in plants. Effects

of salinity seem to appear on entire plant structure including biomass, germination,

seedling growth and reproductive stages [118,119]. In comparison, salt tolerance in some

crops such as Trifolium alexandranium, Medicago sativa, and Trifolium pratense studied

at germination stage and also showed tolerance even at later developmental stages [120].

Similarly, working with safflower [121], different scientists have described that crop

varieties depends on salinity damages in the root tissues or ability to accumulate salts in

the vacuole due to changes in salt tolerance [122]. Wynjones et al. [123] studied that the

higher salt tolerance of Agropyron junceum to that of Agropyron intermedium was linked

to its efficient exclusion of both Na+ and Cl- from their tissues. In another study, Carden

et al. [124] found that the salt tolerant variety kept a 10-fold lower cytosolic Na+ in the

root cortical cells than that of sensitive variety. However, Mansour et al. [125] found that

Na+ accumulation causes reduction in potassium and calcium which remained higher in

salt the tolerant maize cultivar Giza 2 than that of salt susceptible Trihybrid variety [126].

It is well documented that under normal or stress conditions, higher photosynthetic

activity resulted increased plant growth in Gossypium [127], Zea mays [128], Brassica

spp. [129] and Triticum [130]. In addition, salt-induced decrease in photosynthesis due to

opening and closing of stomata has also been well established. Higher addition of Na+ and

Cl- in the leaves tissues also decrease photosynthetic capacity and Na+ content in the

leaves of rice [131] and wheat [132], while high Cl- contents in the citrus [133] and in the

chloroplast of Phaseolous vulgaris. Seemann et al. [134] found that salinity is harmful to

photosynthesis. Consequently, it can be concluded that due to both osmotic and toxic

8

effects of Na+ and Cl-, growth inhibition may occur. However, at early growth stage,

osmotically induced reduction in growth exists at low salinity. More importantly,

photosynthesis is also one of the major helping factors, decrease plant enlargement and

product of photosynthesis. Tolerance to salt stress depends on the mechanism of plant to

exclude or vacuolized in toxic ions of salt. Though, the harmful influence of salinity on

photosynthesizing tissue and overall development varies depends on species type, stress

duration and level of salt.

1.13 Ion analysis

Effects of salinity depend on concentration and plant species exposed to stress.

One of the worst effects of salt stress is the inhibition of plant growth by posing sodium

ion toxicity. The Na+ ion is thought to be harmful to cell functions for many plants when

it is found in the cytosol at certain concentration greater than the tolerable level i.e. 1-10

mM. It can disturb the K+ normal balance, which elicit 50 enzymes or more in the cytosol

[135]. Sodium can displace calcium at higher concentration from the plasma membrane

that changes cell membrane permeability which can be done by escaping potassium from

the cells [136]. Na+ ions being poisonous that harm plant cells posing both ionic and

osmotic stress that decline the productivity and eventually death to the exposed plant may

be occur [137].

Soil irrigation with saline water raises the Na+ content and the interaction of

sodium and calcium, sodium and magnesium, but reduces the calcium, magnesium and

potassium contents, resulting the imbalance of nutrients [138]. Higher levels of sodium

and chloride ions reduce the uptake of potassium and causes potassium deficiency like

symptom in the salinity exposed plants. In plant, the main cause of chlorosis and necrosis

is potassium deficiency [139]. To keep the steady state of osmotic level of cytoplasm and

plasma membranes, normal level of potassium and calcium is very much important.

Wenuxe et al. [140,141] presented that occurrence of higher level of potassium in plant

tissue under salt stress may be due to active potassium uptake and cellular potassium and

sodium in the shoots.

In plant calcium is a signaling molecule that perform main role in mediating

mechanisms that are used for detection and response to various abiotic stresses under

stress environment. Intracellular cytosolic Ca2+ either accumulate or they release by

plants that act as a signal and regulate different physiological processes for the adjustment

of these stresses [142]. It has been detected that Ca2+ limits the Na+ entry in to the plant

9

cells [143]. In Jaya cultivar of rice which is salt sensitive, application of calcium (4 mM)

has been investigated to develop the ratio and limit the cellular levels of sodium [144].

When rice grows under salinity, plants accumulate Na+ and Cl- ions according to

their tolerance capacity. These high Na+ and Cl- concentrations in soils causes water

stress and hyper ionic stress. Plant growth is inhibited by the adverse effects of both

sodium and chlorine because they limit the absorption of other ions and nutrients required

for growth. During salt stress, Na+ competes with K+, Ca2+, Mg2+ and Mn2+ while Cl-

competes with NO3-, PO4

2- and SO42- ions [145].

Mecue et al. [146] stated that interaction of mineral nutrients with salts might

result a significant imbalance in the nutrients and causes their deficiencies in the plants.

Karimi et al. [147] reported that due to excessive accumulation of sodium and chloride,

ionic imbalance found in the cells which reduce the uptake of few other nutrients, like

potassium, calcium and manganese. It has been reported earlier salt stress particularly

affect the leaves by causing leaf senescence and are correlated to the increase level of

toxic ions (Na+ and Cl-) or reduction in the K+ and Ca2+ [148].

1.14 Chlorophyll and total carotenoids

Photosynthetic pigments are considered as the important regulators of

photosynthetic process [149]. The biosynthesis of photosynthetic pigments affected due

the salt stress in the exposed plants [150]. Decrease in chlorophyll contents due to salinity

induced by decaying in the endogenous contents of 5-aminolevulinic acid, that act as an

originator for protochlorophyllide and chlorophyll biosynthesis as well as lower scale of

glutamic acid which is necessary for the production of 5- aminolevulinic acid has also

been observed in plants exposed to salt stress [151]. Fang et al. [152] found that due to

the eradication of phytol the shortage of chlorophyll content in salt stressed plants is

caused due to increase in the enzyme activities. Salinity damaged pigments such as

carotenoids, chlorophylls and enzymes which depend on the period, level of salinity and

plant species [153,154].

Reports shows that salt stress decreases chlorophyll a, b and carotenes in rice

species [155,156] at 200 mM salt stress in which the chlorophyll b is much responsive

than chlorophyll a. High salinity level causes chlorosis in plants and is a common

morphological and physiological characteristic in plants [157]. In the leaf lamina

accumulation of NaCl diminishes photosynthetic process and growth ratio [158].

Response mechanism of plants to higher salt levels is a complex process and comprises

change in the physiology, morphology and metabolism [159]. Ashraf and Harris [160]

10

analyzed that chlorophylls, enzymes and total carotenoids are negatively affected by salt

stress.

Singh et al. [161] studied that chlorophyll a, b and their ratio reduced in checkpea

when exposed to salt stress while, spray of growth regulators like kinetin (10 ppm) and

GA3 (25 ppm) increases chlorophyll contents. Ashraf et al. [162] reported that exposure

to higher concentrations of salt, decrease in the contents of chlorophyll a and b in mung

bean cultivar was observed. It was suggested that high salt stress conditions suppress the

specific enzyme activities responsible for the synthesis of chlorophyll. Hamdia et al.

[163] stated that growth is adversely inhibited by salinity and affected the contents of

chlorophyll and carotenoids in broad bean plants while foliar dose of NaH2PO4 and

NaNO3 greatly alleviated the adverse effects of NaCl resulted an increase in the

photosynthetic pigments. Malibari et al. [164] reported that spray with kinetin (5 ppm)

and abscisic acid (20 ppm) enhanced the chlorophyll content in salt stressed (50, 100 and

150 mM NaCl) wheat plants. Ramanjulu et al. [165] demonstrated that photosynthetic

pigments decreased with increasing salt level in mulberry plants however they found that

the chlorophyll a was diminished more than chlorophyll b.

1.15 Protein and nitrogen contents

It was previously demonstrated that the reduction of contents in total nitrogen and

protein inhibited when exposed the rice (Oryza sativa L.) seedlings to salt stress [166].

Kumar et al. [167] observed that the contents total protein has been reduce of in different

varieties of oat (Avena sativa L.) with rising levels of salt stress. In the shoot of three

chickpea cultivars, salinity caused a significant decrease in the levels of potassium,

magnesium, nitrogen and calcium [168]. Hoque et al. [169] demonstrated that salinity

decreased the development of Pancratinum maritimum by reducing protein content. Afzal

et al. [170] reported that salt stress reduces the growth of Pancratinum maritimum by

decreasing total soluble protein content that can be increased by the application of

exogenous proline that effectively detoxifies H2O2 by enhancing the activities of

peroxidase and catalase in tobacco under salt stress. The severity of salinity can be

reduced significantly with the application of CaCl2. Abdel-Hadye et al. [171] noticed with

increasing levels of salinity decreases the concentration of nitrogen in barley seedlings.

Naseem et al. [172] also stated that salt stress in laboratory grown Cucumber (Cucumis

sativus L.) affect the seedlings growth and physiology. It was observed that nitrogen

contents and total soluble protein decreased with rising levels of salinity. Amirjani [173]

observed that reduction in the total protein contents was not so severe in case when

11

seedlings were treated with 50 and 25 mM of salt concentrations. Increasing salinity

from 100 mM and particularly the 200 mM resulted further decline in the total protein

contents.

1.16 Proline induces the expression of salt stress

The role of proline is to protect plants from oxidative damages and is a compatible

osmolyte and osmoprotectant while, its extra characters in salinity have been less

considered [174-178]. Working with Arabidopsis mutants it was observed that a proline-

deficient mutant when subjected to saline media unable to continue growth [179].

Exogenously applied proline has been reported to produce tolerance against salinity in

somatic embryos. It was found that tobacco cell cultures grown under salt stress improved

by exogenous proline application and was suggested the possible role of proline as an

osmoprotectant for enzymes and membranes against salt inhibition [180, 181]. However,

Nanjo et al. [182] using transgenic Arabidopsis plants with reduced pyrroline-5-

carboxylate syntheses (P5CS) activity, synthesis from glutamate which is the rate limiting

enzyme in proline, confirmed that these plants were not able to develop on saline area and

that exposed to L-proline but not D-proline, could enhance their capability to resist salt

stress. Beside different known roles of proline, it was also incorporated that it plays a

pivotal role during salinity stress. it was found by lyer et al. [183] that salt stress up to

1mM are enough to induce stress-regulated genes such as dehydrins and proline

metabolite, such as pyrroline-5-carboxylate, but not the proline directly confirming that

proline is a protectant inducing stress-protective proteins.

Among the basic abiotic stresses, salt stress is reducing growth, efficiency and

development in the exposed plants. Due to ionic toxicity, this restraint effects, osmotic

inhibition, movement and taking of essential ions disorder, disturb plant cell

physiological and biochemical functions which eventually leads to cell dying [184-186].

Different species of plants possess different percentage of proline accumulation in plants

varies [187]. Addition of proline may be a part of the stress signal affecting adaptive

responses [188].

Accumulation of proline in plants has been proposed due to: (a) increased proline

biosynthesis (b), reduce proline degradation, (c) lower proline use, and (d) protein

degradation [189]. In higher plants, proline is created by means of Glutumate and

ornithine Glu and Orn cycle due to a single bifunctional enzyme, pyrroline-5-carboxylate

synthetase (P5CS) glutamate pathway is catalyzed and gultamic-γ-semialdehyde (GSA) is

produced, continuously changed to pyrroline-5-carboxylate (P5C) and then decreased to

12

proline [190,191]. In many stress conditions particularly drought and salinity, proline

addition is linked with the activity of P5CS, as planned toward the essential and inhibiting

enzyme in the biosynthetic cycle. This would give mechanism of ensuring that the genetic

response to salinity is suitable for the widespread ecological environment conditions

[192].

1.17 ROS and antioxidant

In plants, ROS are generally produced in the apoplast and other cell compartments

normally [193]. In the enlarge region of maize leaf blades ROS formed in the apoplast of

cells indicating its importance for growth and elongation [194]. For modulation of Ca2+

channel activity through regulation of cell expansion, apoplastic ROS is necessary [195].

Moreover, by disturbing the rheological characteristic of cell walls including wall

loosening and stiffening [196]. Common character of the reaction to abiotic and biotic

elicitors in plants is the increased reactive oxygen species [197]. In contrast, the

accumulation of salt in elongation in the spreading region of maize leaves lined with the

reduce (ROS) levels could be inhibit by the accumulation of ROS [198]. The effect of

NaCl was non-osmotic and salt-specific, as incubation in 200 mM sorbitol did not

decrease the contents of reactive oxygen species. Antioxidants, such as ascorbate and

glutathione, both enzymatic and non-enzymatic molecules, limit apoplatitic ROS

concentration. For the apoplastic space, enzymatic antioxidant activities were observed

including dehydroascorbate reductase, glutathione reductase superoxide dismutase SOD,

ascorbate peroxidase, mono dehydroascorbate reductase, and catalase [199].

Plant response to the salt stress increased level of salinity is challenging and

various physiological, morphological and metabolic processes are involved [200].

Morphologically the decreases of plant growth are the most usual sign of stress damage

[201]. Physiological responses have an effect including alteration of ion balance, H2O2

position, mineral nutrition, pigments effectiveness, carbon placement and use commonly

occurs during stress [202-205]. During salinity stress, physiological and metabolic

alteration may be occurring as a result the production of (ROS) such as (O2-), (OH-), (O-

1), and related water [206]. Reactive oxygen species have power to act together with

many cell organelles causing considerable loss to cellular structures and membrane,

resulted limitation of growth in plants [207-209]. By cellular responses some toxic ROS

must be detoxified, if the plant is to continue to exist and develop [210].

Consequently, chronological and immediate action of a many antioxidant enzymes

involves prevention of ROS that include APX, POD, SOD and CAT. To escape the plant

13

from the destruction of oxidative stresses, plants show some defense mechanisms under

stress. Plants have better resistance to damage against high fundamental and induced

antioxidant levels [211]. Generally, defense responses beside abiotic stresses are the

scavenging of ROS [212]. The level of injury due to antioxidant eliminated by ROS

scavenging system [213,214]. In several plants correspondence depends on the balance

between the product of ROS and enzyme activity which in turn determines salinity

tolerance and differentiate the tolerant cultivar with sensitive cultivar. Under salt stress

antioxidant enzymes activities were increased and linked to salt tolerance of many plants

[215-217]. Superoxide dismutase SOD is to be found in different cell compartments and a

major scavenger of superoxide radical. This enzyme can change the superoxide radical

(O2-) to H2O2, which in turn detoxify by ascorbate peroxidase POD at the expense of

oxidizing ascorbate to monohydroascorbate [218]. Hydrogen peroxide H2O2 is also

scavenging by peroxidase POD and catalase CAT by changing it to water and oxygen

[219,220]. Malondialdehyde is measured as a level of lipid per oxidation, has been

thought an indicator and a tool for determination of salt tolerance in plants and salt-

induced oxidation in cell membranes [221]. Particularly in plant sensitive cultivars, lipid

per oxidation ratio was determined to improve with enhance salinity levels [222,223].

During development of plant, there are four major restrictions due to salinity (I)

plant growth and development has been reduced by salinity there by limited H2O uptake,

and hence due to low level of water absorption leads to secondary oxidative stress, (II)

oxidative stress (secondary, unspecific stress), (III) salt stress also leads to ionic toxicity

and (IV) nutrient imbalance [224,225]. High salinity condition also induces limitation of

enzyme activities and the over-accumulation of reactive oxygen species such as H2O2,

superoxide, and hydroxyl radical, resulting in metabolic disorder, lipid peroxidation and

pigments breakdown [226]. To escape from the toxic property of ROS, which plays a key

role in hinder plant development, cells of plant have well-organized anti oxidative

enzymes, such as superoxide dismutase and ascorbate peroxides APX. To neutralize

oxidative damage in plants these enzymes have been confirmed to saline stress [227]. In

other words, salt stress can trigger enhanced activities of antioxidants, plant varieties with

high salt resistance usually showed higher activities of these enzymes [228]. SOD is

considered to be the primary scavenger in the detoxification of active oxygen species in

plant. Its function is to change superoxide to hydrogen peroxide and singlet oxygen

[229]. According to the metal binding in the active site, SOD activity can be divided into

Mn-SOD, Cu/Zn-SOD, or Fe-SOD isoforms. APX has a vital role in detoxifying H2O2,

14

by catalyzing the conversion of H2O2 to H2O [230]. Catalase (CAT) can also decrease

H2O2 to water but it has lesser ability for H2O2 than APX [231].

It is evidence that H2O2 functions as a signaling molecule; although, at higher

concentrations H2O2 is toxic. Levine et al [232], studied a different role for H2O2 under

salt stress in Brassica oleracea roots and found that increased H2O2 production occurred

in response to salt stress. The same experience has occurred in rice plant leaves, along

with the increased activities of SOD and APX. A conceivable clarification for this is, to

stop the toxicity of superoxide radicals, SOD leads to the over production of H2O2 then

the excess of H2O2 functions as salt stress signaling, which induces generation of the

specific APX [234,235]. It has also been observed that rice seedlings tolerance to salt

stress can develop by pre-treating with low levels (<10 mM) of exogenous H2O2, at the

same time, significant increases were found in CAT, APX, SOD, glutathione reductase

(GR) and POD, showing its dual action against H2O2 in salt tolerance. Moreover, salinity

in plants reduces water up take, and water potential and there by depresses mineral uptake

and causing nutrition disorders in the exposed plants. Therefore, for plant adjustment,

saline stress and maintenance of stable levels of intracellular mineral ions is important

[236]. Although, there is a shortage of Fe in the earth’s crust, especially in saline soil its

accessibility to plants remains limited. Fe deficiency reduces the activity of APX and

increases glutathione (GSH) and ascorbate reductase (AR) levels, indicating that Fe

deficiency also inducing secondary oxidative stress in plants [237-239]. In crop

production one of the most worldwide limiting factors especially in saline soil is Zn

deficiency. SOD activities decreased in plant deficient with Zinc and as results in young

leaves chlorosis may occurs [240]. Bowler et al. [241] reported that apoplastic reactive

oxygen species take part in cell expansion regulated through Ca2+ channel activity

modulation [242] which contributing both to wall loosening [243-246] and stiffening

[247,248]. Salinity is a universal restraint in agricultural productivity [249] as leaf

elongation decreases is one of the main salt stress effects [250]. Leaf elongation rates in

grasses is reduced by stress conditions as well as blade elongation region is also affected,

present beneath growing leaf blade, decreasing elemental growth rates and length of the

area [251-254]. Disclosure of Arabidopsis roots to high salt level enhanced ROS

formation in pea plants [255]. In tobacco cell suspension cultures production of both

divalent and monovalent induced O2 d ± through the Nicotinamide adenine dinucleotide

phosphate (NADPH) oxidase activity and a major portion of the O2 d ± was released to

extracellular [256]. In response to saline treatments variation in ROS concentration was

15

also noted under this condition. It is predictable that they would contribute to adaptable

leaf elongation such as possibility has not yet been experimentally tested. In this work, it

was examined whether salinity affects ROS production in the leaf elongation zone and if

this action contributes to regulating leaf elongation under this condition. In plant cells

chloroplasts, mitochondria and peroxisomes are important intracellular generators of

ROS. It is common phenomena now a day that ROS are responsible for different stress-

induced injuries to macromolecules and finally to cell structure [257,258] and for

maintenance of normal growth it is needs to be scavenged. APX, POD, CAT and together

with low-molecular mass scavengers such as glutathione, ascorbate, and proline, formed

in different parts of plant cells, function as most important defense system against ROS.

Catalase, which is in peroxisomes, glyoxysomes and mitochondria is in fact not present in

the chloroplast, dismutase’s mostly by converting the respiratory hydrogen peroxidase

into water and molecular O2 [259]. Peroxidases POD decay hydrogen peroxidase by

oxidation of co-substrates such as phenolic compounds or ROS. Polyphenol oxidase

(PPO) is an enzyme which oxidizes some phenols to chinone. In a number of plants this

enzyme has been studied in relation to injuries, enzymatic browning [260] under biotic

and abiotic stress conditions some alkaloids are also synthesized [261]. Under salinity

stress in different antioxidant enzymes change in the activities have been reported [262,

263]. However, the effects of zinc in the activation of oxygen species have been hardly

investigated. This knowledge can supply information on the mechanism of zinc and non-

zinc applications on plants in saline condition. Lethal effects such as slow down of

photosynthetic rate, injuries to plasma membrane and chlorophyll content, permeability

and other metabolic disorder occurs as result of salinity stress [264,265]. In plants, due to

salinity and water shortage proline accumulation is usually occur and it is often thought to

be concerned with stress resistance mechanisms [266]. In free proline content, some

researcher did not find any huge increase [267,268]. Reactive oxygen species formation is

inhibited by an antioxidant system; low molecular mass antioxidants are again generated

by reactive oxygen species -interacting with enzymes such as catalases, SOD and POD

[269]. Salt stress inducing the plant antioxidant system in response to H2O2 generation

and plays a significant role in induction of stress tolerance [270]. As priming of seeds

with plant derived smoke enhances plant growth subjected to salinity. Therefore, present

investigation aimed to investigate the effects of smoke extracts on maize physiology and

biochemistry and to study the effect of priming with smoke extracts by the alleviation of

salt (NaCl) stress.

16

Hypothesis

It was hypothesized that plant-derived smoke solutions might enhance

physiological and biochemical attributes in maize (Zea mays L.) and priming of maize

seeds with smoke might overcome the impacts of salt stress.

Objectives

To investigate the effect of plant derived smoke solutions on physiological and

biochemical characterization of maize.

To study the effect of priming on alleviation of salt (NaCl) stress through plant-

derived smoke solutions.

17

2. MATERIALS AND METHODS

2.1 Seeds collection

Zea maize (cv. Azam) seed were obtained from Agricultural Research Station

Sarae Naurang, Khyber Pakhtunkhwa Pakistan and stored in paper bags at temperature of

4°C.

2.2 Collection of plant

For preparation of smoke, aerial parts of Cymbopogon jawarancusa were

collected from District Bannu. Plants materials were washed with clean water to remove

dust particles and then shade dried.

Figure 2.2 Plant (Cymbopogon jawarancusa) used for preparation of smoke solutions.

Source google.

2.3 Preparation of smoke solutions

For preparation of aqueous smoke solutions, the method of De Lang and

Booucher [271] was followed with slight modifications. To check the leakage of smoke,

we designed a furnace with an exhausted pipe having window double sided with

aluminum foil. Shade dried plant material weighing 333g burnt in furnace. like water

while the effect of higher dilution was inhibitory so 333 gm of shade dried material of

(Cymbopogon jawarancusa) smoke solution was enhanced seed germination and growth).

The smoke produced was collected in one liter of distilled water in a beaker. The smoke

solution prepared in this way was considered as concentrated smoke solution. It was then

filtered and stored at 4°C in clean sterilized flasks for further use [272, 273].

18

Figure 2.3 Apparatus locally designed for the preparation of Smoke Solutions.

2.4 Dilutions of concentrated smoke solutions

For conducting different experiments, the concentrated smoke solution of C.

jawarancusa was further diluted to 1:100, 1:200, 1:300, 1:400 and 1:500.

2.5 Experimental designed

In order to check the effect of smoke solutions on maize, two sets of experiments

were conducted on biochemical and physiological attributes of maize treated with salt

stress.

2.6 Surface sterilization

Healthy seeds of maize were selected and sterilized with methanol (70%) for 5

minutes. The sterilized grains were then rinsed with distilled water to avoid the effects of

methanol. The grains were air dried and used for experiments.

2.7 Priming of seeds

Maize variety (Cv. Azam) were primed with distilled water (control) and different

concentrations (1:100, 1:200, 1:300, 1:400 and 1:500) of smoke solutions for 24 hours.

The seeds were then air dried and used for germination experiments.

2.8 Germination experiments

Germination experiments were conducted by using sterilized Petri dishes

containing double Whatman No.1 filter papers, to check the effect of different

concentrations of smoke solutions under salt stress condition. Seeds were primed with

smoke solutions and subsequently exposed to salt solutions of 50, 100, 150, 200 and 250

mM. For each treatment, three independent replicates were used having 10 seeds.

19

2.9 Germination test

Seeds were subjected to dark period during germination and provided temperature

of 27±2oC. Up to 3 days, seed germination was recorded after every 12 h. Seeds with

2mm radicals were considered as germinated.

2.10 Root and shoot length

After 10 days, experiments were harvested and root and shoot lengths of maize

seedlings were measured with help of scale in centimeters.

2.11 Root and shoot fresh weight

Fresh weight of roots and shoots was measured with the help of electronic balance

and weights were showed in grams.

2.12 Root and shoot dry weight

For determination of dry weights, the excised shoots and roots were then placed in

oven for 24 hours at 80 °C and measured in grams.

2.13 Sand culture experiment

For experiments using the sand culture, maize grains were primed in smoke

dilutions (1:100, 1:200, 1:300, 1:400 and 1:500) and control was subjected with distilled

water for the period of 24hr followed by surface drying. Twenty seeds were sown in each

replicate and every treatment included three independent replicates in plastic pots having

size of 8 inch. Seedlings were provided with salt solutions of 100, 150, 200 and 250 mM

and for fulfillment of nutritional requirement, Hoagland’s solution was used up to 10

days. After 10 days the experiments were harvested and samples were used for different

parameters related to physiological and biochemical studies.

2.14 Biochemical analysis

To determine different parameters related to biochemical analysis, plant samples

of maize obtained from sand culture experiments were dried in incubator at 80 oC for 24

hr. The dried plant material was grinded to powder form and used for biochemical

analysis.

2.15 Ions determination

Ions were determined according to the standard protocol of AOAC [274] and

Awan & Salim [275] were used with little variations in which 25 mg of each plant same

in powder form was taken and digested in a small beaker by adding concentrated H2SO4

and H2O2 in ratio of 2:1 (v/v) and heated for 15 minutes. To concentrate mineral ions,

liquid in the samples was allowed to evaporate until by remaining small amount of viscus

liquid in each beaker followed by the addition of 20 mL of distilled water. For ion

20

analysis, 100, 150, 200 and 250 ppm standard solutions salts containing sodium,

potassium and calcium were prepared. The concentrations of Na, Ca and K ions in each

sample were measured after standardization of flame photometer.

2.16 Determination of photosynthetic pigment (chlorophyll a and b) and total

carotenoids

Chlorophyll a, b and total carotenoides were determined by following the method

of Lichtenthaler and Wellborn [276]. Leaves samples were taken in Falcon tube of 15 ml

with 25 mg followed by the addition of MgO and 5 ml of methanol. The samples were

homogenized by using the shaker for 2 h and centrifuged at 4000 rpm for 5 minutes. The

supernatant was then taken in a cuvette and absorbance reading was taken by using UV-

VIS spectrophotometer at three different wavelengths (666, 653 and 470 nm). For

determination of photosynthetic pigments i.e. chlorophyll “a”, chlorophyll “b” and total

carotenoides, the following formulas were used.

Ca = 15.65 A666 – 7.340 A653

Cb = 27.05 A653 – 11.21 A666

Cx+c= 1000 A470 – 2.860 Ca – 129.2 Cb / 245

Where;

Ca = Chlorophyll “a”

Cb = Chlorophyll “b”

Cx+c= total carotenoidsoids

2.17 Determination of total nitrogen and total proteins

A 100 mg of dried plant material was digested using digestion mixture consisiting

of 1.0 gram iron sulphate, copper sulphate and potassium sulphate and 2 ml of

concentrated H2SO4. Mixtures were further heated in a beaker on hot plate until it became

colorless. The solution was cooled and added with 20 mL of distilled water. The digested

plant material was transferred to the distillation assembly and 3-4 mL of 30% NaOH

solution was added to it. The change of color of boric acid indicates the completion of

distillation process. The boric acid was titrated with 0.1 N sulfuric acid. The color of

boric acid was changed to violet which is the end point. A blank reagent of distilled water

was equally run and subtracted its volume from that of the sample titration volume.

Nitrogen and protein content was calculated by using the following formula:

N content (ug/g) = (H2SO4 mL /mL blank) × normality ×14.01

Dry weight of sample (g)

21

Total proteins = N2 x 6.25

2.18 Proline determination

In 3% aqueous sulphosalicylic acid, 0.01g/ 0.5 ml the frozen plant material was

homogenized and the residue was removed by centrifugation at 12000 rpm for 10

minutes. The reaction mixture was extracted with 2 ml toluene, mixed vigorously and left

at room temperature for 30 min until separation of the two phases. At room temperature,

the chromophore containing toluene (1ml, upper phase) was warmed and at 520 nm its

optical density was measured using toluene for a blank. The proline concentration was

determined from a standard curve using D-Proline.

2.19 Quantification of TBARS contents and H2O2

For determination of lipid peroxidation and H2O2 as reactive oxygen species

(ROS), 0.5 g of grinded leaves sample of young seedlings was used. Lipid peroxidation

was estimated and was determined as 2-thiobarbituric acid reactive substances (TBARS)

following the method of Zhou and Leul [277]. For evaluation of H2O2, 0.5 g of grounded

samples obtained from leaves of treated seedlings and were crushed with 5.0 ml of TCA

(0.1 %, w/v) in an ice-cold condition and the homogenate was centrifuged at 14,000 rpm

for 20 min [278]. Furthermore, 1 ml of supernatant was added in total 4 ml reaction

mixture, 1 ml phosphate buffer (pH 7.8) and 2 ml KI (1 M) and the absorbance was read

at 390 nm. H2O2 content was determined using an extinction coefficient of 0.28 μMcm-1

and expressed as µmol g-1 FW.

2.20 Total soluble proteins and antioxidants status

In order to determine the total soluble proteins and anti-oxidants like Ascorbate

peroxidase (APX), Superoxide dismutase (SOD), Catalase (CAT) and Peroxidase (POD),

0.5 mg of plant sample was homogenized in 8 ml of 50 mM potassium phosphate buffer

(pH 7.8). The crude extract was centrifuged at 14,000 rpm for 15 minutes at 4 oC and the

supernatant was used for the determination of total soluble protein and different

enzymatic and non-enzymatic antioxidants. Using the method of Bradford [279], total

soluble protein content was determined and bovine serum albumin was used as a

standard.

Using the method of Zhang et al. [280] the activity of SOD was determined

following the inhibition of photochemical reduction due to nitro blue tetrazolium (NBT).

The reaction mixture containing 50 mM potassium phosphate buffer (pH7.8), 13 mM

methionine, 75 mM NBT, 2 mM riboflavin, 0.1 mM EDTA-Na2 and 100 µl of enzyme

22

extract in a 3-ml volume. One unit of SOD activity was measured as the amount of

enzyme required to cause 50% inhibition of the NBT reduction measured at 560 nm.

APX activity was measured in a reaction mixture of 3 ml containing 50 mM

potassium phosphate buffer (pH7.8), 0.1 mM EDTA-Na2, 0.3 mM ascorbic acid, 0.06

mM H2O2 and 100 µl enzyme extract. After addition of H2O2, the change in absorption

was taken at 290 nm 30s [281]. CAT activity was measured according to Aebi [282] with

the use of H2O2 (extinction coefficient 39.4 mM cm-1) for 1 min at A240 in 3ml reaction

mixture containing 50 mM potassium phosphate buffer (pH 7.8), 2 mM EDTA-Na2, 10

mM H2O2 and 100 µl enzyme extract.

POD activity was analyzed by following the method of Zhou and Leul [283] with

minute modifications. The reactant mixture was consisted of 50 mM potassium phosphate

buffer pH7.8), 1% guaiacol, 0.4% H2O2 and 100 ml enzyme extract. Variation due to

guaiacol in absorbance was measured at 470 nm.

2.21 Statistical analysis

By using statistics 9 software, the statistical analysis of the data was performed by

using one way analysis of variance (ANOVA). The fisher’s least significant difference

(LSD) at P ≥ 0.05 level was used to analyze the differences between the means of

different parameters in maize seedlings under both saline and non-saline conditions.

23

RESULTS

PHYSIOLOGICAL PARAMETERS

3.1 Effect of plant-derived smoke solutions on seed germination

During the germination test, some differences were found in germination of

smoke primed and non-primed seeds. It was examined that maize seeds exposed to high

salt concentrations caused reduction in the germination. However, smoke primed seeds

gave highest germination percentage under normal control. Seeds started germination

within 24 h in control as well as smoke primed seeds. Present results revealed that highest

germination percentage (86%) was recorded in the control treatment while in treated

samples highest germination was recorded in 1:400 dilution of smoke after 36 hr (Fig.

3.1). Consequently, maximum germination 90% after 72 hours was recorded in the smoke

primed seeds treated with 50 and 200 mM of salt stress. Among the smoke dilutions,

1:400 of C. jawarancusa showed maximum germination 93% while in saline conditions

the recorded germination percentage was ranging from 53-45 % in salt solutions of 50,

100 and 150 mM, respectively.

Fig 3.1 Effect of smoke solutions (1:100, 1:200, 1:300, 1:400 and1:500) on germination

% in smoke primed seeds under normal and saline (50, 100, 150, 200 and 250 mM)

conditions after 36 hours

24

3.2 Effect of plant-derived smoke solutions on seedling growth

Growth of maize seedlings was influenced upon exposure to different

concentrations of salt. Increase in salt concentrations caused gradual decrease in

root/shoot length. The negative impact of salt stress on plant growth due to high salinity

level was further alleviated by using seed priming. A gradual imcrease in the salts

(NaCl) stress, resulted in gradual decrease in root length. Among which the most

adverse effect was observed in seedlings treated with salt concetration of 100 mM.

Furthermore, these adverse effects were considerably alleviated with smoke through

priming (Fig 3.2). Priming os seeds with 1:300 dilution, showed remarkable length i.e.

7.5 cm and the minimum length 0.5 cm were observed in shoot length subjected to 250

mM of salt stress. Maximum value was recorded in 1:100 smoke primed seeds with 100

mM of salt where length of shoot was recorded as 3.8 cm. Moreover, the shoot length

was reduced to 2, 1.9, 0.9 and 0.5 when seedlings were exposed to salinity levels of

100, 150, 200 and 250 Mm, respectively. Seedlings raised from smoke primed seeds,

showed increased root and shoot length in saline and non-saline treatments at 50 mM

salt stress. Beside these, a decrease at 100 and 150 mM salt stress was found as well

(Fig 3.2). The results indicated that smoke significantly alleviated the negative impacts

of the salt stress in maize seedlings.

25

Fig 3.2 Effect of Plant-derived smoke solutions (1:100, 1:200,1:300,1:400 and 1:500

dilutions) on root and shoot length of smoke primed maize seeds under normal and saline

(50, 100, 150, 200 and 250 mM) conditions.

3.3 Effect of plant-derived smoke on root and shoot fresh and dry weight

At different salinity levels, a decrease was observed both in fresh and dry weights

of non-smoke primed seedlings. Maize seedlings exposed to low level 50 mM of salt

stress did not showed toxic symptoms while higer concetrations depressed the growth

(Fig. 3.3). Maximum shoot fresh weight of 4.16g was observed at 1:200 dilution that

reduced to 0.08g at 250 mM salt stress compared to control 0.3 g. Moreover, 6.7, 6.9, 6.2

and 6.5g of shoot freshweight was observed in 150+1:100, 150+1:300, 150+1:400 and

150+1:500, respectively. Results depicted that increasing salinity level significantly

decreased both fresh and dry weight of shoot as compare to control. Moreover, 0.5, 0.6

and 0.5g of root freshweight was observed in 50+1:100, 50+1:200 and 150+1:400,

respectively. Furthermore, salinity affect root dry weights in maize and a decrease was

observed with increasing salinity level. Maximum root dry weight was found at 50+1:200

(0.6 g) as compare to control 0.3 g. Similarly, 0.3 and 0.4 g of root dry weight was

observed in 100+1:300 and 100+1:400, respectively. With increasing salinity level, dry

weight of root was decreased to 0.15, 0.013, 0.011, 0.09 and 0.07 g upon exposure to salt

concnetrations of 50, 100, 150, 200 and 250 mM, respectively. Maximum shoot dry

weight 3.8 g was found at 150 mM of salt and 1:300 dilution of smoke as compare to

26

control 0.154 g. In the treatments 150+1:200, 150+1:100 and 150+1:500 the observed

shot dry weights were 3.7, 2.8 and 2.6 g, respectively. However, minimum shoot dry

weight 0.052 g was found in seedlings exposed to 250 mM of salt stress while gradual

increase in dry weights were observed with the apllication of decreasing salt

concentrations.

27

Fig 3.3 Effect of smoke solutions (1:100, 1:200,1:300,1:400 and 1:500) on root and Shoot

length; fresh and dry weight (A, B, C and D) in smoke primed maize seeds under normal

and saline (50, 100, 150, 200 and 250 mM) condition.

3.4 Effect of Plant-derived smoke water on different ions contents Na+, K+ and Ca+2

Ions like Na+, K+ and Ca+2 were evaluated and obtained results revealed a clear

difference among the plants raised under different treatments i.e. control, smoked and

saline. Significant decreases were observed in both contents of potassium and calcium

while high level of sodium content found with increasing salt concentrations (Fig. 3.4).

Moreover, higher levels of potassium and calcium and lower value of sodium were

quantified in the seedlings raised under smoke dilutions as compared to control. In

control, the level of Na+ was 20 and 17.8 µg/g in both roots and leaves, respectively. In

plants raised under 1:400, the level of K+ and Ca+2 was found at highest range while,

lowest level of Na+ was observed in both roots and leaves. In case of root, subjected to

28

smoke solution of 1:400, the level of K+, Ca+2 and Na+ were 47.56, 38.65 and 18.8 ug g-1,

respectively as compared to control i.e. 41.5, 33.9 and 31.33 ug g-1, respectively. Present

results also demonstrated an increase in sodium ion while gradual reduction in K+ and

Ca+2 contents in seedlings subjected to increasing salt levels. It was observed the plant

derived smoke solution alleviated the severe effects of salt stress both in roots and leaves

in term of ion analysis. Similarly, in case of leaves, contents of K+, Ca+2 and Na+ were

12.4, 12.1 and 34.3 ug g-1, respectively observed in salt stressed seedlings. Low level of

K+ and Ca+2 while high level of Na+ were observed in plants leaves grown under high salt

content i.e. 250 mM. Overall present results depicted that the adverse effect of salt stress

in leaves were alleviated significantly through seed priming technique before

germination.

29

Fig 3.4 Effect of smoke solutions (1:100 and 1:400 ) on Na+, K+ and Ca+2 concentrations

in smoke primed seeds grown under normal and saline (100 150, 200 and 250 mM)

conditions.

3.5 Effect of plant-derived smoke water on chlorophyll and carotenoidse contents

Present results indicated that the contents of chl (“a” and “b”) in maize seedlings

exposed to non-saline, smoked and saline showed significant variations. Obtained results

indicated that plants rise from smoke (1:100 and 1:400) primed seeds possesses high

chlorophyll (a and b) contents in comparison with control. However, exposure to different

salt levels i.e. 50, 100, 150, 200 and 250Mm showed significant (p ≤ 0.05) decrease in the

chlorophyll a and b (Fig 3.5). In control, the levels of chl (a and b) were 4.14 and 10.76

ug g-1, respectively and were reduced i.e. 3.69, 3.04, 2.2, and 1.35 ug g-1 for chl a and

8.26, 7.85, 5.83 and 3.39 ug g-1 for chl b in gradually increased salt concentrations i.e.

100, 150, 200 and 250 mM, respectively.

Both the contents were found at high rate in seedlings raised from seeds soaked

with smoke dilutions of 1:100 and 1:400, showed significantly (p ≤ 0.05) increase i.e.

7.26 and 6.04 ug g-1 in comparison with untreated control 4.14 and 10.76 ug g-1,

respectively. Results related to biochemical analysis also indicated that exposure to

increasing salt levels 100, 150, 200 and 250 mM reduced the contents of chl a 3.69, 3.04,

2.2, and 1.35 ug g-1 and b 8.26, 7.85, 5.83 and 3.39 ug g-1, respectively. Meanwhile, the

situation was alleviated significantly i.e. chl a 4.0, 3.07, 2.9 and 2.58 ug g-1 and chl b

9.89, 9.51, 7.29 and 4.91 ug g-1, respectively in seedlings obtained from smoke primed

seeds.

Analysis of total carotenoidsoids content showed significant reduction in the

plants exposed to salt stress. Priming with smoke has increased total carotenoids levels

30

significantly i.e. 703 ug g-1 in 1:100 and 655.35 ug g-1 in 1:400 as compared to control i.e.

542.48 ug g-1. Increasing salt stress reduced the carotenoids contents and lowest level i.e.

194.35 ug g-1 was found in plants subjected to 250 mM of salt stress. Seeds primed with

smoke showed significant alleviation in salt stress 100, 150, 200 and 250 mM seedlings

by increased the levels from 455.11, 427.17, 270.63 and 194.35 ug g-1, respectively to

48.5, 455.99, 270.63 and 194.35 ug g-1 (Fig. 3.5).

Fig 3.5 Effect smoke solutions (1:100 and 1:400) on chl a, b and total carotenoids in

smoke primed seeds under normal and saline 100, 150, 200 and 150 mM conditions.

31

3.6 Effect of Plant-derived smoke water on total nitrogen and total protein contents

Biochemical analysis of protein and nitrogen contents in maize was determined in

smoke primed and unprimed seeds exposed to different salt concentrations. Results

revealed a gradual decrease both in nitrogen and protein contents subjected with

increasing salinity levels. Salt stress i.e. 100, 150, 200 and 250 mM, resulted decrease

levels of nitrogen in roots i.e. 61.63, 44.82, 38.01 and 21.2 µg g-1, respectively as well as

70.5, 57.42, 42.02 and 25.21 µg g-1 were observed in leaves with compared to control i.e.

98.4 µg g-1, respectively in root and 120.46 µg g-1 in leaves, respectively. Similar trend

was observed in case of protein contents (Fig 3.6).

Seedlings obtained from smoke primed seeds, showed high nitrogen and protein

contents. Seed primed with 1:400 showed maximum nitrogen contents both in root and

leaves (120.46 µg g-1 and 162.09 µg g-1, respectively and protein levels in roots and

leaves were 780.87 and 1013.06 µg g-1, respectively. Moreover, the present results

indicated that smoke solutions alleviated the inhibitory effect of salt stress.

Fig 3.6 Effect of smoke solutions (1:100 and1:400) on nitrogen and protein content in

smoke primed seeds under normal and saline (100, 150, 200 and 250 mM) conditions.

32

3.7 Proline contents:

Analysis of proline contents revealed a gradual increase with increasing

concentrations of salt. While seed priming technique with smoke dilution reduced the salt

stress by showing low proline contents i.e. in 1:100 it was 0.417 mg g-1 and in 1:400 it

was 0.381 mg g-1 as compared to control i.e. 0.372 mg g-1 (Fig.3.7). Similarly, the

priming of seeds with smoke significantly (p ≤ 0.05) decreased the level of proline as

compared to plant exposed to salt stress. Maximum level of proline 0.6 mg g-1 content

was found in seedlings exposed to 250 mM of salt solution.

Fig 3.7 Effect of smoke solutions (1:100 and 1:400) on nitrogen and protein content in

smoke primed seeds under normal and saline (100, 150, 200 and 250 mM) conditions.

3.8 Total soluble protein contents

Total soluble protein was determined in maize seedlings collected from control,

smoke primed seeds later on subjected to salt stress of different concentrations. With the

exception of 150 mM salt solution, all the other concentrations i.e. 100, 150, 200 and 250

mM reduced the level of TSP as compared with untreated control. Moreover, TSP

contents in maize seedlings raised from smoke primed seeds tended to increase as

priming with 1:400 smoke solution resulted an increase of 1.4-fold as compared with

untreated control. Similarly, the adverse effect of salt stress was significantly (p ≤ 0.05)

alleviated by smoke solutions (1:100 and 1:400) through seed priming and the resulted

1.7-fold increase in 100 mM with 1:100, no increase was observed in 200 mM with 1:100

and a decrease of 0.8-fold in 200 mM with 1:400 was observed. Furthermore, no

difference was observed in samples treated with 250 mM and 1:100 while 1.2-fold

increase was observed in 250 mM and 1:400 dilution (Fig. 3.8).

33

Fig. 3.8 Effect of Plant-derived smoke solutions (1:100, and 1:400 dilutions) on soluble

protein of smoke primed maize seeds under normal and saline ( 100, 150, 200 and

250mM) conditions.

3.9 H2O2 concentration

The effect of smoke solution and salt stress on the activity of (H2O2) as shown in.

(Fig 3.9). Results regarding H2O2 activity showed an increasing trend in the activity of

H2O2 in seedling subjected with increasing levels of salt stress i.e. in 100 mM the activity

was increased 3.4-fold, in 150 mM was 2-fold and in 250 mM a decrease of 0.6-fold was

observed as compared to control. Present experimental results showed significant

differences in H2O2 concentration raised under different treatments i.e. control, smoke

and saline condition. H2O2 was reduced in seedling raised from smoke primed seed and

later on exposed to salt concentrations. Alleviation of salt stress was accomplished with

pretreatment of seeds with smoke solution by priming seeds with 1:400 and later on

exposed to 150 mM of salt stress which enhanced up to 2-fold, priming with 1:100

followed by exposure to 250 mM resulted an increase of 2 fold and 250 mM and 1:400

showed 0.6 fold decrease in the activity of H2O2.

34

Fig. 3.9 Effect of Plant-derived smoke solutions (1:100 and1:400 dilutions) on H2O2

concentration of smoke primed maize seeds under normal and saline (100, 150, 200 and

250 mM) conditions.

3.10 Ascorbate peroxidase (APX) activity of maize under salt stress

Variations in the APX activity was observed in the maize seedlings raised from

smoke and unprimed seeds as well as salt treated and untreated seedlings (Fig. 3.10).

Gradual increase in the APX activity was observed with application of increased salt

levels i.e. exposure to 250 mM of salt stress resulted an increase of 1.3-fold with

compared to untreated control. Smoke solutions at a dilution of 1:100 resulted 1.5-fold

increase and 1:400 resulted 1.4-fold increase with compared to control. Furthermore,

priming with smoke solutions, alleviated the impacts of salt stress and resulted an

increase of 1.4-fold in samples primed with 1:400 smoke solutions and treated with 100

mM of salt stress, 1:400 and 250 mM resulted an increase of 1.3-fold while 250 mM

treatment and priming with 1:400 resulted an increase of 1.4 fold APX activity.

35

Fig. 3.10 Effect of Plant-derived smoke solutions (1:100 and1:400 dilutions) on APX

activity of smoke primed maize seeds under normal and saline (100, 150, 200 and 250

mM) conditions

3.11 Peroxidase activity (POD) of maize under Salt stress

Exposure of maize seedling to salt stress showed gradual increase with increasing

salt concentrations. Results related to percent increase of POD activity showed significant

increase in the activity while exposed to different concentrations of salt solutions i.e. 100,

150, 200 and 250 mM. POD activity was increased up to 1.2-fold in samples treated with

100 mM while treatments of 200 mM and 250 mM of salt showed no significant decrease

or increase in the POD activity. Little increase in POD activity was observed in seedlings

raised from smoke primed seeds i.e. 1.2-fold in 1:100 and 1:400 of smoke solutions.

Similarly, when smoke primed seeds were exposed to salt stress, alleviation was observed

in the activity of POD i.e. 1.2-fold increases in 150 mM and 1:100 primed seeds, no

increase was observed in the samples treated with 200 mM and smoke of 1:400 primed

seeds as well as in 250 mM of salt treated seedlings primed with 1:400 smoke solution.

Fig. 3.11 Effect of Plant-derived smoke solutions (1:100 and 1:400 dilutions) on POD

activity of smoke primed maize seeds under normal and saline (100, 150, 200 and

250mM) conditions.

3.12 Catalase activity (CAT) of maize under Salt stress

The CAT activity was also found under saline condition. In comparison to non-

saline, its average values enhanced in seedling upon exposed to 100 mM and 150 mM of

salt concentrations by 2.2-fold and 2.1-fold (Fig.3.12). Moreover, in seedlings primed

with smoke dilution of 1:400 enhanced CAT activity by 1.1-fold as compared with

control. Furthermore, priming with smoke followed by salt stress showed highest CAT

36

activity i.e. 3-fold in 150 mM and 1: 400, 1.4-fold in 200 mM and 1:100 while 1.4-fold in

250 mM and 1:100 with compared to control. Similarly, an increase of 2.1-fold was

observed in samples treated with 150 mM and 1.3-fold in 200 mM while no increase was

observed in samples treated with 250 mM.

Fig. 3.12 Effect of Plant-derived smoke solutions (1:100 and 1:400 dilutions) on catalase

activity of smoke primed maize seeds under normal and saline (100, 150, 200 and 250

mM) conditions.

3.13 Thiobarbituric acid reactive substances (TBARS) activity

The level of TBARS was observed in maize seedlings exposed salt stress and

smoke solutions. As considered to controls, the TBARS contents of the salt stress clearly

increased in seedlings exposed to 100 mM by 1.2-fold, 150 mM by 1.2-fold and 250 mM

by 1-fold (Fig 3.13). Smoke solution at dilution 1:400 decreased 0.8-fold and 1:100 by

0.8-fold as compared to the control. Similarly, in samples exposed to 150 mM and 1:400

showed an increase of 1.1-fold while treatments of 200 mM and 1:400 shwed no

significant alteration in the level of TBARS.

Fig. 3.13 Effect of Plant-derived smoke solutions (1:100 and 1:400 dilutions) on TBARS

activity of smoke primed maize seeds under normal and saline (100, 150, 200 and 250

mM) conditions.

37

3.14. Correlation amongst the parameters

The correlation among different parameters showed in Table 1 – 3. Statistical correlation

analysis of physiological parameters showed that shoot dry weight is positively and

significantly correlated with root length, root fresh weight, shoot fresh weight and root

dry weight. Similarly root dry weight is significantly correlated with root length, root

fresh weight and shoot fresh weight. Shoot fresh weight is positively correlated with root

length and root fresh weight. Root fresh weight is significantly correlated with root

length.

Table 1. Correlation between physiological parameters

Ger RL SL RFW SFW RDW SDW

Germination (GR) 1 -.268 .201 -.359 -.284 -.256 -.235

Root length (RL) 1 .017 .785** .954** .995** .936**

Shoot Length (SL) 1 -.383 .071 .009 .024

root Fresh Wt. (RFW) 1 .767** .783** .755**

Shoot Fresh Wt. (SFW) 1 .958** .980**

Root Dry Wt. (RDW) 1 .944**

Shoot Dry Wt. (SDW) 1

**. Correlation is significant at the 0.01 level (2-tailed).

Correlation among the biochemical parameters have been incorporated in table 2. The

analysis of correlation data showed that proline is significantly correlated with Na, K, Ca,

N, P and total carotenoids. Total carotenoids are significantly correlated with Na+, K+,

Ca+2, N, and P. Similarly, protein is negatively correlated with Na+, and positively with

K+, Ca+2 and nitrogen. N is negatively correlated with Na+ and positively correlated with

K+ and Ca+2. Ca+2 are negatively correlated with Na+ and positively with K+. Finally, K+

is negatively correlated with Na+.

38

Table 2. Correlation between Biochemical parameters

Na+ K+ Ca+2 N Protein Chl a Chl b

Total

carotene. Prol

Na+ 1 -.812** -.841** -.879** -.779** -.020 -.043 -.845** .835**

K+ 1 .925** .931** .905** .021 .314 .865** -.752**

Ca+2 1 .913** .899** -.076 .258 .860** -.788**

N 1 .918** .031 .356 .851** -.815**

Protein 1 -.019 .462 .847** -.749**

Chl a 1 .140 .239 .071

Chl b 1 .251 -.103

T.Caroten. 1 -.779**

Prol 1

**. Correlation is significant at the 0.01 level (2-tailed).

The overall correlation of physiological and biochemical parameters related data

showed that N in roots is significantly positively correlated with root fresh weight in

leaves, Ca ions in leaves and protein also significantly positively correlate with total

carotenoids in leaves, protein in leaves and in root and Ca in root Ca root positively

correlated with shoot length leaves, ca leaves. Similarly, with total carotenoids and Ca in

roots. K in root significantly correlates with Na in the leaves. Ca in leaves and protein in

leaves. Na in root significantly correlates with Na in the leaves. Proline had a negatively

correlation with germination. Toatl carotenoids have positive correlation with Na in

leaves, K in leaves, and Ca in leaves. Furthermore, Protein in leaves had a positive

relationship with root length, K ion in leaves. Similarly, N level in leaves had a positive

correlation with K ion level in the leaves. Shoot dry weight had a positive correlation with

root length and root fresh weight. Moreover, root dry weight had a positive relationship

with root length while, shoot fresh weight had a positively correlation with root fresh

weight and in turn root fresh weight had a positive relationship with root length.

39

Table 3. Correlation between physiological and Biochemical parameters

*. Correlation is significant at the 0.01 level (2-tailed).

**. Correlation is significant at the 0.01 level (2-tailed).

GR (Germination), RL (Root length), SL (Shoot length), RF (Root fresh weight), SF

(Shoot fresh weight), RD (Root dry weight), SD (Shoot dry weight), Na+ (Leaves), K+

(Leaves), Ca+2 (Leaves), NL (Nitrogen in leaves), PL (Phosphorous in leaves), Pro

(Protein).

40

DISCUSSION

Smoke derived from combustion of plant parts has been reported a metabolic regulator

and play an important role in the seed germination as well as other plant developmental

processes. Recently, smoke is well-known for promoting plant growth and is used for

vigorous plant growth in agriculture industries [284]. Smoke improves plant growth by

enhancing seed germination, root, and shoot growth, flowering production and somatic

embryogenesis, [285,286].

Salinity affects large area around the globe and causing a significant reduction in

the yield of different agricultural crops [287]. Salinity declines the plant growth by

influencing both plant physiology and biochemistry [288]. In the present study,

experiments were designed to check salt induced stress in maize seedlings and its

alleviation by seed priming with smoke solutions on various growth parameters.

Seed germination is considered as an important stage in the life sequence of plants

which determines further increase in the final crop yield [289] (Fig .3.1). Obtained results

revealed that seed germination in maize was adversely affected with increase in the level

of salt while smoke primed seeds showed significant improvement in the overall

germination percentage. Improvement in the seed germination by priming seeds with

smoke solutions could be attributed due to the possible involvement of butanolides by

bringing change in the cell membrane permeability of developing embryo to ionic stress

of salt. Our results are in lined with the previous study of Aslam et al. [290] and Omami

[291] who demonstrated that salt stress negatively affect the overall germination and post

germination growth with increasing level of salt stress [292].

The possible mechanism through which salt stress can affect the growth of plant is

through osmotic imbalance which ultimately reduces the potential of water uptake

resulted reduced plant growth. Furthermore, reduction in the growth might be due to high

accumulation of Na+ ions in shoot and root tissues exposed to salinity while seedlings

raised from smoke primed seeds showed alleviation in the Na+ stress due to presence of

growth regulators in smoke. Moreover, it was previously studied that plants exposed salts

at lower concentration can endure salt stress for longer duration before significant

decrease of root and shoot growth [293]. It was determined that active compound present

in smoke solution shorten the germination period of the seed [294]. Priming of seeds with

smoke increases germination rate, growth and physiology in plants [295]. Such

enhancement credited to nutrient effect on metabolism and enzymatic activates which in

turn promotes growth of the plant. Our results agree with findings of Brown and van

41

Staden [296] which observed that seeds germination can be improved with the application

of more diluted smoke dilutions. Similarly, improved development was found when

smoke solution was applied on Erica arbora species [297] and the same results were

observed in the South African indigenous medicinal plants. De Lange and Boucher [298]

stated that smoke has the capability to enhance seed germination by rupturing seed

dormancy in Audouinia capitata, one of the fynbos species of South Africa.

In arid/semi-arid regions of the globe, salinity is a main ecological restraint to the

yield [299]. Reduction in the yield and growth character was found in maize grown in

saline area. Salinity damages the root and shoots growth of plants [300-301] impaired cell

division due to low movement of reserve foods [302] and damage to the hypocotyls.

Toxic ions like Na+ and Cl- destroy the plant cells [303]. During different developmental

stages exposure of plants to a stressful environment seen to motivate different cytological

changes. [304]. Under high saline conditions root development is susceptible and root

growth decreased [305.306]. It was also suggested the reduction in the seedling growth

due to salt stress could be attributed ionic toxicity [307] or decrease in the absorption of

water [308]. Results also revealed that salt induced stress caused phytotoxicity due to

ionic damages of Na+ and Cl- in maize seedlings and in in lined with the findings of

Chavan and Karadge [309] and Turan et al. [310]. In the present study, salt stress severely

affected shoot and root length with increasing levels of salt stress.

Maize seedlings raised from smoke (1:100) primed seeds when exposed to salts

stress of 100 mM showed alleviation in the adversity of salt stress (Fig. 3.2). Smoke

solution is a potential stimulant that promotes germination and seedling vigor [311].

Smoke soaked seeds enhanced the root and shoot growth could be because of growth

regulator acting like growth enhancing hormones. Earlier studies indicated that smoke

solutions show hormone like characteristic in different species and acting in combination

with the auxins, cytokinins, gibberellins, Abscisic acid ABA and ethylene in different

plant species [312]. Our findings are similar with the findings of Kepezynski et al. [313],

which representing that butenolide also acquire hormone like characteristic and plays an

important role in improvement in the DNA replication, cell prolongation and cell

division. It has been observed that smoke and smoke solution effect the post germination

process resulting improvement in the seedling vigor [314]. In the present investigation

root elongation was observed in seedlings raised from smoke primed seeds even when

exposed to 50 mM of salt stress. However, decrease in the root length at low level of

water potential that could be thought as reasonable response to the salinity and drought

42

[315,316]. Nutrient medium having high concentration of salt causes decrease in the

exposed plant growth [317,318]. Similarly, decrease in the biomass of barley plant has

also been observed upon exposure to salt stress [319]. Plant growth under saline

conditions, decreases in seedling fresh and dry weight at different salinity level. Our

findings are also in correlated with the studies of Naseer et al. [320] who demonstrated

that germination rate and seedling vigor reduced in barley varieties with increasing levels

of salt stress. Upon exposure to sodium and chlorine ions, rice plants showed a significant

decrease in the plant fresh and dry biomasses. Enhancement in growth due smoke

application also suggested a possible involvement of K+ and Ca+ ions that mediated the

adverse effects of Na+ and Cl- in the plants exposed to salinity. Our data are also in

agreement with the studies of Nitika et al [321] who demonstrated that salt stress

decreases the total dry weight in the exposed seedlings.

In present study ions analysis showed that potassium and calcium contents were

decreased in the samples treated with salt stress (Fig. 3.3). The relation of (K+/Na+) was

considerably affected by high level of NaCl application. High level salt stress creates

highly detrimental effects in the exposed plants [322]. The Na+ and Cl- accumulation

interferes the intake of nutrients and in turn causes toxicity in the plants. Na+ decreases

the absorption of micronutrients i.e. K+ and Ca2+ by hinders their availability to the plants.

Yildrim et al. [323] reported that salinity decreases macro nutrients in the plants,

particularly Ca+2 and K+ accessibility and reduces Ca+2 and K+ access to the newly

emergent regions in the plants during initial growth. Greenway et al [324] also reported

that ions imbalance during salinity stress also disturb the intake of some other nutrients

essential for the growth of plants. Furthermore, exposure to increased salt stress resulted a

decline in the availability of both Ca+2 and K+ also stated by Taffouo et al. [325].

Greenway and Munns [326] reported that adverse impact of salinity is related with

increased Na+/ Cl- contents in the exposed plants which in turn influence the intake of

other essentials nutrients. Among the essential nutrients Ca2+ has been revealed decrease

the negative impacts of salinity in plants showing its regulatory roles in the plants

metabolism [327]. Moreover, it has been reported that salinity also declines the contents

of K+ in rice plant tissues [328].

It was observed that reduced Na+ level and increased Ca+2 and K+ ions was the

alleviating effect of smoke priming in maize seedlings. Accumulation of useful ions like

K+ and Ca2+ and reduction in the toxic ions i.e. Na+ and Cl- was considered as

ameliorating impact of smoke on maize seedlings (Fig. 3.4). Taffouo et al. [329] also

43

found that salinity causes reduction in the K+, Ca2+ and increase in the Na+ and Cl-

contents. Ashraf and Rasul [330] also reported that under higher salinity level, inhibition

of enzymes necessary for chlorophyll production occurs that in turn causes instability of

pigments protein complex and chloroplast got damage. Findings of the present results are

in lined with the results of El.Samad et al. [331] who studied that salt stress causes a

delay in the development and injuries in the bean plants. The photosynthetic pigment was

reduced in plants exposed to salt stress. It has been reported that salinity seriously

disturbed the level photosynthetic pigments in the exposed plants [332]. Our findings are

in favor with the findings of Munns [333] who reported that under saline condition a

decline in the chlorophyll contents could be linked to photo inhibition or ROS formation.

With increasing salt level, a gradual decline in the photosynthetic pigments were

observed with the application of 200 mM and 250 Mm of salt concentrations. Similar

trends were investigated by Sheng et al. [334] in maize seedings exposed to salt stress.

The results of Hamida [335] also in lined with our results who investigated that salt stress

affect the overall development of plats and causing injury in the content of photosynthetic

pigments in bean plants. Singh and Dubey [336] also demonstrated that loss of

chlorophyll ‘a’ in susceptible rice cultivars lead to decrease in the total chlorophyll

contents mainly credited to salt stress and also been found in barley [337].

According to Harris et al. [338] reported that smoke treated seeds tend to

germinate with faster rate and plays a pivotal role in drought resistance. Seedling of

raised from smoke primed seeds showed positive significant difference with untreated

seedlings [339]. It could be suggested that smoke and enzyme pool are inter linked and

dependable for the development of photosynthetic pigments that in turn strengthen the

seedlings to minimize the toxic impacts of salts stress.

It was suggested that K+ plays pivotal role in the production of protein [340]. In

saline condition synthesis of protein content become slowly down and loss in the

potassium content leads to decrease in the total protein content in maize. Results revealed

that gradually decrease in total protein and nitrogen content was observed upon exposure

to salinity stress which are in lined with the findings of Naseem et al. [341] who studied a

gradual decline in the contents of nitrogen and protein under elevated level of salt stress

in Cucumis sativus L. Protein synthesis has been considered as a key target of salt toxicity

as also reported by Gulen et al. [342]. Some studies reported that under soil salinity,

protein formation enhanced [343,344], while others reported a decrease [345] or remained

unaffected [346]. Tucker [347] stated that total N contents increased in different parts of

44

tomato with increasing salinity level. With the increase salt concentration N and protein

content were also decreased and exposed to 250 mM resulted highest decline in both the

N and protein contents. Saline condition drastically affects both the N and protein

contents in both root and leave tissues of maize seedlings (Fig 3.6). These findings are in

favor with the findings of Lutts et al. [348] who found that protein biosynthesis severely

reduced under salt stress.

According to Giriga et al [349] synthesis of proline strengthens the plant against

different abiotic stresses. The main defense reaction in plant to maintain the osmotic

pressure in plant is the synthesis and induction of proline that has been reported in salt

tolerant/ susceptible cultivars of numerous crops [350-353]. Saline stress effect the

proline content in different plants like canola, rice and wheat [354]. During the present

study, analysis of variance showed that increase in the salinity level resulted an increase

proline content and are agreed to the findings of Girija et al. [355] who reported that

accumulation of proline in plants may provide energy to increase the stress tolerance

potential in the exposed plants. Seed priming technique with smoke dilution of 1:100 and

1:400 reduced the salt stress by showing low proline contents (Fig.3.7). Similarly, the

priming of seeds with smoke significantly (p ≤ 0.05) decreased the level of proline as

compared to plant exposed to salt stress. High proline accumulation was observed in the

salt tolerant variety of maize as compared to salt sensitive variety [356]. According to

Bandeolue [357] reported that gradual increase in the salinity levels, proline accumulation

has also been raised in plant tissues. In a number reports it has been noticed that proline

accumulation in plants improve with increase in the salt stress [358]. In salt, sensitive and

tolerant mulberry leaves proline accumulation occurs in saline condition. Under salinity

the level of increase of free proline accumulation was upper in the tolerant cultivar

compare to the sensitive variety [359].

Previous studies reported that H2O2 pretreatment to the seeds enhance the process

of seed germination [360,361]. It is possible that H2O2 present in the pericarp promotes

seed germination [362]. Our results showed that H2O2 level was considerable enhanced in

salt exposed seedlings which in turn stimulated the activity of catalase and ascorbate

peroxidase in the seedlings. It was also analyzed that application of salt promotes H2O2

activity many fold as compared to untreated control. Furthermore, alleviation of salt stress

was accomplished with pretreatment of seeds with smoke solution (1:400 and 1:100) and

followed by salt application. It is commonly believed the antioxidant system activated

during seed germination declares a significant increase in the activity of catalase and

45

superoxide dismutase of maize seeds, due to the ROS-induced antioxidant gene

expression [363]. In wheat, improvement in the antioxidants activity was observed during

germination indicating promontory role during germination due to detoxification of H2O2

[364]. It was previously observed that during abiotic stress, H2O2 level increased in the

exposed soybean leaf tissues. Exposure to salt stress caused decline in the water potential

which may be attributed due increased level of H2O2 accumulation in the tissues. Hence,

dryness of tissues occurs by H2O2 mobilize within the cells due lipid peroxidation [365].

(Fig.3.9). in wheat susceptible salt stress, high level of H2O2 was observed with compared

to salt tolerant wheat variety [366]. Study also indicated that exposed of rice leaves to salt

stress did not cause any accumulation of H2O2 which may be credited to the detoxification

mechanisms of the tolerant nature of the rice variety [367]. Effective peroxide content and

lipid peroxidation level enhanced due to the salt treatment from the control of Oryza

sativa L. [368]. Hydrogen peroxide has been shown to enhanced under NaCl stress in

many studies in different plants. In salt susceptible wheat, H2O2 ions have been found to

enhance clearly than in the salt tolerant wheat [369].

APX activity is the main component of the antioxidant system and performs main

function to eradicate H2O2 level in different plant sub cellular domain (Fig.3.10) [370].

During salt stress APX activity considerably increased in the leaves that initiates at mild

salinity level and gradually increased with rise in the salt level. Results also depicted that

exposure to higher level of salt caused many fold increase in the APX activity as

compared to untreated control. Furthermore, application of smoke solutions (1:100 and

1:400) as seed pretreatment alleviated salt stress by increasing the activity of APX. APX

in citrus is the major enzyme that determining salts tolerance and its fundamental activity

much high in salt tolerant varieties. In pea cultivar, high APX activity has been recorded

[371], under salt stress indicating its role in salt tolerance mechanism.

POD activity in higher plants promoted where it is concerned in different

processes, including lignifications, auxin stress, salt tolerance and heavy metal stress

[372]. During growth alteration, POD often plays role as a of metabolism activator.

Effects of salt stress on POD activity in the root and shoot tissues were shown in Fig.

3.11. Results revealed that activity of POD was increased many fold in the maize seedling

upon salt exposure. Moreover, POD activity was also raised when seeds were primed

with 1:400 and 1:100 of smoke solutions. Furthermore, seed raised from smoke priming

followed by salt application showed manifold higher level in the activity of POD. POD

46

isozymes were found to available in variety of plant tissues and is progressively regulated

or affected by different environmental factors [373].

In the detoxification of hydrogen peroxide into water and oxygen, CAT has an

important role which is the most effective antioxidant produced during oxidative stress

[374,375] in Cassia angustifolia L. [376], Zea mays L. [377] and Sesamum indicum

[378]. Different plant species has various CAT activities, metabolic and developmental

position in plant as well as during period and severances of the privileged stress [379]

which also favors the present findings. In tomato CAT activity has been found to increase

under salt stress [380], in soybean, [381], in tobacco [382,383], in cucumber, [384], in

mulberry [385], in potato and rice [386]. Present results also depicted that smoke priming

enhanced the values of CAT in maize seedlings (Fig.3.12). Moreover, stress of salt was

significant alleviated by smoke priming in through increased CAT activity.

For lipids peroxidation MDA is consider as a marker for estimation of lipid

peroxidation that increases in plants when exposed to abiotic stresses. Lipid peroxidation

connected to the antioxidant enzymes activity i.e. with the increase of antioxidants in

plants, tolerance to oxidative stress has been found to increase and the level of MDA is

reduced. In the present investigation, level of MDA was increased upon exposure to salt

stress. In the Alvand amount of MDA increased with the enhanced salt stress. In order to

investigate the tolerance of plants to oxidative stress and the sensitivity of plants to

salinity, lipid peroxidation in terms of MDA can be used as strong biomarker [387]. In

many species, the level of membrane damages different due to the tolerance of plant

species against salt. For example, in rice, in salt stress resulted in no enhancement in lipid

peroxidation and conductivity [388]. Results depicted that MDA contents in the salt

stress seedlings clearly increased many fold upon exposure to 100 mM, 150 mM, 200

mM and 250 mM of salt levels (Fig 3.13). Furthermore, smoke solutions i.e.1:100 and

1:400 decreased MDA contents with compared to salt stressed and control. Present results

clearly depicted the adverse effects of salt stress to maize seedlings and the also

determined the alleviation of salt stress through seed priming with smoke solution.

47

CONCLUSIONS

It is hereby concluded from the observations made during this study that salt stress

affects the maize seedlings by reducing the overall growth and development in the

exposed plants. Moreover, salts stress induced the oxidative stress which in turn activated

the antioxidant system in exposed maize seedling. It was determined that smoke solution

acted as an important remedy for alleviation of salt stress. As salt stress severely affected

all the growth parameters in maize seedlings and this adverse effect were alleviated

successfully by priming of seeds with smoke solutions. Furthermore, priming with

Cymbopogon smoke positively improved the various physiological and biochemical

attributes in the maize seedlings and alleviated the adverse effects of salt stress. Smoke

priming improved the antioxidants enzymes activities which in turn improved the growth

of maize seedling in saline condition. Findings of this study are useful in the amelioration

of salinity affected areas by the application of smoke solution as salinity stress alleviator.

FUTURE PERSPECTIVE

Smoke has the potential to be used for a variety of applications such as

horticultural and agricultural industries, to produce more healthy and vigorous crops. In

future, priming with desirable plant-derived smoke solutions can be applied in saline

fields to minimize the adverse effects of salinity to get better crop production. The use of

smoke-technology might facilitate early harvests and increases in crop productivity, could

assist cultivation of crops in arid and semi-arid regions, and may be a cost-effective and

affordable method of crop improvement which can improve the standard of life of poor

farmers.

48

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